Bok:978 3 319 30906 4

ElenaMikhailovnaEgorova
AslanAmirkhanovichKubatiev
VitalyIvanovichSchvets
Biological
Effects
of Metal
Nanoparticles
Biological Effects of Metal Nanoparticles
Elena Mikhailovna Egorova
Aslan Amirkhanovich Kubatiev
Vitaly Ivanovich Schvets
Biological Effects of Metal

Nanoparticles
123
Elena Mikhailovna Egorova Vitaly Ivanovich Schvets
Institute of General Pathology Moscow State Academy of Fine Chemical
and Pathophysiology Technologies
Moscow Moscow
Russia Russia
Aslan Amirkhanovich Kubatiev

Institute of General Pathology
and Pathophysiology
Moscow
Russia
ISBN 978-3-319-30905-7 ISBN 978-3-319-30906-4 (eBook)

DOI 10.1007/978-3-319-30906-4
Library of Congress Control Number: 2016933474
Springer International Publishing Switzerland 2016

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Contents
Part I Preparation and Characterization of Metal Nanoparticles

in Solutions
1 Methods of the Nanoparticle Preparation in Solutions . . . . . . . . . . . 3
1.1 Introductory Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Principles Used for the Choice of Methods . . . . . . . . . . . . . . . . . 5
1.3 Chemical Synthesis with Traditional Reducing Agents . . . . . . . . . 7
1.3.1 General Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.2 Synthesis in Water Solution . . . . . . . . . . . . . . . . . . . . . . 9
1.3.3 Synthesis in a Two-Phase System and in Non-aqueous
Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
1.3.4 Synthesis in Reverse Micelles . . . . . . . . . . . . . . . . . . . . . 34
1.4 Photo and Radiation-Chemical Synthesis . . . . . . . . . . . . . . . . . . 45
1.5 Electrochemical Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
1.6 Biological Reduction in Water Solution . . . . . . . . . . . . . . . . . . . 60
1.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2 Biochemical Synthesis of Metal Nanoparticles . . . . . . . . . . . . . . . . . 79
2.1 Prerequisites of the Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.1.1 General Information on the Properties of Flavonoids . . . . . 80
2.1.2 AOT/Isooctane Reverse Micelles . . . . . . . . . . . . . . . . . . . 82
2.2 General Scheme of the Synthesis . . . . . . . . . . . . . . . . . . . . . . . . 84
2.3 Peculiarities of the Experimental Procedure . . . . . . . . . . . . . . . . . 84
2.3.1 Preparation of the Flavonoid Micellar Solution . . . . . . . . . 85
2.3.2 Extinction Coefcient Determination for the Flavonoids
in Micellar Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
2.4 Examples of the Nanoparticle Synthesis . . . . . . . . . . . . . . . . . . . 88
2.4.1 Silver Nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
2.4.2 Gold Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
2.4.3 Copper Nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . 103
v
vi Contents
3 Development of the Biochemical Synthesis for Practical

Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
3.1 Determination of the Extinction Coefcient
for Ag Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
3.1.1 Extinction Coefcient of the Complex . . . . . . . . . . . . . . . 111
3.1.2 Extinction Coefcient of the Nanoparticles . . . . . . . . . . . . 113
3.2 The Effect of CQr/CAg Relation on the Formation Rate
and Yield of Silver Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . 114
3.3 Preparation of Small Nanoparticles with Narrow Size
Distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4 Preparation of Metal Nanoparticles in Water Solutions
on the Basis of Biochemical Synthesis . . . . . . . . . . . . . . . . . . . . . . . 125
4.1 Preparation of Nanoparticles as Water Dispersions
by the Transfer from Micellar Solution. . . . . . . . . . . . . . . . . . . . 126
4.2 Properties of Silver and Gold Nanoparticles in Aqueous
Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.2.1 Silver Nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.2.2 Gold Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
4.3 Synthesis of Silver and Copper Nanoparticles in Water
Solution with Natural Stabilizers . . . . . . . . . . . . . . . . . . . . . . . . 133
4.3.1 Silver Nanoparticles Stabilized with Starch . . . . . . . . . . . . 134
4.3.2 Silver Nanoparticles Stabilized with Cyclodextrin . . . . . . . 137
4.3.3 Copper Nanoparticles Stabilized with Starch . . . . . . . . . . . 142
4.3.4 Comparison with the Data Found in Literature . . . . . . . . . 144
5 Materials Modied with Metal Nanoparticles . . . . . . . . . . . . . . . . . 149
5.1 Creation of Liquid-Phase Materials with Metal Nanoparticles . . . . 151
5.2 Solid Materials with Ag and Cu Nanoparticles . . . . . . . . . . . . . . 154
Part II Biological Effects of Metal Nanoparticles

6 The Effect of Metal Nanoparticles on Biological Objects
(Analysis of the Literature). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
6.1 General Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
6.2 Metal Nanoparticles Effects Observed on Microorganisms. . . . . . . 168
6.2.1 Antimicrobial Activity of Ag Nanoparticles . . . . . . . . . . . 178
6.2.2 Antimicrobial Activity of Cu, Au, and Metal Oxide
Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
6.3 On the Mechanism of Antimicrobial Activity of Metal
Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Contents vii
7 Antimicrobial Activity of Nanoparticles Stabilized with Synthetic

Surfactant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
7.1 Ag and Cu Nanoparticles in Liquid Medium . . . . . . . . . . . . . . . . 220
7.1.1 Varnish-Paint Materials with Biocidal Properties . . . . . . . . 220
7.1.2 Water Solutions of Ag Nanoparticles . . . . . . . . . . . . . . . . 225
7.1.3 Solid Materials and Polymer Films
with Ag Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . 228
8 The Effect of Silver Nanoparticles on Some Objects
from the Plants and Fungi Kingdoms. . . . . . . . . . . . . . . . . . . . 235
8.1 Plasmodium of the Acellular Slime Mold Physarum
polycephalum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
8.2 Unicellular Alga Chlorella vulgaris . . . . . . . . . . . . . . . . . . . . . . 245
8.3 Plant Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
9 The Effect of Silver Nanoparticles on Cultured Human Cells . . . . . . 249
9.1 Toxicity of Starch-Stabilized Ag Nanoparticles for HEF Cells . . . . 250
9.2 Toxicity of AOT- and Cyclodextrin-Stabilized Ag
Nanoparticles for UVE Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
9.3 Toxicity of AOT-Stabilized Ag Nanoparticles for HeLa
and U937 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
Inferences from the Results of Our Studies . . . . . . . . . . . . . . . . . . . . . 263
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
About the Authors
Elena Mikhailovna Egorova Doctor of chemical

sciences, head of the laboratory of nanopathology and
biomedical nanotechnologies of the Institute of General
Pathology and Pathophysiology (Russian Academy of
Medical Sciences). Her scientic interests lie in the
eld of physical chemistry of ultradisperse systems,
methods of production and properties of metal
nanoparticles in solutions, biological activity of metal
nanoparticles and nanoparticle-modied materials.
Author of the original method of synthesis of metal
nanoparticles which opens wide perspectives for their
practical application.
Aslan Amirkhanovich Kubatiev Doctor of medical
sciences, professor, academician of the Russian
Academy of Sciences (RAS) and Russian academy of
medical sciences (RAMS), member of RAMS presid-
ium, laureate of the RF State prize, Director and Head
of the department of molecular and cell pathophysiology
of the Institute of general pathology and pathophysiol-
ogy RAMS, Chief of the department of general pathol-
ogy and pathophysiology of Russian medical academy
of postgraduate education of the RF Ministry of Health,
chairman of RAMS Scientic Council for general
pathology and pathophysiology, president of Russian
scientic society of pathophysiologists, chief editor of the journal Pathogenesis.
Specialist in general, molecular and cell pathophysiology, nanobiology,
nanopathology, physiology and pathology of hemostasis.
ix
x About the Authors
Vitaly Ivanovich Schvets Doctor of Chemical

Sciences, Full Professor, Academician of the Russian
Academy of Sciences (RAS), laureate of the USSR
state prize in science and engineering, laureate of the
Prize of the Russian Federation Government in the eld
of education. Head of the Department of innovative
technologies at the Institute of General Pathology
and Pathophysiology of RAMS, Head of the
Department of biotechnology and bionanotechnology
at the Lomonosov Moscow State University of Fine
Chemical Technologies, nowadays Professor of the
Department of biotechnology and industrial pharmacy
at the Moscow Technological University. Area of research interests: development
and production of modern and effective pharmaceuticals based on biologically
active compounds using methods of bionanotechnology.
Abbreviations
AAS Atomic absorption spectroscopy

AA Ascorbic acid
AgNP, SNP Silver nanoparticles
AOT bis(2-ethylhexyl)sulphosuccinate, sodium salt
ASAS Anionic surface active substance
BSA Bovine serum albumin
CD Cyclodextrin
CFU Colony forming units
CTAB Cetyl trimethyl ammonium bromide
DDS Dodecyl sulphate
DLS Dynamic light scattering
ED Electron diffraction
FCC Face-centered cubic (lattice)
FTIR Fourier transform infrared spectroscopy
GA Gallic acid
HAuCl4 Tetrachloroauric acid
HDEHP bis(2-ethylhexyl) phosphoric acid
HDTAB Hexadecyltrimethyl ammonium bromide
HRTEM High Resolution Transmission Electron Microscopy
LD Lethal dose
LPSA Laser photo-stimulated aggregation
Mo Morin
NSP Nanosized particles
PAA Polyacrylic acid
PAAm Poly(amidoamine)
PALA Poly(allylamine)
PCS Photon correlation spectroscopy
PEG Polyethylene glycol
PEI Polyethylene imine
PEOPPOPEO poly(ethylene oxide) and poly(propylene oxide) triblock
copolymers
xi
xii Abbreviations
PNIPAM poly(N-isopropylacrylamide)
PPI-G3 poly(propylene imin) dendrymer
PVA Polyvinyl alcochol
PVP Polyvinylpyrrolidone
QAC Quaternary ammonium compound
Qr Quercetin
RD X-ray diffraction
Ru Rutin
SAS Surface active substance
SAXS Small angle X-ray spectroscopy
SEM Scanning electron microscopy
SPAN80 Sorbitan monooleate (SAS)
TAABr Tetraalkylammonium bromide
TBBT 4,4'-thiobis(benzenethyol)
TEM Transmission electron microscopy
TOAB Tetraoctylammonium bromide
TT Toxicity threshhold
VPM Varnish-paint materials
e(aq) Hydrated electron
max Wavelength at the absorption band maximum
w = [H2O]/[AOT] Hydration extent
Extinction coefcient
l Optical path length
NP, Yield of nanoparticles
CAg, CAOT, CQr Concentration of silver salt, AOT, quercetin, respectively
Introduction
The development of science and technology over the past decade is characterized
by intensive studies on the properties of nanosized objects and by the elaboration of
different ways for their practical application. Analysis and generalization of these
studies as well as a number of successful inculcations of nanoindustry products
suggest [13] that further progress in this direction will help to solve a lot of
problems which faces the mankind, both at present and in future. At the same time,
there is no doubt that the widespread application of nanotechnologies, which
actually means the transition to a new and higher level of scientic and techno-
logical progress, has its reverse side which is fraught with new dangers associated
with manifestations of understudied or not yet known properties and characteristics
of the behavior of nanoscale objects and systems.
One of these dangers quite clearly recognized today is the poor knowledge of
nanoparticles and nanomaterials influence on living organisms, including human
beings. Hence it becomes important to determine the conditions of nanotechnology
products application that are safe for humans, the problem which has been repeatedly
discussed in numerous scientic works [46] and documents of healthcare organi-
zations in different countries including Russian Federation [4, 7, 8]. It is emphasized
that unique properties of nanoparticles, such as small size, large specic surface area,
and high reactivity offer wide opportunities of their application for the good of
society, but may hide simultaneously serious risks for humans. Hence follows that,
to estimate hazards and to reduce possible risks, systematic studies of biological
effects of nanoparticles and nanomaterials are necessary, rst of all determination
of the degree of toxicity to humans upon various practical applications.
These general considerations are supported by the results of bioactivity studies
fullled with nanoparticles of different naturemetals, metal oxides, polymers, and
others. In this book, attention is paid chiefly to metal nanoparticles. The main
reason is that metal nanoparticles are one of the most popular objects of research for
applications in chemistry, engineering and medicine, and they are widely used in
manufacturing both medical products and consumer goods.
As for the medical products, it is possible to state that today metal nanoparticles
have good perspectives in diagnostics and treatment of various diseases (including
xiii
xiv Introduction
infectious and oncological ones), as well as in immunochemical methods of anal-

ysis. In particular, it is shown that silver nanoparticles can be used for production of
different materials with antibacterial properties [912], and gold nanoparticles can
help in early recognition of malignant neoplasms, as well as in the improvement of
efciency and reduction of side effects in radiothermal antitumor therapy [1317].
Researches are conducted aimed to the use of conjugates of gold nanoparticles with
biomolecules for diagnostics of various diseases, targeted drug delivery, and as
biosensors for quantitative analysis of different substances (proteins, peptides, etc.)
in solution [1821]. A unique combination of gold nanoparticles properties, which
opens up a wide range of biomedical applications, gave grounds to the statement
that the Golden Age of biomedical nanotechnologies has come [22].
One of the modern trends in scientic thought suggests that the development of
nanotechnologies can allow to create programmed biocompatible nanoscale struc-
tures (nanorobots) able to perform different functions inside a human body (organ
and tissue control, simulation of protective functions of the immune system, sur-
gical manipulations at the cellular level, etc.), which can be successfully used in
different areas of medicine [23, 24]. Possible applications of different kinds of
nanoparticles, including metal nanoparticles, are actively studied in a new direction
of experimental medicine called Nanomedicine. Since 2004, the journal
NanomedicineNanotechnology, Biology and Medicine is published in English.
At the same time, during the last decade it has been established that penetration
into the human organism of different (especially metal) nanoparticles, either from
the environment or upon the contact with the corresponding materials, can cause
serious diseases (nanopathologies) [4, 5, 25, 26]. Today, it is known that metal
nanoparticles can be present in air, water, food, and household goods produced with
the use of nanoparticles, as well as in medical products (including medicinal cos-
metics), diagnostic pharmaceuticals (e.g., those used in computer and magnetic
resonance tomography [9, 27]) and methods of curing (for instance, in antitumor
radiotherapy).
In our view, special attention should be paid to the penetration of nanoparticles
into the human organism in the form of drugs, since it is the most widespread eld
of their application. Besides, there are reasons to suppose that this way has quite a
long history, since as it is evidenced by some materials of this book (see Sect. 1.6 of
Part I) metal nanoparticles can exist in nature (mainly in plants) and thus be
transferred into drugs manufactured from plant extracts.
Hence follows that, metal nanoparticles should be regarded as a factor present in
the environment and able to exert more or less signicant effect on the human health,
penetrating into an organism in different ways: through mucous membranes of
respiratory passages or digestive tract, through the skin (e.g., with cosmetic prod-
ucts) or through the blood flow after injections (e.g., during MRT-investigation), etc.
Nanoparticles can cause different pathological processes; their character depends on
the degree of nanoparticle penetration into the organism, and its individual reaction
to the nanoparticles with the given set of properties. At present the danger of
nanopathologies is great, though not yet fully recognized, and it will obviously
increase in the future. Elucidation of the causes of nanopathologies and search for
Introduction xv
the means of combating the deceases resulting from the penetration of nanoparticles
become now a subject of new direction in experimental medicine.
Studies on the biological activity of metal nanoparticles are conducted by
examining the influence of nanoparticles or nanoparticle-modied materials on the
viability and/or functional activity of biological objects. Since vital activity of
biological objects takes place in the aqueous environment, nanoparticles can be
used only as aqueous solutions, and modied materials have to retain their specic
properties under the contact with aqueous medium. These conditions impose def-
inite requirements on the methods of nanoparticle preparation, since it is the method
which determines their structure, size, physical and chemical properties, and, what
is especially important, their stabilitythe lifetime in the nanosized state.
Preparation of metal nanoparticles in solutions are most frequently realized by the
chemical methods based on the reduction of metal ions to atoms under the conditions
favorable for the subsequent formation of nanoparticles. It is important for the method
to be suitable for practical application, i.e., it should allow to obtain small-sized metal
nanoparticles in signicant quantities and stable on air; also it should be economically
acceptable (i.e., not requiring signicant energy expenditures, expensive equipment,
additional synthesis, etc.). One of such methods is the biochemical synthesis in
reverse micelles with the use of biological reducing agentsplant pigments from the
group of flavonoids. This method has become the base for a new direction in syn-
thesis, characterization of metal nanoparticles and development of the new ways of
their application [28]. It can be said that this direction issued from the demands of
nanochemical, nanomedical and nanopathological researches aimed, rst of all, at the
solution of practical tasks with the use of metal nanoparticles.
The development of works based on biochemical synthesis allowed producing
nanoparticles of several metals (silver, gold, copper, etc.) stable in nonpolar sol-
vents (in reverse micelles) and in aqueous solution, as well as liquid phase and solid
materials modied by silver and copper nanoparticles. In the course of studies on
synthesis, characterization and practical application of nanoparticles and corre-
sponding materials the main direction of research gradually emerged and was
recognized as the most expedient for further development. That was the study of
biological effects and mechanisms of nanoparticles action on the objects staying on
different levels of organization with the aim to determine the conditions of the safe
use of metal nanoparticles in medicine. These studies allowed to accumulate
extensive experimental data on biological effects of nanoparticles, as well as to
develop requirements, on the one hand, for the nanoparticle solutions suitable for
such experiments, and, on the other hand, for the experimental procedures relevant
to a given type of biological object.
This book presents to the reader the results of researches on the biological effects
of nanoparticles and modied materials prepared by the biochemical synthesis.
The Part I contains the description of the basic chemical methods used for the
preparation of metal nanoparticles in aqueous solutions; considerable attention is
paid to the biological reduction of metal ions in solutions. Also it is shown that
biochemical synthesis can be regarded as one of the methods suitable for systematic
studies of biological activity of nanoparticles. Further in this part the description is
xvi Introduction
given of (1) biochemical synthesis, preparation and characterization of nanoparti-

cles in solutions and (2) some special techniques developed to satisfy the
requirements for the study of nanoparticle effects on biological objects.
The Part II is devoted to the results obtained in studies of biological effects of
metal nanoparticles. It begins with the review of experiments carried out in this eld
in the last 1015 years, mainly on microorganisms. An attempt was made to
determine the influence of the main characteristics of nanoparticles on their
bioactivity; some problems in studies of the mechanisms of their action were
designated and conclusions were made concerning the signicance of the method
used for the nanoparticle preparation, for the further developments in this direction.
In the following chapters, the results are presented, obtained in studies of the effects
of metal (mostly silver) nanoparticles prepared by the biochemical synthesis, on
various biological objectsmicroorganisms, fungus, alga, plant seeds and human
cultured cells. Here attention is paid also to the methodological problems which we
consider to be important basing on our experience accumulated in studies on dif-
ferent objects. In the end of this part, the major results of our studies are summa-
rized and compared with the literary data. Our considerations concerning the
mechanisms of biological effects of nanoparticles are also suggested.
The conclusion briefly summarizes the main problems in studies of the bio-
logical effects of metal nanoparticles and expresses the ideas useful, as we believe,
for the further development in this direction.
The authors are grateful to the organizations and colleagues they collaborated with in
studies of the biological effects of metal nanoparticles. They express also their gratitude to
Drs. L.S. Sosenkova and S.I. Kaba, the employees of the Institute of General Pathology
and Pathophysiology, for their contribution in preparation of this book for publication.
The authors also express hope that this book will be useful both for the
researchers working in the elds of nanotechnology, biology, bionanotechnology
and medicine, and for a more broad circle of the readers interested in the properties
and possible applications of metal nanoparticles.
Part I
Preparation and Characterization of
Metal Nanoparticles in Solutions
Several Words About the Contents of This Part
It is known that studies of the interaction of metal nanoparticles with various

biological systems (microorganisms, cultured mammalian cells et al.) are carried
out by the biologists in the laboratories of the corresponding prole. In most cases
the nanoparticles used in these studies are prepared not in biological, but in
chemical laboratories, the biologists not always having the possibility to choose the
way of nanoparticles preparation the most suitable for the experiment with their
biological object. Additional problems arise from the conventional view accepted
by biologists (including those occupied with toxicological studies of medicinal
remedies) which regards the nanoparticles as one more chemical reagent. Hence the
researchers usually do not pay attention to the fact that nanoparticles represent a
new class of factors having its peculiar features which should be taken into account
for the correct organization of experiment. Therefore the standard procedures used
in toxicological studies may be not applicable in the case of nanoparticles and then,
to obtain the correct result, one should change the experimental procedure.
Methodological problems occurring in studies of the biological effects of metal
nanoparticles will be considered in detail in the next part of this book. In the Part I it
is necessary, as we believe, to give rst the general notion about the methods used
for the preparation of metal nanoparticles in solutions, and then, using biochemical
synthesis as an example, to describe (1) the additional tasks which should be solved
for the creation of nanoparticles or modied material suitable for the experiments
with biological objects and (2) the new possibilities which can be revealed in
studies of the nanoparticles properties and their applications in medicine.
Chapter 1
Methods of the Nanoparticle Preparation
in Solutions
1.1 Introductory Remarks
At present, numerous terms are used with prex nano or without it, both in the
eld of nanotechnologies and in the other elds dealing with preparation and
research of disperse system properties. These terms still do not have generally
accepted denitions, and it often occurs that, to dene the same notion, different
authors use different terms or prex nano is used conformably to the objects
which do not belong to the nanometer range and, in principle, cannot be regarded as
nanoparticles. Therefore, prior to the description of the methods of nanoparticle
synthesis, we found it useful to give the denitions of the terms nanoparticle and
cluster that we consider to be well grounded and they will be used further in this
book.
Characterization of disperse systems by the particle sizes leads to the following
widespread denition: The ultradisperse (nano-) systems are the systems with sizes
of morphological elements (particles, grains, crystallites) less than 100 nm [29,
p. 13]. Similar view is widely used in description of the application of nanoparticles
and nanomaterials in biology and medicine; as an example, we suggest the denition
extracted from the article by Lee and Cho in the collective monograph Biomedical
Nanostructures [30, p. 498]: Nanomaterials are those with structural units being
less than 100 nm in size at least in one dimension. Obvious drawback of such
denition is that since this classication is based only on the particle sizes and does
not take into account the changes in their properties, particles within the range
1100 nm with great difference in size may have signicantly different properties.
As more grounded can be considered the denition which takes into account
changes in the particle properties with diminution of their size. From different
versions of these denitions, the following one can be composed: Nanosized
objects (particles, media, materials) are those that exhibit the so-called size effects,
i.e., the dependence of properties on their size. Also, the parameters of structural
elements of these objects are at least in one dimension comparable to (or less than)
Springer International Publishing Switzerland 2016 3

E.M. Egorova et al., Biological Effects of Metal Nanoparticles,
DOI 10.1007/978-3-319-30906-4_1
4 1 Methods of the Nanoparticle Preparation in Solutions
the correlation radius of some physical or chemical phenomena (e.g., electron or

phonon free path, coherence length in a superconductor, size of magnetic domain or
nucleus of a new phase, etc. [31, p. 18]. Here belong practically all mechanical and
physical properties of a substance. It is known that for nanosized particles, changes
with size of hardness, interatomic lattice spacing, melting temperature, conductiv-
ity, optical and magnetic properties, ionization potentials, and some others have
been detected.
As reported in a number of sources dealing with the nanoparticle size effects
[3134], the size dependence of properties takes place most frequently when the
particle size is less than 100 nm and becomes more noticeable for the sizes below
10 nm. The changes of properties with particle size may differ in sign: Diminution
of the particle size may result in the increase, or decrease, or even non-monotonous
change of a given property. Different kinds of size dependence of properties are
shown in Fig. 1.1.
Each property begins to change at its own characteristic size. It is also important
to note that non-monotonous changes (oscillations) of properties are found only
when the particle size is less than 10 nm. Examples of experimental dependencies
of properties on size for the particles of different metals can be found in the
monographs [29, 33, 35]. It is accepted that nanosized particles may be divided into
two categoriesclusters and nanoparticleswhich differ in particle sizes and/or in
the number of atoms in a particle. As to the denitions of these two types of
particles, different opinions may be found in the literature. According to Pomogailo
et al. [31, p. 18], clusters are particles with the regular structure, having, as a rule,
up to 3840 atoms and often more, with the size of 110 nm, and nanoparticles are
1050 nm in diameter and contain 103106 atoms.
According to Ryzhonkov et al. [29], nanoparticles are the objects of 110 nm in
size, and clusters are the particles with sizes close to the lower limit of nanodi-
mensional range, i.e., about 1 nm. According to these authors, the upper range limit
for nanoparticles can be dened from the ratio of surface and inner atoms.
Fig. 1.1 Changes in material

properties during the size
diminution of its
morphological elements ([29],
Material properties
p. 14). x-axis dimensions of

the morphological elements;
y-axis material properties
10 nm 100 nm
Size of morphological elements
1.1 Introductory Remarks 5
In particular, in chemistry the criterion for the object to be referable to nanoparticles

(and not, for example, to ultradisperse media) is the approximately equal number of
surface and inner atoms. Depending on the shape of a nanoparticle, approximately
103105 atoms correspond to this criterion [29, pp. 1415]. In the monograph by
Sergeev [32], nanoparticles are regarded as the subject of research in nanochemistry,
and as in [29], their upper limit in size is considered to be 10 nm. In the monograph
by Suzdalev [33], all particles with sizes within the range, 1100 nm, are called
clusters.
Further in this chapter, under clusters we shall mean particles of less than 1 nm
in size, including aggregates of several atoms formed at the initial stage of
nanoparticle synthesis.
Summarizing all said above, it is possible to conclude that nanoparticles are the
particles less than 100 nm in size (1 nm = 109 m), which can exhibit special
features of the nanodimensional state of mattersize effects, i.e., the change of
properties with particle size. In particular, size effects become the most prominent
for the particles of less than 10 nm.
Systems composed of such particles (including metal sols) have an extremely
developed specic surface, also surface energy, and reactivity essentially greater
compared to bulk material. As noted by Pomogailo et al. [31, p. 20], specic
properties allow to dene them (nanosized particlesauthors note) as a fth
aggregate state of matter [36]. The study of such systems is the subject of a new
rapidly developing branch of sciencephysical chemistry of nanosized particles
(sometimes called also nanochemistry and physics of clusters).
1.2 Principles Used for the Choice of Methods
In the last 1015 years, a great diversity of methods for the synthesis of metal
nanoparticles has been suggested, including various types and their variations. Such
an impetuous growth of works in this eld was stimulated, on the one hand, by the
growing interest (in many countries also at the state level) to fundamental studies
and applied researches with the use of metal nanoparticles and their compounds
(mostly, oxides and suldes) and, on the other hand, by the improvement of
working and newly developed methods as well as by the studies of nanoparticle and
nanomaterial properties.
The accumulation of data on metal nanoparticle characteristics favored the
widening of their application and helped to realize that in most cases, it was the
method of preparation that determines size range, structure, and properties of the
nanoparticles. Thus, it turns out that the method of nanoparticle preparation plays
an important role in the development of nanoscience and nanotechnologies.
The purpose of this chapter is to describe different methods of the chemical
synthesis of metal nanoparticles and to estimate their advantages and disadvantages
for application in the study of their effects on the biological objects. Careful study
of various classications of the methods under question available from monographs
[29, 3133] led us to the conclusion that, using the information about the chemical
methods given in the sources mentioned and in some other special literature, we
should limit ourselves to the consideration of only those used for the nanoparticle
preparation in liquid (colloid) solutions. The reason is that, rst, biological effects
are studied mainly with nanoparticle solutions and second, these methods are
closely related to biochemical synthesis (and include it as one of the directions).
Therefore, the results of nanoparticle studies obtained by these methods may be
quite lawfully compared with those obtained for nanoparticles prepared by the
biochemical synthesis. The collection of chemical methods composed on the
grounds described above is shown in Fig. 1.2.
As shown from the scheme presented, it comprised all the methods based on the
reduction of metal ions in solutions; they differ in the type of reducing agent and in
the system used for the synthesis. We also included methods of nanoparticle
preparation not only in aqueous, but also in non-aqueous solutions, as well as in
reverse micelles. This is justied because rstly, nanoparticle aqueous solutions are
obtained both by the synthesis directly in aqueous solution and by the transfer from
the nonpolar phase into the aqueous medium and secondly, nanoparticles in a
nonpolar solvent are often used in order to create different liquid-phase and solid
materials containing metal nanoparticles for the further study of their effects on
biological objects.
We consider the methods that use traditional chemical reducing agents,
high-energy radiation sources (radiation-chemical and photochemical), as well as
electrochemical and biological reduction in aqueous solution. It should be emphasized
Molecular
Two-phase system Reverse Reverse
solution +
Liquid/Liquid micelles micelles
stabilizer
Electrode surface + Molecular

metal salt solution + solution +
stabilizer stabilizer Inorganic Organic
Electrochemical Photochemical Radiation-chemical Traditional

reduction on reduction reduction chemical
the cathode Me+ + e (h) Me0 Me + e (aq) Me
+ 0
reducing agents
Reverse Aqueous Biological

micelles Solution reducing agents
Biochemical synthesis
Me+ + e (FL) Me0
Fig. 1.2 Methods of the chemical synthesis of metal nanoparticles in solutions

1.2 Principles Used for the Choice of Methods 7
that we do not pretend on the exhaustive description of methods in each of the

directions mentioned. At the same time, we believe that the review undertaken in this
chapter gives full enough notion of the main features of these methods and elucidates
their advantages and disadvantages, as well as the importance of biochemical syn-
thesis described in the following chapters.
Considering the works in each direction, we focused primarily on the principles
(basic scheme) of synthesis and the parameters of nanoparticles related to their
application in studies of biological effects. Here, we mean the size of nanoparticles,
degree of polydispersity (width of the size distribution), their shape, concentration, and
lifetime in solution. Wherever possible, we took into account also economical aspects
(expenses connected with nanoparticle preparation), since from the point of view of
their application in biology and medicine it is important to study the biological effects
of those nanoparticles which can be used in manufacturing medical products.
1.3 Chemical Synthesis with Traditional Reducing Agents
Chemical synthesis is based on the reduction of metal ions to atoms followed by the
aggregation of atoms and ions with the formation of clusters and metal
nanoparticles.
The synthesis is carried out in molecular (aqueous and non-aqueous) solutions, in
the system of two immiscible liquids or in reverse micellesin the ternary system:
metal salt in aqueous solution/surfactant/nonpolar solvent. In the latter case, the
reduction and nanoparticle formation take place in the water core of micelle formed
by the surfactant molecules. The following reducing agents are used most frequently:
(1) non-organic (hydrazine, sodium borohydride, hypophosphite, etc) or organic
(citrates, formaldehyde, hydroquinone, ascorbic acid, glucose, etc.) substances tradi-
tionally used in chemistry as reducing agents, (2) solvated electron generated by the
ionizing radiation, (3) photoelectrons emerging under exposure to UV or laser radi-
ation, and (4) electrons generated on cathode in the course of electrolysis of solutions
containing salts of the corresponding metals. The corresponding types of synthesis are
called (1) purely chemical synthesis, (2) radiation-chemical synthesis, (3) photo-
chemical synthesis, and (4) electrochemical synthesis.
In each case, the stabilizers are used to prevent the particle aggregation and
provide the sustainable state of the system. As stabilizers most often used are
natural substances (gelatine, agar agar, starch, etc.), synthetic polymers or surfac-
tants, in some cases a reducing agent plays a role of stabilizer. This way is applied
for the preparation of colloidal solutions of metal nanoparticles, or metal sols as
they are often called in colloid chemistry. It is possible to obtain aqueous solutions
(hydrosols) and non-aqueous solutions (organosols). Both are used for developing
practical applications either directly (as solutions) or for the production of different
nanoparticle-modied materials.
It should be noted here that chemical reduction of metal ions in solution can be
used for the preparation of both metal sols and nanopowders. The latter are prepared
mainly by the deposition of nanoparticles from colloidal solutions; synthesis of

metal clusters in zeolite cages is also applied. Depending on the purpose, synthesis
in solution is carried out under different conditions. For the preparation of sols, the
conditions are required (reducing agent to metal ions ratio, pH, stabilizer) allowing
to prevent the aggregation and growth of the particles; in this case, small particles
are formed (usually not exceeding 1015 nm). Preparation of powders is conducted
under the conditions allowing the formation of larger particles. For the production
of highly dispersed nanopowders, deposits of colloid solutions, consisting of
nanoparticle agglomerates, are exposed to different procedures (inert gas ignition,
cryogenic drying, etc.). Nanopowders of metals, alloys, and metal compounds
(including semiconductors) are applied mostly in technics. We shall consider only
the preparation of metal sols. Information about the preparation of nanopowders
based on chemical reduction and deposition from aqueous solutions can be found in
the monographs [29, 34, 37] and references therein, as well as in the original works
[e.g., 37, 38]. In the last years, in studies of the biological activity of metal
nanoparticles, the nanopowders are employed, obtained by plasma technologies.
A brief description of this method is given in Chap. 6 (Sect. 6.1.2).
The regularities of the formation of metal nanoparticles in redox reactions in
liquid media are discussed in detail in the monographs [31, 32, 37]. Here, we give
rst the general considerations, which are the most essential for the preparation of
nanoparticles in solutions and then present the examples of synthesis in aqueous
solutions, two-phase systems, and reverse micelles.
1.3.1 General Considerations
The possibility of reduction of metal ions in solutions can be estimated, in principle,

from the known values of standard oxidationreduction potentials for the corre-
sponding pairs of electrode half-reactions. From the literature available, the most
detailed list of redox potentials for the aqueous solutions of metal-containing sys-
tems is given in [39]. However, as it is noted in [31, p. 192], one should take into
account that standard potentials are determined for the standard values of con-
centration and pH of a solution. However, in the conditions which really exist in the
process of nanoparticle preparation, the reactants concentrations and pH values
(and, consequently, the redox potentials) may differ signicantly from the standard
values. Besides, redox potentials depend (1) on the nature of solvent and (2) on the
peculiarities of aqueous medium; the rst is important for the reactions in
non-aqueous environments and the secondfor the synthesis in reverse micelles.
The properties of the latter will be described in more detail further (Sect. 1.3.4).
There are two main stages in the process of nanoparticle formation in solution:
(1) formation of the germs (clusters) and (2) growth of the germs with the increase
of their mass. Kinetics of this process can be expressed as time dependence of the
extent of metal ion conversion = (C0 Ct)/C0, where C0 Ct is the initial and
current metal ion concentrations, respectively (Fig. 1.3).
1.3 Chemical Synthesis with Traditional Reducing Agents 9
Fig. 1.3 Change of the

reduced metal concentration 1.0
in solution expressed as
dependence of the metal I II III
conversion extent () on time
(t) ([31], p. 202)
0.5
0 11 t
Three main phases are discerned here: Ithe induction period (0), IIthe
acceleration area, and IIIthe attenuation area. The induction period reflects the
initial phase of chemical reduction where a so-called nucleation occurs, that is, the
formation of small aggregates (small clusters) from metal atoms. In this phase, the
extent of metal ion conversion is extremely low. After the completion of induction
period, the formation of the clusters (Mj) is terminated and further particle growth
takes place by the two main mechanisms [31]: incorporation of dissolved metal ions
with their further reduction to atoms on the clusters surface (1.1) or the cluster
aggregation (1.2):
Mj M0 ! Mj 1 M0 ! Mj 2 ! Mn ; 1:1
Mj Mj ! 2Mj Mj ! 3Mj ! kMj : 1:2
Apart from the association of the clusters according to the mechanism (1.2),
aggregates can form through the association of smaller aggregates with a different
number of clusters. Depending on the mechanism predominant in particular con-
ditions, the nanoparticles formed are bigger or smaller and more or less homoge-
neous in their size and shape.
As an example, the mechanism of silver nanoparticle formation will be described
in more detail further in this chapter (Sect. 1.3.4).
1.3.2 Synthesis in Water Solution
Preparation of metal hydrosols by the reduction of metal ions in aqueous solution is

one of the earliest and most well-known methods; it is widely used because of its
simplicity and the possibility to obtain large quantities of the product in many cases
stable for a long time.
It is accepted that it was the article by Faraday published in 1857 [40] that may
be regarded as the rst scientic publication on the formation of metal sols and
study of their properties. This work describes optical properties of gold and other
metal sols and denes the basic conditions necessary for their formation. It is
known that rst stable gold sols obtained by Faraday retain their stability up to the
present day. The samples of these solutions are stored in London, in the Museum of
the Royal Institution of Great Britain [34, p. 65]. Further studies on the preparation,
mechanisms of formation, and properties of the gold sols were conducted by
Zsigmondy [41] and Svedberg [42]. These are the classical works which constitute
the basis for the corresponding eld of science.
Since these classical works, a large pool of data on the methods of preparation
and properties of gold and other metal sols has been accumulated. General infor-
mation on these subjects can be found in the relevant sections of colloid chemistry
(e.g., [43, 44]), in monographs [3134], and in the modern issue on practical nan-
otechnology [45]. A surge of interest in nanotechnology has stimulated the rise and
intensive studies of metal sols, considered now as nanoparticles or nanoscale objects
with a perspective of long-term practical application. A range of metals, reducing
agents, and stabilizers was signicantly widened; new methods of synthesis were
developed, aimed at particular applications. As can be deduced from the literature
available, at present the majority of works is devoted to the synthesis and study of
the properties of noble metal (mainly gold and silver) nanoparticles, as well as
nanoparticles of some transition metals (copper, cobalt, nickel, etc.). General (but
inevitably incomplete) notion of the methods suggested to date for the preparation of
metal sols in aqueous solutions can be derived from the data presented in Table 1.1.
Here, the information is collected on reducing agents, stabilizers, and particle
sizes for silver, gold, and copper nanoparticles. The data are taken from the papers
published mainly in the last 10 years. The more extensive list of publications and
analysis of the results can be found elsewhere [18, 8083].
As mentioned above, the synthesis is conducted using conventional chemical
reducing agents of various types. Basic research methods (used also in other
methods of chemical synthesis, described in this chapter) are listed below. Control
of nanoparticle formation, determination of their concentration, and stability control
are performed by spectrophotometry; sizes are determined by transmission electron
microscopy (TEM), photon correlation spectroscopy (PCS) or dynamic light scat-
tering (DLS); structure is analyzed by electron diffraction (ED), high-resolution
TEM (HRTEM) (for individual nanoparticles) and X-ray diffraction (RD); chemical
composition is found by AAS (atomic absorption spectroscopy) and (for compo-
sition of particle surface) by Fourier transform IR spectroscopy (FTIR).
Until recently, most often used are the well-known inorganic reducing agents
hydrazine (N2H4) and sodium borohydride (NaBH4). Various surfactants, sodium
citrate, and polymers are used as stabilizers; the functions of reducing agent and
stabilizer can also be combined by the use of citrates and natural (starch) or syn-
thetic polymer structures (block copolymers, dendrimers). Size of nanoparticles can
vary within the wide range (from 2 to 100 nm); the size regulation is achieved
mainly by the variation of the reducing agent to metal salt concentration ratio, pH,
and temperature of the reaction mixture. The same factors also affect the shape of
nanoparticles; apart from the spherical particles, it is possible to obtain also trian-
gles, hexagons, rods, plates of various shapes, etc.
Table 1.1 Chemical synthesis of metal nanoparticles in aqueous solution with the use of traditional reducing agents
Metal Reducing Stabilizer Nanoparticle size and shape Note Reference
agent
Au
Citrate Citrate 20 nm, spherical Reduction at 100 C [46]
Polyethylene Polyethylene 1560 nm, spherical 100 C
imine imine
Sugar-persubstituted PAAm 26 nm, spherical, wide size distribution Alkaline medium [47]
dendrimers (sugar balls)
Citrate Citrate 13.4 1.6 nm, spherical 100 C [48]
Ascorbic acid CTAB 25100 nm. Rods, hexagonal, triangles, cubes Au seeds are required [49]
Citrate Citrate Polygonal and hexagonal, wide size distribution pH < 5 [50]
Spherical, narrow size distribution pH > 6
Ellipsoids 40 60 nm, etc. 5 < pH < 6
Citrate Citrate Various morphology including hexagonal plates MW heating [51]
>100 nm
Hydroquinone Benzoquinone 20 nm, spherical Stability depends on pH [52]
PEOPPOPEO block 5100 nm, spherical [53]

copolymers
None CTAB/SDS Icosahedrons, triangles, ellipsoids. Size and shape 180 C, 2 h [54]
depend on XCTAB/(1-XCTAB)
Citrate Citrate Spherical. Size, yield, and stability depend on pH Stable at 5 < pH < 9, aggregate at [55]
pH < 5
Ascorbic acid CTAB Rectangular plates, 50 2570 30 nm Synthesis is triggered by the seeds [56]
formation under -irradiation
(PPI-G3) Spherical. Sizes depend on [PPI-G3]/[Au] ratio In the sunlight [57]
(continued)
11
Table 1.1 (continued)
12

agent
Ag
Sugar-persubstituted PAAm 26 nm, spherical, wide size distribution Alkaline medium [47]
dendrimers
Citrate Citrate 5070 nm, spherical [58]
N2H4, NaBH4 Tween-20 2080 nm, spherical pH 10 [59]
NaBH4 AOT 12 6 nm, spherical, wide size distribution Addition of H3PO4 allows the [60]
transition into organic phase
Starch Starch 1034 nm, spherical, starch-coated 121 C, P > 1 atm, luminescence [61]
(em = 553 nm)
NaBH4 Oleic acid 8 nm, spherical, monodisperse Alkaline medium [62]
NaBH4 PNIPAM 1525 nm, various forms pH = 5.5; CAg = 0.125 mm [63]
Optical and electrical properties
change at PNIPAM phase transition
N2H4 CTAB, SDS, Spherical, 15 nm (CTAB) [64]
Triton X-100 Narrow size distribution
N2H4 Citrate 94 8 nm (base length), triangle [65]
H2 SDS 2060 nm, polydisperse, polymorphic [66]
Formaldehyde Polyethylene 320 nm, spherical 3-nm nanoparticle luminescence, [67]
imine em = 474 nm
Block copolymers Small-sized, spherical [68]
Paracetamol CTAB Spherical, tend to aggregate Alkaline medium [69]
Wide size distribution
NaBH4 Triblock From 7.6 (spherical) to 60 nm, polydisperse 24 h, excess of reduction agent, [70]
copolymers aggregate, em = 300 and 330 nm
Aniline CTAB 1030 nm, spherical Agglomerates [71]
1 Methods of the Nanoparticle Preparation in Solutions
NaBH4 Citrate 5 nm, spherical Size enlargement and shape change [72]
Narrow size distribution within 1 year
(continued)
agent
Cu
N2H4 CTAB, citric 51.2 23.6 nm, mainly cubic In the air, room temperature, [73]
acid stabilized with starch
Ascorbic acid CTAB Various shapes, from spherical (90 nm) to wires Size and shape depend on pH, T, [49]
with diameter of 100250 nm and length 68 m and AA/CTAB ratio
N2H4 PAA 3080 nm, faceted crystals; size depends on PAA In the air, % of Cu and stability [75]
concentration depend on pH
NaBH4 Acetonitrile 50100 nm, spherical Inert atmosphere. Stable, large [76]
quantities
NaBH4 Soybean Mean size is 20 nm, spherical [77]
extract
N2H4 CTAB 515 nm, spherical, size decreases with the increase In the air, high pH (ammonia), [78]
of hydrazine concentration CuNP concentration is up to 0.2 M
NaBH4 C3C6 36 nm, spherical; size depends on the dendrimer [79]
dendrimers structure
Abbreviations PAAm, poly(amidoamine); CTAB, cetyltrimethyl ammonium bromide; PEOPPOPEO, polyethylene oxide and polypropylene oxide triblock
copolymers; SDS, sodium dodecyl sulfate; PPI-G3, polypropylene imine G3 dendrimer; PNIPAM, poly-(N-isopropylacrylamide); PAA, polyacrylic acid
13
We give here a brief description of the peculiarities of nanoparticle preparation

by the reduction with sodium citrate, hydrazine, and sodium borohydride.
In the citrate method, salts of citric acid are applied, usually sodium citrate. This
salt acts both as reducing agent and as stabilizer, so its concentration determines the
reduction rate, growth kinetics, and stability of the nanoparticles. The latter two are
influenced also by citrate oxidation productsacetone dicarbonate and/or sodium
itaconate; structural formulae of the corresponding acids are given below (Eq. 1.3):
1:3
This method is used mainly for the preparation of gold nanoparticles. In the
process of oxidation to acetone dicarbonate, the reduction reaction of metal ions is
described by the following equation [45]:
2AuCl3 3Na3 C6 H5 O7 2Au 3Na2 C5 H4 O5 3CO2 3NaCl 3HCl: 1:4
The synthesis takes place at 100 C, that is, upon boiling of the aqueous solution
of reagent mixture. The process of nanoparticle formation is strongly affected by the
metal ions to reducing agent concentration ratio, pH of the solution, boiling time,
and mixing rate of the reagents. Depending on the synthesis conditions, the
nanoparticles of various sizes and shapes can be obtained. If the citrate concen-
tration is insufcient to stabilize the appearing clusters, particle growth occurs by
means of the aggregation (see above mechanism 1.2) and if the excess of citrate
concentration is sufcientby the atom incorporation into the particle surface
(mechanism 1.1) [45]. Accordingly, in the rst case, the particle size is bigger and
size distribution is wider than in the second case. The rate of reagent mixing affects
the rate of cluster formation and that of nanoparticle growth. Fast (single) injection
of the reducing agent leads to the extremely rapid formation of clusters, followed by
their further slower growth. Due to the high concentration of clusters, the proba-
bility of their aggregation increases, and this results in the bigger average
nanoparticle size. At the gradual injection of citrate, the rate of metal ion reduction
decreases, the processes of cluster formation and nanoparticle growth will run in
parallel, so the probability of aggregation is lower and nanoparticle size is smaller
than in the previous case [45].
Particle size and size distribution width also depend on pH: The average particle
size and degree of polydispersity decrease with increasing pH (in the range 4.56.5)
[50, 55], presumably because of the increased negative surface charge density of
stabilizing citrate shell, which prevents the nanoparticles aggregation. At pH < 5,
the loss of stability may happen, provoked by the nanoparticle aggregation, while at
Fig. 1.4 TEM images of gold nanoparticles obtained via reduction with citrate in aqueous
solution. Reprinted from Ref. [50]. Copyright 2008, with permission from Elsevier
pH > 9decrease in the yield of nanoparticles is possible due to the NaAuO2

formation [55]. The shape of nanoparticles depends on pH [50], as well as on the
way the heating is performed [51]. For example, at pH 4.5, polyhedra and ellipsoids
are formed, at 5 < pH < 6predominantly ellipsoids, and at pH 6.5spherical
particles. Example of the TEM image of nanoparticles obtained in [50] at pH 4.5 is
shown in Fig. 1.4. Comparison of the shape of nanoparticles prepared (providing
that the other conditions are the same) by conventional boiling and heating in a
microwave apparatus shows that in the rst case, spherical particles are formed,
while in the second casethe hexagonal plates [51].
Citrate method is used sometimes also for the synthesis of silver nanoparticles
[45]; however, here citrate is less suitable as reducing agent, since silver is reduced
slower (and oxidized faster) than gold, and nanoparticles are inclined to aggregation
in aqueous solution on air. As a result, the nanoparticles are relatively large (e.g., in
[58]5070 nm) and their yield is low. Therefore, in this case, it is more expedient
to use citrate only as stabilizer in combination with a more effective reducing agent.
Two examples of citrate used in combination with hydrazine or sodium borohydride
[65, 72] are present in Table 1.1. Stabilization with citrate is applied also in silver
reduction by solvated electrons or photoelectrons (see below Sect. 1.4).
Varying the citrate, reducing agent, and silver salt concentration, as well as pH
of the solution, one can change the shape of nanoparticles. For example, as shown
in [65], at the large excess of citrate and equimolar ratio of hydrazine and silver salt
(0.1:0.1 mM), apart from the spherical nanoparticles, the signicant part (22 %) of
triangular nanoparticles is obtained. Such synthesis takes just a few minutes, in
contrast to some other routes applied for the preparation of triangular nanoparticles
(photostimulated reduction [84], boiling in a non-aqueous solvent in the presence of
PVP [85], etc.) where the process takes at least several hours. Along with the 404
410-nm band corresponding to the spherical particles, the absorption spectrum of
solutions produced by the combination of citrate and hydrazine exhibits also the
long-wave bands (672 and 740 nm); according to the authors [65], they reflect the
presence of triangular nanoparticles.
Fig. 1.5 TEM image of

truncated triangular silver
nanoparticles after
centrifugation. Reprinted
from Ref. [65]
The latter can be separated by centrifugation, as they are signicantly larger

(predominant length of the triangle side is 94 8 nm) than spherical particles (5
20 nm). TEM images of such triangular nanoparticles are shown in Fig. 1.5. It is
shown also that spherical nanoparticles turn into triangular ones with time (after
12 months).
Reduction with hydrazine is widely used for the synthesis of nanoparticles of
transition and noble metals. Hydrazine is a strong reducing agent easily soluble in
water and can be used in both acidic and alkaline media. Its redox potential changes
from 0.5 V (pH 3) to 1.15 V (pH 14).
In aqueous solutions, hydrazine behaves as a weak base and is present as
hydrazonium ions bearing one or two positive charges:
N2 H4 H2 O $ N2 H5 OH pK = 6:5; 1:5
N2 H5 H2 O $ N2 H62 OH pK = 15:2: 1:6
Redox potential of hydrazonium ion N2H5+ lies in the range 0.4 V (pH 3)
1.26 V (pH 15) [31]. Reduction of metal ions is carried out usually in weakly acidic
(pH 5) or alkaline solutions. The reaction rate increases with increasing pH.
Many metals (Co, Ni, Rh, Pd, Pt, Cu, Ag, Au) require also the temperature of the
solution to be in the range 5090 C [31, p. 193].
In some cases, to achieve the reduction to the metal, substances are added which
react with hydrazine with formation of the stronger reducing agent; as example of
such kind synthesis of Cu nanoparticles in the presence of citric acid may be
mentioned [73]. Various surfactants and polymers are used as stabilizers. The size
and shape of nanoparticles depend on the metal salt to reducing agent concentration
ratio, on the stabilizer nature and concentration, and on pH and temperature. In
most cases, the particle size decreases with increasing of hydrazine to metal salt
molar ratio; this ratio often exceeds 10:1. The average particle size lies mainly in
the range 35 60 nm. Particles obtained are different in shapespheres [59, 64,
65, 78], triangles [65], cubes, polyhedra [73, 74], and rods [75]. Hydrazine is often
used in the case of the metals easily oxidized on air (e.g., for the synthesis of Cu
nanoparticles), as it allows to avoid the need to carry out the reaction in the absence
of air (in vacuum or in an inert gas atmosphere). However, to ensure stability of the
synthesized nanoparticles, additional measures are necessaryfor example, to
conduct the synthesis in a highly alkaline medium [75] or to introduce additional
stabilizer (sodium silicate) that forms a protective silicon shell preventing the
nanoparticle oxidation [73].
In the last years, water-soluble polymers (PAA, PALAm, PVP, etc.) [74, 75], as
well as block copolymers and dendrimers (branched polymers having active terminal
groups capable of affecting the size and shape of nanoparticles), are often applied as
stabilizers. For example, synthesis with PAA gives particle sizes in the range 30
80 nm, depending on the polymer concentration. At low PAA concentrations,
crystals with pronounced faces are formed and at high concentrationsmainly
spherical nanoparticles. At the synthesis with dendrimer, the size and shape of
particles can strongly depend on its concentration and structure [31, 32]; examples of
such kind are known for reduction both with hydrazine and with borohydride (see
below). Block copolymers and dendrimers with amino or amido groups are used
successfully for the synthesis of nanoparticles as stabilizers and reducing agents
simultaneously [47, 53, 57]. The mechanism of metal nanoparticle stabilization by
polymers in aqueous solutions is considered in detail in monograph [31].
Despite the fact that reduction with hydrazine in aqueous solutions is used for
the preparation of metal nanoparticles for more than 20 years [31], there are only
few data on the mechanism of metal ionshydrazine interaction which underlies
the synthesis of nanoparticles. Among the publications available in the last
10 years, there was only one which proposed a scheme describing the hydrazine
interaction with silver ions [64].
NH2 NH2 Ag !NH2 NH2 Ag 1:7a
NH2 NH2 Ag OH !NH2 NH Ag0 H2 O

1:7b
(Hydrazyl)
NH2 NH Ag !NH NH Ag0

1:7c
(Diimide)
NH NH 2 Ag ! N2 " 2Ag0 H2 " 1:7d
4Ag0 4Ag ! 2 Ag4 2 1:7e

As seen from the reaction sequence presented, it is assumed that complex of

hydrazine with silver ion is initially formed which dissociates into hydrazyl radical,
silver atom, and water. Then, the radical interacts again with silver ion forming
diimide and silver atom. Diimide reduces two more silver ions and is oxidized with
the formation of hydrogen and nitrogen. Finally, silver atoms interact with ions with
the formation of a cluster, which serves as the germ of the future nanoparticle. Thus,
in the reaction with silver ions, hydrazine is oxidized ultimately to nitrogen and
hydrogen (which volatilize as gases) and metallic silver remains in solution in the
form of nanoparticles. In other words, reduction with hydrazine results in the
chemically pure product without any admixture of oxidation products of the
reducing agent. This is considered to be one of the main advantages of the method.
The other advantages are relatively high reaction rate and the ability of hydrazine or
its derivatives to reduce a wide range of metal ions in water solution on air.
This method is applied for the synthesis of metal nanoparticles not only in water
but also in mixed wateralcoholic solutions, in two-phase and reverse micellar
systems (see below), as well as for the preparation of bimetallic nanoparticles, such
as Ni/Co, Ni/Rh, Co/Rh, and others [31, p. 199]. Its disadvantages include high
toxicity of the reducing agent (especially when it is needed in a large excess with
respect to metal ions) and in most cases, low yield of nanoparticlesas a rule, the
nanoparticle concentration in solution is in the range 104103 M. As only
exception may be mentioned the way proposed several years ago allowing to obtain
a high concentration (0.2 M) of Cu nanoparticles 515 nm in size; synthesis is
realized by the reduction in ammoniacal solution in the air at pH 10 [78]. It should
be noted, however, that such concentration of Cu nanoparticles is questionable
since, rstly, at such a high concentration of metal the aggregation of nanoparticles
is highly probable and, secondly, TEM data present in the paper conrm the
preparation of nanoparticles only at the initial (103 M) concentration of metal ions.
Reduction with sodium borohydride is used for the preparation of nanoparticles of
many transition and heavy metals, such as Au, Ag, Cu, Pt, Rh, Pd, Fe, Co, Ni, Cd, Hg,
Pb, and others. Metals of the platinum group can be reduced from oxidation degree not
exceeding 4, and metals of the iron group and the last three from the above-mentioned
metals can be reduced from the double-charged ions. Tetrahydroborates exhibit the
reducing properties in aqueous solutions over a wide range of pH values; the redox
potential of MBH4 compounds changes from 1.24 V in alkaline medium to +0.48 V
in acidic medium [31, p. 195]. Their reduction efciency increases with decreasing pH
as a consequence of the hydrolysis of BH4 anion with the formation of intermediate
products with more pronounced reducing properties.
Reduction with BH4 can be realized if the standard redox potential of metal ion
lies in the range 0.5 V 1.0 V [31, p. 195]. In regard to the mechanism of
reduction, it is known that BH4 anion forms active complexes with metal ions with
bridge bonds of the type MHB (intrasphere mechanism). In such a complex, the
proton transfer occurs, followed by break of the bridge bond, redox reaction, and
break of the MH bond. As a result, borane is formed, which is then hydrolyzed to
form boron hydroxide:
BH3 3H2 O ! BOH3 3H2 : 1:8
Another route is borane decomposition to elemental boron on the surface of a

growing metal catalytically active particle (Fe, Co, Ni, Pt, Ag, Au et al.):
2BH3 ! 2B 3H2 " : 1:9
In this case, boron is deposited on the particle surface and is present therein as an
impurity; its contribution may be quite signicant, leading to the changes in
structure and properties of nanoparticles. For example, nely dispersed powders of
iron, nickel, ruthenium, palladium, and other metals, prepared by precipitation from
aqueous solutions with further reduction with tetrahydroborate, contain spherical
nanoparticles 520 nm in size with boron content equal to 2837 % [31, p. 196].
As in the case of the reduction with hydrazine, to prevent aggregation of the
nanoparticles in solution, a stabilizer should be added that can serve simultaneously
as a reducing agent. Signicance of stabilizer for the synthesis of nanoparticles with
hydrazine and borohydride is well illustrated by the results presented in [59], where
the sizes of Ag particles were compared, prepared with these reducing agents in
aqueous solution at pH 10 without a stabilizer and with the addition of surfactants
(Tween-20, predominantly polyoxyethylene and lauric acid). It was shown that, in
the absence of stabilizer, even with a large excess of the reducing agent, exclusively
large particles are formed, with sizes beyond the nanometer range (Fig. 1.6).
According to SEM data, with borohydride silver particles are produced with the
size of about 0.20.3 microns, whereas with hydrazine the size is about 1 micron
(Fig. 1.7). When stabilizer is used, in the synthesis with borohydride and hydrazine
approximately spherical nanoparticles are formed with sizes in the range 2050 and
5080 nm, respectively.
When low molecular weight stabilizers (AOT, CTAB, citrate, oleic acid) are
used with borohydride, silver nanoparticles are also mainly spherical in shape (see
Table 1.1); in most cases, their size does not exceed 2530 nm and is usually less
than that obtained in the synthesis with hydrazine.
The particle size may be varied here also by changing the borohydride to metal
salt concentration ratio and pH of the solution. However, the cases are known where
a noticeable difference in BH4 to metal ion concentration ratio does not lead to a
signicant change in the particle size. Furthermore, when combination of boro-
hydride with citrate is used, conditions may be created when initially produced
small nanoparticles aggregate slowly with the corresponding shift in the absorption
maximum position. Such an example is given in Table 1.1 [72]. Spherical
monodisperse Ag nanoparticles were prepared with different [BH4]/[Ag+] con-
centration ratios (at the excess of silver ions) and stored for 1 year; system was
monitored by measurements of the absorption spectra. It was found that initially the
nanoparticles are 5 nm in size; this average size suffers no changes with fourfold
variation of the concentration ratio under question. Over time, an increase in the
maximum optical density and a redshift of the absorption band were registered, the
latter indicated to the increase in particle size. At the large excess of silver ions, also
Fig. 1.6 TEM images of silver nanoparticles obtained with a and without b Tween-20 as
stabilizer. Reprinted from Ref. [59]
Fig. 1.7 SEM images of silver nanoparticles obtained by the reduction with hydrazine (a) and
borohydride (b). Reprinted from Ref. [59]
changes in the shape of the particles were observed. These changes were markedly
more pronounced in the solutions stored at room temperature and exposed to light
compared to those stored in the dark at 4 C.
Similarly to the reduction with hydrazine, if stabilization is realized with polymers
(PEI, PNIPAM, PAAm, PAA, PVP, etc.), in most cases the size and shape of
nanoparticles depend on the molecular weight of stabilizer and the stabilizer to metal
ion concentration ratio [63, 70, 74, 75, 79]. As found in the recent years, the use of
synthetic or natural polymers (e.g., starch) either as stabilizers, or as reducing agents
as well, has the additional advantage; namely, they can impart to the nanoparticles
some novel properties which were not detected when stabilization was carried out
with citrate or surfactants. Synthesis of Ag nanoparticles in diluted aqueous solution
of triblock copolymer with oxoethylene and oxopropylene groups (PEOPPOPEO
or Pluronic L64) [70] may serve as an example. Stable nanoparticles were obtained,
with wide size distribution (from 7.6 to 60 nm), which demonstrated the lumines-
cence propertiestwo emission bands (300 and 330 nm) were registered for the
resulting solutions, with the respective excitation bands at 220240 and 260290 nm.
The luminescence of silver nanoparticles was also observed when starch was
utilized both as a reducing agent and as a stabilizer [61]. In this case, the
nanoparticles 1034 nm in size were formed in the starch shell; luminescence of the
solution was registered with 380-nm excitation and 553-nm emission bands.
Luminescence was also observed in the process of synthesis of Ag nanoparticles by
the reduction with formaldehyde and stabilization with PEI [67]. Nanoparticles with
the size of 3 nm emitted at 474 nm. The origin of emission found for Ag
nanoparticles stabilized with polymers is not yet clear; but it is obviously caused by
the chromophore groups formed during the interaction of the polymer active groups
with the particle surface. To our view, it is certainly an interesting effect that can be
used in studies of the mechanism of biological action of silver nanoparticles, for
example, for the determination of their location on the cell surface or in intracellular
medium in experiments on cell cultures.
In addition to the basic chemical reducing agents discussed above, some other agents
are used as well, mainly organic (AA, aniline, paracetamol, formaldehyde), which allow
to perform the synthesis under more mild conditions (e.g., on air at room temperature)
and obtain the higher concentrations of nanoparticles. This makes them more suitable in
view of their supposed practical application. However, still there are limiting factors for
the application of these new reducing agents. Thus, the synthesis with aniline produces
Ag nanoparticles with the size of 1030 nm that easily agglomerate in aqueous solution
and virtually cannot be preserved as isolated particles [71]. Similar problem exists in
reduction with paracetamol [69]. To obtain Au nanoparticles by the reduction of
ascorbic acid, the seeds should be additionally introduced into solution to initiate the
nanoparticle growth [49, 56]. As a result, a preliminary procedure is required for the
preparation of such seeds or it is necessary to use initially a preliminary treatment, for
example, the -irradiation of the solution [56].
It should be noted that the known methods of synthesis with AA (at least for Au
nanoparticles) demonstrate better opportunities for the guided change of the particle
shape than conventional citrate method or synthesis with polymers and dendrimers.
Impressive examples of changing the shape of nanoparticles by varying the com-
binations of Au3+ ions, ascorbic acid, stabilizer, Au seeds, and additives of Ag+ ion
concentrations are given in [49]. Thus, the change in the concentration ratio of AA
and Au3+ ions at the constant stabilizer (CTAB) concentration allows preparing the
nanoparticles of various shapes (Fig. 1.8).
At the [AA]:[Au3+] = 1.6, a mixture of rods, triangles, and squares is formed (a);
the last two shapes and the width of rods are about 100 nm. Increase in this ratio
reduces the percentage of rods and triangles, and initially, predominantly hexagonal
shapes of the same size are formed (b), and further, the cubes with the size of about
80 nm are created (c). Variation of the nuclei concentration along with the constant
concentrations of the other reagents, as in the synthesis of nanoparticles shown in
Fig. 1.8c, gives mostly triangular nanoparticles smaller in size (2530 nm) than in
the previous three cases (Fig. 1.8d). Unfortunately, the concentration of
Fig. 1.8 TEM (inset SEM) images of Au nanoparticles synthesized under different conditions.
Ascorbic acid concentration increases from a to c while Au seed concentration increases from
c to d. Scale bar, 100 nm. Reprinted from Ref. [49]. Copyright 2004, with permission from
American Chemical Society
nanoparticles is small (not more than 2 104 M), and the size is mainly about
100 nm, i.e., at the upper limit of the nanometer range; this does not allow revealing
the size effects observed for the small nanoparticles.
Deserve attention also the attempts to use the unusual stabilizers. For example, in
the synthesis of Cu nanoparticles, it is proposed to use acetonitrile as effective
stabilizer [76], which prevents the nanoparticles from oxidation in the course of
synthesis and allows to obtain the signicant amounts of stable nanoparticles. As
follows from Fig. 1.9 acetonitril provides more efcient protection of the
nanoparticles against oxidation than citrate, as the nanoparticles stabilized with
citrate are oxidized to the greater extent than those stabilized with acetonitrile. Since
the nanoparticle oxidation by the air oxygen represents a problem, especially in the
case of Cu nanoparticles, such a proposal may be helpful. However, the synthesis
procedure in the inert gas atmosphere described in [76] is technically complicated
and the size of nanoparticles (50100 nm) is too large for the studies of size effects.
Among the new trends in the eld of chemical synthesis of nanoparticles in
aqueous solution is noteworthy the development of various types of nanoparticle
Fig. 1.9 Photograph of Cu

nanoparticle solutions
stabilized with citrate (a) and
acetonitrile (b). Reprinted
from ref. [76]. Copyright
2009, with the permission
from Elsevier
transfer from aqueous solution to an organic solvent [60, 62, 8688, and references
therein]. This is conditioned by the expansion of the application area of metal
nanoparticles in technics and the chemical industry where stable nanoparticles in
organic solvent are required. To facilitate the transfer, various ways of hydropho-
bization of the nanoparticle surface are suggested, which provide the complete enough
transition and stability against aggregation in the organic phase. For example, silver
nanoparticles synthesized by borohydride reduction and stabilized by oleic acid have
been transferred to organic solvent after the addition of phosphoric acid to the aqueous
solution [62]. In this case, carboxy anions (ionized carboxyl groups) of oleic acid were
protonated and became neutral; this led to the reorientation of oleic acid molecules and
conversion of the hydrophilic nanoparticle shell into the hydrophobic one. Thanks to
this conversion, the nanoparticles could be extracted almost completely with organic
solvent from the aqueous solution (see Fig. 1.10).
Similar modehydrophobization of the nanoparticle surface by the addition of
phosphoric acidwas successfully used for the transfer of silver nanoparticles
stabilized by anionic surfactant (AOT), which is widely applied for the preparation
of reverse micelles [60] (see below Sect. 1.3.4). Extraction of Au nanoparticles
from aqueous solution prepared by the citrate method was effectively realized by
the introduction of ligands (linear or branched alkanethiols), which are well
adsorbed on the nanoparticle surface [8789].
Fig. 1.10 a Immiscible

layers of the Ag hydrosol
stabilized with oleic acid
(bottom) and cyclohexane
(top) before shaking. b Ag
organosol in cyclohexane
(top) and pure aqueous
solution (bottom) after the
phase transition induced by
carboxy group protonation in
oleic acid. Reprinted from
Ref. [62]. Copyright 2008,
with the permission from
Elsevier
The main problems in the synthesis in aqueous solution are, rstly, prevention of
the nanoparticle aggregation (obtaining of a stable sol), secondly, creation of the
small nanoparticles (less than 20 nm in size) with narrow size distribution, which is
important for a number of their applications, and thirdly, preparation of the large (of
practical signicance) amounts of the solution with high nanoparticle concentration.
The latter condition is important primarily from the economic point of view, since
the real possibilities of industrial application (including production of drugs, wound
dressings, polymers with antibacterial properties, or other materials for medicine)
depend on cost increase resulting from the use of nanoparticles, and this in turn
depends on the volume produced and on the extent of dilution of the nanoparticle
solution which allows to demonstrate the desired effect of the nanoparticles.
1.3.3 Synthesis in a Two-Phase System and in Non-aqueous

Solutions
Along with the reduction in aqueous solution, beginning from the 1990 there took
place the intensive development of studies on the nanoparticle synthesis by the
reduction of metal ions in organic solvent for the preparation of stable organosols.
A detailed description of the methods of preparation, properties, and applications of
organosols (mainly of precious metals) may be found, for instance, in the recently
published review of the Hindu researchers [90]. Interest in these nanoparticles is
conditioned primarily by the application perspectives in technics, rst of all in
catalysis and electronics. For example, it is highly probable that creation of the
two-dimensional or three-dimensional ordered structures of identical metal nano-
sized particles (preferably near the lower limit of the nanoscale) opens new per-
spectives in information storage [32, 34, 91, 92].
It has been established experimentally that, to produce such ordered structures of

nanocrystals (nanocrystalline superlattices), hydrophobic sterically stabilized
nanocrystals can be used because they are able to self-organize on a substrate with
the formation of the densely packed ordered structures simply in the course of
solvent evaporation from the appropriate organosol. For this purpose, it was
important for the particle size distribution in such a sol to be sufciently narrow (the
size dispersion should not exceed 10 % of the average diameter) [93]. So the task
arose of the preparation of stable non-aqueous solutions of metal nanoparticles
several nanometers in size and with a narrow size distribution.
Since, on the one hand, the preparation of such nanoparticles is of interest for the
detection of size effects in studies of their biological activity and, on the other hand,
there are methods of the nanoparticle transport from non-aqueous into aqueous
phase (see one example later in this section), we found it useful to give here a brief
description of this type of synthesis. To make this description more concrete, we
shall dwell on one direction, to our view, the most widespread in this eld.
An important step forward in the preparation of the monodisperse small-sized
metal nanoparticles in organic solvent was made by the two-phase arrested growth
method proposed by M. Brust for the synthesis of 2 nm Au particles, later mod-
ied with the participation of Schiffrin [94, 95]. The method is based on the
reduction of AuCl4 with borohydride in the presence of dodecanethiol (C12H25SH)
in a two-phase water/toluene system. Synthesis scheme includes two basic stages:
(1) transfer of the metal ion from aqueous to organic phase by a quaternary
ammonium compound (QAC) and (2) ion reduction in the presence of long-chain
alkanethiol [96]:

AuCl
4 aq NC8 H17 4 toluene ! NC8 H17 4 AuCl4 toluene: 1:10
mAuCl4 toluene nC12 H25 SHtoluene 3me !

1:11
Aum C12 H25 SHn toluene 4mCl aq:
Metal ion is transferred through the phase boundary owing to the complex for-
mation with QAC, in the original versionwith tetraoctylammonium bromide
(TOAB). Compounds of this type work as the effective carriers of metal ions through
the water/organic solvent boundary and are used therefore in studies of the processes
of metal ion transfer through the cell membrane in liquid/liquid model systems (see
[97]). Metal ions are reduced to atoms in organic phase and then atoms aggregate
with the formation of the clusters and nanoparticles stabilized with dodecanethiol.
The standard synthesis procedure includes the following stages (e.g., [96, 98]):
(1) introduction of HAuCl4 aqueous solution into the reaction vessel, (2) addition of
the predetermined volume of QAC solution in toluene, formation of the two-phase
system, (3) intense shaking of the vessel until the gold is completely extracted from
aqueous solution, this event can be visually controlled by disappearance of the
specic coloration of aqueous phase, (4) addition of dodecanthiol solution in toluene
to the organic phase, and (5) addition of borohydride (in excess relative to the metal
ion concentration) to the reaction mixture. All the operations are conducted on air
under continuous mixing. Some authors point to the importance of maintaining the
phase boundary intact while mixing [99] and of slow introduction of a reducing
agent [48]. The original Brusts synthesis continues for 12 h. In the later versions of
this procedure published by the other authors, the synthesis takes from 4 to 12 h,
depending on the concentration ratio of reagents and the desired particle size.
After the completion of nanoparticle formation, the organic phase is separated
from the water phase and evaporated in vacuum to the small volume; then, the
nanoparticles are precipitated by the excess of ethanol (for complete precipitation,
they are held for several hours at temperatures below freezing), and the precipitate
is ltered, washed on the lter with ethanol (to remove QAC), and dried in vacuum.
The resulting powder is readily soluble in toluene, hexane, or other liquid-saturated
hydrocarbons and may be repeatedly precipitated and redissolved in nonpolar
solvents. Metal nanoparticles are stabilized with alkanethiol or a mixture of alka-
nethiol with unsaturated fatty acid (oleic or lauric acid).
The method is used mainly for the preparation of gold nanoparticles; successful
attempts have been made also to use it for the synthesis of silver and copper
nanoparticles. Characteristics of the system and nanoparticles taken from the chosen
articles published in the last 1015 years are presented in Table 1.2. The synthesis is
conducted most often in toluene/water system [48, 96, 98, 100, 102]; some authors use
saturated hydrocarbons [19, 99, 101, 109] or chloroform [93]. The choice of solvent is
probably determined by the solubility of stabilizer, i.e., by the possibility to provide its
concentration sufcient both to solubilize the reducing agent and to prevent the
nanoparticles from aggregation and oxidation. It is clear that the stabilizer must be
highly effective, since the small size of nanoparticles gives grounds to suggest their
increased tendency to aggregation and high chemical activity. Borohydride is used
almost exclusively as the reducing agent in a large (at least tenfold) excess relative to
metal ions. From the synthesis procedure and other published data, it remains unclear,
however, rstly, whether the borohydride is spent completely or it is still present in the
nanoparticle solution and, if so, in what concentrations, and secondly, whether the
solution contains also the borohydride oxidation products (B(OH)3, BH3, B).
Among the nanoparticle parameters shown in Table 1.2, attention is attracted,
rst of all, to their small sizeit is always less than 20 nm. In most cases, the
average size does not exceed 7 nm, and then, the size dispersion is in the range 0.3
0.7 nm, i.e., the nanoparticle suspensions are almost monodisperse. Studies of the
influence of the reagent concentration ratio on particle size for Au nanoparticles
were undertaken in [96]. It was found that here play a role the ratios of molar
concentrations: [Au]:[C12H25SH], [NaBH4]:[Au], and [TOAB]:[Au]. If the latter
two ratios are held constant, an increase in the ratio [Au]:[C12H25SH] from 1:1 to
6:1 results in the increase in particle size from 1.5 0.3 to 20.2 3.8 nm
(Fig. 1.11).
Particle size was determined by X-ray diffraction measurements of the powders
of precipitated nanoparticles coated with stabilizing shell. The value of 4 nm (twice
the length of dodecanethiol molecule) was subtracted from the measured particle
diameter. As seen from this gure, the experimental dependence of particle size on
Table 1.2 Preparation of organosolsnanoparticle synthesis in a two-phase system
Metal Organic Reducing Nanoparticle size Notes Reference
phase agent/stabilizer and shape
Au
Toluene NaBH4/C12H25SH From 1.5 0.3 nm max = 520545 nm [96]
to 20 3.8 nm Stability decreases with the increase of size
Spherical
Toluene NaBH4/C4H9SH, 1.6 nm, truncated octahedron [100]
NaBH4/ 1.6 nm (40 %) + 2.8 nm (60 %)
TBBT + C7H15SH
Hexane NaBH4/(N(R1)3RII I) 2.6 0.47 nm, max = 520 nm; wall deposition in aqueous phase; [99]
Spherical initial concentration is 0.8 M and decreases
with time
Toluene NaBH4/C12H25SH 1.9 0.7 nm max = 520 nm [48]
5.6 0.4 nm Concentration is 3.5 M
Octahedral
Cyclohexane Formaldehyde <10, spherical, monodisperse Stable [19]
+C12H25NH2/
C12H25NH2
Ag
CHCl3 NaBH4/C12H25SH 6.8 nm, hexagonal, Formation of organized layers during solvent [93]
monodisperse evaporation
Decane NaBH4/(N(R1)3RII I) dmean = 20 nm, spherical [101]
Toluene NaBH4/C12H25SH dmean = 34 nm, size max = 438458 [98]
distribution depends on the Stability depends on the conditions
conditions
(continued)
27
28
Metal Organic Reducing Nanoparticle size Notes Reference

phase agent/stabilizer and shape
Cu
Toluene NaBH4/oleyl amine dmean = 6.06 nm, spherical In the N2 atmosphere [102]
Toluene NaBH4/lauric acid 3.3 0.7 nm, spherical Oxidized in the air; protection by C12H25 SH [19]
and oleic acid
Octane NaBH4/(N(R1)3RII I) 6.6 2 nm, spherical CuO [103]
Abbreviations TBBT, 4,4-thiobis-(benzenethiol); (N(R1)3RII I), NNNtridecyl(3-azo-tridecyltridecan)ammonium iodide; C12H25SH, dodecane thiol
D = 2R40 ()
0 20 40 60 80 100 120 140 160 180 200 220 240
7
4
Au/S
0
20 30 40 50 60 70 80 90 100 110 120 130 140
R()
Fig. 1.11 Size change of Au nanoparticles along with the change in molar concentration ratio
[Au]:[C12H25 SH]. Points are the experimental values (from XRD data), and solid line is
theoretical curve calculated by the authors [96]. Lower horizontal axis corresponds to the
nanoparticle radii including alkyl chains of C12H25SH molecules. Upper horizontal axis
corresponds to the nanoparticle diameters found in the experiment, and they do not include the
C12H25SH chain length. Calculations were made for the xed gold molar fraction (0.001).
Reprinted from Ref. [96]. Copyright 1995, with permission from American Chemical Society
the ratio [Au]:[C12H25SH] (referred to as Au/S) is satisfactorily approximated by

the theoretical curve (based on the thermodynamic approach proposed by the
authors) calculated from the expression for the standard chemical potential (on) of
nanocrystal having n Au atoms, with due regard for the adsorption of a certain
number of dodecanethiol molecules on its surface.
For Ag nanoparticles, the influence of various factors (the [C12H25SH]:[Ag]
concentration ratio, the order of their introduction into the system, and the rate of
reducing agent introduction) on their size, structure, and stability was determined in
[98]. The authors concluded that the characteristics of nanoparticles prepared by
this method are strongly dependent on the synthesis parameters. Stability, structure,
mean particle size, and size distribution may suffer considerable changes as a result
of changes in the sequence of components mixing.
In the case of copper, small-sized (37 nm) nanoparticles can be synthesized;
however, stable nanoparticles cannot be obtained on air, so the synthesis should be
conducted in an inert gas atmosphere [102] or with additional stabilizers, apart from
dodecanethiol. In the example shown in Table 1.2, oleic acid was used as an
additional stabilizer [19]. Substitution of toluene with octane is unlikely to decrease

the risk of nanoparticle oxidation, since in this case only copper oxide nanoparticles
can be produced [103]. For the preparation of stable Cu nanoparticles, another
two-phase system (water/oleic acid) was recently proposed, where oleic acid plays a
role both of a carrier and of a stabilizer [104]. Sodium hypophosphite is used as
reducing agent. In this case, spherical nanoparticles are formed, stable in organic
phase but rather big in size (about 30 nm). It is shown also that stability of Cu
nanoparticles depends on the solvent polarity. In polar solvents (e.g., chloroform),
metallic copper is quickly converted to oxide, while in nonpolar solvents (heptane,
benzene), the nanoparticles are stable on air for a long time [105].
As to the shape of nanoparticles, in contrast to the diversity of forms produced
by the reduction in aqueous solution, synthesis in a two-phase system gives mostly
spherical particles, sometimes polyhedra. Perhaps the reason is that the method has
appeared not so long ago and the appropriate differences in synthesis conditions are
still relatively insignicant.
Summarizing, we can conclude that the use of BrustSchiffrin method allows to
prepare gold, silver, and copper nanoparticles with size of the order of several
nanometers and small size dispersion (the deviation from the mean diameter is less
than 10 %), stable in organic solvents (toluene, liquid saturated hydrocarbons).
These solutions can be concentrated by the solvent evaporation.
It is possible to obtain the nanoparticles also as powders which can be resus-
pended in another organic solvent. Nanoparticles produced by this method can be
successfully used in electronics to create the two-dimensional or three-dimensional
ordered layers on solid substrates. Such layers are formed by self-assembly of the
nanoparticles during solvent evaporation or by the chemical bond formation
between the nanoparticle shell and metal (usually gold) substrate. The rst route can
be illustrated by the two-dimensional superlattice of Ag nanoparticles, obtained by
evaporation of their solution in chloroform on the carbon substrate [93] (Fig. 1.12).
Fig. 1.12 TEM image of the Ag nanocrystal-composed two-dimensional superlattice obtained by

means of evaporation of the nanoparticle solution in chloroform. Reprinted from Ref. [93].
Copyright 1998, with permission from American Chemical Society
Hexagonal silver nanoparticles (6.8 nm in size) after evaporation from the solution
with concentration of 1015 particles/ml form domains with an ordered arrangement
(two-dimensional superlattice) with size of 0.5 0.5 microns. Three-dimensional
superlattices are formed on mica substrate in the similar way. Studies on the effect
of various factors (concentration and size of nanoparticles, density of stabilizer
molecules, degree of faceting, and shape of nanoparticles) allowed to bring
forward the suppositions regarding the driving forces acting in the formation of
such ordered structures (see [106]). The second route may be exemplied by the
monolayers of monodisperse Au nanoparticles with the size of 1.6 nm in the
butanethiol shell, chemically cross-linked to the self-assembled monolayer of 4,4
thiobisbenzenethiol (TBBT) previously deposited on metallic gold [100].
Despite all the attractiveness of such systems obtained on the stage of laboratory
studies, it should be noted that this method has the drawbacks limiting its wide-
spread industrial application.
Firstly, the method is rather complicated technically and can be used almost
exclusively in the laboratory conditions for the preparation of small amounts of
nanoparticles. On account of the strong influence of the minor changes in the
synthesis procedure on the degree of polydispersity and stability of nanoparticles
[98], the reproducibility of monodisperse product cannot be high even if it is
obtained in different research laboratories, not speaking about the pilot plants.
Secondly, at least two of the basic reagents are highly toxic; namely, toluene and
quaternary ammonium compounds used as carriers of metal ions through the phase
boundary. The QACs are well known as potent disinfectants [107, 108] highly toxic
to humans and other living organisms. Thus, with this method of nanoparticle
preparation, the risk increases of personnel poisoning, which makes it difcult to
produce nanoparticles in large quantities even in the laboratory conditions.
Thirdly, as mentioned above, the question remains unresolved about the degree
of nanoparticles contamination with borohydride oxidation products.
Fourthly, nanoparticles are prepared in organic solvents with still restricted set of
stabilizers. Besides, the stability of nanoparticles in solution is low in many cases,
making it difcult to study their properties and develop the possible applications of
such solutions. Therefore, various methods are suggested to increase the
nanoparticle stability in organic solvent, for example, as stabilizers in toluene
alkanethiols are used with varying hydrocarbon chain length or with branched
saturated hydrocarbons [88].
Fifthly, if the nanoparticles should be transferred to the aqueous medium, the
relevant technique is to be developed, this task being connected with additional
technical difculties. In some cases, this problem can be solved by the introduction
of the additional surface-active substance, which is able to solubilize the
nanoparticles in the aqueous solution. Such an example taken from [109] is shown
in Fig. 1.13.
The gure shows photographs of the two-phase system with silver nanoparticles
stabilized with oleic acid, in cyclohexane (a) and after their transfer to the aqueous
phase (b) induced by the addition of cationic surfactant (CTAB). As can be seen
from the corresponding TEM images, transfer to the aqueous phase is accompanied
Fig. 1.13 TEM image of Ag nanoparticles in a cyclohexane and b water. Insets Images of
solutions. Nanoparticles are present in the colored phase. Reprinted from Ref. [109]. Copyright
2008, with permission from Elsevier
by the signicant increase in particle size (according to the authors, from less than 6
to 1050 nm) and broadening of the size distribution.
To produce the nanoparticles small in size and with sufciently narrow size
distribution, as well as to obtain the nanoparticles of various specic shapes
(nanorods, nanowires, etc.), synthesis in nonaqueous solutions, mainly in ethylene
glycol, has been recently applied (the polyol method) [106, 110116]. In this
process, ethylene glycol acts both as a solvent and as a reducing agent. Stabilization
and increase of the yield of anisotropic shapes are commonly achieved by the use of
PVP [110, 114]. Modications of the polyol synthesis are also suggested where
(1) PEG is used as a solvent, reducing agent, and stabilizer [112] and (2) ethylene
glycol is used as a solvent with sodium formate as additional reducing agent and
sodium citrate as a stabilizer [115]. The synthesis is conducted at 140200 C. To
accelerate the particle growth, in some cases the seeds (gold or silver nanopar-
ticles with the size of the order of several nanometers) are preliminarily introduced
into the system. Signicant parameters that have an influence on the rate of for-
mation and the nanoparticle size are temperature and the order and the rate of
introduction of the reagents [114]. The polyol synthesis allows to prepare spherical
Ag nanoparticles with the size of 17 2 nm [116], Co nanoparticles with the size
of 2 nm [115], bimetallic CoPt nanoparticles with the size of 3 nm [112], and stable
Cu nanoparticles with the size of 45 8 nm [114] (Fig. 1.14).
In the latter case, studies by the RD method and the data obtained by
high-resolution TEM (HRTEM) show that there is a layer of amorphous copper
oxide (CuO) on the nanoparticle surface, and chemisorbed PVP is also present.
Figure 1.15 shows nonspherical silver nanoparticles of various shapes produced by
Fig. 1.14 SEM and HRTEM images of Cu nanoparticles. Inset Diffraction pattern for the chosen
TEM area. Reprinted from Ref. [114]
Fig. 1.15 TEM images of the products of polyol synthesis of anisotropic Ag nanoparticles [110]
the polyol synthesis with PVP and with preliminary injection of seeds (Au
nanoparticles) [110]. It is seen that the nanoparticles form agglomerates around a
certain coordinating matrix.
It is assumed that it is PVP that plays a role of such matrix, since it is known that
this polymer is capable of forming complexes with both Ag+ ions and atomic silver
Ag0 [117]. In this case, agglomerate geometry depends on the matrix shape, i.e., on
the conformation of polymer molecules under these conditions. For more infor-
mation about polyol synthesis, see the aforementioned review on the methods of
preparation and properties of organosols [90].
Comparison of the basic characteristics of nanoparticles synthesized in organic
solvents with those prepared by the synthesis in aqueous solutions (described in the
previous section) shows that synthesis in organic solvents allows to obtain the
nanoparticles relatively more stable in solution on air, as well as provides better
control of the nanoparticle size and distribution width. It is important to note that
monodisperse samples of the nanoparticles smaller than 10 nm can be prepared,
convenient for the studies of size effects and biological activity.
1.3.4 Synthesis in Reverse Micelles
The intense development of the studies on the creation and properties of reverse
micellar systems began in the late 1970s to early 1980s of the last century and
almost simultaneously the works appeared devoted to the synthesis of metal
nanoparticles in reverse micelles. The interest to reverse micelles as an experi-
mental system was conditioned by the three main reasons.
The rst reason is the considerable role of the reverse (inverted) lipid micelles in
different biological processes(interaction and fusion of cell membranes, regula-
tion of membrane proteins activity)detected in biophysical studies of the
mechanisms of biological processes at the cellular level and on the model (bilayer)
lipid membranes ([118, 119] and references in these works). The data obtained in
these studies indicated to the good perspectives of reverse micelles for the eluci-
dation of the regularities of some biological processes at the cellular level.
The second reason consists in the realization of the fact that the study of cat-
alytic reactions of enzymes in reverse micelles has obvious advantages in com-
parison with the studies of these reactions in aqueous solutions. The major one is
that enzymatic reactions in reverse micelles and in a biological cell proceed in
similar conditions [119121].
The third reason is that chemical catalysis in reverse micelles was successfully
accomplished in different industrially applicable reactions, thus highlighting the
advantages of reverse micellar system in comparison with molecular solution [122
125]. Achievements in this eld stimulated the attempts to use the reverse micellar
system also for the synthesis of nanoparticles of catalytically active metals (Pt, Co,
Ni, etc.), since it was reasonable to assume that the combination of the advantages
of catalysis on nanosized metals (conditioned, rst of all, by the considerable
increase of their specic surface) with those of catalysis in reverse micelles may
result in a qualitative leap in the effectiveness of many chemical industries.
Results achieved on the initial stage in studies of the structure and properties
internal medium of the reverse micelles, as well as of various reactions in reverse
micelles are collected in the monograph [126]. One of the chapters briefly describes
the composition and some properties of the reverse micellar systems used in metal
nanoparticle synthesis, together with some examples of synthesis and characteristics
(optical spectra and sizes) of Au, Ag, and platinum group metal nanoparticles
obtained with the use of conventional chemical reducing agent (hydrazine) [127].
From these data, it has already become evident that synthesis in reverse micelles
allows to prepare stable metal particles in an organic solvent, the process being
much more simple than the two-phase synthesis and allowing to control the particle
size by changing the hydration extent w = [H2O]/[SAS]. This parameter denes
diameter of the water core, where metal ions are reduced and nanoparticles are
formed (see Fig. 1.16 below). Further progress in studies on the synthesis and
properties of metal nanoparticle in reverse micelles is discussed in several reviews
[8183, 128131]. They consider the methods of nanoparticle preparation using
both conventional chemical reduction and high-energy irradiation (photochemical
and radiation-chemical synthesis). Different modes of synthesis are described in
many articles; the most remarkable growth in number of such publications is
observed in the last 1012 years because of the rapid development of nanotech-
nologies and the corresponding increase of the diversity of nanoparticle applications
in various elds of chemistry, technics, and medicine.

+
+ +
+

d2 +
d1 = 2r
+ +
+ +

Fig. 1.16 Schematic presentation of the reverse micelle formed from an anionic surfactant with
Na+ counterions near polar head groups within the micellar core
In this section, we adduce rst the general information about the main pecu-
liarities of reverse micelles and then briefly describe some examples of the
nanoparticle synthesis with traditional reducing agents. In our view, the examples
can give notion of the compositions of reverse micellar systems, signicant
parameters of the synthesis, and characteristics of the nanoparticles, useful for
comparison with those given in the two previous sections. The photochemical and
radiation-chemical synthesis of metal nanoparticles in reverse micelles with
reduction, respectively, by photo- and solvated electrons will be considered in the
following section.
General information about the reverse micelles. Reverse micelles represent the
ternary systems of the general composition H2O/SAS/nonpolar solvent, where
water is solubilized in a nonpolar solvent by SAS molecules. Thus, typical reverse
micellar system is a solution of water in the nonpolar solvent, where water is
enclosed in reverse micelles, i.e., into the shells consisting of surfactant molecules
(polar heads inside, nonpolar tails outside, Fig. 1.16). The micelle size depends on
the size and structure of surfactant molecules and on the hydration extent, which
determines size of the internal medium (the water core) of a micelle. In most cases,
diameter of the water core is less than 45 nm.
Reduction of the metal ions and further formation of the nanoparticles take place
in the micelle internal medium. At the low hydration extents (w < 10) used con-
ventionally in metal nanoparticle synthesis, water in micellar core possesses the
properties substantially different from those of the bulk water. This is explained by
the ability of surfactant polar heads to bind strongly a number of water molecules
(410 molecules/per one surfactant molecule) [119], so that almost all the water in
the core of micelle turns out to be in the boundary layer subjected to the organizing
influence of polar heads. The lesser the distance from the boundary of polar head
layer, the stronger their influence (and consequently, the rigidity of water structure).
Therefore, the volume of water core is an inhomogeneous medium with respect to
the physical and chemical properties, so that one may speak only about the local
values of the parameters such as viscosity, polarizability, ion activity, etc.
Numerous investigations are devoted to the study of various water properties and
chemical reactions in the core of reverse micelles (see [119, 126, 132143]).
A series of methodological problems connected with the properties of reverse
micellar systems is discussed in [126128, 133, 135, 139141]. In particular, it was
found that water viscosity in the core of micelle is much greater and polarizability is
much less than those of bulk water; [126, 133, 136, 143]. The pH value in the core
of micelle formed by the anionic surfactant is lower and by the cationic surfactant
higher than that of the initial water solution [132, 138, 140]. Water reactivity and
the rate of chemical reactions in reverse micelles are, as a rule, higher than those in
a water solution [119, 133135, 141, 143]. For example, the rate of ketone
iodization in hexane increases by several orders after the addition of a
micelle-forming surfactant [119]. The advantages of reverse micelles for enzyme
catalysis are also known [119, 120122, 126].
The acceleration of reactions in reverse micelles is caused by several factors,
such as the increase of reagent concentration after their binding with micelles, the
unique properties of the internal medium, and rigid xation and orientation of the
reacting molecules [119, 120, 133]. The latter aspect is especially important in the
enzyme catalysis; as noted in [120], high catalytic activity of enzymes in reverse
micelles is explained by the stabilizing influence of micelle matrix on the enzyme
conformation. It is supposed that the internal medium of a micelle freezes the
protein structure, thus neutralizing the unfavorable vibrations and preventing it
from unfolding.
Thanks to the above-mentioned peculiarities of the internal medium and reac-
tions in reverse micelles, they are often called microreactors or nanoreactors
[32, 126, 128, 129, 131]; more and more distinctly realized is their active partici-
pation in the processes taking place in the water core. For example, comparison of
the characteristics of the synthesis products in a molecular solution and reverse
micelles allows to conclude [131] that it is necessary to revise the notion about the
reverse micelles just as passive shells limiting the growth of nanoparticles and
recognize them as playing more active, dynamic role, which opens up new
possibilities of their application.
To obtain micellar solutions, cationic, anionic, and non-ionic surfactants are
employed; a detailed information on the structure and properties of SAS solutions
(including water-in-oil microemulsions, in particular, the reverse micelles) can be
found in [144, 145]. As solvents most frequently used are liquid saturated hydro-
carbons and aliphatic alcohols (hexanol, octanol, etc.).
The choice of surfactantsolvent combination is determined by the possibility to
fabricate stable reverse micelles in the wide enough range of surfactant concen-
trations. The important factor is the geometry of a surfactant molecule, which
determines its ability to form either inverted or direct micelles. For the formation of
reverse micelles, the most appropriate are the molecules with a blunted-cone shape,
where the cross-sectional area of the polar head is smaller than that of the nonpolar
tail (see [129]). This condition is satised, for instance, by the two surfactants
mentioned above (Sect. 1.3.2)anionic, sodium bis(2-ethylhexyl)sulfosuccinate
(aerosol-OT or AOT) and cationic, cetyltrimethyl ammonium bromide (CTAB).
Typical sizes of the water core in AOT reverse micelles used in the biochemical
synthesis, for various hydration extents are given in Chap. 2 (Sect. 2.1.2). Most
often employed are non-ionic surfactants from the hydroxyethylated alcohol group
of the common formula CH3(CH2)i1 (CH2CH2O)jOH, usually designated by CiEj.
Their hydrophilic part consists of the residues of polyethylene glycol chains
including ether and hydroxyl groups. Polarity of one oxoethylene group is signif-
icantly less than that of one polar group of an anionic surfactant; to provide the
necessary degree of hydrophilicity, such molecule should contain 450 oxyethylene
groups, depending on the hydrocarbon tail length [133, 144]. Size of the water
micelles in a given solvent at any chosen hydration extent depends on the surfactant
concentration; as a rule, the SAS concentration is selected in the range which allows
to form micelles of the smallest size. Similarly to the synthesis in water solutions,
mostly hydrazine and sodium borohydride are used as reducing agents.
Usually, the process is carried out according to the standard procedure: First, a
SAS solution is prepared in a corresponding solvent, this solution is used for the
preparation of two micellar solutions by adding the water solutions of metal ions
(a) and a reducing agent (b), and then the reduction reaction is triggered by mixing
of these two micellar solutions; the ion reduction results in the nanoparticle for-
mation as described above (Sect. 1.3.1). The reaction kinetics is determined by the
relation of the reduction rate (nucleation, or cluster formation) to that of intermi-
cellar exchange, which is responsible for the growth of nanoparticles. Usually, the
reduction goes fast enough, and the rate of nanoparticle formation is determined by
the rate of matter exchange between micelles. The average diameter and the width
of size distribution depend on the concentration ratio of the reagents and on the
metal and surfactant ion concentrations. The distribution width can increase in the
course of nanoparticle aggregation through the fusion of micelles with nanoparticles
of various sizes; as a result, the nanoparticles can become bigger in size than the
initial water core of a micelle. These large nanoparticles are stabilized by the
additional adsorption of free surfactant molecules available in the micellar solution.
The diagram illustrating the mechanism of formation of a wide particle size dis-
tribution in micellar solution (for Ag nanoparticles as an example) is shown in
Fig. 1.17. Synthesis in reverse micelles allows to obtain nanoparticles of various
metals in micellar solutions that are often more stable and more resistant to
aggregation and air oxidation than the nanoparticles of the same metals in water
Fig. 1.17 Scheme

illustrating the growth and AgNO3 N2H4
aggregation of Ag
nanoparticles synthesized in
micellar solution (reduction
Collision
with hydrazine). Reprinted Ag
from Ref. [82]. Copyright
2007, with permission from
Exchange
Elsevier
Ag
Aggregation
Ag Ag
Growth
Ag
solutions. As in the case of water solutions, the majority of papers are devoted to
the synthesis of nanoparticles of noble and some transition metals applied in
heterogeneous catalysis. The selected publications on Au, Ag, and Cu nanoparticle
synthesis are presented in Table 1.3.
As seen from the table, synthesis of the gold, silver, or copper nanoparticles
takes place at the hydration extent, w = 115; in most cases, spherical particles are
formed, with sizes below 20 nm. For w < 10, the nanoparticle diameter is less than
10 nm. The increase of hydration extent leads to the increase of both the particle
size and polydispersity.
The rate of formation, yield, and size of nanoparticles depend on the type of
solvent, on the presence of additional surfactants or alcohols (as cosolubilizers of
hydrophilic reagents) and on the type of a reducing agent. Particle size is influenced
also by the SAS concentration. The most signicant is the reducing agent to metal
ion concentration ratio. For example, the increase of hydrazine and metal ion
concentration ratio (at the constant hydration extent) leads to the decrease in size of
Au particles [127], while size of Cu nanoparticles either decreases or remains
constant (depending on w) [156] and that of Ag nanoparticles increases [152]. In the
latter case, the increase of hydrazine concentration provokes also a signicant
widening of particle size distribution, an example of such kind for silver
nanoparticles from [152] is shown in Fig. 1.18.
It turns out that, for the complete reduction of metal ions, it is necessary to use a
large excess (26 mol per mole of metal salt) and high concentration (about 0.2
0.8 M) of the reducing agent [128, 146, 153]. If sodium borohydride is applied in
AOT/isooctane reverse micelles, one has to use much smaller concentrations of the
reducing agent (about 104 M) because of its limited solubility in this micellar
system; hence, the yield of Ag nanoparticles is not great (<11 %) [152]. With equal
or close concentrations of hydrazine and sodium borohydride, the Ag nanoparticles
in AOT/isooctane micelles have equal sizes [159], and the sizes of Au nanoparticles
in non-ionic SAS/octane micelles are found to be differentwith borohydride, the
particle diameter is about 3 times smaller than that with hydrazine (3.5 and 10 nm,
respectively) [159]. This shows that the efciency of a reducing agent depends on
the micellar system composition and the type of metal ion.
In most cases, the nanoparticle stability in micellar solutions is rather highthey
can be stored for many months or even years. However, in some cases, the stability
depends on the amount of water in the system. Thus, for Ag and Cu nanoparticle
synthesis in AOT/isooctane micelles, sometimes the problems arise with the use of
metal salt water solution, because it turns out that the nanoparticles are not stable
(for instance, Ag nanoparticles exist in micellar solution for no longer than an hour)
[152]. To introduce metal ions, the authors used the micellar shells containing the
corresponding salts of the surfactant (AgAOT or Cu(AOT)2); the latter were pre-
liminary synthesized and then used for the preparation of the micellar solution. In
the process of Au nanoparticle synthesis in AOT/toluene micellar solutions, the
Table 1.3 Synthesis of metal nanoparticles in reverse micelles
40
Metal SAS/solvent Reducing w Nanoparticle size Note Reference

agent and shape
Au
Glycerol/AOT/heptane N2H4 1 58.4 nm, spherical [N2H4]/[HAuCl4] = 20.5 [127]
AOT/heptane N2H4 2.23, 30100 nm [N2H4]/[HAuCl4] = 6 [146]
3.38, (AFM data) Nanoparticles form aggregates on the
5.6, support
14.7
AOT/C12E4/isooctane N2H4 Size and shape depend [147]
on the presence of C12E4
and on
[N2H4]/[HAuCl4 ]ratio
AOT/isooctane K2SO3 10 810 nm, spherical Aggregation when d > 8 nm [148]
C12E4/octane N2H4 <8.5 10 1 nm, spherical Narrow size distribution [128]
NaBH4 3.5 1.5 nm, spherical
CTAB/octane+1-butanol NaBH4 rods 10 40 nm; Passivation with C12H25SH [149]
triangles and polyhedral,
2025 nm
AOT/SPAN80/isooctane NaBH4 10 Anisotropic Size and shape depend on NaCl [150]
concentration and T
Na2SO3 Spherical [151]
Triton X-100/cyclohexane <5 nm Narrow size distribution
Triton 520 nm Wide size distribution
X-100/cyclohexane/n-octanol
(continued)
agent and shape
Ag
AOT/isooctane N2H4 Spherical max decreases with increase of [152]
7.5 4.5 27.5 5 nm nanoparticle size; Ag+ ions were
introduced as AgAOT
5 2.7 1 nm
7.5 5 1.7 nm
15 6.5 2.5 nm
AOT/cyclohexane NaBH4 7.5 3 1.1 nm
AOT/dodecane N2H4 Spherical Nanoparticles aggregate when N2H4 [153]
5 1.52 0.83 nm concentration is 0.60.8 M. Lifetime is
less than 24 h.
7.5 3.4 1.3 nm
[N2H4]/[AgNO3] = 3
15 4.98 2.38 nm
AOT/dodecane N2H4 7.5 Spherical [N2H4]/[AgNO3] = 3 [154]
1.6 0.7 nm
(AOT 0.2 M)
3.9 1.05 nm
(AOT 0.4 M)
Rhamnolipid/heptane NaBH4 Spherical, 6.0 1.5 nm Stable for 2 months [155]
(18-3(OH)-18)/n-alkane [86] NaBH4 Spherical, 7.0 1.8 nm Stable for 2 months [86]
(continued)
41
42

agent and shape
Cu
AOT/isooctane N2H4 Spherical Cu2+ ions were introduced as Cu(AOT)2 [156]
1 2.1 0.7 nm [N2H4]/[Cu(AOT)2] = 3
2 2.8 0.7 nm
3 5.2 1 nm
4 7.3 1 nm
5 9.4 1 nm
10 12.6 1.1 nm
15 12.6 1.1 nm
AOT/cyclohexane 1 1 nm
2 1.4 nm
3 3 nm
4 4 nm
5 6 nm
10 7 nm
CTAB/isopropanol [Cu2+]/ Spherical CTAB catalyzes nanoparticle formation [157]
[CTAB]
1/0.25 520 nm
1/0.30 210 nm
HDEHP/cyclohexane N2H4 Spherical, 10 nm Cu2+ ions were introduced as Cu [158]
(DEHP)2
Abbreviations SPAN80, sorbitan monooleate; rhamnolipid, bio-SAA, plant lipid with sugar residue in the polar head; (18-3(OH)-18), 2-hydroxy-1,3-bis
(octadecylmethylammonium)propane dibromide; CTAB, cetyltrimethylammonium bromide; HDEHP, bis(2-ethylhexyl)phosphoric acid
15
W = 7.5 4
Relative Abundance, %
[N2H4] = 7 10 m
10
0
0 12 19 25 31 38 44 50 56 63 69 75 81 88 94 100105112119 125131
Diameter,
15
W = 7.5 2
Relative Abundance, %
[N2H4] = 7 10 m
10
0
0 12 19 25 31 38 44 50 56 63 69 75 81 88 94100105112119125131
Diameter,
Fig. 1.18 TEM images and the corresponding histograms of Ag nanoparticles obtained at the
equal hydration extent and different hydrazine concentrations, 7 104 (top) and 7 102 M
(bottom). Reprinted from Ref. [152]. Copyright 1993, with permission from American Chemical
Society
nanoparticle stability was decreased markedly when metal ions were introduced as
aqueous solutions of HAuCl4 or its salts, and increased, when dry salts were sol-
ubilized in the micellar solution [127, 128]. It was found also that, both with ionic
and non-ionic surfactants, the reaction rate and nanoparticle monodispersity
increased after deoxygenation.
The possibility to prepare metal nanoparticles of different sizes in the given
micellar system using the same reducing agent, as well as application of different
methods for the particle size control, brought into light the two problems actively
discussed in the literature in the last decades (see [81, 128, 159] and references
therein).
The rst one is disagreement of the changes of optical spectra with particle sizes
observed for noble metal nanoparticles, with the Mie theory predictions. It is known
that the Mie theory modied for the description of size effects of metal particles in
the region of small sizes [152, 160, 161] predicts that, the bigger is particle size, the
less is half-width of the absorption band, while its maximum shifts to the longer
wavelengths. Experimental results show in some cases that either the half-width of
the band does not depend on the particle size, or the maximum shifts to the opposite
side than that predicted by the theory, or even both parameters are in contradiction
with the theory [128, 159, 162165].
Various explanations have been suggested for this disagreement in some indi-
vidual cases [128, 165]; however, to date it can be stated that this question remains
unsolved. It is clear therefore that, in the general case, analysis of the nanoparticle
absorption spectra based on the Mie theory does not allow to make any denite
conclusions about the changes in the particle size [128]. Sometimes the researchers
fail to take this point into account; probably, this is one of the reasons of the
signicant dispersion of extinction coefcients reported in the literature for Au
nanoparticles: According to [128] and [48], its values for the nanoparticles with
sizes below 10 nm are about 3 103 and 107108 l/mole cm, respectively.
Another problem is the discrepancy between the results of particle size mea-
surements obtained by TEM and dynamic light scattering (DLS). Studies on
micellar solutions of Au and Ag nanoparticles show [128] that DLS systematically
gives the higher sizes compared to the TEM data. As pointed out by the authors of
[128], this is unlikely to be connected with the presence of large micelles or
surfactant aggregates usually considered as the most probable reason of overstated
size values in DLS measurements. In our view, such a discrepancy may result from
the distortions caused by the metallic origin of nanoparticles; additional arguments
on the subject will be given below (Chap. 2, Sect. 2.4.2). Since measurement of
particle sizes in a solution by DLS has a number of obvious advantages over TEM,
determination of the reasons of these distortions and their elimination represent an
actual task in studies of the metal nanoparticle solutions. However, this problem is
still unsolved, and in spite of the tedious work often needed to obtain the
high-quality electron micrographs, the TEM method still appears to be the most
reliable source of information about the average size and polydispersity of
nanoparticles in solution.
In summary, it can be concluded that synthesis in reverse micelles allows to
obtain small nanoparticles (with sizes less than 20 nm) with narrow enough dis-
tribution (including those suitable for the formation of the two-dimensional ordered
structures on solid substrate), stable in solution for a long time. In comparison with
synthesis in water solution, stability of the nanoparticles increases and a new
parameter appears (the hydration extent) which makes possible a more reliable
control of nanoparticle size. In comparison with synthesis in a two-phase system,
the procedure is simpler and the productivity is considerably higher that allows
producing the large amounts of a nanoparticle solution. The drawbacks of the
synthesis in reverse micelles are (1) application of strong (traditional) chemical
reducing agents in a large excess with respect to metal ions, hence the danger of
contamination of the nanoparticles or nanoparticle-modied materials with
non-metallic impurities, (2) usually small nanoparticle concentration in solution,
and (3) in some cases, one is forced to complicate the procedure (to use additional
syntheses, deoxygenation, etc.) in order to provide the nanoparticle stability.
1.4 Photo and Radiation-Chemical Synthesis 45
1.4 Photo and Radiation-Chemical Synthesis
Apart from the reduction of metal ions in reactions with various reducing agents
(chemical reduction proper), reactions with solvated electrons and other reducing
particles (atoms and radicals) generated in solution under the exposure to
high-energy radiations are used for the synthesis of metal nanoparticles. For this
purpose, mostly sources of the UV radiation (photochemical synthesis, or photol-
ysis) and -radiation (radiation-chemical synthesis, or -radiolysis) are used. In the
photochemical synthesis near ultraviolet (wavelength 200400 nm and energy
below 60 eV) and in the radiation-chemical synthesis, the high energies (60104 eV)
are mainly applied.
Two irradiation methods are usedstationary (continuous) method with smaller
doses or the pulsed one, when an object is exposed to the high dose for a short time.
An important advantage of these methods is, rstly, the possibility of nanoparticle
synthesis in various media (including solid ones, such as lms and polymeric
matrices), and, secondly, the possibility to obtain the product chemically pure,
without the non-metallic impurities incorporated into the nanoparticles or adsorbed
on their surface, which is a common drawback of the chemical reduction proper
[31, 32].
To create metal nanoparticles in the liquid media, molecular (aqueous, aqueous
alcoholic or organic) or micellar solutions of reducible compounds are used. In this
case, organic substances form the short-lived reducing particles acting as secondary
reducing agents. When an aqueous solution is exposed to light, hydrated electrons,
hydrogen atoms, and hydroxyl radicals (OH*) are formed [31, 32, 165]:
H2 O hv ! e
aq H OH
0
1:12
Hydrated electrons and the hydrogen atoms are the effective reductizers.
Standard redox potentials of the pairs H2 O/eaq and H+/H0 are 2.87 and 2.3 V,
respectively [166]. By contrast, the OH* radical is a strong oxidizer, and standard
potential of the pair OH*/OH is +1.9 V [166]. The simultaneous presence of the
hydrogen atoms and OH* radicals stimulate both reduction and oxidation processes.
Depending on the purpose of studies, the conditions are created providing the
predominance of either oxidizing or reducing particles. This is achieved by the
addition of either donors or acceptors of the OH* radicals. In the rst case, solution
is commonly saturated with nitrous oxide, which captures the hydrated electrons
with subsequent formation of OH* radicals [166]:
e
aq N2 O H2 O ! N2 OH OH : 1:13
To create the reducing medium, alcohols, ketones, or salts of organic acids are
introduced into solution. For example, isopropyl alcohol captures both the hydro-
gen atoms and the OH* radicals with the creation of the organic radicals having
reducing properties:
H0 OH CH3 2 CHOH ! H2 H2 O CH3 2 C OH: 1:14
Thus, there are only the reducing particles which remain in solution, namely the
hydrated electron and the organic radical. Standard redox potential of the
(CH3)2C*(OH) radical is equal to 1.39 [166]. Thus, these two particles are capable
of reducing metal ions to atoms [165, 167]:
e
aq Ag

! Ago : 1:15
CH3 2 C OH) + Ag ! CH3 2 COH + Ago : 1:16
Further aggregation of metal atoms and ions results rst in the formation of
clusters and then nanoparticles, as already described above (see the mechanisms
1.11.2). Good results are obtained by the reduction of metal ions in solution
containing high alcohol concentration and saturated (up to much lower concen-
trations) with nitrous oxide [165, 167]. In this case, the OH* radicals created in the
reaction (1.13) are captured by alcohol and form organic radicals according to the
reaction (1.14), with the subsequent reduction of metal ions. In such a way, it is
possible to increase the yield of nanoparticles, since the lifetime of organic radicals
in water solutions exceeds that of the hydrated electron.
To increase the nanoparticle lifetime in water or wateralcoholic solutions,
various stabilizers are used, such as citrates, SAS (DDS, AOT, CiEj, and other), and
polymers (PVA, PVP, PAA and alginates, PEI) [165, 167175]. In some cases, the
additional reducing agents (e.g., fenidon [170]) are introduced. In the recent years,
photochemical synthesis of Au nanoparticles [176] and radiation-chemical syn-
thesis of Ag nanoparticles [177] in water solutions have been successfully
accomplished with stabilization by chitosan, the well-known biodegradable natural
polymer which has good perspectives of application in medicine [178].
Photochemical reduction in solution is most often used to create the nanopar-
ticles of noble and platinum group metals. Recent advances in the synthesis of
metal nanoparticles by means of ion photoreduction in solution, in the increase of
their yield and stability, and in their modications using photochemistry techniques
are described in the review [179]. Of importance here are the radiation source
parameters; accordingly, the methods applied are divided into classic (using
non-monochromatic sources, mainly mercury or xenon lamps with light lters) and
laser ones, using laser light sources. Laser sources with the same or similar
wavelengths are characterized by the high coherence, monochromaticity, and power
[180, 181]; this allows to widen signicantly the potentialities of investigation of
the photochemical reactions including the reduction of metal ions and the
nanoparticle formation [31, 81, 128].
As in the previously described methods of chemical synthesis with the use of
traditional reducing agents, the most actively studied in photochemical synthesis are
Au and Ag nanoparticles. Silver ions are introduced mainly as silver perchlorate,
less often as silver nitrate, and gold ionsas HAuCl4 or its salts. Some examples of
the nanoparticle synthesis illustrating the possibilities of photoreduction in solutions

are given below.
Polydisperse solutions of spherical Ag nanoparticles with sizes in the range 23
67 nm were prepared in the water solution containing ethanol and DDS by UV
irradiation from the high-pressure mercury lamp with a power of 500 W, and the
nanoparticles obtained were stable for more than 8 months [182]. Spherical Ag
nanoparticles with narrow distribution were synthesized by the UV irradiation of a
solution containing Ag+ ions and a rst-generation dendrimer with amino groups
(G1NH2, polyamidoamine), which form a complex with Ag+ ions [183]. The
nanoparticles of 10.5 2.5 nm and 50 nm were formed at silver ion concentration
of 10 mM and 50 mM, respectively (Fig. 1.19). It is assumed that in both cases, the
nanoparticle formation is carried out under organizing action of the dendrimer, with
larger nanoparticles being formed by the aggregation of small particles. Thus, the
possibility of using dendrimers not only for the reduction of metal ions and sta-
bilization of nanoparticles but also for the regulation of their size is demonstrated.
Ag nanoparticles with a size of 46 nm and narrow distribution were synthesized
from solutions of silver diammine ([Ag(NH3)2]+) in the presence of PVP; stability
of the nanoparticles was not less than 6 months [184].
The effect of silver salt composition was also detected: The rate of nanoparticle
formation from diammine nitrate was signicantly higher than that in case if silver
nitrate was used.
Fig. 1.19 TEM images of G1 dendrimer-stabilized nanostructures: a discrete nanoparticles,

b solid nanospheres. Reprinted from Ref. [183]. Copyright 2003, with the permission from
Elsevier
Spherical Au and Pt nanoparticles with an average size of 4.5 and 2.7 nm,
respectively, were prepared by the UV irradiation of metal salt water solutions in
the presence of DDS. Studies of nanoparticle solutions and concentrated solutions
of DDS by small-angle X-ray scattering (SAXS) showed that the nanoparticle
formation occurs in the internal medium of the DDS (direct) micelles and not in the
bulk solution [175]. In the article [174], the possibility to create the nanoparticles of
different shapes was demonstrated. For example, Au nanoparticles in the form of
leaves were prepared by the UV irradiation of the mixture of water solution of
HAuCl4 with alcohol solution of PVP in the presence of Ag+ ions.
In the process of centrifugation of such solution, the nanoparticles gathered in
the ordered flower-like structures (Fig. 1.20). After the ultrasonic treatment, they
dissociated into the foliate particles again. Apart from Ag, Au, and Pt nanopar-
ticles, stable Pd, Rh, and Cu nanoparticles were produced by the UV irradiation of
photosensitive metal complexes in water or wateralcoholic solutions in the pres-
ence of surfactants or electron donors (e.g., anions of carboxylic or amino car-
boxylic acids) [185187].
Fig. 1.20 SEM images of Au nanoparticle samples: a from freshly prepared solutions,
b nanoflowers prepared by centrifugation and redispergation of the a solutions, and c particles
obtained from nanoflowers-containing solution exposed to US irradiation. Scale bar 200 nm.
Reprinted from Ref. [174]. Copyright 2008, with the permission from Elsevier
It should be noted that creation of metal nanoparticles by photochemical (as well

as by radiation-chemical) synthesis has its own peculiarities weakly expressed or
absent in the reduction with traditional chemical reducing agents. Firstly, it was
found that in many cases for photochemical reduction in aqueous or aqueous
alcoholic solutions, the nanoparticle yield and stability on air were signicantly less
than in the absence of oxygen (in vacuum or in inert gas atmosphere) [128, 167].
Secondly, the irradiation time plays an important role in this synthesis. On the one
hand, the yield of nanoparticles may increase with time as a consequence of the
growing concentration of reducing particles in solution [184], and on the other
hand, it was found that the particle size can both decrease and increase with time.
Such effects are observed for Au and Ag nanoparticles with both the lamp sources
and laser irradiation, in the visible and UV range [31, 162, 188190]. For example,
upon irradiation of the aqueous AgClO4 solutions with a mercury lamp (in the
presence of acetone or DDS) during several tens of minutes, from the initially
formed mixture with very wide particle size distribution (10400 nm), a sol is
formed with an average particle size of 1020 nm ([31, p. 210]). Such transfor-
mation is supposed to be associated with the accumulation of reducing particles
(organic radicals), which adsorb on the nanoparticles and thus make them nega-
tively charged. Upon achievement of a certain maximum value, this charge causes
disintegration of the large particles into smaller ones. For Au nanoparticles pro-
duced by the irradiation of alcoholic solutions with a mercury lamp [188190],
gradual widening and subsequent disappearance of the gold surface plasmon band
(523 nm) and the appearance of the other two bands (270 and 840 nm) were
observed. It was suggested that these changes were caused by the nanoparticle
aggregation completed with their full precipitation after 20 h of irradiation.
For Ag nanoparticles in aqueous solutions with stabilizers (prepared both by
photoreduction and with traditional chemical reducing agents), a photostimulated
aggregation was observed, when the concentration of the initially formed small
nanoparticles decreased and aggregates of the larger sizes appeared under irradia-
tion over time [162, 188190]. The aggregation rate depends on the solution
composition and on the wavelength of light: the effect of UV irradiation is stronger
than that of the visible light. In contrast to the aggregation induced by the lamp
sources, specic features of the laser photostimulated aggregation (LFSA) are
conditioned by the fact that, because of the high intensity of the pulsed laser
radiation, it affects not only the coagulation kinetics, but also the properties of the
nanoparticles [159, 162]. Studies on the LFSA mechanism have shown [159] that
the main reason for the aggregation is connected with an increase in the efciency
of the nanoparticle adhesion in the course of collisions. This increase is stipulated
by various factors depending on the type of metal sol stabilization (electrostatic or
steric) and the nature of stabilizer. It was also concluded that the heating of particles
under the influence of a high-intensity pulsed laser beam leads to their destruction
and modication of the physical properties of the dispersion medium, which can
signicantly increase the rate of coagulation.
Thus, it turns out that the light exerts a complex action on the solution studied
and can not only cause the appearance of reducing particles, but also change the
sizes and properties of the created nanoparticles, these changes being the more
signicant, the more powerful and focused is a light source.
Radiation-chemical synthesis of metal nanoparticles in solution is carried out
using -radiation sources (e.g., 60Co). The effect of irradiation depends on various
factorsa source power, irradiation time, solution composition and pH, reagent
concentrations, temperature, etc.; the last four factors may change during the irra-
diation. The radiation yield (the number of active particles generated per 100 eV of
absorbed radiation energy) depends on the radiation dose, which is dened as the
product of the dose rate (usually in kGy/h) and the irradiation time. Initial yield of
the primary reactive particles (ions, electrons, and excited particles) is of the order
of several units (often not more than 10) depending on the medium composition
(mainly on the solvent nature). For example, in water radiolysis, the primary
radiation-chemical yields are [169]:
H2 O !eaq 2:7; H 0:5; OH 2:9; H2 0:5;

1:17
H2 O2 0:7; H 2:7:
The yield of solvated electron for ammonia, alcohol, and normal hydrocarbons is
3.1, 11.8, and 0.10.2, respectively. The experimentally observed radiation yields
vary over a wide range (from 106 to 108 particles per 100 eV) [181] as a result of
subsequent interactions of the primary particles. Reduction of the metal ions is
carried out usually in aqueousalcoholic solutions; main interactions of the primary
particles in solutions are given above (reactions 1.13 and 1.14). Metal atoms and
small clusters created in the reduction process represent the seeds for the
nanoparticle formation. Radiation-chemical synthesis is always performed in
oxygen-free conditions, that is, under vacuum or in an inert gas atmosphere. In
contrast to the photochemical synthesis, spatial distribution of the primary inter-
mediate products is more uniform, and therefore, synthesis of particles with more
narrow size distribution is possible [32, p. 32]. However, creation of stable
nanoparticles in radiation-chemical synthesis is impossible without the stabilizing
additives; the main types of stabilizers have been mentioned above.
In the pulsed mode of irradiation in combination with spectrophotometric
analysis of solutions (pulsed photolysis or radiolysis), it is possible to detect the
appearance of short-lived particles with a small number of metal atoms, created at
various stages of aggregation. For example, in the process of photoreduction of Ag+
ions at the initial time of exposure in the UV absorption spectrum appears the 275
277-nm band, which is attributed to Ag+4 clusters, and also silver nanoparticles are
formed, 23 nm in size [169, 170, 191193]. Study of the early stages of silver
nanoparticle formation by pulsed radiolysis in neutral aqueous solutions (with the
addition of organic compounds) revealed that synthesis of the nanoparticles pro-
ceeds through the formation of Ag0 atoms as well as diatomic Ag+2 and tetraatomic
Ag2+ +
4 ions [169, 194, 195]. It was suggested also that a polyatomic Ag9 cluster
exists with 325-nm absorption band [192]. In some cases, the intermediate forms
(e.g., Ag and Pb clusters [196, 197]) can be stored for several days under exposure
to the ionizing radiation, and similar behavior is probable for the photochemical
reduction [198].
Pulsed radiolysis allowed to obtain ions of various metals in unusual oxidation
states [169]; this is of great interest for elucidation of the mechanisms of metal ion
transformation at the initial stages of metal sol formation.
By means of the stationary or pulsed -radiolysis, the nanoparticles of many
individual metals (Ag, Au, Cu, Pt, Pd, Co, Sn, etc.) and various bimetallic particles
(Au/Ag, Au/Pt, Ag/Pt Ag/Cu, Ag/Pb, etc.) were prepared in aqueousalcoholic
solutions; successful attempts to synthesize trimetallic (Au/Pb/Cd) nanoparticles
[32, pp. 3133] were also reported [31, pp. 214215]. Here, we present some
examples from the works published in the last 1012 years with illustrations that
give a notion about size and shape of the prepared nanoparticles, as well as on the
character of the effects of the main acting factors.
Silver nanoparticles are stabilized with citrate [165], PVA and PAA [172], PVP
[199], and chitosan [177]. In most cases, the nanoparticles are spherical and not
monodisperse, and the sizes and distribution width depend on the metal ions and
stabilizer concentrations, as well as on the radiation dose. With xed concentration
of metal ions and stabilizer, the nanoparticle concentration depends on the radiation
dose. As shown, for example, in [199], at a constant silver nitrate concentration, the
intensity of the nanoparticle absorption band increases with the increase of irradi-
ation dose, i.e., the concentration of nanoparticles is enhanced. The increase of
nanoparticle concentration is observed at different concentrations of Ag+ ions (in
the range 7.4 1041.84 103 M), a slight increase in the ion concentration
resulting in signicant increase in nanoparticle concentration. Increase in the dose
also leads to a small shift of max to the shorter wavelengths (from 410 to 403 nm)
and to a particle size diminution. For example, when the dose is increased from 20
to 60 kGy, the average particle size decreases from 35 to 20 nm with simultaneous
increase in the distribution width and number concentration of the nanoparticles.
This result suggests that largest nanoparticles created during irradiation are the
aggregates of smaller particles and increasing the dose leads to the destruction of
larger nanoparticles with a corresponding increase in the number of particles per
unit volume of solution.
A stabilizer plays an essential role as well. An example of the effect of stabilizer
concentration on the particle size taken from the paper [165] is shown in Fig. 1.21. It
is seen that particle size decreases with the increase in stabilizer (citrate) concen-
tration from 0.5 104 to 0.8 104 M, and then with further increase in its
concentration (up to 1.2 104 M), the average size does not change, but the
distribution becomes more narrow. Aggregates of two or three particles are shown
on the TEM photographs. With further increase up to 5 104 M citrate, an average
particle size and a narrow distribution (10 2 nm) remain unchanged and aggre-
gates of the particles are absent. At the 15 104 M citrate concentration, large
aggregates are formed owing to the instability and coalescence of nanoparticles.
Thus, there is a narrow range of citrate concentrations (1.25 104 M) where
isolated nanoparticles about 10 nm in size are formed with narrow size distribution.
The lower concentrations are not sufcient for stabilization of the small particles,
Fig. 1.21 TEM images of Ag

nanoparticles at different
citrate concentrations.
Reprinted from Ref. [165].
Copyright 1999, with
permission from American
Chemical Society
and hence, their aggregation is observed; at the higher concentrations, citrate exerts
a destabilizing effect on account of the increase of ionic strength. As shows the
high-resolution electron microscopy, in the chosen favorable concentration range
nanoparticles have the form of octahedral or icosahedral crystals. Example of the
icosahedral nanoparticles is shown in Fig. 1.22.
Using the radiation-chemical synthesis, it is possible to control sizes of the
particles prepared by the same or by some other method. For this purpose, the same
metal ions are introduced into solution of previously prepared nanoparticles of a
known size, and then the solution is exposed to ionizing radiation. With this
technique, the size of Ag [165] and Au [167] nanoparticles was increased by several
times. In the latter case, the 15-nm nanoparticles were previously synthesized by the
citrate method, and then certain Au(CN)2 ion concentrations were sequentially
introduced with following irradiation; increase of the particle sizes was achieved by
building up the new atoms on the nanoparticle surface (Fig. 1.23).
Similar technique was applied for the preparation of bimetallic Ag(X)
nanoparticles of different composition, where X is Cu, Pb, and Cd [168, 193, 200],
with a spherical form, with a size of 120 nm, and with core (Ag)/shell structure.
Bimetallic nanoparticles can also be obtained by irradiation of a mixture of two
metal saltsAuAg [172], AgPt, and AuPt [171]. A radiation dose and a type of
stabilizer play an important role. For example, by irradiation of a mixture of Au and
Fig. 1.22 Icosahedral Ag

nanoparticles. Left HRTEM
images of Ag nanoparticles in
different projections at
1.5 104 M of citrate.
Middle and right computer
simulations and calculation
results for multilayer models,
respectively. Reprinted from
with permission from
American Chemical Society
Ag salts with low doses [172], rst Ag+ ions are reduced to atoms, and then these
atoms reduce gold ions with the formation of the gold clusters, and after that, Ag+
ions are reduced on the surface of these clusters. As a result, the nanoparticles
formed consist of a gold core coated with silver. At the high doses, the nanopar-
ticles of a bimetallic alloy are created. After irradiation of a mixture of AgPt and
AuPt salts [171], the nanoparticles of homogeneous structure are produced with
the dimension that depends on the nature of stabilizer: In the presence of PVA, the
nanoparticle size is larger than that in the presence of PAA. In the latter case, small
particles with a diameter of 16 nm are formed.
The combination of chemical reduction with radiation-chemical synthesis allows
to obtain also non-spherical nanoparticles. Thus, it was possible to grow Au
nanoparticles of rectangular shape (Fig. 1.24) from a solution containing Au+ and
Ag+ ions, isopropyl alcohol, CTAB, and ascorbic acid [56]; rst, gold seeds were
created by a short-term -irradiation, and then the created Au1+ ions were reduced
with ascorbic acid. Anisotropic growth of the nanoparticles was favored by the
bilayer structure of CTAB and selective adsorption of Ag+ ions.
Despite the obvious advantages of this method, rst of all, a large variety of
metal ions and their combinations used successfully for the creation of nanoparti-
cles and the possibility to study in detail the mechanism of ion reduction and
nanoparticle formation, the radiation-chemical synthesis has its own limitations.
Firstly, the necessity to deaerate solutions and perform the synthesis in sealed
ampoules complicates the procedure and makes it difcult to produce large quan-
tities of a nanoparticle solution, if necessary for practical application. Secondly, the
stability of the nanoparticles in a solution on air is often not high, since they are
subjected to oxidation, aggregation, and eventually precipitate. Thirdly, similarly to
Fig. 1.23 TEM images that demonstrate redoubling of Au nanoparticle sizes. a citrate-stabilized
initial sol (dav = 15 nm); b, c, and dafter the rst, second, and third stages of the size increase by
the introduction of the additional amounts of gold ions, respectively. Reprinted from Ref. [167].
Copyright 1998, American Chemical Society
photoreduction, in many cases, it is difcult to adjust particle sizes and obtain a

monodisperse solution; polydisperse particles (sometimes with the size of the order
of dozens of nanometers) are often produced, which are not suitable for the studies
of size effects and their practical application.
The advantages of synthesis in reverse micelles noted in the works with con-
ventional chemical reducing agents, in particular, an increase of the nanoparticle
stability during storage in the air and the possibility to control the particle size
stimulated the development of researches in the eld of photo- and radiation-
chemical synthesis in micellar solutions. Ag [8183, 128, 170, 201203], Au
[81, 128], Cu [203], and Pd [203, 204] nanoparticles as well as bimetallic PdAg
and PdAu nanoparticles [204] were prepared with this method. By varying the
ratios of reagents and the hydration extent, it was found possible to increase the
nanoparticle stability in a solution during storage in the air and in some cases to
fabricate smaller nanoparticles with a narrower size distribution than that obtained
Fig. 1.24 TEM image of the

rectangular Au nanoparticles.
Copyright 2011, with the
permission from Elsevier
for the same metal during synthesis in an aqueous solution [81, 82, 128]. Zhang
et al. [81, 128] developed an interesting modication of the synthesis in reverse
micelles, including photoreduction with the application of non-ionogenic surfac-
tants and introduction of metal in the form of dry salt; in such method, it is possible
to signicantly expand the range of chemical reducing agents and improve the
stability of prepared nanoparticles.
However, for photo- and radiation-chemical synthesis in reverse micelles, there
exist methodological limitations associated primarily with the fact that, to create a
sufcient concentration of reducing particles, it is necessary to provide the corre-
sponding concentration of water in the system because the yield of solvated electron
in nonpolar solvents is lower than that in water by more than an order of magnitude.
Since water concentration in reverse micelles is determined by the hydration extent,
this means that synthesis is feasible if w is not below a certain threshold value
corresponding to a minimal acceptable concentration of the hydrated electron. This
value depends on the composition of reverse micellar system; as a rule, it is close to
or is slightly greater than the hydration extent which marks the appearance of free
water in the micellar core. Studies on the properties of the hydrated electron by
pulse radiolysis in AOT reverse micelles in isooctane showed that concentration of
the hydrated electron for w < 15 depends on the hydration extent and decreases
with w diminution [205]. As it is known that AOT can solubilize the largest number
of water molecules among all the micelle-forming surfactants, the threshold w value
should be even smaller for the other SAS. It is also known that in the AOT/n-alkane
system at w < 5, the e(aq) concentration becomes so low that reaction with its
participation cannot be realized [133, 206]. At the same time, for the synthesis of
metal nanoparticles, small hydration extents are used (less than 15, see Sect. 1.3.4),
since this allows to produce the nanoparticles of small size and a sufciently narrow
distribution. Furthermore, positive influence of the medium organization in a water
core at the initial stages of nanoparticle synthesis is efcient only at sufciently low
hydration extents, when properties of the internal medium of micelle (of a
nanoreactor) are essentially different from those of free water. Also, with the
increase of w, the nanoparticle polydispersity increases. Thus, there is a problem of

the choice of such hydration extent that would allow, on the one hand, to create the
solvated electron concentration sufcient for the redox reaction to proceed with an
acceptable rate and, on the other hand, to obtain nanoparticles of a small size and
with narrow distribution. It is clear that the advantages of reverse micelles versus an
aqueous solution can be realized only if this problem is solved successfully.
1.5 Electrochemical Synthesis
Preparation of metal nanoparticles by electrochemical methods is performed by

means of electrocrystallization (electrodeposition) of metals from their salt solu-
tions or melted salts in the course of electrolysis. Electrocrystallization of solutions
is described in detail in [37, 207]. The process is carried out in electrolytic bath
containing the solution with a reducing agent and an ion to be reduced. Under the
influence of continuous current, the reducing agent is oxidized on anode and metal
ion is reduced on cathode. Metal deposit formed on cathode may have a various
structure. This may be a dense layer of microcrystals, or a friable layer of either
crystalline or amorphous particles. The morphology of the deposits depends on the
nature of metal and solvent, metal salt composition, the nature of reducing agent,
concentration of the main reagents and other components of solution, adhesive
properties of the particles deposited on cathode, mixing conditions, current density,
temperature, etc. There are also special requirements for materials and electrode
surface preparation. Thus, the anode material should be resistant to acids and have a
low electrical resistance. Besides, to ensure the purity of a nal product, it is
preferable to use the anode manufactured from a metal that should be reduced in
this process; however, it is not always possible due to technological reasons [29,
p. 67]. Therefore, for example, in the preparation of copper (as a powder) or iron
nanoparticles, one uses, respectively, aluminum or molybdenum anode. For the
cathode, quality and surface roughness are important, as they determine the dis-
persity extent of the resulting metal deposit.
The deposit is periodically removed from the cathode; it is necessary for the
production of a powder of uniform composition and with the dispersity required. In
the process of electrolysis, the electrolyte composition changes continuously in the
vicinity of electrodes by means of the forced circulation of solution in the bath. The
powder removed from the cathode is rinsed and dried. To prevent oxidation of the
product, it is dried in vacuum.
The technique described above allows preparation of approximately 30 metals in
the nanosized state (Au, Ag, Pt, Cu, Fe, Ni, etc.) [29, 31]. Since rening is con-
ducted in the process of electrolysis, the products are characterized by a high degree
of purity. Depending on the conditions, the deposit can be formed as a powder, a
porous sponge, or whiskers (dendrites), which are then easily powdered by
mechanical methods. Sizes of the metal particles vary from 10 thyrsus 20 to more
than 100 nm.
1.5 Electrochemical Synthesis 57
Also, the electrodes can be produced coated with nanosized noble metal alloys,
which can be applied in electrocatalysis. For example, electrodeposition of the Au
Pt alloy nanoparticles on the surface of ITO (indium tin oxide) electrode was
recently performed [208]. The size and shape of nanoparticles depend on the
number of electrodeposition cycles. After 20 cycles, the nanoparticles resemble the
cauliflower (Fig. 1.25). Electrodes with such coatings showed electrocatalytic
activity in nitrite oxidation and oxygen reduction reactions.
When stabilizing additives, such as surface active substances, are introduced into
the electrolyte solution, metal nanoparticles are formed in the bulk of solution [209,
210] (Fig. 1.26).
Anode is fabricated from a reduced metal and cathodefrom the same or a
different metal. Surface-active substances present in solution not only perform the
Fig. 1.25 Typical SEM image of AuPt nanoparticles on the ITO electrode. Nanoparticles have
been obtained in the potential range of 0.11.1 V after 20 cycles in the buffer solution containing
citrate, Na2HPO4 (pH 2.4), as well as 0.12 mM Au and 0.12 mM Pt. Reprinted from Ref. [208].
Copyright 2010, with the permission from Elsevier
Fig. 1.26 Principle scheme

of electrochemical fabrication
of individual metal
M M
nanoparticles in the presence
of dissolved SAS
([31], p. 220)
M
nanoparticle stabilization but also shield the active areas of the cathode surface,
thereby preventing the metal adsorption and deposit formation. Besides, surfactant
molecules can solubilize the nanoparticles deposited on the cathode; thus, they are
transferred back into solution.
In this way, the nanoparticles of Pd and Ni were prepared in TAABr solution
containing mixture of acetone and tetrahydrofuran [209], Au in aqueous solution
containing cationic SASs [211], Pt in aqueous solution in the presence of PVP
[212], Au, Pt, and AuPt in buffer solution of NaH2PO4 + citrate [208], Co, Fe Ni
in aqueous solution at neutral pH [213], and many others. Using a gold anode and a
platinum cathode in aqueous solution with cationic SASs (HDTAB and TOAB), by
electrolysis at 311 K with ultrasound processing, two types of Au nanoparticles
were produced: one of a spherical shape with the size of 10 nm and the other of a
rod shape and the size in the range 80120 nm [211]. A comprehensive information
on the preparation and properties of gold nanorods, including the electrochemical
methods, may be found in the review [214].
A number of various modications of electroreduction of metal ions are also
proposed that allow producing nanoparticles in solution and depositing them on
different surfaces; an extensive literature on electrochemical methods used for the
preparation of metal nanoparticles and nanostructured coatings is given in [215]. As
an example, we mention here a new thermal electrochemical synthesis (TECS),
used to produce Ag nanoparticles with the size of several nanometers, stable in
aqueous solution without stabilizers [216].
The synthesis is conducted at 25100 C, by means of the two silver electrodes
surrounded by dialysis membranes in the form of tubes that are immersed in water
or simple electrolyte solution (Figs. 1.27 and 1.28). Application of dialysis mem-
branes provides the conditions for the synthesis of Ag nanoparticles smaller than
10 nm without stabilizing coatings. It is claimed that these nanoparticles are stable
in aqueous solution. It was shown also that Ag nanoparticles with a broad size
distribution can be converted into monodisperse suspensions by irradiation with
low-power HeNe laser.
There is no doubt that electrochemical synthesis of metal and alloy nanoparticles
is a promising direction representing a research area at the boundary of electro-
chemistry and nanotechnology, allowing to produce materials with useful properties
for application in chemistry, technics, and other elds [29, 31]. The advantages of
this method are (1) the possibility to control the formation and properties of
nanoparticles and nanostructured coatings by varying the parameters of electrolysis,
(2) the possibility to produce chemically pure nanomaterials (rening), and (3) high
productivity, essential for the industrial applications. At the same time, the method
has its disadvantages from economical point of viewit requires the use of special
equipment and very high energy expenditure, so the materials obtained in this way
are relatively expensive and have limited availability for the large-scale application.
The need to use special equipment which is often very expensive is also the
disadvantage of photo- and radiation-chemical synthesis considered in the previous
1.5 Electrochemical Synthesis 59
Fig. 1.27 Mechanism of Ag nanoparticle formation in thermal electrochemical synthesis reactor.

During the progress of electrochemical reaction, black silver oxide accumulates on the anode
surface and then dissociates into ions. At the same time, white powder deposits on the cathode
surface due to ion reduction with the formation of metal nuclei. Afterwards, these nuclei migrate
away from the cathode and grow into the nanoparticles in bulk solution. Reprinted from Ref.
[216]. Copyright 2009, with the permission from Elsevier
Fig. 1.28 Scheme of the thermal electrochemical synthesis reactor. a Electrodes enclosed with
one U-shaped membrane tube with a middle barrier, b electrodes in two separate membrane tubes
with their bottom sealed or tied, and c electrodes embedded into one big membrane tube. Reprinted
from Ref. [216]. Copyright 2009, with the permission from Elsevier
section. This is especially appreciable in the case of radiation-chemical synthesis, as

the source of -radiation, in addition to the high cost, requires also a special pre-
mise, separate operating personnel, and compliance with specic safety regulations.
1.6 Biological Reduction in Water Solution
In the last decade a new direction in synthesis of metal nanoparticles is developed,

which utilizes natural biologically active substances extracted from plants or ani-
mals, aqueous extracts from the living organisms or living organisms able to
synthesize nanoparticles. As recently noted in the literature [217], these methods
have a number of advantages in comparison with the traditional methods using the
basic chemical reducing agents. The major advantages are soft synthesis conditions
and the absence of more or less poisonous side products in a nanoparticle solution,
which often appear due to the presence of basic chemical reducing agents taking
part in the process. For this reason, this biological reduction as a ground for the
preparation of metal nanoparticles is very promising for application in biology,
medicine, and some other elds, where it is important to provide the lowest possible
toxicity of metal nanoparticle solutions for the living organisms. These methods
appear in the literature under various namesgreen synthesis, biosynthesis,
biochemical synthesis, or biological reduction. Sometimes green synthesis
also means the reduction in an aqueous solution using the well-known natural
reducing agents (e.g., sugars) that can be produced articially.
Several years ago, the works by the scientists from India [218, 219] became
widely known, where the formation of gold nanoparticles in geranium leaves was
described, taking place after the injection of water solution containing gold ions.
The process was found to be protractedabout several days were required for the
nanoparticle synthesis. Later on, it turned out that the nanoparticle formation could
be accelerated by the application of water extract from geranium leaves as well as
from many other plants, and also from fungi, cultures of some bacteria species, and
other living organisms. It was established that such nanofactories working in
different types of biological objects serve as the source of substances which
facilitate both the synthesis and stabilization of metal nanoparticles.
As far as we know, for the time being, this method allowed to synthesize only
noble metal nanoparticles, mostly Au and Ag; the relevant data are considered in
the reviews [220, 221] published in the last years. Metal ions have been introduced
as water solutions of HAuCl4 and AgNO3. The notion about the biological objects
used and the main characteristics of nanoparticles obtained can be formed from
Tables 1.4 and 1.5.
The most numerous group is comprised by the nanoparticles synthesized with
the use of water plant extracts. Different parts of plants are employedflowers,
leaves, stems, and roots; also extracts from seeds and fruits are applied (see
Table 1.4).
Table 1.4 Biological reduction in aqueous solution. I. Green plants
Plant part and name/active Metal Nanoparticle shape and size Note Reference
factor
Garcinia mangostana Ag dav = 35 nm Exhibit the antimicrobial activity [222]
leaves/aqueous extract
Acalypha indica leaves/ Ag Cubes with smoothed edges max (1) 440450 nm, [223]
presumably flavonoids lav = 2030 nm max (2) 370380 nm
Exhibit the antimicrobial activity
Mangifera Indica Ag Triangular, hexagonal, spherical, max 439 nm, pH 8 [224]
leaves/aqueous extract dav 20 nm tsyn = 1015 h
Chenopodium album Au, Approximately spherical max (Au) 540 nm [225]
leaves/aqueous extract Ag 10 < dav < 30 nm max (Ag) 460 nm
Olive leaves/aqueous extract Au Triangular, hexagonal: 50100 nm max (1) 535 nm, tsyn 30 min [226]
(luteolin et al.)
1.6 Biological Reduction in Water Solution
Spherical: 13 nm; max (2) 660 nm;

Shape and size depend on the extract ex = 350 nm, em = 425 nm
concentration and pH
Rosa rugosa leaves/aqueous Au, Approximately spherical, wide size max (Au) 578 nm [227]
extract Ag distribution max (Ag) 451 nm
Henna leaves/apiin Au, Au: spherical (1025 nm), triangular Ratio of shapes and dav of a given shape depend on [228]
Ag (4075 nm) apiin concentration; max (o) = 540 nm
Ag: quasi-spherical max () 800 (nm)
dav > 200 nm (39 nm) max (Ag)= 456 nm
Barbated skullcup Au Polyhedral, 530 nm max 540 nm, tsyn 3 h [229]
herb/aqueous extract Stable at least for 3 months
Basil stem and root/flavonoid Ag Polyhedral, 512 nm max 450 nm [230]
(luteolin) Root: 7 4 nm, stem: 7 3 nm
Clove buds/flavonoids Au Irregular shape max 550 nm; tsyn just a few minutes [231]
5100 nm
(continued)
61
62
Plant part and name/active Metal Nanoparticle shape and size Note Reference
factor
Jatropha curcas Ag Spherical, 1050 nm Crystal (fcc) [232]
seeds/aqueous extract
Alfalfa Medicago sativa Ag Spherical, triangular tsyn 2.25 h [233]
seeds/sprouting seed exudate Au lmean = 85108 nm
Lentil Lens culinaris Polyhedral
seeds/sprouting seed exudate
Sugar beet pulp/aqueous Au Polyhedral nanorods, nanowires. max 575600 nm; tsyn 7 h [234]
extract Shape depends on pH
Lemon juice/citric acid Ag Spheres, spheroids, dav < 50 nm 500 < max < 400 nm [235]
Citric acid is supposed to act as stabilizer
1.6 Biological Reduction in Water Solution 63
Table 1.5 Biological reduction in aqueous solution. II. Bacteria, fungi, and other organisms
Organism/active factor Metal Nanoparticle shape Note Reference
and size
Enterobacteria/culture Ag Spherical, d < 20 nm max 420440 nm, [238]
supernatant tsyn 5 min
Staphylococcus Ag d < 100 nm max 420450 nm; showed [239]
aureus/culture antimicrobial activities
supernatant
Fungus Penicillum Ag 23 < dav < 105 nm tsyn 72 h [240]
brevicompactum/growth
medium supernatant
Fungus Fusarium Ag Approximately Stable for several weeks [241]
semitectum /fungus spherical
biomass ltrate 10 < dav < 63 nm
Fungus Aspergillus Ag Spherical, max = 420 nm, tsyn 72 h, [242]
flavus monodisperse protein-stabilized,
Nanoparticles form on dav = 8.92 1.61 nm luminescence, em = 553 nm
the surface
Marine yeast Yarrowia Au Hexagonal, triangular max 540 nm, [243]
lipolytica/nanoparticles tsyn 310 h
are on the surface
Marine sponge Au Spherical, max 526 nm, tsyn = 4 h [244]
Acanthella elongate 7 < d < 20 nm
Silkworm/broin Au, Spherical, Au: max = 524 nm, stable [245]
Ag monodisperse Ag: max = 415421 nm,
Au: dav = 30 nm, aggregate
Ag: dav = 10 nm
In the vast majority of cases, nanoparticles formed have crystal (FCC) structure
and different shapes with wide distribution and sizes from 10 to more than 100 nm.
From the works available, it is only the extract from basil that allowed to produce
nanoparticles with the size of less than 10 nm, though with wide distribution [230].
In a given mode of nanoparticle synthesis, the ratio of different shapes depends on
the metal salt to extract concentration ratio; it is changed usually by changing the
portion of extract solution in a xed volume of water solution with a known
concentration of metal salt. In some cases, the particle sizes also depend on pH of
the solution. As a rule, the process proceeds at the room temperature. The rate of
nanoparticle formation varies from several minutes to several days. After the
nanoparticle formation is over, they usually preserve stability in solution in the air
from several weeks to several months. The absorption bands are often broad, with
flat peaks of low intensity, but clear, well-shaped narrow bands also occur. The
peaks of absorption bands both for silver and for gold are shifted to the longer
wavelength compared with their positions for the nanoparticles from the same
metals prepared by chemical synthesis in aqueous solutions. This can be explained
by the large size of the particles, as well as by adsorption of extract components on
Fig. 1.29 The effect of extract concentration on Au nanoparticle formation. Concentration of

HAuCl4 in all experiments: 1.3 104 M. Extract concentration was varied by the introduction of
different volumes of the extract (0.26.0 mL) into HAuCl4 solution to reach the same total
volume = 10 mL. At the top: photographs of solutions with different extract concentrations
(increases from right to left). Reprinted from Ref. [226]
the particle surface. The nanoparticle solutions have different colors, depending on
the particle size and concentration, particularly wide spectrum of colors and shades
demonstrating solutions of the gold nanoparticles (Fig. 1.29).
In most cases, the active substancesa reducing agent and stabilizerare
unknown. For some extracts [223, 226, 228, 230, 231], they have been identied by
means of the chemical analysis and (or) comparison of FTIR spectrum of an extract
with that of a nanoparticle solution.
It turned out that the reducing agents belong to the flavonoid group, including
those with sugar residues. The stabilizer is either the reducing agent itself [228] or a
derivative of some flavonoids and vegetable proteins with amino or amido groups
[226]. In the synthesis with sugar beet pulp [234] and lemon juice [235], it is
assumed that a reducing agent and a stabilizer are sugars and citric acid or its salts,
respectively. In some cases, the nanoparticle solutions exhibit luminescence [226].
Some examples of nanoparticle synthesis are given in detail below.
Gold nanoparticles can be prepared by mixing HAuCl4 solution with water
extract of olive leaves [226]. Figure 1.29 shows the absorption spectra and colors of
HAuCl4 solutions mixed with different amounts of the extract a few minutes after
the beginning of synthesis.
It is seen that, at the xed concentration of HAuCl4, position and width of the
absorption band depend on the extract concentration in solution. At the small
concentrations (addition of 0.22 ml of the extract), a gradual growth of the band
intensity and the shift from 545 to 530 nm are observed. At the high concentrations
Fig. 1.30 Au nanoparticle formation kinetics. [AuCl4] = 1.3 104 M, 1 mL of the extract in
10 mL of solution. Reprinted from Ref. [226]
(addition of 36 ml of the extract), the band shifts to the longer waves (up to
600 nm). These changes are accompanied with the changes in color: At the small
concentrations, the solution color changes from violet to pink of different intensity,
at the higher concentrations, it becomes reddish purple with various tints, and at the
6 ml added, the solution becomes dark green. Figure 1.30 shows an example of
nanoparticle formation kinetics at the addition of 1 ml of the extract.
The nanoparticle formation is practically accomplished within 20 min from the
beginning of synthesis. Figure 1.31 shows TEM photographs of the nanoparticle
solutions prepared at the different extract concentrations.
At the concentration achieved at 0.5 ml of the extract, a mixture of different
shapes is formedtriangular, hexagonal, and sphericalwith a wide size disper-
sion. At the addition of 5 ml, the nanoparticles are mostly spherical; as reported by
the authors, their size is about 13 nm. Figure 1.32A shows FTIR spectra of the
dried olive leaves and Au nanoparticle solution. The bands of 3409 and 1733 cm1
in the infrared spectrum of the dried leaves are characteristic for OH and C = O
groups, which presumably belong to oleuropein, apigenin-7-glucoside, and/or
luteolin-7-glucoside present in the leaves; the latter two are flavonoids with one of
the protons (at the carbon C7) substituted by a sugar residue. The structural for-
mulae of these substances are shown in Fig. 1.32B. For the nanoparticles obtained,
a luminescence at 425 nm was registered, under the light excitation with the
wavelength of 350 nm.
Similar effect is observed in the synthesis of Au and Ag nanoparticles at the
reduction with apiin (apigenin-7-apiosyl-glucoside) extracted from henna leaves
Fig. 1.31 The dependence of Au nanoparticle size and shape on the extract concentration. a TEM
image at 0.5 ml of the extract demonstrates triangular shapes; b at the same amount of the extract
hexagonal shapes; c magnied triangular particles from photograph a; d shapes of Au
nanoparticles at 5 mL of the extract. Reprinted from Ref. [226]
[228]. Figure 1.33 shows the absorption spectra of Au nanoparticle solutions,

prepared by the addition of apiin in different concentrations to HAuCl4 solution.
Here and in the experiments shown in Fig. 1.34, the total volume of the solution
was 5 ml. Inset in Fig. 1.33 shows the nanoparticle solutions prepared with dif-
ferent apiin amounts (letters on the test tubes correspond to those on the spectra)
20 min after the beginning of the reaction [228]. As in the previous example, color
of the solution changes rst from lilac to pink and then to reddish purple with the
increasing intensity and ends with dark green. At the small concentrations, there are
two bands (540 and 950 nm) in absorption spectrum, and at the high concentration,
there is a single band of 550 nm. Further increase in apiin concentration does not
lead to the increase of the latter band; according to authors, this indicates to the
complete reduction of the gold ions. Such steady-state condition is achieved after
3040 min. Figure 1.34 shows TEM micrographs of the nanoparticle solutions
obtained with the two small apiin concentrations which exhibit two peaks in the
spectrum.
In both cases, two main groups of particles are present in solutionspherical
with diameter below 30 nm and triangular or hexagonal with the size of 6070 nm
Fig. 1.32 A FTIR spectra of a pure olive leaf extract and b Au nanoparticles stabilized with the
extract. B Active substances of the extract assumed on the basis of FTIR spectra. Reprinted from
Ref. [226]
(0.013 ml of apiin) and 4070 nm (0.018 ml of apiin). Relative contribution of the

small particles is greater at the higher apiin concentration. The diffraction pattern
(inset) corresponds to gold monocrystal with FCC lattice. With the increase in apiin
concentration to 0.028 ml, the big particles disappear from the TEM images and
there remain only the particles approximately spherical, with relatively narrow size
distribution, dav = 21 3 nm (Fig. 1.35), with the respective band maximum at
540550 nm.
The comparison of the FTIR spectra of apiin, Au, and Ag nanoparticle solutions
shows that apiin plays a role both as a reducing agent and as a stabilizer. The
nanoparticle formation in the process of apiin interaction with metal ions is
c
Absorbnace
f
1
e
d
a
0
400 600 800 1000
Wavelength, nm
Fig. 1.33 Absorption spectra of Au nanoparticles synthesized at 1 103 M HAuCl4 a and with
addition of various amounts of apiin extract: b 0.013 mL, c 0.018 mL, d 0.022 mL, e 0.028 mL,
f 0.032 mL (at 25 C). Reprinted from Ref. [228]. Copyright 2009, with the permission from
Elsevier
illustrated by the scheme shown in Fig. 1.36. It is assumed that reduction of the
metal ion proceeds through the complex formation with C = O and OH groups of
apiin. During the nanoparticle formation, apiin molecules adsorb on the particle
surface with their polyphenol end. The nanoparticle size and shape depend on the
apiin concentration: At its small concentration, the number of apiin molecules is not
enough to stabilize small spherical nanoparticles, while at the high concentration,
apiin can arrest the growth of nanoparticles and prevent metal atoms from building
up at the certain parts of nanocrystals (presumably, edges and dislocations); as a
result, in the latter case, triangular particles are formed.
In the process of reduction by water solution containing exudates of alfalfa or
lentil germinating seeds, Ag and Au nanoparticles are obtained [233]. According to
the TEM images, in this case nanoparticles formed demonstrate different shapes and
broad size distribution. The example is given in Fig. 1.37. Mixing of 10 mM AgNO3
solution with alfalfa seed exudate in the darkness at 30 C leads to the formation of
spherical and irregular-shaped nanoparticles with the size of 540 nm. Mixing of
Fig. 1.34 TEM images a and the corresponding histograms of Au nanoparticle solutions b at
apiin concentrations 0.013 mL (left) and 0.018 mL (right). [HAuCl4] = 2 103 M. Inset electron
diffraction pattern of a triangular particle. Reprinted from Ref. [228]. Copyright 2009, with the
Fig. 1.35 TEM image of Au

nanoparticles at [HAuCl4]
= 2 103 M and apiin
concentration 0.028 mL.
Fig. 1.36 Formation of Au or Ag nanoparticles in the course of apiin interaction with metal ions.
Fig. 1.37 TEM image of silver nanoparticles prepared at dark using alfalfa seed exudates. Left
initial exudate; right initial exudate diluted with deionized water. C(AgNO3) = 10 mM. Synthesis
takes 2.25 h. Reprinted from Ref. [233], with the permission from the author
AgNO3 solution with the same exudate diluted with deionized water results in the
appearance of bigger nanoparticles (85108 nm), including triangular particles and
irregular-shaped particle aggregates. Composition of the seed exudate is unknown.
Addition of AuCl4 solution to the lentil seed exudate gives spherical and triangular
nanoparticles with wide distribution. The TEM image in this case is similar to that
obtained at the reduction by apiin (Fig. 1.34 above).
Introduction of the gold ions into the macerated aqueous extracellular dried
clove buds solution [231] in normal conditions results in stable irregular-shaped Au
nanoparticles with the size of 5100 nm and wide size distribution. The FTIR
spectroscopic examination of the solution before and after the addition of the gold
salt allows to suggest that the active agents here are the flavonoids extracted from
the buds.
There is also the other group of works, where silver and gold nanoparticles are
synthesized with the use of bacteria supernatants, biomass extracts, or growth
medium supernatants of some fungi and aqueous extracts from some marine
inhabitants (see Table 1.5). The ability of some microorganisms to extract metal
ions from a solution and form metal aggregates of a different size has been known
for a long time and is applied, in particular, for the extraction of precious metals
from industrial wastewaters [80, 236, 237]. However, as far as we know, this ability
has not been detected in malignant bacteria species. Experiments on the synthesis of
Ag nanoparticle using such bacteria cultures pursued two main objectivesrstly,
to develop one more biological method for the synthesis of metal nanoparticle and,
secondly, to nd out whether it is possible to produce an antimicrobial agent
effective against pathogenic microorganisms resistant to antibiotics. The possibility
to synthesize metal nanoparticles with the help of supernatants of pathogenic
bacteria cultures has been demonstrated for a number of enterobacteria (Klebsiella
pneumonia, Escherichia coli, Enterobacter cloacae) [238], Staphylococcus aureus
[239], and Pseudomonas aeruginosa [246]. It turned out that synthesis proceeds
very fastfor example, in case of enterobacteria, the process takes less than 5 min.
Figure 1.38 shows the photograph of Ag nanoparticle solution prepared with
Klebsiella pneumonia supernatant, as well as the TEM image and histogram of the
particle size distribution in this solution.
The nanoparticles are almost spherical, with the size less than 20 nm. With the
use of Staphylococcus, the nanoparticles are no smaller than 100 nm, but in spite of
such a big size, they manifest quite an obvious activity against several
methicillin-resistant bacteria strains (Staphylococcus aureus, Staphylococcus epi-
dermidis, and Streptococcus pyogenes).
Silver nanoparticles have been successfully synthesized with growth medium
supernatant and biomass ltrate of some fungi species. Also, it is possible to obtain
nanoparticles, adsorbed on the surface of a fungus in contact with solution con-
taining Ag+ ions. Several examples are presented in Table 1.5. Here, the synthesis
proceeds very slow, and it takes several days. During synthesis in solutions
(Penicillium brevicompactum, Fusarium semitectum), 10100-nm nanoparticles are
Fig. 1.38 a Klebsiella pneumonia supernatant before (left) and after (right) the addition of
AgNO3 solution. b TEM image and histogram of Ag nanoparticle sizes in solution. Reprinted from
[238]. Copyright 2007, with permission from Elsevier
Fig. 1.39 TEM image a and

size histogram b of Ag
nanoparticles obtained by the
incubation of Aspergillus
flavus mycelium with silver
nitrate solution. Reprinted
from Elsevier
formed with a wide size distribution. Synthesis through the adsorption on a surface
may produce stable small-sized nanoparticles with narrow size distribution. For
example, incubation of mycelium of the fungus Aspergillus flavus with AgNO3
solution and the subsequent removal of nanoparticles from the surface by ultra-
sound allowed to obtain almost monodisperse nanoparticles with dav = 8.92
1.61 nm [242] (Fig. 1.39).
Here, the absorption peak lies at 420 nm. The results of FTIR spectroscopy give
the grounds to assume that in this case, nanoparticles are reduced and stabilized
with proteins, which contain surface amino and amido groups. These nanoparticles
demonstrate the luminescent properties: Light excitation at = 420 nm provokes
the appearance of emission band with = 553 nm (Fig. 1.40).
From the results cited above and a number of other works ([220, 221] and
references therein), one can conclude that biological reduction as the method of
synthesis, at least of noble metal nanoparticles, has a series of advantages over the
methods based on the traditional chemical reducing agents. Firstly, synthesis takes
place in solution in the air at room or slightly higher temperature (3037 C), does
not require any special additional procedures (deaeration, modication of reagents,
etc.) and application of expensive and energy-consuming equipment, and creates
highly stable nanoparticles. Secondly, the efciency (when it can be determined) of
natural reducing agents turns out to be high enough and may be even higher
compared to the chemical reducing agents. For example, in the synthesis of Au
Fig. 1.40 Photoluminescence spectra of Ag nanoparticles synthesized with A. flavus extract.

Maximum in excitation spectrum at 420 nm a coincides with that observed in the absorption
spectrum. Excitation at 420 nm leads to the emission b with maximum at 553 nm. Reprinted from
Ref. [242]. Copyright 2007, with the permission from Elsevier
nanoparticles with apiin extracted from henna leaves [228], the molar concentration
ratio C(apiin):C(HAuCl4) varied from 1:20 to 1:10, and the complete reduction of
gold ions has been achieved. By contrast, to achieve the complete reduction in
aqueous solution with hydrazine or sodium borohydride, a large excess of a
reducing agent has been necessary. Thirdly, as already mentioned above, biological
synthesis is preferable from the ecological point of view, since there are no toxic
reducing agents (or the products of their transformation that appear during the
reaction) and stabilizers in the solution; nanoparticles are stabilized by the natural
stabilizers, which are obviously less toxic for the living organisms. For these rea-
sons, metal nanoparticles prepared by this method may prove to be more acceptable
from economical point of view and more suitable for application in those elds
(rst of all, in medicine, biology, and agriculture), where it is important to provide
minimal toxicity and ensure safety for the environment.
At the same time, to date this biological approach to the nanoparticle synthesis
has, in our opinion, some serious drawbacks. Firstly, in most cases, this synthesis
requires a long time. Secondly, the active agents and their concentrations are often
not known precisely, and even when they can be determined, it is difcult to obtain
reproducible results, since the state of a given living organism and composition of
the corresponding extract can vary depending on the habitat, stage of development
(age), season, and other factors. Thirdly, at the current stage of the research
development, it is difcult to control the size, shape, and distribution of nanopar-
ticles in solution; mostly, nanoparticles with the size of several dozens or even over
100 nm and with wide distribution are produced that can strongly differ in their
shapes. So to investigate the properties of nanoparticles of a denite size and shape,
it is necessary to divide these mixtures into monodisperse fractions, and this is a
complicated task. Fourthly, it is hard to predict whether it is possible to prepare
solutions with high concentration of nanoparticles in the large enough quantities

required for industrial application.
Nevertheless, it is clear that biological synthesis of metal nanoparticles has a
signicant potential as the source of low-toxic and eco-friendly nanomaterials.
Besides, studies in this direction are important for investigation of the mechanisms
of nanoparticle synthesis in living organisms at the lower and higher levels of
organization, including humans. As suppose the authors of one of the works in this
eld [226], the introduction into nanotechnology of the green chemistry principles
is one of the key questions in nanoscience.
1.7 Summary
Taken as a whole, this chapter shows that metal nanoparticles in solutions can be
manufactured by various methods, and each of them has its positive and negative
sides. The choice of the method depends mainly on the supposed application of
nanoparticles and, consequently, on the peculiarities of experimental procedure and
nanoparticle characteristics that are essential for the application desired. Recently,
D.I. Ryzhonkov and coauthors have formulated the general requirements which
should be met by the methods used for the preparation of nanomaterials [29, p. 18]:
The method should be able to produce material with the controlled composition
and reproducible properties;
The method should guarantee the long-term stability of nanomaterials, i.e., rst
of all, to prevent the particles from spontaneous surface oxidation and caking
during the preparation process;
The method must be highly efcient and economic.
The method should provide the production of nanomaterials with a controlled
particle or grain size, their size distribution being narrow enough, if necessary.
The authors conclude further that at present there is no method which fully
satises the whole complex of these requirements [29, p. 19].
As is clear to the reader from the contents of this chapter, this conclusion is true
also for the methods of chemical synthesis described here. In our view, taking into
account the results of studies on the biological activity of nanoparticles and
applications of nanomaterials in medicine and other elds connected with the
nanoparticle effects on the living organisms, one more requirement may be added to
those mentioned above, namely: the method should provide minimal toxicity of
nanoparticles, nanomaterials and conditions of their preparation, as well as ensure
their safe application for plants, animals and humans.
It should be emphasized once more that, in the last years, the requirements of
minimal toxicity and ecological safety of nanoparticle production, including
preparation of metal nanoparticle solutions, come to a foreground due to the
detection of different nanopathologies, as mentioned already in Introduction
section. Among the studies on the chemical synthesis of nanoparticles, this is
1.7 Summary 75
clearly reflected in a rapid growth, in the last 57 years, of a relative contribution of

publications describing various kinds of biological reduction. We have no doubt
that this direction has a great future, since it is important both for the studies on the
mechanisms of biological processes and for the development of various ways of
practical nanoparticle applications. It can be added that, from our point of view,
biological synthesis of nanoparticles can be considered as a direction on the
boundary between bio- and nanotechnology, where biotechnological methods and
techniques (in particular, manufacturing of useful products with the help of plants,
fungi, microorganisms, etc. [247]) are used for the fabrication of nanotechnological
products, that is, of nanoparticles. In other words, the biological approach for metal
nanoparticle preparation can be considered as a kind of bionanotechnology.
In the method of biochemical synthesis used by us, as in the majority of works in
the biological direction, natural plant pigments from the flavonoid group are acting
as reducing agents. As it has been noted above, these substances are more effective
in this quality and, at the same time, less toxic as reagents than the traditional
chemical reducing agents widely employed for the preparation of nanoparticles in
the other, non-biological methods.
However, in our method, the synthesis takes place not in the aqueous solution,
but in reverse micelles. This allows to use the advantages of reverse micelles for the
preparation of small-sized nanoparticles with narrow enough distribution; as shown
above, it is only in the rare cases that such nanoparticles can be obtained by means
of biological reduction. Besides, since in our case not biological extracts, but
substances of the known structure and concentration are used for the reduction of
metal ions, the possibility is provided of the more effective influence on the for-
mation of nanoparticles by varying the signicant parameters, and also a higher
reproducibility of results can be achieved. At the same time, the technological
simplicity and low expenses are retained, as well as high nanoparticle stability, that
is, the advantages of biological reduction compared to the conventional chemical
methods. Certainly, to study the biological effects of nanoparticles, they have to be
transferred from reverse micelles to the aqueous solution; for this purpose, the
corresponding procedure has been developed, as described below (see Chap. 4).
Thus, the biochemical synthesis combines the advantages of natural biological
reducing agents and those of reverse micellar system. So this method can be
regarded, on the one hand, as a constituent part of the biological direction (and
hence of bionanotechnology) and, on the other hand, as a variant of synthesis in
reverse micelles with the application of biological reducing agents. This denes its
position among the methods of chemical synthesis of metal nanoparticles in solu-
tions (see Fig. 1.2).
In conclusion of this chapter, we found it useful to concretize the
above-mentioned general requirements to the methods of nanomaterial preparation
conformably to the studies of their influence on biological objects. Namely, we
propose to supplement these requirements with a set of conditions which should be
fullled for the aqueous solution of metal nanoparticles to be used in studies of the
main biological effects of metal nanoparticles. Under these effects, we mean the
influence of nanoparticle concentration and their main characteristics (size, shape,
surface charge, and composition of a stabilizing shell) on viability and functional

activity of the living organisms. The conditions under question are listed below.
First Nanoparticles are to be stable in aqueous solution, i.e., they must not
disintegrate or aggregate. In other words, their concentration and other character-
istics should remain constant during the period of time necessary to obtain and
reproduce the results of experiment with a biological object. Here, it is not
important whether the nanoparticle synthesis was carried out in an aqueous solution
or in a nonpolar solvent with the subsequent transfer into the aqueous medium.
Second A nanoparticle solution should not contain any other components in the
concentrations that lay obstacles to the determination of pure nanoparticle effects.
This condition arises because, as a rule, apart from the nanoparticles themselves,
their solution can contain some other biologically active components (the unspent
reducing agent and its oxidation products, a stabilizer, etc.). For example, for the
correct determination of the toxicity threshold (of the nanoparticle concentration
corresponding to the beginning of decrease in viability of a living object, see Part II,
Chap. 6), it is important for the solution to be free from the components with
toxicity comparable or larger than that of nanoparticles.
Third To determine the influence of nanoparticle concentration, it must be high
enough in the initially prepared nanoparticle solution, so that dilution of this initial
solution could result in a wide enough range of concentrations, necessary for the
reliable determination of the toxicity threshold and lethal dose.
Forth To determine the influence of nanoparticle size, it is necessary to use no
less than two nanoparticle preparations with narrow size distribution with essen-
tially different mean particle size (at least at two times). It is desirable also for the
average particle sizes to lie below 20 nm, i.e., in the range where the size effects are
clearly expressed.
Fifth To determine the influence of nanoparticle shape, it is necessary to prepare
at least two nanoparticle solutions with the same concentration, but different shape
of nanoparticles and, if possible, with the same characteristic size.
Sixth Studies on the influence of the three nanoparticle parameters mentioned
above (concentration, size, shape) on a given object have to be carried out with
nanoparticles prepared by one and the same method. This is necessary for the
fulllment of the two other important conditionsthe equality (or close similarity)
of (1) surface charge and (2) surface composition of the nanoparticles (if there is a
stabilizing shellof its composition and thickness), both essential for the estima-
tion of their effect on an object under study.
Seventh It is important that the nanoparticles should be produced in the large
quantities, sufcient for their further usage in fabrication of industrial products,
including those intended for the medical applications. Otherwise, studies on the bio-
logical effects of nanoparticles will not be important for their practical application.
Examination of the methods described in this chapter with due regard for the
conditions mentioned above allows to conclude that still there is no method which
satises all these conditions and hence gives the possibility to obtain stable
small-sized nanoparticles with narrow distribution, different in shape, with high
concentrations in the aqueous solution free from the unallowable concentrations of
1.7 Summary 77
other components. It is evident therefore that actually there are two ways available
for investigation of the influence of the named nanoparticle characteristics on their
biological activity: either to use different methods of nanoparticle preparation to
study the influence of one or two characteristics, or to select one method, which
allows to prepare the nanoparticle solutions that meet the necessary requirements
with the help of various modications and additional procedures. As will be seen
from our analysis of the results reported in the literature devoted to the studies of
biological effects of metal nanoparticles (Part II, Chap. 6), in most cases the rst
way is employed by the researchers. Elaboration of the biochemical synthesis
showed that, basing on this method, one can follow the second way, as it is possible
to prepare both aqueous nanoparticle solutions and modied materials suitable for
the studies of their biological activity on various living organisms. The grounds of
this method and techniques used for the preparation of the nanoparticle solutions
and nanoparticle-modied materials are described in the next chapters of this book.
Chapter 2
Biochemical Synthesis of Metal
Nanoparticles
As already mentioned above, biochemical synthesis is based on the reduction of

metal ions in reverse micelles. We realize that the term biochemical synthesis is
not quite correct as it gives rise to an analogy with the processes of biosynthesis that
proceed in living cells and represent one of the subjects studied in biochemistry,
while we actually mean the chemical reaction of the reduction of metal ions to
atoms by biological molecules, which triggers the process of aggregation of atoms
and ions, i.e., the nanoparticle synthesis proper. Similar problem arises at the formal
consideration of names of the methods using biological reducing agents (biore-
duction, green synthesis, biogenic synthesis, also biochemical synthesis),
described in the previous chapter. In our case, from the different names discussed
on the initial stage of the development of this method, it was only the biochemical
synthesis in reverse micelles that was nely established, since it reflects, on the
one hand, the fundamental difference from chemical methods using non-biological
reducing agents in reverse micelles, and on the other hand, difference from the
chemical methods based on the application of biological reducing agents in aqueous
solutions. In the oral presentations and publications, usually the reduced form is
used, namely the biochemical synthesis, with the corresponding explanations on
the principles of this method, if necessary.
In this chapter, we present a brief account of the principles of this method,
followed by the necessary information on the peculiarities of experimental proce-
dure. Then, synthesis of silver, gold, and copper nanoparticles is described, with the
relevant data on the optical spectra and particle sizes.
2.1 Prerequisites of the Method
To provide a more complete notion of the biochemical synthesis, we begin with a

brief description of the flavonoid properties as well as of the internal medium of
reverse micelles, because the combination of these two factors is the main feature of
DOI 10.1007/978-3-319-30906-4_2
80 2 Biochemical Synthesis of Metal Nanoparticles
the method and forms the ground for various applications of stable metal
nanoparticles in solutions.
2.1.1 General Information on the Properties of Flavonoids
Plant pigments of the flavonoid group are low-molecular polyphenolic compounds

with structure formed by the three rings: two aromatic rings (A and B) connected by
heterocycle (C), which contains oxygen and is referred to also as pyrane ring. As an
example, structure of the three flavonoids used by us is shown in Fig. 2.1.
In nature, flavonoids are present in fruits, berries, vegetables, nuts, herbs, and
flowers; some drinks are often mentioned as sources of flavonoids, especially tea and
red wine (see [248, 249]). Many of these compounds are strongly colored, and this is
the origin of bright and diverse coloration of flowers and other plant parts. For the
time being, their function in the plant organisms is not fully elucidated [250]. It is
known that they can protect cell photosynthetic system from the destructive action of
short-wavelength UV radiation and also preserve the plants from various morbic
affections. There exists a great number of flavonoids (more than 4000), divided into
subgroups, that differ by the number and location of substituents (hydroxy groups) at
various carbon atoms (mainly 3, 5, 7, 3, 4, 5) and by the presence (or absence) of
oxygen with double bond at the 4th carbon atom [248250].
Fig. 2.1 Structure of

flavonoid molecules used in
the biochemical synthesis.
1 quercetin; 2 rutin; 3 morin.
Sugar is disaccharide that
consists of rhamnose and
glucose residues. This gure
and gs. 2.2, 2.3, 2.132.20
were reprinted from Advances
in Nanotechnology, vol. 11,
Biochemical synthesis,
optical properties and sizes
of gold nanoparticles,
pp. 119146, Copyright 2014,
author E.M. Egorova, with
permission from Nova
Science Publishers
2.1 Prerequisites of the Method 81
In the method of biochemical synthesis, three flavonoids are used from the
flavonol subgroupquercetin, rutin, and morin. They are the powders (crystallohy-
drates) of yellow color, produced mainly from the peel of onion or from citric
plants, where they are present in relatively large amounts.
Like many other flavonoids, these substances are known for their ability to
chelate metal ions, as well as for the high and versatile biological activity. For
example, quercetin and rutin belong to the vitamin P group and are used in med-
icine for a long time as a means for the reduction of capillary permeability of blood
circulatory system; also, their anti-inflammatory, antioxidant, radioprotective, and
antimutagenic properties are widely recognized. The important advantages of these
substances are their low toxicity for a human organism and wide spectrum of
medicinal effects. There is voluminous literature devoted to the investigation of
mechanisms underlying the action of these flavonoids. In the last years, much
attention is paid to the research of their interaction with animal cells in vitro (in-
cluding the cells of immune system, muscular and neural cells, and tumor cells)
[264] that offer opportunities for the application of these substances as hepato-
protective agents, for combating cancer and a number of other diseases.
According to the literature data, quercetin is studied more intensively than the
other two flavonoids under question. This is probably connected with its more
signicant consumption by a human organism with food products, and hence,
studies on the mechanisms of its action are more urgent [255, 259, 260, 262].
Therefore, its interaction with lipoproteins, nucleic acids, various enzymes, and
other proteins was the subject of thorough investigations by a number of labora-
tories [252, 255, 259, 264].
As noted in literature [260, 265], polyphenolic structure of quercetin makes it
extremely sensitive to the variations of composition and structure of the environ-
ment. It is obvious that biological processes with participation of quercetin and the
other biologically active substances proceed in a living organism in the organized
medium, in conditions that differ signicantly from those which exist in aqueous
solutions in vitro, where, up to the present time, the vast majority of experiments
were performed. Only recently, the publication appeared devoted to the interaction
of quercetin in aqueous solution with direct micelles from anionic or cationic
surfactants [265], considered as a model of plasma membrane or cell organelle,
suitable for the studies of the influence of biological organization on antioxidant
activity and other properties of flavonoids. In our opinion, the reverse micellar
system is more interesting from this point of view because, as shown below,
properties of the internal medium of reverse micelles are more similar to those of
the internal media of biological vesicles or cellular compartments in the living
organisms. However, as far as we know, no attempts have been undertaken so far to
study biologically interesting reactions in reverse micelles with participation of
quercetin and other flavonoids.
Thus, by choosing flavonoids as reducing agents, we became pioneers in the
studies of the flavonoids interaction with metal ions in reverse micelles. According
to the purpose of our work, the main task was to determine the conditions that allow
the formation of metal nanoparticles.
2.1.2 AOT/Isooctane Reverse Micelles
As already mentioned above, in this case the nanoparticle formation is the result of
reduction of metal ions to atoms and further aggregation of atoms and ions in the
internal medium of reverse micelle. Properties of this internal medium, or water
core, primarily depend on its size, which is determined by the hydration extent w =
[H2O]/[SAS]. A brief account of the general information on the water properties in
micellar core is given in Chap. 1 (Sect. 1.3.4).
The anionic SAS used by usaerosol-OT or AOTis most frequently applied
for the preparation of reverse micellar systems, because it is highly soluble in many
nonpolar solvents and allows to obtain stable reverse micelles in a wide range of
SAS concentrations and hydration extents. Also, AOT has a unique capacity for
binding a large amount of water (up to 10 water molecules per one SAS molecule)
without addition of other cosolubilizers. According to the author of [139], this is
conditioned by the high flexibility of the AOT molecular structure. Structure of a
reverse micelle in H2O/AOT/n-alkane system used in our research is shown in
Fig. 2.2 in the general terms.
Formal length of the hydrocarbon part of AOT molecule (at the maximal extension
of hydrocarbon tails) is equal to 89 [136, 139]. Usually, the actual length of this
part in micelle is less due to the bending of hydrocarbon tails. The main parameters
of AOT/isooctane water micelles reported in literature for several hydration extents are
presented in Table 2.1. As seen from the table, water micelles have sizes in the range
37 nm. The experimental values of hydrodynamic radius found by dynamic light
scattering (DLS) technique [137, 139]) are in good agreement with the corresponding
theoretical results calculated from the empirical expression proposed in [142]. Hence,
this expression may be applied for the estimation of micelle size for the other w values
in the range 0 < w < 10. Size of the aqueous core varies approximately in the range of
24 nm; the binded water layer thickness lies within 35 A from the polar heads
boundary [139] and is increasing with the decrease of hydration extent, reaching its
maximum value at w < 4. It is clear therefore that, at so small hydration extents, all the
water in micellar core is binded with all characteristic features mentioned above. For
example, in AOT/isooctane reverse micelles with w = 3.7, that are often used in
biochemical synthesis, microviscosity inside the water core is 200 times higher than
that of bulk water [133]. It is exactly in this medium that synthesis of metal
nanoparticles takes place in our case.
Since the biochemical synthesis is based on electron transfer from the flavonoid
to metal ion, one can expect the manifestations of the features of electron transfer
reactions that proceed in reverse micelles at small hydration extents. The results
obtained in studies of various reactions of the same type in reverse micelles [126,
133, 141] indicate that electron behavior in the internal cavity of reverse micelles
differs essentially from that in the aqueous solution. As was mentioned already in
Chap. 1 (Sect. 1.4), solvation of the electrons generated by pulsed radiolysis in
AOT reverse micelles in isooctane depends on the hydration extent: The smaller is
w, the lower is the rate of solvation, and for w < 5, the electrons are not solvated at
2.1 Prerequisites of the Method 83
Reverse micelle
SAS (AOT)
Water core: Binded water

Ag + e Ag
+ 0
Ag + Ag +... AgNP
0 +
Hydrocarbon
Rh Rw
Fig. 2.2 Structure of AOT reverse micelle in hydrocarbon (in our case, usually in isooctane). Rh
and Rw are hydrodynamic radius and water core radius, respectively. Reduction of metal ions in the
water core is illustrated on Ag+ ion as an example. See text for more details
Table 2.1 Parameters of reverse micelles in water/AOT/isooctane system

w Rh () Rw () Na Reference Rh = 1.5w + 16.4 [142]
0 16.4 9 22 [126, 140]
15 11 [142]
15 [135]
[137]
3.5 *25 [137] 21.65
4 25 10 35 [137] 22
5 *32 [137] 23.9
6 28 14 50 [137] 25.4
8 32 16 72 [139] 28.4
19 115 [126]
18 95 [126]
9 *36 [137] 29.9
Rh hydrodynamic radius; Rw water core radius; Na aggregation number
all [133, 266]. It was found also that in reactions with electron transfer from
aqueous to hydrophobic phase proceeding in AOT reverse micelles, the bound
water at the polar heads layer boundary plays an essential role [141]. As will be
shown below (Sects. 2.3 and 2.4, Chap. 3), specic properties of the internal
medium of reverse micelles, including the influence of bound water, manifest
themselves also in the biochemical synthesis of metal nanoparticles.
2.2 General Scheme of the Synthesis
General principle applied in the nanoparticle synthesis in reverse micelles consists

in mixing the micellar solution of metal ions with that of reducing agent (see
Chap. 1, Sect. 1.3.4). In our case, this principle is implemented by mixing sepa-
rately prepared micellar solutions of flavonoid and metal salt. Micellar solution of
flavonoid is prepared with the use of its concentrated solution in ethanol or pro-
panol. It turned out, however, that, apart from this conventional variant, another
route also exists, namely injection of aqueous metal salt solution directly into the
flavonoid micellar solution. In this case, micellar solution of flavonoid is prepared
by solubilization of the flavonoid taken as dried powder in AOT/liquid hydrocarbon
(n-alkane) solution. In both cases, the nal products are nanoparticles in the water
core of micelles and oxidized flavonoids. For each metal, special work was needed
in order to select the conditions (concentration of reagents, hydration extent, metal
salt composition) that ensure the high rate of formation and stability of nanopar-
ticles, as well as high enough degree of transformation of the metal ions into
nanoparticles. According to the observations made on the initial stage of work on
biochemical synthesis, as a rule, with the conventional method, the rate of formation
and yield of nanoparticles were lower compared to those obtained with the second
route, so afterward the nanoparticles were prepared almost exclusively by the latter
way. Owing to the peculiarities of optical properties inherent both to the complexes
of flavonoids with metal ions and to the nanoparticles, process of nanoparticle
formation is accompanied by the characteristic changes of color, thus allowing a
visual control of the onset and (approximately) of the rate of synthesis.
2.3 Peculiarities of the Experimental Procedure
Whereas our method has some differences from the conventional procedure fol-
lowed in the nanoparticle synthesis in reverse micelles, prior to the description of
examples of the nanoparticle synthesis, we found it reasonable to give the necessary
information on the most essential peculiarities of this experiment. Here belong
(1) preparation of the flavonoid micellar solution, (2) determination of the flavonoid
extinction coefcient in micellar solution, and (3) determination of the stabilizer
(AOT) concentration in the water nanoparticle solution obtained from their micellar
2.3 Peculiarities of the Experimental Procedure 85
solution. Below in this section, we present a brief description of the procedures used
in the preparation of the flavonoid micellar solution and estimation of its extinction
coefcient; determination of the AOT concentration in water solution of the
nanoparticles will be elucidated in Chap. 4 devoted to the preparation of metal
nanoparticles in water solutions.
2.3.1 Preparation of the Flavonoid Micellar Solution
Micellar solutions of flavonoids are prepared by the procedure described in [202,

267, 268]. At rst, AOT solution in liquid hydrocarbon is prepared; hexane, heptane,
octane, isooctane, and decane can be used as solvents [269]. Experience has shown,
however, that isooctane is the most suitable for the nanoparticle synthesis, both from
methodological and economical point of view. Then, flavonoid (taken as powder) is
solubilized in the AOT solution. The AOT concentration in isooctane lies in the
range 0.10.3 M, depending on the metal used for the nanoparticle synthesis. In our
standard synthesis procedure, the AOT concentration is 0.135 M. For applications
that require the lower AOT concentration in micellar or aqueous solution, AOT
concentration in isooctane can be reduced; however, in such cases, the additional
control is necessary, to check the stability and size of nanoparticles in micellar and
water solutions, as well as the AOT concentration in water nanoparticle solution.
AOT and flavonoids were preliminary dried to remove the hydration water, if it
was necessary. Concentration of the flavonoids in micellar solution was determined
from their optical absorption spectra; for this purpose, the corresponding extinction
coefcients had been found in separate experiments.
2.3.2 Extinction Coefcient Determination

for the Flavonoids in Micellar Solution [268]
Similarly to their wateralcoholic or water solutions, micellar solutions of the fla-

vonoids used in our work have two main adsorption bands in the UV region
(Fig. 2.3): band I and band II positioned at 360380 and 240270 nm, respectively.
The absorption bands stem from the * transitions in the two chromophore
systems: band Iin the B ring conjugated with three-carbon fragment of the C ring,
band IIin the A ring conjugated with the C ring. The corresponding resonant
structures for quercetin as an example are shown in Fig. 2.4 [265, 270].
Comparison of absorption spectra of quercetin (Qr) in aqueous solution (phos-
phate buffer) and in micellar solution in AOT/isooctane revealed different behavior
of the two main bands: absorption in the short-wavelength band II was almost the
same in the two solutions, while intensity of the long-wavelength band I in micellar
solution was signicantly higher than in the aqueous one.
1.5
1.0
Optical density
Ru
II
I
0.5
Mo
Qr
0.0
250 300 350 400 450 500 550
Wavelength, nm
Fig. 2.3 Optical absorption spectra of flavonoidsquercetin (Qr), rutin (Ru), and morin (Mo)
in micellar solutions. I and IItwo main absorption bands. Spectra shown here and in the other
gures within this part of the book were measured in 1-mm cuvettes
An example for the 50 M Qr solution is shown in Fig. 2.5a. Picture of such

kind is observed for various quercetin concentrations in the range studied
(25100 M). Thus, it turned out that different parts of the flavonoid molecule have
various degrees of sensitivity to the changes in external conditions; namely, A ring
demonstrates a weak reaction, while B ring reacts signicantly stronger.
Hence follows that Qr molecule is positioned inside reverse micelle in such a
way that surroundings of the A ring are less different from those in aqueous solution
than surroundings of the B ring. Because of its structure, the molecule cannot be
located in the region of AOT hydrophobic tails, but only in the polar part of micelle,
in the layer of AOT polar headgroups, on its boundary with the polar core or inside
the polar core. Since polar core is the most similar in structure to the water solution,
the A ring is likely to be located in the polar core (or at least is oriented toward it),
and the B ring is located in the region with more rigid structurein the layer of
AOT polar headgroups or on its interface with the polar core.
Owing to the closeness of absorption values in the band II maximum (DIImax ) for
water and micellar solutions with equal Qr concentration, it is possible to use these
values for the determination of Qr extinction coefcient (). The II values in series
of 810 experiments were found from the expression II = DIImax /CQr * l, where CQr
2.3 Peculiarities of the Experimental Procedure 87
Fig. 2.4 Resonance structures in quercetin molecule corresponding to the band I (h1) and band II
(h2) in optical absorption spectrum [261, 270]. Reprinted from Ref. [261]. Copyright 2006, with
is quercetin concentration and l is optical path length. For water and micellar solutions,
they were equal to (1.79 0.07) 104 and (1.81 0.08) 104 L/mol cm, respec-
tively. Hence, the average value is II = (1.8 0.1) 104 L/mol cm.
Using this extinction coefcient, we compared further the spectra obtained for
micellar solutions with constant quercetin concentration and different water content.
Figure 2.5b shows the spectra obtained for 50 M Qr solutions, prepared by sol-
ubilization of Qr powder in anhydrous AOT solution in isooctane (curve 2) and in
AOT solution in isooctane with preliminary added water, with w = 20 (curve 1). It
is seen that absorption in the band I for anhydrous (dry) solution is signicantly
higher than that for the water-containing solution. These two cases differ in that
micelles containing quercetin are formed by AOT molecules with different
hydration of the polar heads, so it is possible to assume that electron transitions in
the B ring of quercetin suffer a strong influence of the water molecules binded with
AOT polar headgroups. It is possible, therefore, that this part of quercetin molecule
in the dry micelle is located in the AOT polar headgroups layer and forms with
them hydrogen bonds through its OH groups and molecules of the binded water.
In Qr micellar solution with the given water content (with the given AOT
hydration extent), absorption in the band I depends only on the flavonoid con-
centration, and hence, one can single out two extinction coefcients determined
from the absorption in band I and band III and II, respectively. In the particular
case of dry solution, the extinction coefcient in band I is maximal and equal to
(2.8 0.1) 104 L/mol cm. At the small hydration extents, (w = 35) I = (2.26
0.07) 104 L/mol cm.
Extinction coefcient for rutin was determined from spectrophotometric mea-
surements of micellar solutions with several rutin concentrations. These solutions
(a) (b)
0.16
0.12
0.14 2
2
0.10 0.12
Optical density
Optical density
0.08 1 0.10 1
0.08
0.06
0.06
0.04
0.04
0.02
0.02
0.00 0.00
200 300 400 500 600 200 300 400 500 600
Wavelength, nm Wavelength, nm
Fig. 2.5 Spectra of 50 M Qr solutions in various media: a 100 mM sodium phosphate buffer (1)
and micellar solution obtained by the introduction of Qr solution in isopropanol into AOT solution
in isooctane (2); b Micellar solutions obtained by Qr solubilization in isooctane with preliminarily
added water to the hydration extent w = 20 (1) and in the dry AOT solution in isooctane (2)
were prepared by the introduction of rutin alcoholic solutions into the AOT solution
in isooctane; the alcohol concentration in micellar solution did not exceed 1 %.
From the results of 5 independent measurements, the extinction coefcients for
band I (366 nm) and band II (258 nm) were found to be (2.32 0.13) 104 and
(3.26 0.33) 104 L/mol cm, respectively.
2.4 Examples of the Nanoparticle Synthesis
Aqueous solutions of metal salts were prepared on deionized water obtained from
the device Vodoley (production of Chimelectronica, Moscow). For the
preparation of complex salts (silver diammine nitrate, copper tertammine nitrate,
and salts of other metals), 10 or 27 % ammonium hydroxide solutions were added
to the metal salt solution (for details see [28]).
Addition of metal salt water solutions to the micellar solutions of flavonoids,
after shaking for several minutes, leads to the drastic changes in color and
absorption spectra of these solutions that reflect different stages of flavonoids
interaction with metal ions, which is completed by the formation of nanoparticles.
Synthesis of nanoparticles was performed mainly in the quercetin solutions; also,
the experiments with all three flavonoids were conducted to study the effect of
differences in their structure on the nanoparticle formation process. Measurements
of absorption spectra were performed on the spectrophotometers Helios-a
(production of Thermo Electronics, Great Britain) or Shimadzu UV-2600
2.4 Examples of the Nanoparticle Synthesis 89
(Shimadzu, Japan). Particle sizes in solution were characterized by the dynamic

light scattering (DLS) technique (known also as photon correlation spectroscopy,
PCS) on the devices Horiba LB-550 (Horiba, Japan) or ZetaPALS (Brookhaven
Instruments, USA). Electron microphotographs were obtained by means of the
transmission electron microscope LEO912 AB OMEGA (Carl Zeiss, Germany).
Materials for the microscopy were prepared immediately before the measurements,
by placing a drop of solution on the copper grid with preliminary deposited polymer
lm. The microphotographs obtained were used for plotting the size distribution
histograms; the number of particles for each sample was no less than 350.
The biochemical synthesis allows to prepare the micellar solutions of silver,
gold, copper, zinc, cobalt, and nickel nanoparticles. In the context of this edition,
we considered it to be expedient to present the examples of optical spectra and
particle sizes obtained for micellar nanoparticle solutions of three metalssilver,
gold, and copper. Silver and copper nanoparticles were applied in studies of the
biological effects (see Part II), and gold nanoparticles are of interest for the com-
parison of their characteristics with those of the nanoparticles prepared by the
biological reduction in aqueous solution (see Part I, Chap. 4).
2.4.1 Silver Nanoparticles
Addition of silver nitrate aqueous solution to the Qr micellar solution, after shaking
for 13 min, leads to the sudden change of the solution colorfrom colorless to
pale-yellow or red-brown of different intensity, or even almost black, depending on
the nanoparticle concentration. In the absorption spectrum, the two Qr bands dis-
appear and appears instead a new band in the visible region with maximum in the
range 420440 nm, typical for silver nanoparticles in reverse micelles (see [152]).
This new band increases gradually, and optical density in the band maximum
(Dmax) reaches its maximum value (D0max ) in 14 days; the rate of this increase and
the D0max value depend on the chosen parameters of the system (silver salt com-
position, reagents concentration ratio, etc.). During the next weeks, the deviation
from D0max does not exceed 10 % so this value of Dmax is usually considered as
corresponding to the stationary stage or completion of the process of nanoparticle
formation. Figure 2.6 shows the characteristic form of absorption spectrum regis-
tered for the micellar solution of silver nanoparticles on the stationary stage. The
intense color of such a solution is retained for a long time (up to several years), thus
making possible the visual control of the nanoparticle stability. As an illustration,
Fig. 2.7 shows the appearance of micellar solutions with different concentrations of
silver nanoparticles.
The rate of formation, yield, and size of nanoparticles depends on several factors,
such as silver salt to quercetin concentration ratio, AOT concentration, hydration
extent, and silver salt composition; results of the corresponding experiments will be
described in Chap. 3. Here, we present just one example (Fig. 2.8): the typical
change of absorption spectra that reflects the nanoparticle formation kinetics (at all
423 nm
1.6
1.4
AgNP
1.2
Optical density
1.0
0.8
Qr
0.6
0.4
0.2
300 400 500 600 700 800

Wavelength, nm
Fig. 2.6 Characteristic form of the optical spectrum obtained for micellar solution of Ag
nanoparticles (AgNP). Reagents concentrations: CAg = 3 mM, CQr = 0.265 mM, CAOT = 0.135 M,
w = 3.7. For comparison, spectrum of the initial quercetin solution (Qr) is shown
Fig. 2.7 Micellar solutions

of silver nanoparticles.
Nanoparticle concentration
decreases from left to right
(color)
other conditions being equal) observed for different silver salts, namely nitrate and
diammine nitrate (Ag(NH3)2 NO3). As seen from this Figure, in both cases a
well-formed band of nanoparticles can be observed after 3040 min; however, the
rate of the band increase (i.e., the rate of nanoparticle formation) and the Dmax value
(a) (b)
1.8 3 weeks 2 weeks
3.0 24 hours
4 days
1.6 3 hours
24 hours
3 hours 2.5 40 min
1.4
40 min
Optical density
Optical density
1.2 2.0
1.0
1.5
0.8
0.6 1.0
0.4
0.5
0.2
0.0 0.0
200 300 400 500 600 700 200 300 400 500 600 700
Wavelength, nm Wavelength, nm
Fig. 2.8 Formation kinetics of silver nanoparticles from silver nitrate (a) and silver diammine
nitrate (b) solutions. CAg = 3 mM, CQr = 0.236 mM, CAOT = 0.15 M, w = 3.7. Insets show the
time interval from the introduction of silver salt solution
(which reflects the degree of ion conversion or the yield of nanoparticles) are
signicantly higher for ammonium salt. This difference is distinctly observed at
silver salt concentrations in micellar solution in the range of several millimoles. Our
study on the origin of this difference allowed to suggest [28] that it could issue from
the signicant deviations from ideality in the AgNO3 water solution introduced into
the Qr micellar solution in the course of nanoparticle synthesis. To test the effect of
non-ideality of the AgNO3 solution, optical densities in the nanoparticle band
maximum were calculated with correction for the activity coefcient (Dcmax ):
Dcmax Dmax c 2:1
where is the activity coefcient, and D*max is optical density calculated for the
concentrations of silver nitrate in micellar solution by the BouguerLambertBeer
law (see also Chap. 3 below). In other words, Dmax values for each salt concen-
tration correspond to the maximum possible yield of nanoparticles, or to the degree
of conversion of silver ions equal to 1.
The Dcmax values found from Eq. 2.1 (theoretical curve) were compared with
experimental D0max values obtained experimentally for several silver nitrate con-
centrations (in the range 0.020.3 M) in its initial water solution. The results are
shown in Fig. 2.9b. It is seen that there is quite satisfactory agreement with the
theory in the whole range studied. For comparison, the Dmax dependence on ionic
strength of the initial silver salt solution is also shown, calculated for the ideal case
( = 1), as well as experimental data points obtained for the Ag(NH3)2 NO3 con-
centrations in its initial solution in the decimolar range. It is clear that (1) signicant
deviations for silver nitrate begin from 0.1 M and (2) for diammine nitrate there is a
good agreement with theoretical results for the ideal case.
(a) (b)
Dmax(th)
0.90 3.0
0.85 2.5
0.80 2.0 DCmax(th)
0.75 1.5
Dmax
0.70 1.0
0.65 0.5
0.60 0.0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.05 0.10 0.15 0.20 0.25 0.30
Ionic strength, M Ionic strength, M
Fig. 2.9 Manifestation of the non-ideality observed for water solution of silver nitrate. a The
dependence of activity coefcient () on AgNO3 concentration in water solution calculated from
the DebyeHkkel theory [281] in the range 0.010.1 M and from the GibbsDughem equation in
the range 0.10.3 M [282]. b Comparison of theoretical (lines) and experimental (dots)
dependencies of Dmax at the stationary stage on silver salt concentration in the initial solution.
Solid line refers to the ideal solution ( = 1), dashed lineto the solution where varies as shown
in (a). Points nitrate, diammine nitrate
Hence follows that the most probable cause of the acceleration of nanoparticle
formation with the use of ammoniac silver salt (for silver salt concentration in the
initial water solution, C O. M) may be the increase of effective concentration of
the added metal ions due to the difference in activity coefcients of the two tested salts.
In the course of studies on the mechanism of silver nanoparticle formation in
micellar solution, the problem arouse of determination of the intermediate stages of
Qr interaction with Ag+ ions. As seen from the adsorption spectra of micellar
solutions of silver nanoparticles in the standard biochemical synthesis procedure
(see Fig. 2.8), here the quick formation and increase of the nanoparticle band
intensity are observed, so that it is impossible to discern the intermediate stages.
This problem was solved in studies of the quercetin interaction with silver ions at
low concentrations of the reagents [28, 268]. It was shown that at the rst stage, the
[Ag+ Qr] complex formation takes place, which manifests itself in the noticeable
growth of the 295-nm band, present in the original Qr spectrum as a small bump
between the two main bands I and II (see Fig. 2.3). Theoretical estimates of the
enthalpy of complex formation between quercetin and metal ions [271], as well as
our researches on the influence of flavonoid structure on the nanoparticle formation
process [28], give grounds to believe that this complex is formed by binding of
silver ions with the oxygen atoms located at C3 and C4 atoms in quercetin mole-
cule. Then, this complex dissociates with the formation of the oxidized quercetin
and silver atoms; association of silver atoms and ions leads to the formation of
nanoparticles which can be observed in the spectrum by the characteristic band in
the range 420440 nm. Isolation of the band corresponding to the complex and
determination of its extinction coefcient will be briefly considered later, along with
the description of the procedure used for the determination of extinction coefcient
of silver nanoparticles in micellar solution (Chap. 3, Sect. 3.1.2).
Figure 2.10 shows electron microphotographs and the corresponding histograms
of particle size distribution on the stationary stage (one day after the beginning of
synthesis) for the nanoparticles prepared from silver nitrate and silver diammine
nitrate with the equal initial salt concentration and hydration extent. It is seen that
the nanoparticles produced have different size: In the Gaussian approximation, the
average size is 15 nm for nitrate and 21 nm for ammoniac salt.
Analysis of electron diffraction patterns shows that nanoparticles have a crystal
structure with lattice parameters close to those of gold crystals. Example in
Fig. 2.11 shows the diffraction patterns for the crystal gold sample (a) and silver
nanoparticle preparation obtained from the ammoniac salt (b). It can be seen that
diffraction patterns are practically identical; less clear image in the case of
nanoparticles is explained by much lower density of the metal. Silver nanoparticles
may have different shapes: In the case of ammoniac salt, the shape is approximately
(a)
25
20
% of particles
15
10
0
6 7 8 9 10 1112 13 14 15 1617 1819 20 2122 23
100 nm Particle diameter, nm
(b)
50
40
% of particles
30
20
10
0
200 nm 16 18 20 22 24 26
Particle diameter, nm
Fig. 2.10 Electron micrographs and size distribution histograms for the nanoparticles (on the
stationary stage) obtained from nitrate (a) and diammine nitrate (b) at silver salt concentration
3 mM and w = 3.7
(a) (b)
500 Pixel 20 m
Fig. 2.11 Electron diffraction patterns for the standard sample of bulk gold (a) and for the silver
nanoparticles obtained from ammoniac salt (b)
spherical, whereas in the case of silver nitrate, also triangles and hexagonal particles
are formed (Fig. 2.10).
According to our observations, this difference is connected not with the metal
salt composition but rather with the different rate of nanoparticle formation: in the
case of diammine nitrate, reduction of the rate of nanoparticle formation also leads
to the appearance of crystalline particles of different shapes. It should be noted also
that comparison of the electron micrographs obtained for one and the same solution
at different times from the beginning of synthesis can detect changes in the average
size and shape of nanoparticles. These changes probably reflect the crystallization
processes, as well as nanoparticle aggregation and dissociation of these aggregates
occurring in the system until the equilibrium is established.
Example of a histogram obtained by PCS for micellar solutions of silver
nanoparticles is shown in Fig. 2.12. Measurements give a single peak with the
average size in the range 1026 nm; the peak position depends on the nanoparticle
Fig. 2.12 Typical particle size distribution in micellar solution of silver nanoparticles measured
by PCS technique with the help of Horiba LB-550. Average particle size20.3 nm
concentration in solution. Similar result for these solutions was obtained on the
device Zetasizer Nano ZS (manufactured by Malvern, Great Britain), which also
implements the PCS method.
Comparison with TEM data indicates that, as a rule, measurements by PCS in
solution give the average nanoparticle size somewhat greater than that obtained by
electron microscopy. As will be seen later, the same is observed in the size mea-
surements of gold nanoparticles. Discussion of the possible reasons of such dis-
crepancy is given in the following section.
2.4.2 Gold Nanoparticles
Changes in the spectra of Qr micellar solutions after the addition of HAuCI4 (also
referred to below as golden acid, GA) depend on the Qr/GA molar ratio. Here, we
give examples of the results obtained with the excess of Qr (in the range
1 < Qr/GA < 2.5) and then with the equimolar ratio of reagents or with the excess
of GA (0.25 < Qr/GA < 1).
At the excess of Qr, addition of HAuCI4 water solution leads to the rapid
changes in the color of solution. First bright-red (rather ruby) color appears and then
(in 12 min) the color acquires the purple tint; its intensity increases in the sub-
sequent 1015 min. Afterwards, no color changes take place and such a red-purple
solution remains stable for a long time (up to several years).
Photograph of the red-purple solutions is shown in Fig. 2.13. Typical changes of
the optical spectra at the Qr excess are shown in Fig. 2.14. In the rst several
minutes, the band I of quercetin decreases and simultaneously the increase of band
295 nm and appearance of the weak band in the visible region are observed, in the
range 535540 nm. Band II suffers practically no changes. In the subsequent 15
30 min, band I continues to decrease and nally disappears, and the two other
bands become more intense, the band in the visible region acquiring a distinct
maximum at 537540 nm. This latter band lies in the well-known region charac-
teristic for the absorption of colloidal gold, between 500 and 540 nm, depending on
the particle size [46, 48, 128, 228].
This band suffers insignicant changes in the next 24 days and then remains
unchanged and reproducible for several years. However, its intensity is low com-
pared with that observed for the silver nanoparticles: Usually, Dmax does not exceed
0.240.26 for the measurements in 1 mm cuvette. Similar spectra changes take
place after the addition of HAuC14 to morin micellar solution. The only difference
is that solubilization of this flavonoid in AOT/isooctane solution goes more slowly,
and therefore, additional time is needed to reach the concentration level required for
the necessary rate of the nanoparticle synthesis. The spectra changes shown here are
similar to those mentioned above in the description of the mechanism of silver
nanoparticle formation (see also [268]): On the rst stage, the [Qr Au3+] com-
plex is formed, and this event is reflected in the spectrum by the increased intensity
of 295 nm band. Then, this complex dissociates with the formation of atomic Au
Fig. 2.13 Photograph of the gold nanoparticles in micellar solutions. Concentration increases
from left to right
and oxidized Qr. Metal atoms play a role of seeds for the subsequent nanoparticle
formation by means of the association of metal atoms and ions. As in the case of
silver, the complex may be formed through the coherence of metal ions with
oxygens at C and C4 atoms. Also, the probability exists of the ions binding with
oxygens at C4 and C5 atoms. The latter version issues from the estimates of
(1) enthalpies of the Qr complexes with metal ions and (2) sizes of the cavities
between the oxygen atoms at CC4 and C4C5 positions [271].
If GA concentration exceeds that of Qr, the process of nanoparticle formation
provokes the spectra changes as shown in Fig. 2.15. Immediately after the addition
of HAuC14, the ruby color also appears and, after 23 min, increases the intensity
of 295 nm band, indicative of the complex formation. However, the red color
quickly darkens, and after 1520 min, metal is precipitated, and the solution
acquires a golden color, characteristic for the HAuCI4 solution, testifying to the
destruction of both complex and nanoparticles. We regarded this as a consequence
of the quercetin incapability of the effective reduction of Au3+ ions in the acidic
medium inside the reverse micelles at the high HAuC14 concentrations used in this
case. This problem was solved by the increase of pH; for this purpose, 23 min
after the addition of HAuC14, water solution of ammonium hydroxide was added to
the system to the concentration 0.010.03 M. Addition of NH4OH leads rst to the
appearance of the nanoparticle band and that of the gold ions in water solutions
(313315 nm). Hence follows that the solution contains free Au3+ ions in addition
1.5
1.0
Optical density
Qr
30 min
2 min
0.5
5 days
3 months
0.0
250 300 350 400 450 500 550 600 650 700 750
Wavelength, nm
Fig. 2.14 Gold nanoparticle formation in micellar solution with the excess of Qr. Optical spectra
measured in different time intervals after the introduction of aqueous HAuCl4 solution. C
(HAuCl4) = 1.55 104 M, w = 2. Initial Qr concentration3.89 104 M
to those participating in the complex with Qr or transformed into the nanoparticles.

After 34 h, the Au3+ band decreases, and at the same time, the band at 537
540 nm increases, showing to the increase of gold nanoparticle concentration. The
growth of this band continues for several days (see inset in Fig. 2.15), until the
maximum optical density Dmax = 0.40.5 is established. Thus, here a higher con-
centration of nanoparticles is achieved, in comparison with the previous case.
Particle sizes were determined by TEM and PCS methods. Examples of the
electron micrographs of Au nanoparticles in micellar solutions are shown in
Figs. 2.16 and 2.17. Images shown in Figs. 2.16a and 2.17a are obtained for the
nanoparticles synthesized at the excess of Qr and those in Figs. 2.16b and 2.17b
at the excess of GA. The nanoparticles exhibit a wide variety of shapes: hexagonal,
pentagonal, triangle, cylindrical, and spherical shapes are present. Relatively small
density of the particles per unit area in Fig. 2.16 is accounted for by the dilution of
the nanoparticle solution for the microscopy measurements; dilution was necessary
to avoid artifacts caused by the high AOT concentration in micellar solution. At the
higher magnication (Fig. 2.17), it is seen that particles have rounded corners, but
nevertheless some of them undoubtedly look like crystals.
2.5
0.75
3 h 40 min
1 day
Qr 2 days
2.0 30 min 7 days
10 min
Optical density
0.50
537 nm
1.5
0.25
1.0
0.00
400 500 600 700
0.5 3 h 40 min
10 min
0.0
300 400 500 600 700
Wavelength, nm
Fig. 2.15 Gold nanoparticle formation in micellar solution at the excess of HAuCl4. Optical
spectra measured in different time intervals after the introduction of aqueous HAuCl4 solution. C
(HAuCl4) = 1.944 103 M, w = 4. Initial Qr concentration1.04 103 M
(a) (b)
200 nm 100 nm
Fig. 2.16 Electron micrographs of gold nanoparticles at the excess of Qr (a) and at that of GA (b)
(a) (b)
20 nm 20 nm
Fig. 2.17 Electron micrographs of gold nanoparticles at the higher magnication. Excess of Qr
(a) and excess of GA (b)
The electron diffraction analysis shows that nanoparticles actually have crystal
structure with the parameters corresponding to FCC (face centered cubic) structure
of the bulk gold. Typical diffraction pattern of the gold nanoparticles is shown in
Fig. 2.18a. Comparison with the diffraction pattern of a standard crystal gold
sample (Fig. 2.18b) shows that reflexes are practically identical, though for
nanoparticles they are less expressed because of the lower density of the substance.
(a) (b)
20 m 500 Pixel
Fig. 2.18 Electron diffraction patterns for gold nanoparticles in micellar solution (a) and for a
standard sample of crystalline gold (b)
Size distributions for the particles produced at the excess of Qr and GA are shown
in Fig. 2.19a and b, respectively. In both cases, the majority of nanoparticles (over
80 %) have sizes in the range 725 nm. The Gaussian approximation results in the
average sizes of 19.6 1.3 nm (Fig. 2.19a) and 21.5 5.5 nm (Fig. 2.19b).
So in both cases, we obtain similar average size of the particles, but the distri-
bution at the excess of GA is somewhat wider than that at the excess of Qr. The data
shown above were obtained for the relatively young particles (24 weeks after
the beginning of synthesis). Control of the same solutions by the TEM method in
the course of long-term storage had shown that, for both types of solutions, size
distribution undergoes similar changes. Examples of the histograms obtained a year
after the beginning of synthesis are shown in Fig. 2.20.
For the excess of Qr, we obtain the average diameter of 16.3 8.5 nm
(Fig. 2.20a) and for the excess of GA20.2 9.9 nm (Fig. 2.20b). That is, the
average size slightly decreases with time or remains practically constant, while the
size distribution becomes signicantly wider in both cases. This is probably the
consequence of slow processes of the particles association and dissociation taking
place in solution.
Another interesting observation may be made concerning the contribution of
triangular particles in our micellar solutions. As known from the data available in
literature on the biological activity of silver nanoparticles, their toxicity may depend
on the particles shape, and there are reasons to assume that triangular particles are
more toxic than spherical ones [272]. Also the data exist that indicate to the sig-
nicant difference in biological activity of the triangular and spherical gold
nanoparticles [18, 273]. In view of the possible practical applications of our gold
nanoparticles in medicine, we found it useful to evaluate the contribution of tri-
angular nanoparticles and its variation with time.
(a) (b)
30 12
25 10
% of particles
% of particles
20 8
15 6
10 4
5 2
0 0
5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Particle size, nm Particle size, nm
Fig. 2.19 Particle size distribution obtained from TEM data for the young micellar solutions of
gold nanoparticles at the excess of Qr (a) and at that of GA (b)
(a) (b) 16
25 14
% of particles
% of particles
20 12
10
15 8
10 6
4
5
2
0 0
5 10 15 20 25 30 35 10 20 30 40 50
Particle diameter, nm Particle diameter, nm
Fig. 2.20 Particle size distribution obtained from TEM data for the old micellar solutions of gold
nanoparticles at the Qr excess (a) and at the GA excess (b)
It turned out that, for hydration extents 2 < w < 4, the contribution of triangular
particles in the micellar solutions depends on the Qr/GA ratio. At the Qr excess (in
the range of 2 < Qr/GA < 2.5) for the young solutions, we obtained signicantly
more triangles (1520 %) than at the excess of GA (46 % for Qr/GA = 0.5). In our
view, this difference is connected with the different rate of synthesis as a conse-
quence of different pH of the medium: at the excess of Qr, synthesis takes place in
the more acidic media, where the reduction proceeds slower than at the higher pH
resulting from the addition of ammonia solution.
The same reason is likely to be responsible for the diminution of contribution of
the non-spherical particles (including triangles) at the basic pH observed in [226]
for the gold nanoparticles prepared in water solution by the reduction with plant
extract (see Chap. 1, Sect. 1.6). It seems probable, therefore, that it is possible to
avoid the formation of triangular nanoparticles by the acceleration of synthesis.
This observation may be useful for the preparation of nanoparticles non-toxic for
biological objects.
In both types of solutions, portion of the triangular particles decreased with time:
After the storage for one year for Qr/GA 2 and Qr/GA 0.5, it was found equal to
710 and 23 %, respectively. Thus, for the synthesis procedure and solution
parameters used in our experiments, a decrease of the triangles contribution is
independent on the ratio of main reagents and acidity of the medium. So it is possible
to expect that our old preparations will be less toxic than the young ones.
Measurements of the particle sizes in solution by PCS method give the results
similar to those shown above for Ag nanoparticles. There is also one peak in the
histogram, the average particle size is larger than that obtained from the TEM data
for the same solution, and this difference is more marked than in the case of silver
nanoparticles. A typical histogram for the young solution prepared at the excess
of Qr is shown in Fig. 2.21. Average size of the particles is equal to 42.1 nm; the
standard deviation is 2.5 nm. Comparison with the histogram based on the TEM
Fig. 2.21 Particle size distribution obtained by PCS technique for the young micellar solutions
of gold nanoparticles at the excess of Qr. Average particle size42.1 nm
data (see Fig. 2.19a) shows that the average size and the standard deviation mea-
sured by PCS are approximately 2 times greater than those obtained by TEM.
The same difference was observed for nanoparticles synthesized at the excess of
GA. Similar discrepancy between the PCS and electron microscopy results was
detected in [128] for the gold nanoparticles synthesized both in aqueous solution
(reduction with citrate) and in reverse micelles (reduction with hydrazine and other
non-biological reducing agents).
Such an increase of the average diameter (or hydrodynamic radius) in com-
parison with the electron microscopy data was observed not only for gold
nanoparticles but also for the particles of different natureliposomes, latexes, and
metal oxides [274]. This was assumed to be connected mainly with one of the PCS
disadvantages, i.e., its extreme sensitivity to the presence of large particles that
scatter light most stronglyas is generally known, the scattering intensity is pro-
portional to r6, where r is particle radius. Therefore, even a small numerical con-
tribution of the large particles can cause a signicant increase of the measured
average particle size.
In our opinion, apart from the high sensitivity of PCS method to the presence of
large particles, the observed discrepancy may be provoked by another cause.
Firstly, the error is possible because of the deviation of the true particle shape from
spherical one assumed in calculations of a particle hydrodynamic radius by the
StokesEinstein equation, and secondly, as noted also in [128], the error may
happen due to the proximity of the nanoparticle absorption band to the laser beam
wavelength (in the device Horiba LB-550 this wavelength is 633.4 nm). In our
case, there are two lines of indirect evidence in favor of the latter version: (1) the
difference between the PCS and TEM data is greater for gold nanoparticles than for
silver ones that could be expected since the absorption band of gold nanoparticles is
more close to the laser wavelength and (2) our results obtained on the same device
for uncolored solutions which do not absorb in the visible region. For example, for
the standard polystyrene latex particles with the size of 20 nm, Horiba LB-550
gives 18.2 1.8 nm, i.e., there is a good agreement for the particles with almost the
same average size as that determined for the gold nanoparticles by TEM.
Control of the particle size in micellar solutions for Au nanoparticles in various
time intervals after the beginning of synthesis had shown that, unlike the results
obtained by electron microscopy, the average particle size increases with time up to
5060 nm. Besides, in some cases, the bimodal distribution is observed, where one
peak is close to the average size determined by TEM, and the other one lies beyond
the nanosized region (exceeds 100 nm). Result of such kind obtained half a year
after the synthesis of nanoparticles at the excess of HAuC14 is shown in Fig. 2.22.
More than 60 % of the particles have a size in the range 1723 nm (average
21.6 nm) and approximately 40 %in the range 170200 nm. If this is not the
consequence of the well-known problems with reliability of the analysis of the
autocorrelation function for polydisperse samples (see, e.g., [275]), one can suggest
that the larger particles responsible for the discrepancy with TEM data in the
young solutions gradually aggregate and increase in number, so that they nally
appear in the histogram as a separate peak. It should be added that such large
particles can be of nonmetallic nature; for example, in our case, aggregates of the
AOT micelles may be present, containing (or not containing) Qr or HAuC14.
2.4.3 Copper Nanoparticles
Spectrophotometric studies of the copper ion interaction with quercetin in micellar

solutions had shown that, after the addition of aqueous solutions of simple salts
(sulfates or nitrates), a bathochromic shift of the Qr band was observed testifying to
the complex formation where copper ion is associated with oxygens of the catechol
group (binded with C and C4 atoms in the ring B, see Fig. 2.1); similar situation
takes place in the Cu2+ ion interaction with Qr and other flavonoids in water or
wateralcoholic solutions [257, 262, 264, 276]. Also it is known that, apart from the
formation of complexes, in its molecular solutions, Qr can be oxidized with the
formation of the products absorbing at the wavelengths 330335 nm and also of Cu+
ions [257, 262]. Further oxidation of quercetin and reduction of Cu+ ions to atoms in
micellar solution could lead to the nanoparticle formation that can be detected by the
appearance of characteristic band in the range of 550570 nm. It turned out, how-
ever, that the process did not proceed further and the nanoparticle formation was not
observed. We supposed that the reason was the instability of Cu+ ions which did not
Fig. 2.22 Particle size distribution obtained by PCS technique for the old micellar solutions of
gold nanoparticles at the excess of GA
allow the complex formation with quercetin, where the reduction to atom could be
realized. Similar problem arouse, for instance, in studies of metal ion reduction by
the hydrated electron in aqueous solution [169, 277], or in the synthesis of copper
nanoparticles in aqueous solution in the presence of polymer [278]. In both cases,
special efforts were undertaken to stabilize Cu+ ions by addition of the agents
forming stable complex with these ions.
Following the same logic, and taking in account also that univalent copper ions
form stable complexes with ammonia [168, 278], instead of the simple salts, we used
ammoniac copper salts obtained by the introduction of ammonium hydroxide into
sulfate or nitrate water solutions, resulting in the formation of complex cations [Cu
(NH3)4]2+. We assumed that, after the reduction of these ions in the complex with
quercetin, single-charged cations formed will be stable enough to realize the next
stage, i.e., its reduction to atom with the subsequent formation of nanoparticles.
Indeed, addition of ammoniac copper salts [Cu(NH3)4]SO4 or [Cu(NH3)4]NO3 to the
Qr micellar solution gave rise rst to the complex formation and then to the
appearance of nanoparticles. These events were reflected in the corresponding
changes of Qr absorption spectra in micellar solution. Typical picture observed for the
hydration extent 3.7 and ratio C0Cu :C0Qr 10:1, corresponding to the maximum yield
of copper nanoparticles, is shown in Fig. 2.23. At rst, shift of the band I takes
place which indicates to the complex formation; then, this band disappears, and the
band appears corresponding to the copper nanoparticles (545555 nm). The com-
plex dissociates with the formation of the two products: oxidized quercetin (band
320330 nm) and nanoparticles. The synthesis goes fast enough under standard
conditions, so that already in 2 min the band I of original quercetin is not visible,
and a weak absorption appears in the band of nanoparticles. As in the case of silver,
1.0
0.8 Qr
Optical density
0.6
0.4
1
0.2 2
0.0
200 300 400 500 600 700 800 900 1000
Wavelength, nm
Fig. 2.23 Spectrum changes of Qr micellar solution in 2 min (1), and 30 min (2) after the
introduction of ammoniac copper salt. C0Cu = 3.4 mM, w = 3.7 C0Qr = 0.32 mM
to reveal the stages preceding the nanoparticle formation, the process should be
slowed down by reducing the reagent concentration and/or the hydration extent.
Example of such kind for the ratio CCu:CQr = 5.5:1 and hydration extent 3.7 is
shown in Fig. 2.24. In several minutes after the addition of Cu(NH3)4SO4 water
solution, changes of Qr spectrum are observed, testifying to the formation of
quercetin complex with [Cu(NH3)4]2+ ions; the band maximum of the complex lies
here around 450 nm. Then, intensity of this band begins to fall, with the simulta-
neous increase of absorption at < 400 nm, and also the band is revealed with
maximum at 310320 nm. There is isobestic point around 400 nm that indicates to
the direct connection between the diminution of complex concentration and the
increase of concentration of the component that absorbs at 310320 nm. At the
same time, absorption increases in the region of 540600 nm where the band of
copper nanoparticles is formed under different system parameters (see Fig. 2.23).
Micellar solution of copper nanoparticles has copper-red color; the same color is
inherent to the solutions of copper nanoparticles obtained with traditional chemical
reducing agents (see, e.g., Chap. 1, Sect. 1.3.2, Fig. 1.9). The band maximum lies at
550 5 nm, i.e., in the absorption region characteristic for these nanoparticles (550
570 nm) [156, 278, 279]. Typical absorption spectrum is shown in Fig. 2.25.
Comparison with the spectrum of copper nanoparticles produced by the reduction
with hydrazine in reverse micelles at the close hydration extent (w = 4) [156] shows
that the maximum position is almost the same, but in our case, the nanoparticle band
is more distinctly expressed, probably owing to the higher level of conversion of the
copper atoms and, consequently, to a lower optical density in the UV region due to
the lower concentration of the non-reduced metal ions. Absorption at 800 nm (that
characterizes the copper oxide concentration in solution) [156] is negligible in both
1.0
Qr
+Cu(NH3)4SO4, 2 min
0.8 8 min
30 min
Optical density
2 h 30 min
0.6
0.4
0.2
0.0
200 300 400 500 600 700 800 900
Wavelength, nm
Fig. 2.24 Cu nanoparticle formation in micellar solution C0Cu = 1.64 mM, w = 3.7,
C0Qr = 0.29 mM
Fig. 2.25 Absorption 0.30

spectrum of the copper
nanoparticles obtained by
biochemical synthesis (CuQr) 0.25
at the stationary stage,
w = 3.7. For comparison,
Optical density
0.20 CuH
spectrum is shown of the
copper nanoparticles obtained
in reverse micelles (w = 4) by 0.15
the reduction with hydrazine
(CuH) [156]
0.10
CuQr
0.05
0.00
350 400 450 500 550 600 650 700 750 800
Wavelength, nm
cases; this shows that in biochemical synthesis, the nanoparticle oxidation by the
atmospheric oxygen is as small as in the synthesis with traditional chemical reducing
agent.
It turned out to be extremely difcult to obtain the high-quality TEM images for
copper nanoparticles, presumably on account of the small size of the particles and their
lower electronic density compared to the previous cases and also probably due to the
imperfect procedure used for the sample preparation for microscopy. For today, one of
Fig. 2.26 Electron

micrograph of the Cu
nanoparticles in micellar
solution. Average particle size
does not exceed 15 nm
100 nm
the most successful attempts is shown in Fig. 2.26. Here, one can see numerous small
particles with sizes not exceeding 15 nm, embedded in the AOT clot.
The rate of formation and yield of Cu nanoparticles for the same concentrations
of reagents and hydration extent are less than those obtained for silver and gold.
This is conditioned presumably by the lower degree of quercetin binding with
copper ions, detected in studies of metal ion adsorption from aqueous solution by
polymer membranes containing quercetin [280].
The PCS analysis of copper nanoparticles in micellar solutions gives a bimodal
distribution with large portion of small particles (with the size of several
nanometers). Monomodal distribution corresponding to the electron microscopy
data could not be obtained in this case, probably due to the low nanoparticle
concentration and, accordingly, low intensity of the useful signal.
Copper nanoparticles in micellar solution are stable for at least several months,
which allows to use them successfully for the modication of liquid-phase and solid
materials (see Chap. 5).
Chapter 3
Development of the Biochemical Synthesis
for Practical Applications
Development of our works on the synthesis of metal nanoparticles included studies

of the influence of different factors on the characteristics of nanoparticle solutions;
in these studies, we are guided primarily by aspiration for the achievement of
practical results [28]. Therefore, the main purpose of our experiments was to
determine the conditions providing fulllment of the main requirements from
potential customers interested in the application of nanoparticle solutions for the
modication of their products or for the creation of new materials. The main
requirements are formulated as follows: production of the large quantities of
solutions with high concentration of small nanoparticles which have a narrow size
distribution and a long-time stability in the air. As turned out afterwards, these
general requirements essential for practical application may be actually addressed to
any method used for the nanoparticle preparation (see Chap. 1, Sect. 1.7). Thus, the
following characteristics of nanoparticles are found to be the most signicant for us:
(1) the rate of formation and yield of nanoparticles that determine, respectively, the
time required to produce a large amount of nanoparticle solution and nanoparticle
concentration, (2) the nanoparticle size, and (3) stability of nanoparticle solutions in
the air. By stability we mean here the preservation of nanoparticle concentration
and sizes obtained at the stationary stage, after the completion of synthesis.
Recent progress in nanotechnologies, in particular a more profound under-
standing of the effects provoked by the nanoparticles in living organisms, brought
forward one more general requirement, namely, minimal toxicity and ecological
safety of nanoparticles and nanomaterials. Besides, while working out various
applications of the nanoparticles produced by biochemical synthesis, we came
gradually to the conclusion that our main efforts should be concentrated on the
studies of nanoparticle biological activity for their applications in biology and
medicine. This implies primarily the creation of medical products and materials
with antimicrobial activity and possibly also the development of new methods of
diagnostics and nanoparticle visualization for the researches on cellular level.
Hence, the need arouse in the preparation of not only micellar, but also aqueous
nanoparticle solutions and, apart from the general requirements valid for the
DOI 10.1007/978-3-319-30906-4_3
110 3 Development of the Biochemical Synthesis for Practical Applications
different applications of nanoparticles, additional requirements for their solutions

used in biomedical applications were also formulated (see again Chap. 1, Sect. 1.7).
In the course of studies in this direction, the techniques have been developed
based on the biochemical synthesis and allowing the preparation of nanoparticles in
aqueous solutions, either by the transfer of nanoparticles from micellar solutions or
by the synthesis directly in aqueous solution with application of the same natural
reducing agent, i.e., quercetin (see Chap. 4). Thus, our studies were carried out with
two main objects: micellar solutions and aqueous solutions of nanoparticles.
With micellar solutions, the influence of different factors on the rate of forma-
tion, yield, sizes, and stability of nanoparticles was investigated. The results of
these works were used for the preparation of aqueous solutions and in studies of
various nanoparticle properties. Studies of the catalytic properties of silver, copper,
cobalt, and nickel nanoparticles were realized on micellar solutions. The data
obtained in these studies are not included in this edition, but they can be found in
the other sources [9, 28, 283]. Aqueous solutions were used in the research of
adsorption properties and biological action of silver nanoparticles; the latter
direction included both their antimicrobial activity and their toxicity toward the
other living organisms (Part II, Chaps. 79). Studies on the adsorption properties
led to the creation of various solid-state materials with nanoparticles deposited on
their surface (Chap. 5), which were used also in the researches of antimicrobial and
catalytic activity of the nanoparticles. The results on antimicrobial activity of the
nanomaterials are presented in Chap. 7.
This chapter includes results of our works fullled with aim to solve the three
tasks essential for the further researches of the biological action of silver
nanoparticles: (1) determination of nanoparticle concentration in micellar solution,
(2) preparation of micellar solutions with high concentration of nanoparticles, and
(3) preparation of solutions containing small-sized nanoparticles (below 20 nm)
with narrow distribution for the studies of size effects on biological objects. To
solve the rst task, we determined the extinction coefcient for silver nanoparticles
in micellar solution; the second task was solved in the course of studies of the
influence of reducing agent/metal ion concentration ratio on the rate of formation
and yield of nanoparticles; and the third task required investigation of the influence
of silver salt and stabilizer concentrations and that of hydration extent on the
average size and size distribution width of nanoparticles.
3.1 Determination of the Extinction Coefcient

for Ag Nanoparticles
It is known that nanoparticle concentration is one of the main parameters used both
in the studies of biological activity or other properties of nanoparticles and for the
manufacturing of materials modied by nanoparticles. However, despite a large
number of publications on the synthesis of metal nanoparticles in solutions, the
extinction coefcient of nanoparticles for the majority of metals is not known,
3.1 Determination of the Extinction Coefcient for Ag Nanoparticles 111
because it is usually difcult to determine precisely what part of the ions present in
solution is converted to nanoparticles, and what part is present in another form, not
reflected in the spectrum of nanoparticle solution. In many cases, it is assumed
therefore that the yield is equal to 100 %; that is, all of the metal ions had been
transformed to nanoparticles. Hence follows that the nanoparticle concentration is
equal to the concentration of metal salt introduced into solution [48, 128, 172];
accordingly, there are no problems with the determination of the extinction coef-
cient. Nevertheless it is evident that metal can exist in the nanoparticle solution also in
the form of unreduced ions, metal oxide, or metal ion complex with reducing agent,
these forms being undetectable in the spectrum. For example, as shown in [111], in
aqueous solution of Ag nanoparticles prepared by the radiation-chemical method,
silver is present also as oxide microcrystals which do not absorb in the visible region.
For silver, gold, and copper nanoparticles, the extinction coefcients may be found in
the literature, but these values can be dependent on the method of nanoparticle
preparation, and therefore, for the reliable determination of the nanoparticle con-
centration, it is desirable to nd the extinction coefcients for the particular experi-
mental system. This work was accomplished by us for the silver nanoparticles.
Taking into account that in our case synthesis of Ag nanoparticles proceeds
through the formation of quercetin complex with Ag+ ions, it seems reasonable to
suggest that this complex also can be present in the nanoparticle solution. Thus,
determination of the extinction coefcient of Ag nanoparticles includes at least two
stages: rst, determination of extinction coefcient for the complex and second,
determination of this coefcient for the nanoparticles.
3.1.1 Extinction Coefcient of the Complex
To determine the extinction coefcient of the complex, it is necessary to separate

the stage of its formation in the form as clear as possible, i.e., to obtain the solution
where only the complex is present, but the nanoparticles are not yet produced.
Separation of this stage could be realized in the conditions where Qr interaction
with the Ag+ ions proceeds with the rate low enough for the complex absorption
band (295 nm) to be observed without the simultaneous appearance of the
nanoparticle absorption band (see also Chap. 2, Sect. 2.4.1). It turned out to be
impossible for the Qr micellar solution prepared from the dried (preliminary
dehydrated) AOT usually applied for the standard nanoparticle solutions. The
problem was that even with extremely small Qr and silver ion concentrations, the
bands of the initial Qr still remain; those of both complex and nanoparticles are also
visible and the appearance of a faint yellow-brown color is observed, caused by the
nanoparticle absorption in the visible region (at 420430 nm). To overcome this
difculty, we used the small concentrations of reagents and prepared the Qr solution
using slightly hydrated AOT (obtained by means of the preliminary addition of
water into the dried AOT/isooctane solution to the hydration extent w < 3).
Introduction of Ag+ ions into thus prepared Qr micellar solution results in the
0.3 0
C (Qr)
1
Optical density
0.2
0.1
2 t
C (Qr)

0.0
200 300 400 500 600
Wavelength, nm
Fig. 3.1 Determination of the extinction coefcient for AgQr complex in micellar solution.
1spectrum of the initial Qr solution; 2nal stage of the complex formation. C0(Qr) and Ct(Qr)
are quercetin concentrations, respectively, in the initial solution and at the nal stage of complex
formation. is the optical density difference measured at kImax between spectrum 2 and extension
of the complex band in the range 350400 nm
gradual decrease of the intensity of both Qr absorption bands with simultaneous

increase of the 295 nm band intensity. This process ends with the formation of the
distinct band at 295 nm and of the small shoulder in the range of 370380 nm
conditioned by the remaining Qr (Fig. 3.1). During these changes in the spectrum,
the solution remains colorless and the nanoparticle band is absent. At the higher Qr
and Ag+ ion concentrations used in the standard synthesis procedure in micellar
solutions with weakly hydrated AOT, also the nanoparticle band is formed and the
characteristic reddish brown color appears. However, with the other conditions the
same, the synthesis goes signicantly slower, and the nanoparticle concentration is
smaller than in the micellar solutions prepared with dried AOT. This points to
the important role of water in the core of reverse micelle in the process of
nanoparticle formation.
The spectra of the type shown in Fig. 3.1 are used for the determination of
extinction coefcient of the complex according to the equation which follows from
the BouguerLambertBeer law [43]:
.
e Dmax 295 C0Qr CtQr l0 3:1
where C0Qr and CtQr are the initial and nal Qr concentrations, respectively; 10 is the
optical path length. The CtQr value can be determined graphically from the difference
of optical densities (D) for the wavelength equal to max of the band I of Qr (kImax ),
using previously found extinction coefcient for this band at the corresponding
3.1 Determination of the Extinction Coefcient for Ag Nanoparticles 113
AOT hydration extent: * = 2.26 104 L/mol cm (see Chap. 2, Sect. 2.3.2 and
[268]). From ve determinations at different silver and quercetin concentrations, we
obtained * = (1.98 0.05) 104 L/mol cm. Thus, extinction coefcient for the
product with absorption band 295 nm proved to be similar to that found for quercetin.
To determine the Qr/Ag+ ratio in the complex, we calculated the maximal
(equilibrium) amount of quercetin transferred to the complex after introduction of
the equal or smaller amount of Ag+ ions. For this purpose, the spectra were used,
obtained for the small concentrations of reagents several days after the introduction
of silver ions, when the changes of absorption bands terminated. In all cases
studied, the amount of Qr transferred to the complex was almost equal to the initial
concentration of Ag+ ions. Hence, Qr and Ag+ ions are present in the complex in
equimolar ratio (for details see [268]).
3.1.2 Extinction Coefcient of the Nanoparticles
Extinction coefcient of Ag nanoparticles was determined with the use of the

extinction coefcient and stichiometry of the complex [Ag+ Qr]. For this
purpose, experiment was carried out in the conditions when quercetin is completely
transferred to the complex, and silver ions are either associated in the complex or
present in solution as nanoparticles. Such situation is realized for the small Qr
concentrations and certain excess of Ag+ ions; the latter should be small enough for
the free silver ions to be absent in solution after the nanoparticle formation and, at
the same time, large enough for the formation of the intense nanoparticle band,
which does not overlap with that of the complex (Fig.3.2). Extinction coefcient for
the nanoparticles was determined from the equation:

eNP Dmax kNP
max C AgNO3 s l0 3:2
where Dmax(kNP max ) is optical density in nanoparticle band maximum, and C

(AgNO3 (s)) is the concentration of Ag+ ions present in the form of nanoparticles.
From the data obtained in several experiments with different Qr to Ag+ ion
concentration ratio, the extinction coefcient for Ag nanoparticles was found as
NP = (1.03 0.08) 104 L/mol cm [268]. Similar though somewhat lower NP
values (78 103 L/mol cm) were obtained for micellar and aqueous solutions of
silver nanoparticles synthesized with other reducing agents [152, 172]. Extinction
coefcient determined by us was conrmed also by the comparison of experimental
spectra of silver nanoparticles with those calculated from the Mie theory for the
particle sizes determined on the stationary stage by PCS [268]. As shown in Fig. 3.2,
the growth of nanoparticle band is accompanied by reduction of the band at 295 nm;
it is quite natural since the increase of nanoparticle concentration must correlate with
decay of the Qr complex with silver ions. Thus, our data disagree with the opinion
expressed in literature [284] that closely positioned adsorption band (298 ) is
related to the products of Qr oxidation with metal ions and hydrogen peroxide.
Fig. 3.2 To the extinction

coefcient determination for 0.30
silver nanoparticles. 2
Quercetin concentration in the
initial solution is 69 M. 0.25
1 and 2nanoparticle
formation, respectively, in 2
Optical density
0.20 1
and 6 days after the
introduction of AgNO3 to the
concentration of 300 M
0.15
Qr
0.10
0.05
0.00
200 300 400 500 600 700
Wavelength, nm
Determination of the extinction coefcients of the complex and Ag nanoparticles

allows to nd their concentrations in micellar solution prepared at the different
system parameters (AOT and reagents concentrations, hydration extent), as well as
changes of the nanoparticle concentration in solution due to the adsorption on solid
materials.
3.2 The Effect of CQr/CAg Relation on the Formation Rate

and Yield of Silver Nanoparticles
According to our data, the rate of formation and yield of nanoparticles depend on
metal salt composition and on metal salt to reducing agent (flavonoid) concentration
ratio. The influence of metal salt composition on the rate and yield of silver
nanoparticles is discussed in Chap. 2. It was found that the use of ammoniac silver
salt (diammine nitrate) signicantly accelerates the process of synthesis and
increases the nanoparticle concentration in solution compared to the results
obtained with simple silver nitrate. It was shown also that this difference is con-
nected, most likely, with different degree of the deviation from ideality that exists in
the initial aqueous metal salt solutions used for the preparation of nanoparticles.
The advantage of diammine nitrate was found useful further for the preparation of
silver nanoparticles not only in micellar but also in water solutions, and not only
with AOT but also with some other stabilizers (Chap. 4). Here, we shall consider
the influence of metal salt to flavonoid concentration ratio on the formation rate and
yield of silver nanoparticles.
3.2 The Effect of CQr/CAg Relation on the Formation Rate 115
The rate of nanoparticle formation was determined from the changes with time
of absorption spectra after the introduction of metal salt aqueous solution into the
Qr micellar solution, which marks the beginning of synthesis; in other words, series
of the spectra were used that reflected the kinetics of nanoparticle formation of the
type shown in Fig. 2.8. From the spectrum changes with time, optical density
variations in the maximum of adsorption band (Dmax(t)) were found, which reflect
the changes in nanoparticle concentration.
The yield of nanoparticles (NP) was dened as the ratio of the maximal
nanoparticle concentration achieved, i.e., of the nanoparticle concentration on the
stationary stage (C0NP ), to the concentration of metal ions introduced into solution:

bNP C0NP =CAg 100 % 3:3
Concentration of nanoparticles was found from the BouguerLambertBeer law

using the value of optical density on the stationary stage:

CNP D0max eNP l0 3:4
where NP is the nanoparticle extinction coefcient, 10 is the optical path length.

Here, CNP is expressed in moles (in the units of molar concentration equivalent to
the concentration of metal salt). If a mass concentration is preferable (i.e., weight of
the metal present in the form of nanoparticles), it may be determined by recalcu-
lation from molar concentration using the metal atomic weight.
The effect of quercetin and silver concentrations on the rate of formation and
yield of nanoparticles was determined either by the variation of quercetin con-
centration or by the variation of silver concentration, at the constant concentrations
of the second reagent and AOT, and hydration extent. Influence on the nanoparticle
formation rate was estimated from the analysis of Dmax(t) dependences obtained for
different reducing agent to metal ion concentration ratios, i.e., for the different
values of CQr/CAg.
An example of such dependence obtained by the variation of quercetin con-
centration is shown in Fig. 3.3. It is seen that the rate of nanoparticle formation
increases with the increase of the ratio CQr/CAg. After 13 days, the stationary stage
is achieved, where practically no changes of Dmax value are observed. The yield of
nanoparticles was determined usually in 36 days after the beginning of synthesis.
In the experiment shown in Fig. 3.3, the NP values determined in 3 days after the
synthesis, for CQr/CAg = 0.0245 (1), 0.0442 (2) and 0.083 (3) were equal to 35.7,
62, and 100 %, respectively.
Figure 3.4 presents the result of similar experiment but with the variation of
silver ion concentration. As shown in gure, the nanoparticle concentration
increases with the increase of silver concentration; insert shows the corresponding
change in the yield of nanoparticles with variation of the CQr/CAg ratio. The yield
equal to 100 % is observed for the CQr/CAg 0.1, i.e., beginning from the value
close to that corresponding to the 100 % yield in experiments with variation of Qr
6 3
4 2
Dmax
3
1
2
0
0 10 20 30 40 50 60 70 80
t, hours
Fig. 3.3 Influence of the quercetin to silver salt concentration ratio on the nanoparticle formation
rate. CAOT = 0.08 M, w = 3.7. CQr/CAg is equal to 0.0245 (1), 0.0442 (2), and 0.083 (3)
100
4.0 7 5-6 90
3.5 4 80
, %
70
3.0 60
3
Optical density
50
2.5
40
2.0 30
0.0 0.1 0.2 0.3 0.4 0.5 0.6
C/Qr CAg
1.5
2
1.0
1
0.5
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 3.4 The effect of silver salt concentration on the nanoparticle formation. Spectra obtained in
7 days after the introduction of aqueous silver salt solution into the quercetin micellar solution.
CQr = 0.265 mM, CAOT = 0.08 M, w = 3.7. Silver concentration in solution, CAg, is 0.5 mM (1);
1 mM (2); 2.4 mM (3); 4 mM (4); 6 mM (5); 7.4 mM (6); 10 mM (7). Inset shows the
dependence of Ag nanoparticle yield on CQr/CAg ratio
3.2 The Effect of CQr/CAg Relation on the Formation Rate 117
concentration. In the other pairs of similar experiments, at different values of Qr and

Ag+ ion concentrations, the same results have been obtained: 100 % yield or the
complete reduction of silver ions was registered for the quercetin to silver ion
concentration ratio no less than 0.1. Hence follows that, for the complete
reduction of silver ions, no less than 1 mol of Qr per 10 mol of silver salt is
required. In other words, one molecule of Qr is able to reduce up to ten Ag+ ions.
Qualitatively similar pictureacceleration of the process and increase in the
yield of nanoparticles with the increase of the reducing agent to metal ion con-
centration ratiois observed at the reduction with traditional chemical reducing
agents in aqueous solutions or reverse micelles (see Chap. 1). However, there is a
considerable difference in the concentration ratio under questionwith hydrazine
or sodium borohydride, this ratio is by approximately two orders higher than in our
case. As we already mentioned above (Chap. 1, Sect. 1.6), similar difference is
revealed also at the comparison of traditional reducing agents with biological one
(apiin) similar in its structure to flavonoids, when synthesis is performed in aqueous
solutions. Thus, there are reasons to believe that efciency of biological reducing
agents (at least of flavonoids and the related substances) is much higher than that
observed for traditional chemical reducing agents in the nanoparticle synthesis both
in aqueous solution and in reverse micelles. The origin of such difference yet
remains unclear; one may suppose only that it is connected with different mecha-
nism of the reducing agent interaction with metal ions.
The particularity feature of quercetin noted above allowed us to obtain high
concentrations of nanoparticles, by orders of magnitude more than those available
for the traditional chemical reducing agents. The data of the similar studies with
aqueous solutions of silver and copper nanoparticles are presented in Chap. 4.
3.3 Preparation of Small Nanoparticles with Narrow Size

Distribution
The main task of this series of experiments was the preparation of silver
nanoparticles of different average size and with narrow size distribution that allow
to study the size dependence of nanoparticle properties (primarily of the biological
action). We considered that it was important to study the size range below 20 nm,
where the so-called size effects were observed ([31, 32, 35] and Chap. 1, Sect. 1.1).
Accordingly, one could expect that it was just in this range that the effect of particle
size would be most distinctly expressed. To solve this task, we varied the param-
eters of reverse micellar system which affect the particle size. The results of this
work are described in detail in [285].
Our starting point in this research was the standard micellar solution of Ag
nanoparticles used in various practical applications. To prepare such solution, the
parameters were selected allowing to obtain reproducibly micellar solutions with
the nanoparticle concentration of the order of several millimoles (of several
50 dav = 9.3 nm
= 1.7 nm
40
% of particles
30
20
10
0
5 6 7 8 9 10 11 12
50 nm particle size, nm
Fig. 3.5 Electron micrograph and particle size distribution for the standard micellar solution of
silver nanoparticles at w = 3.7, CAg = 7.4 mM, CAOT = 0.135 M. dav is the mean particle size,
is the standard deviation
hundreds mg/l) and an average particle size in the range 910 nm. An example of
electron micrograph and corresponding size distribution for such standard solution
is shown in Fig. 3.5.
The particles are spherical, and average size is 9.3 nm with deviation 1.7 nm.
Analysis of the electron diffraction patterns indicates that nanoparticles have crystal
structure with parameters of FCC lattice similar to those of the gold crystals.
To change the average particle size, we varied the AOT concentration, the silver
salt concentration, and the hydration extent. As known from the literature, variation
of AOT concentration and hydration extent leads to the change of water core size in
reverse micelles [139, 142, 286] and, consequently, to the changes of the conditions
of nanoparticle formation which affect the particle sizes. Variation of silver salt
concentration has an influence on the particle size through the intensity changes of
nanocluster aggregation process [82, 153]. The effect of these parameters was
estimated by the comparison of optical spectra and particle sizes (from TEM data)
for the solutions studied on the stationary stage, in 7 days after the beginning of
synthesis.
The influence of AOT concentration is shown in Figs. 3.6 and 3.7. A decrease of
AOT concentration (Fig. 3.6) provokes insignicant reduction of the nanoparticle
concentration, more noticeable for the lowest AOT concentration. Besides, there is
a small bathochromic shift of the absorption band (from 433 to 440 nm) for the two
lower AOT concentrations; according to the Mie theory, this gave grounds to
assume the increase of the average particle size [152,172].
To test this assumption, we determined further the particle size in solution with
CAOT = 0.08 M and compared it with that in the original standard solution. As
shown in Figs. 3.5 and 3.7, at the decrease of AOT concentration from 0.135 M to
0.08 M, the average particle size did not increase, but perhaps even decreased
slightly (dav = 8.4 1.6 nm). Thus, in this case the increase of particle size
3.3 Preparation of Small Nanoparticles with Narrow Size Distribution 119
4.0 1
3.5 2 3
3.0
Optical density
2.5
2.0
1.5
1.0
0.5
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 3.6 The effect of AOT concentration on the nanoparticle formation. Parameters of micellar
solution: CAg = 7.4 mM, w = 3.7, CAOT (M): 0.135 (1), 0.08 (2), and 0.042 (3)
50 dav = 8.4 nm
= 1.6 nm
40
% of particles
30
20
10
0
5 6 7 8 9 10 11 12
Fig. 3.7 Electron micrograph and particle size distribution for the micellar solution of silver
nanoparticles at w = 3.7, CAg = 7.4 mM, CAOT = 0.08 M
assumed because of the redshift of adsorption band was not conrmed by the
electron microscopy data.
The influence of silver salt concentration on the absorption spectra of
nanoparticles is shown in Fig. 3.4. Reduction of silver concentration from 7.4 to
0.5 mM leads to the signicant decrease of nanoparticle concentration and to the
noticeable shift of the absorption band maximum to the shorter wavelengthsfrom
438 to 420 nmthat suggests a decrease of particle size. As shown by TEM of the
45
dav = 4.6 nm
40
= 1.8 nm
35
30
% of particles
25
20
15
10
5
0
2 3 4 5 6 7 8 9 10 11
nanoparticles at w = 3.7, CAg = 1 mM, CAOT = 0.08 M
solution with CAg = 1 mM (Fig. 3.8), in this case (in agreement with the Mie
theory) shift of the maximum position to the shorter wavelength is really associated
with signicant decrease of the average particle size (to 4.6 nm) with deviation
(1.8 nm) almost the same as in the standard solution.
We suggested further that the discrepancy with the Mie theory, revealed in the
previous experiment, is caused by the too large concentration of silver ions, which
does not allow to detect the effect of AOT concentration. This supposition issued
from the following considerations. It is obvious that the rate of formation and
subsequent aggregation of the nanoparticles is the higher, the greater is concen-
tration of silver ions. The effect of AOT concentration in the aggregation process
can manifest itself in that the AOT molecules adsorb on the surface of nanoparticles
and thus prevent them from aggregation, hence the decrease in the average particle
size.
The rate of AOT adsorption will increase with increasing AOT concentration,
and this will lead to the decrease of average particle size. However, this effect can
become noticeable only when the rate of AOT adsorption is comparable with that of
nanoparticle aggregation, i.e., at the low enough concentration of silver ions.
To verify this reasoning, the influence of AOT concentration on the particle size
had been determined for the solution with CAg = 1 mM. As shown in Fig. 3.9, as in
the case of CAg = 7.4 nM, the decrease of AOT concentration is accompanied with
the bathochromic shift of the adsorption band. The TEM images of the solution
with CAg = 1 mM and CAOT = 0.015 M are shown in Fig. 3.10.
Analysis of the histogram gives dav = (7.7 1.13) nm; that is, a signicant
increase of the average size is observed in comparison with solution where
CAOT = 0.08 M. This means that average size of the particles may be increased by
reducing the AOT concentration, but this parameter is working at the small enough
concentrations of silver ions. It should be added that, apparently, in this case the
1.2 1
2
1.0
3
0.8
Optical density
4
0.6
0.4
0.2
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 3.9 The influence of AOT concentration on the nanoparticle formation at CAg = 1 mM,
w = 3.7. CAOT (M): 0.135 (1), 0.08 (2), 0.04 (3), and 0.015 (4)
70
dav = 7.7 nm
60 = 1.13 nm
50
% of particles
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
nanoparticles at w = 3.7, CAg = 1 mM, CAOT = 0.015 M
bathochromic shift of the nanoparticle absorption band reflects the increase of

particle size, in accordance with the Mie theory.
The influence of hydration extent has been studied on the solutions with
CAg = 1 mM, CAOT = 0.08 M. Figure 3.11 shows the spectra of nanoparticle
solutions obtained at the hydration extent w = 3.7, 5, and 10. It is seen that in this
range the increase of hydration extent exerts practically no influence on the
nanoparticle concentration, but leads to the 67 nm shift of the band to the shorter
1.2
1.0
0.8
Optical density
0.6 2 3
0.4
0.2
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 3.11 Spectra of micellar solution at different hydration extents w: 3.7 (1), 5 (2), and 10 (3).
CAg = 1 mM, CAOT = 0.08 M
dav = 10.35 nm
25 = 2.5 nm
20
% of particles
15
10
0
6 7 8 9 10 11 12 13 14
Fig. 3.12 Electron micrograph and particle size distribution for micellar solution at w = 10,
CAg = 1 mM, CAOT = 0.08 M
wavelengths. Though this shift, according to the Mie theory, can be interpreted as
the result of particle size diminution, such an effect is unlikely to occur in this case,
since it is known that at the nanoparticle synthesis in reverse micelles, an increase
of w, on the contrary, results in an increase of particle size (e.g., [82, 127, 129,
153]). Similar effect of the increase of hydration extent is observed in our case.
Figure 3.12 shows the electron micrograph and size distribution for the solution
with w = 10. The particle average size increased by approximately 3 times in
Table 3.1 Nanoparticle sizes and concentrations, AOT concentration, and hydration extent in
micellar solutions of silver nanoparticles
Solution dav (nm) (nm) CAOT (M) w CNP (mM) CNP (particles/mL)
number
1 9.3 1.7 0.135 3.7 3.7 9.1 1013
2 8.4 1.6 0.08 3.7 3.61 1.2 1014
3 7.7 1.13 0.015 3.7 1 4.4 1013
4 4.6 1.8 0.08 3.7 1 2.1 1014
5 10.35 2.5 0.08 10 1 1.8 1013
comparison with the solution with w = 3.7 and is equal to 10.35 nm; however, the
distribution width also increased, since the standard deviation became equal to
2.5 nm.
Similar tendency to the increase in polydispersity with increasing hydration
extent for micellar solutions of Ag nanoparticles was observed by the other authors
(e.g., [153]). Presumably, this is connected with increase in the portion of less
rigidly structured water in micellar core; as a consequence, the exchange of micellar
contents by collisions is facilitated. As a result, increases the frequency of colli-
sions, fusions, and ssions of micelles containing the growing nanoparticles, hence
the increase in the size distribution width (Chap. 1, Fig. 1.17).
To sum up, in the course of these studies the system parameters were deter-
mined, allowing to prepare several kinds of micellar solutions with the average
particle size in the range 4.610.5 nm, with size distribution close to monodisperse
one (deviation from average size is less than 2.5 nm). Parameters of these solutions
are summarized in Table 3.1. Variants of the solutions can be used to study the
influence of particle size, AOT concentration, and number concentration of the
particles. From micellar solutions, aqueous solutions can be prepared with the same
particle sizes, for the studies of biological activity of the nanoparticles.
Characteristics of the aqueous solutions of silver nanoparticles produced by the
transfer from micellar solution, as well as description of the synthesis of silver and
copper nanoparticles in aqueous solution by reduction with quercetin will be given
in the following chapter.
Chapter 4
Preparation of Metal Nanoparticles
in Water Solutions on the Basis
of Biochemical Synthesis
Micellar solutions of metal nanoparticles may be applied both as additives to the

liquid-phase materials (e.g., to the varnishpaint materials (VPM) based on organic
solvents), and for the nanoparticle deposition on the surface of solid-state materials
for the production of modied materials with various special properties. Examples
of such materials will be presented in Chap. 5.
At the same time, in the working out of various nanoparticle applications it was
found important to prepare stable nanoparticle dispersions not only in organic sol-
vents (in our casein liquid hydrocarbons), but also in the polar media, primarily in
water. For example, silver nanoparticles in the form of aqueous dispersion are more
preferable for the use in production of water-soluble paints or new cosmetics with
antibacterial properties. Water dispersions are more convenient also for the creation
of technologies suitable for the production of carbon or polymeric materials with
deposited silver nanoparticles, useful for the water purication from bacterial con-
taminations in different reservoirs or ltering devices [287, 288]. Finally, there are
all grounds to believe [18, 21, 31, 32, 289, 290] that stable metal nanoparticles in
aqueous solutions will nd practical applications in biology and medicine. It is only
on water dispersions that studies on the mechanisms of biological action of metal
nanoparticles can be realized, which are of great importance for the development of
nanoparticle applications in medicine, as well as for the determination of toxicity of
various nanomaterials for humans and other living organisms.
For the studies of biological effects and development of nanoparticle application
in aqueous media, we created the procedures allowing the preparation of aqueous
nanoparticle solutions. The task was to obtain stable small-sized nanoparticles in
high enough concentration in aqueous solution, and with minimal concentration or
total absence of toxic impurities. The works were conducted in the two directions:
(1) preparation of the aqueous solutions by the transfer of nanoparticles from
micellar solutions and (2) synthesis of the nanoparticles directly in aqueous solution
with application of the same reducing agent (quercetin), but with the other stabi-
lizer. In the latter case, instead of synthetic surfactant we used two natural stabi-
lizers (starch and cyclodextrin).
DOI 10.1007/978-3-319-30906-4_4
126 4 Preparation of Metal Nanoparticles in Water Solutions
In this chapter, we give a brief description of the corresponding procedures and

examples of the optical spectra and size measurement for each of these directions.
For silver nanoparticles synthesized with natural stabilizers, the synthesis param-
eters and nanoparticle characteristics are compared with the relevant data present in
literature.
4.1 Preparation of Nanoparticles as Water Dispersions

by the Transfer from Micellar Solution
The procedure used for preparation of the water nanoparticle solution by the
transfer from micellar solution was developed for silver nanoparticles and was
applied later on for the gold, copper, and zinc nanoparticles; a detailed description
of the procedure is given elsewhere [288, 291]. First the two-phase micellar
solution/water system was prepared, and then, the transfer of nanoparticles was
performed by one of the two ways: either by centrifugation of the two-phase system
or by mixing the micellar solution with water and subsequent phase separation.
For thus-prepared nanoparticle solutions, apart from the concentration and size
of nanoparticles, the important parameter is also AOT concentration, because this
surfactant can be toxic for biological objects and its concentration should be
minimized for some of the nanoparticle applications (e.g., as additives to the cos-
metic products), and also for the studies of biological effects. For the determination
of this parameter, we used the well-known standard procedure accepted for the
measurement of anionic SAS concentration in water and stated in the document:
Determination of the molar concentration of anionic surfactants in the concen-
tration range 0.0150.25 mg/l by spectrophotometrical methods (GOST R
51211-98). Conformably to our case, this technique was modied to be suitable for
the higher SAS concentrations (up to 15 mg/l). With the use of the modied
technique the procedures have been developed that allowed the lowering of AOT
concentration in aqueous dispersion of silver nanoparticles and thus to eliminate the
side effects of this surfactant and to reveal just the effect of nanoparticles on
biological objects (see Chaps. 79).
Centrifugation was performed on the laboratory centrifuge (T52.I.MLW,
Germany) in the tubes with the diameter of 14 mm. Initially, the tubes were lled
with water, and then, micellar solution was slowly introduced from above until the
system was formed with clearly dened interface between the phases. After the
one-step centrifugation (5300 rpm, 15 min), a small part of the nanoparticles is
transferred from micellar solution into the water phase. To increase the nanoparticle
concentration, thus obtained aqueous solution can be subjected to the subsequent
centrifugations, taking fresh portions of the initial micellar solution as organic
phase.
This route allows to prepare the aqueous nanoparticle dispersion with small
AOT concentration, but the nanoparticle concentration will be small as well. As
4.1 Preparation of Nanoparticles as Water Dispersions 127
shown in [291], even after the three-step centrifugation, each step with addition of
the fresh micellar solution, silver nanoparticle concentration in the resulting
aqueous solution did not exceed 60 % of that in the initial micellar solution.
Besides, with centrifugation procedure it is technically complicated to produce
sufciently large amounts of the nanoparticle solution required for the practical
applications. Therefore, in studies of the aqueous nanoparticle dispersions and for
the development of practical applications mainly the second way was applied of the
two mentioned above.
Preparation of aqueous solutions by mixing of micellar solution with water and
the subsequent settling includes several operations aimed to achieve the most
complete (1) extraction of the nanoparticles from micellar solution and (2) removal
of the AOT excess from the aqueous dispersion of nanoparticles. Depending on the
required volume of nanoparticle aqueous solution, mixing can be fullled either by
handshaking or by a mixer with electrical drive. In this procedure, the most sig-
nicant parameters are pH and ionic composition of water used as aqueous phase,
the intensity of mixing, temperature, and some other factors. The technology
applied for the preparation of aqueous nanoparticle dispersion is described in one of
our patents [288]. With the help of this technology, aqueous solutions of silver
nanoparticles were obtained; additional improvements were introduced for the
production of gold, copper, and zinc nanoparticles in aqueous solutions. It was
established also that, by the subsequent dialysis against water, it was possible to
signicantly diminish the AOT concentration in nanoparticle aqueous solution
without a decrease of nanoparticle concentration.
4.2 Properties of Silver and Gold Nanoparticles

in Aqueous Solution
Owing to the strong coloration of the nanoparticle solutions, to the rst approxi-
mation transfer of nanoparticles to the aqueous medium may be controlled visually,
by changes of the color intensity of organic and aqueous phases, namely the col-
oration of organic phase is fading while that of the aqueous phase becomes more
intensive. The absorption spectra of both phases demonstrate a decrease of the
nanoparticle band in organic phase, as well as formation and then growth of this
band in the aqueous phase till the complete extraction of nanoparticles from
micellar solution.
4.2.1 Silver Nanoparticles
Typical change of the absorption spectrum of silver nanoparticles after the transfer
from organic to aqueous phase is shown in Fig. 4.1.
4.0
3.5
= 411 nm
= 427 nm
3.0
2.5
Optical density
2.0
1.5
1.0
0.5
0.0
300 400 500 600 700
Wavelength, nm
Fig. 4.1 Typical absorption spectra of silver nanoparticles in standard micellar solution (dashed
line) and in aqueous solution obtained by the transfer from this micellar solution (solid line). This
gure and Fig. 4.2 were reprinted from Silver nanoparticles: optical properties, characterization
and applications, pp. 221258, copyright 2010, author E.M. Egorova, with permission from Nova
Science Publishers
Maximum of the nanoparticle absorption band in aqueous solution lies in the

range 400420 nm, in agreement with maximum positions for silver nanoparticles
in aqueous solutions reported in literature (e.g., [111, 172, 292]). The nanoparticle
absorption band in aqueous solution is shifted to the shorter wavelength for 16
20 nm from its position in micellar solution. Similar shift of the absorption bands
was noted in literature [152, 293]; it is conditioned by the change of medium
polarity near the nanoparticle surface (in this case caused by penetration of the
water molecules into the micellar core) upon the transfer to the water phase. Optical
density at the band maximum in aqueous solution after the complete extraction of
nanoparticles is greater than that measured in micellar solution, as in the example
shown in Fig. 4.1. Since it is clear that the extent of particle transfer to the aqueous
phase cannot exceed 100 %, hence follows that extinction coefcient in the aqueous
solution is somewhat higher than that in micellar solution. Average value found
from the similar data for 810 initial nanoparticle concentrations in micellar solu-
tion is as follows: eW
NP 1:15 0:06 10 L=mol cm.
4
Example of the electron micrograph and size distribution is shown in Fig. 4.2.
Average particle size given by the TEM is practically equal to that found for the
appropriate micellar solutions, but the size distribution is often wider. Electron
4.2 Properties of Silver and Gold Nanoparticles in Aqueous Solution 129
dav = 10.5 3.5 nm (83.2%)

16
14
12
% of particles
10
8
6
4
2
0
5 10 15 20
50 nm Particle size, nm
Fig. 4.2 Electron micrograph and size distribution in aqueous solution of silver nanoparticles.
Mean particle size is 10.5 3.5 nm
99.0 100.0
Undersize
, %
0.0 0.0
1.0 10.0 100.0 1000 6000
Diameter, nm
Fig. 4.3 Example of the particle size measurement in aqueous silver nanoparticle solution
obtained by PCS technique on Horiba LB 550
diffraction patterns indicate that here remains the FCC structure, the same as that
detected for these nanoparticles in micellar solution. As in the case of micellar
solutions, in PCS measurements the particles sizes are usually larger than those
determined from the electron micrographs.
Examples of the results of size measurements obtained for silver nanoparticles in
aqueous solutions prepared from the standard micellar solution, fullled on the
devices Horiba LB550 and Zetasizer Nano ZS, are shown, respectively, in Figs. 4.3
and 4.4. Average particle size is larger than that determined by TEM by approxi-
mately 10 nm (Fig. 4.3) and 14 nm (Fig. 4.4). It is possible that, apart from the
reasons mentioned above (see Chap. 2, Sect. 2.4), this increase in the average size
is caused also by the presence of AOT aggregates transferred from micellar to the
aqueous solution along with the nanoparticles. AOT concentration in aqueous
solution varies from several dozens to units of millimoles, depending on the AOT
concentration in micellar solution and some details of the procedure applied in
particular case. The lowest AOT concentration (CAOT = 1 mM) was achieved by
Size Distribution by Intensity

20
Intensity (%)
15
10
0
1 10 100 1000 10 000
Size (d, nm)
Fig. 4.4 Particle size histogram obtained for the aqueous silver nanoparticle solution.
Measurement by PCS technique using Zetasizer Nano ZS. Mean particle size: 24.1 nm
dialysis of the aqueous nanoparticle solution with concentration of this surfactant

not exceeding 40 mM. This AOT concentration (1 mM) is close to the minimal
possible, i.e., to that necessary for the stabilization of nanoparticles. As will be seen
from the results of studies of the biological effects of silver nanoparticles on cells
in vitro (Chap. 9), in such a solution no signs of AOT toxicity are detected.
4.2.2 Gold Nanoparticles
Typical change of the absorption spectrum of Au nanoparticles observed after the

transfer to aqueous phase is shown in Fig. 4.5. The Dmax value of the nanoparticle
absorption band in aqueous solution is smaller than in the initial micellar solution.
In contrast to the situation taking place with Ag nanoparticles, here the 100 %
transfer of the nanoparticles to aqueous phase is not achieved. Probably, this is
connected with the higher AOT concentration in micellar solution, which compli-
cates the procedure by increasing the number of extraction and subsequent phase
separation steps necessary to remove the AOT excess from the aqueous solution of
nanoparticles.
Another possible reason follows from a noticeable decrease in the average particle
size in aqueous solution due to the increase in the portion of very small particles.
Example of particle size distribution after the transfer of nanoparticles to aqueous
phase is shown in Fig. 4.6. As seen from the electron micrograph (Fig. 4.6a), in the
aqueous solution formation of a large number of 23 nm particles is observed,
presumably due to the destruction of the bigger particles present in the initial micellar
solution. As a result, we obtain a considerable shift of the average particle size from
20 nm in micellar solution to approximately 2 nm in aqueous solution (Fig. 4.6b).
According to the Mie theory predictions for metal particles with the size much
smaller than wavelength of the incident light [50, 51, 55], conrmed by the results of
experiments with metal nanoparticles in solutions [51, 292], a decrease in particle
Fig. 4.5 Optical spectra of gold nanoparticles in micellar solution and after their transfer to the
aqueous solution. This gure and Figs. 4.6, 4.7 and 4.8 were reprinted from Advances in
Nanotechnology, vol. 11, Biochemical synthesis, optical properties and sizes of gold
nanoparticles, pp. 119146, copyright 2014, author E.M. Egorova, with permission from Nova
Science Publishers
40
dav = 2.05 1.64 nm
% of particles
30
20
10
0
6 10 22 30
Fig. 4.6 Electron micrograph and size distribution histogram for gold nanoparticles in the
young aqueous solutions (2 weeks after the transfer from micellar solution). Mean size: 2.05
1.64 nm. Transfer to the aqueous phase from the young micellar solution obtained at the
excess of HAuCl4
Fig. 4.7 Diffraction pattern of the Au nanoparticles in aqueous solution
size results in diminution of the optical density of absorption band. It should be noted
that, despite the substantial change of the average size, Au nanoparticles still have
crystal structure similar to that of massive gold samples. This is seen from the
comparison of diffraction patterns obtained for the nanoparticles in aqueous solution
(Fig. 4.7) and for the crystal gold (Chap. 2, Fig. 2.18b).
Results of the measurements of particle sizes by the PCS technique are quali-
tatively similar to those obtained for micellar solutions. In the young samples,
there is a single peak shifted to the larger sizes compared to the TEM data, and in
the old samples, usually two peaks are present, with bigger (more intensive) peak
positioned close to the average size given by TEM, and smaller peak corresponds
with the sizes exceeding 100 nm. The single peak in the young solutions lies
most frequently in the range 3040 nm; the larger peak in the old solutions is
positioned around 10 nm. Example of the histogram for the old solution is shown
in Fig. 4.8. The distribution was measured in six months after the preparation of
aqueous solution by the transfer from micellar solution of the nanoparticles syn-
thesized at the excess of HAuCl4. The corresponding electron micrograph is shown
in Fig. 4.6. As shown in Fig. 4.8, the bigger peak (more than 50 % of the particles)
lies around 8 nm, and the smaller one (approximately 43 % of the particles)
around 150 nm. The latter peak may be of the same origin as in the old micellar
74.0 100.0
Undersize
q, %
0.0 0.0
1.0 10.0 100.0 1000 6000
Diameter, nm
Fig. 4.8 The nanoparticle size distribution measured by PCS technique for the old aqueous
solutions (half a year after the transfer from micellar solution)
solution, with the only difference that in aqueous solution the larger particles are
likely to represent the AOT aggregates of various structures formed in water by this
surfactant [294].
As for the shape of nanoparticles in aqueous solution, it is less distinctly
expressed, smoothed, probably due to the larger thickness of AOT shell sur-
rounding each particle. Besides, for very small particles it is difcult to discern the
shape, actually all of them look like spherical ones. Since here the small particles
prevail, the contribution of spherical particles turns out to be more, and that of
triangular particlesless sizable than that in micellar solution. Such sizes and
distribution are advantageous for the medical applications since there are reasons to
suggest that, rstly, small particles have a higher antimicrobial activity [295], and,
secondly, spherical nanoparticles are supposed to be less toxic compared to the
triangular ones [272]. The AOT concentration of AOT here varies in the same range
(from several units to several dozens of millimoles) as in the aqueous solutions of
silver nanoparticles. By means of dialysis aqueous solutions of Au nanoparticles
have been prepared with AOT concentration of 13 mM; as in the case of Ag
nanoparticles, this solves the problem of AOT toxicity in studies of the biological
activity of nanoparticles in aqueous media.
Gold and silver nanoparticles in water solution prepared by the transfer from
micellar solution are stable for several years under storage in the air at room
temperature. Studies on the biological action of silver nanoparticles are considered
in the Part II of this book.
4.3 Synthesis of Silver and Copper Nanoparticles in Water

Solution with Natural Stabilizers
Apart from the transfer from micellar solution, for the studies of biological activity
of nanoparticles the modications of biochemical synthesis have been developed
allowing the nanoparticle preparation directly in water solution with the use of
natural stabilizers. Owing to these modications, we managed to solve the problem

of the stabilizer toxicity, important for the nanoparticles stabilized by AOT;
therefore, it was possible to avoid the corresponding dialysis procedures required
for the minimization of AOT concentration in aqueous solutions. Here belong the
procedures used in preparation of (1) silver nanoparticles stabilized with starch,
(2) silver nanoparticles stabilized with cyclodextrin and (3) copper nanoparticles
stabilized with starch. Below, we give rst the generalized scheme of nanoparticle
synthesis in aqueous solution comprising the three variants mentioned, and then,
the relevant absorption spectra and particle sizes for each case will be presented.
4.3.1 Silver Nanoparticles Stabilized with Starch
Typical procedure applied for the preparation of silver nanoparticles in water

solution of starch is depicted in Fig. 4.9.
First starch is dissolved in distilled water, then alcoholic solution of quercetin is
added to the Qr concentration required; synthesis of nanoparticles is initiated by the
introduction of silver salt solution. The latter is prepared by the addition of
ammonium hydroxide to silver nitrate water solution as described in Chap. 2. The
process of nanoparticle formation at the silver diammine nitrate concentration of
1 mM is reflected in the spectra changes shown in Fig. 4.10. There are two
absorption bands registered for quercetin in aqueous starch solution 256 and
365 nm, both are characteristic for flavonoids [26, 270]. After the introduction of
Silver nitrate
(AgNO3)
Water-soluble starch
or -cyclodextrin Deionized water
t
Silver nitrate
aqueous solution 27% ammonia
Distilled water aqueous solution
(AgNO3)
Reducing agent Reducing agent Complex silver ammoniac

(Qr) alcohol and stabilizer salt aqueous solution
solution aqueous solution [Ag(NH3)2]NO3
Silver nanoparticles
Fig. 4.9 General scheme used in the synthesis of starch- or cyclodextrin-stabilized silver
nanoparticles
4.3 Synthesis of Silver and Copper Nanoparticles 135
1.4
7 days
2 months
1 day
1.2
2 hours
1.0 1 hour
30 min
Optical density
0.8 15 min
5 min
0.6
0.4
0.2 Qr
0.01 % starch
0.0
250 300 350 400 450 500 550 600
Wavelength, nm
Fig. 4.10 Formation kinetics of silver nanoparticles. Parameters of the solution: Cstarch = 0.01 %,
CQr = 0.1 mM, CAg = 1 mM. The initial spectrum of quercetin in starch aqueous solution is
shown at the bottom
silver salt, the solution color changes from pale yellow to yellow-brown, which
grows more intensive with time. The changes of color are caused by the increase of
nanoparticle concentration in solution. As seen from the gure, instead of quercetin
spectrum a new band appears with the maximum in the range of 410415 nm,
characteristic for silver nanoparticles in aqueous solutions [58, 111, 172, 291].
The process of nanoparticle formation takes no more than one day; the solution
remains stable for no less than one year. The nanoparticle concentration in these
solutions was determined with the use of the extinction coefcient for silver
nanoparticles in water solution produced by the transfer from micellar one
( = 1.15 104 L/mol cm).
Figure 4.11 shows the spectra of nanoparticle solutions on the stationary stage
for different quercetin to silver ions concentration ratios, CQr/CAg, together with the
corresponding dependence of the nanoparticle yield. It is seen that here the picture
observed is similar to that taking place in the synthesis in reverse micelles: The
maximal yield is 100 % and is achieved for the CQr/CAg 0.1, that is, for the
complete reduction of silver ions no less than 1 M of Qr per 10 M of metal salt is
required. Hence follows that the efciency of quercetin as a reducing agent suffered
no changes because of the transfer from reverse micelles to the water solution.
Figure 4.12 presents the electron microphgraph and histogram of particle size
distribution for the water solution of silver nanoparticles stabilized with starch; the
synthesis parameters are the same as those indicated in Fig. 4.11.
100
2.5 2.4 mM 2.8 mM
95
2.2 mM
90
, %
2.0 85
1.6 mM
80
Optical density
1.5 75
1.2 mM
70
1 mM 0.0 0.1 0.2 0.3 0.4 0.5
1.0 0.8 mM CQr /CAg
0.5 0.4 mM
0.2 mM
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 4.11 The effect of quercetin to silver ion concentration ratio on the absorbance band intensity
and yield of the starch-stabilized Ag nanoparticles. CQr = 0.1 mM, Cstarch = 0.05 %. Silver salt
concentrations are shown above the curves. Inset: the dependence of nanoparticle yield on CQr/CAg
ratio
25 dav = 7 5 nm (89.2%)
20
% of particles
15
10
0
5 10 15 20
Fig. 4.12 Electron micrograph and size distribution histogram for the silver nanoparticles
obtained by reduction in potato starch aqueous solution. CQr = 0.1 mM, CAg = 1 mM
As seen from Fig. 4.12, this stabilizer allows to produce the nanoparticles of
approximately spherical shape; the average size, dav 7.0 nm, standard deviation
= 5.0 nm. Here the size distribution is usually wider compared to the
nanoparticle aqueous solutions prepared by the transfer from micellar solutions.
(a) (b)
20 m
Fig. 4.13 a Electron diffraction pattern for the silver nanoparticles obtained by reduction in potato
starch aqueous solution. b A large-scale image of the same nanoparticles. Inset shows the
diffraction pattern of an individual particle
Comparison of the data on the particle size obtained for the different types of
synthesis (for the same synthesis parameters) is presented in the following section.
Analysis of the nanoparticle solutions by means of electron diffraction shows
(Fig. 4.13) that here, as in the synthesis in reverse micelles, the nanoparticles have a
crystalline structure characteristic for the bulk gold crystal.
For the starch-stabilized silver nanoparticles, the toxicity was determined toward
the human cultured cells. The results of these experiments are presented in Chap. 9.
4.3.2 Silver Nanoparticles Stabilized with Cyclodextrin
Synthesis of the nanoparticles in water solution was carried out with one more
natural stabilizera starch-like substance from the group of cyclodextrins, obtained
by enzymatic degradation of one of the starch components (amylase) [296]. Below,
we give a brief description of structure and properties of cyclodextrins, followed by
the spectra and sizes of nanoparticles stabilized with the chosen representative of
this group.
General information on the structure and properties of cyclodextrins.
Cyclodextrins (CDs) are cyclic oligosaccharides consisting of six, seven, or eight -
Dglucopyranose units linked with -1,4-glycosidic bonds into a cone-like
structure [296] (Fig. 4.14). Structure of these substances represents a basket with
internal hydrophobic cavity and outer hydrophilic surface; therefore, the CD can
form inclusion complexes with various organic, inorganic and biological sub-
strates; besides, they are highly soluble in water, stable in the wide pH range, and
are non-toxic to humans. Owing to these properties, they nd application in food
Fig. 4.14 Structural formulae of cyclodextrins
industry, cosmetics, and pharmaceutics. In the last two decades, prospects appeared
of the CD application in modern nanotechnologies, including preparation of metal
and semiconductor nanoparticles coated with organic monolayers. Utilization of
CD offers wide opportunities for the modication of nanoparticle surface applied, in
particular, for the transfer between the aqueous and organic medium. More detailed
information on the production, properties, and applications of cyclodextrins may be
found in [21, 289, 296, 297].
CDs were successfully applied for the synthesis of iron oxide (magnetite) [298,
299], zinc and nickel oxides [300], gold, platinum, and palladium oxides [301, 302]
nanoparticles. Here, the possibility is demonstrated of the formation both of
inclusion complexes between -CD and metal oxide [299] and of the nanoparticle
shell from -CD molecules [288]; the latter mode of synthesis was suggested for the
application in medical diagnostics and therapy. Synthesis of zinc and nickel oxides
nanoparticles was carried out by thermal decomposition of inclusion complexes
between metal salts and -CD. It was shown that application of inclusion com-
plexes exerts a substantial influence on the nanoparticle shape and size distribution
[300]. Gold nanoparticles were obtained by means of HAuCl4 reduction with
sodium citrate or sodium borohydride in the presence of -, -, and -CD. It was
found also that the presence of CD accelerates the nanoparticle formation, and the
increase of concentration leads to the diminution of average particle size: from 12
15 to 46 nm with citrate and from 68 to 24 nm with borohydride; at the same
time, no changes of size distribution were observed. This result was conrmed by
the blueshift of absorption maximum. It was established also that thus-prepared
gold nanoparticles remain stable for three months [301]. Surface modication of Pt
and Pd nanoparticles with -CD allows to produce water solutions of monodisperse
particles with the size of 14.1 2.2 nm (Pt) and 15.6 1.3 nm (Pd) [302]. As point
out the authors of this work, such new materials have prospects of applications in
green chemistry, as they exhibit selective catalytic activity that can be controlled
by molecular recognition, inherent to the surface-attached CD molecules.
In our work, we used -CD kindly supplied by State Research Institute of Starch
Products. The characteristics of this CD form are summarized in Table 4.1.
Table 4.1 Properties of -cyclodextrina

Property -cyclodextrin
Number of glucose residues in the macrocycle 7
Molecular weight, Da 1135
Outer torus diameter, 15.3
Inner diameter of the torus cavity, 6.6
Torus height, 7.9
Volume of the inner cavity 3 262
Solubility in water at 25 C, g/100 mL 1.85
pK (in potentiometry) 12.202
Decomposition point, C 299
a
Compiled from the data given in [289], p. 54
Optical properties and sizes of Ag nanoparticles. Synthesis of silver

nanoparticles followed the same scheme as that used with starch stabilization. At
the beginning, 1 mM -CD was dissolved in the distilled water at 5060 C. Then,
alcoholic Qr solution was added at the same temperature to the concentration of
0.1 mM, the alcohol concentration not exceeding 1 %. Synthesis was initiated by
the addition of [Ag(NH3)2]N03 aqueous solution to the concentration of (0.13)
mM. This provokes a sharp change in colorfrom pale yellow to red-brown; in the
absorption spectrum, the two Qr bands disappear and appears instead the intense
band at 400420 nm, characteristic for Ag nanoparticles in aqueous medium.
Changes in the absorption spectrum with time after the addition of silver salt to the
concentration of 1 mM is shown in Fig. 4.15. As shown in the gure, the
nanoparticle formation is completed on the whole in twenty four hours after the
beginning of synthesis. Optical density remains practically constant in the course of
several months. As in the case of stabilization with starch, the nanoparticle con-
centration was determined with the use of extinction coefcient measured for the
aqueous solutions prepared by the transfer of nanoparticles from micellar solution.
The effect of quercetin to silver ions concentration ratio on the yield of nanopar-
ticles is illustrated in Fig. 4.16, which shows the spectra of nanoparticle solutions at
the stationary stage, measured for the synthesis realized at the xed Qr concen-
tration and various concentrations of silver salt.
As follows from the dependence (CQr/CAg), here a 100 % yield of nanopar-
ticles is also achieved for CQr/CAg > 0.1, that is, when there is no more than 10 mol
of silver salt per 1 mol of Qr. Thus, the reducing ability of quercetin at the synthesis
in aqueous solution does not change when starch (as stabilizer) is replaced by
cyclodextrin. The nanoparticle sizes were determined by TEM and PCS techniques.
An example of electron micrograph and the corresponding histogram is presented in
Fig. 4.17, and electron diffraction pattern is shown in Fig. 4.18.
1.2 7 days
1 day
3 hours
1.0 2 hours
1.5 hours
55 min
0.8
30 min
Optical density
0.6
0.4
0.2 Qr
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 4.15 Changes of absorption spectrum during the formation of -cyclodextrin-stabilized silver
nanoparticles
100
2.6
3 mM 95
2.4
2.5 mM
2.2 90
, %
2.0 85
2 mM
Optical density
1.8 80
1.6
1.5 mM 75
1.4
70
1.2 1 mM
1.0 0.0 0.2 0.4 0.6 0.8 1.0
0.8 CQr/CAg
0.5 mM
0.6
0.4 0.3 mM
0.2 0.1 mM
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 4.16 The effect of silver salt concentration on the nanoparticle formation. Spectra were
obtained in 7 days. CQr = 0.1 mM, CCD = 1 mM. Silver concentrations in solution are indicated
above the curves. Inset: the dependence of nanoparticle yield on CQr/CAg ratio
dmean = 12.5 3.5 nm

25
20
% of particles
15
10
0
100 nm 2 4 6 8 10 12 14 16 18 20 22 24 26
Particle size, nm
Fig. 4.17 TEM micrograph and particle size distribution for CAg = 1 mM, CCD = 1 mM,
CQr = 0.1 mM
Fig. 4.18 Electron

diffraction pattern for the
CD-stabilized silver
nanoparticles in aqueous
solution
20 m
Table 4.2 Sizes of Ag nanoparticles synthesized in different conditions with the equal quercetin
and silver ion concentrations (CQr = 0.1 mM, CAg = 1 mM)
Mode of synthesis Nanoparticle sizes (by TEM) (nm)
In micellar solution, stabilization with AOT 5.5 2.9
In aqueous solution, stabilization with starch 7.0 5
In aqueous solution, stabilization with cyclodextrin 12.5 3.5
The nanoparticles are approximately spherical in shape and have crystal struc-
ture with the same FCC lattice as that observed for the other types of synthesis
described above. However, as seen from the data shown in Table 4.2, for the same
Qr and silver concentrations, in this case the average particle size is bigger than at
the synthesis in reverse micelles and in aqueous solutions with starch stabilization.
4.3.3 Copper Nanoparticles Stabilized with Starch
To study the action of copper nanoparticles on the biological objects and to develop
the applications in medicine, synthesis of these nanoparticles in aqueous solution
was realized with starch as stabilizer. The synthesis was carried out according to the
scheme used in the synthesis of silver nanoparticles (see Fig. 4.9). At rst, the
starch aqueous solution was prepared, and then, the Qr alcoholic solution was
added; after that, synthesis was initiated by the introduction of copper salt
(tetraammine nitrate, Cu(NH3)4S04). Changes in the spectrum of quercetin in the
starch solution after the addition of copper salt are illustrated in Fig. 4.19.
2.0
1.8
1.6
1.4
Qr
1.2
Optical density
1.0
0.8
1 day
2 min
0.6
8 min
2 hours
0.4
[Cu(NH3)4]SO4 8 days
0.2
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 4.19 Changes of absorption spectra in the course of copper nanoparticle formation after the
copper salt introduction to CCu = 2 mM. CQr = 1 mM, Cstarch = 0.1 %. Spectrum of the copper
tetraamine sulfate aqueous solution is also shown
As in the course of synthesis in micellar solution, the process begins with the
complex formation between quercetin and metal ions, with characteristic band at
450 nm, and then this complex dissociates with formation of the Qr oxidation
product (maximum at 310320 nm); at the same time grows the absorption at
> 550 nm. Gradually (in 78 days), the band is formed with a maximum at 550
560 nm, typical for the copper nanoparticles [28, 303306]. By varying the starch,
reducing agent and copper salt concentrations optimal conditions were determined
that provided the maximum yield and stability of nanoparticles. The starch con-
centration was varied in the range 0.010.5 weight %, quercetin to tetraammine
sulfate concentration ratioin the range 1:11:4. It was found that maximum yield
(determined in this case from the optical density in the nanoparticle band maximum)
is achieved at the starch concentration 0.1 %, CQr = 1 mM and CCu = 2 mM, i.e. at
CQr/CCu = 1:2. This nanoparticle band remains practically unchanged at least for a
month.
Figure 4.20 shows the spectrum of copper nanoparticles prepared with these
system parameters. For comparison, the spectrum is shown of the copper
nanoparticles in micellar solution with similar value of optical density in the band
maximum. It is seen that this band is more distinctly expressed in micellar solution,
but its Dmax value is smaller than that measured at the synthesis in aqueous solution.
Taking the extinction coefcient of copper nanoparticles available from literature
3 103 L/mol cm [168], for the micellar and aqueous solution the yield of
nanoparticles will be equal to 24 and 25 %, respectively.
Fig. 4.20 Spectra of the copper nanoparticles in starch and micellar solution
40 dmean = 3.4 0.8 nm

35
30
% of particles
25
20
15
10
5
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Fig. 4.21 Electron micrograph and size distribution histogram for the starch-stabilized copper
nanoparticles. CQr = 1 mM, CCu = 2 mM
Sizes of the copper nanoparticles stabilized with starch were determined by

TEM. Figure 4.21 shows the electron micrograph and size distribution histogram
for the optimal synthesis conditions.
4.3.4 Comparison with the Data Found in Literature
As already mentioned above, in the last decade substantial efforts were made for the
development of the methods of nanoparticle synthesis in aqueous solution with the
use of only natural reducing agents and stabilizers, in order to avoid the toxic effects
dangerous for humans and the environment. In this group of green chemistry
methods, attention is paid mainly to Ag nanoparticles because, rstly, they are the
most required for the biomedical applications, and secondly, in this case the synthesis
is relatively less labor-consuming. Judging from the literature available, the most
widespread stabilizer in this eld is starch. In this connection, we found it useful to
compare our mode of starch-stabilized silver nanoparticle preparation with the other
variants of the synthesis of Ag nanoparticles with the same stabilizer, reported in
literature [61, 306311]. Most often synthesis is carried out in alkaline medium with
glucose [307310] or borohydride [311] as reducing agents; it is possible also to use
starch simultaneously as reducing agent and stabilizer [61], as well as to use starch as
reducing agent in combination with sodium citrate as stabilizer [312].
Table 4.3 shows the parameters of synthesis and nanoparticle characteristics
taken from several articles published in the last 10 years. For comparison, we added
the corresponding data for the nanoparticles produced by our variant of the bio-
chemical synthesis. As follows from the analysis of this table, the nanoparticle
concentration achieved in the cited works does not exceed 0.33 mM; that is, it is at
least three times lower than that obtained by our method. Also, in the other
methods, always the equimolar ratio or the excess of reducing agent is required; by
Table 4.3 Synthesis parameters and characteristics of the aqueous solutions of starch-stabilized Ag nanoparticlesa
Parameter or Ortega-Arroyo Vasileva et al. Raveendran et al. Vigneshwaran et al. [61] Raveendran et al. Biochemical synthesis
characteristic et al. [310] [309] [307] [308] in aqueous solution
Cstarch, % 1.7 0.13 0.17 1 0.2 0.05
T, C 80 30 40 121 100 5060
pH 12 >7 Neutral Neutral 910
CAg, mM 1.67 0.31 1.67 1 0.5 1
CRed b, mM 800c (glucose) 0.94 (glucose) 2.14 (glucose) 1 (starch) 1.25 (glucose) 0.1 (quercetin)
CRed/CAg at 479 3 1.28 1 2.5 0.1
Do max
CAgNP, mM 0.23 0.26 0.329 0.217 0.21 1
max, %d 14 84 19.7 21.7 42 100
Synthesis 3h 30 min 20 h 5 min 1 min 24 hf
duratione
4.3 Synthesis of Silver and Copper Nanoparticles
Additional 4.7 mM Synthesis in inert Synthesis in the autoclave Synthesis in the

procedures NaOH, gas atmosphere at P > 1 atm (15 psi) microwave oven
sonication
Nanoparticle 4.79 2.77 14.4 3.3 5.3 2.6 23 13 5.9 2 75
size, nm 3.69 2.13
Nanoparticle Not reported Not reported Stable Not reported No less than a year
lifetime
a
All the nanoparticles are spherical and have crystalline structure
b
Concentration of the reducing agents
c
This concentration is given in the article. Our calculations based on the procedure description give glucose concentration equal to 2.45 mM
d
Calculated by us from the spectra available for (Ag) = 1.15 104 L/(mol cm)
e
From the data given by the authors; information concerning further spectra changes is not available
f
Time required for the achievement of stationary stage (constant Dmax value in the nanoparticles band)
145
contrast, in the synthesis with quercetin reduction of the large excess of metal ions
can be realized. Hence follows that the use of quercetin allows simultaneously
increasing the nanoparticle concentration in solution and decreasing the con-
sumption of reducing agent, the possibility important from the economical point of
view. In most cases, the yield of nanoparticles is considerably less than 100 %. An
example of such kind taken from one of the recent publications [310] is presented in
Fig. 4.22. Assuming the extinction coefcient for Ag nanoparticles in aqueous
solution to be 1.15 104 L/mol cm, in this case we obtain the maximal nanopar-
ticle concentration equal to 2.70/1.15 104 = 2.35 104 M, or 0.235 mM. So, the
yield of nanoparticles is (0.235/1.67) 100 % 14 %.
The time spent on the synthesis of nanoparticles in the above articles varies from
1 min [308] to 20 h [307]. Apart from the differences in the procedures, such a
large spread in time may be connected with the different criteria used by the authors
for evaluation of the synthesis duration. In our case, the synthesis duration was
estimated as the time necessary to achieve the stationary condition (when the optical
density in the maximum of nanoparticle absorption band remains practically con-
stant); therefore, the time duration turns out to be the greatest (24 h). Since in the
publications considered in the table the spectra changes after the synthesis time
3.0
2.5
2.0
Absobance, u.a.
1.5
1.0
0.5
0.0
300 400 500 600 700 800

Wavelength, nm
Fig. 4.22 Spectra of silver nanoparticles prepared at pH12, 80 C and glucose concentration of
0.8 M. CAgNO3 = 1.67 mM, optical path length 1 cm. Reprinted from Ortega-Arroyo et al. [310].
Copyright 2013, with permission from Elsevier
indicated are not shown, it remains unclear, whether the stationary conditions had
been reached or it will take much more time than indicated by the authors.
In our experiments, synthesis is carried out in the air under mild heating and
without additional influences, except for mixing with conventional magnetic stirrer.
Almost in all the other works additional procedures were required: the removal of
oxygen [307], additional mechanical treatment (ultrasound [309], synthesis under
pressure [61]), or heating in a microwave oven [308]. All this obviously leads to the
additional expenses, which is a disadvantage from the economical point of view.
The average size of the nanoparticles is close to or higher than that produced by
our method; but in our case the size distribution is wider, and this seems to be our
only disadvantage to date.
It should be noted also a signicant difference in the concentration of carbo-
hydrates (starch and glucose) between our version and the other shown in the table.
Glucose is not used in our case, and the starch consumption at the reduction with
quercetin is not larger than 0.05 %, while in all other cases the starch concentration
is not less than 2 times larger. This factor, in our opinion, is essential for mani-
festation of the antimicrobial properties of nanoparticles. Since the aqueous solution
of carbohydrates is known to be a favorable medium for microbial growth, in case
of their large concentration and simultaneously, at a low concentration of silver
nanoparticles, antimicrobial activity (in any case, during the prolonged storage)
may be insufcient even for inhibiting the growth of microorganisms in the
nanoparticle solution, not speaking about their use as an antibacterial agent in
medical products. Therefore, for the sufciently pronounced antimicrobial action it
may be necessary to use here much higher concentrations of nanoparticles than
those required at the stabilization with synthetic surfactant. This argument is con-
rmed by the data reported in [312], where the antimicrobial effect of silver
nanoparticles stabilized with starch was observed at the nanoparticle concentration
of 2 mg/ml, which is by 2 or 3 orders of magnitude higher than the working
concentrations found in studies of the antimicrobial activity of silver nanoparticles
produced with other stabilizers (Chaps. 6 and 7).
In general, it is clear that the choice between synthetic surfactant and natural
stabilizers used here depends on what is considered as more essential for the
achievement of the goal pursued in a study of nanoparticle action on the selected
biological object: elimination of the toxic effect of synthetic surfactant or the ability
to reproducibly obtain smaller nanoparticles with less broad distribution, and also to
achieve a high antimicrobial activity at substantially lower concentrations of
nanoparticles. The signicance of the latter factor will be discussed in Chap. 6.
Chapter 5
Materials Modied with Metal
Nanoparticles
Investigations of the biological activity of metal nanoparticles, as well as

application-oriented studies, have been performed using both nanoparticle solutions
and nanoparticle-modied liquid and solid-state materials. The works with solu-
tions of silver and copper nanoparticles have been performed with due regard for
the possible applications shown in Fig. 5.1. Attention was paid mainly to the
studies of antimicrobial activity and toxicity of the nanoparticles. Also, experiments
were carried out for the determination of catalytic activity of the nanoparticles in
some industrially valuable reactions: copper nanoparticles in dichlorobutene iso-
merization, which is a stage in the rubber synthesis, and silver nanoparticles in
methanol to formaldehyde conversion. Modication of materials with nanoparticles
followed one of the two routes: The nanoparticles were either introduced into a
liquid-phase system or adsorbed onto a solid substrate. The former approach was
employed to incorporate nanoparticles into varnishpaint materials, cosmetics and
detergents, as well as into polymer lms. To modify the paints based on organic
solvent, micellar nanoparticle solutions were employed, whereas for water-based
paints and other water-based systems, aqueous nanoparticle solutions were used.
For the modication of solid-state materials, either micellar or aqueous solutions
were applied, depending on the nature of substrate surface.
This chapter represents a brief summary of the methods used for the modication
of materials intended for biomedical applications. Description of the synthesis
techniques and data on the catalytic activity of the modied materials may be found
elsewhere [9, 28]. The following part of this book contains the results of studies on
the biological activity of materials modied with silver and copper nanoparticles.

DOI 10.1007/978-3-319-30906-4_5
150 5 Materials Modied with Metal Nanoparticles
Formaldehyde Rubber
preparation synthesis Disinfectants
Catalytic Antimicrobial
properties properties
Metal nanoparticles
in solutions
Micellar Aqueous
solutions solutions
Modified materials
Solid Liquid-phase
Fabrics and
nonvowens VPM
Adsorbents Cosmetics
Carbon Silica gel

materials Detergents
Synthetic Biodegradable
Polymers
Fig. 5.1 Possible applications of metal nanoparticles (see text for details)
5.1 Creation of Liquid-Phase Materials with Metal Nanoparticles 151
5.1 Creation of Liquid-Phase Materials with Metal

Nanoparticles
Here, we shall dwell upon the two directions of those related to the liquid-phase
materials in Fig. 5.1, namely on varnishpaint materials (VPM) and biodegradable
polymer, where the most valuable results had been obtained.
Varnishpaint materials with metal nanoparticles. We would remind that, in the
last decades, in the paint production, considerable attention is paid to the bioactive
coatings. Such coatings are formed from VPMs, impregnating compounds, and
polymers by means of the addition of various biocides as active components.
Usually, the biocides are added in order to obtain the polymer compositions with
special properties useful for the protection of wood against molds or bugs, pro-
tection of coatings against fungi, conservation of water-based compositions, and for
the other purposes. Despite the widespread application and popularity, such
bioactive compositions had serious disadvantages that stimulated new researches in
this eld.
Firstly, the biocides applied were characterized by a high specicity, that is, they
were active against only one denite group of microorganisms. Hence, their pro-
tective action was far from being complete enough to solve the problem and that
was undesirable from the practical point of view. Besides, they were directed, in
principal, against microbes dangerous for the paint compositions and painted
materials, but could not work against the pathogens dangerous for humans.
Meanwhile, the level of environmental pollution by bacteria or viruses was con-
stantly enhanced, and therefore, the importance was realized of the creation of
paints and coatings active against a broad spectrum of pathogen microorganisms.
At the same time, it was clear that some of the known disinfectants (e.g., containing
chlorine) cannot be used for these purposes because their chemical properties do not
allow introducing them into paints and coatings.
Secondly, there is a serious problem of environmental pollution. From this point
of view, a large number of the currently used bioactive coatings cannot be con-
sidered as satisfactory because, apart from the contamination by the solvents pre-
sent in these compositions, they are not neutral themselves and often are toxic
toward the surrounding media. List of the chemical biocides commonly used as
components of paints can be found in advertising materials of VPM manufacturers
(e.g., [313]).
A little more than 10 year ago, poly(hexamethylene) guanidine (PHMG) and its
derivatives were proposed as a biocide additives to paints with a broad spectrum of
action. This chemical biocide has demonstrated a high degree of bacteria and
viruses inactivation on the painted surfaces, as well as considerable sporicidal and
fungicidal activity. This allowed using such paints for interior decoration of various
premises crowded with people and thus having a high level of microbial contam-
ination. The application of this biocide had been started in manufacturing of paints,
and such products are still offered on the market (e.g., [314]). However, for the
paints containing both PHMG and the other chemical biocides, the problem remains
of their toxicity and pollution of the environment.
It was assumed that these disadvantages could be eliminated with no loss in the
spectrum of antimicrobial activity by using a biocide of different nature, such as
metal nanoparticles. Therefore, the technology has been elaborated allowing the
production of paints containing silver nanoparticles. Nanoparticle solutions were
introduced into paints as micellar or aqueous solutions; then, these paints were
tested on physical and mechanical properties, as well as on the antimicrobial
activity. For this purpose, water-based paints or those with organic solvents were
used, commonly applied for construction works in residential areas, medical, sports
and childrens institutions, catering enterprises, and other institutions where it is
important to decrease the concentration of bacteria dangerous for humans.
The antimicrobial activity tests of paints had shown that nanoparticle additives
are efcient against the broad spectrum of microorganisms at a very low metal
concentrationhigh bactericidal activity was observed for the silver concentration
in the range 15 104 %. Also, the coating material with biocidal properties was
proposed, containing both PHMG salts and silver nanoparticles [315], which
demonstrated the high bactericidal, virucidal, and fungicidal activity at a substan-
tially lower PHMG concentration compared to the same paint without nanoparti-
cles. This allowed to signicantly decrease the paint toxicity without a loss of its
biocidal activity.
Copper nanoparticles were used for the modication of paints applied for the
ship bulge coating needed to prevent its fouling by various marine and river
organisms (algae, crustaceans, fungi, and other inhabitants of seas and rivers) [316].
This biofouling is a source of various problems, from the reduced fuel efciency to
the loss of structure stability. The common practice accepted for the prevention of
the growth of marine organisms is the surface coating by special paints. As with the
paints used in construction works, here the biocide properties are provided by the
introduction of highly toxic chemical substances. In view of the strengthening of
the requirements to the application of eco-friendly varnishpaint coatings, espe-
cially for the bioprotection of ships and constructions working in contact with water
(e.g., for bridges), a search for the new methods of manufacturing of the low-toxic
biocide coatings becomes very important.
At present, in the VPM coatings meant for the coloring of underwater part of
ships, as a biocide cuprous oxide is used, introduced into the VPM in very large
amounts (5060 % of the overall composition). This leads to the deterioration of
mechanical and protective properties of the coatings. Besides, cuprous oxide has a
low efciency against some types of sea and river organisms, and hence, it should
be used in combination with other highly toxic biocides. Experiments with the
paints containing the additions of copper nanoparticles demonstrated that
antifouling effect of nanoparticle additives manifests itself at the copper concen-
tration of 23 104 %. Thus, replacement of cuprous oxide with the nanoparticles
allows decreasing the copper concentration by many orders with preservation of the
paint biological activity.
5.1 Creation of Liquid-Phase Materials with Metal Nanoparticles 153
The results of tests on the biocidal activity obtained for the paints containing
nanoparticles are described in Chap. 7.
About the creation of lms from biodegradable polymer with silver nanoparti-
cles. It is well known that creation and development of applications of
biodegradable polymers, which undergo destruction under the influence of natural
environmental (microbiological or biochemical) processes, become now extremely
important both for medicine and for the solution of environmental pollution
problems. In medicine, the materials are used, subjected to degradation in a human
organism due to the hydrolysis by various enzymes. For example, constantly
widens the application of biodegradable materials for surgical stitches; good pro-
spects have such polymers for the creation of nanocontainers for the targeted
delivery of drugs and implants, which can be gradually replaced with the natural
tissue of an organism [317320].
Among the most promising biodegradable polymers for medical applications, an
important role play chitin and chitosannatural polysaccharides produced from the
chitin shells of crustaceans and shellshes. They possess a low toxicity, biocom-
patibility, lm-forming capacity, and curative properties; in particular, thanks to
their wound-healing ability they are used already in clinical practice [321, 324].
Polysaccharide structure of these polymers provides biocompatibility, and the
presence of reactive functional groups allows the strengthening of biological
activity by means of the synthesis of various chemically modied derivatives [321,
325]. Recently, the tendency appeared to the creation and research of the properties
of chitosan compositions with metal ions and nanoparticles, mainly silver and
copper [326, 327]. These works are aimed at the fabrication of biodegradable
materials, where the useful, mainly bactericidal, properties of this natural polymer
are combined with antimicrobial properties of nanoparticles and thus a strong
therapeutic effect can be achieved. This possibility is indicated, for example, by the
data reported in [328]; it was shown here that modication of chitin bers with the
complex of polyvinyl pyrrolidone and nely dispersed silver (product Poviargol)
leads to a signicant increase of antimicrobial activity of the resulting material.
To create polymer compositions with silver nanoparticles suitable for the pro-
duction of bactericidal lms, we studied the interaction of the aqueous solutions of
AOT-stabilized silver nanoparticles with chitin derivative (carboxymethyl chitin,
CMC). The work was fullled in cooperation with A.V. Topchiev Institute of
Petrochemical Synthesis and Kosygin Moscow State Textile University. The choice
of CMC was conditioned by its high solubility in water at neutral pH, while
chitosan is soluble mainly in acetic acid [321]. At the same time, Ag nanoparticles
in AOT shell, bearing anionic sulfo groups, are stable in neutral and alkaline
medium due to the electrostatic repulsion of AOT polar headgropus, while in acidic
medium, they tend to aggregate and precipitate. We studied the CMC mixtures with
Ag nanoparticles at different ratios of components, by spectrophotometry and PCS
techniques; a detailed description of the research and lm preparation procedures is
given in [329]. As a result, the polymer lms have been produced, with low silver
CHIT10 0.03% AG
0 1 2 3 4 5 6 7 8 9 cm
Fig. 5.2 Photograph of CMC lms without (left) and with (right) Ag nanoparticles
nanoparticles concentration (0.030.06 mass%); the lms acquired a weak yellow

brown color testifying to the stability of nanoparticles. Example of such lm is
shown in Fig. 5.2.
The lms prepared from CMC-Ag nanoparticles mixtures demonstrated a high
antimicrobial activity against two kinds of pathogenic bacteria, this activity being
considerably stronger than that observed for the control lms from CMC without
the nanoparticles. Method used for the testing of antibacterial activity and the
results obtained are presented in Chap. 7.
5.2 Solid Materials with Ag and Cu Nanoparticles
Deposition of nanoparticles on the surface of solid materials was performed by

means of adsorption from micellar or water solution; the purpose was to produce
various modied materials with special properties for the application in medicine
and technics. In these studies, we used the micellar and aqueous solutions of
AOT-stabilized silver nanoparticles, as well as micellar solutions of copper
nanoparticles. For each type of material, the conditions were selected that provided
the high enough adsorption rate and surface density of the nanoparticles and also
ensured the stability of the coverage under the supposed operating conditions. Type
of solution used for the deposition of nanoparticles depends on the properties of the
adsorbent surface. Here, the adsorption process is governed mainly by the regu-
larities known from the theory of adsorption: polar additives from the nonpolar
medium are actively adsorbed on hydrophilic surfaces, while nonpolar additives
from the polar medium are actively adsorbed on hydrophobic surfaces [330]. From
this point of view, a micellar solution with nanoparticles is the nonpolar medium,
5.2 Solid Materials with Ag and Cu Nanoparticles 155
where nanoparticles in reverse micelles represent a polar component; such solutions

were used for the deposition on glass, metals, textiles, metal oxide powders, silica
gel, and other materials with polar groups on the surface. Aqueous solution con-
taining nanoparticles is the polar medium where the nanoparticles in AOT micelles
can be regarded as nonpolar component. From such solutions, the nanoparticles
were well adsorbed on activated carbon, carbon clothes, as well on the other
materials with the surface which possessed predominantly hydrophobic properties.
Control of the adsorption rate was performed by spectrophotometric measure-
ments of the changes in intensity of the nanoparticles absorption band in solution
used for the deposition on the particular material. The amount of adsorbed
nanoparticles was estimated as amount of the deposited metal per unit weight of the
material (usually in mg/g) or (if the specic surface area for a sample was known)
as amount of the metal per unit surface area (mg/cm2). If the nanoparticles adsorbed
from the solution with narrow particle size distribution, the density of coverage was
also determined, as the number of nanoparticles per unit surface area of the sample.
Works on the modication of solid materials were performed with silver and
copper nanoparticles stabilized by AOT. Until recently, the most required were the
materials, modied with silver nanoparticles, because they had good prospects of
application in manufacturing of different products for medicine and other areas,
where their antimicrobial properties can be applied. Besides, silver nanoparticles
deposited on solid carriers are of great interest as efcient catalysts for the chemical
industry. With the use of Ag nanoparticles the following modied materials were
obtained: (1) activated carbon and polyamide membranes for the ltering devices
used in water purication, (2) silica gel for the removal of microbial contaminants
from hydrocarbons and other nonpolar liquids, and also for the possible use in
production of formaldehyde from methanol, (3) different types of textiles for the
manufacturing of clothes and other products with biocidal properties, (4) aluminium
and titanium oxide powders for the application in paints as antimicrobial compo-
nents, and some other materials with different densities of the coverage, (5) wood
samples for elucidation of the possibility to use the nanoparticle solutions as
antiseptics for the treatment of furniture and other wood products. Also silica gel
and alumina oxide beads with copper nanoparticles were prepared for the study of
the catalytic activity; a high catalytic activity of copper nanoparticles deposited on
alumina oxide was detected in the isomerization of dichlorobutenes [9].
Deposition of silver nanoparticles gives rise to the characteristic red-brown or
yellow coloration, its intensity depends on the density of coverage. Deposition of
the copper nanoparticles causes the appearance of slightly bluish or brown color.
The photographs of different materials with deposited silver or copper
nanoparticles are shown in Figs. 5.3, 5.4, 5.5, and 5.6.
To choose the optimal conditions providing the rate of adsorption and coverage
density desired, as well as to achieve a high degree of nanoparticle extraction from
solution, the studies were performed allowing to determine the influence on these
parameters of different factors, such as AOT and nanoparticles concentration in
micellar or aqueous solution, special properties of the adsorbent surface, its origin
(for example, for activated carbonthe method of its manufacturing), and others.
(a) (b) (c)
Fig. 5.3 Silica gel without nanoparticles (a) and with silver (b) and copper (c) nanoparticles
Fig. 5.4 Polyamide

membranes with silver
nanoparticles
The practical signicance of such studies can be illustrated by the two examples:
production of silica gel with the nanoparticles deposited from micellar solution and
activated carbon with silver nanoparticles deposited from aqueous solution.
As found in the works aimed at the creation of silica gel modied by
nanoparticles, adsorption of the particles depends on the water content in silica gel,
as well as from the hydration extent and concentration of both AOT and
nanoparticles in micellar solution. In particular, it turned out that for the efcient
adsorption of nanoparticles, it was necessary to wet the silica gel surface. As found
in the preliminary experiments, the rate and maximal quantity of nanoparticles
adsorption depend on the water content in silica gel; it was established that the
maximal adsorption rate is achieved at the water content no less than 15 mass%,
Fig. 5.5 Fabrics with deposited silver nanoparticles: cotton (left) and velvet (right). For
comparison, samples without nanoparticles are also shown: for cottonin the center, for velvet
the last on the left
Fig. 5.6 Aluminum oxide powder (left) and wood sample (right). For comparison, samples
without nanoparticles are shown on the right in each photograph
while the water content in original samples did not exceed 7 mass%. The samples
studied were saturated with water vapor by exposure in humid atmosphere at ele-
vated temperature. After wetting of silica gel to the water content of 1520 %,
incubation of the adsorbent for 6 h with the nanoparticle solution allowed to
achieve the adsorption close to the maximal possible value (approximately 90 % of
nanoparticles were extracted from solution). It was found also that the less is AOT
concentration, the higher is the rate of nanoparticles adsorption. In the AOT con-
centration range studied (822 mM), the optimal was its lowest concentration equal
to 8 mM.
Figure 5.7 shows the changes of nanoparticle concentration in the process of
adsorption from standard micellar solution on the untreated surface, as well as from
the solution with optimized AOT and nanoparticles concentrations on the pretreated
(wetted) silica gel surface. In the rst case, less than 50 % of nanoparticles are
deposited in 2 weeks, whereas in the second case, practically full extraction of
(a) 1.3
Original 3 days
1.2
1.1 7 days
D0 D
1.0
10 days
0.9
Optical density
0.8
14 days
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
300 350 400 450 500 550 600
Wavelength, nm
(b)
0.8 Original
0.7
D0 D
0.6
Optical density
0.5
2 hours
0.4
0.3
0.2 4 hours
0.1 6 hours
0.0
300 350 400 450 500 550 600
Wavelength, nm
Fig. 5.7 Concentration change of silver nanoparticles in micellar solution in the course of
adsorption from the standard solution onto silica gel with untreated surface (top) and from the
solution with optimized composition onto the pretreated surface (bottom)
nanoparticles from the solution is achieved in 6 h. Similar surface pretreatment was

performed also for the deposition of copper nanoparticles. It is known that for the
application of modied silica gel, as well as of the other modied adsorbents,
density of a surface coverage with nanoparticles is considered to be the important
parameter [11, 331333]; however, it is difcult to nd the specic surface area
necessary for the determination of this parameter, so as a rule, it is unknown. In our
studies of the nanoparticles adsorption on silica gel, we managed to nd the specic
surface area from the results of the measurements of AOT adsorption isotherm from
micellar solution. The value obtained was 330 m2/g, that is, in the range 300
500 m2/g usually determined for the adsorbents of this kind [334]. Hence, we could
nd that the density of coverage with silver nanoparticles in our modied silica gel
samples was equal to (24) 1017 particles/m2.
A few words should be added about the possible mechanism of nanoparticle
adsorption from micellar solution on the silica gel. Analysis of the whole pool of
results allows to suppose that xation of a nanoparticle on the silica gel surface
occurs owing to the formation of hydrogen bonds between the binded water layer
on the adsorbent and that near the nanoparticle surface. As a result, the particle
becomes rmly bound to the surface of silica gel and cannot be washed during the
further incubation with isooctane as could be expected in case if the layer of soluble
in isooctane AOT molecules existed between the nanoparticle and the adsorbent
surface. Verication of this supposition will be the subject of future researches.
Important also were the preliminary studies undertaken for the fabrication of
activated carbon modied with silver nanoparticles. Figure 5.8 shows the time
dependence of the portion of adsorbed nanoparticles for the carbons of different
origin. As seen from the gure, the adsorption capacity is essentially different for
the samples studied; this can be connected both with the difference in specic
surfaces and peculiarities of surface active groups. It was found that in the increase
of the extent of nanoparticles extraction from solution, essential role belongs to the
right choice of the solution volume to adsorbent weight ratio as well as to AOT and
nanoparticles concentrations. Besides, from the point of view of practical appli-
cation, it is important to know size of the granules and their resistance to
mechanical loads, for example, against pressure caused by a water flow.
Fig. 5.8 The time 100

dependence of the percentage
of adsorbed nanoparticles for 80
different carbons at solution
D/D , %
volume to carbon mass ratio 60

5:1 (mL/g): 1-coconut
charcoal; 2-birch charcoal; 40
AG-3 charcoal
20
0
0 10 20 30 40 5
time, h
1 2 3
Fig. 5.9 Ag nanoparticle 1.0 Original (D0)
D0 D
adsorption onto the 5 min
15 min
polyamide membrane. The 20 min
optical density changes in the 0.8
nanoparticles band maximum
Optical density
take place within 20 min after
0.6
the beginning of experiment;
afterwards no optical density
changes are observed 0.4
0.2
0.0
350 400 450 500
Wavelength, nm
Proceeding from the whole complex of results obtained in our studies, the
charcoal AG-3 was recognized as optimal for the further usage. As will be shown
below (Chap. 7), such a charcoal modied by silver nanoparticles exhibits a high
antimicrobial activity.
Almost all materials with the deposited nanoparticles were tested for the bac-
tericidal activity; the procedure applied depended on the supposed practical
application. For example, the activated charcoal and polyamide membranes meant
for water purication from microbial contaminations were tested according to the
procedures developed for the appropriate ltering devices. Titanium and aluminum
oxide powders were tested as components of paints. Different kinds of textiles with
antimicrobial properties, intended for the manufacturing of clothes or other prod-
ucts, were tested in the conditions allowing their contact with surface of a medium
containing bacteria cultures. Descriptions of the relevant experiments and the
corresponding results are presented in Chap. 7.
An interesting peculiarity was revealed in our tests of the materials modied
with nanoparticlesthese materials manifested the essential antimicrobial activity
even in those cases when, according to our data, the nanoparticle adsorption was
small, mainly because of the small specic surface value of the material under
study. For instance, adsorption of silver nanoparticles on the polyamide membrane
used in the ltering equipment for water purication, made up not more than 15 %
of the initial nanoparticle concentration in aqueous solution (Fig. 5.9). But it was
quite enough for the strong antimicrobial effect. In experiments with membranes
manufactured by different technologies, it was found also that the nanoparticle
adsorption depends on the membrane surface charge caused by the dissociation of
ionogenic groups: adsorption was observed on the membranes with cationic groups
(bearing positive charge), while on the negatively charged membranes with anionic
groups, the adsorption was practically absent. This can be regarded as manifestation
of electrostatic interactions between the membrane surface and micelles containing
the nanoparticles, since the micellar shell is negatively charged due to the disso-
ciation of anionic groups present in AOT molecules.
Part II
Biological Effects of Metal Nanoparticles
Chapter 6
The Effect of Metal Nanoparticles
on Biological Objects (Analysis
of the Literature)
6.1 General Information
cIt is beyond doubt today that studies on the biological activity of metal nanopar-
ticles are topical and necessary, rst, for improving the remedies, diagnostics, and
therapies currently available and for designing the new ones, that is, for nanome-
dicine; second, for understanding the causes of diseases provoked by the penetration
of nanoparticles into the human body (nanopathologies) and for their treatment; and
third, for establishing the well-grounded allowable ranges for concentrations and
sizes of nanoparticles contained in water, air, and various materials humans come in
contact with (for nanosafety). Currently known routes of metal nanoparticle pene-
tration into the human body are shown in Fig. 6.1. As shown, metal nanoparticles
occur both in the environment and in the consumer goods, including food, and are
found in medical products, such as drugs, dietary supplements, and certain reagents
used in modern diagnostic tests. The fact that an important role has been realized for
the effects nanoparticles exert on the human body and the environment is reflected,
in particular, in that nanotoxicology has been isolated as a new toxicology eld in
the past decade to focus on the problems of nanosafety and, partly, nanopathology
[1, 335]. Main nanotoxicology research areas as formulated in the review [1] are
shown in Fig. 6.2. The discipline considers the routes whereby nanoparticles pen-
etrate into the human body, their distribution in the body, the influences of
physicochemical characteristics of nanoparticles, the molecular mechanisms of
nanoparticle effects, and other problems. The journal Nanotoxicology has been
published since 2006 to discuss, apart from the above issues, the techniques
employed in nanoparticles toxicology researches on various objects, the possibility
of using computer models to predict the toxic effects, and the methods to evaluate
the risk of using nanoparticles and nanomaterials in medicine and other elds.

DOI 10.1007/978-3-319-30906-4_6
164 6 The Effect of Metal Nanoparticles on Biological
Natural background Products of industries

and military actions
Air
Food products Remedies
Reagents for
Medicine diagnostics
Industrial goods Food supplements
Water
Soil Water supply

systems
Fig. 6.1 Ways of metal nanoparticle penetration into a human organism
The problems of ensuring nanosafety have also received particular attention in

Russia. Guidelines for evaluating the safety of nanomaterials have been developed
via joined efforts of several relevant research institutes [336]. This comprehensive
document describes the principles of designing and conducting studies in the eld,
necessary equipment, requirements imposed on test systems, techniques to inves-
tigate the basic properties of nanomaterials, methods of toxicology testing in ani-
mals, and other essential information in order to ensure expert evaluation of the
conditions for the safe use of nanomaterials. The document has been approved and
put into effect by the Federal Service for the Oversight of Consumer Protection and
Welfare (Order no. 280 dated October 12, 2007).
Ecological aspects of nanotoxicology are also recognized as important. Relevant
studies focus on the environmental distribution of metal (primarily silver)
nanoparticles, their concentrations and forms occurring in water and soil, the ways
they interact with organisms living in reservoirs and rivers, the response of the
organisms to nanoparticles entering the body, etc. [337, 338].
6.1 General Information 165
Size/Shape
Surface area Surface chemistry
Physicochemical
Government determinants Respiratory tract
Industry Regulatory issue Routes of

Skin
exposure
Academy
Gastrointestinal Tract
Nanotoxicology
Mutagenesis
Clearance
Genotoxicity Biodistribution
Opsonization
Chromosomal
Aberrations Molecular
determinants
Oxidative
Inflammation stress
Fig. 6.2 Main components of nanotoxicological studies. Reprinted from Ref. [1]. Copyright
2012, with the permission from Elsevier
Studies are performed mainly with Au, Ag, and Cu nanoparticles and three types
of objects: microorganisms, cultured cells, and multicellular organisms, including
sh and mammals (rats and mice) (Fig. 6.3).
There are also few data on the results obtained on algae and green plants [338]
and the effects of nanoparticles and silver nanoparticle-containing materials
observed in humans [339341]. In the latter case, silver-containing drugs taken
orally as aqueous solutions or applied as lotions may lead to argyria, which is a
phenomenon known in medicine and is characterized by grayish blue or brown
pigmentation of the skin, hair, nails, and mucous membranes caused by the irre-
versible accumulation of metal silver. In the Russian literature, Lopatina et al. [342]
have described in detail the clinical signs of argyria and the toxic effects of excess
silver in the human body. The article emphasized that self-medication with
advertised silver solutions is dangerous and that the processes underlying their
pathological effects are poorly understood, so the necessity exists to investigate the
mechanisms of colloid silver penetration into the skin and other organs.
A vast literature is available for biological activity of metal nanoparticles in all of
the three types of biological objects; an overview of the results is possible to elicit
from several monographs and reviews [18, 80, 289, 335, 337, 343347]; and
detailed information, from many original articles cited therein. Another review of
Bacteria Mammals Fishes
Microorganisms Vertebrates
Main types
Viruses of Animals
bioobjects
Invertebrates
Cultured cells
Normal Tumor
Fig. 6.3 Main types of objects used in studies of the biological effects of metal nanoparticles
the relevant literature would certainly be useful because intense development of

research and new data accumulation always provide an opportunity to gain deeper
insight, to re-evaluate the importance of particular ndings and to pose new
problems. Yet such a review could be published rather as separate edition and is
apparently beyond the scope of this book. We have the other objective here.
As already noted in Part I of this book while discussing the requirements for a
nanoparticle solution to be suitable for testing for biological activity (Chap. 1,
Sect. 1.7), two basic ways can be followed, in principle, to study how various
parameters of nanoparticles influence their effect on a given biological object,
namely either different methods should be used to obtain nanoparticles, or one
method should be modied conformably to the testing of different nanoparticle
parameters. Accordingly, two approaches, or research strategies, are possible
horizontal and vertical [348]. A horizontal strategy means that one object or objects
from the same level of biological organization (microorganisms, plant or animal
cells, tissues, or multicellular organisms of a certain type) are used to study par-
ticular metal nanoparticles that vary in size and shape and are obtained by different
methods. A vertical strategy means that studies are performed with different objects
or at different organization levels and involve nanoparticles that are the same in size
and shape and are obtained by one and the same method. The horizontal strategy
makes it possible to detect the effects that are due to the difference in the parameters
of nanoparticles. The vertical strategy allows us to register the effects conditioned
by the variation in specic properties of biological objects. Judging from the aims
pursued in the relevant publications, up to now the horizontal strategy has been
6.1 General Information 167
followed in the vast majority of studies in the eld, i.e., the nanoparticles which
were obtained by one or different methods and had the same or different parameters
were applied for examination of the objects staying on the same organization level.
The objective of our analysis in this review was to try to understand, rst, how
efcient is this research strategy in elucidation the effects of basic parameters (size,
shape, surface charge, and the stabilizing shell composition) of nanoparticles on
their biological activity and, second, whether the strategy makes it possible to
select, on reasonable grounds, the nanoparticles best suitable for particular appli-
cations in medicine and bionanotechnology. Chapter 6 of this part of the book
describes the results of our analysis. Further chapters consider the ndings that have
been made with nanoparticles obtained by biochemical synthesis and tested for
effect on the objects of various organization levels. Conclusions drawn from the
experimental data complete this part.
In our analysis, we followed the plan outlined below. First, relevant studies were
divided into three groups according to the object types mentioned above (mi-
croorganisms, cells, or multicellular organisms). For each group, we have chosen
the criteria characterizing the strength of an effect nanoparticles exert on the vital
activity of an object. Note here that the choice of the method to quantify the
biological action of nanoparticles when evaluating the doseresponse relationships
has been a matter of discussion [337]. Conformably to nanoparticles, three variants
mass, number, and surface area basedwere considered by these authors
for expressing the nanoparticle dose, which is measured, respectively, as mass,
number, and surface area of nanoparticles in a given volume, mass, or object. The
doseeffect curves obtained for TiO2 nanoparticles in experiments with rats and
mice described in [337] showed that the surface area variant is the most con-
venient, while the mass variant is the least acceptable because in the latter particle
size variation is disregarded. It is clear, however, that the difference between the
two variants becomes signicant when the dose is measured in terms of particle
mass without indicating the particle size.
Judging from the literature available, in the majority of studies focusing on the
biological effects of metal nanoparticles, mass concentrations of nanoparticles of a
known size have been indicated as the basic criterion. Accordingly, for the con-
venience of data comparisons, we selected the nanoparticle mass as a measure of
dose, bearing in mind the possibility of transfer to the number or surface area
variant when necessary. Mass concentrations corresponding to (1) the toxicity
threshold (at which pathological signs become detectable) and (2) the lethal effect
were used as criteria characterizing the biological effect. We have previously
demonstrated their suitability in studies of the biological effects of silver
nanoparticles on the objects of various organization levels [348].
Then, the results were generally reviewed in each group of studies. Only one of
the three groups, studies on microorganisms, proved adequate for our aimeval-
uation of the influence of nanoparticle characteristics on the above-mentioned
criteria. Studies of the group were the most numerous, involved the broadest range
of objects, and contained sufcient data on nanoparticle characteristics and their

effects on biological activity. Therefore, we restricted this review of published data
to the analysis of nanoparticle effects on microorganisms; the main problems of
methodology issuing from this analysis are also indicated. Then, we describe in
brief the mechanisms of the antimicrobial effect of nanoparticles that have been
discussed in the literature, noting also the gaps that arose from the main problems
mentioned. A summary of our review is given in the last section of this chapter.
6.2 Metal Nanoparticles Effects Observed

on Microorganisms
Although the effects metal ions exert on cells and higher organisms have been
studied for a long time (e.g., see [80, 236]), data on the biological activity of metal
nanoparticles have been accumulated mostly in the past 1012 years. Up to the late
1990s, the majority of publications focused on metal biosorption and biomineral-
ization by bacterial cells [80, 349], which are processes that underlie extraction of
precious (Au and Ag) or certain technical (Cu, Fe, Ni, etc.) metals from soil waters
or industrial wastes. Some data on the effect of metal ions and colloidal metals on
living organisms were obtained with unicellular algae [237]. The results were used
to consider various events that accompany nanoparticle interactions with the cell
membrane, meaning mostly that of a bacterial cell. The interaction was thought to
include processes of at least three types: (1) colloidal metal particles adhesion on
the cell surface owing to electrostatic chemisorption forces; (2) the cell response to
adhesion of a metal particle so that the particle dissolves as its atoms are oxidized,
and new metal particles of a larger size subsequently form via association of atoms
and ions; and (3) microbial cells form aggregates at sites of nanoparticle adhesion.
In addition, it was found that interactions of metal nanoparticles substantially differ
between living and dead cells and that the cell surface charge, which may vary
among cell membrane regions, plays an important role in the interactions [349].
A role of the surface charge was similarly noted in studies of the interactions of
Ag+, Cu2+, and Au3+ ions with unicellular algae [237].
A noticeable jump in the number of publications devoted to the biological
effects of nanoparticles started from the early 2000, when it became necessary to
ensure the safe use of nanotechnology products (see above). In the majority of
studies, the interaction of nanoparticles with microorganisms, mostly bacteria, was
investigated. The results obtained in the eld have been summarized in several
recent reviews [7, 343, 346, 347, 350]. Most studies were aimed at designing
nanoparticle-based remedies to treat infectious diseases, infections as complications
of wounds, burns, or surgery and other cases requiring the use of antiseptics.
Relatively few studies considered the ecological aspects of the effects of
nanoparticles, including their interactions with microorganisms living in soil and
natural water reservoirs and rivers [1, 338].
6.2 Metal Nanoparticles Effects Observed on Microorganisms 169
As far as can be seen from the literature under consideration, Ag nanoparticles

were used in the vast majority of studies. There are also data on Cu, Au, and metal
oxide (CuO, ZnO, Al2O3, and Fe3O4) nanoparticles. Studies were performed with
nanoparticles obtained via reduction in an aqueous solution with conventional
chemical agents (N2H4, NaBH4, H2, ascorbic acid and gallic acid), UV irradiation,
biological reducers (plant and fungal extracts), as well as with commercial
nanoparticles preparations purchased from various companies. The particle size
varied within a broad range from 3 to 100 nm or a greater size; polydisperse
samples with a wide particle size distribution were used most often, as solutions or
powders. The effect of nanoparticles was studied mostly on widespread pathogens,
including bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa, etc.), viruses (HIV, hepatitis viruses, etc.), and fungi (Candida,
Aspergillus, etc.). Experiments with bacteria employed conventional tests for sen-
sitivity to antibiotics and other chemical agents, both on agar media (estimation of
the growth inhibition zone around a disk or a well with an agent or the decrease in
colony number on a nanoparticle-containing medium) and in liquid cultures (de-
termination of the lag time and growth rate in the presence of various concentra-
tions of nanoparticles).
The following criteria are used to quantify and to compare the efciency of
nanoparticles.
Minimal inhibitory concentration (MIC). Denitions of the criterion differ
among the studies. In the works [351, 352], the MIC was understood as the minimal
concentration of a material that inhibits the growth of a microorganism as compared
to a control culture. In the other studies [295, 353, 354], the MIC was dened as the
minimal nanoparticle concentration that completely inhibits bacterial growth.
The MIC dened in such a way was used as a state standard with reference to the
National Committee for Clinical Laboratory Standards (NCCLS) in [353].
However, the period of time was not specied for cell incubation with an agent,
although it is of importance for estimating nanoparticle activity. Because the
incubation period greatly varied among the studies, additional problems arise in
comparing the MIC values reported.
Minimal bactericidal concentration (MBC) is the lowest concentration at which
an agent kills 99.9 % of bacteria [351]. Note that for certain authors [355] the
concentration that causes virtually complete death of bacteria (no growth in 7 days)
is dened as MIC (!).
Bacterial susceptibility constant is determined as Z = ln(N/N0)C, where N and
N0 are the CFU counts on agar plates (dishes) with and without nanoparticles,
respectively, and C is the nanoparticle concentration (g/mL) [356].
Bacteriostatic and bactericidal concentrations are, respectively, the concentra-
tions at which growth inhibition and complete death of bacteria are observed
[357, 358].
The MIC is most commonly used [295, 351, 353355, etc.]. However, as noted
above, what is meant by the term MIC differs among the studies by different
50 (a) (b)
Number of E. coli colonies, %
40
(c) (d)
30
20
10
0
10 20 30 40 50 60
Concentration of silver nanoparticles, g/ml
Fig. 6.4 Number of E. coli colonies at different concentrations of Ag nanoparticles expressed as a

percent of control (nanoparticle-free medium). Inset shows Petri dishes after incubation without
nanoparticles (a) and with nanoparticle concentrations (g/mL): 10 (b), 20 (c) and 50 (d). Initial
cell concentration is 105 CFU/mL. Reprinted from Ref. [360]. Copyright 2004, with the
researchers and even by the same researchers in some cases (e.g., see [355, 359]),
complicating comparisons of the results from the corresponding studies. We think it
is reasonable to use two criteria that make it possible to distinguish between the
early inhibition and complete death of bacteria. In our terms, these criteria are the
nanoparticle concentrations that correspond, respectively, to a toxicity threshold
and lethal dose. From the above-mentioned criteria used in the literature, the toxicity
threshold (TT) corresponds to the MIC (understood as the minimal concentration
that suppresses the microbial growth as compared to a control culture) [352, 352] or
a bacteriostatic concentration [357, 358]. The lethal dose (LD) is the concentration
that causes complete death of bacteria; the LD corresponds to the MBC, to bac-
tericidal concentration, or the MIC understood as the minimal concentration that
completely kills bacteria [295, 353, 354].
The determination of the above criteria may be illustrated by the study [360]
where Ag nanoparticles sized 12.3 4.2 nm and stabilized with an anionic sur-
factant (Daxad 19, sodium salt of naphthalene sulfonate formaldehyde condensate
[360, 361]) were tested for their effect on E. coli growth on agar media and in liquid
suspensions.
Nanoparticles were obtained as a powder, resuspended in deionized water to a
desired concentration by sonication before use, and added to a bacterial culture at
the dilutions desired. Agar cultures were incubated at 37 C for 24 h, and colonies
were counted. The growth rates of bacteria in liquid cultures without (control) and
with nanoparticles were determined by measuring the optical density at 650 nm
(D650 = 0.1 corresponds to 108 CFU/mL) every 30 min. The colony number
dependence on the nanoparticle concentration in LuriaBertani (LB) agar and
photographs of several Petri dishes are shown in Fig. 6.4.
The number of colonies already decreased by 70 % relative to the control at
10 g/mL nanoparticles, i.e., the TT did not exceed 10 g/mL, and the LD was
approximately 50 g/mL. As follows from the colony number dependence on the
initial cell concentration at the nanoparticle concentration 20 g/mL, both TT and
LD increase with the increasing bacteria concentration and decrease with the
decreasing cell concentration (within the range (0.12) 105 CFU/mL). For
instance, the LD decreased to 20 g/mL at 104 CFU/mL. Bacterial growth curves
obtained in the liquid LB medium at various nanoparticle concentrations are shown
in Fig. 6.5. It is seen that nanoparticles increased the lag phase and decreased the
maximal cell concentration. As follows from these results, the TT lies within the
range 1050 g/mL, and the LD is higher than 100 g/mL. Thus, both criteria
differ for the cultures grown on an agar medium and in liquid suspension. As
explained by the authors, this difference is conditioned by the partial nanoparticle
agglomeration in the liquid medium, so that their effective concentration is higher
than in the agar medium.
2.0
1.5
9
Bacterial cell number 10
1.0
0.5
0.0
0 2 4 6 8 10
Time, h
Fig. 6.5 Growth curve for E. coli in LB medium at initial cell concentration 107 CFU/mL in the
presence of different nanoparticle concentrations (g/mL): () 0, () 10, () 50, and () 100.
In experiments with suspensions, mostly the total bacterial cell count was
determined (by optical density, OD at 595650 nm); in some cases, living and dead
cells were separately counted with the use of fluorescent labels [362]. In several
studies, the standard microdilution assay was used to estimate the MIC [347, 353,
354]. Nanoparticle solutions in specied dilutions and a cell suspension with a
known cell concentration were mixed in wells and incubated for a certain period of
time, and the minimal nanoparticle concentration causing complete death of bac-
teria was determined.
A general review of experimental results shows that silver nanoparticles do
possess antibacterial, antiviral [363], and antifungal [364, 365] activities and may be
effective against certain disease vectors, for instance, malaria mosquito larvae [366].
In the course of analysis of the literature, a number of factors were found to affect
the TT and LD of nanoparticles. Apart from the main characteristics of nanopar-
ticles, important roles are played by (1) the method used to obtain nanoparticles,
which determines the form (a solution or a powder) tested for antimicrobial activity
and the presence or absence of a stabilizing agent; (2) the bacterial species; (3) the
bacterial cell concentration; (4) the strain of a given bacterial species; and (5) the
composition of a culture medium. Therefore, it is not surprising that, in spite of the
abundance of the relevant literature, we could not nd even two publications where
all of the factors affecting the criteria used were the same. A correct quantitative
comparison of the concentrations characterizing the antimicrobial effect of
nanoparticles was thereby impossible. Nevertheless, certain observations and con-
clusions could be made as to how the main characteristics of nanoparticles influence
their antibacterial and antiviral activities. Such a work may help to identify the
combinations of characteristics that ensure maximal efcacy of nanoparticles as an
antimicrobial agent and is therefore of importance because it is clear that it is just a
high efciency which serves as the necessary argument to substantiate a broad
application of nanoparticles as curative means.
To illustrate the situation in the eld, excerpts from the results of several studies
of antibacterial properties for Ag and other metal nanoparticles are collected in
Tables 6.1, 6.2, 6.3. Certainly, we had no intention to fully elucidate the results
achieved in the eld; however, as we believe, the data presented in the tables give
full enough notion of the main methods, the range of microbial species, and the
possibilities to reliably estimate the nanoparticle concentrations corresponding to
the TT and LD as two quantitative efcacy criteria for particular bacterial species. It
should be added that reliable estimates of these concentrations are important for
evaluating the toxicity of nanoparticles for cells and the whole body in animals and
humans; the lower these concentrations are, the greater is the chance to create a
nanoparticle-based remedy that is effective against the corresponding infection and
does not provoke substantial adverse reactions (side effects).
Table 6.1 Characteristics and data on antimicrobial activity of silver nanoparticles
Synthesis Reducing Size, nm Shape Microorganism Experimental TT, g/mL LD, Reference
method agent/Stabilizer species, cells procedure g/mL
concentration
TC Ascorbic acid/ 12.3 4.2 Spherical E.coli (Presque Incubation with cells 50 (105) [360]
Daxad 19 Charge () Isle Cultures) in Petri dishes for 10 20 (104)
104105 CFU/mL 24 h
(107 CFU/mL) Cell growth in liquid 1050 >100
suspensions
TC NaBH4/Citrate 9.2 2.8 ND E.coli 12 Oxidized NP with 3b (9.2 nm) 5.4 (MIC) [295]
Charge () MG1655 (Genetic Ag+ ions on the
Stock Center, USA) surface. Incubation
with cell suspension
Citrate/Citrate 62 18 108 CFU/mL for 10 min (Ag0 -are 30 (62 nm) ND
(OD650 = 0.1) not effective)
TC NaBH4/Citrate E.coli ATCC10536 Incubation with the <125 (107) 1000 (107) [272]
(seeds) 105107 CFU/mL cells in Petri dishes 60 (105)
Citrate/Citrate 39 Spherical for 24 h
Ascorbic acid/ (192 16) Rods 125 (107) Not
CTAB achieved
6.2 Metal Nanoparticles Effects Observed on Microorganisms
+ NaOH 40 Triangular < 100 (107) 500 (107)

Spherical Cell growth in liquid <0.012 (107) 0.5
medium in rotary
Triangular shaker (244 rpm) <0.06 (105) 0.5
<0.012 (107) 0.025
TC NaBH4/PVA 14 6 ND E.coli PHL628 Incubation with the 0.3 10 [362]
(1040) cells in medium for
24 h
(continued)
173
174
concentration
TC NaBH4 + H2/No 3.32 1.129 Spherical E.coli Incubation with cell [351]
stabilizer (2.2610.34) ATCC25922 suspension in the 40 60
ATCC10536 rotary shaker 180 220
Presumably, ATCC 8739 (200 rpm) to prevent 280 280
Ag0 MTCC1302 NP aggregation and 120 200
St. aureus sedimentation
ATCC6538P 120 160
ATCC25923 120 160
ATCC29213 120 160
B. subtilis
6
ATCC6633 40 60
103104 CFU/mL
TC GA/GA + NaOH 7 Spherical E.coli Incubation with cell [353]
pH 11 ATCC25922 suspension for 24 h 6.25
() ? St. aureus
ATCC25923 7.5
Here and below,
105 CFU/mL
GA/GA + NH4OH 29 Spherical E.coli
pH 10 ATCC25922 13.02
() St. aureus
ATCC25923 16.67
GA, UV/GA 89 Polyhedrons E.coli
Neutral pH ATCC25922 11.79
St. aureus
ATCC25923 33.71
(continued)
The Effect of Metal Nanoparticles on Biological
concentration
TC N2H4 and 9 2 (citrate Spherical E. coli CCM 3954 NP incubation with 14.38 for [354]
citrate/SDS C1) Aggregated P. aeruginosa CCM cell suspension for all strains
14 5 (citrate Polydisperse 3953 24 h 28.77 for
C2 = 2C1) Non-oxidized St. aureus CCM all strains
(Ag0) 3953
St. aureus MRSA
(105106 CFU/mL)
24 6 E. coli CCM 3954 258.89
(850) P. aeruginosa
(SDS) CCM 3953 6.74
St. aureus CCM 258.89
Charge (-) 3953
St. aureus MRSA 258.89
(105106 CFU/mL)
30 7 215.74 for
(citrate + SDS) all strains
TC 812 ND E. coli Incubation with cell 16a [364]
NaBH4/SDS + PVP
K12 NCTC 10538 suspension for 24 h for all
2.7 107 CFU/mL strains
P. aeruginosa
ATCC 15442
1.7 107 CFU/mL
St. aureus
ATCC 6538
1.5 107 CFU/mL
C. albicans
ATCC 10231
5 106 CFU/mL
175
(continued)
176
concentration
PC e
aq ethanol=SDS 45 10 Spherical P. aeruginosa Inhibition zone 13 5 [357, 358]
Polydisperse. Isolated from a determination in Petri
plant dishes
Charge () 107 CFU/mL
Incubation with 15
bacteria suspension
for 24 h
106 CFU/mL
6
RC eaq/Chitosan 7 Spherical St. aureus Incubation with cell 5 [382]

Charge (+) suspension
B Acalpha indica leaf 2030 Spherical E. coli Incubation with cell 10 [383]
extract V. cholerae suspension
B Nelumbo Lucifera 2580 Mostly spherical A. subpictus Incubation with larva 0.3125 5 [384]
(Sacred Lotus) and other forms mosquito larvae suspension for 24 h
leaf extract
C. quinquefasciatus 0.3125 5
(20/mL)
(continued)
concentration
NT, Inc. Powder from carbon 21 18 Polyhedrons E. coli Incubation with cell 25 [368]
matrix suspended in (cuboctahedrons, S. typhus suspension for 75
water using UV icosahedrons P. aeruginosa 30 min 50
treatment et al.) V. cholera 75
5 108 CFU/mL
(OD595 = 0.5)
HNC Ltd. ND 5 (polydisp.) ND (spherical) E. coli ATCC8739 Incubation with cell 1.25 10 [355, 359]
1.000 ppm 107 CFU/mL suspension for 6 h
(1 mg/mL, St. aureus ATCC under shaking 1.252.5 20
or 0.01 mM) 6538P (150 rpm)
107 CFU/mL
ABC ND 40 ND E. coli Incubation in agar Z = 0.0236c* 70 [356]
Nanotech B. subtilis medium Z = 0.0622 70
Co Ltd 2 102 CFU/mL mL/g
Notes TT, toxicity threshold; LD, lethal dose; TC, PC, RC, B, (method of synthesis), respectively, traditional chemical, photochemical, radiation chemical, biological; NT,
Nanotechnology; HNC, Huzheng Nanotechnology Company (Shanghai); ATCC, American Type Culture Collection; ND, not determined; PVA, polyvinyl alcohol; NP,
nanoparticles; GA, gallic acid; SDS, sodium dodecyl sulfate; PVP, polyvinyl pyrrolidone
a
Determined in accordance with European standards for antibacterial and antifungal activities of chemical disinfectants (decrease of microorganism concentration by 5
and 4 orders, respectively; see references from [364])
b
Calculated by us from the bacterial concentrations versus nanoparticle concentrations dependencies after 15 h of incubation, as given in the work
c
Z = ln(N/N0)Csusceptibility coefcient proposed by authors as characteristic for bacterial susceptibility; for explanation of the formula, see text
177
Table 6.2 Comparison of antimicrobial activities of the silver nanoparticles stabilized with SDS,
PVP, and their mixtures [364]
AgNP + stabilizer Exposure, h Pathogens causing hospital-acquired
infections
E. coli St. aureus C. albicans
AgNP (0.0016 %) + PVP 1 <0.39 <0.43 <0.57
2 <0.39 <0.43 <0.57
4 <1.08 <0.43 <0.57
24 >5.06 <0.43 <0.57
AgNP (0.0016 %) + SDS 1 >5.06 2.73 >4.19
2 >5.06 5.01 >4.19
4 >5.06 >5.18 >4.19
24 >5.06 >5.18 >4.19
AgNP (0.0016 %) + PVP + SDS 1 >5.06 1.57 >4.19
2 >5.06 2.07 >4.19
4 >5.06 4.35 >4.19
24 >5.06 >5.18 >4.19
Initial amount of the microorganism (control for 7.45 7.17 6.68
the culture), lg
6.2.1 Antimicrobial Activity of Ag Nanoparticles
In agreement with the aim of our analysis, we considered the data on how the main
characteristics of nanoparticlesthe size, shape, surface charge, and the nature of a
stabilizing agentaffect the toxicity threshold and lethal dose. It is obvious that to
evaluate the effect of the nanoparticle size, the effects of the three other charac-
teristics on biological activity should be eliminated. Therefore, the following
conditions are to be satised in an experiment:
(1) Nanoparticles of different (at least two) sizes in the range 1100 nm should be
obtained by the same method and should be the same in shape, surface charge,
and composition of the stabilizing shell (if the latter exists);
(2) The nanoparticle solutions differing in mean particle size should have narrow
enough size distribution, so that their standard deviations do not overlap.
(3) The effect of nanoparticles differing in size should be investigated by the same
method, on the same bacterial strain of the same species, and at the same
bacterial cell concentrations.
From the literature available, and according to the particle sizes reported by the
authors, there are only two publications [295, 353] that meet the requirements listed
above. Two more articles [354, 367] can be added if a partial overlap of the particle
size distributions is disregarded. In these works, the nanoparticles were obtained by
traditional chemical methods and their sizes were changed by varying the reagents
concentrations or by using an additional reducing agent.
Table 6.3 Characteristics and data on antimicrobial activity of copper, gold, and metal oxide nanoparticles
Method of Reducing Mean size, Shape Microorganism Experimental procedure TT, g/mL LD, g/mL Reference
synthesis or agent/Stabilizer or nm species, cell (MIC) (MBC)
source of other data concentration
nanoparticles
CuO
Nanotechnology ND 100 Spherical E. coli Incubation in agar medium Z = 0.0574 60 [356]
Inc. B. subtilis Z = 0.0734 60
2 102 CFU/mL mL/g
TC NaBH4 + H2/no 9.25 1.79 Spherical E. coli Incubation with cell [351]
stabilizer ATCC25922 suspension in the orbital 140 160
ATCC10536 shaker (200 rpm) to prevent 220 260
ATCC 8739 NP aggregation and 200 230
MTCC1302 sedimentation, 280 300
St. aureus measurement of OD and
ATCC6538P plating on agar mediumb 120 160
ATCC25923 120 160
ATC29213 120 160
B. subtilis
ATCC6633 20 40
103104 CFU/mL
SarPCCa Powder, suspension in 3040 ND St. aureus Incubation with cell <1(96 %)c 1 [389]
Plasma chemistry 0.9 % NaCl 10 clinical strains suspension for 30 min,

3 105 CFU/mL followed by plating on agar
medium
High-temperature Powder in distillate, 47119 ND E. coli AB1157d Inhibition zone diameter 0.5 (70 %)c 110 [390]
condensation sonication E. coli K12 and effect of NP on
method wild bacteria growth
St. albus NDd
(continued)
179
180
nanoparticles
Intrinsic Materials Plasma technology 22.494.8 Rectangular E. coli Incubation with cell [392]
Ltd ECTC 9001 suspension in the orbital 250
St. aureus shaker (200 rpm) for 24 h
(Golden) 2500
St. aureus
(Oxford) 100
St. epidermidis
SE-51 2500
St. epidermidis
SE-4 2500
6
P. aeruginosa
PAOI 5000
5 107 CFU/mL
Other oxides
Al2O3 Dry powder 172 Spherical E. coli Incubation with cell [393]
Sigma-Aldrich (aggregates NCIM 2666 suspension in the orbital >1000
in water) 108 CFU/mL shaker (200 rpm)
ZnO Dry powder 8 ND St. aureus N315 Incubation with cell 40 (8 nm) [391]
Nanoscale 5070 St. epidermidis suspension in the orbital for all strains
Materials Inc., 1487 shaker (200 rpm)(b)
Sigma-Aldrich St. pyogenes
NZ131
B. subtilis 168 1200
5 107 CFU/mL (5070 nm)
(OD = 0.040.06) for all strains
(continued)
nanoparticles
Au
TC Citrate/PEG 46 ND E. coli Incubation with cell No inhibition [387]
(Spherical) pBR322JM105 suspension in the shaker up to
(180 rpm) 4.4 104 g/mL
TC NaBH4 + dextrose/ND 5 ND E. coli Incubation with cell No inhibition up [388]
109 CFU/mL suspension to 100 g/mL
Notes ND, not determined; NP, nanoparticles; PEG, polyethylene glycol
a
SarPCC, Saratov plasma chemistry complex, an afliate of the State Scientic Center of the Russian Federation State Research Institute for Chemistry and Technology of Organoelement
Compounds (Moscow)
b
Determination of bacterial growth rate through optical density change in suspension (OD600650)
c
In brackets: percent of the dead cells
d
Museum strains
181
In the study [295], nanoparticles with mean sizes of 9.2 and 62 nm were
obtained by the citrate method in an inert gas atmosphere with subsequent oxidation
by blowing through with oxygen and were tested for effect on the E. coli growth
rate. Oxidation was performed because nanoparticles synthesized in the inert
atmosphere had no antimicrobial activity, presumably because they consisted only
of neutral Ag atoms. The oxidation led to the appearance of Ag+ ions on the
nanoparticle surface; as a result, the nanoparticles acquired the antimicrobial
properties. As shown in Table 6.1, the TT value determined by us from the bacterial
growth rate in suspension after incubation with nanoparticles as a function of the
nanoparticle concentration was substantially lower for the smaller nanoparticles.
Hence follows that, the smaller the size, the higher is the toxicity of Ag nanopar-
ticles, as many times noted in the literature with reference to the study in question.
The effect of particle size on the MIC (corresponding to the complete death of
bacteria) was studied in [353]. Nanoparticles of various sizes were obtained by
silver ion reduction in solution with gallic acid combined with alkali (pH 1011) or
with additional UV irradiation. Gallic acid and/or its oxidation products served as
stabilizing agents. Preparation of Ag nanoparticles sized 7 (a), 29 (b), and 89 (c) nm
with a narrow distribution was reported. However, the data from the publication
make it possible to question if the particle sizes have been reported correctly. For
instance, electron micrographs presented and the corresponding histograms
obtained by PCS (Fig. 6.6) show that apart from the particles of approximately the
same size (in the range 2040 nm) contained in the sample (b), the two other
samples do not contain the particles with the sizes declared. First, sample (a) dis-
plays a broad particle size distribution with particles of less than 7 nm in diameter
prevailing. Second, sample (c) contains only particles of more than 100 nm in size
(outside the nanoscale range), as is also evident from the results of size measure-
ments shown in the photograph.
The mean particle sizes reported were obtained by PCS, which is known from
the literature to yield mean sizes other than those inferred from electron
micrograph-based histograms (e.g., see [28, 128, 348] and Part I). All these cir-
cumstances indicate that the mean sizes reported are not the actual mean sizes in at
least two out of the three samples. Moreover, as reported by the authors, the particle
shape was differentpolyhedral for the sample (c), while spherical particles were
contained in the samples (a) and (b). Finally, the composition of sample (c) is
questionable because, unlike the two other samples, its absorption spectrum is a
broad band with a flat peak at about 500 nm and appreciable absorption throughout
the visible range (Fig. 6.7). Judging from the spectrum, components of an unknown
nature were likely to occur in sample (c) along with Ag particles detected by TEM.
Thus, if sample (c) is excluded from the analysis as differing in particle shape
and, probably, composition from the two other samples, the effect of particle size on
antimicrobial activity can be evaluated using only the two samples, one with a mean
particle size of no more than 7 nm and a broad size distribution and the other with a
mean size of 29 nm and a narrow distribution. The LD was established for each
sample with the same E. coli and St. aureus strains (Table 6.1). The LDs of the
smaller nanoparticles were lower than those of the larger particles, suggesting
(a) (b)
(c)
Fig. 6.6 Electron micrographs and particle size histograms (in insets) obtained for samples of
silver nanoparticles with the size of 7 (a), 29 (b), and 89 nm (c) by means of PCS technique (by
the Zetasizer Nano ZS device). Reprinted from Ref. [353]. Copyright 2008, with the permission
from Springer
higher toxicity for the former. The result agrees with the TT estimates obtained on
E. coli for nanoparticles of different sizes [295].
In the study [354], nanoparticles were obtained via (1) reduction with a
hydrazinecitrate mixture and citrate stabilization at two citrate concentrations,
(2) hydrazine reduction and SDS stabilization, and (3) hydrazinecitrate reduction
and SDS stabilization. The nanoparticles were washed with deionized water under
nitrogen flow to prevent oxidation. Nanoparticle powders obtained by freeze drying
Fig. 6.7 Absorption spectra for silver nanoparticles. Reprinted from Ref. [353]. Copyright 2008,
with the permission from Springer
were used in experiments; a nanopowder was resuspended in deionized water and

homogenized in an ultrasonic bath. The nanoparticles were reported to be (1) 9 2
and 14 5 nm at 1 and 2 mM citrate, respectively; (2) 24 6 nm; and (3) 30
7 nm. Although the standard deviation intervals overlap, a set of samples with
these mean sizes is, at rst glance, of interest for studying the effect the nanoparticle
size exerts on their biological activity, since a region close to the lower margin of
the nanoscale range is covered. The rst two sizes are of particular interest, because
they fall within the range 120 nm, wherein size effects should be expected. In this
connection, it was important to verify the particle sizes and it was easy to perform
because TEM images and the corresponding histograms were given in this paper.
The TEM images and histograms are reproduced in Fig. 6.8 for the rst two
samples with the mean particle sizes 9 and 14 nm.
As can be seen by the reader, for the rst case (9 nm) the image is extremely
poor in quality and is difcult to use for estimating the particle size. Yet, the size
was established by the authors since a corresponding histogram is presented,
showing a broad size distribution (617 nm). In the second case (14 nm), the
histogram actually shows two particle populations, one including particles of 2
12 nm which represent at least 60 % of the sample, and the other including particles
greater than 22 nm, but no more than 30 nm. As reported by the authors, particles
of 14 nm were absent from the sample. Hence, two nanoparticle solutions with
broad size distributions were actually tested, although the distribution was narrower
in one case (9 nm) than in the other (14 nm). Judging from the histograms, the most
prevalent sizes in the second sample (24 nm) were lower than in the rst one (6
9 nm); therefore, in testing the effect of the particle size, the samples should be
arranged in the opposite order. Nanoparticle aggregates might also occur in the
solutions because a shoulder is seen in the long-wavelength range of their
(a) (b)
(a) 80
60
Frequency
40
20
0
6 8 9 11 13 17
Diameter, nm
(b) 80
60
Frequency
40
20
0
4 7 12 22 24
Diameter, nm
Fig. 6.8 TEM images and particle size histograms for samples with mean size of 9 (a) and 14 nm
(b). Reprinted from Ref. [354]. Copyright 2012, with the permission from Elsevier
absorption spectra (Fig. 6.9). Antibacterial activity assays of the two samples
(Table 6.1) showed that the LD of the particles with the (now relative) mean size
9 nm was half that of the particles with the (relative) size 14 nm. The result agrees
with the data from the two articles discussed above, but can hardly be considered as
providing independent evidence for the higher toxicity of smaller nanoparticles
because of, softly speaking, the uncertainty in particle sizes.
3.6
3.4
3.2 (a)
3.0 (b)
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.00
190.0 250 300 350 400 450 500 550 600 650 700.0
, nm
Fig. 6.9 Absorption spectra for solutions of the Ag nanoparticles with mean size of 9 (a) and
14 nm (b) obtained by hydrazine reduction with citrate stabilization at citrate concentration 1
(a) and 2 mM (b). Reprinted from Ref. [354]. Copyright 2012, with the permission from Elsevier
As for the other two nanoparticle samples examined in the study, their his-
tograms and absorption spectra are similarly indicative of a broad size distribution
and possible aggregation of nanoparticles, the size range being even greater (8
50 nm) than in the two previous cases. In our view, the LD of these nanoparticles
was found to be substantially (more than one order of magnitude) higher because
they were more prone to aggregation and precipitation when incubated with bac-
terial suspension, rather than because their size (more exactly, size range) was
greater.
Uncertainty in particle sizes does not allow drawing denitive conclusions also
from the work [367], wherein antibacterial activity was assayed for Ag nanopar-
ticles sized 25, 35, 44, and 50 nm and obtained via reduction of ammoniac silver
salt with mono- and disaccharides. MIC (and MBC) values reported for several
bacterial species show that, in the majority of cases, a stronger effect was observed
for the smaller particles. However, the electron micrographs available and particle
size measurements obtained by PCS indicate that, in reality, the nanoparticles had a
broad size distribution and a high extent of aggregation so that it was impossible to
construct histograms and estimate the mean size from TEM data. The PCS-derived
sizes were therefore reported, while ignoring the known fact that PCS usually
overestimates the particle size as compared to TEM (see Sect. 1.3.4, Chap. 1; Sect.
2.4, Chap. 2; and Sect. 4.2, Chap. 4). Hence, it remains unclear whether the dif-
ference in particle size between the samples was reliable; if so, how great the
difference was; and whether the biocidal effect was actually related to the particle
size.
Thus, with the last study excluded, the results of the three other publications,
where the size of silver nanoparticles was tested for effect on their antimicrobial
activity in conditions mostly meeting the requirements listed above, showed that
toxicity of nanoparticles tends to increase with the decreasing particle size, at least
for the two pathogenic bacteria, E. coli and St. aureus. Speaking more precisely,
nanoparticles of no more than 1012 nm may exert a stronger bactericidal effect as
compared to nanoparticles sized more than 20 nm. It should be emphasized that, in
our opinion, the available data are still insufcient for a denitive conclusion to be
made about the relationship between the bactericidal effect and the particle size; one
can speak rather about a tendency observed in some experiments.
It is important to add here that even this tendency has not been demonstrated
conclusively, taking into account that the same mass concentrations were used for
nanoparticles of different sizes and were obtained by similarly diluting stock
solutions of the same nanoparticle concentrations. A problem arises because when
the difference in mean particle size is great (especially in the study [295], where the
results seem to be the most reliable), the same mass concentrations correspond to
substantially different number concentrations. A difference in number concentration
and therefore in total surface area of nanoparticles may play a considerable role in
their biological activity, for instance, by determining a difference in the rate of
medium saturation with Ag+ ions or in total area of nanoparticle contacts with the
bacterial cell surface. The effect of particle size alone is therefore impossible to
isolate with the above experimental design. In other words, two factors, the number
concentration (total surface area) and the size proper, actually affect antimicrobial
activity when we consider the above-mentioned tendency to higher antimicrobial
activity at a smaller particle size. When number concentrations are the same, a
difference in particle size may be important for the bactericidal effect, e.g., smaller
nanoparticles are capable of penetrating into the cell, while larger nanoparticles are
only capable of interacting with cell surface structures. It is clear also that specic
size-related effects can be assumed for nanoparticles of less than 10 nm based on
the size dependences of their physical properties and chemical activity [35].
Thus, from methodological point of view, when studying the effect of particle
size on antimicrobial activity of nanoparticles, for the correct experimental design
comparison should be made of antimicrobial activities for the same number con-
centrations, but not mass concentrations of nanoparticles. A difference in mass
concentration can be quite appreciable in this case. For instance, consider the
nanoparticles of two sizes examined in [295] at 3 g/mL (the TT of 9.2-nm
nanoparticles). The number concentrations of the 9.2- and 62-nm nanoparticles
were 7.5 1011 and 2.25 109 particles/mL, respectively. Then, the mass con-
centration of the 62-nm nanoparticles that is equivalent to their number
concentration 7.5 1011 particles/mL is (7.5 1011/2.25 109) 3 = 1000 g/mL,

or 1 mg/mL (compare to 3 g/mL !).
Let us hope that further studies with silver nanoparticle solutions that meet the
necessary requirements and have the same number concentrations for differently
sized nanoparticles will give the grounds for more denite conclusions about the
influence of particle size on its antimicrobial activity. As shown in above examples
and other data from Table 6.1, in the majority of experiments to date polydisperse
solutions have been used, which are in principle unsuitable for studying the effect of
the particle size. Apparently, the major problem here is that it is extremely difcult
to obtain monodisperse solutions that differ only in nanoparticle size, being similar
in all other main characteristics.
Let us try now to conclude about the effects of the other nanoparticle charac-
teristics from the known published results, including those collected in Table 6.1.
The effect of the shape of silver nanoparticles on their antibacterial activity is
obviously possible to determine in conditions which exclude the effects of other
important characteristics, by analogy with the above requirements for studying the
effect of the nanoparticle size. In this case, it is important for the nanoparticles
differing in shape to be similar in size, for their charges to be the same in sign and
similar in magnitude, and for their stabilizing coatings to be the same in
composition.
The effect of the nanoparticle shape was studied on E. coli growing both on an
agar medium and in liquid suspension [272] (see Table 6.1). Three nanoparticle
shapesspherical, rod shaped, and truncated triangularwere examined, and
truncated triangular nanoparticles were found to possess the highest activity, pre-
sumably because an active high-atom-density {111} facet (the truncation plane)
occurs in their crystal (FCC) lattice. However, an analysis of the data presented
shows that there are grounds for doubting the adequacy of the conclusions made.
First, it is striking that the spherical and triangular particles are similar in size (39
and 40 nm, respectively), while the rod-like nanoparticles obtained in the study are
incomparable in size with the two other nanoparticle samples (Fig. 6.10). The
difference can be expressed quantitatively in terms of number concentration. At the
same mass concentration, e.g., 100 g/mL (one of the concentrations used in the
study), number concentrations of the spherical- and rod-like nanoparticles were,
respectively, 3.075 1017 and 4.914 1015, differing approximately by two orders
of magnitude. Hence, only the spherical and triangular nanoparticles can be com-
pared to estimate the effect of the shape on activity.
Yet another problem arises with these particles. As reported by the authors,
differently shaped nanoparticles were obtained by different methods: reduction and
stabilization with citrate in the case of the spherical nanoparticles and reduction
with ascorbic acid and stabilization with CTAB in the case of the rod-like and
triangular particles. In the former case, the stabilizing shell was negatively charged
and in the latter case it was positively charged, as a result of dissociation of acidic
and basic ionogenic groups, respectively, in a cell-containing medium (at a neutral
pH) The nanoparticles therefore differed not only in shape, but also in the sign of
their surface charge. Since the bacterial cell membrane is negatively charged,
Fig. 6.10 TEM image of (c)

puried nanorods. Reprinted
from American Society for
Microbiology
50 nm
nanoparticles with a negative surface charge (spherical) can be expected to have a

lower efcacy than nanoparticles with a positive surface charge (triangular) as a
result of electrostatic interactions. Thus, higher activity observed for the triangular
nanoparticles can hardly be attributed to certainty to their shape alone because their
positive charge might play an important role. It is also noteworthy that the TT and
LD values strikingly differ between experiments on an agar medium and in liquid
suspension. Substantially lower estimates obtained for both TT and LD in liquid
suspensions need explanation. Taking into account that, from Table 6.1, this study
is the only one reporting so low TT values from bacterial growth experiments, it is
possible to assume that particular features of the experimental design can play their
role.
In the majority of other publications, only spherical nanoparticles were tested.
There is evidence also on the antimicrobial properties of the mixtures of polyhedral
nanoparticles (cuboctahedra, icosahedra, etc.) [353, 368], but polydisperse samples
were used in both cases, and data are missing that would allow comparisons of the
bactericidal effect for the mixtures and spherical particle samples. Thus, it is still
unclear at present how the shape of nanoparticles affects their antimicrobial activity.
The effect of the surface charge might be detectable provided, apparently, that
the particles are identical in size, shape, and composition of the stabilizing shell (if
any). In the general case, for particles in aqueous solutions the surface charge of the
stabilizing coating should be distinguished from that of the nanoparticle itself, i.e.,
of the bare or uncapped nanoparticle. The sign and magnitude of the particle
charge in solution can be estimated by measuring the -potential. The charge may
appreciably differ in both magnitude and sign between uncapped and coated
nanoparticles; such examples have been described for nanoparticles of various
natures in the review [350]. In most cases, nanoparticles are obtained by a stabilizer
that bears ionizable groups, and their charge depends on their pK and pH of the
medium. It should be noted in this connection that the above requirement that the
stabilizing coating compositions be the same needs comments. The requirement is
possible to fulll, in principle, by using nanoparticles obtained with the same
stabilizing agent and experimentally testing them at different pH values to ensure a
difference in surface charge. Yet such an experiment may be difcult to perform
because pH of the medium considerably affects activity of the test microorganisms.
Hence, in studying the effect of surface charge for stabilized nanoparticles, it is
admissible to use stabilizers that differ in nature and pK of the ionizable groups, but
are as structurally similar as possible to ensure a minimal difference in thickness
and chemical properties of the nanoparticle coatings.
Stabilizers with acidic groups (citrate, SDS, AOT, GA, AA, etc.) are negatively
charged at neutral pH, while those with basic groups (CTAB, PEI, etc.) are posi-
tively charged. In studies of the biological effects of nanoparticles, the stabilizers
with acidic groups are mostly used, i.e., in the majority of cases, nanoparticles are
negatively charged in solution. The influence of surface charge on the antibacterial
activity of nanoparticles can manifest itself in two ways. First, when incubated with
cells, nanoparticles can aggregate in the medium as their charge is neutralized by
positively charged metal ions adsorbed on their surface. Second, electrostatic
interactions arise between the nanoparticles and the cell membrane, which is known
to bear a negative charge. Aggregation of silver nanoparticles added to the E. coli
growth medium containing sodium, calcium, and other metal salts was observed in
[295, 360]. As noted by the authors in [295], the aggregation resulted in that the
nanoparticles had lost their bactericidal activity; to prevent aggregation in the
medium, the nanoparticles were preliminarily treated with bovine serum albumin
(BSA), which is adsorbed on the particle surface and protected the particles from
electrolyte-induced coagulation.
Only scarce data are available for the -potential of stabilized silver nanoparticles
tested for antimicrobial activity [360, 361, 369]. In the study mentioned above [360],
Ag nanoparticles sized 12.3 4.2 nm and stabilized with an anionic surfactant were
tested for their effect on E. coli growth on an agar medium and in liquid suspensions.
Earlier measurements with silver nanoparticles sized 60 5 nm and obtained by the
same method showed that the -potential in 1 mM KCl changes from 27 to
48 mV in the pH range 210 [360, 361]. Hence follows that the nanoparticles used
in experiments with cells were negative in charge. In the study [369], the -potential
was measured for silver nanoparticles obtained by radiation-chemical synthesis and
stabilized with chitosan. The stabilizing coating was positively charged in this case
as a result of protonation of chitosan amino groups, and accordingly, the -potential
changed from +28 to +3 mV in the pH range 29.
Unfortunately, it is impossible in both the above cases to estimate the surface
charge from the reported -potential values by using the relationships (the potential
versus distance from the surface dependence or the GouyChapman equation)
known from the electrical double-layer theory [370, 371] modied conformably to
ionogenic group-bearing membranes [372, 373]. The problem is that standard
software incorporated in the corresponding instruments (manufactured by Malvern,
Coulter Electronics, or Brookhaven) usually calculates the -potential from the

electrophoretic mobility measured by PCS without the correction for the relaxation
effect, applicable for the measurements of nanoparticles. At the same time, the
correction is essential in view of the small nanoparticle size, because a more or less
substantial error in the -potential value is otherwise inevitable, as was shown, for
instance, in [374376]. Such errors are likely to occur in the relevant studies since
the -potential is usually calculated from the Smoluchowski equation, and the
resulting values (sm) may considerably differ from the true -potential. According
to our experience in the eld, an optimal way to introduce the relaxation correction
in calculations of the potential from electrophoretic mobility measurements is
suggested by one of the equations of the Dukhin theory of electrophoresis [377]. It
is essential to introduce the relaxation correction based on the Dukhin theory, as we
have demonstrated when studying the electrophoretic mobility of liposomes as a
function of the ionic strength of an electrolyte solution (see [378, 379] and refer-
ences therein). But the correct values of the -potential and, therefore, of the surface
charge density can only be obtained from the measured electrophoretic mobility
values, which are absent in the studies considered above.
As follows from the above arguments, it is still poorly understood how the
surface charge of nanoparticles affects their antimicrobial activity.
The effect of the stabilizing shell composition on biological activity of metal
nanoparticles is of interest primarily for developing medical applications of
nanoparticles. It is important in this case that the stabilizing agent could, on the one
hand, efciently prevent oxidation and aggregation of nanoparticles to ensure their
storage in solution for a sufciently long period of time and, on the other hand, be
minimally toxic for a human organism. At the same time, when nanoparticles are to
be used as a remedy, the stabilizing agent should not considerably reduce
nanoparticle activity against the target disease. In particular, antimicrobial activity
should not be considerably reduced for the nanoparticles to be effective against
infectious diseases or complications. Thus, studies on the influence of the nature of
a stabilizing agent on the antimicrobial properties of silver and other metal
nanoparticles is an essential step in developing effective and reasonably safe
medicines.
To detect experimentally the effect exerted by the nature of a stabilizing agent,
nanoparticles similar in size, shape, and surface charge should be used. In the
literature available, this condition is best satised in the study of antimicrobial
activity for silver nanoparticles stabilized with SDS, PVP, and their mixtures [364].
The silver nanoparticles were 812 nm in size, most likely spherical in shape, and
were obtained by sodium borohydride reduction in an aqueous solution. True, the
surface charge of the stabilizing shell could differ in this case because SDS is
negatively charged, while PVP may bear a weak positive charge, as far as can be
judged from the monomer structure. Nanoparticle solutions were incubated for
various periods of time with the bacteria E. coli, St. aureus, and P. aeruginosa and
the fungus Candida albicans, which were used in suspension at approximately
107 CFU/mL. The effect of nanoparticles was estimated from a decrease in
microbial concentration and expressed as logarithm of the initial to nal cell
concentration ratio (the reduction coefcient, R). Main results are summarized in
Table 6.2. As shown, the PVP-stabilized nanoparticles were virtually inactive upon
incubation for several hours, while the SDS-stabilized particles displayed a strong
antimicrobial effect. A similar result was obtained for St. aureus and C. albicans
after 24 h of incubation. Solutions of the stabilizers without nanoparticles did not
show antimicrobial activity in control experiments.
According to the authors of that study, the difference in effect between the two
stabilizing agents arises because SDS may facilitate activation of the antimicrobial
action of nanoparticles, while PVP exerts a dual effect, acting, on the one hand, as
an antidote to silver and, on the other hand, stabilizing colloidal silver solutions.
The effect of SDS, as the authors believe, is connected with the capability of
surfactants to reversibly adsorb on the cell surface, altering the normal function of
the cell membrane. At the same time, though efcient as a stabilizer, PVP seems to
exert an inactivating effect on nanoparticles. Stabilization with a SDSPVP mixture
was therefore found to be an optimal variant, although nanoparticle activity was
somewhat lower than upon stabilization with SDS alone, possibly because PVP
neutralized the SDS effect on the cell. Yet, the nanoparticle stability increased in
this case; as was demonstrated in [364], such nanoparticle solutions retain
high-level antimicrobial activity for at least one month, which is essential for their
use for medical purposes.
As indirect evidence which conrms that SDS stabilization increases the bac-
tericidal effect of silver nanoparticles could be regarded the relatively low TT and
LD values found by the authors of [354, 357, 358] when studying the effect of
nanoparticles on P. aeruginosa upon incubation both on an agar medium and in a
liquid suspension (Table 6.1). An inactivating effect of PVP might be responsible
for a substantial difference in antiviral activity and cytotoxicity between uncapped
and stabilized silver nanoparticles in the study [363], where the effect was examined
for nanoparticles isolated from a carbon matrix and for those stabilized with PVP
and BSA. The uncapped nanoparticles were shown to be far more active, sug-
gesting a considerable inhibitory effect on the nanoparticle interaction with cells
and virus particles for the stabilizing agents. High activity of uncapped nanopar-
ticles toward E. coli and St. aureus was similarly observed in [355, 359] upon
incubation with cells in liquid suspensions.
According to the authors of [363], the inhibitory effect of PVP and BSA is
determined by the mechanism of their interactions with the nanoparticle surface.
The authors noted that PVP is a linear polymer that stabilizes nanoparticles via
pyrrolidone ring adsorption on the nanoparticle surface; according to IR spec-
troscopy and X-ray photoelectron spectroscopy (XRS) data, adsorption is accom-
panied by bonding with nitrogen and oxygen atoms of the pyrrolidone ring [380].
BSA is one polypeptide chain and consists of amino acid residues with sulfur-,
oxygen-, and nitrogen-containing groups [381], which are capable of binding to the
nanoparticle surface; the tightest bonds can form with the thiol groups of cysteine
residues. As a result, BSA ensures reliable steric stabilization of nanoparticles,
protecting them from aggregation in the culture medium and, at the same time,
reducing their antimicrobial activity. It is possible, however, that differences in size
and shape also play a role in the study of antiviral activity for nanoparticles
described in [363]. The uncapped nanoparticles used in the work were a polydis-
perse mixture of particles varying in shape (polyhedrons, spheres, and rod-like
particles) with a size of 21 18 nm, while the particles stabilized with PVP and
BSA were approximately spherical in shape and more homogenous in size, 6.53
2.41 and 3.12 2.00 nm, respectively. Taking into account the above-mentioned
tendency to higher bactericidal activity at a lower particle size, it is possible to
assume that potentially higher activities of the stabilized nanoparticle samples
might be masked by the stabilizers. In other words, the requirements essential for
a correct evaluation of the effect one of the four main characteristics of nanopar-
ticles exerts on their biological activity were not met in this case, like in a number
of the other cases described above.
To summarize, it is clear that in the general case, a stabilizing shell is not just an
inert coating that prevents nanoparticles from aggregation without affecting their
biological activity. On the contrary, the stabilizing agent can substantially affect the
interaction of nanoparticles with the medium and the cell membrane because its
structure and chemical properties determine both the mechanism of its binding to
the nanoparticle surface and the surface charge of the stabilizing coating; that is, the
factors that may increase or decrease biological activity of nanoparticles. The
factors play no role when uncapped particles are used as a powder in experiments.
This variant was used in [351], where silver and copper nanoparticles were obtained
by borohydride reduction in a hydrogen flow without a stabilizer and in experi-
ments with silver nanoparticles isolated from a carbon matrix by sonication and/or
exposure to an electron beam in TEM [355, 359, 363, 368]. Nanoparticles
embedded in a carbon matrix are produced by several companies outside Russia
(Nanotechnologies Inc.; Nanotech Co, Ltd.; Huzheng Nanotechnology Company;
see Table 6.1). Yet other problems arise with their use. First, as far as can be
inferred from relevant publications, a broad size range and a variety of shapes are
characteristics for these nanoparticles so that the effects of size and shape on
biological activity are infeasible to evaluate. Second, the particles rapidly aggregate
and precipitate in water or a culture medium, so that in experiments with liquid
suspensions continuous agitation (rotation) is essential to perform, potentially
affecting the cell response. This factor is probably responsible for the high values
reported for the TT and LD in [351]. Third, a protective coating should be applied
on these nanoparticles when they are intended for medical use, in order to prevent
their adverse effects on the body. Hence, the results from studying the effects of
such nanoparticles on bacteria, viruses, or animal cells cannot be directly used to
create medical products, e.g., curative remedies.
Apart from conclusions regarding the effects of the main characteristics of
nanoparticles discussed above, the data from Table 6.1 allow several observations as
to how the effect of nanoparticles differs among different bacterial species or dif-
ferent strains of one species. Data from several studies where two or more bacterial
species were used to evaluate the effect of silver nanoparticles [353356, 359, 363,
368] make it possible to conclude that E. coli is more sensitive to these nanoparticles
than St. aureus [353355, 359] and several other microorganisms [368] and that the
difference is seen at different nanoparticle sizes and with different stabilizing agents.
The study [356] showed that vegetative B. subtilis cells are more sensitive to
nanoparticles than E. coli is. Testing several E. coli strains in [351] made it possible
to observe a higher sensitivity for strain ATCC25922, although it remains unclear
whether the result is specic to the nanoparticle size or experimental conditions
used. Apart from the above-mentioned features of uncapped nanoparticles, which
were used in the study, such specicity is possible because substantially different
results were reported for the same E. coli and St. aureus strains in [353], where the
LD values were more than one order of magnitude lower than in [351]. It can be
added that the difference in results obtained for one bacterial species or one strain in
different publications, apart from the other factors, may be determined by the dif-
ferences in the composition of culture media
The problems identied in the discussion of studies focusing on antimicrobial
activity of silver nanoparticles pertain well to nanoparticles of other metals and
metal oxides, which are considered in the section below.
6.2.2 Antimicrobial Activity of Cu, Au, and Metal Oxide

Nanoparticles
Apart from silver nanoparticles, the effect on microorganisms was studied for
nanoparticles of other metals, such as copper [351, 385, 386], gold [387, 388] and
copper, iron, zinc, and aluminum oxides [356, 389392]. Data from some of the
studies are summarized in Table 6.3. Gold nanoparticles are obtained in solution by
traditional chemical methods. Copper nanoparticles are obtained by chemical
reduction of a copper salt in solution [351, 386] or in submicron sepiolite (mag-
nesium phyllosilicate) particles [385]. The procedure yields nanoparticles with a
small mean size (25 or *9 nm) and a narrow size distribution. The main problem
in this case is that copper is readily oxidized in air so that the nanoparticles have a
metal core and an oxide shell on the surface. In the study [351], an oxide shell on
the surface was detected by energy-dispersive X-ray spectroscopy (EDS) for
nanoparticles obtained by borohydride reduction in solution in a hydrogen atmo-
sphere. However, purely metal Cu nanoparticles are possible to obtain by ethylene
glycol reduction from copper acetate hydrate (CuAc2 2H2O), as was demonstrated
in a recent study by the other researchers [386]. Apart from EDS, the absorption
spectrum of the resulting nanoparticles in solution (Fig. 6.11) testies to the
absence of copper oxide, having a distinct band of metal nanoparticles with a
maximum at 580 nm. Antimicrobial activity of the nanoparticles was determined
from the diameter d of the growth inhibition zone forming around a well with a
nanoparticle solution on an agar medium. The nanoparticles were found to be
effective against four bacterial and three fungal species. The highest sensitivity was
observed for the bacteria E. coli and St. aureus and the fungus C. albicans
(d = 21 26 mm), while P aeruginosa was the least sensitive (d < 5 mm). A high
sensitivity was observed for B. subtilis as well.
2.5
580 nm
2
Intensity, a.u.
1.5
0.5
0
0 200 400 600 800 1000
Wavelength, nm
Fig. 6.11 Absorption spectrum of Cu nanoparticle solution. Reprinted from Ref. [386]. Copyright
Methods based on high-temperature evaporation and subsequent vapor con-

densation are also commonly used to obtain copper nanoparticles to be tested for
antimicrobial activity; here belongs, for example, a variant that includes evapora-
tion in a plasma flow at a temperature of 50006000 K followed by the vapor
condensation [389, 392]. A general scheme of the method is shown in Fig. 6.12,
which is cited from [392].
The method is used to obtain nanoparticles of copper, copper oxides, and other
metals in the form of powders varying in particle size. Outside Russia, such
powders are manufactured by various commercial nanotechnology companies;
some of them are listed in Table 6.3. In Russia, original variants of similar methods
based on the same principles are used to obtain metal and metal oxide nanopow-
ders. Two of the variants are presented in Table 6.3 [389, 390].
In biological activity assays, a powder is usually suspended in deionized or
distilled water via sonication, and then used for measurements of (1) the diameter of
a growth inhibition zone around a disk with nanoparticles on a Petri dish, (2) the
number of viable colonies on a Petri dish after incubation at various nanoparticle
concentrations in an agar medium, or (3) the optical density of a liquid culture
medium to determine the effect of nanoparticles on the growth of bacteria. The usage
of nanoparticle powders for such studies has its drawbacks, as was noted in the
previous section for silver nanoparticles supplied in a carbon matrix. Moreover, most
Fig. 6.12 Scheme of a

nanoparticle plasma synthesis Feed
equipment. Reprinted from system
Feedstock
Elsevier
Vaporised
Cooled &
Modificatio n
Nano
product
likely copper oxide, rather than metal copper is tested for interaction with cells in
this case because a more or less thick oxide shell forms on the nanoparticle surface.
This assumption is, in fact, supported by the data reported in the relevant studies.
The particle sizes and phase compositions of nanopowders were presented in the
publication [390] (Table 6.4); copper oxide occurs in a higher or lower proportion
in all of the nanoparticle samples, forming an oxide lm or a shell of a varying
thickness on the nanoparticle surface.
A difference in phase composition between copper and copper oxide nanopar-
ticles obtained by the same method was clearly demonstrated in the study [394]
(Fig. 6.13).
As shown in the gure, the two nanoparticle samples actually differ in the
proportion of metal copper and copper oxides (CuO and Cu2O). Since there are all
Table 6.4 Size and phase composition of copper nanoparticles [390]

Sample Size of copper Content of Other forms of copper Thickness of
No nanoparticles, crystalline Form Content, % oxide lm,
nm copper, % of nm
copper
1 103.0 2.0 96.0 4.5 CuO 4.0 0.4 6
2 47.0 0.6 84.0 4.2 CuO 16.0 1.6 6
3 86.8 0.9 94.0 4.3 CuO 6.0 0.6 10
4 119.0 1.0 0.50 0.02 CuO 90.45 6.15 a
CuO 9.05 0.85

5 103.0 2.0 71.4 4.0 CuO 28.6 0.8 6
a
Nanoparticles were completely oxidized, except for the core containing (0.5 0.02) % of
crystalline copper
Copper oxide nanoparticles Copper nanoparticles
23%
50%
77%
49.5% Cu
Cu2O
CuO
Fig. 6.13 Phase composition comparison for copper and copper oxide nanoparticles using XRD
analysis [394]
grounds to believe that surface interactions play a signicant role in the effect
nanoparticles exert on bacterial cells (see Sect. 6.2 below), studies where copper
nanoparticles obtained in the form of oxidized powders were tested for antimi-
crobial activity can be combined in one copper oxide nanoparticle group [351, 356,
389, 390, 392], which is presented in Table 6.3.
The results obtained by copper oxide show that the TT and LD values reported
for nanoparticles in the works [351, 356, 392] amount to tens, hundreds, or even
thousands of micrograms per milliliter, exceeding those of silver nanoparticles by
more than one order of magnitude in the majority of cases (Table 6.1); i.e., bac-
tericidal activity of these copper oxide nanoparticles is far lower. The same order of
magnitude is observed for the oxide concentrations regardless of the nanoparticle
size, bacterial cell concentration, species, or strain, as shown in Table 6.3.
Considerably higher LDs (MBCs) of copper and several metal oxide (ZnO, Cu2O,
and CuO) nanoparticles compared with silver nanoparticles were registered also in
the work [392] for nanoparticles obtained by plasma technology. The same is true
for zinc oxide and aluminum oxide nanoparticles obtained as powders via similar
technology in other studies [391, 393].
At the same time, in the articles [389, 390], activity at approximately 1
10 g/mL was reported for copper (oxide) nanoparticles, being comparable with
activity of effective Ag nanoparticles and substantially higher than activities
obtained in the previous group of studies with copper oxide particles. As shown in
[389], copper nanoparticles 3040 nm in size obtained by plasma chemical syn-
thesis and, incubated with St. aureus cells in suspension for only 30 min, killed
96 % of cells when used at 1 g/mL and 99 % of cells at 10 g/mL. Almost
complete inhibition of E. coli growth in a liquid medium at 110 g/mL
nanoparticles was reported also in the dissertation [390]. High activity of copper
nanoparticle powders synthesized by a similar method was observed in the study

[395], where the nanoparticles were tested for activity toward Staphylococcus
strains (St. epidermidis and St. salivarius strains isolated from saliva samples taken
from patients) and a museum collection E. coli strain; i.e., nanoparticles used at
10 g/mL caused 85 % cell death in the case of the Staphylococcus isolates (from
healthy patients) and 89 % cell death in the case of E. coli.
The cause of such a striking difference between the above results and those
obtained by the authors working beyond the border remains unclear. However,
taking into account that the works outside Russia were devoted almost exclusively
to the studies of museum strains, while in Russia mostly clinical strains were
examined, one can suppose that the latter are on average more sensitive to
nanoparticles. In this connection, it seems reasonable to undertake the task of the
comparison of sensitivity for museum and wild strains of a broad range of
bacteria species because it is obvious that if substantial differences are observed,
just the data for clinical strains should be used for the creation of nanoparticle-based
antimicrobial medicines, and data for soil and water environmental strainswhen
evaluating the ecological safety of nanoparticles. The necessity to pay great
attention to the difference between natural and museum strains of microorganisms
follows, in particular, from the conclusion made more than 10 years ago in the
monograph [396, p. 101]: there are many objects in nature that can not be
compared with known ones; organisms isolated into pure cultures are few in nature.
Therefore, a systematic error is introduced in studying biodiversity of the microbial
world by cultural methods, and the entire validation concept based on the idea of a
pure culture from an acknowledged collection got wrecked; i.e., it is possible to
say and difcult to refute that the existing bacterial taxonomy as it is presented in
Bergeys manual based on bacteria made legitimate according to the Code of
Nomenclature of Bacteria is a classication of artifacts.
Among the results obtained with oxides, it is of interest to note a rare rectangular
shape of CuO nanoparticles synthesized in the work [392] (Fig. 6.14) and data on
the influence of zinc oxide nanoparticles size on their bactericidal activity.
According to [391], ZnO nanoparticles completely inhibit the bacterial growth at the
concentration of 40 g/mL, the effect being much the same with different species.
As an example, growth curves for two bacterial species are shown in Fig. 6.15.
A series of experiments with St. aureus showed that nanoparticles with the size
of 8 nm are more active than larger particles (5070 nm). However here, like in a
similar study with Ag nanoparticles [295] discussed above, a substantial difference
in number concentration of nanoparticles was disregarded; hence, only a tendency
can be considered for the present. A denitive conclusion will be possible when
activity is compared for different mass concentrations of differently sized
nanoparticles; i.e., a due correction is made for the difference in number concen-
tration. The nanoparticles were additionally found to be light sensitive, being more
effective in the light than in the dark.
At the same time, as was demonstrated in [393], Al2O3 nanoparticles were
almost fully deprived of antimicrobial activity toward several bacterial species. As
explained by the authors, the reason was that the nanoparticles are capable of
Fig. 6.14 TEM image of CuO nanoparticles. Reprinted fromRef. [392]. Copyright 2009,with the
permission fromElsevier
(a) 2.000 (b)
1.500
OD600nm
1.000
0.500
0.000
0 1 2 4 6 8 10 0 1 2 4 6 8 10
Time, h
Fig. 6.15 Growth curves for St. Aureus (a) and S. epidermidis 1487 (b) in the presence of 8 nm
ZnO nanoparticles at concentrations (g/mL): ()0; ()2; ()5; and ()10. Reprinted
from Ref. [391]. Copyright 2008, with the permission from Oxford University Press
preventing the generation of reactive oxygen species (ROS) and thereby protect
cells from oxidative stress. In view of the considerable extent of aggregation
observed for the nanopowder in water (Fig. 6.16), an additional role, as we believe,
could play precipitation of the nanoparticle aggregates and consequently, a low
nanoparticle concentration in the solution. It is possible, therefore, that the
Acc. V Spot Magn Det WD

10.0 kV 4.0 84234x SE 6.0
1 m
Fig. 6.16 SEM image of aluminum oxide nanoparticles. Reprinted from Ref. [393] Copyright
conclusion that aluminum oxide nanoparticles are safe to use in household products
(ceramic cookware) and environmentally friendly [393] requires verication in
additional experiments with isolated nanoparticles that do not aggregate in solution.
The experiments with gold nanoparticles are far fewer than those made with
silver nanoparticles, probably because antimicrobial activity can hardly be expected
for the former in view of the well-known chemically inert character of gold. Indeed,
total absence of bactericidal activity toward E. coli was reported for Au nanopar-
ticles obtained by the citrate method [387] or by reduction with borohydride and
dextrose [388]. An example of testing Au nanoparticles for the effect on E. coli
growth is shown in Fig. 6.17.
However, it is possible that in this case the role of the inert nature of gold is not
so much important as that of the stabilizing coating. Thus, the nanoparticles syn-
thesized by the citrate method were stabilized not only with citrate, but also with
polyethylene glycol (PEG). As noted by the authors in [387], here the aggregation
and precipitation of nanoparticles were not observed at a high (0.2 M) metal cation
concentration in the LB medium, though it was quite expectable for citrate stabi-
lization. The result allows to conclude that PEG efciently isolated the nanoparti-
cles from the contact not only with the medium, but also with the cells. Similarly,
efcient isolation might be provided in the other case by dextrose, which acts as
both a reducer and a stabilizer which forms a dense protective shell on a
nanoparticle, such as other starch-like substances (see Chap. 4, Part I). The TEM
image obtained for the gold nanoparticles in [388] (Fig. 6.18) is indicative of this
possibility.
In this gure, rather than nanoparticles, a polymeric structure in which the
nanoparticles are embedded or wrapped is seen quite well; it is also difcult to
understand how the mean particle size (5 nm) indicated in the gure caption (cited
1.8 1.8 E. coli

1.6 1.6 Au 25 g/ml
1.4 1.4
OD at 600 nm
OD at 600 nm
Au 50 g/ml
1.2 1.2 Au 75 g/ml
1 1 Au 100 g/ml
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0 0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Time, h Time, h
Fig. 6.17 Comparison of the E. coli growth curves in normal medium and in the presence of Au
nanoparticles, testifying to the non-toxicity of nanoparticle [388]
20 nm
Fig. 6.18 TEM image of Au nanoparticles suggesting that nanoparticles have a size of about
5 nm [388]
from [388]) could be determined using such images. Besides, large nanoparticle
aggregates might predominantly occur in their aqueous solution to additionally
reduce their biological activity. Such a possibility follows from the particle sizes
reported to be >100 nm in solution as measured by PCS. These considerations
indicate that though the Au nanoparticles can indeed be biologically inactive, the
negative results of the above two studies are not sufcient for the denite
conclusion that an antimicrobial effect is absent toward E. coli, the more so toward
other bacteria species.
These results demonstrate once more how great an effect the stabilizing agent
can exert on antimicrobial activity of nanoparticles. A further illustration is pro-
vided by the work [397] where the -potential has been measured for Au
nanoparticles with various stabilizers used when nanoparticles are to be tested for
biological effects. In particular, a negative charge ( * 30 mV) was observed for
citrate-stabilized nanoparticles, while CTAB adsorption on their surface changed
the charge to positive ( * +23 mV). The change in the sign of the surface charge
is likely to affect biological, including antimicrobial, activities of Au nanoparticles.
To summarize, the analysis of the results of evaluating the effects of silver, gold,
copper, and metal oxide nanoparticles on microorganisms shows that the data
currently available are still insufcient for denitive conclusions as to how any of
the four main characteristics of nanoparticles affects their antimicrobial activity. In
our opinion, the only inference possible today is that the bactericidal effect tends to
increase with the decreasing particle size. Nevertheless, valuable observations made
in the relevant studies give materials for suppositions concerning the mechanism
underlying the antimicrobial activity of metal nanoparticles. A brief review of the
observations and the mechanisms discussed in the literature is given in the fol-
lowing section.
6.3 On the Mechanism of Antimicrobial Activity of Metal

Nanoparticles
As mentioned above, in studies of the past century nanoparticle adhesion to the cell
surface and adhesion-associated interactions of nanoparticles with each other and
with the cell membrane were considered to be the main events that occur during
interactions of colloidal metals with bacterial cells [80, 236, 237, 349]. An
important role in inhibiting bacteria was attributed to metal ions released from
nanoparticles into the medium near the membrane surface; for instance, a specic
binding of Ag+ ions with SH groups of surface and membrane proteins or, at high
nanoparticle concentrations, of internal cell structures were considered as respon-
sible for the inhibition of cell respiration and nutrition [80, 236]. Viewed in this
way, metal nanoparticles were regarded primarily as a source of ions, which acted
as a main factor that stipulates nanoparticle toxicity.
In the past 1012 years, studies of antimicrobial activity for various, including
metal, nanoparticles yielded data that help to better understand the mechanisms of
nanoparticle action, including their absorption, the role of metal ions, and specic
effects of nanoparticles. The results, their consequences for the medical use of
nanoparticles, and the role nanoparticles play in natural processes have been
summarized in several reviews [5, 338, 343, 350, 398400]. The mechanism of
action has mostly been discussed for Ag nanoparticles [338, 343, 399 and 400]
6.3 On the Mechanism of Antimicrobial Activity of Metal Nanoparticles 203
because just these nanoparticles are the subjects of the majority of studies. As noted in
the above and many other publications, the cause is that antibiotic-resistant bacterial
strains are increasing in number, and a pressing problem is to create a new type of
medicines (antiseptics) that are unlikely to induce resistance in microorganisms. Metals
are considered as promising candidates in searching for such remedies; of particular
interest are silver with its long-known bactericidal properties and silver nanoparticles as
an agent potentially more effective than the solid metal. Copper and metal oxide (MgO,
ZnO, Al2O3, and TiO2 [350]) nanoparticles have also been examined; examples of the
relevant studies have been considered in the previous section.
The mechanism of antimicrobial activity of nanoparticles is still far from being
fully understood, but it is possible to describe various events accompanying the
nanoparticles interaction with microorganisms in an aqueous medium using the
experimental data available. Microscopic studies have shown that antimicrobial
effect of Ag nanoparticles is accompanied with their adsorption on the cell surface,
impairment of the cell membrane integrity, and penetration into the cell in some
cases. These changes have been observed in experiments with E. coli [353, 359,
360, 368, 401], St. aureus [401], P. aeruginosa [358, 368], Salmonella typhus,
Vibrio cholerae, [368], nitrifying bacteria [362], etc. Example micrographs of cells
with adsorbed nanoparticles and associated structural alterations of the cell mem-
brane are shown in Figs. 6.19, 6.20, 6.21, 6.22 and 6.23.
Adsorption of nanoparticle aggregates on the cell surface was observed in
experiments with E. coli (Fig. 6.19); the membrane structure was altered, and
characteristic pits formed in the cell wall (Fig. 6.20), presumably reflecting cell
attempts to neutralize the effect of nanoparticles.
Pits and lesions in the E. coli membrane were reported in [355] as well, and
nanoparticles were additionally shown to damage membrane vesicles formed of cell
membrane fragments (Fig. 6.21). Disruption of the E. coli membrane by Ag
nanoparticles provokes the increase in its permeability; the internal cell compo-
nents, including sugars and proteins, are released; and respiratory chain enzymes
are inhibited to an extent increasing with the increasing nanoparticle concentration.
It is still unclear whether lipopolysaccharide- or protein-rich regions of the bacterial
outer membrane (cell wall) are responsible for nanoparticle penetration [355].
Experiments with St. aureus similarly showed that substantial alterations occur
both on and within the bacterial cell upon incubation with nanoparticles (Fig. 6.22)
[359].
Light DNA threads are visible within the normal cell on a TEM micrograph
(Fig. 6.22a); the threads are reduced to dots or disappear in cells incubated with
nanoparticles for 6 h (Fig. 6.22c), suggesting DNA condensation. Dramatic changes
occur in cell appearance, i.e., normal cells grown for 12 h have a smooth surface and
retain their coccal morphology (Fig. 6.22b), while after 12 h of incubation with
nanoparticles the cells are lysed, their membranes completely disrupted, and their
contents released (Fig. 6.22d). Cell disruption and release of the cell contents were
observed also in experiments with P. aeruginosa (Fig. 6.23) [358].
Based on the results of the above-mentioned and other similar studies, it is
possible to conclude that the interaction of silver nanoparticles with
1 m
Fig. 6.19 TEM micrograph of E. coli after incubation in liquid medium with Ag nanoparticles (at
50 g/mL for 1 h). Reprinted from Ref. [360]. Copyright 2004, with the permission from Elsevier
microorganisms includes (1) nanoparticles adsorption on the surface of a bacterial

cell or a virus particle; (2) structural and morphological changes resulting in the
nanoparticle adsorption; (3) penetration of nanoparticles into the cell; and (4) in-
hibition of cell functions (nutrition, respiration, and division), leading to cell death.
Metal ions released by nanoparticles and lipid peroxidation due to the generation
of ROSreactive oxygen species (superoxide anion radical *O2 and hydroxyl
radical OH*)upon nanoparticle absorption on the cell surface are considered as
playing a main role in membrane disruption and cell death (for information on
reactive species, see also Chap. 1, Sect. 1.4). The role of electrostatic interactions in
(a)
(b)
Fig. 6.20 SEM images of initial E. coli culture (a) and after incubation with 50 g/mL of Ag
nanoparticles for 4 h (b). Reprinted from Ref. [360]. Copyright 2004, with the permission from
Elsevier
nanoparticle adsorption has been discussed. A general notion of the processes

proceeding during the interaction of nanoparticles with bacteria is described by the
scheme cited from the review [350] (Fig. 6.24).
It is supposed that antibacterial activity of nanoparticles is based on lipid per-
oxidation and ROS production, occurring upon their contact with the cell surface.
The contact formation is controlled by the boundary forces, primarily electrostatic
interactions, namely by attraction and repulsion, respectively, of positively (1a) or
negatively (1b) charged nanoparticles and the negatively charged cell membrane.
Fig. 6.21 TEM image of (a)

vesicles formed by membrane
fragments of E. coli: (a) intact
vesicles; (b) after incubation
with 10 g/mL of silver
nanoparticles. Reprinted from
Springer
100 nm
(b)
100 nm
Processes that change the charge of nanoparticles are capable of indirectly affecting
their interaction with the bacterial cell (1c). Once in contact with the cell,
nanoparticles cause irreversible disruption of the cell membrane by inducing ROS
production (2d) and penetrate into the cell interior (2e). Cell disruption may be
hindered by exopolymers (2f), which include saccharides and proteins that are
secreted by the cell and are often found in bacterial biolms. In the environment, a
contact of nanoparticles with the cell membrane may be hindered by various
physicochemical processes, such as aggregation and precipitation of nanoparticles
at a higher ionic strength (3g), complexation with natural substances (e.g., humic
acids), and adsorption on mineral particles in soil (3i).
(a) (b)
(c) (d)
Fig. 6.22 The effect of Ag nanoparticles on St. Aureus [359]. For details, see text. Reprinted from
Ref. [359]. Copyright 2010, with the permission from Springer
An active role assumed for ROS is supported by experiments where antioxidants

capable of ROS binding alleviated or abolished the biocidal effect of silver
nanoparticles [358, 362, 402]. The results of an example experiment are shown in
Fig. 6.25 cited from [358].
In this experiment, N-Acetylcellulose (NAC) and ascorbic acid (AA) were used
as ROS scavengers. As shown in the gure, the two antioxidants efciently protect
the cells from nanoparticles. Upon exposure to 4 g/mL of Ag nanoparticles,
100 % of cells remained viable in the presence of AA and about 70 % in the
presence of NAC, while 100 % of P. aeruginosa cells were killed in their absence.
The result indicates that ROS take part in biocidal activity of nanoparticles. At the
same time, factors other than ROS may also play a role in nanoparticle toxicity, as
was noted in [362]. It is well-known that metal ions are assumed to act as such a
factor.
(a) (b)
Fig. 6.23 SEM image of P. aeruginosa bacterium before (a) and after (b) incubation with silver
nanoparticles. Reprinted from Ref. [358]. Copyright 2010, with the permission from Springer
Fig. 6.24 Basic mechanisms

of antibacterial activities of 1 + (c)
+ +
nanoparticles. See text for + +
+ + +
explanations. Reprinted from
Ref. [350]. Copyright 2008, (a) (b)
Springer
2
(d) (f)
(e)
ROS
3 (h)
(g) (i)
(a)
(b)
AgNPs + AA
AgNPs + NAC
AgNPs
AA
NAC
0 50 100 150 200 250

Number of viable cells (CFU)
Fig. 6.25 Effect of antioxidants on bactericidal activities of Ag nanoparticles when interacting

with P. aeruginosa. a Bacterial growth in Petri dishes: top left control culture; top right Ag
nanoparticles; bottom left Ag nanoparticles +10 mM NAC; bottom right Ag nanoparticles
+10 mM AA. b The number of viable cells in the presence of antioxidants and 4 g/mL of Ag
nanoparticles [358]. Reprinted from Ref. [358]. Copyright 2010, with the permission from
Springer
A role of metal ions was checked by (1) studying the effect of silver ions used at
the same concentrations as in experiments with nanoparticles [295, 368] and
(2) estimating the concentrations of metal ions released into the medium by
nanoparticles. [351, 368]. Discrepant results were obtained in either case. In the
study [295] performed with E. coli, Ag+ ions exerted a considerable bactericidal
effect, being similar in LD to nanoparticles, while nanoparticle adsorption on the cell
surface was absent. However, another study with the same bacterium [368] showed
that nanoparticles were intensely adsorbed on cells and that silver ions and silver
nanoparticles greatly differed in effect, i.e., a DNA-rich zone formed in the central
region of the cell upon exposure to silver ions, while nanoparticles did not exert this
effect, but penetrated into the cell and were regularly spread through its interior.
The concentrations of silver ions released by nanoparticles into the culture
medium were measured in [351] and proved to be approximately one order of
magnitude higher than the concentrations of ions released by nanoparticles into the
equimolar NaNO3 solution as found in [368].
According to the authors of the study [351], the ndings show that both
nanoparticles and metal ions released are responsible for the bactericidal effect of
Ag and Cu nanoparticles; ion binding to the negatively charged cell wall and
bonding with sulfhydryl and/or phosphate groups of proteins leads to membrane
disruption and cell death. At the same time, the authors of the work [368] did not
nd any ground to assume an appreciable role for silver ions in the effect of
nanoparticles.
Such discrepancies might arise because different methods were used to obtain
nanoparticles, i.e., evaporation and condensation in a carbon matrix to produce
uncapped nanoparticles in [368] and chemical reduction in solution in [295, 351].
As a result, the nanoparticles differed in surface properties (surface charge) and
shape (judging from the TEM images present in these publications).
Thus, for today there are grounds to believe that both metal ions and ROS may
mediate the bactericidal effect of silver nanoparticles. However, it remains unclear
whether electrostatic interactions really play a key role in nanoparticle adsorption
on the bacterial cell (Fig. 6.24). First, as pointed out by several authors [350, 360,
et al.], nanoparticles with a negatively charged stabilizing coating in many cases
were found to possess a high bactericidal activity. Second, there is still no con-
vincing evidence that a positive charge ensures a higher antimicrobial activity of
nanoparticles as compared to a negative charge, all other conditions being the same.
As noted above when discussing Table 6.1 (Sect. 6.1.1), for the reliable determi-
nation of the role of surface charge, further experiments should be performed to
satisfy the necessary conditions, namely that the nanoparticle preparations studied
should be different only in the magnitude and/or sign of the surface charge.
Concerning the causes of the influence of nanoparticles size on their antimi-
crobial activity, the additional interesting results were obtained in a study of the
effect Ag nanoparticles isolated from a carbon matrix exert on virus (HIV) particles
and bacteria. Nanoparticles of about 5 nm were selectively adsorbed on E. coli cells
from a polydisperse sample with a particles size of 21 18 nm; the small
nanoparticles were found to penetrate into the cell and to spread through its inner
volume, the events being associated with size-specic local changes in the electron
structure of the nanoparticles [368]. As detected in the same study, only
nanoparticles of 110 nm were adsorbed from a polydisperse sample in experi-
ments with all bacteria, including E. coli, P. aeruginosa, S. typhus, and V cholerae.
Selective adsorption of similarly sized Ag nanoparticles was observed earlier on
HIV particles by the same research team [363] (Fig. 6.26). According to the authors
of [368], the nding suggests higher biocidal activity for nanoparticles with sizes
close to the lower limit of the nanoscale range, in line with the above-mentioned
tendency to an increase in antimicrobial activity with the decreasing size of
nanoparticles based on the results reported in [295, 353, 354].
However, it is evident from Table 6.1 that this conclusion disagrees with the
results of other studies where silver nanoparticles of about 3 nm were less active
[351] and those of 45 10 nm were more active [358] than the particles examined
in [368], as judged from the TT and LD values. The difference might be connected
with different methods used to obtain nanoparticles, like with the discrepancies
considered above when discussing the role of ions.
Taken as a whole, studies on the mechanism of nanoparticles action can be
divided into two groups differing in the extent of agreement between the results
(a)
20 nm
Fig. 6.26 HAADF scanning TEM image of the HIV virion with adsorbed BSA-conjugated Ag
nanoparticles [363]
obtained by different researchers: (1) studies on the processes (adsorption, mem-

brane disruption, etc.) registered by microscopic or biochemical methods which
yield mainly consistent results and (2) elucidation of the causes of these processes
(including the roles of metal ions, ROS, and nanoparticle size and charge), where
the discrepancies arise between the results reported by different research teams. It is
easy to see that the discrepancies are the natural consequences of the absence of
reliable conclusions regarding the effects the main characteristics of nanoparticles
exert on their antimicrobial activity, as was demonstrated above while discussing
the experimental data summarized in Tables 6.1 and 6.3. We think that the situation
stems mainly from underestimation of the importance of the method used for the
nanoparticle preparation, for achieving the aim of an experiment.
A similar conclusion can be drawn from the results of metal nanoparticle testing
for cytotoxicity on animal and human cultured cells. Judging from the literature
available, in the majority of cases the nanoparticle size has been considered to be a
crucial parameter, while little attention has been paid to the other characteristics
important for biological activity. As illustration may serve Table 6.5, where the
data are summarized on the main characteristics of silver nanoparticles tested for
cytotoxicity for several past years. As shown in the table, nanoparticles obtained by
different methods were used in the studies; the majority of samples were com-
mercial powders produced by plasma technology. Even the particle size has not
been checked in some cases [403, 404], not to mention that the shape, the surface
charge, and the composition and properties of the surface have been disregarded in
organization of experiments. Meanwhile, as mentioned above, the size and shape of
particles in the powders can vary greatly, so that it is difcult to be certain that the
particles are mostly similar in shape and fall within the nanoscale range in size.
For instance, TEM images have been given in [405] for two nanoparticle samples
examined, which were obtained from different manufacturers (Fig. 6.27): One
sample (on the left) contained aggregates where the mean size and shape of particles
cannot be determined, and the other represented a polydisperse system with particle
sizes varying from less than 10 nm to more than 100 nm (at the magnication
indicated). In other cases, nanoparticles were small in size and had a narrow size
distribution in the original sample, but aggregated in the culture medium; e.g., in the
work [407], interaction with cells was actually tested for aggregates of an arbitrary
shape and a size of 100300 nm, instead of the particles indicated as being 510 nm
in size, so that it is not feasible to elucidate the mechanism of the nanoparticles
action or the possibility of their medical application.
Several studies were aimed at clarifying the effect on cytotoxicity of the particle
size [409] or shape and the nature of a stabilizing coating [410]. However, in the
former case, PVP was used as a stabilizer, which was shown to substantially reduce
antimicrobial activity of silver nanoparticles in experiments with microorganisms
[364], so that the effect of the size was hardly accessible, the more so because
differences in the number concentrations of nanoparticles were disregarded, like in
the other similar experiments. In the latter case, the effect was compared for
PVP-stabilized nanoparticles of different shapes (spherical and triangular); the
Table 6.5 The effect of silver nanoparticles on cultured animal and human cells
Method/ Characteristics of Cells Reference
Manufacturer nanoparticles
Size, nm Shape
Charge Shell
Plasma 15 ND Spermatogonial stem [403]
technology/ ND ND cells of C18-4 germ
Air Force Research line
Laboratory
ND/Air Force 15 ND Rat liver cells [404]
Research ND BRL #A (ATCC,
Laboratory 100 ND CRL-1442)
ND/Ching-Tai 1100 ND Mouse broblasts [405]
Resin, Sun-Lun ND ND NH3T3
International
Biotechnology
ND/dispersed after 620 ND Normal human lung [406]
lyophilization ND Starch broblasts NMR90
Human glioblastoma
U251
ND/Nanopoly 510 Spherical Human hepatoma [407]
(Seoul, Korea) ND ND HepG2
Aggregates
ND/Kyoto Nano 710 ND Human hepatoma [408]
Chemical, Ltd ND PEI HepG2
ND/Huzheng 5 Spherical Human cells A549, [409]
Nano Technology, ND PVP SGC-7901, HepG2,
Ltd., CC 20 Spherical MCF-7
(reduction ND PVP
with sodium 50 Spherical
hypophosphite) ND PVP
Citrate + NaBH4 30 Spherical Human skin [410]
ND PVP keratinocytes
ND Citrate HaCat
Triangular
PVP
particles were similarly inactive that was quite expectable with this stabilizing
agent. However, it does not follow from the result that the biological effect is
independent of the particle shape. In [410], the effects of spherical nanoparticles
obtained with different stabilizing agents, PVP, and citrate, were also compared.
The nanoparticles stabilized with citrate were found to be far more toxic than the
PVP-coated nanoparticles; yet, an influence of the nature of a stabilizer is impos-
sible to infer from the nding because the difference in the magnitude (and, pos-
sibly, in the sign) of the particle charge is unknown for the coatings.
Another problem arises when the aim is to compare the antimicrobial properties
and cytotoxicity for a given nanoparticle sample in order to evaluate whether the
(a) (b)
100 nm 40 K 20 nm 100 K
Fig. 6.27 TEM images of silver nanoparticle samples used in the work [405]. a The sample from
Chig-Tai Resin, 40,000; b The sample from Sun Lun International Biotechnology, 100,000.
Reprinted from Ref. [405] Copyright 2008, with the permission from Elsevier
working nanoparticle concentrations established in experiments with bacteria are

dangerous for mammalian cells. Such a comparison should be performed, for
instance, when creating a medicine on the basis of silver nanoparticles, to ensure
that the nanoparticle concentration chosen for the medicine produces the expected
therapeutic effect and is safe enough for use. The problem is that cytotoxicity
testing has not been performed in the vast majority of studies evaluating the
antimicrobial properties for nanoparticles of a given kind. We are aware of only two
publications which reported the results obtained on cells for Ag nanoparticles with
known antimicrobial activity [411, 412]. According to these two publications,
cytotoxicity is observed at nanoparticle concentrations higher than required for a
considerable antimicrobial effect, as has been estimated for nanoparticles of 720
nm with broblasts and of no more than 50 nm with mesenchymal stem cells. The
nding indicates that silver nanoparticles can be safely used as an antibacterial
agent in the relevant diseases.
All other studies with microorganisms and animal cells utilized nanoparticles
differing in originobtained by different methods and often differed in size, to say
nothing of the shape, surface charge, and the way used for stabilization (or its
absence). As shown, for instance, in Tables 6.1 and 6.5, even commercial
nanoparticles were purchased, as a rule, from different manufacturers to use for the
studies with bacteria and mammalian cells. The studies [355, 359] (bacteria) and
[409] (cultured human cells) are the only exception, utilizing nanoparticles of the
same size (5 nm) from one manufacturer (Huzheng Nanotechnology Company).
Yet, the effects on bacteria and human cells cannot be compared even in this case
because in experiments with bacteria nanoparticles were used without a stabilizer,
while in experiments with cells they were stabilized with PVP, which substantially
reduces the biological activity of nanoparticles.
6.4 Summary 215
6.4 Summary
In general, we can conclude that intense development of nanotechnologies posi-

tively affected the related biomedical research area where biological effects are
investigated for nanoscale particles and materials, including metal nanoparticles, on
objects of various complexity levels. Our analysis of the results obtained in
studying the effects of metal nanoparticles on microorganisms makes it clear that
the ranges of microbial species and methods to produce nanoparticles have greatly
expanded over the past decade, and the technical level of experiments have grown
higher, i.e., new possibilities of microscopic methods have been used and the
methods to analyze the composition and structure of nanoparticles improved, as
well as the methods to study biochemical reactions as indicators of cell viability.
However, as is often the case, technological progress has given rise to new prob-
lems, and the results of scientic research in the eld can hardly be expected to nd
full-scale application without their solution.
The main problem is, in our opinion, that a rapid growth in number of the
methods to obtain metal nanoparticles which is fully enough reflected, as we hope,
in Chap. 1 of this book, is accompanied by the underestimation of the specic
character of nanoparticles as a factor that affects a given biological object. Under
the specic character, we mean here that biological activity of nanoparticles
depends on their main characteristics: the size, shape, surface charge, the compo-
sition and structure of the stabilizing coating; a substantial role of each of the
characteristics has become especially evident in the recent years. More exactly, for
the correct determination of the working concentration for given metal nanoparti-
cles against a given microorganism, a particular combination of the main charac-
teristics is of importance, and this combination is determined by the method used to
obtain the nanoparticles. As can be seen on the example of silver nanoparticles, in
the vast majority of cases the effect on a particular microbial species has been tested
for nanoparticles obtained by different methods; this circumstance could be a
(possibly key) factor responsible for the differences in values reported for the
toxicity threshold and lethal dose as main parameters of nanoparticle activity
(Table 6.1). Other factors related to the biological aspect of experiments can be
added, including the medium composition, the assay (experimental conditions)
employed, and the strain origin.
As a result, actually it turns out to be rather difcult to establish the working
concentration for given metal nanoparticles against a given microbial species
because the results of laboratory experiments are valid only for particular cases, that
is, for nanoparticles with a certain combination of characteristics, that have been
tested in certain conditions with a certain microbial species and/or strain; such
results cannot be mechanically extrapolated to some or another application of
nanoparticles in sanitary or medicine.
What answers are now possible for the questions posed when formulating the
objective of our literature analysis? We can state that data currently available in the
literature are not comprehensive enough both for the determination of influence of
the main nanoparticle characteristics (the size, shape, surface charge, and the
composition of the stabilizing coating) on their biological activity and for the
grounded choice of the nanoparticles best suitable for a particular application in
medicine or bionanotechnology. There is no ground, however, for thinking that this
is the consequence of the inefciency of horizontal approach to investigation; the
reason may be, rst of all, in that insufcient attention is payed to the method-
ological requirements necessary for the correct solution of the task confronting the
researchers. Specically, to detect the effect for one of the four main characteristics
of nanoparticles, it is essential to make sure that the other three characteristics
remain constant. We are certain that this requirement is possible to satisfy by using
the potential of nanoparticle production methods in a purpose-oriented manner and
correcting the experimental conditions and that the questions that are unclear now
will thereby be claried in the nearest future.
In view of the above arguments in favor of the importance of the method used
for nanoparticle preparation, we found it expedient to utter some general consid-
erations on the potentialities of various methods, as we hope they may be useful for
further developments in the eld discussed here. Our literature analysis shows that,
to a rst approximation, the methods employed can be divided into two groups:
(1) nanoparticle preparation by chemical reduction in solution and (2) nanoparticle
preparation by high-temperature evaporation and subsequent condensation.
The rst group includes primarily traditional methods of synthesis in an aqueous
solution (see Chap. 1), which yield nanoparticles that have a stabilizing shell and a
size from several units to several tens of nanometers. This makes it possible to vary
the nature of a stabilizing agent and its surface charge and thus to change the
antimicrobial activity of nanoparticles in accordance with a given purpose. For
instance, a bactericidal effect of nanoparticles can be decreased or increased by
using as stabilizer, respectively, a polymer or a low-molecular-weight anionic
surfactant [364]. A relatively narrow distribution is possible to achieve in some
cases for small nanoparticles (up to 15 nm) [295, 351, 360, 364], which is essential
for studying the size influence on the antimicrobial effect. To study the influence of
the shape, nanoparticles that are similar in size and differ in shape can be obtained
by selecting the conditions of their synthesis [272, 399]. Thus, chemical reduction
provides an opportunity to establish a relationship between important characteristics
of nanoparticles and their effect on microorganisms, which is essential for under-
standing the mechanism of nanoparticlecell interactions, that is, for the creation of
scientic grounds for the subsequent applications. At the same time, special
knowledge and experience are necessary for producing nanoparticles by these
methods, and it is not always possible to correctly combine in one study preparation
of nanoparticles with desirable characteristics and their testing for antimicrobial
activity, so that in the end a reliable result could be obtained. Examples of the
problems arising here have been described in the previous sections.
The second group nds increasing application in microbiological studies over
several past years. Apart from the fact that a broader set of methods becomes
available as nanotechnology industry grows and expands the range of its products, a
possible cause is that biologists involved in relevant researches try to simplify the
6.4 Summary 217
experimental procedures by using commercial nanoparticles from various institu-

tions or companies specializing in their manufacture. Commercial preparations are
especially important to use when a study is aimed at the creation of a product for
medicine (e.g., a remedy) because the aim requires the nanoparticles to be con-
tinuously produced in the necessary amounts. However, poorer quality of
nanoparticles is an inevitable cost of this simplication, since commercial prepa-
rations are usually polydisperse samples, the particle size varies from several tens to
several hundreds of nanometers, and the particle surface is oxidized or unprotected
with a stabilizer, so that the particles rapidly aggregate and precipitate in solution,
and this generates additional problems in carrying out experiments, as has been
noted above. When oxidized nanoparticles are used, activity is actually determined
for metal oxides, but not for metals in a nanosized state, and this is not always
acceptable in terms of their efciency and safe use.
In summary, it is clear that choice of the method for nanoparticle preparation
plays an important role in studying their antimicrobial activity. The choice should
be determined, in principle, rst, by the aim pursued in a studyeither elucidation
of the general regularities of the nanoparticle interaction with microorganisms or
solution of applied problems and second, by the extent of compliance of the method
with the necessary requirements listed at the end of Chap. 1 of this book. From this
point of view, an optimal method is one that makes it possible to pursue both the
aims mentioned and satises the above requirements as fully as possible.
Our long-term experience in studies of the biological effects of metal nanopar-
ticles has shown that our method of biochemical synthesis may become, with time,
one of those which deserve to be regarded as optimal. Results described in the
further chapters of this book will help the reader to judge whether our opinion is
grounded.
Chapter 7
Antimicrobial Activity of Nanoparticles
Stabilized with Synthetic Surfactant
As was noted in the previous chapter, up to now the horizontal strategy has
dominated in the studies of biological activity of metal nanoparticles; i.e., various
metal (mostly silver, gold, and copper) nanoparticles have been used in studies on
one biological object or different objects of the same organization level (e.g., dif-
ferent bacterial species or different bacterial strains of one species). Unquestionable
progress in the eld has been achieved primarily owing to improvements in the
technical means, allowing visualization of nanoparticles and the processes of their
interaction with microorganisms and mammalian cells, as well as analysis of the
nanoparticle structure, composition, and surface properties. However, it is still
impossible to reveal, rst, the effects of the main characteristics (the size, shape,
surface charge, and the composition of a stabilizing coating) of nanoparticles on
their biological activity and, second, the roles the corresponding metal ions and
reactive oxygen species play in this activity. In our opinion, advances in solving
these problems by following the horizontal strategy are possible to make by
eliminating at least one of the obstacles, namely differences in nanoparticle char-
acteristics that result from differences in methods used to obtain nanoparticles; i.e.,
nanoparticles with a denite set of characteristics are to be used. Besides, it may be
useful to follow the vertical strategy with such nanoparticles, that is, to study
objects staying on different organization levels and thereby to ascertain the specic
responses of objects differing in complexity, from bacteria to mammals.
This and the subsequent chapters present the results of our attempts to translate
these possibilities into action, using silver and copper nanoparticles obtained by
biochemical synthesis in micellar solution and also aqueous solutions of silver
nanoparticles coated with synthetic (anionic surfactant, AOT) and natural
(water-soluble starch or cyclodextrin) stabilizers.
In this chapter, we describe the results of testing the antimicrobial properties of
Ag and Cu nanoparticle solutions and nanoparticle-modied materials. Silver
nanoparticles were used as a standard micellar solution and aqueous solutions

DOI 10.1007/978-3-319-30906-4_7
220 7 Antimicrobial Activity of Nanoparticles
prepared by the transfer from this micellar solution; here, the nanoparticles were
910 4 nm in size. Cu nanoparticles were used as a micellar solution, and the
mean particle size was no more than 15 nm. Further chapters include the data on the
toxic effects (registered as viability changes) of aqueous solutions of silver
nanoparticles, obtained in experiments with various living organisms. In addition,
we studied the influence of the particle size and particle charge. The size effect was
observed for plasmodium of a lower fungus, using aqueous dispersions of silver
nanoparticles, which were small in size, had a narrow size distribution, and were
obtained as described in Chap. 3 (Sect. 3.1.2). The charge effect was determined for
human cultured cells treated with nanoparticles similar in size and shape, but dif-
ferent in zeta potential, measured in conditions when the zeta potential really
reflects a surface charge density of the particles.
7.1 Ag and Cu Nanoparticles in Liquid Medium
Antimicrobial activity testing of silver nanoparticles was carried out in the Sysin
Institute (Russian Academy of Medical Sciences), Gamaleya Institute (Russian
Academy of Medical Sciences), Institute of Genetics and Selection of Industrial
Microorganisms (State Research Center), and Moscow City Center of Disinfection.
Biocidal effect testing of copper nanoparticles was performed in the Gamaleya
Institute (Russian Academy of Medical Sciences) and Institute of Paints with
Experimental Engineering afliated with Plant Viktoriya (in the Laboratory of
Varnish-Paint Technologies). The results were presented as reports, conference pre-
sentations, and published articles [9, 10, 316, 329, 413, 414]; the most complete
collection of the results obtained in the studies of the effects of silver nanoparticles in
micellar and aqueous solutions may be found in the review [348] and dissertation [28].
7.1.1 Varnish-Paint Materials with Biocidal Properties
Micellar and aqueous solutions of silver nanoparticles were used as minor additions
to VPMs of various compositions based, respectively, on organic solvents and water.
The resulting modied paints were used to spread on samples of wood or other
materials; after drying, bacterial suspensions were placed onto the painted surfaces,
the surfaces were washed at various time intervals, and viable bacteria were counted
in the washing fluids. The paints with silver nanoparticles were shown to possess
pronounced bactericidal activity toward several pathogenic bacteria often found in
everyday life, while the control paints without nanoparticles did not display such an
effect [9, 10]. Example testing results are given in Tables 7.1 and 7.2. As shown in
7.1 Ag and Cu Nanoparticles in Liquid Medium 221
Table 7.1 Dynamics of bactericidal effects of water-based paint containing silver nanoparticles
(AgNPs) on various bacteria strains.* Data from the Gamaleya Institute
Strain tested Control culture (+) Log of living bacteria numbers on the surface of
and paint with painted samples at different time intervals (hours)
AgNPs () after the culture application
0 1.0 2.0 4.0
Escherichia coli + 6.2 0.1 6.2 0.1 6.2 0.1 6.2 0.2
ATCC 25922 4.8 0.2 0 0 0
Salmonella + 6.1 0.1 6.1 0.1 6.0 0.2 6.0 0.2
typhimurium 5.1 0.2 0 0 0
TMLR 66
Salmonella + 6.0 0.1 6.1 0.2 6.0 0.1 6.0 0.1
typhi Ty 2 4.6 0.1 0 0 0
Shigella flexneri + 6.2 0.2 6.2 0.2 6.2 0.2 6.1 0.1
516 4.5 0.1 0 0 0
Staphylococcus + 6.0 0.1 6.0 0.1 6.0 0.1 6.0 0.1
aureus Wood 46 5.4 0.1 0 0 0
Enterococcus + 6.1 0.1 6.1 0.1 6.1 0.1 6.1 0.1
faecalis CG 110 4.9 0.1 0 0 0
Listeria + 6.2 0.2 6.2 0.2 6.2 0.1 6.1 0.1
monocytogenes 4.6 0.1 0 0 0
EGD
Pseudomonas + 6.1 0.1 6.1 0.1 6.0 0.1 6.0 0.1
aeruginosa 508 5.6 0.2 3.4 0.2 1.2 0.2 0
*Nanoparticle concentration was 2.1 gAg/mL of paint
Table 7.1, after culture placement on the surface covered with a paint containing a
minor addition of a micellar solution of nanoparticles, complete death of all bacteria
except for Pseudomonas aeruginosa was observed as early as in 1 h. Even the most
resistant P. aeruginosa cells died in 4 h. The results of the tests showed that the
bactericidal effect of modied paints is preserved for at least two months. Other
experiments additionally demonstrated that nanoparticles contained in paints possess
a higher biocidal activity toward bacteria than toward fungi and spores.
Table 7.2 summarizes the results obtained with enamel and water-based emul-
sion paint to characterize the effect nanoparticles exert on the bacterium E. coli and
the coliphage MS-2 (a virus that infects this bacterium). A higher extent of inac-
tivation after a particular exposure time on nanoparticle-containing paints compared
with a control was observed for both the bacterium and the virus; the effect was
more distinct at short exposition times (0.52 h). The results of the above and other
laboratory tests made it possible to create paint compositions with silver
nanoparticles for testing in schools, hospitals, and one of the Moscow jails; studies
Table 7.2 Biocidal effect of enamel and water-based emulsion paint modied with silver
nanoparticles (AgNP).* Data from the Sysin Institute
Microflora Exposure time Enamel % of Emulsion paint % of
inactivation inactivation
Control +AgNP Control +AgNP
E. coli 0.5 h 0 0 24.08 84.63
1h 14.29 31.83 18.85 98.22
3h 100 100 99.71 99.07
24 h 100 100 99.99 99.90
MS-2 phage 2h 92.4 99.9 0.44 53.62
24 h 99.5 99.9 59.08 91.42
3 days 100 100 100 100
7 days 100 100 100 100
*Nanoparticle concentration was 1.62 gAg/mL of paint
were performed in cooperation with the private company AOZT Lakma-Imeks.

The results of testing carried out in the pretrial detention center Matrosskaya
Tishina are summarized in Table 7.3 [10, 415].
Various internal surfaces (the walls, ceiling, etc.) of two jail cells were painted
with the same paints of various brands; silver nanoparticles were added to the paints
in one cell, and the other cell was used as a control. Washing fluids were obtained
from the painted surfaces of the two cells at certain time intervals and tested for the
presence of various microorganisms. It is seen that the addition of nanoparticles to
paints appreciably reduced the total microbial count, that is, the total microbial
contamination of the painted surfaces in the cell. The effect of nanoparticles toward
bacteria and viruses (coliphages) was greater than toward fungi and spore-forming
microorganisms, like in the laboratory tests.
It should also be noted that, in contrast to the majority of other biocidal agents
widely used in paint industry, Ag nanoparticles are substantially less toxic to
humans and more environmentally friendly, as was conrmed by the corresponding
hygienic certicates. Hence, silver nanoparticle-containing paints and coatings hold
great promise for use in health care, childcare, sports, penitentiary, public catering
institutions, and other places with higher-level infection contamination.
Paints with silver nanoparticles were protected by a patent [315]. It was shown
also that it is possible to add nanoparticles to paints by spreading on powdered paint
components, e.g., aluminum oxide (see Chap. 5, Fig. 5.6).
Micellar solutions of copper nanoparticles showed positive results in testing of
modied paints [316, 416] used to protect machinery working under water, such as
ship hulls and constructions installed in river or marine waters (water supply pipes
and oil platforms), from biofouling, that is, attachment of various organisms:
mussels, small crustaceans, calcareous tube worms, and marine algae. Biofouling
Table 7.3 Effect of the paints containing silver nanoparticles on different microorganisms on the painted surfaces of cells in the pretrial detention
center Matrosskaya tishinaa
Object Sampling Index
point TMNb E. coli Staphylococci Fungi Sporous Viruses Coliphages
Total Aureus Total Mold Yeast-like
number number
Control Door 500 0 3000 0 6500 5000 1500 10 150
Toilet room 2200 30 660 20 12000 6000 6000 100 + 400
7.1 Ag and Cu Nanoparticles in Liquid Medium
Wall 100 0 100 0 4000 3000 1000 20 100

Ceiling 200 0 500 0 5000 3000 2000 10 150
AgNP Door 10 0 100 0 3000 1000 2000 20 20
Toilet room 200 15 2300 10 6500 2500 4000 100 + 50
Wall 10 0 100 0 1200 200 1000 0 20
Ceiling 20 0 300 0 4000 3000 1000 0 30
a
In columnssurface concentration of microorganisms (cells/100 cm2). Nanoparticle concentration was 6.4 gAg/mL of paint
b
Total microbial number
223
poses many problems, from a decrease in the efciency of fuel use to loss of
construction stability. A common practice is to control marine biofouling by cov-
ering the construction surfaces with special paints, which can be divided into bio-
cidal and those which prevent deposition of the biological contaminations. Biocidal
properties are conventionally ensured by adding highly toxic chemicals to paints.
Since more stringent requirements to use environmentally friendly paints have been
imposed, especially for bioprotection of vessels and constructions exploited in water,
searching for new means to create low-toxic biocidal coatings is a pressing problem.
Cuprous oxide is used up till now as a biocidal agent in paints to cover the
underwater parts of vessel hulls. Cuprous oxide is added to paints in large amounts,
approximately 5060 % of the total formulation. This deteriorates the solidity and
protective properties of coatings. Moreover, cuprous oxide is low efcient against
particular marine and river organisms and should be used in combination with other
highly toxic biocides.
The objective of our work was to nd out whether copper nanoparticles, which
possess pronounced biocidal and catalytic activities, may be used as a less toxic
addition to paints.
Micellar solutions with the Cu nanoparticle concentration 2.2 mM were added to
the khaki Kornika anticorrosion epoxide composition and the black UR-1524
enamel at 2 % by volume (2.8 g/mL of paint). The paints were chosen for
modication with Cu nanoparticles because they are recommended for use in
protecting machinery working under water and have been included in the guidelines
on vessel painting with paints made in Russia and abroad. The Kornika and
UR-1524 coatings are highly waterproof, but lack biofouling resistance. The paints
therefore cannot be used alone and are combined with biocidal coatings; hence, the
painting procedure becomes more laborious and expensive.
Copper nanoparticles were added to ready-to-use compositions prior to diluting
them to a working viscosity with a solvent. The materials were thoroughly mixed
after adding and let to stay for 20 min. Two enamel layers of 100 2 m each were
used for coating the prepared steel samples by pneumatic spraying. Drying was
carried out in ambient conditions according to the paint specications.
The coating appearance did not change after adding a micellar solution of copper
nanoparticles; the surface was smooth and even and lacked defects, indicating that
the micellar solution of copper nanoparticles was well compatible with the original
paints (Fig. 7.1).
The modied coatings were tested for mechanical properties (adhesion and
impact resistance), resistance to salt water, and resistance to biofouling. The
mechanical properties by the two parameters and the resistance to salt water of the
nanoparticle-modied paints were found to be no inferior or even superior to those
of the non-modied samples. To evaluate the biocidal properties of the paints, eld
testing was performed in the river Pazha, Moscow region. Biofouling with
Cladophora algae was not observed on copper nanoparticle-containing samples
Fig. 7.1 Samples of biocide-containing coatings. From left to right: cuprous oxide; copper
nanoparticles (enamel UR-1524); copper nanoparticles (Kornika)
over three months of eld testing. Control (non-modied) samples were affected by
biofouling in as early as one month of exposure.
The results make it possible to conclude that, after proper testing with river or
marine vessels, epoxide and polyurethane paints modied with copper nanoparti-
cles may provide a more efcient biofouling protection for vessel hulls as compared
with current coatings.
Taken together, the results of testing paints modied with Ag and Cu
nanoparticles allow us to establish the nanoparticle concentration range that ensures
sufciently high antimicrobial activity against a broad range of pathogenic bacteria:
The range is 17 g/mL of paint.
7.1.2 Water Solutions of Ag Nanoparticles
Aqueous solutions of silver nanoparticles were tested both as additions to paints and
as agents added to aqueous solutions containing suspensions of pathogenic bacteria
of various strains in various concentrations. In the latter case, the effect of
nanoparticles was evaluated by taking samples at various time points and com-
paring the CFU counts with those of control solutions, which contained bacteria
without nanoparticles. In all cases, substantial antibacterial or antiviral activity was
observed for the nanoparticle solutions [10]. As an example, Fig. 7.2 shows the
results obtained with E. coli in an aqueous medium [348].
100
Inactivation level, %
95
90
0 5 10 15 20 25 30 35
C(AgNP), g/ml
Fig. 7.2 The effect of silver nanoparticles on E. coli inactivation level in aqueous medium. AgNP
concentration was varied by dilution of the nanoparticle stock water solution; initial nanoparticles
and AOT concentration were 60.5 gAg/mL and 20 mM, respectively. Suspension was incubated
at each AgNP concentration for 30 min, then the nanoparticles were inactivated. Initial
concentration of bacterial cells3 108 CFU/mL. Data from the Institute of Genetics and
Selection of Industrial Microorganisms. This gure and Fig. 7.3 were reprinted from Silver
nanoparticles: optical properties, characterization and applications, pp. 221258, copyright 2010,
author E.M. Egorova, with permission from Nova Science Publishers
At the high initial bacteria concentration (3 x 108 cells/mL) and short exposure
time, high inactivation level (90100 %) was provided in a wide range of dilutions
(up to 75 fold) of the aqueous stock solution of nanoparticles.
Similar experiments were carried out in the Sysin Institute to compare the effect
on the bacterium E. coli and phage MS-2 between nanoparticles and Ag+ ions
(added as silver nitrate) in the corresponding concentrations. Bacterial and phage
suspensions in an aqueous medium were prepared, and each of them was incubated
with either Ag nanoparticles or silver nitrate in an equivalent concentration. As was
observed with the three chosen nanoparticle concentrations (10.8, 6.5, and
3.2 g/mL), Ag nanoparticles were more effective than Ag+ ions. Figure 7.3 shows
the results obtained with 6.5 g/mL nanoparticles with E. coli (a) and phage MS-2
(b). With bacteria, a greater effect of nanoparticles was apparent after 30-min
incubation and less distinct after 1 h, while their effect was almost indistinguishable
from that of silver ions after 24 h, inactivation being 100 %. Phage death in
response to nanoparticles occurred far more rapidly than in response to silver ions
used at the same concentration in solution, although the highest inactivation level
(98.2 %) did not reach 100 %; the nding means that the virus is less sensitive to
the effect of nanoparticles as compared with the bacterium.
High-level bactericidal activity of nanoparticles added to an aqueous medium
was demonstrated also in trials carried out with E. coli and St. aureus at the
Moscow City Center of Disinfection [414] (Table 7.4). For the two bacteria species,
the lower limit of nanoparticle concentration in an aqueous medium that caused
100 % death of bacteria in 30-min incubation was found to be 2.88 g/mL. Data
Fig. 7.3 Inactivation (a)

dynamics for E. coli (a) and 6.0
MS-2 phage (b) in aqueous AgNP
5.5
medium after the introduction AgNO3
5.0
of silver nanoparticles and
Ag+ ions. Nanoparticle 4.5
CFU/mL, % of initial
(AgNP) and ion (AgNO3) 4.0
concentrations: 0.06 mM 3.5
(6.5 gAg/mL). Initial 3.0
bacteria concentration 2.5
3.5 105 CFU/mL and MS-2 2.0
particles50,000/mL. Data 1.5
from the Sysin Institute
1.0
0.5
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Incubation time, hours
(b)
60
50
AgNP
MS-2 particles, % of initial
AgNO3
40
30
20
10
0
1.0 1.5 2.0 2.5 3.0 3.5 4.0
Incubation time, hours
from these and several other studies make it possible to conclude that aqueous
solutions of Ag nanoparticles possess strong antimicrobial activity and can be
considered as a basis for the creation of new type disinfectants that are more
efcient and safer to humans than common disinfectants based on chlorine, its
derivatives, or quaternary ammonium compounds.
It is expedient to compare our results with published data in the cases where the
particle sizes and experimental procedure allow correct comparisons. In the literature
available, such a comparison is possible with the MIC value obtained for E. coli in
the study [295] (see also Table 6.1). The minimal inhibitory concentration of Ag
nanoparticles of the same size (9.2 2 nm) was found as 5.4 g/mL in [295], being
similar to our LD value, that is, to the minimal concentration necessary for 100 %
bacterial death (5 g/mL, Fig. 7.2). Taking into account that the nanoparticles
examined in [295] were protected with a protein (BSA) from coagulation in the
Table 7.4 The effect of aqueous silver nanoparticle solution (AgNP) on Escherichia coli 1257
and Staphylococcus aureus 906 strains in aqueous medium.* Data from Moscow Disinfection
Center
Bacterium Exposure (h) Bacterial growth at different AgNP concentrations. Upper line
% of initial nanoparticle concentration. Lower linenanoparticle
concentration in the medium, g/mL
3 2 1.5 1.0 0.75 0.5 0.25 0.125
(6.5) (4.32) (2.88) (2.16) (1.44) (1.08) (0.54) (0.27)
E. coli 0.5 + + + + +
1257 1.0 + + + + +
2.0 + + + + +
24.0 + + +
St. aureus 0.5 + + + + +
906 1.0 + + + + +
2.0 + + + + +
24.0 + + +
*Initial concentration of AgNPs and AOT in the stock solution216 gAg/mL and 30 mM,
respectively. Initial concentration of bacterial cells2 105 CFU/mL. Antibacterial activity tests
were performed by means of bacteria incubation with AgNPs at different dilutions of their stock
solution. () and (+)absence and presence of bacteria, respectively
medium, that is, they had a coating other than the AOT bilayer used in our exper-
iments, it is possible to conclude that, in this case, the nature of a stabilizing coating
is not crucial for antibacterial activity of nanoparticles, at least toward E. coli. Hence
follows also that toxicity of our stabilizer, which is a synthetic anionic surfactant,
does not play a considerable role in the antibacterial effect of nanoparticles at the
stock solution dilutions used in our experiments. This conclusion disagrees with
other data obtained with nanoparticles that were similar in size, but were obtained
with other (different) stabilizer or without a stabilizer. For instance, in the work
[364], the biocidal effect on E. coli in the experiments with Ag nanoparticles sized
812 nm was higher at stabilization with anionic surfactant (SDS) than with PVP.
A substantial difference in biocidal activity between Ag nanoparticles with and
without a stabilizing coating was observed with the human immunodeciency virus
[363]. The effect of stabilizer was considered in more detail in Chap. 6 (Sect. 6.1.1).
7.1.3 Solid Materials and Polymer Films with Ag

Nanoparticles
Tests for antibacterial activity were performed for Ag nanoparticle-containing

fabrics [10, 414], activated carbon [413], metal plates, polyamide membranes, and
polymeric lms based on a chitin derivative [329].
Fabric (wool, cotton, flaxen, etc.) samples were placed in an aqueous medium
containing E. coli cells or onto the surface of an agar medium with E. coli cells in
Petri dishes. The antimicrobial effect of a fabric sample was estimated from the
extent of bacterial growth inhibition after incubation for various periods of time as
compared with control samples of the same fabric, but without nanoparticles. In
both cases, fabric samples with Ag nanoparticles were shown to exert a strong
inhibitory effect on the bacterial growth. The results of an experiment performed in
Petri dishes are shown in Fig. 7.4.
Three silver nanoparticle-containing wool samples (stained, 50 mg Ag per gram
fabric) and two control (unstained) samples were placed onto the surface of a
culture medium containing E. coli cells. Two nanoparticle-containing samples and
one control sample were removed after 24 h incubation at 37 C (Fig. 7.4a). As is
seen, the agar medium remained transparent under the nanoparticle-containing
samples, testifying to the absence of bacterial growth, while the remaining medium
surface (including the region under the control sample) was covered with an even
lawn of bacterial colonies, which was conrmed by a microscopic examination.
Haloes are seen around the nanoparticle-containing samples, pointing to the
avoidance of an adverse environment as a bacterial response. The transparent
segments from which the nanoparticle-containing fabric samples had been removed
remained much the same in size after one week incubation of the Petri dish
(Fig. 7.4b), indicating that biocidal activity persisted, although the fabric sample
was absent. At the same time, a substantial growth in colony number was observed
(a) (b)
3 4
4
3
2
Wool 5
Wool 5
2 1
1
Fig. 7.4 Bactericidal effect of fabrics with silver nanoparticles. The experiment in Petri dishes.
a 24 h after placing of fabric samples on the agar surface with E. coli cells that have just been
introduced. 1 Control (non-modied) sample; 2 Sample of the same fabric impregnated with
nanoparticles; 3,4 Clear medium segments (no bacterial growth observed) after the removal of
fabric samples with nanoparticles; 5 Dense lawn of E. coli that have grown up after removal of
the control sample. b The same Petri dish 7 days after the removal of fabric samples. Clear sites
(3 and 4) became colonized with bacteria, but their concentration here is substantially lower than in
the control segment 5. Data from the Institute of Genetics and Selection of Industrial
Microorganisms
in the segment from which the control sample had been removed. Similar results
were obtained with other fabrics.
In addition, washing conditions were selected so that fabrics almost fully pre-
served the initial nanoparticle coverage density. It is clear, therefore, that further
studies in the eld may allow designing fabrics and manufacturing products for use
in medicine (e.g., medical gowns or uniforms with biocidal properties) or for
common use where their biocidal properties are advantageous.
Activated carbon impregnated with silver nanoparticles was tested in columns;
water from the water pipe was inoculated with E. coli or coliphage MS-2 to con-
centrations appreciably higher than the allowable ones and then was continuously
passed through the columns for one week. Water samples were collected at the
outlet and seeded onto a culture medium, and CFUs were counted. A similar
procedure was carried out in parallel with carbon without nanoparticles.
A substantial reduction in viable microbial count was observed for nanoparticle-
impregnated carbon compared with the control, and the reduction was stable
throughout the period of the experiment. The nding demonstrates that nanopar-
ticles are not washed out from carbon by a water flow, in contrast to silver
salt-impregnated carbons, which are characterized by a rapid washing out of silver
ions [417]. Based on the testing results, activated carbon possessing biocidal
properties was protected by a RF patent [288].
Metal plates covered with Ag nanoparticles were tested for antimicrobial activity
toward Legionella pneumophila Philadelphia strain. This bacterium causes le-
gionelliosis, which is a severe disease that is often caused by breathing air passed
through an air-conditioning system. The plates were made of stainless steel because
this material is used to manufacture pure water tanks that are built in air-conditioning
systems and predominantly accumulate L. pneumophila to high concentrations. The
nanoparticles were deposited by means of adsorption from micellar solution with
various initial nanoparticle concentrations, the nanoparticle density on the plate
surface being very small, as followed from the low adsorption values (see Chap. 5,
Fig. 5.9). It was established that complete death of bacteria occurred on the plate
surface with nanoparticles already after one hour of incubation, whereas the initial
bacterial concentration (107 cells/mL) did not decrease even in one day in control
experiments with plates without nanoparticles (Table 7.5).
As another control, bacteria were placed on the plates incubated with
AOT/isooctane solutions; here, the bacterial concentration was found to slightly
decrease in 1 h, but the decrease was many orders of magnitude lower than that
observed on the plates with nanoparticles. Thus, nanoparticle treatment of metal
tanks in air-conditioning systems can be expected to substantially reduce the risk of
legionelliosis.
Microltration polyamide membranes with Ag nanoparticles were obtained via
nanoparticle adsorption from aqueous solutions. Membranes for the study were
provided by the research and production company Technolter (Vladimir), which
manufactures lter devices for producing ultrapure water to be used in medicine
and biotechnology. Membranes modied with silver nanoparticles showed positive
results in microbiological tests performed at the Institute of Medical Polymers
Table 7.5 The effect of metal plates coated with silver nanoparticles on bacterium Legionella
pneumophila.* Data from the Gamaleya Institute
Sample Composition of micellar solution used 0.5 h 1h 24 h
for the plate treatment
1 AOT/isooctane, C0 1.0 107 1.0 106 0
2 AOT/isooctane, C0/5 1.0 107
1.0 106 0
3 Ag/AOT/isooctane, C0 1.0 105
0 0
4 Ag/AOT/isooctane, C0/5 5.0 104 10 0
5 Control 1.0 107 1.0 107 9.0 106
*Initial AgNPs and AOT concentrations (C0) in micellar solution1 and 135 mM, respectively
(Moscow). Antimicrobial activity of membranes was estimated from the numbers of

colonies and microorganisms that grew on a membrane at various time points after
ltering tap water. The testing results are summarized in Tables 7.6 and 7.7. Results
obtained with standard Millipore polymeric membranes in the same conditions are
shown for comparison. As shown in Table 7.6, colony growth was not observed in
the two rst days after ltration, and the number of colonies did not exceed 10 on
the third day on silver nanoparticle-modied membranes with various nanoparticle
Table 7.6 Results of microbiological tests for polyamide membranes with deposited silver
nanoparticlesgrowth of the colonies on a membrane surface in various time intervals after
ltration of water from the water pipe.* Data from the Institute of Medical Polymers (Moscow)
Sample Sample Name Filtration Colony Colony Colony count on
time for count on count on the 3rd day
50 ml of the 1st day the 2nd
water, min day
1 MMPA+0.2 4 0 0 5 of the rst type,
membrane, AgNPs 10 of the second
coverage density type
0.33 mgAg/kg
2 MMPA+0.2 2.5 0 0 5 of the rst type,
1.16 mgAg/kg
3 MMPA+0.2 3.5 0 0 3 of the rst type,
2.22 mgAg/kg
4 MMPA+0.2 24 0 0 5 of the rst type,
2.55 mgAg/kg
5 HA membrane by 0.5 27 of the 49 of the 55 of the rst type,
Millipore, rst type rst type a few of the second
0.45 m type
*The 1st colony type is a round convex yellow colony with even borders
The 2nd colony type is an unstructured colorless opaque colony
Table 7.7 Control of bacterial growth on the ltration membranes modied with silver
nanoparticles. Data from the Institute of Medical Polymers (Moscow)
Sample Sample name Bacterial growth with time Growth
1 day 24 days 46 days 78 days 910 days under the
membrane
tested
1 Membrane with None None None 3 No new Clean
AgNPs, coverage
density 0.16
mgAg/g
2 Membrane with None None None 7 No new Clean
AgNPs, coverage
density 0.33
mgAg/g
3 Membrane, with None None None 2 No new Clean
AgNPs, coverage
density
2.22 mgAg/g
4 GS (Millipore, 34 40 49 Growth Growth 2
0.22 m) Continues stopped
coverage densities at various flow rates. The result testies to substantially higher
antimicrobial activity of our polymer membranes compared with Millipore mem-
branes. The conclusion was further conrmed in an experiment where bacterial
growth on membranes was checked over 10 days (Table 7.7). Based on the results,
it is possible to expect that polyamide membranes impregnated with silver
nanoparticles will be introduced as elements of lter devices in industrial
production.
Polymeric lms with Ag nanoparticles were obtained by adding minor amounts
of an aqueous solution of nanoparticles to an aqueous solution of a biodegradable
polymer (carboxymethyl chitin, CMC); the study was carried out in collaboration
with Topchiev Institute of Petrochemical Synthesis (Russian Academy of Sciences)
and Kosygin Moscow State Textile University [329]. The assumption was that
silver nanoparticles would improve the known antimicrobial properties of chitin and
chitosan polymers to produce more efcient wound dressing materials. The
nanoparticle contents in the lms were 0.03 and 0.06 % (by weight); the lm color
consequently changed to pale yellow (Fig. 5.2, color inert), which made it possible
to visually verify the nanoparticle stability in the system.
The lms were tested for antimicrobial activity toward Staphylococcus aureus
and Salmonella typhimurium at the Gamaleya Institute. Doctor of Science (Med.)
B.I. Marakusha developed a special procedure for these experiments; the procedure
is described in detail elsewhere [329]. The results are summarized in Table 7.8.
As is seen, lms with minor additions of silver nanoparticles possess substantial
antimicrobial activity toward the bacteria in question at high bacterial cell con-
centrations in suspensions contacting the lm (104 and 106 CFU/mL), the activity
being signicantly higher than that observed for lms of the same polymer, but
Table 7.8 Dynamics of interaction of Salmonella and Staphylococcus strains taken in different
concentrations with CMC (carboxymethyl chitin) lms containing or not containing AgNPs. Data
of the Gamaleya Institute
Strain tested, dose Indication of lms with different Lg of the number of
silver contents living bacteria in tested
lms at time intervals, h
1 3 6 24
Salmonella typhimurium Initial culture 6.1 6.1 6.1 6.1
TMLR66 CMC A, 0 % AgNPs 6.1 5.7 5.2 2.8
106 CFU/mL
CMC.B, 0.03 % AgNPs 5.0 4.0 2.7 0
CMC.C, 0.06 % AgNPs 0 0 0 0
Staphylococcus aureus Initial culture 6.0 6.0 6.0 6.0
Wood 46 CMC A, 0 % AgNPs 6.0 5.8 4.9 3.2
106 CFU/mL
CMC.B, 0.03 % AgNPs 5.0 4.1 3.0 0
CMC.C, 0.06 % AgNPs 0 0 0 0
Salmonella typhimurium Initial culture 4.0 4.0 4.0 4.0
TMLR66 CMC A, 0 % AgNPs 4.0 3.7 3.4 1.5
104 CFU/mL
CMC.B, 0.03 % AgNPs 2.8 1.8 0 0
CMC.C, 0.06 % AgNPs 0 0 0 0
Staphylococcus aureus Initial culture 4.0 4.0 4.0 4.0
Wood 46 CMC A, 0 % AgNPs 4.0 3.8 3.3 1.6
104 CFU/mL
CMC.B, 0.03 % AgNPs 2.9 1.9 0 0
CMC.C, 0.06 % AgNPs 0 0 0 0
without nanoparticles. The higher the silver nanoparticle concentration in the lm,
the greater was its bactericidal effect. Films containing 0.06 % nanoparticles caused
100 % bacterial death in as early as 1 h after application. The results demonstrate
that the lms under study possess pronounced bactericidal activity. The nding
indicated that our polymeric material may nd application in medicine (e.g., for the
treatment of skin lesions).
Taken as a whole, studies of the antimicrobial properties of nanoparticles in
various media showed that the nanoparticles display antibacterial and antiviral
activities both in liquid media, including aqueous solutions and liquid composite
materials, such as paints or polymeric lms, and on solid surfaces (carbon, fabrics,
and metal).
Chapter 8
The Effect of Silver Nanoparticles on Some
Objects from the Plants and Fungi
Kingdoms
In the previous chapter, we described the results of studying the biocidal effect on
microorganisms, obtained for metal nanoparticles synthesized in our laboratory.
Now, we turn to the experiments on biological objects staying on the higher
organization levels. First of all, it should be noted that experiments with microor-
ganisms are aimed primarily at producing new effective means to combat various
infections, while experiments with higher-level living systems have a variety of
objectives, which are determined by the intricate complex of processes associated
with the effect of nanoparticles. Two main lines of research can be discriminated
here, in accordance with the two types of effects metal nanoparticles exert on the
human body, as has been mentioned in Introduction, that is, positive (therapeutic)
effects and negative effects (nanopathologies). In the rst line, studies are devoted
primarily to the improvement of the current diagnostics and drugs and production of
new ones with the use of metal nanoparticles. In the second line, toxicology studies
are performed; their purpose is to understand the character of the negative effects
exerted by nanoparticles and to establish the conditions ensuring the safe use of
various nanoparticle-containing materials.
As already mentioned in Chap. 6 when analyzing the literary data on the biological
effects of nanoparticles, the toxicology studies have come to be especially urgent in the
past years because, on the one hand, metal nanoparticles are a topical subject of
applied studies in various elds of chemistry, engineering, and medicine, and on the
other hand, they are nding increasing application in the production of consumer
goods (cosmetics, clothes, household appliances, toys, etc.). Therefore, toxicity of
nanoparticles used in solutions or as components of various materials is of immense
importance to estimate in studies of the biological effects of metal nanoparticles on
plants, animals, and humans. Apart from yielding data that are essential for estab-
lishing the standards ensuring the safe use of nanoparticles and nanoparticle-modied
materials, toxicity testing may provide useful information both for the better under-
standing of the mechanisms underlying the nanoparticle action on living organisms

DOI 10.1007/978-3-319-30906-4_8
236 8 The Effect of Silver Nanoparticles on Some Objects
and hence their curative and adverse effects, and for the development of various
applications of nanoparticles in biology, biotechnology, and medicine.
Our studies were performed with silver nanoparticles. We worked mostly with
standard aqueous solutions of nanoparticles sized 910 4 nm; in some special
cases, we used smaller nanoparticles with a narrow distribution, obtained as
described in Chap. 3 (Sect. 3.3), as well as larger nanoparticles with a mean size of
13 nm. Chapter 8 presents the data on their toxic effects on biological systems
varying in organization level, including specimens of the kingdoms Fungi and
Plants. Chapter 9 is devoted to the studies on cytotoxicity and the mechanism of
nanoparticle action for normal and tumor human cell cultures.
Investigating the effects, including toxic ones, metal nanoparticles exert on the
functions of living systems staying on various organization levels, is now a new
research eld, which has both achievements and problems, the latter being related
primarily to the specics of nanoparticles as a research subject and the consequent
technical difculties. An extremely important role plays here the choice of the
method used to obtain nanoparticles, which is to ensure that the solution of
nanoparticles used to study their effects on biological systems meets the necessary
requirements (Chap. 1, Sect. 1.7). As is evident from this set of requirements, for a
successful solution of the tasks posed here, it is essential for the metal nanoparticles
to have reproducible characteristics and be stable in aqueous solutions with minimal
concentrations of other biologically active components, thus allowing experiments
with correct controls and minimal possible side effects. To study how the size of
nanoparticles affects their biological activity, it is essential also that the method
makes it possible to obtain nanoparticles of at least two different mean sizes with a
narrow distribution, the sizes preferably falling in the range 120 nm, where the
size effects are most likely to occur.
The method of biochemical synthesis holds great promise in this respect, pro-
viding an opportunity to obtain stable aqueous solutions of nanoparticles of a given
mean size, a narrow distribution, and a known, low enough concentration of a
stabilizing agent, which can be tested independently for its biological effect in
control experiments.
In this chapter, we briefly describe the results of studying the toxic effects for
aqueous solutions of AOT-stabilized silver nanoparticles in experiments with
plasmodium of the lower fungus, unicellular alga, and plant seeds. In all experi-
ments, the effects of aqueous nanoparticle solutions were compared with those of
aqueous solutions containing AOT in the same concentrations as in the nanoparticle
solutions and the effects of Ag+ ions, which were added in the form of aqueous
solutions of silver nitrate. Thus, it was possible, rst, to isolate the biological effect
of silver nanoparticles from the total effect of nanoparticles and AOT and, second,
to determine whether the nanoparticle effect is due to the silver ions released by the
nanoparticles, a question important for elucidating the mechanism of nanoparticle
action. Apart from AOT-stabilized nanoparticles, in studies with human and
mammalian cells in vitro, the starch- and cyclodextrin-stabilized nanoparticles were
used. The objective was to nd out whether the toxic effect of nanoparticles
changes when a natural stabilizing agent is applied instead of a synthetic one.
8.1 Plasmodium of the Acellular Slime Mold Physarum polycephalum 237
8.1 Plasmodium of the Acellular Slime Mold Physarum

polycephalum
Studies were carried out in collaboration with the Institute of Theoretical and
Experimental Biophysics of Russian Academy of Sciences (Pushchino, Moscow
region). The Physarum polycephalum plasmodium is regarded as a convenient test
model for the studies of the chemotaxis phenomena, that is, of a cell movement in
response to adverse chemical factors of the environment. The plasmodium is a
multinuclear protoplasm surrounded by a common membrane and is capable of
unlimited growth and amoeboid movements (Fig. 8.1).
Submersed in a liquid medium, a plasmodium disperses into microplasmodia,
which are rounded multinuclear cells 100200 m in size. In both macroplas-
modium and microplasmodia, protoplasm is differentiated into a relatively sta-
tionary ectoplasm and a fluid endoplasm, which streams through ectoplasmic
channels and tubular strands, which pass through the ectoplasm. The direction and
velocity of endoplasmic flow change according to a changing pressure gradient,
which is produced via ectoplasm contractions coordinated along the plasmodium
body [418]. Contractions are provided by myosin oligomers interacting with actin
laments attached to the membrane [419]. The period of force autowaves and
shuttle streaming of the endoplasm depends on the physiological state of the
plasmodium and varies in the range of 15 min [420]. Each plasmodium fragment
is capable of restoration of the plasma membrane integrity and recommencement of
contractile and locomotive activities. This circumstance makes it possible to use
strands excised from a plasmodium to measure the force and lm fragments of a
standard size and shape to evaluate chemotaxis.
Fig. 8.1 Photograph of the

plasmodium growing on
nutritional medium. This
gure and gs. 8.28.6 were
reprinted from Ref. [427]
permission from Nova
Science Publishers
The Physarum polycephalum plasmodium is one of the classical models for

studying non-muscular motility, and its chemotactic behavior is well understood
[418422]. In particular, it had been shown that substances stimulating negative
taxis (repellents) increase the period of contractile activity and reduce the area of
plasmodium spreading on agar gel [421, 423, 424]. These characteristic features of
the plasmodium allow detection of not only a lethal effect, as in studies with many
other models, but also changes in its behavior in mild conditions, at sublethal
nanoparticle concentrations. Thus, as with other models capable of chemotaxis, a
plasmodium test is a sensitive tool in studies of the biological activity of silver and
other metal nanoparticles.
The effects of nanoparticles on the plasmodium viability and locomotive activity
were studied in our experiments. An inhibitory effect (abiotic or repellent activity,
which characterizes the extent of toxicity) was compared for aqueous solutions of
Ag nanoparticles, AOT, and Ag+ ions used in the corresponding concentrations;
experiments were performed in aqueous solutions and on agar medium in Petri
dishes and on plates.
The experimental procedure is detailed in [425427]. Briefly, in viability tests,
microplasmodia were incubated with test agents for 1 h in a growth medium, in a
nitrate buffer containing the salt components of the growth medium, or in a control
solution (10 mM HEPES, 0.1 mM CaC12, pH 7) wherein calcium nitrate was used
in place of calcium chloride to prevent Ag+ ions from precipitating in the form of
AgCl. Incubation was carried out in conical flasks with continuous agitation on a
shaker. A release of the yellow plasmodial pigment into the solution was considered
as indicator of the necrotic death of microplasmodia during incubation. The viability
of protoplasm strands after the incubation with test substances was inferred from the
resumption of locomotive activity after transferring the strands onto 2 % agar gel.
In chemotactic tests, the plasmodium response to a test agent in solution was
determined from the changes in the period of longitudinal force autowaves gen-
erated by isolated strands in isometric conditions. The force was recorded using a
highly sensitive tensiometer with an electromechanical converter, which makes it
possible to preset a controllable stretching of the strand. Strand sections of
approximately 4 mm in length and 300500 m in diameter were mounted hori-
zontally on arms of a tension sensor with agar and placed in a cuvette with a control
solution. Elastic properties of the ectoplasm were estimated from the fast-phase
amplitude of the force response to a stepwise change in strand length [428].
A chemotactic response to test substances added to 2 % agar gel was estimated
from the area of spreading on the substrate, using standard samples 4 mm in
diameter. Samples were excised together with the support from a smooth region of
the frontal zone of a large plasmodium with the help of a steel cutting punch and
placed in a Petri dish onto a 23-mm layer of agar gel containing a test substance.
Direct comparisons of chemotactic efciency were performed using paired agar gel
plates containing test substances or their combinations. A plasmodium was placed
at the interface, and the difference in repellent activity was inferred from the
direction of its migration [422]. In view of the photosensitivity of silver com-
pounds, experiments were carried out in the dark at 20 C. Images of spreading and
migrating plasmodia were recorded in digital form with an Astra 6700 UMAX
scanner (UK).
Experiments were performed in aqueous solutions and on agar substrates. The
effects of nanoparticles on the microplasmodium viability and strand auto-oscillation
pattern were studied in aqueous solutions. Experiments on agar gel were performed
to estimate the biocidal effects by repellent activity in temporal (a decrease in the
area of spreading) and spatial (directional locomotive response) tests.
In aqueous solutions, the viability testing showed that microplasmodium death
(membrane impairment, cell discoloration, and release of the yellow pigment)
occurs after 1-h incubation at a nanoparticle concentration of 105 M
(1.08 gAg/mL) both in the standard growth medium or salt medium and in control
solution. At 106 M (0.108 gAg/mL) nanoparticles, microplasmodia fully pre-
serve both the integrity of the outer membrane and the capability of movements.
Measuring the auto-oscillations of macroplasmodium strands showed that force
autowaves generated by isolated protoplasmic strands ceased within several min-
utes of incubation in the presence of 104 M (10.8 gAg/mL) silver nanoparticles
or the corresponding AOT concentration and persisted for more than 1 h in an
AgNO3 solution (Fig. 8.2). Strand relaxation or loss of tension was not responsible
for the termination of oscillations because a 20 % stepwise increase in strand length
25
20
15 AgNO3
10
5
0
25
20
15 AOT
Force, mg
10
5
0
25
20
15 SNP
10
5
0
0 10 20 30 40 50 60 70 80 90 100
Time, min
Fig. 8.2 Effects of Ag nanoparticles (SNP), AOT, and AgNO3 on plasmodium motive activity in
aqueous solution. See text for explanations
did not restore them. The strand response to stretching indicates that the strands
preserved their elastic properties [425]. After 1-h incubation with nanoparticles or
AOT, but not AgNO3, strands dismounted from the tensiometer arms and washed
with the control solution lost the capabilities of attaching to a substrate and
migrating. Thus, 1-h incubation with 104 M AgNO3 did not reveal a considerable
effect of silver ions, while Ag nanoparticles and AOT used in matching concen-
trations acted similarly rapidly provoking termination of the force autowaves and
subsequent death of the plasmodium.
When the nanoparticle concentration was reduced to 3.24 g/mL, oscillations
similarly stopped within the rst 1020 min after adding nanoparticles. Oscillations
temporarily reappeared with an appreciably greater period in response to stretching
and ceased again as stretching was stopped. The effect did not result from plas-
modium death because contractile activity of the strands was completely restored
after washing out nanoparticles (Fig. 8.3a). As was evident from the yellow strand
color (the bottom row in Fig. 8.3b), the strands remained viable for approximately
1 h of incubation in the control solution with this concentration of nanoparticles,
(a)
(b)
5' 10' 15' 20' 30' 40'
15' 20' 25' 35' 40' 50'
Fig. 8.3 Effect of Ag nanoparticles (SNP) on plasmodium viability: dependence on their

concentration and incubation time. a Reversibility of auto-oscillation termination induced by the
introduction of 3.24 g/mL nanoparticles. b Viability of the plasmodium at the incubation with
10.8 (upper row) and 3.24 g/mL (lower row) of nanoparticles. Timing marks indicate the
incubation time in the nanoparticle solution; the photographs were taken in 4 h after the washout
and placing the strands on agar layer. Scale bar, 1 cm
without displaying apoptosis-related signs of pigment loss or cell separation into

fragments. Moreover, the stands started spreading on the agar substrate 4 h after
washing from nanoparticles.
The reversibility of the nanoparticle effect makes it possible to assume that
plasmodium organelles essential for viability, primarily mitochondria, remained
intact. Although nanoparticle-induced changes in their functional activity cannot be
excluded, the changes must be reversible and incapable of causing cell death. It
remains unclear what regions of the cell membrane are targeted by nanoparticles.
When the AgNO3, AOT, and nanoparticle concentrations were 1.08 g/mL, i.e.,
one order of magnitude lower than the concentration leading to plasmodium death,
protoplasmic strands remained viable after 3-h or longer incubation in solutions
with additions of nanoparticles, AOT, AgNO3, or a mixture of AOT with AgNO3.
Isometric force measurements in the presence of these agents showed that the
oscillation period increased, as is characteristic of repellent action; the effect varied
in extent and the rate of development. Namely, higher repellent activity was
observed for nanoparticles compared with AOT, being undetectable when testing is
performed at lethal concentrations. With a longer observation period, a slow
developing and relatively weak effect was revealed for AgNO3.
We considered it useful to compare the lethal and sublethal nanoparticle con-
centrations estimated for the plasmodium with those obtained when studying tox-
icity of silver nanoparticles in bacteria and cultured animal cells in liquid media.
Comparisons included only data on the nanoparticles similar in size (less than
20 nm), obtained by biochemical synthesis (in the case of bacteria) [348] and other
methods [295, 403, 411]. The results are summarized in Table 8.1.
Table 8.1 Lethal and sublethal doses of silver nanoparticles estimated for various bio-objects in
aqueous solution
Object Lethal doses, g/mL Sublethal doses, g/mL Reference
Microplasmodia 1.08 0.108 < C(AgNP) < 1.08 [425, 427]
Necrosis
Macroplasmodia 10.8 1.08b C(AgNP) 3.24c [425, 427]
Necrosis
Bacterial cellsa 5, 2.88 [348]
E. coli 5.4 [295]
St. aureus 2.88 [348]
Spermatogonial 8.75 2.50 [403]
stem cells (MTT EC50) (LDH EC50)
Sharp decrease of Slight increase of LDH
mitochondrial functions leakage; apoptosis occurs
and cell viability rather than necrosis
HT-1080 cells 12.5 6.25, oxidative stress [411]
Necrosis 0.78, apoptosis
A431 cells 12.5 6.25, oxidative stress
1.56, apoptosis
a
100 % death after 30-min exposure
b
Twofold increase of oscillation period
c
Reversible oscillation suppression
Sublethal concentrations are understood here as the concentrations that inhibit

vital functions, but do not cause death; i.e., these are the toxicity threshold con-
centrations considered in Chap. 6. It is shown from the table that the lethal con-
centrations established for the macroplasmodium are similar to those for animal
cells [403, 411] and somewhat higher than found for bacteria [295, 348]. However,
a lower difference might be observed in the latter case if the macroplasmodium is
tested at nanoparticle concentrations lower than 10.8 g/mL (but higher than 3.
24 g/mL). At the same time, this testing might expand the sublethal concentration
range. Judging from the lethal concentration estimates available now, the Physarum
macroplasmodium is as sensitive to the nanoparticle effect as bacterial (E. coli and
St. aureus) and certain animal cells are. The lethal concentrations obtained for
microplasmodia are one order of magnitude lower. The difference is probably
related to the increase in the plasma membrane area to cell volume ratio upon the
transition from a macroplasmodium to microplasmodia. It is also possible that the
capability of repairing cell membrane lesions is limited in microplasmodia as a
result of a substantially lower cell volume [429].
In the case of agar gel, several series of experiments on Petri dishes and plates
showed that nanoparticles used at concentrations that do not cause plasmodium
death (105 M or 1.08 gmL and lower concentrations) are more toxic than Ag+
ions, AOT, and their combinations used at the concentrations corresponding to
those introduced with aqueous nanoparticle solutions. Example results of the
experiments are shown in Figs. 8.4 and 8.5.
In experiments carried out in Petri dishes, the areas of spreading were compared
in various periods of time (3 and 6 h) after placing plasmodia on media containing
nanoparticles, AOT, and Ag+ ions. As shown in Fig. 8.4, plasmodium growth was
Control AgNO3 AOT AgNO3+AOT SNP
Fig. 8.4 Spreading of plasmodia on the agar plates in control and in the presence of Ag
nanoparticles (SNP), AgNO3, and AOT in 6 h after placing of plasmodia on the agar surface.
Concentrations: top row, AgNO3105 M, AOT2.5 103%, SNP105 M; bottom row,
AgNO3104 M, AOT2.5 102%, SNP104 M. Scale bar, 1 cm
AgNO3
+ AOT
SNP
Fig. 8.5 Spatial test for comparison of the repellent activity (negative taxis of plasmodia away
from the stronger agent) by the number of samples showing this reaction. Comparison was made
for the action of nanoparticles (SNP, bottom) and AOT + AgNO3 (top) aqueous solutions. Reagent
concentrations: AgNO3105 M; AOT2.5 103%; SNP105 M. 10 h after placing of
plasmodia germs at the border between plates; scale bar, 1 cm
completely suppressed in all cases at the nanoparticle concentration 104 M, tes-

tifying to the plasmodium death. At the nanoparticle concentration 105 M, plas-
modium grows; its growth considerably differs between the nanoparticle-containing
medium and the three media containing silver nitrate, AOT, and their mixture in the
corresponding concentrations. It is clear also that the pure control without any agent
is virtually indistinguishable from the case with AgNO3 and only slightly differs
from the two cases with AOT. The result supports the earlier conclusion that
nanoparticles are more efcient than Ag+ ions used at the same concentrations,
which was made in studies with E. coli and coliphages [10, 28, 348, 414] (see also
Chap. 7). The experiments additionally showed that nanoparticles exert a greater
inhibitory effect as compared with AOT and a mixture of AOT with silver ions.
Another conclusion from the experiments is that sublethal concentrations are
expedient to the use for substances to be tested for their effect.
At the same time, the resolution of this temporal test proved to be insufcient for
detecting a trustworthy difference in the area of plasmodium spreading between
AOT and the mixture of AOT with silver nitrate. To check for such a difference,
spatial tests on plates were used. A plasmodium germ was placed at the interface
between plates with a medium containing a solution of nanoparticles and AOT or a
mixture of AOT with silver nitrate, and the area of plasmodium spreading was
estimated on both sides of the interface after a certain incubation period. Figure 8.5
shows the result of an experiment where the effect was compared between
nanoparticles and a mixture of AOT with silver ions. The taxis direction clearly
demonstrates far greater repellent activity of nanoparticles.
The above and other similar experiments showed that when an aqueous solution
of silver nanoparticles or those of Ag+ ions and AOT at the corresponding con-
centrations are added to a culture medium, the plasmodium growth is inhibited to a
greater extent in the case of nanoparticles. Taken together, the experimental data
made it possible to arrange the agents in the following order by comparative ef-
ciency: AgNO3 << AOT < AgNO3 + AOT << Ag nanoparticles. Thus, plasmod-
ium chemotactic assays demonstrated that the toxic effect of silver increases
manifold in the case of nanoparticles and that the increase cannot be reduced to a
destabilizing effect exerted on the cell membrane by the surfactant (AOT) that
forms a micellar shell. Besides, a higher efciency of nanoparticles compared with
silver ions gives grounds to suppose that the biological effect of nanoparticles
cannot be reduced to the effect of silver ions, but possibly implicates another
mechanism wherein metal nanoparticles themselves play an important role.
To elucidate the effect of the particle size, experiments were carried out with
aqueous solutions of nanoparticles of different sizes with narrow distributions, 5.5
2.0 nm (SNP4) and 9.2 2.7 nm (SNP5). Information on the methods to obtain
such nanoparticles is presented in Chap. 3 (Sect. 3.3). A negative reaction of the
plasmodium was found to be greater in the case of smaller nanoparticles, the total
silver nanoparticle concentration in the medium being the same for either sample.
The result is shown in Fig. 8.6. A plasmodium germ was placed at the interface
between two agar strips (separated by a 1-mm gap), which contained nanoparticles
of different sizes. As is shown, the plasmodium grew almost exclusively toward the
medium with larger nanoparticles (SNP5); i.e., smaller nanoparticles (SNP4) were
distinctly avoided [427]. The nding agrees with the reports present in the literature
that smaller silver nanoparticles are more toxic (see Chap. 6, Sect. 6.1.1). However,
it is also possible in our case, like in the cases described in the literature, that the
cell response is determined by the difference in nanoparticle number per unit vol-
ume of the medium, rather than by the particle size. The number concentrations
differed by approximately one order of magnitude in our experiments, being
1.48 1014 particles/mL for SNP4 and 3.16 1013 particles/mL for SNP5.
Taken together, the results obtained in the above studies indicate that silver
nanoparticles are promising not only as a bactericidal agent, but also as a fungicidal
one, although they may be potentially hazardous for animal cells. Hence follows
that the nanoparticles may be put to good use in treating various infectious diseases,
SNP4
SNP5
SNP4
Fig. 8.6 Plasmodium response on the presence of silver nanoparticles with different sizes
but with caution in view of the risk of damaging healthy tissues. It is also important
to bear in mind that chemotaxis is directly involved in the immune response and
tissue regeneration. Since similar mechanisms of chemotaxis are employed by
molds and animal cells [430, 431], the low threshold nanoparticle concentrations
established in our experiments for the plasmodium avoidance reaction indicate that
directional migration of animal cells along a gradient of bacterial peptides,
cytokines, and growth factors may be substantially altered by the opposite effect of
nanoparticles. For this reason, the effect of nanoparticles on chemotactic behavior
of cells in tissues targeted by treatment should be assessed when elaborating
methods for clinical application of nanoparticles.
Our results also demonstrate that chemotaxis is a highly sensitive test to study
nanoparticle toxicity. The data obtained with the Physarum polycephalum plas-
modium give grounds to conclude that similar tests can be recommended for
detecting trace amounts of harmful substances with the use of various prokaryotic
or eukaryotic cells capable of chemotaxis.
8.2 Unicellular Alga Chlorella vulgaris
Toxicity of Ag nanoparticles for living cultured cells of the microalga Chlorella

vulgaris beier (hereinafter referred to as chlorella) was studied in collaboration with
the Engineering Center of the RF Ministry of Natural Resources and Ecology (with
the Biotesting Laboratory, Izhevsk). Studies were performed by microelec-
trophoresis, using a Tsito-Ekspert instrument system, developed for the rapid
assessment of the toxicity of surface, drinking, and puried wastewaters by
biotesting. The toxicity of nanoparticles was deduced from the change in the
amplitude of cell oscillations in an alternating-current electric eld, the change
being due to a decrease in the cell surface charge, which is indicative of cell
viability. Algal cells were incubated in distilled water with nanoparticles at
nanoparticle concentrations varying from 108 to 104 M (0.0010810.8 g/mL)
for a given period of time, and the toxicity index was estimated as T = (Ac Ace11)/
Ac, where Ac and Acell are the mean oscillation amplitudes obtained for control (cells
without nanoparticles) and test suspensions, respectively. Independently, the toxi-
city was determined for Ag+ ions and an Ag+ + AOT mixture used in the corre-
sponding concentrations, as in the above experiments with the plasmodium.
For comparisons, the toxicity of nanoparticles was additionally determined with
E. coli by luminometry and was inferred from the change in the intensity of
luminescence of bacterial cells carrying luminescent probes. The toxicity was
characterized in this case by the index T = (IC Iexp)/IC, where IC and Iexp are the
luminescence intensities measured for control and test cell suspensions,
respectively.
The results of measurements performed with chlorella and bacteria are shown in
Fig. 8.7.
1.0 EcoliAgNP
ChlAgNP
ChlAgNO
ChlAgAOT
0.8
Toxicity (T)
0.6
0.4
0.2
0.0
10(8) 10(7) 10(6) 10(5) 10(4)
Concentration (M)
Fig. 8.7 Toxicity of nanoparticles (AgNP), AgNO3, and AOT + AgNO3 mixture for Chlorella
vulgaris alga cells and nanoparticle toxicity for E. coli. Incubation time, 25 min. This gure was
reprinted from Ref. [348] Copyright 2010, with the permission from Nova Science Publishers
For chlorella, nanoparticle toxicity initially increased with the increasing

nanoparticle concentrations and then remained constant in the range from
0.108 g/mL (106 M) to 10.8 g/mL (104 M); the toxicity level achieved was
approximately half the maximal level (T = 1). At all but one concentration
examined (104 M), nanoparticles were more toxic for chlorella than Ag+ ions and
their mixtures with AOT used at the same concentrations as in the nanoparticle
solution. The nding demonstrates that the toxicity of nanoparticles cannot be
reduced to the effect of ions or both ions and AOT, which were present in the
nanoparticle solution. The conclusion agrees with the data obtained in experiments
with the plasmodium and, in the case of Ag+ ions, in studies of antimicrobial
activity of silver nanoparticles with E. coli (Chap. 7, Fig. 7.2). Similar toxicity
obtained for the three agents at the highest concentration (104 M) may be
explained, most likely, by the loss of cell viability, as was observed with the
plasmodium at the same nanoparticle concentration.
A comparison with the data on the effect nanoparticles exert on E. coli according
to luminescence measurements showed, rst, that the maximal toxicity for chlorella
is achieved at lower nanoparticle concentrations and, second, that this maximal
toxicity is approximately half as high as that found for E. coli. The nding possibly
indicates that chlorella is less sensitive to the effect of nanoparticles than E. coli. In
addition, the nding makes it possible to assume different mechanisms for the
interactions of Ag nanoparticles with algal and bacterial cells, i.e., the chlorella cell
wall is composed of three layers [237] and may protect the cell from nanoparticle
penetrating into its interior, while the bacterial membrane may be more permeable
to nanoparticles. It should also be noted that the results obtained for E. coli
demonstrate a considerable increase in toxic effect at 1.08 g/mL and complete cell
8.2 Unicellular Alga Chlorella vulgaris 247
death at 10.8 g/mL, in line with the working concentrations of Ag nanoparticles

active toward E. coli in an aqueous solution, including both concentrations reported
in the literature [272, 295, 402] and our values obtained in experiments described
above (Chap. 7, Sect. 7.1.2).
8.3 Plant Seeds
The effect of Ag nanoparticles on seed germinating ability was tested with seeds of
Arabidopsis thaliana and soybean Glycine max; data were obtained in the Vavilov
Institute of General Genetics (Russian Academy of Sciences). The two plants are
well-known models used to study the effects of various factors on the genetic
characteristics of plant organisms. Testing silver nanoparticles for effect on seed
germinating ability was considered as a rst step of research, which would make it
possible to estimate the toxicity for an aqueous solution of nanoparticles, as it is
important for selecting the working concentrations for further experiments. The
effect was compared for an aqueous solution of nanoparticles sized 9 6 nm and
AOT solutions used in the corresponding concentrations. The nanoparticle and
AOT concentrations in a stock solution were 5 103 and 15 103 M, respec-
tively. Seeds were preliminarily soaked in distilled water for 24 h, then were flood
with nanoparticle or AOT solutions, incubated for 24 h, washed with distilled
water, and let to germinate in standard conditions. The presence of AOT had
virtually no effect on the seed germinating ability; i.e., a cytolytic effect was absent.
In the case of nanoparticles, a decrease in germinating ability was observed only at
concentrations exceeding 1 103 M (108 g/mL), while the effect was absent at
the lower concentrations.
The data from these experiments demonstrate that an aqueous dispersion of
silver nanoparticles is more toxic than an AOT solution at the highest concentra-
tions of the range examined, in agreement with the toxicity order established with
the plasmodium. Another conclusion is that an aqueous solution of silver
nanoparticles is not toxic for the seeds in question in a broad concentration range
(from 5 107 to 1 103 M). The nding indicates that the solution may be used
to treat seeds in order to protect them from pathogenic bacteria. In contrast to the
results obtained with the plasmodium and E. coli, Ag+ ions were observed to exert a
strong cytotoxic effect on seeds, but the causes of the effect are still unclear.
Chapter 9
The Effect of Silver Nanoparticles
on Cultured Human Cells
Studies on the effect of silver nanoparticles on human cells in vitro were undertaken
with two main purposes: rst, to determine the quantitative characteristics of the
nanoparticle toxicity and second, to gain, if possible, additional information on the
mechanism of nanoparticles action on living systems. As quantitative characteristics
we estimated the toxicity threshold (TT), i.e., minimal nanoparticle concentration
which provokes the toxic response (see also Sect. 6.1), and the half-inhibitory
nanoparticle concentration (IC50) used conventionally in studies of the toxicity of
chemical substances (including nanoparticles) toward biological objects. To better
understand the processes taking place in the nanoparticlecell interaction, we
compared the influence of nanoparticle solutions with those of silver ions and/or
stabilizer solutions in the relevant concentrations. Some efforts were applied also to
move on in understanding of the role of nanoparticle shell, including its surface
charge, in a cell response.
Experiments were carried out with normal and malignant cell cultures; the for-
mer were represented by human embryo broblasts (HEF) and umbilical vein
endothelium (UVE) cells, and the latter were the HeLa and U937 cancer cell lines.
In both cases, we hoped to use the results for the development of medical appli-
cations of the nanoparticles. With normal cells, we applied the nanoparticles syn-
thesized with natural stabilizers (starch and -cyclodextrin), to nd out whether
they can serve as components of the medicines designed for combating infectious or
cardiovascular deceases. With cancer cells, we aspired to elucidate the possibilities
for the creation of an effective remedy against oncological deceases.
Experimental procedure was organized similarly to those used previously in
studies on microorganisms (Chap. 7), lower fungus and plant seeds (Chap. 8). Cells
were incubated with different nanoparticle concentrations; after the incubation, cell
viability was estimated by MTT test. The effect of nanoparticle solutions was
compared with that of a stabilizer and (for HEF, HeLa and U937 cells) also with
that of silver ions in the relevant concentrations. To obtain the reliable results,
preliminary studies were required for the choice of conditions necessary to avoid at

DOI 10.1007/978-3-319-30906-4_9
250 9 The Effect of Silver Nanoparticles on Cultured Human Cells
least some of the experimental errors known from literature. The most essential
methodological points are elucidated in the sections presented below.
Studies were performed in the laboratory of molecular and cellular biology of the
Institute of General Pathology and Pathophysiology (IGPP) and in the laboratory of
tissue cultures of the D.I. Ivanovski Institute of Virology (IvIV). Below, we
describe the results obtained in the following sets of experiments:
(1) Determination of cytotoxicity for starch-stabilized silver nanoparticles on HEF
cells by MTT test; comparison with the effect of stabilizer and silver ions
(IvIV);
(2) Determination of cytotoxicity for -cyclodextrin-stabilized silver nanoparticles
on UVE cells by MTT test, morphology studies, and cytofluorimetry (regis-
tration of apoptotic and necrotic cells); comparison with the effect of stabilizer;
studies on the mechanism of nanoparticlecell interactionexamination of the
surface charge effect, including -potential measurements in the conditions
providing that its values reflect the virtual surface charge density of
nanoparticle stabilizing shell (IGPP);
(3) Determination of cytotoxicity for AOT-stabilized silver nanoparticles on HeLa
and U937 cells by MTT test, cytofluorimetry (registration of apoptotic and
necrotic cells); comparison with the effects of stabilizer and silver ions;
visualization of the nanoparticle penetration into a cell interior by laser
microscopy technique (IGPP).
9.1 Toxicity of Starch-Stabilized Ag Nanoparticles

for HEF Cells
Experiments with broblasts were carried out with nanoparticles stabilized with
water-soluble starch; the preparation procedure, electron micrographs, and particle
size distribution are given in Chap. 4 (Sect. 4.3.1). The nanoparticles were spher-
ical, 7 5 nm in size; concentration of the nanoparticles and starch in the stock
solution was 1 mM (108 g/mL) and 0.01 %, respectively.
The nanoparticle solution was diluted with Igla medium without serum, and then
introduced into the wells with cells to the concentrations (g/mL): 0.54, 1.08, and
2.16. Cells were incubated for 24 h and the mitochondrial activity was estimated by
the standard procedure used in MTT assay (e.g., [404, 405, 407] and literature
therein). To reveal the separate effects of stabilizer and silver ions, cells were
incubated with additions of the 0.0.1 % starch and 1 mM AgNO3 aqueous solutions
in the same dilutions as those used with nanoparticle solution. Control cell culture
(without nanoparticles, starch, or silver ions) was also incubated in the serum-free
medium. A detailed description of experimental procedure is given elsewhere [432].
The results obtained with HEF cells are shown in Fig. 9.1.
It is seen that the nanoparticle toxicity manifests itself beginning from the
concentration 1.08 g/mL, which may be regarded as toxicity threshold. That is,
9.1 Toxicity of Starch-Stabilized Ag Nanoparticles for HEF Cells 251
Fig. 9.1 Cytotoxicity of Ag 110

nanoparticles stabilized with 100 Starch
starch as studied on HEF 90
cells. Results of MTT assay
for the nanoparticles (AgNP) 80
OD, % of control
and the corresponding 70
concentrations of Ag+ ions 60
(AgNO3) and starch AgNP
50
40
30
20
10
AgNO3
0
10
0.0 0.5 1.0 1.5 2.0 2.5
CAgNP, mcg/mL
the TT value is virtually equal to that found for plasmodium (Sect. 8.1).
Comparison with the relevant data for toxicity measurements with AOT-stabilized
silver nanoparticles on bacteria in aqueous suspensions (Fig. 7.2 and Table 7.4)
shows that, rst, the TT is close to the minimal nanoparticle concentration used in
experiments with bacterium E. coli in aqueous suspensionas follows from Fig. 7.
2, incubation with 0.8 g/mL of nanoparticles leads to 92 % inactivation of bacteria
cells; consequently, here the toxicity threshold is likely to be lower than 0.8 g/mL,
i.e., denitely lower than that found for the starch-stabilized nanoparticles with
HEF cells. Second, in the similar tests with E. coli and St. aureus (Table 7.4), after
24 h of incubation the lethal dose was 1.44 g/mL for both bacteria species, so the
TT value is supposed to be lower. Both results indicate that probably the
nanoparticle toxicity for bacteria may prove to be higher than for HEF cells and this
makes it possible to regard the nanoparticles as candidates for creating the
antimicrobial remedies. However, this supposition requires verication in the cor-
responding experiments with starch-stabilized silver nanoparticles on bacteria, since
AOT as stabilizer can contribute to the toxicity of nanoparticle solution, unlike
starch; as clear from Fig. 9.1, this natural stabilizer does not exhibit toxicity in the
whole concentration range studied with human cells.
When the nanoparticle concentration is increased to 2.16 g/mL, the decrease in
mitochondrial activity is observed but by no more than 50 % of control. At the
same time, a much more essential suppression of mitochondrial activity is regis-
tered at the incubation with Ag+ ions in equivalent concentrations, up to the
complete loss of vital activity. Consequently, in contrast to bacteria and plas-
modium, the HEF cells are much more sensitive to the action of silver ions than to
that of silver nanoparticles. One may infer, therefore, that the toxic effect observed
with nanoparticle solutions is conditioned partly by the action of silver ions released
from the nanoparticle surface. As will be shown later in this chapter, this result does
not agree with the data obtained for cancer (HeLa) cells with similar adhesive
properties. Hence, it is possible also that, in the case of broblasts, we face the
peculiarities of the cell nature and/or of the medium composition, which favor the
higher toxicity of Ag+ ions compared to the other cell cultures.
9.2 Toxicity of AOT- and Cyclodextrin-Stabilized Ag

Nanoparticles for UVE Cells
The experiments on UVE cells were aimed at elucidating the influence of

nanoparticle surface charge on their toxicity toward normal human cells. Studies
were performed on endothelium cells (EA.hy926 line) with the two nanoparticle
preparationssilver nanoparticles stabilized with AOT and those stabilized with -
cyclodextrin (CD). Preparation of water solutions of the CD-stabilized nanoparti-
cles is briefly described in this book (Sect. 4.3.2) A detailed description of
experimental procedures and results of this study may be found in our recent
publication [433]. Here, we include the points which are important, as we believe,
for the correct performance of experiment and for the better illustration of the
signicance of nanoparticle surface properties in its interaction with living cells.
Both AOT- and CD-stabilized nanoparticles were negatively charged, but their
charge differed in magnitude, as found by measurements of their electrophoretic
mobility. As followed from the modern theories of electrophoresis, for the case of
small charged particles in aqueous solutions one should be aware of the fact that
true -potentials may differ from those calculated from the measured mobilities
through the Smoluchowski equation (sm) (see [374379] and Chap. 6). Taking into
account this circumstance, to nd the reliable sm values (and hence to estimate
correctly the relation of surface charges for the two nanoparticle preparations), the
mobilities should have been measured in conditions ensuring that difference in sm
values really reflects the difference in surface charge for the two nanoparticle
preparations studied. These conditions imply that the two nanoparticle preparations
are equal (or very close) in their mean size and their mobilities are measured in
solutions with equal ionic strength.
At the same time, as followed from the methodological requirements discussed
above in this book (Chap. 1, Sect. 1.7 and Chap. 6, Sect. 6.1), to obtain the reliable
results on the influence of one of the four main nanoparticle characteristics on its
interaction with biological object, it is necessary that the other three remain con-
stant. Conformably to the influence of nanoparticle surface charge, this means that
the two chosen nanoparticle preparations are to be different only in their surface
charge, but should be similar in size, form, as well as composition and properties of
a stabilizing shell other than its surface charge.
In our case, difference in surface charge was stipulated by the difference in the
nature of stabilizing shell, so the similarity of its compositions could not be pro-
vided. As mentioned in this connection in Chap. 6, this requirement can hardly be
fullled in most cases in studies of the surface charge effects because, in practice,
difference in surface charge of coated nanoparticles can be achieved by using
9.2 Toxicity of AOT- and Cyclodextrin-Stabilized Ag Nanoparticles for UVE Cells 253
different stabilizers. Therefore, we made every effort to the fulllment of the other
conditions, including both the similarity of size and form and the additional
requirement of the equality of ionic strength in mobility measurements.
In view of all said above, it seems reasonable to present here the data allowing
comparison of the nanoparticles used for the toxicity measurements. Figure 9.2 shows
the electron micrographs and the corresponding size distributions obtained for the AOT-
and CD-stabilized nanoparticles in aqueous solutions. It is shown that both kinds of
nanoparticles are spherical and have equal mean size (13.2 nm). True, the size distri-
bution is wider for CD-stabilized nanoparticles. The sm values were determined from
mobility measurements. The equality of ionic strengths was achieved through the
conductivity measurements; the mobilities were measured in solutions with conduc-
tivity within 440 40 S/cm. The sm values for AOT- and CD-stabilized nanoparticles
were found as 89.42 4.32 mV and 35.93 2.52 mV, respectively (Fig. 9.3).
Thus, we could be sure that the AOT-stabilized nanoparticles had a negative charge,
which was more than twice as large as that of CD-stabilized nanoparticles.
Endothelium cells were incubated with AOT-AgNPs and CD-AgNPs solutions
added to the medium in wells to the nal concentrations 0.5, 1.0, 1.5, 2.0, and
2.5 g/mL. To check the potential toxic effects of stabilizers, cells were also
incubated with AOT and CD water solutions in dilutions corresponding to those of
(a) (b)
100 nm 100 nm
18 (a) Size:
18 (b) Size:
16 13.2 4.72 nm 16 13.2 7.97 nm
14 14
%of particles
12 12
% of particles
10 10
8 8
6 6
4 4
2 2
0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Nanoparticle diameter, nm Nanoparticle diameter, nm
Fig. 9.2 TEM images and size distribution histograms of silver nanoparticles stabilized with AOT
(a) and -cyclodextrin (b)
(a) (b)
1.0 1.0
0.8 0.8
0.6 0.6
Power
Power
0.4 0.4
0.2 0.2
0.0 0.0
-160 -80 0 80 180 -160 -80 0 80 160
Zeta Potential (mV) Zeta Potential (mV)
Fig. 9.3 Zeta potentials (sm) obtained by PCS measurements on ZetaPals instrument for the stock
solutions of AOT-stabilized (a) and -CD-stabilized (b) Ag NPs. The conductivity control was
applied for providing the equality of ionic strength
Ag NPs in wells. The incubation time was 24 h with all agents added. The MTT
assay was performed according to the standard protocol; the mitochondrial activity
was inferred from the optical density of formazan band at 450 nm.
In the parallel set of experiments, the portion of apoptotic and necrotic cells after
incubation at each nanoparticle concentration was determined by means of flow
cytofluorimetry. An Annexin V-FITC Apoptosis Detection Kit I was used; the
procedure was realized following the protocol supplied by the manufacturer. Cells
were incubated in the plates wells for 24 h. At the transfer of cells to cytometry
tubes, caution was taken to avoid the loss of self-detached cells present in the
incubation medium. Before the cytofluorimetric analysis, cells were incubated in
the tubes with annexin and propidium iodide (PI), then diluted with binding buffer,
and fluorescence levels of FITC and PI were measured in FACSCalibur flow
cytometer (BD Biosciences, USA).
120
AOT
100 -CD
Cell viability, % of control
AgNPs-AOT
AgNPs--CD
80
60
40
20
0
0.0 0.5 1.0 1.5 2.0 2.5
Concentration of Ag NPs, g/mL
Fig. 9.4 Viability of EA.hy926 cells after 24 h of incubation with AOT-stabilized and -
CD-stabilized AgNPs at various nanoparticle concentrations (MTT assay). Cells were also
incubated with corresponding nanoparticle stabilizers (AOT and -CD) in the relevant
concentrations, to determine their contribution to the nanoparticle toxicity
The influence of nanoparticles on the cell viability estimated by MTT assay is

illustrated in Fig. 9.4. The results are presented, obtained both with the two
nanoparticles preparations and with the relevant stabilizers. First of all, it is clear
that a noticeable difference exists between the effect of AOT-AgNPs and
CD-AgNPs. The former provoke a decrease in viability to 70 % of control at
1 g/ml and a sharp fall to 20 % of control at the higher nanoparticle concentra-
tions, while the latter does not induce noticeable viability changes, which remain at
the 80 % of control in the whole concentration range. The results indicate to much
higher toxicity of the AOT-stabilized nanoparticles; here, the toxicity threshold is
1 g/ml, and the half-inhibitory concentration IC50 = 1.3 g/ml, while for
CD-AgNPs the IC50 is obviously greater than 2.5 g/ml.
As for the effect of stabilizers, it is seen that AOT provokes a sharp decrease in
cell viability beginning from its concentration present in the 2 g/ml nanoparticle
solution. This means that AOT exerts a toxic action, though less strong than that of
the nanoparticles. A more detailed comparison of the nanoparticles action with that
of AOT solution [433] makes it possible to conclude that the nanoparticles are more
toxic than AOT in the range 0.51.5 g/mL, with difference most strongly
expressed (70 % in cell viability) and minimal AOT contribution at 1.5 g/mL. At
the same time, CD has no influence on cell viability.
This different effect of the two nanoparticle preparations on cell viability
observed in MTT test is further supported by flow cytofluorimetric analysis, which
makes it possible to detalize cell viability changes by nding the portion of
apoptotic and necrotic cells at each nanoparticles concentration (Fig. 9.5). In
agreement with viability data, with AOT-AgNPs obvious decrease in the contri-
bution of viable cells is registered at the two highest nanoparticles concentrations, a
decrease in viability beginning from the higher nanoparticle concentration
(2.0 g/mL) than that found by MTT assay (1.5 g/mL). This probably indicates
that changes in mitochondrial activity take place earlier than cells fall into apop-
tosis. In other words, mitochondrial functions turn out to be more strongly affected
by the toxic action of nanoparticles than proapoptotic functional elements within
the cell. As shown in Fig. 9.5, with CD-AgNPs no apoptotic or necrotic cells are
detected in the whole concentration range considered, in accordance with the MTT
results.
Evidence in favor of similar difference between the two kinds of nanoparticles
was obtained also in our investigations of cell morphology changes by phase
contrast microscopy.
Photographs obtained for the cells incubated with the same nanoparticle con-
centrations showed that, again, with AOT-AgNPs drastic changes of cell mor-
phology took place at the concentrations which correlated with those corresponding
to the viability decrease in MTT assay and also to the noticeable growth in number
of apoptotic cells in cytofluorimetric measurements. Example of such kind is pre-
sented in Fig. 9.6. It is seen that incubation with AOT-AgNPs at 2.0 g/mL leads to
the formation of apoptotic bodies and clearly expressed destruction of cells (b), in
contrast to the incubation with lower nanoparticles concentration (a, 0.5 g/mL),
AOT-stabilized Ag NPs
100
90
80
70
% of total cells
60
Living cells
50
Early apoptosis
40 Late apoptosis
30 Necrosis
20
10
0
0.0 0.5 1.0 1.5 2.0 2.5
-cyclodextrin-stabilized Ag NPs
100
90
80
70
% of total cells
60
Living cells
50
Early apoptosis
40 Late apoptosis
30 Necrosis
20
10
0
0.0 0.5 1.0 1.5 2.0 2.5
Fig. 9.5 Early and late apoptosis and necrosis of EA.hy926 cells detected by flow cytofluorimetry
after 24 h of incubation with various concentrations of AOT- and CD-stabilized AgNPs. Events
expressed in percentages (total amount of cells = 100 % of events, corresponding to 15,000 cells)
where practically intact cells can be observed. Meanwhile, incubation with

CD-AgNPs exerts practically no influence on cell morphology (data not shown).
The whole complex of data leads to the conclusion that the AOT-stabilized silver
nanoparticles possess a higher toxicity toward endothelium cells than those stabi-
lized with CD. Since the two kinds of nanoparticles differ in sm values and hence,
in surface charge density, and also in the toxic action of stabilizer, AOT being toxic
in contrast to CD, the difference in the nanoparticles toxicity may result partly from
the difference in the nanoparticles surface charge density and partly from that in the
effect of stabilizer. In the case of AOT-AgNPs, the contribution of stabilizer into the
overall toxicity is minimal at 1.5 g/mL of nanoparticles; judging from our
experimental results, at this concentration the toxicity of nanoparticle solution is
Fig. 9.6 Phase contrast (a)

micrographs of EA.hy926
cells after 24 h of incubation
with AOT-stabilized silver
nanoparticles
(magnication 200). Control
cells were incubated
with deionized water. At
0.51.5 g/mL, cells stayed
normal and were adhered to
the well bottom without any
changes in shape; example
(a) is shown for 0.5 g/mL.
Cells incubated with
2.0 g/mL (b) had alterations
in viable morphology
presented as cell detachment,
formation of apoptotic bodies (b)
and shrinkage; confluence of
the cell monolayer was
strongly decreased
(Microscope Eclipse TE
2000-U (Nikon, Japan)
equipped with a CoolSNAP
Pro digital camera (Media
Cybernetics, USA))
conditioned virtually by the nanoparticles themselves and may be attributed to the

electrostatic interactions more strongly expressed than those taking place with
CD-AgNPs. To put it in a more general form, biological activity exhibited by the
nanoparticles of the same size and shape depends on the composition and surface
charge of the stabilizing shell, the contribution of each factor being dependent on
the nanoparticle concentration.
To sum up, we found that AgNPs with bigger negative charge are more toxic
than those with smaller negative charge. This inference disagrees with the
assumption that a more negative particle charge should result in a decrease of the
nanoparticle toxicity due to a decrease of electrostatic repulsion between
nanoparticles and cell surface. Similar disagreement between the nanoparticles
negative zeta potential and their quite measurable toxic action was discussed
conformably to the bactericidal action of negatively charged TiO2 nanoparticles
[350]. By contrast, obvious difference in bactericidal activity (on Gram-positive
bacillus species) was reported for negatively and positively charged AgNPs [434],
namely positively charged nanoparticles were essentially more toxic than those
negatively charged. Similar conclusion was made from experiments with AgNPs on
normal and malignant cells [435, 436].
A detailed discussion of the problems arising in comparison with the results
obtained in studies on the effect of nanoparticle surface charge on their interaction
with cells in culture is present in our recent publications [433, 437]. The main
hindrance is that the correct comparison can be made only with the nanoparticle
preparations of the same size and form, and with the sm values which reflect true
charge density of a nanoparticle surface. These conditions are rarely, if at all,
satised because of either the difference in the results of TEM and DLS mea-
surements of particle sizes or conventional neglect of correction for the relaxation
effect in estimation of zeta potential from mobility measurements, or both. It is
important to note also that, judging both from the literature available and our
results, to elucidate the role of electrostatic interactions, it is necessary to separate
the contribution of surface charge and chemical properties of a stabilizing coating
into a nanoparticlecell interaction.
9.3 Toxicity of AOT-Stabilized Ag Nanoparticles

for HeLa and U937 Cells
Studies were carried out with two types of malignant cells: HeLa (adhesive cells)
and U937 (suspension cells), both supplied by the Institute of Cytology of RAS.
Here, we used the AOT-stabilized nanoparticles, as they were supposed to possess a
higher toxicity than those synthesized with natural stabilizers. The nanoparticle
aqueous stock solutions with initial AgNPs and AOT concentration equal to 1 mM
(108 g/mL) and 2 mM, respectively, were obtained by the transfer from micellar
solution as described in Chap. 4 (Sect. 4.2.1). The nanoparticles were spherical,
with mean size of 13.4 4.7 nm. Toxicity of the nanoparticles was estimated in the
wider range 0.58.0 g/mL by MTT test and flow cytofluorimetry technique after 4
and 24 h of incubation with cells. The nanoparticles were introduced by dilution of
their stock solution to the concentrations desired. Details of experimental procedure
may be found in our original publication [438]. To separate the effect of
nanoparticles from those of AOT and silver ions, in parallel experiments cells were
incubated with AOT and AgNO3 water solutions introduced to the concentrations
corresponding to those present in the nanoparticle solutions at the same dilutions.
Figure 9.7 presents the results of MTT assay obtained after 24 h of incubation
with HeLa (a) and U937 (b) cells. It is seen that for HeLa incubated with
nanoparticles the viability decreases beginning from 2.0 g/mL; similar behavior is
observed after 4-h incubation. Hence, the TT value is 2.0 g/mL in this case and
IC50 is approximately 1.5 g/mL. As is clear also from Fig. 9.7a, both AOT and
silver ions do not exhibit noticeable toxic effect in the concentration range studied.
It is worth noting also that an increase in viability is observed at the lowest AOT
9.3 Toxicity of AOT-Stabilized Ag Nanoparticles for HeLa and U937 Cells 259
(a) (b)
Ag ions Ag ions
140 140
AOT AOT
AgNPs AgNPs
120 120

100 100
80 80
60 60
40 40
20 20
0 0
0.5 1.0 2.0 4.0 8.0 0.5 1.0 2.0 4.0 8.0
Concentration of AgNPs, g/mL Concentration of AgNPs, g/mL
Fig. 9.7 Viability of HeLa (a) and U937 (b) cells after 24 h of incubation with AOT-stabilized
AgNPs, Ag+ ions, and AOT at various nanoparticle concentrations in the range 0.58 g/mL
(MTT assay)
and nanoparticles concentrations, presumably showing to the stimulation of cell

growth by these agents below the toxicity threshold. Similar observations were
made in studies on the same cell lines with AOT-stabilized nanoparticles smaller in
size (9.2 2 nm) performed in IGPP by A.A. Moskovtzev and co-authors (un-
published data). In our opinion, this phenomenon is probably a manifestation of the
stimulating effects of low and super-low doses registered previously in experiments
with various biologically active substances [439, 440].
For U937 cells (Fig. 9.7b), a clearly expressed toxic effect takes place already at
the smallest nanoparticle and silver ion concentrations, while AOT is less toxic for
these cells almost in the whole concentration range under consideration. For
nanoparticles, the TT value is 0.5 g/mL and IC50 is approximately 0.8 g/mL. The
latter is twice as large as that found for Ag+ ions; that is, here the nanoparticles are
less toxic than silver ions; the same was observed after 4 h of incubation. The toxic
action of AOT is stronger than that observed with HeLa cells; however, it is
obviously less pronounced than that of silver ions at all concentrations studied
except for the maximal one (8.0 g/mL). At the incubation with nanoparticles, there
is an unexpected increase in cell viability at this maximal concentration. As we
believe, this may be the consequence of nanoparticles aggregation in the cell
medium, resulting in a decrease of active nanoparticle concentration.
Comparison of the data obtained by MTT assay for both cell lines shows that
HeLa cells are less sensitive to the toxic action of silver ions than U937 cells. To
our view, this may be conditioned by the difference in properties inherent to these
cells. Since HeLa are adhesive cells, at the incubation their surface is partly
adherent to the well and therefore is less available to silver ions, while U937, as
suspension cells, are dispersed in the bulk solution so that silver ions can attack
their whole surface. Besides, U937 cells are smaller in size than HeLa, hence at the
same number of cells per unit volume of suspension, their total surface area is larger
and the silver ions are more effective.
Studies by means of flow cytofluorimetry technique after 24 h incubation
revealed that, with HeLa cells the percentage of viable, apoptotic and necrotic cells
correlated with changes in cell viability found in MTT test at the corresponding
nanoparticle concentrations. With U937 cells, an unexpectedly low portion of
viable cells and high percent of necrotic cells were registered at the two lowest
nanoparticle concentrations (0.5 and 1.0 g/mL), in spite of the high viability
observed for this concentration in MTT assay. At the two highest concentrations
(4.0 and 8.0 g/mL), no living cells were found; the majority of cells were in the
late apoptotic state, but almost no necrotic cells were present. This may be con-
nected with nanoparticle aggregation and the resulting decrease in the concentration
of active nanoparticles, capable of interaction with cell surface, in agreement with
the MTT data on the increase in cell viability at 8.0 g/mL of nanoparticles. The
other possible reason may be cell proliferation in the course of 24 h of incubation at
this nanoparticle concentration.
To verify our assumption about the nanoparticles aggregation at their high
concentrations in a plate well during incubation with cells, we undertook the
attempts to measure the particle sizes by PCS technique directly in the cell culture
media after the introduction of nanoparticles solution to the relevant concentrations.
However, it turned out that this experiment could not be performed because the
dynamic light scattering technique cannot distinguish between the aggregates of
nanoparticles and those formed by the other medium components or by their
combinations with nanoparticles. Therefore, we fullled these measurements using
PBS (phosphate buffer solution) as the nearest possible model of cell medium.
Particle sizes were determined in PBS (without cells) at the 4.0 and 8.0 g/mL of
nanoparticles after 1 h and 2 h of incubation at 37 C. It was found that
nanoparticles form aggregates after 1 h of incubation; they manifest themselves in
bimodal distribution with one peak of low intensity (smaller size) and the second of
high intensity (bigger size). The particle sizes depended on the nanoparticle con-
centration and varied from 60100 nm for 4 g/mL to 10001500 nm for 8 g/mL.
After 1 h of incubation, no further size changes were observed. A more detailed
information on this point is given in [438].
From the data obtained in this set of experiments followed that the nanoparticle
aggregation (and the corresponding decrease in toxicity) could really take place at
their high concentrations in the culture medium. Aggregation of silver nanoparticles
in cell culture media was detected in similar studies on nanoparticlecell interac-
tions known from literature [441, 442]. In our case of AOT-stabilized nanoparticles,
the aggregation is stimulated, most likely, by the cation adsorption on the nega-
tively charged headgroups of stabilizer present on the nanoparticle surface.
Comparison with the data on cell viability response to silver nanoparticles
available from literature was possible for HeLa cells after 24 h of incubation; the TT
value was used as the main criterion, since it could be easily determined from the
viability versus nanoparticle concentration plots present in the publications con-
sidered. It was found [438] that the TT obtained by us (2 g/mL) was noticeably less
9.3 Toxicity of AOT-Stabilized Ag Nanoparticles for HeLa and U937 Cells 261
Fig. 9.8 Visualization of the

aggregates of AgNPs (shining
points indicated by white
arrows) inside the HeLa cells.
Laser confocal scanning
microscopy (IGPP)
than those reported for the same cells by some other authors [441, 443], but close to
that found for A431 cells [411] and HepG2 cells [444] with nanoparticles of similar
size, and not essentially higher than the TT values registered for the other cell types
with nanoparticles different in size [411, 441, 443]. It is possible to infer that, for the
time being, particle size determined in an initial nanoparticle solution is not
important for their effect on cell viability; as the likely cause one may regard the
nanoparticles aggregation noted by some researchers [407, 442] and evidenced also
in our work. It is worth adding that as issues from our observations and the results
present in literature mentioned above, it seems that the aggregation does not prevent
nanoparticles from being active toward biological cells.
The latter inference may be illustrated by the data recently obtained on mes-
enchymal stem cells [412]; it was demonstrated that in spite of the aggregation in
culture medium, the nanoparticles are able to penetrate into the inner cell structures,
including nucleus, and to provoke the consequent cytotoxic and genotoxic
phenomena.
As one more illustration, we present here the image of HeLa cells after incu-
bation with silver nanoparticles (Fig. 9.8, color insert), where the presence of
nanoparticle aggregates was detected inside the cells by means of laser confocal
microscopy. The aggregates are visualized as shining points indicated by white
arrows. Examples of such kind testify once more to the good perspectives of
modern laser microscopy techniques in studies of the mechanisms of nanoparticle
interaction with biological objects on cellular and subcellular levels.
In summary, our experimental results obtained with AOT-stabilized silver
nanoparticles testify to their high toxicity toward cultured cancer cells.
Consequently, the nanoparticles can be considered as possible anticancer agents
useful for application against the corresponding oncological deceases.
Inferences from the Results of Our Studies
Taken as a whole, our studies on the action of aqueous solutions of silver

nanoparticles coated with synthetic anionic surfactant (AOT) or natural stabilizing
shell (starch or -cyclodextrin) on the biological objects give grounds for the fol-
lowing conclusions:
First, it is clear that AOT-stabilized nanoparticles with sizes within 9 6 nm
represent a strong toxic agent, which is capable of suppressing the viability of a
wide spectrum of microorganisms as well as of some other living systems,
including fungus, plant organisms, and cultured human cells. Toxic effect on
microorganisms is registered both in an aqueous medium (with nanoparticles
introduced as water solution) and in testing of the liquid-phase or solid materials
modied with nanoparticles by means of small additions of nanoparticle solutions
or nanoparticle adsorption on solid surfaces.
The extent of toxicity for the aqueous solutions of nanoparticles depends on their
concentration in an aqueous medium. Minimal nanoparticle concentration corre-
sponding to the beginning of viability decrease (the toxicity threshold, TT) varies
for the objects different in nature. For microorganisms, the TT value lies in the
range 13 g/mL, while their total death takes place at 510 g/mL (Chap. 7).
Similar TT and lethal doses were found for macroplasmodium of acellular slim
mold (Physarum polycephalum), but much lower values were obtained for its
microplasmodium and unicellular alga; however, much higher ones were found for
plant seeds (Chap. 8). The TT values within 0.52 g/mL were determined for
human cells in vitro (Chap. 9). Basing on the experimental results, it is possible to
arrange the objects studied in the following sequence by their sensitivity (in TT
values) to the effect of nanoparticles:
Plant seeds (>108 g/mL) << bacteria + macroplasmodium + human cells
(0.53 g/mL) < microplasmodium + chlorella (0.108 g/mL)
Our preliminary inference from this sequence is that the extent of nanoparticle
toxicity may be related to the surface properties of an object studied: the less sen-
sitive are the plant seeds with the most rigid external shell, while the most sensitive
are the microplasmodium cells with the most thin and delicate cell membrane. This
points to the essential role of nanoparticle adsorption in the process of their inter-
action with biological object. In this connection, it seems doubtful that unicellular

DOI 10.1007/978-3-319-30906-4
264 Inferences from the Results of Our Studies
alga really possess such a high sensitivity, since it is protected by a three-layer cell
wall and, as noted in description of the relevant experiment (Sect. 8.2), demonstrates
a lower sensitivity compared to the bacterium E. coli. Probably, its high sensitivity is
conditioned by the peculiarities of microelectrophoresis technique applied in this
case to estimate the nanoparticle toxicity.
Second, AOT as synthetic surfactant is also toxic for biological objects; conse-
quently, to isolate a pure effect of nanoparticles, it is necessary to minimize the
AOT concentration in a nanoparticle solution and to carry out the parallel experi-
ments with AOT water solution diluted to the relevant concentration. The results of
such experiments show that, for all objects studied, toxic effect of nanoparticles
exceeds that of AOT at its concentration equal to that introduced with the nanoparticle
solution, and so it was possible to determine the range of nanoparticle concentrations
where the AOT toxicity is negligible, so that the biological effect observed was
provoked only by the nanoparticles. For example, in experiments with plasmodium at
the nanoparticle concentrations not exceeding 1.08 g/mL (105 M) the AOT toxi-
city can be neglected, while the effect of nanoparticles is quite distinctly expressed.
The preliminary procedures decreasing the AOT concentration in the nanoparticle
stock solution make it possible to increase the working nanoparticle concentrations
corresponding with the absence of AOT toxicity. This was the case in studies of the
HeLa and U937 cultured cells, where an actually pure toxic action of nanoparticles
was observed at their concentrations of 12 g/mL (Chap. 9, Sect. 9.3).
Third, substitution of AOT for the natural stabilizers (starch or cyclodextrin)
allows the preparation of nanoparticle solution with stabilizer nontoxic toward
human cultured cells (at least for broblasts and endothelium cells studied). In this
case, a decreased toxicity of silver nanoparticles to these normal cells is also
detected, in contrast to what is observed for the AOT-stabilized nanoparticles.
A low toxicity of silver nanoparticles coated with starch was showed also in
experiments with sh embryos [1]. At the same time, a high antimicrobial activity
of our silver nanoparticles stabilized with cyclodextrin was found in the recent
experiments performed in collaboration with Coletex, a science production
company specializing in the development and production of various materials for
medical use. Hence follows that further progress in these studies may result in
determination of the concentration range where the cyclodextrin- or
starch-stabilized silver nanoparticles possess simultaneously a strong enough bac-
tericidal activity and a low enough cytotoxicity, thus ensuring the conditions for
their safe application as antimicrobial remedy.
Henceforth, it is supposed to apply the experimental procedures useful for the
fabrication of the above-mentioned nanoparticles in testing some other stabilizers
that promise to be acceptable for the creation of efcient medical means. Besides,
comparison of the results obtained for the nanoparticles with sizes within 120 nm
prepared with different stabilizers will be useful, as we believe, for gaining a deeper
insight into the role of stabilizing shell in biological activity of metal nanoparticles
in the range where their size effects are observed.
Fourth, in the majority of cases studied the effect of nanoparticles exceeds
signicantly that of silver ions in equivalent concentrations. This means that, in
Inferences from the Results of Our Studies 265
these cases, the nanoparticle action proceeds by a mechanism different from that
assumed for silver ions. Similar conclusion was made in the studies of silver
nanoparticle interaction with bacteria [24] and sh embryos [1].
At the same time for the three of our objectsplant seeds, broblasts and U937
cellsa completely different picture was observed: toxicity of silver ions was
almost equal (U937) or noticeably higher (plant seeds and broblasts) than that
exhibited by the nanoparticles, testifying to the active role of ions in biological
action of silver nanoparticles. Active participation of ions released by the
nanoparticles into solution was established also in some studies on bacteria species
[5, 6]. Thus, for the time being there is evidence in favor both of ionic mechanism
and a leading role of the nanoparticles themselves in their biological activity. It
seems probable that, in reality, both mechanisms can work, but the contribution
each of them makes depends on the object studied and experimental conditions
used by the researchers. In any case, it is clear that today there is no grounds for the
denite conclusion on this point: the question still has to be solved in the course of
future investigations.
Fifth, as follows from our experiments and also some literary data, it is rea-
sonable to pay more attention to the role of stabilizing coating in the toxic action of
silver nanoparticles. On the one hand, the nanoparticles sized below 20 nm exhibit
the toxicity at their equal or close concentrations in aqueous medium toward var-
ious biological objectsbacteria, lower fungus, cultured human and animal cells
independently on the shell composition and the way of nanoparticle preparation. In
other words, it turns out that the biological effect of nanoparticle issues from their
own activity, independent or weakly dependent on the presence and nature of a
coating. On the other hand, as concluded in our recent review of the studies on the
effect of silver nanoparticles on cultured cancer cells [7], for the nanoparticles
obtained by bioreduction, a composition and chemical properties of surface coating
are likely to play a signicant role in the toxic action of nanoparticles. The dif-
ference in surface coating may manifest itself, in particular, in the different
mechanism of nanoparticlecell interaction for the nanoparticles capped with
polyphenols (synthesis in plant extracts) and those capped with bacterial proteins
(synthesis in bacterial extracts). So it appears that, as with the choice between the
role of ions and nanoparticles, the problem with the actual contribution of
nanoparticle surface coating is to be recognized as occupying its lawful place in
studies of the nanoparticle interaction with biological objects.
Conclusion
At the end of this work, we would like to emphasize the most essential points which
ensue from the results of studies on the biological effects of metal nanoparticles
considered here. First of all, the fact is established that silver nanoparticles possess a
high biological activity toward the living organisms staying on different levels of
organization. This allows to regard these nanoparticles as a new effective means, a
kind of double-edged tool which can bring both a great benet and a great damage,
depending on the way it is used. The most extensive knowledge is achieved in
studies of the antibacterial properties of nanoparticles and modied materials. Here,
the aspiratory results have been obtained, which open the real possibilities to create
varnishpaint and lter materials with biocidal properties, medicines, and various
materials for medical application and other products. Denite hopes give also the
results of the tests of antimicrobial properties made with copper nanoparticle and
nanoparticle-containing materials. In the last 1015 years, the number of methods
allowing to obtain nanoparticles was markedly enhanced and the spectrum of the
objects studied was widened signicantly. Great progress was made in application
of the modern analytical methods used for the determination of nanoparticles
structure and composition, the composition of culturing media, also in perfection of
the microscopic techniques for the visualization of processes taking place in the
course of nanoparticlesanimal cells interaction.
At the same time, as noted in several reviews [15] and issues also from the
contents of this book, in spite of the intensive studies undertaken in the last years,
still much remains to be elucidated. There are white points in the knowledge about
the influence of the main characteristics of silver nanoparticles (size, form, surface
charge, the nature of stabilizer) on their antimicrobial activity, in the understanding
of the processes allowing to realize the nanoparticles effect on microorganisms, as
well as in the studies of the nanoparticles toxicity toward living cells and human or
animal organism. Several examples are presented below.
Among the main characteristics of nanoparticles, the influence of their surface
charge is actually not investigated; at the same time, there is evidence showing to
the essential role of electrostatic interactions in the biological activity of nanopar-
ticles [5, 6]. As for the mechanisms of their action, the results obtained at present do
not allow to clear out whether the toxic effects observed on microorganisms and

DOI 10.1007/978-3-319-30906-4
268 Conclusion
animal cells are caused by the metal ions released from the nanoparticles or by the
action of the nanoparticles themselves in the process of their adsorption on cell
membrane, penetration into the cell interior, and interaction with intracellular
components. There is evidence in favor of each of these mechanisms; it is possible
also that they act in combination, and each makes different contribution depending
on the object studied and experimental conditions. It is unclear also, whether the
nanoparticles activity suffers marked changes with time and to what extent it
depends on preservation conditions. There are only few data on the stability of their
antimicrobial activities [7] and the enhancement of their cytotoxicity with time
(within 23 months) because of their dissociation into ions [8].
There is no doubt that, for the practical use of nanoparticles and evaluation of the
danger caused by their release to the environment, it is extremely important to
reveal the nanoparticles activity changes with time. But it is evident also that
determination of the conditions providing simultaneously a high biocidal activity
and low enough cytotoxicity of nanoparticles is closely connected with the choice
of method used for their preparation. Besides, it requires the systematic investi-
gation of influence of the main characteristics of nanoparticles on their activities
against the denite bacteria species and cells, relevant to the chosen way of
application.
The lack of information mentioned above and some other questions may be
regarded probably as quite natural taking into account that at present only the initial
stage of the works is actually considered, when mainly the accumulation of
experimental data takes place and the existing pool of evidence is not enough for
the formulation of general regularities. Meanwhile, the intensive development of
nanotechnologies makes its high demands to the investigators of the nanoparticles
and it becomes more and more clear that, without the distinct understanding of the
processes underlying the biological effects of silver (and other metal) nanoparticles,
one can hardly hope to suggest the well-grounded recommendations for the safe use
of nanoparticles, both as native solutions or powders and as components of liquid or
solid materials.
The development of such recommendations becomes very urgent today, since in
our country silver and other metal nanoparticle and nanoparticle-containing mate-
rials are manufactured already by private companies which offer their production
(or that from the foreign sources) for the general consumption. Such offers may be
easily found in the Web, for example, on sites nanotech.ru, kvilmoskva.ru, and
tech-textil.ru. This testies to the importance of thorough investigations of the
biological effects and mechanism of nanoparticle action because, apart from the
positive results, the application of such production with insufciently studied
properties may also have highly negative consequences.
Analysis of the literature undertaken by us as well as the experience acquired in
the course of experimental work on various biological objects allows to put forth
several considerations on methodology of the studies under question, which may be
useful both for choosing the strategy of experiment and for the progress in
understanding of the mechanism of nanoparticle action on the living organisms.
Conclusion 269
First of all, it is worth to pay attention to the peculiarities of metal nanoparticles

as the object of our studies. At the early stage of the works with these nanoparticles,
undertaken mainly on microorganisms, the nanoparticles were usually regarded as a
certain set of metallic particles (in the simplest caseof the balls of denite size),
with properties depending only on their size irrespective of the way of their
preparation. In other words, one supposed that size is the only meaning parameter
for the nanoparticles of a given metal. As the data were accumulated, it became
clear that biological activity of the nanoparticles depends also on their form and
surface properties. The latter include two componentssurface composition (i.e.,
composition of a stabilizing shell of the naked nanoparticle surface) and surface
charge. That is, there are at least three more meaning parametersform, surface
composition and charge, the latter two depending on the method of nanoparticle
preparation. So it turned out that metal nanoparticles represent a rather complicated
object, whose biological activity depends on its origin, that is, on the method of
preparation.
To elucidate a mechanism of the nanoparticles action, it is important to detect
the influence of each of the four parameters mentioned above on their biological
activity. From the methodological point of view, it is obvious that, to estimate the
effect of a given parameter, it is necessary that three other remained constant.
Analysis of the literature shows, however, that, as a rule, this condition is not
satised in studies aimed to nd the effect of one parameter, so that the results
obtained cannot be regarded as quite reliable. Examples of such kind are given in
Chap. 6. Besides, in studies of the particle size effects the additional parameter
appears, namely the numerical particles concentration (the number of particles in
unit volume). This means that, for the particles of different sizes, their biological
activity should be compared at their equal numerical (but not mass) concentration.
It should be added that, the greater is difference in particle sizes, the greater is
difference in mass concentrations corresponding with the equal numerical con-
centrations. As a result, it turns out to be necessary to check the results obtained so
far with the observance of the above-mentioned condition but for different
numerical concentrations. Therefore for the time being, conclusion about the higher
biocidal activity of the smaller nanoparticles cannot be considered as quite reliable.
As we believe, at present one can only speak about the tendency, which still needs
to be conrmed by experiments.
It is worth noting also that, to our opinion, studies on the role of electrostatic
interactions in biological activity of nanoparticles requires the revision of the
presently working notion about -potential obtained by means of PCS technique, as
an adequate characteristic of the particle surface charge. As was explained in
Chap. 6 (Sect. 6.1.1), for the correct determination of surface charge density from
electrophoretic mobility measurements, calculation of -potential should be made
with correction for the relaxation effect. Unfortunately, in the appropriate modern
devices this correction is either absent (calculation by the Smoluchowski equation)
or introduced for the narrow potential range (calculation by the Henry equation).
That is why the -potential values obtained in such devices may differ signicantly
from the true -potentials which can be used for the estimation of surface charge
270 Conclusion
density. The resulting error in surface charge density may be signicant in mea-
surements on the nanoparticles with sizes 110 nm in solutions of low ionic
strength. The importance of the correction for the relaxation effect was clearly
demonstrated on examples taken from measurements on lipid membranes [9, 10].
All said above emphasizes the paramount importance of the creation of
well-grounded scientic foundation for the building of the whole system of notions
on the regularities which determine the biological activity of metal nanoparticles.
Such system could serve as a kind of framework for the accumulation of experi-
mental data and, as grand total, formation of the valuable scientic direction with
wide spectrum of applications in medicine, biology, biotechnology, and other elds.
Creation of the scientic foundation obviously requires the subsequent widening of
the knowledge about the formation, properties, and interactions of metal
nanoparticles with various biological objects. Here could be useful, as we believe,
investigation of the nanoparticle interactions with biological molecules and vesicles
existing in living cells. Besides, it would be reasonable to consider the possibility of
modeling the biological action of nanoparticles on lipid membranes, taking into
account the experience accumulated in the relevant eld of research [11]. One could
draw in also the modern theoretical approaches for the description of processes
taking place in liquid media with participation of nanoparticles, including their
aggregation and desaggregation, changes of form, structure, and surface composi-
tion of a growing particle. To our view, of special value in this respect are the
approaches developed by member correspondent of RAS, Prof. Melikhov [12].
In the foundation of the direction under question, its denite place can occupy,
as we believe, the biochemical synthesis of metal nanoparticles in reverse micelles
with the use of biological reducing agents, since this method opens new possibil-
ities for the fundamental studies of the processes taking place in living organisms. It
is known that, at low hydration extents, the internal medium of reverse micelles
possesses properties very close to those of organelles and compartments in a living
cell. Thus, the fact established by us, of metal nanoparticle formation through the
flavonoids interaction with metal ions in reverse micelles, demonstrates the prin-
cipal possibility of clusters and nanoparticle formation in living organisms through
the interaction of the same type. Hence follows that reverse micellar system may
serve as an adequate model in studies on the mechanisms of biological activity of
flavonoids, including their interactions with metal ions. Taking into account also
that, as have been recently ascertained, metal nanoparticles can be synthesized with
the help of plant extracts containing flavonoids (Chap. 1, Sect. 1.6), there are
reasons to assume that biochemical synthesis in reverse micelles will allow to
elucidate the regularities guiding the metal nanoparticle formation in plants. Studies
undertaken in this direction may give the grounds for valuable practical applications
for instance, for the creation of new effective means of metal extraction from soil
waters or for the enhancement of seed germination for agricultural plants.
All said above about the signicance of scientic foundation certainly does not
mean that we call for the neglect of the increasing role of applied studies, in our
case in the eld of biomedical applications of metal nanoparticles. It is important,
however, that practical requirements should not be transformed to a single focus for
Conclusion 271
the concentration of scientic researches, because this often leads nally to the
unjustied expenditure of time and forces with minimal helpful yield. We would
like to believe that, in studies of the biological effects of metal nanoparticles, it will
be possible to nd reasonable combination of the leading role of scientic thought
with its practical embodiments, and this direction will become one of the examples
testifying to the great signicance of science as a way of acquiring knowledge about
the world for the benet of humanity.
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ElenaMikhailovnaEgorova

Biological Effects of Metal

Aslan Amirkhanovich Kubatiev

ISBN 978-3-319-30905-7 ISBN 978-3-319-30906-4 (eBook)

Library of Congress Control Number: 2016933474

Springer International Publishing Switzerland 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Part I Preparation and Characterization of Metal Nanoparticles

3 Development of the Biochemical Synthesis for Practical

Part II Biological Effects of Metal Nanoparticles

7 Antimicrobial Activity of Nanoparticles Stabilized with Synthetic

Inferences from the Results of Our Studies . . . . . . . . . . . . . . . . . . . . . 263

Elena Mikhailovna Egorova Doctor of chemical

Vitaly Ivanovich Schvets Doctor of Chemical

AAS Atomic absorption spectroscopy

infectious and oncological ones), as well as in immunochemical methods of anal-

given of (1) biochemical synthesis, preparation and characterization of nanoparti-

Several Words About the Contents of This Part

It is known that studies of the interaction of metal nanoparticles with various

1.1 Introductory Remarks

Springer International Publishing Switzerland 2016 3

the correlation radius of some physical or chemical phenomena (e.g., electron or

Fig. 1.1 Changes in material

p. 14). x-axis dimensions of

In particular, in chemistry the criterion for the object to be referable to nanoparticles

1.2 Principles Used for the Choice of Methods

Electrode surface + Molecular

Electrochemical Photochemical Radiation-chemical Traditional

Reverse Aqueous Biological

Fig. 1.2 Methods of the chemical synthesis of metal nanoparticles in solutions

that we do not pretend on the exhaustive description of methods in each of the

1.3 Chemical Synthesis with Traditional Reducing Agents

mainly by the deposition of nanoparticles from colloidal solutions; synthesis of

1.3.1 General Considerations

The possibility of reduction of metal ions in solutions can be estimated, in principle,

Fig. 1.3 Change of the

Mj Mj ! 2Mj Mj ! 3Mj ! kMj : 1:2

1.3.2 Synthesis in Water Solution

Preparation of metal hydrosols by the reduction of metal ions in aqueous solution is

PEOPPOPEO block 5100 nm, spherical [53]

Metal Reducing Stabilizer Nanoparticle size and shape Note Reference

We give here a brief description of the peculiarities of nanoparticle preparation

2AuCl3 3Na3 C6 H5 O7 2Au 3Na2 C5 H4 O5 3CO2 3NaCl 3HCl: 1:4

pH > 9decrease in the yield of nanoparticles is possible due to the NaAuO2

Fig. 1.5 TEM image of

The latter can be separated by centrifugation, as they are signicantly larger

N2 H4 H2 O $ N2 H5 OH pK = 6:5; 1:5

N2 H5 H2 O $ N2 H62 OH pK = 15:2: 1:6

NH2 NH2 Ag !NH2 NH2 Ag 1:7a

NH2 NH2 Ag OH !NH2 NH Ag0 H2 O

NH2 NH Ag !NH NH Ag0

NH NH 2 Ag ! N2 " 2Ag0 H2 " 1:7d

4Ag0 4Ag ! 2 Ag4 2 1:7e

As seen from the reaction sequence presented, it is assumed that complex of

BH3 3H2 O ! BOH3 3H2 : 1:8

Another route is borane decomposition to elemental boron on the surface of a

2BH3 ! 2B 3H2 " : 1:9

Fig. 1.9 Photograph of Cu

Fig. 1.10 a Immiscible

1.3.3 Synthesis in a Two-Phase System and in Non-aqueous

It has been established experimentally that, to produce such ordered structures of

mAuCl4 toluene nC12 H25 SHtoluene 3me !