Journal of Controlled Release 70 (2001) 1–20

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Review

Biodegradable polymeric nanoparticles as drug delivery devices

Kumaresh S. Soppimath a , Tejraj M. Aminabhavi a , *, Anandrao R. Kulkarni a ,
Walter E. Rudzinski b
a
Department of Chemistry, Polymer Research Group, Karnatak University, Dharwad 580 003, India
b
Department of Chemistry, Southwest Texas State University, San Marcos, TX 78666, USA

Received 12 June 2000; accepted 28 September 2000

Abstract

This review presents the most outstanding contributions in the field of biodegradable polymeric nanoparticles used as drug
delivery systems. Methods of preparation, drug loading and drug release are covered. The most important findings on surface
modification methods as well as surface characterization are covered from 1990 through mid-2000.  2001 Elsevier
Science B.V. All rights reserved.

Keywords: Nanoparticle; Method of preparation; Surface modification; Drug delivery; Drug targeting

1. Introduction and reduce the toxicity or side effects [2]. However,

Over the past few decades, there has been consid-
erable interest in developing biodegradable
nanoparticles (NPs) as effective drug delivery de-
vices. Various polymers have been used in drug
delivery research as they can effectively deliver the
drug to a target site and thus increase the therapeutic
benefit, while minimizing side effects [1]. The
controlled release (CR) of pharmacologically active
agents to the specific site of action at the therapeu-
tically optimal rate and dose regimen has been a
major goal in designing such devices. Liposomes
have been used as potential drug carriers instead of
conventional dosage forms because of their unique
advantages which include ability to protect drugs
from degradation, target the drug to the site of action
*Corresponding author. Fax: 191-836-747-884.
E-mail address: rrist@bgl.vsnl.net.in (T.M. Aminabhavi).
developmental work on liposomes has been limited
due to inherent problems such as low encapsulation
efficiency, rapid leakage of water-soluble drug in the
presence of blood components and poor storage
stability. On the other hand, polymeric NPs offer
some specific advantages over liposomes. For in-
stance, NPs help to increase the stability of drugs /
proteins and possess useful CR properties.
Nanoparticles generally vary in size from 10 to
1000 nm. The drug is dissolved, entrapped, encapsu-
lated or attached to a NP matrix and depending upon
the method of preparation, nanoparticles, nanos-
pheres or nanocapsules can be obtained. Nanocap-
sules are vesicular systems in which the drug is
confined to a cavity surrounded by a unique polymer
membrane, while nanospheres are matrix systems in
which the drug is physically and uniformly dis-
persed. In recent years, biodegradable polymeric NPs
have attracted considerable attention as potential

0168-3659 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0168-3659( 00 )00339-4
2 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
drug delivery devices in view of their applications in
the CR of drugs, their ability to target particular
organs / tissues, as carriers of DNA in gene therapy,
and in their ability to deliver proteins, peptides and
genes through a peroral route of administration [3,4].
Some general aspects on micro- and nanoparticles
have been reviewed earlier [1,5–11]. A majority of
these reviews have dealt with the NPs of poly( D,L-
lactide), poly(lactic acid) PLA, poly( D,L-glycolide)
PLG, poly(lactide-co-glycolide), PLGA, and poly-
(cyanoacrylate) PCA. The present review details the
latest developments on the above mentioned poly-
mers as well as NPs based on chitosan, gelatin,
sodium alginate and other hydrophilic / biodegradable
polymers. Surface modification aspects are also
covered in more detail. The PLA, PLG and PLGA
polymers being tissue-compatible have been used
earlier as CR formulations in parentral and implanta-
tion drug delivery applications [12–14]. In addition,
poly(e-caprolactone), PCL, which was first reported
by Pitt et al. [15,16] for the CR of steroids and
narcotic antagonists as well as to deliver opthalmic
drugs [17], and poly(alkylcyanoacrylate), PACA, are
now being developed as NPs. In addition, less
frequently used polymers like poly(methylidene
malonate) [18], gelatin [19], chitosan [20] and so-
dium alginate [21] will also be included in this
review. The important published literature within the
period 1990–2000 is critically reviewed. The review
does not cover the entire literature within this period,
but the reader is advised to go to the original
literature in order to get more details.
2. Preparation of nanoparticles
Conventionally, NPs have been prepared mainly
by two methods: (i) dispersion of the preformed
polymers; and (ii) polymerization of monomers.
2.1. Dispersion of preformed polymers
Several methods have been suggested to prepare
biodegradable NPs from PLA, PLG, PLGA and
poly(e-caprolactone) by dispersing the preformed
polymers [22–25].
2.1.1. Solvent evaporation method
In this method, the polymer is dissolved in an
organic solvent like dichloromethane, chloroform or
ethyl acetate. The drug is dissolved or dispersed into
the preformed polymer solution, and this mixture is
then emulsified into an aqueous solution to make an
oil (O) in water (W) i.e., O / W emulsion by using a
surfactant / emulsifying agent like gelatin, poly(vinyl
alcohol), polysorbate-80, poloxamer-188, etc. After
the formation of a stable emulsion, the organic
solvent is evaporated either by increasing the tem-
perature / under pressure or by continuous stirring.
The effect of process variables on the properties of
NPs was discussed earlier [26]. The W/ O / W method
has also been used to prepare the water-soluble
drug-loaded NPs [27]. Both the above methods use a
high-speed homogenization or sonication. However,
these procedures are good for a laboratory-scale
operation, but for a large-scale pilot production,
alternative methods using low-energy emulsification
are required. In this pursuit, following approaches
have been attempted.
2.1.2. Spontaneous emulsification /solvent diffusion
method
In a modified version of the solvent evaporation
method [28–30] the water-soluble solvent like ace-
tone or methanol along with the water insoluble
organic solvent like dichloromethane or chloroform
were used as an oil phase. Due to the spontaneous
diffusion of water-soluble solvent (acetone or metha-
nol), an interfacial turbulence is created between two
phases leading to the formation of smaller particles.
As the concentration of water-soluble solvent (ace-
tone) increases, a considerable decrease in particle
size can be achieved.
2.1.3. Salting out /emulsification–diffusion method
The methods discussed above require the use of
organic solvents, which are hazardous to the environ-
ment as well as to the physiological system [31]. The
US FDA has specified the residual amount of organic
solvents in injectable colloidal systems [32,33]. In
order to meet these requirements, Allemann and
co-workers have developed two methods of prepar-
ing NPs. The first one is a salting-out method [34,35]
while the second one is the emulsification–solvent
diffusion technique [36,37].
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
2.1.4. Production of NPs using supercritical fluid
technology
Production of NPs with the desired physicochemi-
cal properties to facilitate the targeted drug delivery
has been a topic of renewed interest in pharma-
ceutical industries. Conventional methods like sol-
vent evaporation, coacervation and in situ polymeri-
zation often require the use of toxic solvents and / or
surfactants. Therefore, research efforts have been
directed to develop the environmentally safer en-
capsulation methods to produce the drug-loaded
micron and submicron size particles. If solvent
impurities remain in the drug-loaded NPs, then these
become toxic and may degrade the pharmaceuticals
within the polymer matrix. Supercritical fluids have
now became the attractive alternatives because these
are environmentally friendly solvents and the method
can be profitably used to process particles in high
purity and without any trace amount of the organic
solvent. Literature on the production of drug-loaded
microparticles using supercritical fluids is enormous
[38–44]. However, comparatively much less have
been investigated to produce NPs [39,40]. It is
beyond the scope of the present review to give an
entire coverage on supercritical fluid technology; we
will discuss only two of the most commonly used
methods of producing micro- or nanoparticles.
In the rapid expansion of supercritical solution
(RESS) method the solute of interest is solubilized in
a supercritical fluid and the solution is expanded
through a nozzle. Thus, the solvent power of super-
critical fluid dramatically decreases and the solute
eventually precipitates. This technique is clean be-
cause the precipitated solute is completely solvent-
free. Unfortunately, most polymers exhibit little or
no solubility in supercritical fluids, thus making the
technique less of practical interest. RESS was very
popular in the late 80s and early 90s for particle
production of bioerodible drug-loaded polymers like
PLA. A uniform distribution of drug inside the
polymer matrix can be achieved by this method for
low molecular mass (,10 000) polymers. However,
the RESS method cannot be used for high molecular
mass polymers due to their limited solubility in
supercritical fluids. For these reasons, much less
information is found in the literature over the past
6–7 years on this technique [41,43].
In the supercritical anti-solvent (SAS) method
3
[44], the solution is charged with the supercritical
fluid in the precipitation vessel containing solute of
interest in an organic solvent. At high pressures,
enough anti-solvent will enter into the liquid phase
so that the solvent power will be lowered and the
solute precipitates. After precipitation, when the final
operating pressure is reached, the anti-solvent flows
through the vessel so as to strip the residual solvent.
When the solvent content has been reduced to the
desired level, the vessel is depressurized and the
solid product is collected. A schematic of the SAS
method is shown in Fig. 1. In a modified version of
the SAS technique [39], the solid of interest is first
dissolved in a suitable solvent and then this solution
is rapidly introduced into the supercritical fluid
through a narrow nozzle. The supercritical fluid
completely extracts the solvent, causing the super-
critical fluid insoluble solid to precipitate as fine
particles. This method, also called as gas anti-solvent
(GAS) technique, has been successfully used to
produce microparticles as well as NPs.
2.1.5. Polymerization methods
Nanoparticles can also be prepared by polymeri-
zation of monomers. Poly(alkylcyanoacrylate)s,
PACA, being biodegradable, have been used as tissue
adhesives in surgery since these are well tolerated in
vivo [45,46]. This has prompted intense research
activity to study polymerization reactions. Couvreur
et al. [47,48] reported the production of NPs (|200
nm diameter) by polymerizing mechanically the
Fig. 1. Schematic diagram of the SAS method: PV1 and PV2 are
two volumetric pumps, N is nozzle, P is precipitation vessel, MV
is micrometric valve and EV is expansion vessel.
4 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20

dispersed methyl or ethyl cyanoacrylate in aqueous

acidic medium in the presence of polysorbate-20 as a
surfactant without irradiation or an initiator. Here,
the cyanoacrylic monomer is added to an aqueous
solution of a surface-active agent (polymerization
medium) under vigorous mechanical stirring to poly-
merize alkylcyanoacrylate at ambient temperature.
Drug is dissolved in the polymerization medium
either before the addition of the monomer or at the
end of the polymerization reaction. The NP suspen-
sion is then purified by ultracentrifugation or by
resuspending the particles in an isotonic surfactant-
free medium. The mechanism of polymerization of
PACA monomer is given below.

Polymerization follows the anionic mechanism,
since it is initiated in the presence of nucleophilic
initiators like OH 2 , CH 3 O 2 and CH 3 COO 2 leading
to the formation of NPs of low molecular mass due
to rapid polymerization. Such NPs are degraded very
fast. In order to circumvent this problem and to
produce higher molecular mass as well as stable
NPs, polymerization must be carried out in an acidic
medium (pH 1.0–3.5). After dispersing the monomer
in an aqueous acidic medium containing surfactant
and stabilizer, polymerization is continued for 3–4 h
by increasing the pH of the medium to obtain the
desired products.
During polymerization, various stabilizers like
dextran-70, dextran-40, dextran-10, poloxamer-188,
-184, -237, etc are added. In addition, some surfac-
tants like polysorbate-20, -40 or -80 are also used.
Particle size and molecular mass of NPs depend
upon the type and concentration of the stabilizer
and / or surfactant used. A schematic representation
for the production of poly(alkylcyanoacrylate) NPs is
shown in Fig. 2. The size and molecular mass of NPs
depend upon the pH of the polymerization medium
Fig. 2. Schematic representation for the production of poly-
(alkylcyanoacrylate) nanoparticles by anion polymerization.
[49], but NP production is not possible above a pH
of 3.0, probably due to the aggregation and stepwise
molecular mass increase at lower pH. Other factors
that influence the formation of NPs include the
concentration of monomer and the speed of stirring.
The NPs of PACA have gained wide popularity in
recent years despite some major drawbacks such as
use of low pH (around 2) and cytotoxicity [50]. This
has lead to the synthesis of new dialkyl-methylidene
malonic acid ester monomers [51] and the NPs of
poly(methylidenemalonate), PDEMM were prepared,
and these were found to be non-biodegradable both
in vitro and in vivo [52,53]. To overcome this
problem, new derivatives of PDEMM were prepared
i.e., ethyl-2-ethoxycarbonylmethylenoxycarbonyl
acrylate. NPs from these monomers were prepared
by the same methods as those adopted for the
preparation of PACA NPs by anionic polymerization
[54]. The pH of the polymerization medium critically
influenced the physicochemical properties of NPs,
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
but the minimum sized NPs were produced in the pH
range of 5.5–6.0 when compared to pH 2.0 and pH
7.6 for the PBCA and PDEMM, respectively [55].
The reaction scheme for the synthesis of PMM NPs
is given below.
An attempt was also made to reduce the formation
of oligomer and to increase the yield of PMM 2.1.2.
[56]. The process variables like pH, concentration of
surfactant and monomer concentration have been
monitored to produce NPs with higher molecular
mass [57]. Recently, the preparation of ethyl-2
(ethoxycarbonyl) ethyl methylene malonoate-co-
ethylene oxide have been reported [58]; these poly-
mers are associated with both the hydrophilic and
hydrophobic functionalities and they may be better
polymers to prepare the long-circulating NPs. The
hydrophilic NPs ,100 nm and narrow size dis-
tribution were prepared by using the aqueous core of
the reverse micellar droplets as nanoreactors [59,60].
Other polymerization methods were also reported in
the literature for the development of acrylic based
NPs but these are not discussed since they are not
biodegradable.
2.1.6. NPs prepared from hydrophilic polymers
Other than the commonly-used synthetic hydro-
phobic polymers, active research is now focused on
the preparation of NPs using hydrophilic polymers
like chitosan, sodium alginate, gelatin, etc. Different
methods have been adopted to prepare NPs from the
hydrophilic polymers. Several hydrophobic–hydro-
philic carriers having limited protein-loading capaci-
ty have been prepared by using organic solvents
[61,62]. Calvo and coworkers [63–66] have reported
a method to prepare hydrophilic chitosan NPs. The
5
preparation method involves ionic gelation, with a
mixture of two aqueous phases, of which one
contains chitosan and a diblock copolymer of ethyl-
ene oxide (EO), and the other contains a polyanion
sodium tripolyphosphate (TPP). In this method, the
positively charged amino group of chitosan interacts
with the negatively charged TPP. The size (200–
1000 nm) and zeta potential (120 mv and 160 mv)
of the NPs produced were modulated by varying the
composition of chitosan with the PEO–PPO diblock
polymer. These NPs have shown good association
with proteins, such as bovine serum albumin, tetanus
toxoid and diptaheria toxoid [63,64], insulin [65] as
well as oligonucleotide [66].
Mao and co-workers [67,68] prepared the DNA–
chitosan NPs by a complex coacervation technique
and used for the oral gene delivery. The complex
coacervation technique was also used to prepare the
DNA–gelatin NPs [69]. The chitosan NPs are proved
to be better carriers than the gelatin-based NPs for
loading the immunological and antineoplastic pro-
teins [70]. The chitosan NPs were also produced by
the emulsion coacervation method [71]. In this
method, chitosan and the drug to be loaded were
dissolved in water and water-in-oil emulsion pre-
pared in liquid paraffin using an emulsifying agent.
To this stable emulsion, another emulsion of NaOH
in liquid paraffin was added. When in contact with
NaOH, chitosan NPs were produced by the coacerva-
tion of the polymer. Alginate-based NPs were also
developed and used for the delivery of oligonucleo-
tides [21,72].
Novel biodegradable polyesters, consisting of
short poly(lactone) chains grafted onto poly(vinyl
alcohol) (PVA) or charge-modified sulfobutyl-PVA
(SB-PVA) were prepared by bulk melt polymeri-
zation of lactide and glycolide in the presence of
different core polyols. The modified backbones were
obtained by reacting the activated PVA with the
sulfobutyl groups. By carefully adjusting the poly-
mer composition, novel class of water-soluble comb-
like polyesters were prepared. These polymers un-
dergo spontaneous self-assembling to produce NPs,
which form the stable complexes with a number of
proteins such as human serum albumin, titanous
toxoid and cytocrome C. However, the development
of NPs from such polymers does not require the use
of solvents or surfactants [73–75].
6 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
3. Drug loading
A successful NP system may be the one, which
has a high loading capacity to reduce the quantity of
the carrier required for administration. Drug loading
into the NPs is achieved by two methods: one, by
incorporating the drug at the time of NP production
or secondly, by adsorbing the drug after the forma-
tion of NPs by incubating them in the drug solution.
It is thus evident that a large amount of drug can be
entrapped by the incorporation method when com-
pared to the adsorption [76,77]. Adsorption iso-
therms for the NP/ drug delivery system give vital
information on the best possible formulation, the
drug binding capacity onto the surface of NPs and
the amount of drug adsorbed. For instance, Couvreur
et al. [78] reported the adsorption of two antineoplas-
tic drugs viz, dactinimycin and methotrexate onto
poly(methylcyanoacrylate) and poly-
(ethylcyanoacrylate). It was observed that methotrex-
ate was bound to the NPs to a lesser extent than
dactinimycin. Generally, in the case of PACA, it is
observed that longer the alkyl chain length higher the
affinity for the drugs. The capacity of adsorption is
thus related to the hydrophobicity of the polymer and
the specific area of the NPs. In case of entrapment
method, an increase in concentration of the mono-
mer, increases the association of drug, but a reverse
trend is observed with the drug concentration in the
dispersed solution. This observation was further
substantiated by Radwan [79] who studied the effect
of monomer concentration on % drug loading. These
results indicate that there is a need to optimize the
amount of monomer available for the drug entrap-
ment.
The type of surface-active materials and stabilizers
has an effect on drug loading [80]. Chukwu et al.
[81] studied the adsorption of different psycho-
pharmacological agents onto NPs of poly-
(isobutylcyanoacrylate), PIBCA, in the pH range
between 2.0 and 7.4. Adsorption of drugs onto NPs
followed the Langmuir mechanism [82,83]. In
another study [84], a dielectric method was used to
investigate the adsorption of b-blockers onto PIBCA
NPs. In this method, the NP suspensions were taken
into a capacitance cell, exposed to a high-frequency
field (10 MHz) and the complex impedance was
measured. This technique is rapid and inexpensive
when compared to chromatographic methods, which
require ultracentrifugation.
In addition to adsorption and incorporation, a new
method of drug loading for the water-soluble drugs
was proposed by Yoo et al. [85]. In this method, drug
was chemically conjugated into NPs. The conjugated
doxorubicin–PLGA and doxorubicin-loaded PLGA
NPs were prepared by the spontaneous emulsion–
solvent diffusion method. The encapsulation ef-
ficiency of 96.6% and 3.5% loading of doxorubicin–
PLGA conjugate have been achieved. For the un-
conjugated doxorubicin, these values were, respec-
tively 6.7% and 0.3% (w / w).
4. Drug release
Drug release from NPs and subsequent biodegra-
dation are important for developing the successful
formulations. The release rates of NPs depend upon:
(i) desorption of the surface-bound / adsorbed drug;
(ii) diffusion through the NP matrix; (iii) diffusion
(in case of nanocapsules) through the polymer wall;
(iv) NP matrix erosion; and (v) a combined erosion /
diffusion process. Thus, diffusion and biodegradation
govern the process of drug release.
Methods to study the in vitro release are: (i)
side-by-side diffusion cells with artificial or bio-
logical membranes; (ii) dialysis bag diffusion tech-
nique; (iii) reverse dialysis sac technique; (iv) ultra-
centrifugation; (v) ultrafiltration; or (vi) centrifugal
ultrafiltration technique. Despite the continuous ef-
forts in this direction, there are still some technical
difficulties to study in vitro drug release from NPs
[86,87]. These are attributed to the separation of NPs
from the release media. In order to separate NPs and
to avoid the tedious and time-consuming separation
techniques, dialysis has been used; here, the suspen-
sion of NPs is added to the dialysis bags / tubes of
different molecular mass cut-off. These bags are then
incubated in the dissolution medium [88–90].
Another technique involves the use of a diffusion
cell consisting of donor and acceptor compartments;
this technique was used to separate through the
artificial / biological membranes [91]. In this method,
kinetic study was not performed under the perfect
sink conditions, because the NPs were not directly
diluted in the release media, but were separated from
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
the release media through the membrane. Thus, the
amount of drug in the release media did not reflect
the real amount released. In order to avoid the
enclosure of NPs in the dialysis bag, Leavy and
Benita [92] used a reverse dialysis technique for the
O / W emulsion. In this method, NPs were added
directly into the dissolution medium. The same
technique was adopted by Calvo et al. [17] for the
evaluation of NPs, nanocapsules and nanoemulsions.
However, the method is not very sensitive for
studying the rapid release formulations; but can only
be used for the release of formulations having the
release time longer than 1 h [93].
Release profiles of the drugs from NPs depend
upon the nature of the delivery system. In the case of
a matrix device, drug is uniformly distributed / dis-
solved in the matrix and the release occurs by
diffusion or erosion of the matrix. If the diffusion of
the drug is faster than matrix degradation, then the
mechanism of drug release occurs mainly by diffu-
sion, otherwise it depends upon degradation [28].
Rapid initial release is attributed to the fraction of
the drug which is adsorbed or weakly bound to the
large surface area of the NPs, than to the drug
incorporated in NPs. Following the dilution of the
dissolution media under perfect sink conditions the
drug partition showed an increase due to the immedi-
ate release phase. Later, an exponential delayed
release rate is observed probably due to the drug
diffusion from the matrix [94,95,35,17,28]. Release
in the matrix type of NPs follows the first-order
kinetics [90,79].
Recently, Polakovic et al. [96] theoretically
studied the release of PLA NPs loaded with varying
amounts (7–32% w / w) of lidocane. Two models
were used to study the drug release: (i) by crystal
dissolution and (ii) by diffusion through the polymer
matrix. When the drug loading is ,10% (w / w) (the
drug is molecularly dispersed), the release kinetics
shows a better fit to the diffusion model. The
existence of lidocane crystals at higher concentration
(.10%) is observed. Since the drug should dissolve
first from the crystals and then diffuse from the
matrix, the overall release mechanism could be
described by the dissolution model.
In the case of nanocapsules (reservoir-type drug-
delivery systems) the drug core is coated with the
polymer and the release occurs by diffusion of the
7
drug from the core across the polymeric barrier
layer. Hence, theoretically, the drug release should
follow the zero-order kinetics. Calvo et al. [17]
obtained almost the similar release profiles for
indomethacin from both NPs and nanocapsules. This
indicated that the polymer coating does not show any
barrier properties for the drug release. The drug
release from the nanocapsule takes place mainly by
the partitioning of the drug; however, the main factor
controlling the release is the volume of the aqueous
medium. For instance, with higher dilution of the
dissolution media, a faster and complete release of
the drug takes place. However, Lu et al. [97]
reported that the release of bovine serum albumin
from PLA nanocapsule depends upon the molecular
mass of the polymer, which indicates that the release
may not occur by partitioning of the drug, but may
be due to diffusion across the polymer coating.
The method of drug incorporation into NPs has
also shown an effect on drug release. Fresta et al.
[90] reported a higher burst up to 60–70% for the
NPs loaded with drug by adsorption; here, the burst
effect is less and the remaining drug release is quite
slow. This study demonstrated that the incorporation
method has shown better sustained release charac-
teristics. When the drug is chemically conjugated
with PLGA NPs, the release took place over 25 days,
whereas with those NPs containing unconjugated
free drug, a rapid release in about 5 days occurred
[85]. Here, the CR properties have been attributed to
chemical degradation of the conjugated PLGA,
which permitted water solubilization and subsequent
release of the drug-conjugated PLGA oligomers into
the medium. In case of drug release from hydrogel
NPs, release occurs mainly due to swelling, which
can be controlled by either adding the hydrophilic
functional groups or by monitoring cross-linking of
the matrix.
5. Surface properties of NPs
5.1. Protein adsorption and phagocytosis of NPs
Plasma protein adsorption and phagocytosis of
NPs is a subject that has been widely studied in
recent years. When the NPs are administered in-
travenously they are easily recognized by the body
8 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
immune systems, which are then cleared from the
circulation. Apart from the size of NPs, their surface
hydrophobicity determines the amount of adsorbed
blood components, mainly proteins (opsonins). These
will determine the in-vivo fate of NPs [98,99].
Binding of these opsonins onto the surface of NPs,
called opsonization, acts as a bridge between NPs
and phagocytes. Hence, for a qualitative and quan-
titative understanding of the interaction of blood
proteins with NPs, it is necessary to design long-
circulating NPs by surface modification.
In a study by Allemann et al. [100], it was
reported that when the PLA NPs are incubated in
human plasma and serum, the IgG was found to be
the major protein along with albumin, apolipopro-
tein-E, which were adsorbed on the surface. Compli-
ment C 3 components (part of immune system used
for the recognition of foreign surfaces) were also
adsorbed onto the surface of NPs after incubation in
the serum reaching the level of antibody IgG. Blunk
et al. [101] studied the kinetics of protein adsorption
onto polystyrene NPs and confirmed that albumin
and fibrinogen were adsorbed in a highly diluted
plasma (0.08 and 0.8%). However, in the plasma of
high concentration (80%), proteins were displaced
within seconds or even fractions of a second. The
study indicated that apolipoproteins A-I, C-III, E and
J were the major proteins adsorbed onto NPs.
A two-dimensional polyacrylamide gel electropho-
resis (2-DPAGE) was used to estimate quantitatively
the interaction of plasma proteins with iron oxide
NPs in the presence of plasma proteins stabilized by
polysaccharide. Particles incubated in the plasma
were separated and were then washed with different
washing media. The protein adsorbed on NPs was
then estimated by 2-DPAGE. By this, it was found
that fibrinogen, IgG and albumin were the major
plasma proteins adsorbed onto NP surface [102,103].
In another study by Luck et al. [104], the interaction
of proteins with NPs was shown to depend upon the
method of NP preparation. For example, the amount
of several apolipoproteins in plasma protein adsorp-
tion patterns of the spray-dried PLGA and PLA NPs
were distinctly higher than when compared to the
adsorption patterns of the particles produced by W/
O / W emulsion technique. Some adsorbed proteins
were found to be specific for particles produced by
the same method. The presence of hydrophilic chain
of poly(oxyethylene) in the polymer has drastically
decreased the protein adsorption when compared to
the pure polyesters.
In another study by the same group of researchers
[105], an attempt was made to correlate the ad-
sorption results with the in-vivo circulation of NPs.
The di-block and multi-block copolymers of PEG
were used as model polymers to show the decrease
in adsorption of proteins; these NPs have shown
long-circulating properties. The reduced liver uptake
of NPs was dependent on the molecular mass and
surface density of PEG. The in-vitro protein rejection
properties of the PEG-coated poly-
(alkylcyanoacrylate) NPs were investigated after
when the freeze fracture of NPs were pre-incubated
with fibrinogen as model blood protein [106]. The
decrease in protein adsorption onto PEG-coated NPs
was evident by 2-DPAGE after incubating them in
human serum. The NPs were also long-circulating as
proved from in-vivo tests.
5.2. Surface characterization methods
Many techniques have been developed and used to
study the surface modification of NPs. The efficiency
of surface modification can be measured either by
estimating the surface charge, density of the func-
tional groups or an increase in surface hydrophilicity.
One method used to measure the surface modi-
fication is to determine zeta potential (j) of the
aqueous suspension containing NPs. In this method,
the mobility of charged particles is monitored by
applying an electrical potential. The zeta potential
values may be positive or negative depending upon
the nature of the polymer or the material used for
surface modification. The extent of surface hydro-
philicity can then be predicted from the values of j .
This is a widely used technique to understand the
surface charges of NPs.
Another commonly used technique is electron
spectroscopy for chemical analysis, ESCA, also
called X-ray photoelectron spectroscopy (XPS). This
technique is based on the emission of electrons from
materials, in response to irradiation by photons of
sufficient energy, to cause ionization of the core-
level electrons. These electrons are emitted at ener-
gies characteristic of the atoms from which they are
emitted. Since photons have low penetration energy,
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
only those electrons pertaining to atoms at or near
the surface (up to 100 A) ˚ escape and these can be
counted. For each atom type, the number of electrons
emitted is related to the number of atoms of a
particular type of atom. Using this technique, surface
elemental analysis was performed [107].
In another technique, the surface hydrophobicity
of NPs can be directly measured by hydrophobic
interaction chromatography. This technique involves
the column chromatography, which is able to sepa-
rate materials based on the interaction with a hydro-
phobic gel matrix [108]. The nanoparticle and the gel
interaction is a function of surface hydrophobicity of
NPs. Propyl agarose gel is used as a stationary phase
and elution of NPs can be achieved by using the
phosphate buffer. Eluent samples can be collected
and the optical density measured spectrophotomet-
rically at 400 nm. The gel matrix can then be washed
to remove the NPs.
5.3. Methods of surface modification
Surface modification of biodegradable and long-
circulating polymeric NPs has been achieved mainly
by two methods: (i) surface coating with hydrophilic
polymers / surfactants; and (ii) development of bio-
degradable copolymers with hydrophilic segments.
Some of the widely used surface-coating materials
are: polyethylene glycol (PEG), polyethylene oxide
(PEO), poloxamer, poloxamine, polysorbate (Tween-
80) and lauryl ethers (Brij-35).
5.3.1. PEG and PEO-coated NPs
PEG-coated NPs have received a lot of attention.
Gref et al. [109] described the one step method to
prepare the PEG-coated NPs using amphiphilic
PEG–polyester diblock copolymer as the starting
material and showed that the protective coating
affecting against the phagocytes depends upon den-
sity and molecular mass of PEG. They also studied
the biodistribution of covalently-attached PEG–
PLGA NPs. The protective effect of PEG on carriers
like liposomes, NPs and micelles was studied by
Torchilin [11] in terms of the statistical behavior of
polymers. A mechanism was proposed which as-
sumes that the surface-grafted chains of flexible and
hydrophilic polymers form dense conformational
clouds thus preventing other polymers from inter-
9
action with the surface even at lower concentrations
of the protecting polymeric layer. The biological
consequences of steric protection of drug carriers
with the surface-grafted polymers have been dis-
cussed and clinical applications of the long-circulat-
ing NPs have been studied [10]. A theoretical model
of repulsion of proteins from the solid substrate was
proposed by Joen et al. [110]. The steric repulsion,
van der Waals attractions and hydrophobic inter-
action free energy have been correlated. The model
provides a basis for the prevention of opsonin
deposition. High surface density and long chain-
lengths of PEG are necessary for low protein ad-
sorption. However, surface density has a greater
effect than the chain-length on steric repulsion and
van der Waals attraction.
Bazile et al. [111] developed the NPs based on
methoxy PEG–PLA i.e., Me-PEG–PLA and blends
of PLA with Me-PEG–PLA. These NPs, labeled by
introducing 14 C-labeled PLA in the formulation were
more slowly captured by the cultured THP-1 mono-
cytes when compared to pluronic F68-coated PLA
NPs. The half-life of Me-PEG–PLA NPs was im-
proved by a factor of 180 (360 min) when compared
to the uncoated and F68-coated NPs. Even though, a
high amount of radioactivity was located in the heart
and blood vessels due to particle circulation, in other
phagocytic organs, radioactivity was found even
after 6 h of i.v. administration indicating a delay in
phagocytosis. Tobio et al. [112] observed much
greater penetration of tetanus toxoid (TT) encapsu-
lated PEG–PLA NPs than PLA NPs after nasal
administration. A high persisting radioactivity was
found in body compartments up to 8 h after the
introduction of 125 I TT-loaded NPs.
Gref et al. [113] reported the preparation of blend
NPs of PLA with monomethoxy polyoxyethylene
(MPOE) by solvent evaporation method using so-
dium cholate surfactant. The zeta potentials mea-
sured at various concentrations of NaCl varied from
255 mV for PLA to 0 mV for blends depending
upon the composition of MPOE in the NPs. The zeta
potential increased with an increasing amount of
MPOE suggesting that the MPOE chains that are
present on the surface of NPs mask the ionized
COO 2 end-group of PLA. These results are sup-
ported by a phagocytosis study on the monocytes.
When MPOE content in the blend is greater than
10 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20

2–3%, the MPOE chain adopts a brush-like configu-
ration forming a sterically-uncharged barrier, thereby
reducing the zeta potential and phagocytosis.
Govender et al. [114] examined the drug-encapsu-
lation characteristics of PLA–PLA NPs using
procaine hydrochloride, a water-soluble drug. The
PLA–PEG NPs were produced by the nano-precipi-
tation technique. The drug-entrapment efficiencies of
these NPs were compared with those of PLGA NPs.
Kim et al. [115] used the ESCA method to evaluate
the presence of PEG on the surface of indomethacin-
loaded Me-PEG–PLA NPs. The in-vitro cytotoxicity
of these NPs did not show any remarkable cytotox-
icity against the normal human fibroblast cells.
The optimum surface density of PEG on NPs
plays an important role in steric repulsion. These
NPs have shown a lower accumulation in the liver,
but the observed high spleen uptake is due to the
removal of PEG coating from the surface of NPs, an
important property in spleen targeting [116]. In
addition, the distance between PEG chains on the
surface of NPs is critical to avoid the adsorption of
plasma proteins. For instance, a decrease in the
distance between PEG chains on the surface from 6.2
to 5.1 nm drastically decreases the adsorption of
apolipoproteins up to 90%. This further confirms that
the density of hydrophilic segment on the surface of
NPs is important in opsonization. However, any
further decrease in this distance did not show
significant effects on the adsorption of plasma pro-
teins [117].
Peracchia et al. [118] used the emulsification and
solvent evaporation method to prepare the diblock
Me-PEG–PLA copolymeric NPs containing 20 and
33% of lidocaine. They confirmed high-density of
the surface PEG by ESCA. However, the size of NPs
produced by the block copolymer was twice as high
as those of PLGA NPs. This was attributed to an
increase in the chain length of PEG. Peracchia et al.
[119,120] reported the chemical coupling of PEG
with PBCA NPs prepared by emulsion polymeri-
zation in the presence of PEG, Me-PEG and (Me) 2 -
PEG. Polymerization was possible only in the pres-
ence of PEG and Me-PEG as hydroxyl group was
necessary for polymerization and association of PEG
on the surface of NPs. Higher PEG density was
observed on the surface of NPs when Me-PEG was
used. A decrease in hydrophobicity was observed for
the PEG coated NPs. Peracchia et al. [121] also
prepared the methoxy PEG cyanoacrylate–hexadecyl
cyanoacrylate amphiphilic NPs by polymer precipi-
tation or by solvent evaporation method; the PEG
coating was confirmed by XPS. The particles ex-
hibited a reduced cytotoxicity and enhanced degra-
dation. NPs prepared in the presence of PEGs have
shown some advantages in preventing opsonization
and thereby avoiding the MPS uptake [122]. This
mechanism is explained in Fig. 3.
PEO-surface modified systems have received an
increased attention in recent years. Jaeghere et al.
[123] studied the freeze-dried PEO-surface modified
NPs as a function of PEO chain length and surface
density to avoid the MPS uptake. NPs were produced
by salting-out method using the blends of PLA and
PLA–PEO copolymers. In an effort to study the
effect of surface density of PEO on the compliment
consumption, Vittaz et al. [124] used the diblock
polymer of PLA and polyethylene oxide (PLA–
PEO). It was found that as the PEO density on the
surface of the NPs increases, a decrease in compli-
ment consumption is observed due to steric repulsion
of the surface to proteins. A preliminary study was
made on the synthesis of amphiphilic PEO–PPO–
Fig. 3. Effect of surface PEG density and its conformation on the
opsonization process: (A) opsonization takes place when the
density is low, (B) opsonization is not possible at higher surface
density and (C) when both the end groups of PEG participate in
surface modification.
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
PEO block copolymers (Plunoric) and poly(e-cap-
rolactone) by bulk polymerization [125]. The size of
the NPs prepared varied from 116 to 196 nm,
depending upon the type of copolymer used.
5.3.2. Poloxamine and poloxamer coated NPs
Poloxamer and poloxamine have been widely used
in surface coating studies. Storm et al. [126] pre-
sented an overview of the advances made up to 1995
on the surface modification of NPs to oppose the
MPS uptake. In a study by Illum and Davis
[127,128], poloxamer and poloxamine were used as
the coating materials to prepare the long-circulating
NPs of polystyrene and poly(methyl methacrylate). A
prolonged circulation time and reduction in liver
uptake in rabbits was found for the poloxamine-
coated polystyrene NPs (60 nm size) when compared
to the uncoated NPs of the same size. A decrease in
hepatic uptake of about 20% for the NPs prepared
with poloxamer-188 and about 40% for the NPs
coated with poloxamer-338 was observed. Rabbit
131
experiments with I-labelled polystyrene
poloxamer-407 coated NPs showed the superior
performance over that of poloxamer-338 to avoid the
hepatospleenic uptake.
Rudt and Muller [129] studied the uptake of
surface modified poloxamine-coated polystyrene NPs
and found extremely low levels of uptake for 100 nm
size NPs. Compared to poloxamer, poloxamine was
more effective as a coating material to avoid the liver
capture of rabbits [130]. Moghimi and Gray [131]
developed the long-circulating poloxamine-908
coated polystyrene NPs (60 nm) that are resistant to
MPS uptake. The time interval of administration was
important in maintaining the long circulation time.
Spleen-capture study of the fluorescent-labeled
polystyrene NPs coated with poloxamer 407 or
poloxamine 908 was made [132] on two rodent
species viz., mouse and rat in order to assess the
effect of coating on their intraspleenic distribution.
Two fluorescent polystyrene NPs used were:
Estapor  (FX-010, 185 nm, Prolabo, France) and
Fluoresbrite  (Plain YG, 260 nm, Polysciences,
UK). A fluorimetric investigation indicated that the
Fluoresbrite  NPs were more efficiently trapped by
the spleen than the Estapor  -based NPs in mice and
rats. Results indicate an increase in the size of NPs
after coating the Estapor  NPs. Different spleen
11
uptake pathways are observed for NPs depending
upon their surface characteristics and the rodent
species. Coating was effective in stimulating the
spleen uptake in rats and mice. Spleen uptake of
Fluoresbrite  NPs was higher than Estapor  NPs,
probably due to differences in the surface charac-
teristics of NPs.
Polystyrene-latex nanospheres (PSL-NS, mean
diameter, 85 nm) were coated with lactosyl-poly-
styrene (LPS, high affinity to hepatocytes) to evalu-
ate their targeting characteristics to hepatocytes and
PSL-NS surfaces [133]. Hepatocytes were adhered
specifically with the LPS-coated dishes made of the
same materials as PSL-NS. Flow cytometry inves-
tigations showed that the LPS-coated fluorescein-
isothiocyanate (FITC)–PSL-NS were taken up by
hepatocytes when compared to the noncoated FITC–
PSL-NS as a control. These findings indicated that
LPS–PSL-NS could target to hepatocytes. The sur-
face of LPS on PSL-NS showed higher hydrophil-
icity than PEG-6000, Tween-80, poloxamer-407 and
poloxamer-908, which indicated that LPS–PSL-NS
may avoid the reticuloendothelial system capture and
have a long plasma duration after the in-vivo i.v.
adminstration. Plasma coagulation can be prevented
by the addition of 0.1% of PVA in LPS–PSL-NS
solution when LPS–PSL-NS were injected. The
LPS–PSL-NS were the potential hepatocyte targeting
carriers for the injectable formulations.
5.3.3. Cyclodextrin /carbohydrate coated NPs
Carbohydrates were also found to avoid the MPS
uptake when coated on the surface of NPs. To avoid
the MPS uptake the coated NPs with carbohydrate
was reported by Cho et al. [134]. The NPs of PLA
and poly( L-lysine)-grafted-polysaccharide were also
developed for the delivery of DNA [135] and these
were found to be resistant against self-aggregation
and nonspecific adsorption of the serum proteins.
Recently, Duchene et al. [136] used amphiphilic
cyclodextrin NPs to increase the loading of water-
soluble drugs and bioavailability of the poorly water-
soluble drugs intended for targeted delivery by the
oral or parenteral route. In order to further increase
the loading capacity, PIBCA NPs were loaded with
the natural or hydroxypropyl cyclodextrins. The
loading capacity increased with an increase in stabili-
12 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20

ty constant of the inclusion drug / parent g-cyclo-
dextrin and with a decrease in water solubility.
The PIBCA NPs were prepared by the anionic
polymerization of isobutylcyanoacrylate in 0.01 M
HCl containing 1% poloxamer-188 and in the pres-
ence of cyclodextrins. The size, zeta potential and
cyclodextrin content were influenced by the nature of
cyclodextrin. The smallest size particles were ob-
tained from hydroxypropyl b-cyclodextrin, but the
highest cyclodextrin content was obtained for b-
cyclodextrin. The cyclodextrin NPs or the polymeric
NPs containing cyclodextrin were useful in targeting
the water-insoluble drugs through oral or parentral
route. The presence of cyclodexrins in these NPs has
drastically reduced the surface negativity probably
due to their hydrophilicity; hence, the cyclodextrin
coated NPs may help in avoiding the MPS.
biological membrane was studied and an increase in
the permeation of polysorbate-80 coated NPs
occurred [91]. A schematic representation of the
increased drug permeation from the polysorbate-80
coated NPs through the biological membrane is
shown in Fig. 4.
Borchardt et al. [142] studied the uptake of
polysorbate-80 coated poly(methyl methacrylate)
(PMMA) NPs by bovine brain microvessel endo-
thelial cell monolayers. These NPs showed an in-
creased uptake by the endothelial cells of the BBB.
Troster et al. [143] demonstrated a nine-fold increase
in the accumulation of radioactivity in the brain area
after i.v. administration of polysorbate-80 coated
14
C-PMMA NPs. A recent study by Steiniger et al.
[144] suggested that polysorbate-80 coated poly-

5.3.4. Polysorbate-coated NPs to penetrate the

blood–brain barrier
Targeting drugs to the brain by crossing the
blood–brain barrier (BBB) has been a challenge. In
this pursuit, many attempts have been made to
develop novel drug delivery systems. BBB is formed
by the tight endothelial cell junctions of the capil-
laries within the brain, which limits the ability of
many drugs to penetrate through the brain tissue in
order to enter the central nervous system (CNS). It is
known that many regulators of the brain functions
such as cytokines, transferrin, enkephalins, endor-
phins or delta sleep inducing peptides pass through
BBB from the vessels into brain [137,138] as well as
some excitatory and depressant amino acids, pene-
trate poorly through BBB. The poor BBB penetration
of such substances makes the problem of drug
targeting to the brain highly pertinent. However, the
surface modified NPs have been used to deliver the
anti-inflammatory drugs acting on the CNS because
these can pass through BBB [139,140].
The mechanism of enhancement of drug transport
from the coated NPs through BBB is due to the
number of mechanisms: (i) by binding the NPs to the
inner endothelial lining of the brain capillaries and
subsequently, particles deliver drugs to the brain by
providing a large concentration gradient, thus en-
hancing the passive diffusion; (ii) brain endothelial Fig. 4. The schematic representation of the drug uptake through
uptake by phagocytosis [141]. The effect of surfac- biological membrane from (A) free drug and (B) polysorbate-80
tant coated NPs on drug permeation across the coated nanoparticle bound drug [91,141].
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
(alkylcyanoacrylate) NPs are superior over the un-
coated NPs to transport drugs across BBB.
Recently, investigations have been carried out
[145] with PBCA NPs as well as with the non-
biodegradable polystyrene (PS) NPs (200 nm in
diameter) to study the transport of analgesic peptide,
dalargin to the brain. Its entry into the CNS of the
mice was evaluated using the tail-flick procedure.
Locomotor activity measurements were performed to
compare the toxicity of NPs. BBB permeability of
PBCA NPs was studied in-vitro using a co-culture of
bovine brain capillary endothelial cells and rat
astrocytes. Dalargin associated with PBCA NPs and
polysorbate-80 induced a potent and prolonged anal-
gesia, which was not observed by using polystyrene
NPs, but not using the PBCA NPs. Locomotor
activity dramatically decreased in the mice dosed
with PBCA NPs, but not with the polystyrene NPs.
The in-vitro and in-vivo results suggested that the
PBCA NPs induce a nonspecific opening of the BBB
in the presence of polysorbate-80 allowing the
transport of dalargin into the CNS. Although
polysorbate-80 coated PBCA NPs are useful in
increasing the penetration of drugs into the CNS,
potential therapeutic applications are limited because
of the high systemic NP concentration needed to
deliver drugs to the CNS.
In an effort to deliver anticancer drugs to the brain
using NPs, Gulyaev et al. [146] demonstrated that
the brain concentration of systemically administered
doxorubicin was enhanced by more than 60-fold by
binding it to polysorbate-80 coated PBCA NPs.
Doxorubicin was selected as a model drug due to its
potent antitumor activity and because the drug is not
able to cross the BBB by i.v. injection. Polysorbate-
80 coated NPs reached the brain intact and released
the drug after endocytosis by the brain blood vessel
endothelial cells. High brain concentrations achieved
in this study suggested a significant improvement in
the treatment of brain tumors.
Alyautdin et al. [147] studied the efficiency of
polysorbate-80 coated PBCA NPs in crossing BBB
to deliver the water-insoluble analgesic drug, loper-
mide, in mice. Intravenous injection of the
polysorbate-80 coated NPs resulted in a long and
significant analgesic effect, which was measured by
the tail flick method, while the uncoated NPs were
unable to produce analgesia. Alyautdin et al. [141]
13
also reported an increased analgesic activity of
dalargin-loaded PBCA NPs coated with polysorbate-
80 when administered in mice. In a further study,
Alyautdin et al. [148] found that BBB crossing was
observed for low molecular mass and polar hydro-
philic drug like tuborcurarin after loading it into
polysorbate-80 coated PBCA NPs. An in-vivo per-
fused rat brain was used along with the simultaneous
recording of an electroencephalogram (EEG) since
the drug induces epileptic form seizures. An i.v.
injection of the NPs demonstrated the appearance of
EEG seizures 5 min after the administration.
Schroeder et al. [149] studied the transport of
dalargin, kytorphin (centrally-acting analgesics) and
amitriptyline (antidipressant)-loaded PBCA NPs
coated with polysorbate-80 across the BBB. In-vivo
analgesic activity carried out in mice showed a
drastic enhancement of analgesia for the drug-loaded
NPs coated with polysorbate-80. The amitriptylin
concentration in the brain increased, but the con-
centration in serum decreased for dextran-stabilized
polysorbate-80 coated NPs. These results indicate
that the surface modification of NPs by coating with
polysorbate-80 is effective in drug delivery through
BBB.
6. Delivery of proteins and peptides using NPs
Peptide drugs are attracting, as their role in
physiopathology is better understood and because of
the progress made in biotechnology and bioengineer-
ing. Particularly, the development of DNA-recombi-
nant technology has made these compounds available
on large scale than in the past. However, the use of
peptide in medicine is partly limited by their rapid
degradation by proteolytic enzymes in the gastroin-
testinal tract; thus, they need to be administered
through the parentral route. The biological half-life
of peptides is short and needs frequent administra-
tions. On the other hand, their transport across
biological barriers is poor due to poor diffusivity and
lower partition coefficients. In this pursuit, the
particulate biodegradable delivery systems have been
proposed for the safe and controlled parentral ad-
ministration of peptides [150].
Proteins and peptides are unstable in PLGA
because of the hydrophobicity and acidity of PLGA
14 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
[151]. Another problem is the fast burst release of
protein drugs from the PLGA matrices. In order to
circumvent these problems, different approaches
have been explored to modify the properties of
PLGA matrices by using the hydrogel NPs
[152,153]. This has prompted the development of
novel protein delivery systems. In these studies,
bovine serum albumin (BSA) was encapsulated first
in PVA NPs, which were then loaded into PLGA
microspheres using the phase separation method. The
protein loaded PLGA–PVA composite NPs were
then characterized and were having the nonporous
surface to release BSA for over 2 months. In a recent
study by Gasper et al. [154], it was shown that the
presence of end carboxyl group in PLGA resulted in
a high protein loading of up to 4.86 mass% and the
release continued for about 20 days. On the other
hand, the presence of esterified carboxyl end groups
in PLGA led to a lower loading (2.65 mass%) of
proteins and a release of up to 14 days.
The release kinetics and in-vivo effects of NPs
containing PGDF-Receptor b (PDGFRb) tyrphostin
inhibitor, AG-1295, AG-1295-loaded PLA NPs were
prepared by the spontaneous emulsification / solvent
displacement technique [155]. The in-vitro release
rate and the impact of drug / polymer ratio on the size
of NPs were determined. It was shown that by
modulating the formulation variables, release kinet-
ics and particle size were tailor-made to address the
clinical needs. A novel pulmonary delivery system of
PLGA nanosphere (400 nm size) encapsulating the
physiologically active peptide was developed by
Kawashima et al. [156]. These were prepared by
using the modified emulsion solvent diffusion meth-
od in water. The aqueous dispersions of PLGA
nanospheres administered pulmonarily to guinea pig
via nebulization reduced significantly the blood
glucose level for over 48 h when compared to the
nebulized aqueous solution of insulin as a reference.
About 85% of the drug was released from the
nanospheres during the initial burst, followed by a
prolonged release of the remaining drug for few
hours in saline solution at 378C.
Zinc insulin was successfully encapsulated in
various polyester and polyanhydride nanosphere
formulations using the phase inversion nanoencapsu-
lation technique [157]. The encapsulated insulin
maintained its biological activity and was able to
release insulin from the nanospheres over a span of 6
h. The 1.6% zinc insulin in PLGA with fumaric
anhydride oligomer and iron oxide additives was
shown to be active orally and was able to control the
plasma glucose levels. A number of properties of this
formulation, including size, release kinetics, bioadhe-
siveness and ability to traverse the gastrointestinal
epithelium have contributed to its oral efficacy.
In recent years, greater advances have been made
particularly by the research group of Professor
Robert Langer at MIT (USA) on the development of
gene delivery systems. However, a discussion on
these systems is beyond the scope of this review.
7. Conclusions
The use of biodegradable polymers for the CR of
therapeutic agents is now well established. Although
currently there are only a small number of commer-
cially available products that utilize this technology
(e.g., Lupron Depot  ), these polymers have great
utility for the CR of several drugs like vaccines,
human growth hormone, insulin, anti-tumor agents,
contraceptives and also vaccines. Long circulation of
drugs in the body is the key in successful drug
delivery and drug targeting to the site of action.
Many polymeric NPs have been developed for this
purpose. Certainly, surface modification is useful in
achieving these goals. From the polymer chemistry
viewpoint, it is important to synthesize newer poly-
mers and copolymers to match the hydrophilic and
hydrophobic properties. Production of NPs using the
environmentally friendly processes like supercritical
fluids is quite a promising area of research to
develop the products that are free from the unwanted
toxic residual solvents. Although many important
goals have been reached in achieving stabilization of
drugs in circulation, yet more investigations are
needed to develop the newer materials in this area.
Acknowledgements
We immensely thank the Council of Scientific and
Industrial Research, New Delhi, India [Grant [
80(0025)97 / EMR-II] for a major financial support
of this study. Dr. Walter E. Rudzinski thanks the
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
Southwest Texas State University, San Marcos for a
research enhancement grant. We also thank Dr. M.I.
Aralaguppi for his assistance.
References
[1] J. Kreuter, Nanoparticles, in: J. Kreuter (Ed.), Colloidal Drug
Delivery Systems, Marcel Dekker, New York, 1994, pp.
219–342.
[2] C.G. Knight (Ed.), Liposomes From Physical Structure To
Therapeutic Applications, Elsevier, Amsterdam, 1981.
[3] R. Langer, Biomaterials in drug delivery and tissue engineer-
ing: One laboratory’s experience, Acc. Chem. Res. 33 (2000)
94–101.
[4] R.P. Lanza, R. Langer, W.L. Chick, Principles of Tissue
Engineering, in: Academic Press, Austin, TX, 1997, pp.
405–427.
[5] L.B, Peppas, Recent advances on the use of biodegradable
microparticles and nanoparticles in the controlled drug
delivery, Int. J. Pharm. 116 (1995) 1–9.
[6] A. Zimmer, J. Kreuter, Microspheres and nanoparticles used
in ocular drug delivery systems, Adv. Drug. Deliv. Rev. 16
(1995) 61–73.
[7] P. Couvreur, L. Grislain, V. Lenaerts, F. Brasseur, P. Guiot,
in: P. Guiot, P. Couvreur (Eds.), Polymeric Nanoparticles and
Microspheres, CRC Press, Boca Raton, Florida, 1986.
[8] D.F. Raney, Biomimetic transport, rational drug delivery,
Biochem. Pharmacol. 59 (2000) 105–114.
[9] K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakes-
sheff, Polymeric systems for controlled drug release, Chem.
Rev. 99 (1999) 3181–3198.
[10] C. Monfardini, F.M. Veronese, Stabilization of substances in
circulation, Bioconjug. Chem. 9 (1998) 418–450.
[11] V.P. Torchilin, Polymer-coated long-circulating microparticu-
late pharmaceuticals, J. Microencapsul. 15 (1998) 1–19.
[12] D.L. Wise, T.D. Fellman, J.E. Sanderson, R.L. Wentworth,
Lactide / glycolide acid polymers, in: G. Geregoriadis (Ed.),
Drug Carriers in Biology and Medicine, Academic, London,
1979, pp. 237–270.
[13] T.M. Jackanicz, H.A. Nash, D.L. Wise, J.B. Gregory, Poly
lactic acid as a biodegradable carrier for contraceptive
steroids, Contraception 8 (1973) 227–234.
[14] L.C. Andersson, D.L. Wise, J.F. Howes, An injectable
sustained release fertility control system, Contraception 13
(1976) 375–384.
[15] C.G. Pitt, M.M. Gratzi, A.R. Jeffcot, R. Zweidinger, A.
Schindler, Sustained release drug delivery systems II: factors
affecting release rate for poly(e-caprolactone) and related
biodegradable polyesters, J. Pharm. Sci. 68 (1979) 1534–
1538.
[16] C.G. Pitt, T.A. Marks, A. Schindler, Biodegradable drug
delivery systems based on aliphatic polyesters: application to
contraceptives and narcotic antagonists, in: R. Baker (Ed.),
Controlled Release of Bioactive Materials, Academic, New
York, 1980, pp. 19–43.
15
[17] P. Calvo, J.L. Vila-Jato, M.J. Alonso, Comparative in vitro
evaluation of several colloidal systems, nanoparticles,
nanocapsules and nanoemulsions as ocular drug carriers, J.
Pharm. Sci. 85 (1996) 530–536.
[18] F. Lescure, C. Seguin, P. Breon, P. Bourrinet, D. Roy, P.
Couvreur, Preparation and characterization of novel poly-
(methylidene malonate 2.1.2.)-made nanoparticles, Pharm.
Res. 9 (1994) 1270–1277.
[19] C.A. Farrugia, M.J. Grover, Gelatin behavior in dilute
aqueous solutions: Designing a nanoparticulate formulations,
J. Pharm. Pharmacol. 51 (1999) 643–649.
[20] R. Fernandez-Urrusuno, P. Calvo, C. Remunan-Lopez, J.L.
Villa-Jato, M.J. Alonso, Enhancement of nasal absorption of
insulin using chitosan nanopartilces, Pharm. Res. 16 (1999)
1576–1581.
[21] I.C. Aynie, C. Vauthier, E. Fattal, M. Foulquier, P. Couvreur,
Alginate nanoparticles as a novel carrier for antisense
oligonucleotide, in: J.E. Diederichs, R. Muler (Eds.), Future
Strategies of Drug Delivery With Particulate Systems, Med-
pharm Scientific Publisher, Stuttgart, 1998, pp. 5–10.
[22] C. Vauthier, S. Beanabbou, G. Spenlehauer, M. Veillard, P.
Couvreur, Methodology of ultradispersed polymer system,
S.T.P. Pharm. Sci. 1 (1991) 109–116.
[23] E. Allemann, R. Gurnay, E. Doelker, Drug-loaded nanopar-
ticles-Preparation methods and drug targeting issues, Eur. J.
Pharm. 39 (1993) 173–191.
[24] P. Couvreur, C. Dubernet, F. Puisieux, Controlled drug
delivery with nanoparticles: Current possibilities and future
trends, Eur. J. Pharm. 41 (1995) 2–13.
[25] M.J. Alonso, Nanoparticulate drug carrier technology, in: C.
Cohen, H. Bernstein (Eds.), Microparticulte Systems For the
Delivery of Proteins and Vaccines, Marcel Dekker, New
York, 1996, pp. 203–242.
[26] P.D. Scholes, A.G.A. Coombes, L. Illum, S.S. Davis, M. Vert,
M.C. Davies, The preparation of sub-500 nm poly(lactide-
co-glycolide) microspheres for site-specific drug delivery, J.
Control. Rel. 25 (1993) 145–153.
[27] M.F. Zambaux, F. Bonneaux, R. Gref, P. Maincent, E.
Dellacherie, M.J. Alonso, P. Labrude, C. Vigneron, Influence
of experimental parameters on the characteristics of poly(lac-
tic acid) nanoparticles prepared by double emulsion method,
J. Control. Rel. 50 (1998) 31–40.
[28] T. Niwa, H. Takeuchi, T. Hino, N. Kunou, Y. Kawashima,
Preparations of biodegradable nanospheres of water-soluble
and insoluble drugs with D,L-lactide / glycolide copolymer by
a novel spontaneous emulsification solvent diffusion method
and the drug release behavior, J. Control. Rel. 25 (1993)
89–98.
[29] P. Wehrle, B. Magenheim, S. Benita, The Influence of
process parameters on the PLA nanoparticle size distribution
evaluated by means of factorial design, J. Pharm. Biopharm.
41 (1995) 19–26.
[30] H. Murakami, H. Yoshino, M. Mizobe, M. Kobayashi, H.
Takeuchi, Y. Kawashima, Preparation of poly( D,L-lactide-co-
glycolide) latex for surface modifying material by a double
coacervation method, Proced. Intern. Symp. Control. Rel.
Bioact. Mater. 23 (1996) 361–362.
[31] D.T. Birnbaum, J.D. Kosmala, D.B. Henthorn, L.B. Peppas,
16 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
Controlled release of b-estradiol from PLAGA microparti-
cles: The effect of organic phase solvent on encapsulation
and release, J. Control. Rel. 65 (2000) 375–387.
[32] R. Bodmeier, J.W. McGinity, Solvent selection in the prepa-
ration of poly( D,L-lactide) microspheres prepared by the
solvent evaporation method, Int. J. Pharm. 43 (1988) 179–
186.
[33] R. Arshaday, Preparation of porous and nonporous bio-
degradable polymeric hollow microspheres, J. Control. Rel.
17 (1991) 1–22.
[34] E. Allemann, R. Gurnay, E. Doelker, Preparation of aqueous
polymeric nanodispersions by a reversible salting-out pro-
cess: influence of process parameters on particle size, Int. J.
Pharm. 87 (1992) 247–253.
[35] E. Allemann, J.C. Leroux, R. Gurnay, E. Doelker, Invitro
extended-release properties of drug-loaded poly( D,L-lactic)
acid nanoparticles produced by a salting-out procedure,
Pharm. Res. 10 (1993) 1732–1737.
[36] J.C. Leroux, E. Allemann, E. Doelker, R. Gurnay, New
approach for the preparation of nanoparticles by an emulsifi-
cation–diffusion method, Eur. J. Pharm. Biopharm. 41
(1995) 14–18.
[37] G.D. Quintanar, Q.A. Ganem, E. Allemann, H. Fessi, E.
Doelker, Influence of the stabilizer coating layer on the
purification and freeze drying of poly ( DL-lactic acid)
nanoparticles prepared by emulsification-diffusion technique,
J. Microencapsulation 15 (1998) 107–119.
[38] J.W. Tom, P.G. Debenedetti, Particle formation with super-
critical fluids — a review, J. Aerosol Sci. 22 (1991) 555–
584.
[39] T.W. Randolph, A.D. Randolph, M. Mebes, S. Yeung, Sub-
micron-sized biodegradable particles of poly( L-lactic acid)
via the gas antisolvent spray precipitation process, Biotech-
nol. Prog. 9 (1993) 429–435.
[40] L. Benedetti, A. Bertucco, M. Lora, P. Pallado, in: Atti del 38
Congresso I fluidi Supercriticai e le Loro Applicazioni, I.
Kikic and P. Alessi (Eds.), Trieste, 1995, p. 221.
[41] K. Mishima, K. Matsuyama, D. Tanabe, S. Yamauchi,
Microencapsulation of proteins by rapid expansion of super-
critical solution with a nonsolvent, AIChE J. 46 (2000)
857–865.
[42] J.W. Tom, P.G. Debenedetti, Formation of bioerodiable
polymeric microspheres and microparticles by rapid expan-
sion of supercritical solution, Biotechnol. Prog. 7 (1991)
403–411.
[43] J.W. Tom, P.G. Debenedetti, R. Jerome, Preparation of
poly( L-lactic acid) and composite poly( L-lactic acid)-pyrene
by rapid expansion of supercritical solution, J. Supercrit.
Fluids 7 (1994) 9–29.
[44] S. Mawson, K.P. Johnston, J.R. Combes, J.M. DeSimone,
Formation of poly(1,1,2,2-tetrahydroperfluorodecyl acrylate)
submicron fibers and particles from supercritical carbon
dioxide solutions, Macromolecules 28 (1994) 3182–3191.
[45] K.C. Pani, G. Gladieux, G. Brandes, R.K. Kulkarni, F.
Leonarda, The degradation of n-butyl alpha-cyanoacrylate
tissue adhesive, II, Surgery 63 (1968) 481–489.
[46] F. Leonarda, Hemostatic application of alpha cyanoacrylates:
bonding mechanism and physiological degradation of bonds,
in: R.S. Manly (Ed.), Adhesion in Biological Systems,
Academic, New York, 1970, pp. 185–199.
[47] P. Couvreur, B. Kante, M. Roland, Les perspectives d’utilisa-
tion des formes microdisperses ´ comme vecteurs intracel-
lulaires, Pharm. Acta Helv. 53 (1978) 341–347.
[48] P. Couvreur, B. Kante, M. Roland, P. Goit, P. Bauduin, P.
Speiser, Polycyanoacrylate nanocapsules as potential
lysosomotropic carriers: preparation, morphology and sorp-
tive properties, J. Pharm. Pharmacol. 31 (1979) 331–332.
[49] N. Behan, C. Birkinshaw, N. Clarke, A study of the factors
affecting the formation of poly(n-butylcyanoacrylate)
nanoparticles, Proced. Intern. Symp. Control. Rel. Bioact.
Mater. 26 (1999) 1134–1135.
[50] C. Lherm, R.H. Muller, F. Puiseux, P. Couvreur,
Alkylcynoacrylate drug carriers: II. Cytotoxicity of
cyanoacrylate nanoparticles with different alkyl chain length,
Int. J. Pharm. 84 (1992) 13–22.
[51] J.-L. De Keyser, C.J.C. De Cock, J.H. Poupaert, P. Dumont,
Synthesis of 14 C labeled acrylic derivatives: diethyl [3- 14 C]
methylidenemalonate and isobutyl [3- 14 C] cyanoacrylate, J.
Lable. Comp. Radiopharm. 27 (1989) 909–916.
[52] J.-L. De Keyser, J.H. Poupaert, P. Dumont, Poly(diethyl
methylidenemalonate) nanoparticles as a potential drug car-
rier: preparation, distribution and elimination after intraven-
ous and peroral administration to mice, J. Pharm. Sci. 80
(1991) 67–70.
[53] T.K.M. Mabela, J.H. Poupaert, P. Dumont, A. Haemers,
Development of poly(dialkyl methylidenemalonate)
nanoparticles as drug carriers, Int. J. Pharm. 92 (1993)
71–79.
[54] P. Breton, D. Roy, L. Marchal-Heussler, C. Seguin, P.
Couvreur, F. Lescure, New poly(methylidene malonate
2.1.2) nanoparticles: Recent developments, in: G. Gre-
goriadis, B. McCormack, G. Poste (Eds.), Targeting of
Drugs, Advances in System Constructs, Vol. 4, Plenum Press,
New York, 1994, pp. 161–172.
[55] F. Lescure, C. Seguin, P. Breton, P. Bourrinet, D. Roy, P.
Couvreur, Preparation and charecterization of novel poly-
(methylidene malonoate 2.1.2.)-made nanoparticles, Pharm.
Res. 11 (1994) 1270–1277.
[56] P. Breton, X. Guillon, D. Roy, F. Lescure, G. Riess, N. Bru,
C. Roques-Carmes, Physico-chemical characterization, prep-
aration and performance of poly(methylidene malonate 2.1.2)
nanoparticles, Biomaterials 19 (1998) 271–281.
[57] N. Bru-Magniez, X. Guillon, P. Breton, P. Couvreur, F.
Lescure, C. Roques-Carmes, G. Riess, Method for preparing
malonate methylidene nanoparticles, nanoparticles optionally
containing one or several biologically active molecules,
International Patent PCT WO 98 / 18455, 1998.
[58] N. Bru-Magniez, V. Larras, G. Riess, P. Breton, P. Couvreur,
C. Roques-Carmes, Novel surfactant copolymers based on
methylidene malonate, International Patent PCT WO 99 /
38898, 1999.
[59] A.N. Mitra, P.K. Ghosh, S. Sahoo, pH and thermoresponsive
hydrogel nanoparticles, Proc. Intern. Symp. Control. Rel.
Bioact. Mater. 25 (1998) 234–235.
[60] A.N. Mitra, P.K. Ghosh, S. Sahoo, Long circulating RES
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
evading hydrophilic nanoparticles, Proc. Intern. Symp. Con-
trol. Rel. Bioact Mater. 25 (1998) 168–169.
[61] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V.P.
Torchilin, R. Langer, Biodegradable long circulating poly-
meric nanospheres, Science 18 (1994) 1600–1603.
[62] M. Amiji, K. Park, in: S.W. Shalaby, Y. Ikada, R. Langer, J.
Williams, (Eds.), Polymers of Biological Significance, ACS
Symp. Ser. 540, Washington, DC, 1994.
[63] P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso,
Novel hydrophilic chitosan and chitosan / polyethylene oxide
nanoparticles as protein carriers, J. Appl. Polym. Sci. 63
(1997) 125–132.
[64] P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso,
Chitosan and chitosan / ethylene oxide–propylene oxide
block copolymer nanoparticles as novel carriers for proteins
and vaccines, Pharm. Res. 14 (1997) 1431–1436.
[65] R. Fernandez-urrusuno, P. Calvo, C. Remunan-Lopez, J.L.
Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of
insulin using chitosan nanoparticles, Pharm. Res. 16 (1999)
1576–1591.
[66] P. Calvo A.S. Boughaba, M. Appel, E. Fattal, M.J. Alonso, P.
Couvreur, Oligonucleotide–chitosan nanoparticles as new
gene therapy vector, Proc. 2nd World Meeting APGI /APV
Paris, 1998, pp. 1111–1112.
[67] H.-Q. Mao, K. Ray, S.M. Walsh, J.T. August, K.W. Leong,
DNA–chitosan nanoparticles for the gene delivery, Proc.
Intern. Symp. Control. Release. Bioact. Mater. 23 (1996)
401–402.
[68] K. Ray, H.-Q. Mao, K.Y. Lin, S.-K. Huang, K.W. Leong,
Oral immunization with DNA–chitosan nanoparticles, Proc.
Intern. Symp. Control. Release. Bioact. Mater. 26 (1999)
348–349.
[69] V.L. Truong-Le, H. Mao, S. Walsh, K.W. Leong, J.T. August,
Delivery of DNA vaccine using gelatin–DNA nanoparticles,
Proc. Intern. Symp. Control. Rel. Bioact. Mater. 24 (1997)
39–40.
[70] X.-X. Tian, M.J. Groves, Formulation and biological activity
of antineoplastic proteoglycans derived from Mycobacterium
vaccae in chitosan nanoparticles, J. Pharm. Pharmacol. 51
(1999) 151–157.
[71] H. Tokumitsu, H. Ichikawa, Y. Fuukumori, Chitosan–
gadopenteic acid complex nanoparticles for gadolinium
neutron-capture therapy of cancer: preparation by novel
emulsion–droplet coalescence technique and characteriza-
tion, Pharm. Res. 16 (1999) 1830–1835.
[72] C. Vautheir, I. Aynie, P. Couvreur, E. Fattal, Pharmacokinetic
and tissue disposition of oligonucleotide associated with
alginate nanoparticles, Proc. Intern. Symp. Control. Rel.
Bioact. Mater. 25 (1998) 228–229.
[73] T. Jung, A. Breitenbach, T. Kissel, Sulfobutylated poly(vinyl
alcohol)-graft-poly(lactide-co-glycolide) facilitate the prepa-
ration of small negatively charged biodegradable nanos-
pheres for protein delivery, J. Control. Rel. 67 (2000) 157–
169.
[74] A. Breitenbach, G. Nykamp, T. Kissel, Self-assembling
colloidal carriers for protein delivery: nanoparticulate poly-
mer protein conjugates with novel water soluble biodegra-
17
dable comb polyesters, Proc. Intern. Symp. Control. Release.
Bioact. Mater. 26 (1999) 159–160.
[75] M.A. Breitenbach, W. Kamm, K.-D. Hungere, E. Hund, T.
Kissel, Oral and nasal administration of tetanus toxoid
loaded nanoparticles consisting of novel charged biodegra-
dable polyesters for mucosal vaccination, Proc. Intern. Symp.
Control. Release. Bioact. Mater. 26 (1999) 348–349.
[76] M.J. Alenso, C. Losa, P. Calvo, J.L. Vila-Jato, Approaches to
improve the association of amikacin sulphate to poly-
(cyanoacrylate) nanoparticles, Int. J. Pharm. 68 (1991) 69–
76.
[77] M. Ueda, A. Iwara, J. Kreuter, Influence of the preparation
methods on the drying release behavior of loperamide-loaded
nanoparticles, J. Microencapsulation 15 (1998) 361–372.
[78] P. Couvreur, B. Kante, M. Roland, P. Speiser, Adsorption of
antineoplastic drugs to polyalkylcyanoacrylate nanoparticles
and their release in calf serum, J. Pharm. Sci. 68 (1979)
1521–1523.
[79] M.A. Radwan, In vitro evaluation of poly-
isobutylcyanoacrylate nanoparticles as a controlled drug
carrier for theophylline, Drug Dev. Ind. Pharm. 21 (1995)
2371–2375.
[80] M.A. Egea, F. Gamisani, J. Valero, M.E. Garcia, M.L.
Garcia, Entrapment of cisplatin into biodegradable poly-
alkylcyanoacrylate nanoparticles, Farmaco 49 (1994) 211–
217.
[81] K. Chukwu, U. Ishroder, P. Sommerfeld, B.A. Sabel, Load-
ing some psychopharmacologic agents onto poly(butyl-
cyanoacrylate) nanoparticles-a means of targeting to the
brain and improving therapeutic efficiency, Proced. Intern.
Symp. Control. Rel. Bioact. Mater. 26 (1999) 1148–1149.
[82] J. Vora, N. Bapat, M. Boroujerdi, Investigation of the relative
affinity of doxorubicin for neutral and negatively charged
particulate carriers, Drug Dev. Ind. Pharm. 19 (1993) 759–
771.
[83] J. Martin, P. Macchi, M. Hoffman, P. Maincent, Study of the
binding mechanisms of drugs onto polyalkylcyanoacrylate
nanoparticles, J. Pharm. Belg. 49 (1994) 498–508.
[84] `
E. Benoit, O. Prot, P. Maincent, J. Bessiere, Adsorption of
beta-blocker onto polyisobutylcyanoacrylate nanoparticles
measured by depletion and dielectric method, Pharm. Res. 11
(1994) 585–588.
[85] H.S. Yoo, J.E. Oh, K.H. Lee, T.G. Park, Biodegradable
nanoparticles containing doxorubicin–PLGA conjugate for
sustained release, Pharm. Res. 16 (1999) 1114–1118.
[86] C. Washington, Drug release from microdisperse systems. A
critical review, Int. J. Pharm. 58 (1990) 1–12.
[87] B. Magenheim, S. Benita, Nanoparticle characterization: a
comprehensive physicochemical approach, S.T.P. Pharm. Sci.
1 (1991) 221–224.
[88] M.S. El-Samaligly, P. Rohdewald, H.A. Mohmoud, Poly-
alkylcyanoacrylate nanocapsules, J. Pharm. Pharmacol. 38
(1986) 216–218.
[89] A.M. LeRay, M. Vert, J.C. Gautier, J.P. Benoit, End-chain
radiolabeling and in vitro stability studies of radiolabeled
poly(hydroxy) nanoparticles, J. Pharm. Sci. 83 (1994) 845–
851.
[90] M. Fresta, G. Puglisi, G. Giammona, G. Cavallaro, N.
18 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
Micali, P.M. Furneri, Pefloxacin mesilate- and ofloxacin-
loaded polyethylcyanoacrylate nanoparticles; characterization
of the colloidal drug carrier formulation, J. Pharm. Sci. 84
(1995) 895–901.
[91] G. Cavallaro, M. Fresta, G. Giammona, G. Puglisi, A. Villari,
Entrapment of b-lactams antibiotics in poly-
ethylcyanoacrylate nanoparticles: studies on the possible in
vivo application of this colloidal delivery system, Int. J.
Pharm. 111 (1994) 31–41.
[92] M.Y. Leavy, S. Benita, Drug release from submicron O / W
emulsion: A new in vitro kinetic evaluation model, Int. J.
Pharm. 66 (1990) 29–37.
[93] R. Jalil, J.R. Nixon, Biodegradable poly(lactic acid) and
poly(lactide-co-glycolide) microcapsules: problems associ-
ated with preparative techniques and release properties, J.
Microencapsul. 7 (1990) 297–325.
[94] B. Magenheim, M.Y. Levy, S. Benita, A new in vitro
technique for the evaluation of drug release profiles from
colloidal carriers-ultrafiltration technique at low pressure, Int.
J. Pharm. 94 (1993) 115–123.
[95] J.M. Rodrigues Jr, H. Fessa, C. Bories, F. Pusieux, J.-Ph.
Devissaguet, Premaquine-loaded poly(lactide) nanoparticles:
physicochemical study and acute tolerance in mice, Int. J.
Pharm. 126 (1995) 253–260.
[96] M. Polakovic, T. Gorner, R. Gref, E. Dellacherie, Lidocaine
loaded biodegradable nanospheres. II. modeling of drug
release, J. Control. Rel. 60 (1999) 169–177.
[97] Z. Lu, J. Bei, S. Wang, A method of the preparation of
polymeric nanocapsules without stabilizer, J. Control. Rel.
61 (1999) 107–112.
[98] H. Muller, K.M. Willis, Surface modification of i.v. inject-
able biodegradable nanoparticles with poloxamer polymers
and poloxamine 908, Int. J. Pharm. 89 (1993) 25–31.
[99] C.J. Van Oss, Phagocytosis as a surface phenomenon, Annu.
Rev. Microbiol. 32 (1978) 19–39.
[100] E. Allemann, G. Patricia, J.C. Leroux, B. Luc, R. Gurnay,
Kinetics of blood component-adsorption on poly( D,L-lac-
tide) nanoparticles: Evidence of compliment C 3 component
involvement, J. Biomed. Mater. Res. 37 (1997) 229–234.
[101] T. Blunk, M. Luck, ¨ A. Calvoer, D.F. Hochstrasser, J.C.
Sanchez, B.W. Muller, R.H. Muller, ¨ Kinetics of plasma
protein adsorption on model particles for controlled drug
delivery and drug targeting, Eur. J. Pharm. Biopharm. 42
(1996) 262–268.
[102] K. Thode, M. Luck, ¨ W. Semmler, R.H. Muller, ¨ M. Kresse,
Determination of plasma protein adsorption on magnetic
iron oxide: sample preparation, Pharm. Res. 14 (1997)
905–910.
[103] K. Thode, M. Luck, ¨ W. Semmler, R.H. Muller, ¨ M. Kresse,
Influence of sample preparation on plasma protein ad-
sorption patterns on polysaccharide-stabilized iron oxide
particles and end-terminal microsequencing of unknown
proteins, J. Drug Target. 5 (1997) 35–43.
¨
[104] M. Luck, K.F. Pistel, Y. Li, T. Blunk, R.H. Muller, ¨ T.
Kissel, Plasma protein adsorption on biodegradable micro-
spheres consisting of poly( D,L-lactide-co-glycolide), poly( L-
lactide) or ABA triblock copolymers containing poly(oxy-
ethylene), J. Control. Rel. 55 (1998) 107–120.
¨
[105] R. Gref, A. Domb, P. Quellec, T. Blunk, R.H. Muller, J.M.
Verbavatz, R. Langer, The controlled intravenous delivery
of drugs using PEG-coated sterically stabilized nanos-
pheres, Adv. Drug Deliv. Rev. 16 (1995) 215–233.
[106] M.T. Peracchia, S. Harnisch, H. Pinto-Alphandary, A.
Gulik, J.C. Dedieu, D. Desmaele,¨ J. d’Angelo, R.H. Muller,
¨
P. Couvreur, Visualization of in vitro protein-rejecting
properties of PEGylated stealth  polycyanoacrylate
nanoparticles, Biomaterials 20 (1999) 1269–1275.
[107] B.D. Ratner, A.B. Johnston, T.J. Lenk, Biomaterial surface,
J. Biomed. Mat. Res. Appl. Biomat. 21 (1987) 59–90.
[108] H. Carstensen, B.W. Muller, R.H. Muller, Adsorption of
ethoxylated surfactants on nanoparticles. I. Characterization
by hydrophilic interaction chromatography, Int. J. Pharm.
67 (1991) 29–37.
[109] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, R.
Langer, Biodegradable long-circulating polymeric
nanoparticles, Science 263 (1994) 1600–1603.
[110] S.I. Joen, J.H. Lee, J.D. Andrade, P.G. de Gennes, Protein-
surface interactions in the presence of polyethylene oxide. I.
Simplified theory, J. Colloid. Interf. Sci. 142 (1991) 149–
158.
[111] D. Bazile, C. Prud Homme, M. Bassoullet, M. Marlard, G.
Spenlehauer, M. Veillard, Stealth Me-PEG–PLA nanoparti-
cles avoid uptake by the mononuclear phagocyte system, J.
Pharm. Sci. 84 (1995) 493–498.
[112] M. Tobio, R. Gref, A. Sanchez, R. Langer, M.T. Alonso,
Stealth PLA–PEG nanoparticles as protein carriers for nasal
administration, Pharm. Res. 15 (1998) 270–275.
[113] R. Gref, G. Miralles, E. Dellacherie, Polyoxyethylene-
coated nanospheres: effect of coating on zeta potential and
phagocytosis, Polymer Int. 48 (1999) 251–256.
[114] T. Govender, T. Riley, S. Stolnik, M.C. Garnett, L. Illum,
S.S. Davis, PLA–PEG nanoparticles for site specific deliv-
ery: drug incorporation studies, J. Control. Rel. 64 (2000)
269–347.
[115] S.Y. Kim, I.G. Shin, Y.M. Lee, Preparation and characteriza-
tion of biodegradable nanospheres composed of methoxy
poly(ethylene glycol) and DL-lactide block copolymer as
novel drug carriers, J. Control. Rel. 56 (1998) 197–208.
[116] M.T. Peracchia, E. Fattal, D. Desmaele, M. Bensard, J.P.
Noel, J.M. Gomis, M. Appel, J. d’Angelo, P. Couvreur,
Stealth  PEGylated polycyanoacrylate nanoparticles for
intravenous administration and spleenic targeting, J. Con-
trol. Rel. 60 (1999) 121–128.
[117] A. Gessnerl, B.R. Paulke, R.H. Muller, Plasma protein
adsorption on poly(ethylene-glycol) (PEG) modified poly-
styrene nanoparticles: influence of PEG surface density,
Proc. Intern. Symp. Control. Rel. Bioact. Mater. 26 (1999)
597–598.
[118] M.T. Peracchia, R. Gref, Y. Minamitake, A. Domb, R.
Langer, PEG-coated injectable nanospheres prepared from
amphiphilic diblock and multiblock copolymers: investiga-
tion of drug encapsulation and release characteristics, J.
Control. Rel. 46 (1997) 223–231.
[119] M.T. Peracchia, C. Vauthier, F. Puisieux, P. Couvreur,
PEG–PIBCA nanoparticles with different PEG-coating
configurations formed by chemical coupling of PEG,
 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
Proced. Intern. Symp. Control. Rel. Bioact. Mater. 23
(1996) 395–396.
[120] M.T. Peracchia, C. Vauthier, F. Puisieux, P. Couvreur,
Development of sterically stabilized poly(isobutyl-2-
cyanoacrylate) nanoparticles by chemical coupling of poly-
(ethylene glycol), J. Biomed. Mater. Res. 34 (1997) 317–
326.
[121] M.T. Peracchia, C. Vauthier, D. Desmaele, A. Gulik, J.C.
Dedieu, M. Demoy, J. d’Angelo, P. Couvreur, PEGylated
nanoparticles from a novel methoxypolyethylene glycol
cyanoacrylate–hexadecyl cyanoacrylate amphiphilic co-
polymer, Pharm. Res. 15 (1998) 550–556.
[122] M.T. Peracchia, C. Vauthier, C. Passirani, P. Couvreur, D.
Labarre, Compliment consumption by poly(ethylene glycol)
in different conformations chemically coupled to
poly(isobutyl-2-cyanoacrylate) nanoparticles, Life Sci. 61
(1997) 741–761.
[123] F.D. Jaeghere, E. Allemann, J.C. Leroux, W. Stevels, J.
Feijen, E. Doelker, R. Gurny, Formulation and lyoprotec-
tion of poly(lactic acid-co-ethylene oxide) nanoparticles:
Influence on the physical stability and in vitro cell uptake,
Pharm. Res. 16 (1999) 859–866.
[124] M. Vittaz, D. Razile, G. Spenlehauer, T. Verrecchia, M.
Veillard, F. Puisieux, D. Labarre, Effect of PEO surface
density on long-circulating PLA–PEO nanoparticles which
are very low complement activators, Biomaterials 17
(1996) 1575–1581.
[125] J.C. Ha, S.Y. Kim, Y.M. Lee, Poly(ethylene oxide)–poly-
(propylene oxide)–poly(ethylene oxide) (Plunoric) / poly(e-
caprolactone) (PCL) amphiphilic block copolymeric nanos-
pheres I. Preparation and characterization, J. Control. Rel.
62 (1999) 381–392.
[126] S. Storm, S.O. Belliot, T. Daemen, D.D. Lasic, Surface
modification of nanoparticles to oppose uptake by the
mononuclear system, Adv. Drug Deliv. Rev. 17 (1995)
31–48.
[127] L. Illum, S.S. Davis, Effect of the nonionic surfactant
poloxamer 338 on the fate and deposition of polystyrene
microspheres following intravenous administration, J.
Pharm. Sci. 72 (1983) 1086–1089.
[128] L. Illum, S.S. Davis, The organ uptake of intravenously
administered colloidal particles can be altered using a
non-ionic surfactant (poloxamer 338), FEBS Letts. 167
(1984) 79–82.
[129] S. Rudt, R.H. Muller, In vitro phagocytosis assay of nano-
and microparticles by chemiluminescence. III. Uptake of
differently sized surface-modified particles, and its correla-
tion to particle properties and in vivo distribution, Eur. J.
Pharm. Sci. 1 (1993) 31–39.
[130] L. Illum, S.S. Davis, R.H. Muller, E. Mak, P. West, The
organ distribution and circulation time of intravenously
injected colloidal carriers sterically stabilized with a block
copolymer poloxamine 908, Int. J. Pharm. 89 (1993) 25–
31.
[131] S.M. Moghimi, T.A. Gray, Single dose of iv injected
poloxamine coated long-circulating particle triggers macro-
phage clearance of subsequent doses in rats, Clin. Sci. 93
(1997) 371–379.
19
[132] M. Demoy, J.P. Andreux, C. Weingarten, B. Gouritin, V.
Guilloux, P. Couvreur, Spleen capture of nanoparticles:
Influence of animal species and surface characteristics,
Pharm. Res. 16 (1999) 37–41.
[133] T. Shinoda, A. Maeda, S. Kojima, S. Kagatani, Y. Konno,
T. Sonobe, T. Akaike, Nanosphere coated with lactosyl-
polystyrene polymer as a targeting carrier to hepatocytes,
Drug Deliv. 6 (1999) 147–151.
[134] C.S. Cho, Y.I. Jeong, T. Ishihara, R. Takei, J.U. Park, K.H.
Park, A. Maruyama, T. Akaike, Simple preparation of
nanoparticles coated with carbohydrate-carrying polymers,
Biomaterials 18 (1997) 323–326.
[135] A. Maruyama, T. Ishihara, S.W. Kim, T. Akaike, Nanoparti-
cle DNA carrier with poly( L-lysine) grafted polysaccharide
copolymer and poly( D,L-lactic acid), Bioconjug. Chem. 8
(1997) 735–742.
[136] D. Duchene, D. Wouessidjewe, G. Ponchel, Cyclodextrins
and carrier systems, J. Control. Rel. 62 (1999) 263–268.
[137] S. Raeissi, K. Audus, In vitro characterization of blood–
brain barrier permeability to delta sleep-inducing peptide, J.
Pharm. Pharmacol. 41 (1989) 848–852.
[138] B. Zlokovich, The in vivo approaches for studying peptide
interaction at the blood–brain barrier, Peptides 10 (1989)
249–254.
[139] U. Schroeder, B.A. Sabel, Nanoparticles, a drug carrier
system to pass the blood–brain barrier, permit central
analgesic effects of i.v. dalargin injections, Brain Res. 710
(1996) 121–124.
[140] U. Schroeder, P. Sommerfeld, B.A. Sabel, A efficacy of oral
dalargin-loaded nanoparticle delivery across the blood–
brain barrier, Peptide 19 (1998) 777–780.
[141] R. Alyautdin, D. Gothier, V. Petrov, D. Kharkevich, J.
Kreuter, Analgesic activity of the hexapeptide dalargin
adsorbed on the surface of polysorbate 80-coated poly(butyl
cyanoacrylate) nanoparticles, Eur. J. Pharm. Biopharm. 41
(1995) 44–48.
[142] G. Borchardt, L.A. Kenneth, S. Fenlin, J. Kreuter, Uptake
of surfactant-coated poly(methyl methacrylate)-nanoparti-
cles by bovine brain microvessel endothelial cell mono-
layers, Int. J. Pharmacol. 110 (1994) 29–35.
[143] S.D. Troster, U. Muller, J. Kreuter, Modification of the
body distribution of poly(methyl methacrylate) nanoparti-
cles in rats by coating with surfactants, Int. J. Pharm. 61
(1990) 85–100.
[144] S. Steiniger, D. Zenker, H.V. Briesen, D. Begley, J. Kreuter,
The influence of polysorbate 80-coated nanoparticles on
bovine brain capillary endothelial cells in vitro, Proced.
Intern. Symp. Control. Rel. Bioact. Mater. 26 (1999) 790–
791.
[145] J.C. Olivier, L. Fenart, R. Chauvet, C. Pariat, R. Cecchelli,
W. Couet, Indirect evidence that drug brain targeting using
polysorbate 80-coated polybutylcyanoacrylate nanoparticles
is related to toxicity, Pharm. Res. 16 (1999) 1836–1841.
[146] A.E. Gulyaev, S.E. Gelperina, I.N. Skidan, A.S. Antropov,
G.Y. Kivman, J. Kreuter, Significant transport of doxorubi-
cin into the brain with polysorbate 80-coated nanoparticles,
Pharm. Res. 16 (1999) 1564–1569.
[147] R.N. Alyautdin, V.E. Petrov, K. Langer, A. Berthold, D.A.
20 K.S. Soppimath et al. / Journal of Controlled Release 70 (2001) 1 – 20
Kharkevich, J. Kreuter, Delivery of lopermide across the
blood–brain barrier with polysorbate 80-coated poly-
butylcyanoacrylate nanoparticles, Pharm. Res. 14 (1997)
25–328.
[148] R.N. Alyautdin, E.B. Tezikov, P. Ramga, D.A. Kharkevich,
D.J. Begley, J. Kreuter, Significant entry of tubocurarin into
the brain of rats by adsorption to polysorbate 80-coated
polybutylcyanoacrylate nanoparticles: in situ brain perfu-
sion study, J. Microencapsulation 15 (1998) 67–74.
[149] U. Schroeder, P. Sommerfeld, S. Ulrich, B.A. Sabel,
Nanoparticle technology for delivery of drugs across the
blood–brain-barrier, J. Pharm. Sci. 87 (1998) 1305–1307.
[150] P. Couvreur, F. Puiseux, Nano- and microparticles for the
delivery of peptides and proteins, Adv. Drug Deliv. Rev. 5
(1993) 141–162.
[151] T. Uchida, A. Yagi, Y. Oda, Y. Dakada, S. Goto, Instability
of bovine insulin in poly(lactide-co-glycolide) (PLGA)
microspheres, Chem. Pharm. Bull. 44 (1996) 235–236.
[152] J.K. Lin, N. Wang, X.S. Wu, A novel biodegradable system
based on gelatin NPs and poly(lactic-co-glycolic acid)
microspheres for protein and peptide drug delivery, J.
Pharm. Sci. 86 (1997) 891–895.
[153] N. Wang, X.S. Wu, J.K. Li, A heterogeneously structured
composite based on poly(lactic-co-glycolic acid) micro-
spheres and poly(vinyl alcohol) hydrogel nanoparticles for
long-term protein drug delivery, Pharm. Res. 16 (1999)
1430–1435.
[154] R.M.M. Gasper, D. Blanco, M.E. Cruz, M.J. Alonso,
Formulation of L-aspargainase-loaded poly(lactide-co-gly-
colide) nanoparticles: influence of polymer properties on
enzyme loading, activity and in vitro release, J. Control.
Rel. 52 (1998) 53–62.
[155] I. Fishbein, M. Chorny, L. Rabinovich, S. Banai, I. Gati, G.
Golomb, Nanoparticulate delivery system of a tyrphostin
for the treatment of restenosis, J. Control. Rel. 65 (2000)
221–229.
[156] Y. Kawashima, H. Yamamoto, H. Takeuchi, S. Fujioka, T.
Hino, Pulmonary delivery of insulin with nebulized DL-
lactide / glycolide copolymer (PLGA) nanospheres to
prolong hypoglycemic effect, J. Control. Rel. 62 (1999)
279–287.
[157] G.P. Carino, J.S. Jacob, E. Mathiowitz, Nanosphere based
oral insulin delivery, J. Control. Rel. 65 (2000) 261–269.