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Nano-Graphene Oxide: A Potential

Multifunctional Platform for Cancer Therapy

ARTICLE JULY 2014

DOI: 10.1002/adhm.201300023 Source: PubMed

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University of Aveiro University of Aveiro
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Nano-Graphene Oxide: A Potential Multifunctional

Platform for Cancer Therapy
Gil Gonalves,* Mercedes Vila,* Mara-Teresa Portols, Mara Vallet-Regi, Jos Gracio,
and Paula Alexandrina A. P. Marques*

wide range of areas. The 2D structure of

Nano-GO is a graphene derivative with a 2D atomic layer of sp2 bonded
carbon atoms in hexagonal conformation together with sp3 domains with
carbon atoms linked to oxygen functional groups. The supremacy of nano-GO
resides essentially in its own intrinsic chemical and physical structure, which
confers an extraordinary chemical versatility, high aspect ratio and unusual
physical properties. The chemical versatility of nano-GO arises from the
oxygen functional groups on the carbon structure that make possible its rela-
tively easy functionalization, under mild conditions, with organic molecules
or biological structures in covalent or non-covalent linkage. The synergistic
effects resulting from the assembly of well-defined structures at nano-GO sur-
face, in addition to its intrinsic optical, mechanical and electronic properties,
allow the development of new multifunctional hybrid materials with a high
potential in multimodal cancer therapy. Herein, a comprehensive review of the
fundamental properties of nano-GO requirements for cancer therapy and the
first developments of nano-GO as a platform for this purpose is presented.
1. Introduction to Nano-GO
Currently, graphene is the carbon allotrope generating the
greatest interest, not only from the point of view of academic
curiosity, but also considering its potential applications in a
Dr. G. Gonalves, Prof. J. Gracio, Dr. P. A. A. P. Marques
TEMA-NRD, Mechanical Engineering Department and
Aveiro Institute of Nanotechnology (AIN)
University of Aveiro
3810-193 Aveiro, Portugal
E-mail: ggoncalves@ua.pt; paulam@ua.pt
Dr. M. Vila, Prof. M. Vallet-Regi
Department of Inorganic and Bioinorganic Chemistry
Faculty of Pharmacy
Universidad Complutense de Madrid
28040 Madrid, Spain
Dr. M. Vila, Prof. M. Vallet-Regi
Networking Research Center on Bioengineering
Biomaterials and Nanomedicine
CIBER-BBN, Spain
E-mail: mvila@ucm.es
Prof. M.-T. Portols ments C/O on the GO depends of the level of oxidation, as the
Department of Biochemistry and Molecular Biology I
Faculty of Chemistry
Universidad Complutense de Madrid
28040 Madrid, Spain
DOI: 10.1002/adhm.201300023
carbon atoms arranged in a hexagonal lat-
tice allows unique physical and chemical
properties to be obtained; specific surface
area (2630 m2 g1), high charge mobility
(200000 cm2 v1s1), high Youngs mod-
ulus (1.0 TPa), high thermal conduc-
tivity (5000 Wm1 K1) and high optical
transmittance ( 97.7%).[1] The synthesis
of high quality graphene can be per-
formed through various approaches,
namely chemical vapour deposition,[25]
arc discharge[68] and epitaxial growth
in SiC.[911] Graphene can be also pre-
pared from the wet chemical exfoliation
of graphite, the most widely adopted
approach when large scale production is
needed due to its technological simplicity,
high efficiency and low cost procedure.[12]
The exfoliation of graphite in solution
requires an extensive oxidation of the aro-
matic structure in order to weaken the Van der Walls inter-
actions between the stacked carbon planes. Notably, graphite
oxide can be completely exfoliated to produce aqueous colloid
suspensions of few-layer carbon sheets by sonication or by
magnetic stirring in an oxidative medium. The intermediate
species obtained through these chemical methods is desig-
nated as graphene oxide (GO), a planar carbon structure with
high density of oxygen functional groups, that subsequently
can be reduced into graphene typically by chemical[13] or
thermal[14] methods.
GO was initially considered as just an intermediate on the
processing of graphene; however, studies of their unique phys-
ical/chemical properties made it a very interesting material for
various types of applications.[15] Nano-GO is obtained from GO
by converting the micrometric lateral dimensions of the GO
sheets to nanometric size (below 100 nm) by applying ultra-
sound energy. Pan et al.[16] showed that the lateral size of GO
sheets can be controlled by the degree of oxidation (defects)
that allows the formation and propagation of cracks during
sonication treatment. The stoichiometry ratio between the ele-
composition and structure do not change significantly with
the preparation method used after achieving some threshold
oxidation degree (TOD). The TOD id between the values,
1.8<TOD<2.5, where the most common value is that approxi-
mately 2.[1719]

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Today impressive achievements have been made at the cross
Gil Gonalves graduated in
section of nanotechnology and biotechnology by employing
2003 in Industrial Chemistry at
carbon nanomaterials, where GO has been one of the most
University of Aveiro (Portugal)
promising nanoparticles proposed for these applications in the
and obtained a Master degree
last few years. Its unique morphology, surface chemistry and
in Materials Science (EMMS,
structure can be the key point for the development of new mul-
Joint European Masters
tifunctional materials for cancer therapy.
Programme) in 2008, his
The surface chemistry of GO is highly versatile, because the
thesis was based on polymers
presence of the oxygen functional groups allows the use of sev-
nanocomposites from renew-
eral functionalization approaches.[20,21] Acylation is among the
able resources. In 2012 he
most common approaches to promote covalent attachment of
received his PhD in Mechanical
molecular precursors to the GO surface.[2226] Regarding non-
Engineering at the University of
covalent functionalization, the most usual is the stacking
Aveiro with a thesis dedicated
and/or hydrophobic interactions with the aromatic structure
to nanocomposite materials for biomedical application. Now,
of GO.[2730] Functionalized GO with unique and specific con-
his research interests are dedicated to the development of
trolled properties has been used to build up biological plat-
new carbon nanoplatforms for multimodal cancer therapy.
forms, biosensors and biodevices.[3134]
The GO aromatic structure allows strong light absorption
ability in the near-infrared (NIR) range (700900 nm), which Mercedes Vila graduated
is commonly called therapeutic window[35] as it is a non-inva- in Physics at Universidad
sive, harmless and skin penetrating irradiation. This property is Autnoma de Madrid (Spain),
particularly attractive for the induction of cellular hyperthermia and received her PhD in
in tumour treatments as a minimally invasive alternative to sur- Materials Physics (2003,
gery (Photothermal therapy)[36]. Materials Science Institute of
Curiously, graphene showed interesting photoluminescent Madrid, CSIC). She performed 5
properties without any type of surface modification. Since gra- years of post-doctoral research
phene has no band gap, photoluminescence is not expected at Aveiro University (Portugal),
from relaxed charge carriers. However, Lui et al.[37] observed and since 2008 she is a Ramon
significant light emission from graphene under excitation by y Cajal researcher at the
ultrashort (30-fs) laser pulses, that was found to occur across Universidad Complutense de
the visible spectral range. Photoluminescence is also observed Madrid. Her research interests include the design, synthesis
in GO that can shift from red to blue emission depending on and application of bioceramic materials for bone tissue engi-
the degree of oxidation, that means dependence from the pro- neering and carbon based nanosystems for cancer therapies.
portion between sp2 and sp3 carbon atoms.[3840] The authors
suggest that this behaviour can be attributed to electron-hole
recombination from two different types of excited states. This Paula Marques is Assistant
unusual property could make graphene and GO potential Researcher at the University
imaging agents on biological systems. of Aveiro (UA) Portugal, being
Recent research studies showed that functionalized graphene the Scientific Coordinator of
and GO have enormous potential as sensors for early cancer the Nanotechnology Research
detection.[41] The low limit detection for proteins such as Cyclin Division of the Centre for
A2[42,43] or telomerase,[44] which are usually overexpressed in Technology and Automation
many types of cancers, allows to prognostic aggressiveness, sur- (TEMA) at this University. She
vival and chemotherapy response for the different types of cancer. graduated in Chemistry (UA,
Regarding the biocompatibility of graphene derivatives, the 1994), completed a masters in
in vitro[4548] and in vivo[46,49] studies developed so far are still in Physics and Chemistry Teaching
a preliminary stage. In vitro studies demonstrated that the gra- (UA, 1997) and a doctorate in
phene can induce the generation of reactive oxygen species in Materials Science Engineering (UA, 2003). Paulas main field
neural pheochromocytoma derived PC12 cells by time/concen- of research is dedicated to the engineering and development
tration dependence and also cause apoptosis for concentrations of new nanostructured materials. In particular, she is working
up to 10 g/mL.[45] In the case of GO, the induction of apop- on the preparation and study of graphene based nanocompos-
tosis was also observed in human fibroblasts for concentrations ites with applications in the biomaterials and biosensors area.
of 50 g/mL.[46] In human lung epithelial cells, GO doses over
50 g/mL showed no toxicity in vitro,[48] but higher GO con-
centrations cause a dose-dependent oxidative stress in these
cells and a slight loss of cell viability. In fact, most studies agree A few in vitro studies were already performed in order to
that GO promotes cytotoxicity mainly through generating reac- compare the biocompatibility between single wall carbon nano-
tive oxygen species (ROS) in a dose-dependent manner which tubes (SWCNT) and graphene. Agarwal et al. reported that
induces oxidative stress.[5052] SWCNT cause inhibition on cell proliferation (neuroendocrine

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PC12 cells, oligodendroglia cells and osteoblasts) in contrast to
reduced GO.[53] Zhang et al. found higher lactate dehydroge-
nase levels for neural phaeochromocytoma-derived PC12 cells
for SWCNT as compared to the graphene.[45] The results sug-
gest high dependence of the nanotopography, aspect ratio and
shape of carbon nanostructures.[45,53] Other parameters such
as hydrophilic character,[54] dosage levels[46] and lateral dimen-
sions[55] of graphene derivatives make a decisive contribution to
the assessment of biocompatibility.
In vivo tests in mice model showed that, after intravenous
administration, GO doses of 0.1 and 0.25 mg did not exhibit
toxicity. However, GO doses of 0.4 mg exhibited chronic tox-
icity. Carefully analysis showed that administration of high
doses of GO leads to the lung granuloma formation.[46] How-
ever, other studies showed no toxicity after three months of
injection of GO (20 mg/Kg) in this animal model.[56] Those
results suggest that graphene derivatives have a multitude of
chemical structures and morphologies, which requires long
term toxicity/biocompatibility (dose and specificity on different
tissues) studies in order to further validate these materials in
biomedical applications.
The main propose of this review is to discuss the intrinsic
properties of nano-GO that makes this nanomaterial so prom-
ising for the development of new therapeutic agents for cancer
therapy. In fact, nano-GO shows enormous potentialities
derived from its simple chemical structure and morphology.
However, it is necessary to bear in mind the challenges that
arise when trying to develop a material for therapeutic applica-
tions in humans. Herein, important biological details and syn-
thesis approaches are reviewed in order to summarize the key
concepts for developing new engineered nano-GO that could
act efficiently and effectively in the detection and treatment of
tumours.
2. Fundamental Concepts on Nanoparticle
Cancer Therapy
The clinical application of nanoparticles has the potential to
make paradigm-changing impacts on the detection, treatment,
and prevention of cancer. The design of nanoparticles repre-
sents a new hope in the development of new therapeutic agents
for cancer treatment. Here, we discuss the most relevant phys-
icochemical properties of nanoparticle platforms that make
them effective in their interaction with biological structures. A
discussion of the basic biological structure of tumours, as well
as the relevant properties of nanoparticles on cancer therapy
will be discussed in this section. These fundamental concepts
are of extreme relevance to understand the factors which give
nano-GO huge potential as a nanoparticle for cancer therapy.
2.1. Cancer Biology
Engineering new nanoparticles for cancer therapy must take
into account strategies to overcome the biological barriers, from
the mode of administration (inhalation, oral, intravenous etc)
to the organ and cellular level. Critical issues include rapid
filtration in the kidney and clearance via reticulo-endothelial
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system (RES), as well as transport from the bloodstream to
target cells within tissues.[57] At cellular level, the nanoparticles
must cross the cell membrane and, in some cases, the nuclear
membrane.
An improved knowledge of the cancer biology, including
cancer microenvironment, signalling pathways and evolution
of metastasis, has resulted in clear advances in cancer therapy.
However, due to the complexity of tumour progression, tumour
composition, blood vessel structures, and drug resistance
mechanisms, most of the current therapies have provided lim-
ited extension of survival time across multiple cancer types. The
development of blood vessels is an essential step in the growth
of a tumour. Without vessels, tumours cannot grow to be larger
than a small fraction of an inch. When the area around the cells
in a tumour starts to get too far from a blood vessel, the oxygen
and nutrient levels begin to go down. A decrease in oxygen is
also called hypoxia. Hypoxia triggers changes in the behaviour
of the tumour cells which start producing (or cause nearby
cells to produce) growth factors that stimulate the formation
of blood vessels. The tumour is able to overcome the diffusion
limitation by increasing the surrounding vasculature, this event
is called angiogenesis. It results in abnormal blood and lym-
phatic networks and vascular barriers. The increased internal
pressure causes an outward convective interstitial fluid flow,
which decreases drug diffusion to the centre of the tumour.
However, drugs and nanoparticles that gain interstitial access
to the tumour have higher retention times than in normal tis-
sues. This is called enhanced permeability and retention effect
(EPR).
From the biological point of view tumours can be structurally
divided in three different regions: periphery, seminecrotic and
necrotic core, each one with metabolically compromised micro-
environment (Figure 1a).[58] The unstable state of tumours can
be attributed to accumulated solid stress,[59,60] abnormal blood
vessel networks,[6163] elevated fluid pressure,[64,65] and a dense
interstitial structure.[66,67]
The transport barriers to drug delivery arise from those
abnormal characteristics of the tumour microenvironment.
Actually, several nanoparticles were already designed to take
advantage of the EPR effect;[68,69] the concept is referred to as
passive targeting, however this effect just starts to be observed
on tumours with volume higher than 2 mm3.[70] Three impor-
tant pharmacokinetic steps govern the diffusion of nanoparti-
cles into tumours cells (Figure 1b and c).[71] Overall, the nan-
oparticle internalization on the tumour is low and heteroge-
neous relatively to the different tumour regions, where a high
retention rate is usually observed on the peritumor tissue.
Besides the treatment of the primary tumour, one of the
most challenging facets in cancer therapy is the early detection
and treatment of metastasis and secondary tumours. Cancer
metastasis consists on the spread of cancer cells from the pri-
mary tumour on affected organ to the blood stream leading to
the formation of secondary tumours on other receptor organs
(Figure 2). Indeed, the rate of survival is high (> 90%) when
cancer is limited to one organ whereas there is only 20%
5-years survival rate when cancer cells metastasize.[72]
The therapy of metastatic cells remains a challenge. Nev-
ertheless metastatic cells have the particularity of always
expressing a specific endogenous surface protein.[72] To take
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of nanoparticles varies from drug delivery
systems, imaging agents to theranostics
(combination of therapeutics and diagnosis).
The nanoparticle systems based on inor-
ganic structures that are approved for cancer
therapy are very few and composed of
superparamagnetic iron oxide nanoparticles
coated with dextran or carboxydextran for
bioimaging agents (ferumoxides and ferucar-
botran).[86] A few other nanoparticles based
on inorganic structures are under clinical
trials like gold nanoparticles (CYT-6091,
Aurimmune), quantum dots (Bioconjugated
nanoparticles) and iron oxide nanoparticles
(Combidex, Ferumoxtran-10).[87] In fact, inor-
ganic nanoparticles offer great potential for
future clinical applications in imaging, diag-
nosis and therapeutics of cancer, however,
their possible toxicity and accumulation on
cells and organs could be some important
limitations to their use.[88]

2.3. Nanoparticle Behavior in Biological

Systems
Figure 1. Transport in tumors. (a) A schematic of drug transport steps overlaid on an image of
the tumor margin of an orthotopic mammary tumor in a mouse that depicts delivery of a low- Nanoparticles have high surface to volume
molecular-weight fluorescent probe after 30 min. Overall, delivery is heterogeneous and poor, ratios when compared with larger particles
with retention of the probe in the peritumor tissue. Examples of vascular, transvascular, and and the manipulation of their surface at
interstitial transport steps are depicted. (b) Transport steps in a tumor tissue unit consisting quasi-atomic level provides incomparable
of blood vessels and the surrounding tissue. (c) General properties of the tumor microenviron- freedom to modify fundamental properties
ment, including drug delivery heterogeneity. Three regions of tumorsthe periphery, semine- such as solubility, diffusivity, blood circula-
crotic region, and necrotic coreare delineated along with their characteristics. Reproduced
with permission.[71] Copyright 2011, Annual Reviews.
tion half-life, drug release characteristics and
immunogenicity. In this context, the control
of nanoparticles properties can dictate the
advantage of this feature, the nanoparticle surface could be tai- success on the biodistribution and circulation time in vivo.[89,90]
lored with specific target ligands, like peptides[7375] or antibody In fact, the body response mechanism to foreign nanoparticles
conjugates[7678] to target the tumour cells with minimal side is a result of a complex interplay of factors that can be funda-
effects on normal cells. This approach was already developed mentally attributed to the nanoparticles properties. We desig-
with magnetic[79,80] and polymeric[81] nanoplatforms showing nated these fundamental properties of the nanoparticles that
a high success for recognition and treatment of circulating control the biological interactions as the 3Ss factor: Size, Shape
cancer cells from the bloodstream. and Surface Chemistry (Figure 4).

2.3.1. Nanoparticle size

2.2. Nanoparticles on Clinical Uses
Nanotechnology started having some impact on biomedicine
half a century ago, and during this period of time, approxi-
mately 40 different types of nanoparticles have completed the
extremely exigent journey from laboratory to clinical trials
as exemplified in Figure 3.[82,83] Many other nanoparticles
are currently under various stages of preclinical and clinical
development.[84]
The first generation of nanopharmaceuticals (this sub-
discipline was defined as the science and technology of nano-
metric size scale complex systems) in clinical use or under
clinical development for cancer therapies can be classified on
the following materials families: liposomes, polymer nano-
particles, polymer micelles and dendrimers.[85] The application
The plasma membrane is the interface between cells and their
harsh environment. Endocytosis encompasses diverse mecha-
nisms by which cells internalize macromolecules and particles
into transport vesicles derived from the plasma membrane. It
is well known that the size of the particles can determine the
biological mechanism of internalization on cells.[9193] It is also
important to emphasize that the mechanism of internalization of
nanoparticles into cells is also dependent on the cell type. Thus,
phagocytic cells (macrophages)[94] usually internalize bigger par-
ticles by phagocytosis and non-phagocytic cells (osteoblasts, fibro-
blasts, ovarian, endothelial cells, ) favour the internalization of
smaller particles by using more than one pathway of internaliza-
tion, although it is assumed that particles up to 200 nm are inter-
nalized mainly by (receptor-mediated) endocytosis.[95,96]

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system. The results showed that nanoparticle

size can also affect the process of surface
protein adsorption in blood plasma (opsoni-
sation) which can contribute to a faster or
slower elimination from the organism.[101103]
Nanoparticles with a size range between
2100 nm can change the signalling pro-
cesses of cells for essential basic functions
including cell death.[104] These results reveal
the potentialities to control the cellular
response at the nanoscale level, but also evi-
dence the potential biological risks.

2.3.2. Nanoparticle shape

Figure 2. Metastatic spread to different organs. Blood flow patterns can predict the specific tively cilindrical and planar geometries) by
regions of metastases in approximately two-thirds of cancers. Reproduced with permission.
Copyright 2011, Nature Publishing Group.
Indeed, it has been demonstrated that the size of the nano-
particles has a great influence in terms of biodistribution in
organs[97,98] and cells[99] and also on the level of penetration
into tumours.[100] The influence of nanoparticle size has also
been investigated concerning the effects on the innate immune
Nanoparticle shape also has an important
influence on the biodistribution and cellular
internalization dynamics of the nanoparticles
into cells. Studies demonstrated that nano-
particles with similar volumes but with dif-
ferent shapes were internalized at very different
rates.[81] There were extreme cases of nano-
particles shapes that led to inhibition of cellular
internalization.[105] A recent review by Bussy
et al[106] discusses different cellular uptake
mechanisms of carbon nanotubes (CNTs) and
graphene (two carbon allotropes with respec-
[ 72]
the cell membrane. The results show that the
shape of carbon allotropes can really control the
type of cellular interactions and internalization.
Understanding how shape can affect the biodistribution
of intravascularly injected particles is, thus, of fundamental
importance for optimal design of new nanomaterials for cancer
therapy. Quantitative in vivo studies performed suggest that
nanoscale extravasational competence is heterogeneous and

Figure 3. Time line of clinical stage nanomedicine. Liposomes, controlled release polymeric systems for macromolecules, dendrimers, targeted-
PEGylated liposomes, first FDA approved liposome (DOXIL), long circulating poly(lactic-co-glycolic acid)-polyethyleneglycol (PLGA-PEG) nanoparti-
cles, iron oxide MRI contrast agent nanoparticles (Ferumoxide), protein based drug delivery system (Abraxane; nab technologyt), polymeric micelle
nanoparticles (Genexol-PM),targeted cyclodextrin-polymer hybrid nanoparticles (CALAA-01), targeted polymeric nanoparticles (BIND-014; Accurint
Technology), fully integrated polymeric nanoparticle vaccines (SEL-068, tSVPt Technology) Reproduced with permission.[84] Copyright 2012, Royal
Society of Chemistry.

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Figure 4. Physical and chemical properties of the nanoparticles that
affect their performance, both in vitro and in vivo. The 3Ss factor (size,
shape and surface chemistry) governs the interaction of nanoparticles
with biological structures.
highly dependent on nanoparticle geometry and on the biodis-
tribution in vivo on the diverse organs and tumours.[107,108] In
fact, the shape of nanoparticles also affects the circulation time
on bloodstream, for example, Geng et al.[81] observed that the
filomicelles persisted in the circulation ten times longer than
the spherical micelles.
Theoretical studies using mathematical modelling with in
vitro and in vivo experimental data, allowed the optimization
of mesoporous silica particles parameters for example specific
geometries and sizes to maximize localization within tumour
vasculate while minimizing RES uptake.[109] Design maps
have been generated to be used as a preliminary reference for
choosing the properties of the nanoparticle as a function of
physiological parameters, such as the wall shear stress and the
receptors surface density, at the site of desired adhesion within
the target vasculature.
2.3.3. Nanoparticle surface chemistry
The surface chemistry of nanoparticles is another fundamental
parameter in the biological interaction with tissues, normal and
tumoural cells. In spite of what has been achieved so far, a com-
plete understanding of how biological structures interact with
nanomaterials at the molecular level is lacking. However, it is
well known that the surface chemistry of nanoparticles effec-
tively controls their interaction with biological components,
from blood vessels to the organs, cells and cellular components.
The free functional groups at the nanoparticles surface have
a tremendous impact on the cellular uptake and cellular biodis-
tribution.[99] It was also verified that the surface charge of nano-
particles has also an important contribution on cellular uptake
(rate and amount) and cellular biodistribution.[110,111]
The surface charge of the nanoparticles is, likewise, an
important parameter that stimulates the EPR effect on tumours.
Some studies demonstrated that slightly negative[112] or posi-
tive[113] nanoparticles showed higher internalization rates.
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Current methods to increase the circulation time of nanopar-
ticles have focused almost exclusively on reducing the opsonisa-
tion process, by rendering the particle surface more hydrophilic
or by reducing the surface charge. The surface of nanoparticles
can be tailored with polymers that increase the circulation half-
time. Polyethylene glycol (PEG) has been the most widely used
polymer so far for this application because it can enhance the
solubility and plasma stability of proteins and reduce immuno-
genicity.[100,114] Moreover, the PEG chemical structure effects on
cell uptake was also assessed by comparing internalization of a
branched PEG ligand to the PEG linear version, and it has been
found that the linear version is internalized in a greater propor-
tion than the branched one.[115]
Several multifunctional systems have been developed based
on the surface manipulation of nanoparticles providing new tools
for the diagnosis and treatment of cancer disease. The ability to
tailor the nanoparticle surface with conjugated fluorescent units
makes these materials excellent candidates for cell sensing.[116]
Using this sensor array, it was possible to obtain highly repro-
ducible characteristic patterns from different cell types enabling
the identification of cell types and cancer states.[116,117] Cur-
rent trends in nanomedicine involve incorporation of targeting
moieties that are highly specific for receptors overexpressed in
tumoural cells (e.g., folic acid, proteins and antibodies).[104,118]
2.4. Design Nanoparticles for Cancer Therapy
The nanoparticles must be designed for the intended function:
recognition, imaging, or treatment of cancer cells. To design
new nanoparticles for cancer therapy it is necessary to clearly
identify what are the expected different levels of action and
subsequently to define which chemical structures will be neces-
sary to achieve the predetermined objectives. This is part of a
multidisciplinary group of intervenients (Figure 5).[119] After the
Figure 5. Role of nanobiotechnology in the management of cancer. This
is a scheme of the role of nanobiotechnology and its interaction with
other technologies and approaches used in the management of cancer.
Reproduced with permission.[119] Copyright 2010, BioMed Central.
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and folic acid), peptides sequences and

monoclonal antibodies.[122]
Davis et al. describe that the use of nano-
particles as a platform on cancer therapy has
several advantages with respect to other types
of therapeutic modalities.[124]
So far, some challenges still arise in the
use of nanoparticles as a therapeutic agent.
It is still necessary to do extensive research
work to understand the behaviour of the
nanoparticles in the human body and to rec-
ognise which are the risks associated with
possible toxic effects. Indeed, a series of
questions needs to be answered. Neverthe-
less, there are various types of nanoparticles
for cancer therapy already approved or under
Figure 6. Design project for assembly chemical structures in order to develop multifunctional clinical studies.
nanoplatforms for cancer therapy.

correct definition of the role to be played by the nanoparticles 3. Key Points of Nano-GO on Cancer Therapy
in the human body it is possible to consider the different syn-
Nano-GO is obtained after exfoliation of graphite in a strong
thesis mechanism and chemical structures. There are some
oxidant medium, followed by ultrasonication.[125] Nano-GO is
authors that call this area of knowledge of nanomedicine as
simply defined as a plane of carbon atoms in hexagonal struc-
Nano-oncology.[119]
ture with carboxylic and carbonyl groups on the edges and
Historically, the evolution of nanoparticle design can be
hydroxyl and epoxy groups in the basal planes. It holds a great
divided in three different generations.[89] The first genera-
potential for bio-engineering as nanoparticle vector, since its
tion of nanoparticles was based on basic surface chemistry
oxidized state provides negative surface charge and reactive
studies, only concerned with biocompatibility and toxicity.
sites for functionalization.[31]
The second generation of nanoparticles consisted on the opti-
One of the key parameters that makes nano-GO one of the
mization of the surface chemistry in order to increase the
most interesting materials for biological applications is its high
biological stability and targeting specific biological locations.
aspect ratio. The 2D structure of carbon atoms with dimensions
The third generation was described as nanoparticles that can
below 100 nm and atomic thickness, allows obtaining high
respond to biological (pH, oxidant medium, etc.) or external
values of specific surface area (Figure 7). This property is very
stimuli (temperature, light, etc.) in order to target and deliver
important because it allows a high rate of functionalization and
on the specific locations and improve the biological efficiency
maximizes the interaction with biological systems. In fact, the
(Figure 6).
planar and flexible structure of nano-GO, is also an additional
Conventional methods of preparing multifunctional nano-
factor that makes graphene a high-potential material for the
particles involve a series of chemical processes whereby the
interaction with biological structures.
nanoparticles core is initially formed, followed by the function-
As discussed in the previous sections, the 3Ss factor of the
alization of ligands (drugs, imaging agents, DNA, etc.) to the
therapeutic nanoparticles plays a crucial role in tumoural tissue
surface of the nanoparticles (Figure 6). These ligands can be,
extravasation and interstitial transport. Studies have shown
for example, small molecule drugs like Doxorubicin (DOX),
that macromolecules with linear, semi-flexible configurations
hydroxycamptothecin (HCPT), Paditaxel (PTX), Cisplatin, Car-
boplatin, Irinotecan, Gefitnib.
Strategies for delivering nanoparticles to cancerous tissue
have been focused on passive and active targeting.[120] As
has been explained before, the passive targeting approach on
cancer therapy is based on the non-selective process of EPR,
which allows the higher accumulation of macromolecules
in tumoural tissues than in normal tissues. Active targeting
approaches are based on recent methods of targeting the
nanoparticles, which consist of the surface functionalization
of nanoparticles with specific ligands that are highly selective
in binding to specific receptors that are overexpressed on the
surface of cancer cells (Figure 6).[121123] A few examples of
molecular targeting used to recognise specific types of cells Figure 7. 2D structure of nano-GO with lateral dimensions below 100 nm
and increase the biodistribution are: small molecules (biotin and atomic thickness.

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like nano-GO, diffuse more efficiently in the interstitial matrix
than do comparable sized, rigid spherical particles.[126] Yue et
al. studied the role of the lateral dimension of GO in the regu-
lation of cellular responses showing that, in comparison with
nanosized GO, micro-sized GO showed divergent intracellular
locations and induced much stronger inflammation response
in six cell types.[55]
Nano-GO acts as the next generation of carbon materials
intended to be used as anticancer therapeutic agents. One
important aspect of nano-GO, which derives from its aromatic
chemical structure, is the strong optical absorbance in NIR
(therapeutic window, 700900 nm range) that makes it an excel-
lent candidate for tumour cells ablation by photothermal treat-
ment (Hyperthermia).[36,127129] Acik et al. described a strong
infrared-absorption of reduced GO at 800 cm1 due to the
oxygen edge.[130] This new phenomenon opens the door to tai-
loring giant infrared absorption at different spectral positions
by modifying the nature of the edge termination.
Moreover, the upconversion fluorescence property of nano-
GO allows them to be excited in the NIR region without inter-
ference auto-fluorescence from tissues, making bio-detection
and bio-imaging efficient.[131136]
The photoluminescence of nano-GO is becoming a subject
of research for optical bioimaging.[137] It has been demonstrated
that strong photoluminescence from two-photon excitation can
be induced in nano-GO by an ultrafast pulsed laser.[138] Peng
et al. demonstrated that the photoluminescence of the nano-GO
can be tailored through varying the size of the nano-sheets.[139]
The application of this technique to cancer cell imaging has
already been investigated with different types of cells, gastric
cancer cell line (AGS),[140] BT549 breast cancer cells line[141] and
MG-63 human osteosarcoma cell line[135]. Photoluminescence
of nano-GO was also investigated along with the potential appli-
cation on photothermal cancer cell therapy. Li et al. observed
that intensive microbubbling can be induced by irradiation at
a laser power lower than 4 mW in the presence of nano-GO,
which causes instant cell damage.[140]
The nano-GO aromatic molecular structure and the richness
of its surface functional groups allow functionalization with
small molecules or biostructures to build-up new hybrid systems
that can perform in all stages of the cancer therapy: recognition
imaging, and treatment of cancer cells (Figure 8). The chemical
versatility of nano-GO is, surely, a key point for the combina-
tion of several diagnosis and therapy techniques on the same
system, usually defined as a multifunctional nano-platform.
Figure 8. Schematic representations of different approaches for the functionalization of nano-
GO in order to obtain multifunctional platforms for cancer therapy.
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These approaches have already been explored with carbon
nanotubes: both single- (SWCNTs) and multi-walled
(MWCNTs) carbon nanotubes. However, some limitations on
the control of the synthesis parameters, such as the presence
of metallic impurities and homogeneous size and length dis-
tributions, demonstrated some ambiguity in studies of possible
toxic effects.[142] In addition, their morphology, similar to that
of asbestos, which is known to exhibit deleterious effects with
the induction of mesothelioma,[143] has limited their potential
biological applications.
Non-targeted nano-GO can be of particular interest for
the treatment of primary tumours by taking advantage of the
enhanced permeability and retention effect.[68] However, one
of the biggest challenges in the cancer therapy context is the
combat of metastatic cells, because they can spread in the
bloodstream or be mixed with healthy cells developing sec-
ondary tumours. Nevertheless, as has been already mentioned,
metastatic cells have the particularity of expressing always a
specific endogenous surface protein.[72] Taking advantage of this
feature, the non-targeted nano-GO could be further improved
by tailoring its surface with specific target ligands, like peptides
or antibody conjugates. Those targeted nanomaterials can be
used to detect, visualize, and destroy cancer cells with minimal
side effects on normal cells and monitoring treatment effects
in real time (Figure 8).[144,145] Nano-GO has been also proposed
as a drug delivery vehicle for anti-cancer drugs[145149] or bio-
imaging agents with many molecular imaging techniques,
including magnetic resonance imaging (MRI), optical, photoa-
coustic and radionuclide-based imaging (Figure 8).[150153]
All the referred characteristics of nano-GO highlight its
potential and point out the importance of further exploring
this nanomaterial in the emerging area of nanomedicine. This
review focuses on the engineering of multifunctional nano-
GO platforms able to respond to particular demands in cancer
therapy.
4. Developments on Nano-GO Platforms
for Cancer Therapy
The nano-GO potentialities have been explored by several
research groups worldwide with the hope of finding realistic
procedures to fight one of the most dramatic diseases. In this
section the recent approaches developed on nano-GO based
systems as therapeutic agents on cancer are reviewed.
4.1. Nano-GO Functionalized with
Poliethyleneglycol
When a material is placed in contact with
human body fluids and tissues, the coagu-
lation of proteins occurs on its surface,
allowing its recognition by the immune
systems complementary proteins and mac-
rophages, leading to fast elimination by the
liver.[154,155] In fact, the triggering of immu-
nological processes leads to a reduction of
the circulation half-life of nanoparticles in
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the blood stream, which culminates in a reduction of biodis-

tribution and often the impossibility of achieving the specific
targets.
Nanoparticles can be engineered to either avoid immune
system recognition or to specifically inhibit the immune
responses. Surface-modification of nanoparticles with PEG,
PEGylation, is promising as a method to improve the stability
and in vivo performance, thus preventing the opsonization of
particles by the macrophages of the RES and increasing their
cytoplasmic transport.[156,157] The protective mechanism of
action of PEG is based on the formation of a dense, hydrophilic
shell of long flexible chains at nanoparticle surface (non-fouling
surfaces) that reduces the hydrophobic interactions with the
RES.[158] The steric repulsion resulting from a loss of entropy
of the bound PEG chains and the low interfacial energy of PEG
in water contributes to the extraordinary physiological proper-
ties.[159] PEG offers the advantage of being non-toxic and non-
immunogenic, leading to the approval by the United States Food
and Drug Administration (FDA) for internal use in humans.

4.1.1. In vitro toxicity and biodistribution of PEGylated nano-GO

Pegylation is already a recognized approach used to increase
the biocompatibility and reduce thrombogenicity of nano-
GO.[56] Although nano-GO colloids are soluble in water, they
need further functionalization with PEG that render the mate-
rial dispersible and highly stable in aqueous solution and facili-
tate the cell internalization. Recently, some in vitro studies of
nano-GO functionalized with PEG were performed in order
to access the cyto- and biocompatibility. For example, Sayan et
al. reported the study of cytotoxicity of nano-GO modified with
PEG-DSPE(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-
N-[amino(polyethylene glycol)]) with four different types of
cells: Henrietta Lacks cells (HeLa) derived from cervical cancer
tissue, National Institute of Health 3T3 mouse fibroblast cells
(NIH-3T3), Sloan Kettering breast cancer cells (SKBR3) and
Michigan cancer foundation-7 breast cancer cells (MCF7). The
results suggested that the studied nano-GO hybrids have dif-
ferential cytotoxic effects on the four cell lines, being HeLa the
one that exhibited greater toxicity compared to the other cell
lines. PEG-modified nano-GO was also studied for A549 cell
line (human lung adenocarcinoma epithelial cell line), and the
results suggests no cytotoxicity effects.[160]
Our group studied the nano-GO uptake kinetics in different
cell lines (human Saos-2 osteoblasts, murine L929 fibroblasts,
murine RAW-264.7 macrophages and murine MC3T3-E1 pre-
osteoblasts) and the results revealed differences in the agents
uptake amount and speed as a function of the type of cell
involved.[115] The osteoblast-like cells nano-GO uptake was
higher and faster without resulting in greater cell membrane
damage (Figure 9). Moreover, the dependence on the com-
monly used PEG nature (linear or branched) also influences
the viability and cell uptake speed.[115]
Further studies demonstrated that, after cell internaliza-
tion, nano-GO is preferentially localized on F-actin filaments
inducing cell-cycle alterations (Figure 10), apoptosis and oxi-
dative stress in these cell types.[161] The observed nano-GOs
effects should be considered in further studies focused on pho-
tothermal cancer therapy as a synergistic factor.
Figure 9. Incorporation of 1-GOs and 6-GOs labeled with FITC by cultured
human Saos-2 osteoblasts, murine L929 fibroblasts, murine RAW-264.7
macrophages and murine MC3T3-E1 preosteoblasts after a 1 day treat-
ment. Controls represent the basal fluorescence values in the absence of
GOs. (b) Effect of 1-GOs and 6-GOs on proliferation of cultured human
Saos-2 osteoblasts, murine L929 fibroblasts, murine RAW-264.7 mac-
rophages and murine MC3T3-E1 preosteoblasts after a 1 day treatment.
The cell number is given as a percentage relative to the controls (100%)
carried out in the absence of material. Reproduced with permission.[115]
Copyright 2012, IOP Publishing.
4.1.2. PEGylated nano-GO for photothermal therapy
In vivo studies (mice model) of pegylated nano-GO have also
been performed in order to evaluate its biodistribution and tox-
icity. Yang et al.[56] showed that nano-GO functionalized with
PEG mainly accumulates in the reticuloendothelial system
including the liver and spleen after intravenous administration
and can be gradually cleared up, most problably by both renal
and faecal excretion. Additional in vivo studies found some very
interesting behaviour of Nano-GO-PEG nanoparticles by intra-
venous administration, including highly efficient tumour pas-
sive targeting and relatively low retention in reticuloendothelial
systems (Figure 11).[162] They further studied the in vivo photo-
therapy in the NIR region and the results showed an efficient
tumour ablation after intravenous administration of modified
nano-GO at low-power NIR laser irradiation on the tumour.[162]
No significant toxicity at tested doses to the treated mice was
evidenced by blood biochemistry, hematological analysis and
histological examinations,[56,162] which suggests a great poten-
tial of developed materials in cancer treatment.

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this strategy can be used with other types of
insoluble drug molecules including CPT ana-
logues and Iressa (geftinib) to increase the
effects of chemotherapy. Dong et al. inves-
tigated the potentialities of nano-GO-PEG
system on the delivery of Zinc phthalocyanine
(photosensitizer for photodynamic therapy)
towards MCF-7 carcinoma cell line.[164] The
results showed a drug loading efficiency
up to 14 wt% and high phototoxicity under
Xe light irradiation. Wen et al. developed a
new nano-GO disulfide-linked PEG system
for DOX delivery that is able to respond to
redox environments by detachment of PEG
chains.[165] In vitro studies with HeLa cells
showed after internalization that DOX is 1.55
times effectively released by nano-GO dis-
sulfide linked PEG then nano-GO-PEG sys-
tems. They believe that the incorporation of
stable PEG shell on nano-GO/DOX system
can create a diffusion barrier that decreases
the drug release. Dembereldorj et al. inves-
tigated both in vitro and in vivo glutathione-
triggered DOX realease in real time from
nano-GO-PEG platform.[166] In vitro studies
on A549 cells demonstrated a drug delivery
efficiency of 23.5 wt% from nano-GO-PEG
platforms after treatment with 2 mM glu-
tathione within 15 min. They also observed
Figure 10. Morphology evaluation by confocal microscopy of cultured human Saos-2 osteo- an increase of 2.5 times in vitro drug release
blasts after 1 day treatment with GOs. Cells were stained with DAPI (A) for the visualiza- on cells by externally triggering glutathione
tion of the cell nuclei in blue, Rhodamine phalloidin (B) for the visualization of cytoplasmic ethyl ester rather than endogeneous glu-
F-actin filaments in red and FITC-GOs for the visualization of GOs in green (C). Figure 10D
tathione. In vivo studies demonstrated the
shows the whole composition with the three stainings revealing that FITC-GOs colocalizes with
rhodamine phalloidin resulting in yellow/orange coloration. Reproduced with permission.[161] possibility to monitor the real-time release
Copyright 2013, Elsevier. of DOX by fluorescence images after an
external trigger with glutathione.

Reduced nano-GO with a strong optical absorption in the

NIR region due to the higher restructuration of the aromatic
structure combined with non-covalent bond PEG shell, showed
a remarkably improvement in the in vivo photothermal cancer
treatment. The results obtained an efficacy of 100% on tumour
elimination after intravenous injection at lower power density
(0.15W/cm2).[163] This new approach showed a higher efficiency
in the in vivo tumour treatment when compared with non-
reduced pegylated nano-GO.
4.1.3. PEGylated nano-GO for drug delivery
Nano-GO modified with PEG has been also studied as a drug
carrier due to its excellent stability on physiological fluids, since
it is well known that aromatic drugs are water insoluble. Liu et
al. showed that Pegylated nano-GO can be non-covalent modi-
fied with hydrophobic drugs like SN38, a camptohecin (CPT)
analog.[146] Their results demonstrated a higher efficacy of this
new system NGO-PEG-SN38 for in vitro cell HCT-116 (human
4.1.4. PEGylated nano-GO for imaging research
A significant number of research groups have been exploring
the nano-GO-PEG nanomaterials for in vivo and in vitro
imaging by functionalization of terminal PEG amine groups
with different organic dyes (fluorescent labelling).[115,150] Peng
et al developed a new nano-GO-PEG platform functionalized
with fluorescein for intracellular imaging. The results showed
that new nanocomposite material has high rates of internali-
zation on HeLa cells and exhibits excellent pH-tunable fluo-
rescent properties.[150] Our studies showed that nano-GO-PEG
functionalized with fluorescein isocyanate (FITC) allowed the
detection of the biodistribution during in vitro cell internali-
zation (Saos-2 osteoblasts, L929 fibroblasts, RAW-264.7 mac-
rophages and MC3T3-E1 preosteoblasts).[115,161]
Another approach for the development of new nano-GO plat-
forms for bioimaging consist on the incorporation of different
atoms on its structural defect, for example,125I,[56]] 64Cu[167] and
66Ga[168] (radiolabeling). The nano-GO platforms modified with

colon cancer cell line) killing when compared with irinotecan these different atoms were investigated with serial noninvasive
(CPT-11), a FDA approved water soluble SN38 prodrug used positron emission tomography imaging and biodistribution,
for the treatment of colon cancer. The authors postulate that showing very promising results during the in vitro or in vivo

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carriers for the delivery of genes or oligo-

nucleotides, also designated as vectors. PEI
and its derivatives have been recognized as
some of the most effective and powerful cati-
onic gene delivery vectors due to their high
cationic-charge density at physiological pH
that improves the DNA and RNA binding
and condensation efficiency. These factors
lead to osmotic swelling and burst of the
endosmosis to release the loaded DNA or
RNA into the nucleus or cytoplasm, respec-
tively, when the amine groups of PEI are
protonated.[169,170]
The challenge of cancer gene therapy
resides on the development of efficient gene
vectors that can protect oligonucleotides
from enzymatic cleavage and enable cel-
lular uptake with high efficiency. Nano-GO
has been developed as a novel gene delivery
nano-vector with low cytotoxicity and high
transfection efficiency. That is promising for
future applications for cancer based gene
therapy. The surface modification of nano-
GO with PEI can be performed by one of
two different approaches: covalent linking
via amide bond formation[171] or by non-
covalent linking based on electrostatic and
hydrogen interactions.[172] Several new sys-
tems have been developed for gene therapy
based on nano-GO-PEI with DNA[171174] or
RNA[175177], showing high transfection effi-
ciency and low cytotoxicity in vitro. Feng
et al.[174] proved that the resultant nanoplat-
forms are able to further bind with plasmid
DNA for intracellular transfection of the
enhanced green fluorescence protein gene
in HeLa cells. Chen et al.[171] verified that
transfection efficiency on the nano-platform
is higher than PEI alone. Bioactivity analysis
Figure 11. Semiquantitative biodistribution analysis of NGS-PEG-Cy7. 4T1 tumour-bearing of the nanoplatforms showed that intracel-
mice were sacrificed at various time points post NGS-PEG-Cy7 injection with major organs col- lular tracking of Cy3-labelled pGL-3 could
lected for fluorescence imaging. (a) Spectrally resolved ex vivo fluorescence images of organs effectively deliver plasmid DNA into cells
before injection and 1, 6, and 24 h after injection of NGS-PEG-Cy7. SK: skin, M: muscle, I: intes-
tine, H: heart, LU: lung, LI: liver, K: kidney, SP: spleen, ST: stomach, and T: tumour. (b) Semi-
and is preferentially located on the nucleus.
[173]
quantitative biodistribution of NGS-PEG-Cy7 in mice determined by the averaged fluorescence Zhou et al. reported a new nano-GO-PEI
intensity of each organ (after subtraction. by the fluorescence intensity of each organ before system with high transfection efficiencies
injection). Error bars were based on three mice per group. Reproduced with permission.[162] combined with very low cytotoxicity. They
Copyright 2010, American Chemical Society. noted that plasmid DNA could be trans-
fected into mammalian cell lines with up to
studies. By comparing the results, radiolabeling has been dem- 95% efficiency (90% viability) and into zebrafish embryos with
onstrated to be a much more sensitive method and accurately 90% efficiency (high viability) compared to efficiencies of 30%
allows tracking the labelled substances in vivo in a quantitative or lower for established transfection technologies available
manner (Figure 12). (Figure 13).
With the aim of increasing the effectiveness of nano-GO
platforms, Fang et al.,[176] developed PEI-grafted GO nano-car-
4.2. Nano-GO Functionalized with Polyethylenimine - Gene rier for the simultaneous delivery of siRNA and DOX. On the
Transfection contrary to what they were expecting, the co-delivery of siRNA
and DOX do not show synergistic effects in the treatment of
The surface modification of nano-GO with polyethylen- cancer cells, most likely due to the slow down regulation of pro-
imine (PEI) has, as a primary objective, the development of tein expression by Bcl-2-targeted siRNA compared with rapid

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Figure 12. In vivo PET/CT imaging of 66 Ga-labeled GO conjugates in 4T1 tumour-bearing mice. (A) Serial coronal PET images of 4T1 tumour-bearing
mice at different time points post-injection of 66Ga-NOTA-GO-TRC105, 66Ga-NOTA-GO, or 66Ga-NOTA-GO-TRC105 at 2 h after a blocking dose of
TRC105 (denoted as blocking). (B) Representative PET/CT images of 66Ga-NOTA-GO-TRC105 in 4T1 tumour-bearing mice at 3 h post-injection.
Tumours are indicated by arrowheads. In vivo PET/CT imaging of 64Cu-labeled GO conjugates in 4T1 murine breast tumour-bearing mice. (a) Serial
coronal PET images of 4T1 tumour-bearing mice at different time points postinjection of 64Cu-NOTA-GO-TRC105, 64Cu-NOTA-GO, or 64Cu-NOTA-
GO-TRC105 after a preinjected blocking dose of TRC105. (b) RepresentativePET/CT images of 64Cu-NOTA-GO-TRC105 in 4T1 tumour-bearing mice at
16 h postinjection. (A) and (B) reproduced with permission.[167] Copyright 2012, American Chemical Society. (a) and (b) reproduced with permission.
Copyright[168] 2012, Elsevier.

4.3. Nano-GO Functionalized with Others

Biopolymers - Drug Delivery

Nano-GO can also be functionalized with

other types of highly hydrophilic and biocom-
patible polymers. A new approach is based on
the surface functionalization of nano-GO with
well-known poly(vinyl alcohol) (PVA) that
allows non-covalent bonding of camptothecin
for anticancer drug delivery. The results show
a higher cytotoxity activity of hybrid materials
when compared to free camptothecin.[178]
Nano-GO can also be functionalised with
gelatin (GE),[179] pluronic F127 (PF127)[148]
or chitosan (CS)[147] to further increase its
stability in water and various physiological
Figure 13. Fluorescence images of PEI-g-USGO in zebrafish using a pEGFP reporter gene fluids. In the case of nano-GO-gelatin hybrid
6 h after injection. (a) Overview image, (b)(e) confocal images at low resolution, (f)(h) the anticancer drug DOX was loaded by
confocal images at high resolution; fluorescence images of pEGFP- transfection into zebrafish physiorsorption, whereas gelatin can mediate
embryos, using PEI-g-USGO (a)(c), lipofectamine (d)(f), and PEI (60 kDa) (g)(i) as the
transfection agent, injected in the interlayer (6 h post-transfection), (a,d,g: overview image;
the drug release in vitro. The nanocomposite
b,c,e,f,h,i: confocal images. Reproduced with permission.[173] Copyright 2012, Springer. nano-GO-gelatin-DOX system showed a high
toxicity for MCF-7 cells, while the nano-GO-
gelatin was not toxic under the same experi-
DOX-DNA interactions. In fact, they demonstrated that the mental conditions for concentrations higher than 200 g mL1.
sequential delivery of siRNA and DOX by the PEI-GO nanoplat- The nano-GO-PF127 hybrid is found to also encapsulate DOX
form leads to a significantly increased efficiency in cancer HeLa by physiorsorption with ultrahigh drug-loading efficiency (289%
cells therapy. (w/w)) and exhibits a pH responsive drug release behaviour.

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The covalent functionalization of nano-GO with chitosan (GO-
CS) exhibits an interesting potential as a drug delivery platform
(CPT) with high cytoxicity to HepG2 and HeLa cells when com-
pared with to the drug alone. Further studies demonstrate that
cationic GO-CS nanoplatform can be also applied as a vector for
gene therapy due to the ability to condense plasmid DNA on its
surface; the in vitro studies with HeLa cells exhibit reasonable
transfection efficiency.
4.4. Multifunctional Nano-GO Platforms
The new challenge of nano-GO is to combine the different
therapy approaches available, such as chemotherapy, hyper-
thermia (photothermal therapy), oxygen photodynamic therapy,
etc. with imaging (optical, magnetic, radioactive, etc.) and tar-
geting agents (active or passive), in order to develop a multi-
functional graphene based nanoplatform with synergistic
effects for a more efficient cancer therapy. The possibility of
combining all these agents and properties is huge and just
depends on imagination, and the knowledge of bio-structures
and material properties, in order to determine more efficient
approaches for treating each specific type of tumour and cancer
cells. In the following sections, some approaches previously
developed for the engineering of multifunctional nano-GO plat-
forms for cancer therapy will be discussed.
4.4.1. Organic nano-GO plaforms
The intrinsic properties of nano-GO combined with specific
molecules or bio-structures can achieve synergistic effects
that allow the development of more efficient multifunctional
Figure 14. Thermal images of vials containing pellets of control nontreated U87MG cells, cells
treated by nano-rGO-RGD, and cells treated by nano-rGO-RAD, respectively, after 8 min of
irradiation with an 808 nm laser at a power of 15.3 W/cm2 (a). Cell pellet temperature versus rGO-RGD platforms; however after NIR irra-
time during irradiation and (c) cell viability 24 h post irradiation (b). Reproduced with permis-
sion.[127] Copyright 2011, American Chemical Society.
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nanoplatforms for cancer therapy. Sun et al.[144] covalently
functionalized the nano-GO surface with PEG to increase the
biological stability of the system and to take advantage of the
intrinsic photoluminescence of nano-GO to obtain imaging
live cell agent in the NIR with little background. Subsequently,
they targeted the nano-GO platform with antibody (anti-CD20)
to increase the cell selectivity, loading it with anticancer drug
DOX to promote chemotherapy effects in the system. The in
vivo results showed a higher selectivity and effectiveness on
the cancer cells death by these multifunctional nanoplatforms,
in comparison to the drug alone or the nanoplatform without
targeting units. This nano-GO platform allows the combina-
tion of photothermal therapy and chemotherapy in the same
system. Zhang et al.[128] introduced this concept to increase the
therapeutic efficacy on cancer treatment, by developing the new
system nano-GO modified with PEG and loaded with DOX.
The in vivo combined treatments demonstrate a synergistic
effect on the elimination of the tumours without weight loss or
recurrence of tumours.
A new nano-GO multimodal drug delivery carrier platform
with active molecular targeting was developed by Zhang et al.[145].
The assembly of nano-GO platform was based on the covalent
bonding of folic acid (FA) molecules to its surface, thus allowing
it to specifically target MCF-7 cells (human breast cancer cells
with FA receptors). Nanoplatform nano-GO-FA was further
functionalized with two anticancer drugs, DOX and CPT via
stacking and hydrophobic interactions. The new nanoplatform
showed a high level of targeting efficiency to MCF-7 cells, and
remarkably high cytotoxicity effects when compared to nano-GO
loaded with either DOX or CPT only. The multifunctional nano-
GO platform developed by Shen et al.[180] involves the surface
modification of GO with PEG and posterior modification with
the targeting molecule of FA. This system
is further complemented with gadolinium
(Gd3+) as a magnetic resonance imaging probe
and loaded with DOX as an anticancer drug.
The resultant hybrid material is denominated
GO-PEG-FA/Gd/DOX. In vitro MRI testing
of the new multifunctional nano-GO showed
a superior targeting imaging efficiency over
Gd3+ free materials and demonstrated drug
release (DOX) effectiveness under the tumour
physiological conditions (pH 5.5), resulting
in a clear cytotoxic effect for HepG2 cells.
Robinson et al.[127] performed the surface
modification of reduced nano-GO with amphi-
philic PEG by covalent bonding in order to
increase the physiological stability. Afterwards,
they targeted the system with peptide Arg-Gly-
Asp (RGD) or control peptide (RAD) motif in
order increase the selectivity to the U87MG
cells. Reduced nano-GO was chosen as sub-
strate instead of non-reduced because it exhib-
ited 6-fold higher NIR absorption, resulting in
more efficiency on cell photothermal ablation.
In vitro tests showed low cytotoxicity of nano-
diation, they showed a high efficacy for pho-
toablation of cells (Figure 14).
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Photodynamic therapy (PDT) has emerged as an alternative combination with nano-GO, thereby developing materials for
and promising non-invasive treatment for different types of cancer therapy with high performance. Yang et al. described
cancer, which involves the uptake of photosensitizers (PSs) by one of the first attempts to build up a multifunctional nano-GO
cancer cells followed by irradiation. One of the photosensitizer platform with Fe3O4, further loaded with DOX.[186] These hybrid
molecules most used is Chlorin e6 (Ce6), due to its capacity materials showed interesting magnetic properties and pH-trig-
to generate cytotoxic singlet oxygen when exposed under light gered controlled magnetic behaviour that makes them a prom-
excitation. However the lower solubility on biological fluids ising candidate for controlled targeted drug delivery. Recently
remains a problem. The combination of the photosensitizer they further developed a nano-GO platform by including a tar-
Ce6 with GO nanocarriers previously modified with targeting geting molecule of FA.[187] In vitro studies showed an increase
molecule FA, can be achieved by simple non-covalent interac- of the specificity of nanoplatforms to SK3 cells and high toxicity
tions (hydrophobic and stacking).[181] The resultant system to Hela cells. Gollavelli et al.[188] developed a nano-GO platform
can efficiently deliver Ce6 to the tumour due to high affinity with in situ growth of iron oxide nanoparticles by microwave
of the targeting molecules to MGC803 cells and can increase functionalized with polyacrylic acid (PAA) bridge for linking
the photodynamic efficiency upon irradiation. Tian et al.[182] the fluorescein o-methacrylate (FMA), to increase the hydro-
observed that the combination of GO with Ce6 can result in a philic behaviour and add fluorescent property. In vitro studies
controlled drug delivery system. They used the photothermal with HeLa cells showed that nanoplatforms locate only in the
effect of GO to deliver Ce6 molecules in specific locations, in an cytoplasm and evidence low cytotoxic effects.
efficient way, enhancing the PDT against cancer cells. Another important application of nano-GO platforms with
iron oxide nanoparticles is for cellular magnetic resonance
4.4.2. Hybrid nano-GO platforms imaging (MRI). Chen et al.[189] performed the surface modifi-
cation of GO with iron oxide nanoparticles by using a spacer
Inorganic nanoparticles have received increased attention in agent 1-ethyl-3(3-dimethylaminopropyl) carbodiimide. The
the last few years due to their high potential for diagnostic and nanocomposite materials showed a good physiological stability,
therapeutic systems in the oncologic area. They have demon- low toxicity and good internalization on HeLa cells. In addition,
strated a clear potential in wideranging fields of cancer therapy imaging studies demonstrated significant enhance of cellular
such as tumour targeting, imaging, hyperthermia therapy and MRI when compared with iron oxide nanoparticles alone.
drug delivery.[87,183] Indeed, iron oxide[184] and gold[185] nanopar- A truely multifunctional nano-GO platform with iron oxide
ticles are the ones most highlighted in the various approaches nanoparticles was recently developed and was explored in all
for the treatment of cancer due to their intrinsic properties. its potential. Yang et al.[190] assembled a new nano-GO hybrid
Several organic/inorganic hybrid materials based on these material based on the surface modification of GO with iron
kinds of nanoparticles are already in preclinical stages or even oxide nanoparticles and posterior noncovalent functionaliza-
in marketing.[83] tion with PEG that resulted in a nanoplatform with excellent
In view of the success achieved by these nanoparticles, some physiological stability, strong NIR optical absorbance and
authors have tried to obtain synergistic effects through their superparamagnetic properties that has been used as a thera-
nostic agent for in vivo triple modal imaging
(flurescence, photoacoustic and magnetic
resonance) and effective photothermal abla-
tion.[190] The results showed high efficiency
tumour imaging in vivo of the nano-GO plat-
form using external radioactive or fluores-
cent labels and intrinsic optical and magnetic
properties and triple-modal fluorescence/
MR/PAT due to the high passive tumour
accumulation (Figure 15). The photothermal
in vivo treatment showed an efficient tumour
ablation at low laser power density. Both in
vivo and in vitro toxicity studies did not show
evidence of cross effects under the level of
experimental conditions.
Nano-GO modified with gold nanoparticles
can also be an interesting multifunctional
Figure 15. Blood circulation, biodistribution and multimodal imaging. a) The blood circula- nanoplatform for the cancer imaging and
tion of 125 I-RGOIONPPEG. The pharmacokinetics of RGOIONPPEG followed the two- therapy, however at the moment the potenti-
compartment model. b) Biodistribution of 125 I-RGOIONPPEG in 4T1 tumour-bearing ality of these materials is not fully explored.
mice. High tumour uptake of RGOIONPPEG was observed. ce) Multimodal imaging of Wang et al.[191] performed the surface modi-
4T1 tumour-bearing mice after intravenous injection of RGOIONPPEG: c) Fluorescence
imaging using Cy5 labeled RGOIONPPEG; d,e) T2-weigted MR imaging (d) and photoa-
fication of GO by electrostatic interactions
coustic imaging (e) using RGOIONPPEG. All the images showed that RGOIONPPEG with dodecanethiol-CTAB-capped gold nano-
could passively accumulate in the tumour after intravenous injection. Error bars in (a,b) were particles with RGO subsequently loaded with
based on four mice per group. Reproduced with permission.[190] DOX. In vitro results showed that these new

Adv. Healthcare Mater. 2013, 2, 10721090 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com 1085
 www.advhealthmat.de
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REVIEW

MCF-7 cells irradiated at NIR wavelength at

the power of 2 Wcm2 for 4 min (Figure 16).
They verified the efficiency of the nanoplat-
forms in cell killing due to the synergistic
effects of NIR absorption by GO/QDs. It was
also observed that cell viability showed a cor-
relation with fluorescence intensity, which
would allow the possibility of precise motori-
zation of photothermal therapy.

5. Trends and Challenges of

Nano-GO for Cancer Therapy
This review gives an overview of the prop-
erties of nano-GO that make it an excellent
candidate as a therapeutic agent for cancer
treatment. In addition, a critical discus-
sion has been provided on the current uses
of nano-GO as a platform for multimodal
cancer therapy by the self-assembly of the
most diverse chemical or biological struc-
tures. Recent works highlight the huge
potential applications of graphene and gra-
phene derivatives in biomedicine.[33,34]
Nano-GO and its derivatives include a
large variety of nanostructures with variable
characteristics including size, shape, and
surface properties. Consequently, due to the
resultant immunotoxicological heterogeneity,
it is very complicated to provide a catch-all
immune safety evaluation. Variations may
occur such as interactions with immune
Figure 16. Schematic illustration of QD-rGO under irradiation (left) causing cell death and cells, different inflammatory responses, dif-
diminished QD fluorescence (right). b) Before irradiation, the cells incubated with the FA-QD- ferent antigen-specific hypersensitivity reac-
US-rGO were viable (green) and the internalized QDs were fluorescent (red). The live cells were tions, etc. This is the reason why opposite
stained with propidium to appear in green. c) After irradiation (808 nm for 4 min, in region left and conflictingndings have commonly been
of the marker curve), essentially all of the cells were killed and the fluorescence was absent.
reported for similar nanosystems.
d) The viability of cells incubated with FA-QD-US-rGO (38 nm) and FA-QD-S-rGO (260 nm)
versus the duration of the irradiation. e) Cell death and normalized fluorescence intensity for Nano-GO presents a panoply of features
cells incubated with FA-QD-US-rGO after irradiation for various duration. Reproduced with which make it to stand out as one of the
permission.[153] most desired platforms for cancer therapy.
Intrinsic nano-GO properties such as high
aspect ratio, surface chemistry richness, and
nanoplatforms can cause inhibition of HepG2 cells and allow unusual physical properties can make it a powerful weapon as
cellular imaging due to the surface plasmon band of gold nano- a therapeutic agent. The photoluminescence and NIR absorp-
particles (absorption band at 520 nm). The authors described tion evidenced by the nano-GO structure allow its use as an
the potentialities of this new system for multimodal probe and imaging and photothermal therapy agent. The high aspect ratio
as a drug carrier for targeting. combined with the chemical surface versatility of nano-GO
Semiconductor quantum dots (QDs) are high potential bio- facilitates the assembly of various structures from biopolymers
materials, due to brighter fluorescence, lower photoleaching, to inorganic nanoparticles, biostructures, small molecules and
high resistance to chemical degradation and potential to per- atoms with relevance for different phases of cancer therapy.
form photothermal therapy; however they exhibit a high toxicity. In fact, nano-GO can be viewed as a LEGO piece that allows
In order to reduce the levels of toxicity Hu et al.[192] developed a thousands of possible combinations, which are limited solely
new multifunctional based nano-GO platform with QDs by use by imagination, knowledge of biological structure (tumours
of a spacer agent (an amphiphilic polypeptide, poly(L-lysine)) and cancer cells) and materials properties. Indeed nano-GO
and targeted with FA. In vitro studies with HeLa and MCF-7 can offer the possibility of development of multifunctional
cells demonstrated internalization by cells and revealed insig- nanoplatforms with the opportunity to combine all the different
nificant cytotoxicity, with approximately 92% of cell viability. stages of cancer therapy: recognition, imaging and treatment.
The authors also evaluated the photothermal therapy effects on An unexplored field of the nano-GO platforms is the assembly

1086 wileyonlinelibrary.com 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Healthcare Mater. 2013, 2, 10721090

www.MaterialsViews.com
of smart materials for the development of intelligent nano-
systems that can improve the targeting efficiency and therapy
efficacy through biological or external stimuli-responses.
The works described in this review effectively demonstrate
that multifunctional nano-GO platforms can combine high
targeting efficiency, good imaging contrast and extraordinary
therapy efficacy for cancer cells and tumours. In fact, it has
been shown that the use of nano-GO as a drug delivery carrier
may be useful to deliver very efficient drugs, which have pre-
viously failed due to solubility issues, in a targeted molecular
form. This fact would open the door to completely new treat-
ment options.
The future research work in nano-GO field requires a deep
understanding of the interaction between cells and nano-GO
as well as an integration of the immunological, metabolic and
pharmacological processes to provide insight into how cells
respond to nano-GO. Thus, it has become necessary to iden-
tify a map of potential toxicities for each material, including
relevant physiology, biodistribution, movement of materials
through tissues, phagocytosis, opsomization and endocytosis
and establish a risk/benefit considerations for every system.
To sum up, there is currently no definitive conclusion that
can be made regarding the potentialities or risks of nano-GO
systems. In fact, while there are still many opportunities pre-
sented by nano-GO in the development of multifunctional
platforms for cancer therapy, it is also very highly important
to assimilate knowledge about nano-GO interaction with bio-
logical tissues: normal or carcinogenic. Overall, an improved
specificity to tumours and cancer cells leading to an increase
of success rate of treatments, and a decrease in the number of
operations or the risk of postoperative intervention, is expected
from the design of nano-GO therapeutic agents. This would
allow shorter recovery times and a diminution of side effects
for patients. In general, the ultimate goal of nano-GO research
as a therapeutic agent in cancer therapy will be the improve-
ment of life quality, life expectancies and a reduction in the
global cost of treatments of oncologic patients.
Received: January 18, 2013
Published online: March 22, 2013
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