reviews
Graphene
Behavior and Toxicity of Graphene and Its Functionalized
Derivatives in Biological Systems
Kai Yang, Yingjie Li, Xiaofang Tan, Rui Peng,* and Zhuang Liu*
From the Contents
1. Introduction ......................................... 1493
2. Bacterial Toxicity of Graphene and
Graphene Oxide ................................... 1493
3. In Vitro Interactions with
Mammalian Cells ................................. 1494
4. Interactions Between Graphene-Based Sub-
strates and Cells .................................. 1496
5. In Vivo Behavior and
Toxicology in Animals ........................... 1497
6. Summary ...............................................1501
1492 wileyonlinelibrary.com © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Graphene, as a class of 2D carbon nanomaterial, has
attracted tremendous interest in different areas in recent
years including biomedicine. The toxicity and behavior
of graphene in biological systems are thus important
fundamental issues that require significant attention.
In this article, the toxicity of graphene is reviewed by
describing the behavior of graphene and its derivatives
in microorganisms, cells, and animals. Despite certain
inconsistencies in several detailed experimental results
and hypotheses of toxicity mechanisms, results from
numerous reports all agree that the physicochemical
properties such as surface functional groups, charges,
coatings, sizes, and structural defects of graphene may
affect its in vitro/in vivo behavior as well as its toxicity
in biological systems. It is hoped that this review article
will provide an overview understanding of the impacts,
behavior, and toxicology of graphene and its derivatives
in various biological systems.
small 2013, 9, No. 9–10, 1492–1503
Behavior and Toxicity of Graphene and Its Functionalized Derivatives
1. Introduction
Since 2004, graphene has become a ‘superstar’ in materials
science and shown great promise in a wide range of fields
including energy storage, nanoelectronic devices, batteries,
transparent conductors, and many others.[1–6] Owing to its
unique 2D shape, together with fascinating physical and
chemical properties, graphene has also aroused substantial
attention in biomedicine. Functionalized graphene has been
used as a novel carrier for drug and gene delivery,[7–19] cell
and tumor imaging,[8,20–23] as well as cancer photothermal
therapy.[8,20,21,24–26] Various groups have also reported that
graphene could be used as a biosensor platform to detect
various biomolecules via different mechanisms.[27–33] More
recently, inorganic nanoparticles have been grown on the
surface of graphene to obtain graphene-based nanocompos-
ites for multi-modal bio-imaging and imaging guided cancer
therapy.[21,23,34–37]
Despite these exciting results using graphene in biomedi-
cine, there may still be a long way to go before really applying
this class of materials in a clinic. The potential toxicity of
graphene and its derivatives in biological systems at dif-
ferent levels from bacteria and mammalian cells to animals,
has generated growing debate in recent years. In addition, the
increasing usage of graphene-based materials in both aca-
demic labs and industry also demands better understanding
of their potential negative impacts on human health.
Currently, several different groups have studied the influ-
ence of graphene-based materials on bacterial growth, unfor-
tunately observing inconsistent results likely due to different
experimental conditions and material preparations.[38–40]
Numerous papers have investigated the interactions between
graphene-based materials with various classes of mamma-
lian cells to understand the mechanisms and regulating fac-
tors of their in vitro toxicity.[9,41–47] As a single-atom thin film,
graphene has also been developed as a substrate to control
the growth, differentiation, and functions of specific cell
types such as stem cells.[48–55] Moreover, a number of groups
including our group have studied the in vivo biodistribution
and toxicology of graphene and its functionalized deriva-
tives in animals via different administration routes. Although
pristine graphene and graphene oxide (GO) tend to be toxic
to mice in a dose-dependent manner,[56–58] functionalized
nano-GO (e.g., by biocompatible polymer coating) exhibits a
much-reduced in vitro and in vivo toxicity.[59,60] This article
is a comprehensive review to summarize the recent studies
related to the nanotoxicology of graphene-based materials,
as well as their interactions with, and behavior in, different
levels of biological systems.
2. Bacterial Toxicity of Graphene
and Graphene Oxide
Depending on the compositions of cell walls, bacteria can
be either Gram-negative or Gram-positive, exhibiting dif-
ferent antibiotic susceptibilities. Antibacterial materials are
widely used in daily life to protect public health. With the
large-scale use of traditional antibiotics, there are increasing
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variations of microbial populations caused by drug resistance.
The development of nanotechnology provides new potential
opportunities to solve this problem. Antibacterial properties
of nanomaterials including silver nanoparticles,[61] titanium
oxide nanoparticles,[62] and carbon nanotubes (CNTs)[63,64]
have been explored in the past years. More recently, the
potential use of graphene in the antibacterial field has also
attracted considerable interest.[38–40,65]
Graphene has been found to be a promising candidate
as an antibacterial material owning to its bacterial toxicity.
In 2010, Huang and co-workers[39] for the first time explored
the antibacterial properties of GO by studying the interac-
tion of Gram-negative bacteria, Escherichia coli (E. coli)
DH5a, with GO. They found that GO at a concentration of
85 μg/mL could remarkably suppress the growth of E. coli
after 2 h incubation at 37 °C with an inhibition rate of over
90% (Figure 1a,b), while presenting low cytotoxicity to
mammalian cells. Transmission electron microscope (TEM)
images showed that antibacterial properties were attributed
to cell membrane damage induced by GO (Figure 1c,d),
leading to leakage of the cytoplasm. They further found that
macroscopic GO papers prepared by vacuum filtration of
the GO suspension could effectively restrain the growth of
E. coli.[39]
Similar to the above-mentioned work, Akhavan et al.[40]
selected Gram-negative E. coli and Gram-positive Staphylo-
coccus aureus (S. aureus) as model bacteria to investigate the
bacterial toxicity of GO and reduced grapheme oxide (RGO)
nanowalls prepared by depositing GO or RGO on stain-
less steel substrates. It was found that GO nanowalls could
cause cell membrane damage of bacteria by direct contact
with the very sharp edges of the nanowalls. Additionally, the
cell membrane of Gram-positive S. aureus without an outer
membrane was more severely damaged as compared with the
Gram-negative E. coli. Owing to their more sharpened edges
and a better charge-transfer ability between RGO nanowalls
and bacteria, RGO nanowalls showed stronger antibacterial
activity than GO nanowalls. Later, Liu et al.[38] studied the
antibacterial mechanism of graphene by exploring interac-
tions between four types of graphene-based materials with
E. coli. They found that GO showed the strongest antibacte-
rial activity among all materials under similar concentrations
and incubation conditions, followed by RGO, graphite, and
graphite oxide. Their antibacterial mechanisms were attrib-
uted to the synergy of the membrane stress and oxidative
stress induced by interactions between bacteria and materials,
similar to the three-step cytotoxicity mechanism proposed by
Vecitis et al.[66] for carbon nanotubes.
K. Yang, Y. J. Li, X. F. Tan, Prof. R. Peng, Prof. Z. Liu
Jiangsu Key Laboratory for Carbon-Based
Functional Materials & Devices
Institute of Functional Nano & Soft
Materials Laboratory (FUNSOM)
Soochow University
Suzhou, Jiangsu 215123, China
E-mail: rpeng@suda.edu.cn; zliu@suda.edu.cn
DOI: 10.1002/smll.201201417
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 reviews
The results mentioned above seem to support the idea
of graphene as a promising antibacterial material with low
mammalian cell cytotoxicity. However, recent studies have
indicated that GO may lack any antibacterial effects.[67] It was
reported that GO alone showed no noticeable antimicrobial
effect against E. coli or Pseudomonas aeruginosa (P. aerugi-
nosa) bacteria, though Ag nanoparticle-modified GO could
effectively inhibit bacterial growth. Interestingly, a recent
work by Ruiz et al.[68] reported that GO presented neither
intrinsic antibacterial functions nor cytotoxicity properties to
mammalian cells and, instead, could act as an enhancer to cel-
lular growth. They found that GO could produce a dramatic
increase in microbial growth by enhancing attachment, pro-
liferation, and biofilm formation instead of inhibiting their
growth. Ag-decorated GO films, but not bare GO, showed
strong antibacterial activity against E. coli.[69–71]
For these controversial findings, the different synthetic
methods, sizes, structures, and surface treatments of GO may
affect its biological behavior. Obviously, a lot more careful
studies should be carried out to understand the detailed
mechanisms and controlling factors regarding the interac-
tions between graphene-based materials with microbes.
3. In Vitro Interactions with Mammalian Cells
Given the diverse inspiring biomedical applications of
graphene and its derivatives, systematic evaluations of their
potential toxicity to mammalian cells become critically
important. We here summarize cytotoxicity investigations of
Figure 1. Antibacterial activity of GO. (a) Metabolic activity of E. coli incubation with 20 and
85 mg/mL of GO at 37 °C for 2 h. (b) Antibacterial activity of 85 mg/mL GO against E. coli DH5 documented that the generation and elim-
cells. (c,d) TEM images of untreated E. coli (c) and E. coli exposed to GO nanosheets (d) at ination of ROS is dynamically balanced
37 °C for 2 h. Reproduced with permission.[39] Copyright 2010, American Chemical Society.
1494 www.small-journal.com © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
K. Yang et al.
Zhuang Liu received his BS degree from
Peking University (China) in 2004 and a PhD
from Stanford University (USA) in 2008. In
2009, he joined the Institute Functional Nano
& Soft Materials (FUNSOM) at Soochow
University in China as a principal investiga-
tor. Dr. Liu’s research in the past few years
has mostly focused on the development of
functional nanomaterials including carbon
nanomaterials, upconverting nanoparticles,
and other composite nanostructures, for
application in biomedical imaging, drug de-
livery, phototherapy of cancer, and stem cell
research. The awards Dr. Liu has received
include an MRS silver award (USA) in 2008 and a SCOPUS young researcher award
(China) in 2012.
pristine and functionalized graphene on different cell types
including immortalized cell lines, macrophages, and blood
components, hoping to provide a guideline for later in-depth
studies.
3.1. The Cytotoxicity of Pristine Graphene and GO in Cell
Cultures
The toxicity of graphene or GO sheets to different cell lines
has been evaluated in monolayer cultures of lung epithe-
lial cells, fibroblasts, and neuronal cells. A comprehensive
examination by Chang et al.[72] uncovered neither obvious
cytotoxicity nor significant cellular uptake of GO in A549
adenocarcinomic human epithelial cells
at low GO concentrations. However, high
concentrations of GO could induce oxida-
tive stress that slightly reduced the viabili-
ties of cells. A number of other studies
suggested that, compared to GO, RGO or
pristine graphene had less dispensability
and appeared to be more toxic to cells,[43,46]
also via an oxidative stress mechanism. In
a recent study, Zhang et al.[73] compared
the toxicity of different types of carbon
nanomaterials including nanodiamonds,
carbon nanotubes, and GO to HeLa cells.
Their results indicated that although GO
showed the lowest cell uptake ratio com-
pared to the other two types of carbon
nanomaterials, all three materials exhib-
ited a dose-dependent toxicity. The gener-
ation of reactive oxygen species (ROS) to
induce oxidative stress was again found to
be a mechanism leading to the toxicity of
carbon nanotubes and GO. Thus, genera-
tion of ROS, which is critical characteristic
of oxidative stress inside cells, appears to
be an important cause of in vitro toxicity
for graphene-based materials. It is well
inside cells, and disturbing the balance
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Behavior and Toxicity of Graphene and Its Functionalized Derivatives

may induce intracellular protein inactivation, lipid peroxida- pristine graphene, as verified by the stimulated transcription
tion, dysfunction of mitochondria, and eventually apoptosis activity of the Smads proteins, a small family of eukaryotic
or necrosis.[74,75] transcription regulators. As the result, Bim and Bax, two
Macrophages are one of the critical components in medi- pro-apoptotic members of the Bcl-2 family, were further acti-
ating phagocytosis as well as in alerting the rest of the immune vated, inducing the permeabilization of the mitochondrial
system against invaders to elicit an immune response.[76] outer membrane and relocalization of the mitochondrial pro-
Macrophage uptake is also one major elimination route to apoptotic factors into the cytosol, where final initiation of the
clean up nanomaterials after they are systemically adminis- caspase cascade happened. Although different from the sig-
tered in vivo.[77,78] Sasidharan et al.[41] had found that hydro- naling pathways of CNTs reported in previous studies,[80–82]
phobic pristine graphene largely retained on the cell surfaces both materials could induce ROS generation and trigger the
of RAW 264.7, a macrophage cell line, and induced oxidative mitochondrial apoptotic pathway, resulting in mitochondrial
stress together with an abnormal stretched morphology of damage. This study offers an improved understanding of the
F-actin filopodial extensions at concentrations above 75 μg/ molecular mechanism of cytotoxicity induced by graphene.
mL. ROS-mediated toxic effects of pristine graphene may be To understand interactions between nanomaterials and
attributed to strong hydrophobic interactions with the cell blood cells is important for better design of intravenously
membrane, leading to the hindrance of essential nutrients and administered nanomaterials used in biomedicine. Sasidharan
protein uptake into cells. The cytoskeleton is critical in phago- et al.[41] demonstrated that both pristine and functionalized
cytic defense mechanisms of macrophages and the highly graphene obtained by nitric acid oxidation exhibit excellent
stressed conditions may lead to the inhibition of actin proteins hemocompatibility with red blood cells and platelets, and
and subsequent cytoskeletal dysfunction. In
contrast, functionalized graphene obtained
by nitric acid oxidation showed excellent
intracellular uptake without visible adverse
effects on the integrity of the cytoskeletal
architecture and filopodial extensions and,
moreover, much reduced oxidative stress-
induced cytotoxicity. Interestingly, mem-
brane integrity analysis indicates that no
physical damage to the plasma membrane
can be induced by either pristine graphene
or functionalized graphene, which is dif-
ferent from the case of CNTs.
Recently, another report by Li et al.[42]
carefully studied the toxicity mecha-
nism of pristine graphene in RAW 264.7
marcophage cells at the molecular level
(Figure 2). The signaling pathways of
mitogen-activated protein kinase (MAPK)
as well as transforming growth factor beta
(TGF-β) were found to be activated in
pristine graphene-treated cells, further
activating the downstream Bcl-2 protein
family to initiate execution of mitochon-
drial-related apoptosis (Figure 2). MAPK
cascades, known to be critical in cell
proliferation, differentiation, and apop-
tosis, were regarded to be stress-sensitive
and involved in apoptotic cell death in
oxidative conditions.[79] All three phos-
phorylated kinases including extracel-
lular signal-regulated kinase (ERK), p38
mitogen-activated protein kinase (p38),
and c-Jun N-terminal kinase (JNK), were
found to be significantly up-regulated, indi-
cating the activation of the three MAPK
signal pathways upon pristine graphene
treatment. Another key component of cell
apoptosis, especially in immune-related Figure 2. The signal pathway of graphene induces cell apoptosis. Reproduced with
cells—TGF-β—was also activated by permission.[42] Copyright 2012, Elsevier.

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 reviews
caused negligible alteration of cytokine expression. No pre-
mature immune cell activation or suppression was induced
by both graphene systems at the maximum dose of
75 μg/mL after 72 h incubation. Paul et al.[83] demonstrated
similar results that graphene was compatible with blood and
induced no platelet or complement activation. In contrast,
Singh et al.[84] revealed that GO produced by Hammer’s
method evoked strong aggregation of platelets through the
activation of non-receptor protein tyrosine kinases of the Src
family in platelets, and release of calcium from intracellular
stores. Consistently, GO was found to trigger extensive pulmo-
nary thromboembolism in mice after intravenous administra-
tion. Significantly, RGO showed largely attenuated activation
of platelets, which may be correlated to difference in surface
charge distribution from GO. In a follow-up study by the
same group,[44] amine-modified graphene (G-NH2) showed
no thrombotoxic property, as it induced neither stimulatory
effects on human platelets, nor pulmonary thromboembolism
in mice following intravenous administration. G-NH2 was
also found to induce no obvious hemolysis, maintaining the
integrity of erythrocytes, another key component in blood.
In another report by Liao et al., GO with small sizes showed
strong hemolytic activity, which could be nearly eliminated if
GO was coated with chitosan.[43] Therefore, there is no doubt
that the interactions between graphene-based materials with
blood components are highly dependent on the surface chem-
istry of graphene. For the development of graphene-based
nanomedicine, it is important to adopt appropriate surface
coatings on nanomaterials to minimize their negative impacts
to blood cells as well as all to other important cell types.
3.2. The Cytotoxicity of Functionalized GO
As shown in many reports and discussed above, pristine
graphene or uncoated GO exhibit dose-dependent toxicity
to various types of cells. In contrast, surface modification of
GO with hydrophobic macromolecules such as chitosan,[43,85]
tween,[86] artificial peroxidase,[87] PEG,[88] dextran,[89] and
even proteins,[90] has been reported to remarkably decrease
its cytotoxic effects. Fetal bovine serum in cell culture
medium has been reported to largely attenuate the toxicity of
GO in A549 cells, as presented by Hu et al.[90] Via both elec-
trostatic and hydrophobic interactions, GO could adsorb pro-
teins, which then impede the direct interaction of GO with
cells to reduce cytotoxicity of GO. We and others have also
found in a large number of studies that coating of GO with
biocompatible polymers such as PEG or dextran[88,89] could
offer GO excellent solubility and stability in physiological
solutions, attenuating its direct interactions with cell mem-
branes, reducing nonspecific binding with other functional
biomolecules, and resulting in much lower in vitro cytotox-
icity to cells.
In brief, pristine graphene and uncoated GO show an
obvious negative impact on various types of cells via a
number of mechanisms such as membrane damage, ROS
generation, and alterations of expression levels of several key
genes related to apoptosis. To our delight, functionalized GO
derivatives with appropriate surface coatings exhibit much
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K. Yang et al.
lower cytotoxicities, as separately evidenced in many reports.
Although researchers have gathered a large amount of infor-
mation regarding the in vitro toxicity of graphene and its
derivatives, many aspects, including how exactly the surface
chemistry and sizes control their interactions with cells, their
long-term fate inside cells, as well as their detailed toxicity
mechanism at the molecular level, remain to be explored in
future studies.
4. Interactions Between Graphene-Based
Substrates and Cells
Different from the above-mentioned studies in which
graphene-based materials are added into cell cultures in solu-
tion, motivated by the unique mechanical, electronic, and
optical properties of graphene, a number of groups have also
developed graphene-based substrates for potential applica-
tions in tissue engineering. Therefore, to understand how cells
interact with a graphene-based substrate becomes important
in this area.
Mesenchymal stem cells (MSCs), known as multipotent
progenitor cells derived from adult bone marrow, are able to
differentiate into various cell lineages such as adipocytes, oste-
oblasts, and chondrocytes, and have shown promising appli-
cations in tissue repair and cell therapies.[91] By manipulating
the microenvironment via material mechanics,[92] substrate
topography,[93] and soluble growth factors,[94] the differen-
tiation of MSCs can be induced in a controlled manner. Lee
et al.[54] elucidated that graphene and GO sheets could act as
efficient preconcentration platforms for osteogenic inducers,
namely, dexamethasone and β-glycerolphosphate, to accel-
erate the adhesion, proliferation, and differentiation of MSCs.
As shown in Figure 3, a significantly higher density and more
normal spindle-shaped morphology of cells on graphene and
GO substrates were observed than on a polydimethylsiloxane
(PDMS) substrate. Compared to the chemically induced oste-
ogenic differentiation on polystyrene tissue culture dishes
that require 21 days to complete, the MSCs on graphene
exhibited extensive mineralization by only day 12 owing to
the π–π stacking interactions between aromatic rings in the
inducer molecules and the graphene basal plane.
In a different study, Nayak et al.[49] also confirmed a con-
trolled and accelerated osteogenic differentiation of MSCs
induced by the graphene substrate, with a rate comparable to
that induced by a common growth factor, BMP-2. The pres-
ence of graphene drastically enhances the calcium deposits
on polyethylene terephthalate (PET) and PDMS substrates
which are reported to be less favorable toward osteoblasts.[49]
The lateral stress of graphene is of significance in providing
the right amount of local cytoskeletal tension, which leads
to the formation of strong anchor points of cytoskeleton.
Another factor to be considered is often the substrate stiff-
ness[95] and strain which strongly influences cell differentia-
tion. In addition, in two other separate studies, it was found
that the oxygen content in GO films could be an important
factor that regulates cell growth and differentiation.[52,96]
Compared to RGO with its reduced oxygen content, GO
appeared to be more favorable for cell adhesion, growth, and
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Behavior and Toxicity of Graphene and Its Functionalized Derivatives
Figure 3. Mesenchymal stem cells incubated with PDMS, graphene (G), and GO substrates.
The cell morphology was visualized by staining F-actin fibers with rhodamine-phalloidin.
Scale bars are 100 mm. Reproduced with permission.[54] Copyright 2011, American Chemical
Society.
differentiation. To summarize, the ability of graphene and
GO to accelerate stem cell differentiation without affecting
the physiological conditions of the microenvironment is
expected to be desirable in applications of tissue engineering
and regenerative medicine.
The differentiation of human neural stem cells (hNSCs)
specifically toward neurons is critical in brain repair and
neural regeneration. Park et al.[48] found that a graphene sub-
strate significantly enhanced cell adhesion and neurite out-
growth, and further induced differentiation of hNSCs more
toward neurons than glial cells. As indicated in Figure 4, the
graphene substrate was mostly occupied by differentiated
hNSCs with neurite outgrowths, while hNSCs on glass were
gradually detached during three weeks. After 1 month of dif-
ferentiation, more neurons than glial cells were found on the
graphene region as confirmed by immunostaining of differ-
entiated cells. In another study by Li et al.,[50] it was revealed
that, compared with tissue culture polystyrene (TCPS) sub-
strates, graphene films with excellent biocompatibility signifi-
cantly promoted neurite sprouting and outgrowth of mouse
hippocampal neurons, especially during the early develop-
mental phase. Enhanced growth associate protein-43 (GAP-
43) expression, which is closely related to the boost of neurite
sprouting and outgrowth, was observed when graphene films
were used as the substrate. An advantage of using graphene
film as the neurite cell growth substrate compared with other
traditional ones is its high electron conductivity, which allows
graphene to serve as a stimulating electrode to strengthen the
small 2013, 9, No. 9–10, 1492–1503 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
electrical coupling with differentiated neu-
rons without much chemical reaction.[51]
Although pristine graphene or
uncoated GO, when added into cell cultures
in the solution phase, have been found to
be toxic to cells, the interactions between
cells and graphene-based cell-growth sub-
strates are very different. Instead of nega-
tively affecting cell viability or growth in
cell cultures, graphene as a substrate may
provide a particular strain or ripples, as
well as enrichment of certain cell growth
factors, favorable for the adhesion, prolif-
eration, and differentiation of certain stem
cells, and useful for potential applications
as a tissue engineering scaffold.
5. In Vivo Behavior and
Toxicology in Animals
5.1. In Vivo Behavior of Graphene
Derivatives
In order to understand the potential in
vivo toxicity of graphene, the behavior
of graphene in animals should be studied
first. Unlike single-walled carbon nano-
tubes (SWNTs), graphene does not pos-
sess the strong resonance Raman signals
used to monitor the in vivo biodistribution of SWNTs.[97–99]
To track graphene in vivo, external labeling methods have
to be used. In our earlier work, we studied the in vivo bio-
distribution of PEGylated nano-GO (nGO-PEG) with ultra-
small sizes by fluorescent labeling and imaging. The results
showed that nGO-PEG exhibits efficient tumor passive tar-
geting owing to the enhanced permeability and retention
(EPR) effects of cancerous tumors.[59] However, the fluores-
cent labeling method has many intrinsic limitations such as
photobleaching and quenching of fluorescent dyes, and thus
is not an ideal approach to accurately monitor the long-term
behavior of graphene in animals.
Radiolabeling, as a quantitative and sensitive method,
has been widely used to study the in vivo behavior of var-
ious nanomaterials in animals.[89,100–104] The in vivo bio-
distribution of graphene and its derivatives has also been
studied by a radiolableing method.[24,59,105,106] Huang and
co-workers[56] studied the in vivo behavior of GO without
surface coating by labeling GO with 188Re, and found that
after intravenous (i.v.) injection 188Re-labeled GO (188Re-
GO) was predominantly deposited in the lung for a long
time without much excretion. Cui et al. also found that i.v.
injected GO without surface modification mainly accumu-
lated in the lung.[58]
In our recent work, we studied the long-term in vivo
biodistribution of nGO-PEG labeled with 125I.[59] It was
found that after i.v. injection 125I-nGO-PEG mainly accu-
mulated in the reticuloendothelial system (RES) such as
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 reviews K. Yang et al.

To further understand how sizes and

surface coatings affect the in vivo behavior
of GO, we synthesized three GO derivatives
with different sizes and surface coatings,
including ultra-small nGO-PEG (∼23 nm)
with covalent PEG coating, as well as
ultra-small nRGO-PEG (∼27 nm) and
larger RGO-PEG (65 nm) with non-
covalent PEGylation (Figure 6a–d). Since
the non-covalent surface PEG coating by
an amphiphilic polymer was in this case
more condensed compared to covalent
PEG conjugation, nRGO-PEG and RGO-
PEG with non-covalent PEG coatings
showed much longer blood circulation com-
pared to nGO-PEG (Figure 6e), leading
to a remarkably increased tumor uptake
for the first two GO derivatives due to the
ERP effect (Figure 6f).[24] The size of
nanomaterials also played an important
role in regulating their in vivo behavior.
Compared to RGO-PEG with bigger
sizes, ultra-small nGO-PEG and nRGO-
PEG exhibited significantly reduced RES
accumulation (Figure 6f). Thereafter,
Figure 4. Human neural stem cell (hNSC) growth and differentiation on a graphene substrate. nRGO-PEG with the optimized surface
(a) Bright-field images of hNSCs differentiated on the surface of glass and graphene for coating and size was chosen as a photo-
3 days, 2 weeks, and 3 weeks. (b) Bright-field images (top row) and fluorescence images thermal agent to kill the tumor, achieving
(bottom row) of hNSCs differentiated on a glass (left) and graphene film (right) for 1 month.
excellent in vivo treatment efficacy at
In fluorescence images, cells were stained with GFAP (red) for astroglial cells, TUJ1 (green) for
neural cells, and DAPI (blue) for cell nuclei. All scale bars represent 200 μm. (c) The numbers ultra-low laser power (0.15 W/cm2). This
of hNSCs on graphene and glass after 1 month differentiation counted an area of 0.64 mm2. work suggests that finely tuning the sur-
(d) Percentages of astroglial cells (stained by GFAP) and neural cells (stained by TUJ1) on face chemistry of graphene is important to
glass and graphene substrates. The graphene substrate promoted hNSCs differentiation optimize its in vivo pharmacokinetics and
towards neural cells more than astroglial cells. Reproduced with permission.[48] Copyright biodistribution for the desired biomedical
2011, Wiley-VCH Verlag GmbH & Co.
application.

the liver and spleen, in which the uptake levels gradually
decreased (Figure 5a,b). Unlike the as-made GO, no appre-
ciable lung uptake of nGO-PEG was observed. To confirm
that the observed time-dependent decrease in RES uptake
was indeed owing to the excretion of nGO-PEG but not the
detachment of radiolabels, we carefully examined haema-
toxylin and eosin (H&E) stained liver slices. Large numbers
of black spots, which were aggregates of nGO-PEG, were
observed at early time points after injection of nGO-PEG,
but gradually disappeared over time (Figure 5c–f), con-
sistent with the biodistribution data based on radioactivity
measurements. High radioactivities were found in both
urine and faecal samples, suggesting that nGO-PEG with
ultra-small sizes in the range of 10–30 nm (sheet diameter)
might be cleared out through both renal and faecal excre-
tions (Figure 5g). In a separate study, our group used another
biocompatible hydrophilic polymer, dextran, to function-
alize GO. The in vivo behavior of dextran-coated GO (GO-
DEX) was studied using an 125I-labeling method. Our results
showed that GO-DEX also mainly accumulates in the RES
organs at early time points after intravenous injection, and
could be gradually excreted over time.[89]
5.2. Toxicity of GO and Functionalized GO after Intravenous
Injection
In vivo toxicology evaluation in animal experiments is a crit-
ical step before clinical use of any drug formulation, and it
also provides valuable information regarding the exposure
risk of nanomaterials. In the past decade, a large number of
reports have uncovered that CNTs, as a ‘sister’ of graphene,
if without appropriate surface modification, may induce
granuloma formation, inflammation and pulmonary tox-
icity.[44,84,107–111] Other groups have reported that the toxicity
of CNTs is largely controlled by their surface chemistry, and
would be largely decreased by functionalizing CNTs with bio-
compatible polymers such as PEG.[17,44,59,89] Known from the
literature, similar to CNTs, the in vivo toxicity of graphene-
based materials is also closely associated with their surface
coatings.
Recently, Dash and co-workers[59] reported that GO
could induce high thrombogenicity in mice and evoked a
strong aggregatory response in human platelets. This finding
indicates that GO, without surface modification, may induce
blood clots after intravenous injection. Huang and Cui also

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Behavior and Toxicity of Graphene and Its Functionalized Derivatives

Figure 5. Biodistribution and clearance of nGO-PEG. (a) Time-dependent biodistribution of 125I-labeled nGO-PEG in mice after i.v. injection.
(b) 125I-nGO-PEG levels in the liver and spleen over time. (c–e) H&E stained liver slices from the untreated control mice (c) and nGO-PEG injected
mice at 3 days (d) and 20 days (e) post injection (p.i.) brown-black spots were observed in the livers of mice 3 days after injection of nGO-PEG.
(f) Statistics of averaged black spot numbers per image field in liver slices at various times post injection of nGO-PEG. (g) Radioactivity levels in
urine and faeces in the first week after injection of 125I-nGO-PEG. Error bars in the above data were based on standard deviations of 4–5 mice per
group. Reproduced with permission.[59] Copyright 2011, American Chemical Society.

found that pristine GO without a surface coating after i.v. Therefore, in order to improve the biocompatibility of
injection mainly accumulated in the lung for a long period of graphene, surface modification is critical. Up to now, poly-
time, inducing pulmonary edema and granuloma formation mers (e.g., PEG, Dextran, and chitosan) and functional
in the lung.[56,58] groups (e.g., amine and carboxyl) on the surface of

Figure 6. Size and surface coating regulated in vivo behavior of graphene derivatives. (a) A scheme to show the preparation of nGO-PEG, nRGO-PEG,
and RGO-PEG with different sizes and surface PEG coatings. (b–d) Atomic force microscope (AFM) images of nGO-PEG, RGO-PEG and nRGO-PEG.
Inset are corresponding photos of their solutions. (e,f) The blood circulation and biodistribution of graphene derivants of 125I-labeled nGO-PEG,
RGO-PEG and nRGO-PEG after i.v. injection. Reproduced with permission.[24] Copyright 2012, Elsevier.

small 2013, 9, No. 9–10, 1492–1503 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-journal.com 1499
 reviews K. Yang et al.

Figure 7. Blood chemistry and hematology data of female Balb/c mice i.v. injected with nGO-PEG at a dose of 20 mg/kg collected at 3, 20, 40, and
90 days p.i. Age-matched control untreated mice were sacrificed at 3, 40, and 90 days (3d-CK, 40d-CK, and 90d-CK). Hepatic function indicators
including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and the ratio of albumin and globulin
(A/G) were measured. Blood urea nitrogen (BUN) was an indicator of kidney function. Gray areas in the figures present the reference ranges for
healthy female Balb/c mice. No appreciable toxicity was noticed in nGO-PEG injected mice from the above data. Reproduced with permission.[59]
Copyright 2011, American Chemical Society.

graphene have been studied to decrease the in vivo tox-
icity of graphene.[97,99,100,112–114] In our work, we systemati-
cally studied the potential long-term toxicity of i.v. injected
nGO-PEG at a dose of 20 mg/kg to mice. The blood of
treated mice was collected after different time points post
injection (p.i.) for blood biochemistry and hematology tests.
The results showed that nGO-PEG treated groups at dif-
ferent times p.i. appeared to be normal compared with the
control groups and in good agreement with the reference
normal ranges (Figure 7). No noticeable organ damage or
inflammation was observed, suggesting no obvious toxicity
caused by i.v. injected nGO-PEG to mice at our tested
dose.[24] In addition, our group also studied the short-term
toxicity of GO-DEX and found that GO-DEX did not
induce obvious toxicity in treated animals.[89] Beside our
studies, in a recent work by Gollavelli et al., polyacrylic
acid (PAA) was used to functionalize GO to reduce its tox-
icity to cells and zebrafish.[23] Therefore, well-designed sur-
face modifications of graphene could effectively decrease
its in vivo toxicity.
5.3. Pulmonary Toxicity
Many groups have reported that pristine CNTs could
induce severe pulmonary toxicity and inflammation of
mice.[107,108,110,115] However, Mutlu et al.[60] found that well-
dispersed SWNTs by Pluronic F 108NF after intratracheal
instillation would be engulfed by macrophages and gradu-
ally cleared over time, without rendering obvious pulmonary
toxicity. Not to our surprise, Dash and co-workers found
that GO could induce extensive pulmonary thromboem-
bolism in mice.[84] Schinwald et al. further uncovered that
few-layer graphene with diameters up to 25 μm would
deposit beyond the ciliated airways after inhalation, and
induce high levels of inflammation in the mouse lung.[116]
Interestingly, in another recent work, Mutlu and co-workers
studied the potential pulmonary toxicity of various forms
of graphene.[60] Solutions of aggregated graphene, Pluronic
dispersed graphene, and GO were injected directly into
the lungs of mice. It was found that GO could induce the
mitochondrial generation of ROS, activate inflammatory

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Behavior and Toxicity of Graphene and Its Functionalized Derivatives

Figure 8. Pulmonary toxicity induced by different forms of graphene. Mice were treated with Pluronic dispersed graphene (GD), aggregated
graphene (GA), and GO by intratracheal instillation. The lungs were examined 21 days after treatment. (a) Photomicrographs of paraffin blocks of
the lung after sectioning. (b–d) Photomicrographs of lung sections at 1× (b), 50× (c), and 200× (d). (e) The percentage of terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) positive nuclei in paraffin-embedded lung sections. (f) Total lung collagen determined by picrosirius red
precipitation of whole lung homogenates. (g) In vitro data showing the generation of ROS and (h) the resulting DNA fragmentation in an alveolar
macrophage cell line. Water (-) or water with 2% Pluronic (-)P were used as negative controls. Reproduced with permission.[60] Copyright 2011,
American Chemical Society.

and apoptotic pathways, and also result in severe and
persistent lung injury. However, the mice treated with
aggregated graphene and dispersed graphene showed no
obvious lung injury. In order to further study potential pul-
monary toxicity, this group of researchers measured lung
fibrosis in mice treated with GO, aggregated graphene
or Pluronic-dispersed graphene, and then examined tri-
chrome-stained lung sections. It was found that aggregated
graphene induced patchy fibrosis in mice, while no obvious
fibrosis of mice treated with Pluronic-dispersed graphene
was observed. Although GO-induced lung inflammation
persisted 21 days after administration, it did not induce
lung fibrosis production in treated mice (Figure 8). They
thus concluded that well-dispersed graphene, unlike GO or
aggregated graphene, would induce minimal toxicity in the
lung. Therefore, the pulmonary interactions and toxicity of
graphene, similar to that of CNTs, are also controlled by
the surface functional groups, coatings, and dispersion state
of the material.
6. Summary
The biomedical applications of graphene have been inten-
sively studied by many groups around the world in recent
years. However, the further biological application of
graphene, similar to many other inorganic nanomaterials, has
been often challenged by concerns regarding its potential
toxicity. Understanding the interactions between graphene
and biological systems and the toxicity of graphene in vitro
and in vivo are of utmost importance for further develop-
ment of graphene-based nanomedicine, as well as providing
safety guidance to all researchers working with this new type
of nanomaterials. In this article, we have reviewed the tox-
icity of graphene by describing the behavior of graphene
and its derivatives in the microorganisms, cells, and animals.
Despite certain inconsistencies in several detailed experi-
mental results and hypotheses of the toxicity mechanisms,
numerous reports agree that physicochemical properties
such as the surface functional groups, charges, coatings, sizes,
and structural defects of graphene may affect its in vitro/
in vivo behavior as well as its toxicity to biological systems.
Nano-graphene, with ultra-small sizes, biocompatible surface
coatings, excellent dispersibility and stability in physiological
environments, appears to be much less harmful in vitro to
cells and in vivo to animals. How to abolish the toxicity and
accelerate excretion or even degradation of functionalized
graphene in biological systems, however, is a challenging
task for our future research. Furthermore, more systematic
investigations are still highly demanded to fully understand
the biological effects and address safety concerns before the
practical application of any graphene-based materials in the
clinic.

small 2013, 9, No. 9–10, 1492–1503 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.small-journal.com 1501
 reviews
Acknowledgements
This work was supported by the National Basic Research Program
of China (973 Program, 2012CB932601 and 2011CB911002),
National Science Foundation of China (NSFC, 51002100,
51132006, 31070707, 91027039), and a project funded by the
Priority Academic Program Development of Jiangsu Higher Edu-
cation Institutions (PAPD).
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Received: June 23, 2012
Published online: September 17, 2012
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