Biomaterials 33 (2012) 2206e2214

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

The influence of surface chemistry and size of nanoscale graphene oxide

on photothermal therapy of cancer using ultra-low laser power
Kai Yang a, Jianmei Wan b, Shuai Zhang a, Bo Tian a, Youjiu Zhang b, Zhuang Liu a, *
a
Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou,
Jiangsu 215123, China
b
School of Radiation Medicine and Public Health, Soochow University, Suzhou, Jiangsu 215123, China

a r t i c l e i n f o a b s t r a c t

Article history: Photothermal therapy as a physical treatment approach to destruct cancer has emerged as an alternative
Received 5 November 2011 of currently used cancer therapies. Previously we have shown that polyethylene glycol (PEG) function-
Accepted 22 November 2011 alized nano-graphene oxide (nGO-PEG) with strong optical absorption in the near-infrared (NIR) region
Available online 12 December 2011
was a powerful photothermal agent for in vivo cancer treatment. In this work, by using ultra-small
reduced graphene oxide (nRGO) with non-covalent PEG coating, we study how sizes and surface
Keywords:
chemistry affect the in vivo behaviors of graphene, and remarkably improve the performance of
Nano-reduced graphene oxide
graphene-based in vivo photothermal cancer treatment. Owing to the enhanced NIR absorbance and
Non-covalent functionalization
Size effect
highly efficient tumor passive targeting of nRGO-PEG, excellent in vivo treatment efficacy with 100% of
Photothermal therapy tumor elimination is observed after intravenous injection of nRGO-PEG and the followed 808 nm laser
Ultra-low laser power irradiation, the power density (0.15 W/cm2, 5 min) of which is an order of magnitude lower than that
Cancer treatment usually applied for in vivo tumor ablation using many other nanomaterials. All mice after treatment
survive over a period of 100 days without a single death or any obvious sign of side effect. Our results
highlight that both surface chemistry and sizes are critical to the in vivo performance of graphene, and
show the promise of using optimized nano-graphene for ultra-effective photothermal treatment, which
may potentially be combined with other therapeutic approaches to assist our fight against cancer.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction achieved with an ultra-low laser power, we could avoid unneces-

Photothermal therapy (PTT) using photo-absorbing agents to
generate heat from optical energy has been widely developed as
a minimally invasive treatment methodology in comparison with
other cancer therapies such as surgery, chemotherapy and radio-
therapy. In past years, a wide range of nanomaterials with strong
optical absorption in the near-infrared (NIR) tissue transparency
window, such as gold nanorods [1], nanoshells [2,3], nanocages
[4,5], as well as carbon nanotubes [6e9], have been developed as
photothermal agents for PTT treatment of cancer. However, one of
major limitations of using PTT for clinical cancer treatment is the
limited light penetration depth that leads to the ineffective ablation
of large tumors or tumors deeply located inside the body, and the
relatively high optical power (usually 2e4 W/cm2 for gold nano-
materials) [1e5,10e13] that required to obtain sufficient heating for
cancer cell killing. Thus, if effective PTT cancer destruction can be
* Corresponding author.
E-mail address: zliu@suda.edu.cn (Z. Liu).
sary heating of normal tissues, and importantly, may be able to
ablate larger or certain internal tumors by the phototherapy since
such a lower optical power could still be delivered to tumors even
after substantial light absorption and scattering by the covering
tissues.
Graphene, a class of two-dimensional carbon nanomaterials
with fascinating physical and chemical properties, has attracted
tremendous interests in many different fields including biomedi-
cine [14e19]. A large number of groups have developed a variety of
graphene-based biosensing platforms for detection of biological
molecules via different mechanisms [20e26]. We and others have
used functionalized nano-graphene as drug carriers for in vitro
intracellular delivery of anticancer chemotherapy drugs and genes
[16,17,27,28]. Recently, the in vitro and in vivo toxicology of gra-
phene oxide (GO) has also received significant attention. It is now
generally accepted that the in vitro cellular toxicity of graphene is
closely related to its surface functionalization, showing improved
biocompatibility for those with better coatings [29,30]. In a recent
study, we uncovered that covalently PEGylated nano-GO (nGO-
PEG) did not induce appreciable toxicity at our tested dose (20 mg/

0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.11.064
 K. Yang et al. / Biomaterials 33 (2012) 2206e2214
kg) to mice in a period of 3 months and could be gradually excreted
by both renal and fecal excretion [31].
Phototherapies with graphene have also shown promise in
a number of in vitro and in vivo studies [18,32e34]. In a previous
work by our group, nGO-PEG that exhibit high NIR absorbance was
used for effective photothermal therapy of tumors in a mouse
model. In that work, the tumors of mice intravenously injected with
nGO-PEG (2 mg/ml) were completely eliminated by the NIR laser
irradiation (808 nm, 2 W/cm2) [18]. The phtotothermal effect of
nGO-PEG could also be used to enhance the intracellular delivery of
photosensitizers for improved photodynamic therapy [35].
Recently, Dai and co-workers reported that ultra-small reduced
nano-GO (nRGO) with even higher NIR absorbance could act as
a highly efficient targeted photothermal agent for in vitro selective
cancer cell killing [36]. Herein, by using non-covalently PEGylated
nRGO (nRGO-PEG) with the optimized surface coating and size, we
further improve the graphene-based in vivo PTT cancer treatment
in animal experiments, and achieve excellent in vivo treatment
efficacy. The power density (0.15 W/cm2, 5 min) used in this work is
an order of magnitude lower than that usually applied for in vivo
tumor ablation using various gold nanostructures [1,2,4,5,10e13].
Our nRGO-PEG with ultra-small sizes and non-covalent PEG
coating appears to be an extremely effective agent for in vivo PTT
ablation of cancers.
2. Experimental section
2.1. Synthesis of reduced graphene oxide
Graphene oxide was synthesized by the modified Hummer
method, using flake expandable graphite as the original material
according to our previous protocol [16,18,37]. To prepare reduced
graphene oxide, hydrazine hydrate (w1 ml) was added to the GO
solution (10 ml) at a concentration w10 mg/ml and heated in an oil
bath at 95
C under stirring for 24 h [41]. The resulting solution was
filtrated to remove hydrazine hydrate and washed with deionized
water, yielding an insoluble RGO black solid.
PEG grafted poly(maleic anhydride-alt-1-octadecene) (C18PMH-
PEG5000) was synthesized following our previously reported
procedure [8,38]. In brief, 1 M equivalent (eq.) of 5 k mPEG-NH2
(PEG Bio, China) were reacted with poly(maleic anhydride-alt-1-
octadecene) (PMHC18, SigmaeAldrich) at the PEG: MHC18 mono-
mer molar ratio of 1:1 in 2 ml dichloromethane for one day in the
presence of 2 eq. of N-(3-Dimethylaminopropyl)- N0 -ethyl-
carbodiimide hydrochloride (EDC, SigmaeAldrich) and 4 eq. trie-
thylamine. The reaction solution was blown-dry by nitrogen,
yielding a solid product which was then dissolved in water. The
amphiphilic polymer solution was dialyzed against water using
a 14 kDa molecular weight cut-off (MWCO) membrane and then
lyophilized.
To functionalize RGO, 5 ml of C18PMH-PEG5000 (2 mg/ml) was
mixed with 1 mg RGO and sonicated for 90min. The suspension was
then centrifugated at 14,800 rpm for 3 h to remove any unstable
aggregates. The supernatant was collected and washed through
100 nm filter membrane to remove excess C18PMH-PEG.
The covalently functionalized nGO-PEG was synthesized
following our established protocol [16,18,31,37]. GO aqueous
suspension (5 ml) at a concentration of w3 mg/ml was sonicated
for about 30 min to give a clear solution. NaOH (0.12 g/ml) was
added to the GO suspension and heated at 40
C for 4 h. The
resulting solution was neutralized, purified by repeated rinsing and
centrifugation. A solution of 6-arm-polyethylene glycol-amine
(Sunbio Inc.) (3 mg/ml) was added to the base treated GO solu-
tion (0.5 mg/ml) and the mixture was sonicated for 5 min. N-(3-
dimethylaminopropyl-N0 -ethylcarbodiimide) hydrochloride (EDC,
2207
from Fluka Inc.) was then added to the mixture in two equal
portions to give a final concentration of 1 mg/ml totally. The reac-
tion was allowed overnight, yielding a nGO-PEG solution which
was purified by filtration through 100 kDa MWCO filters
(Millipore).
Ultra-small nRGO-PEG was synthesized using the same proce-
dure as the preparation of RGO-PEG, except that nGO-PEG was used
instead of GO as the starting material.
125
2.2. I labeling
nGO-PEG, RGO-PEG, and nRGO-PEG were labeled by 125I using
a standard chloramine-T oxidation method according to our
previous protocol [31,39]. A mixture of 200 ml GO-derivatives
(2 mg/ml), w300 mCi Na125I and 100 ml of 4 mg/mL chloramine-T
(SigmaeAldrich) was reacted in a pH 7.5 phosphate buffer
(0.05 M) for 30 min at room temperature. The labeled GO-
derivatives were purified by centrifugation filtration through
Amicon filters (MWCO ¼ 100 kDa) to remove excess 125I until no
detachable gamma activity in the filtrate solution. A radiolabeling
yield of w50% was achieved.
2.3. Tumor models
4T1 murine breast cancer cells were cultured in RPMI-1640
supplemented with 10% FBS and 1% penicillin-streptomycin. Balb/
c mice were obtained from Nanjing Peng Sheng Biological Tech-
nology Co, Ltd and performed under protocols approved by Soo-
chow University Laboratory Animal Center. The 4T1 tumor models
were generated by subcutaneous injection of 2  106 cells in 50 ml
PBS into the right shoulder of female BALB/c mice. The mice were
used for treatment when the tumor volume reached w50 mm3.
2.4. Blood circulation and biodistribution study
Healthy female Balb/c mice were intravenously injected with
125
I-nGO-PEG, 125I-RGO-PEG, and 125I-nRGO-PEG (100 ml of 20 mCi
per mouse, a dose of 4 mg/kg). Blood circulation was measured by
drawing w10 ml of blood from the tail of balb/c mice. The radio-
activity in blood was measured by gamma counter (Science and
Technology Institute of China in Jia Branch Innovation Co., Ltd.). In
order to test the tumor uptake, mice bearing 4T1 tumor were
injected with 125I-labeled GO-derivatives at the same dose and
sacrificed 2 days after injection. The major organs were weighed
and collected for gamma counting.
2.5. Photothermal therapy
Female Balb/c mice bearing 4T1 tumors were injected with
200ml of 2 mg/ml nRGO-PEG or nGO-PEG. For the laser treatment
groups, the tumors of mice were irradiated by an optical fiber
coupled 808 nm NIR laser (Hi-Tech Optoelectronics Co., Ltd. Beijing,
China) at the power density of 0.15 W/cm2 for 5 min. The exact
optical powers of lasers used in our experiments were calibrated
and measured by an LPE-1B laser power/energy meter (Physcience
OptoElectronics Co. Ltd. Beijing). The tumor sizes were measured by
a caliper every the other day and calculated as the volume ¼ (tumor
length)  (tumor width)2/2. Relative tumor volumes were calcu-
lated as V/V0 (V0 was the tumor volume when the treatment was
initiated).
2.6. Histology examinations
Control untreated and nRGO-PEG treated mice were sacrificed
100 days after treatment. Major organs of those mice were
2208 K. Yang et al. / Biomaterials 33 (2012) 2206e2214

collected, fixed in 4% formalin, conducted with paraffin embedded
sections, stained with hematoxylin and eosin (H&E), and examined
under a digital microscope (Leica QWin).
3. Results and discussion
3.1. Preparation and characterization of GO-derivatives with
different sizes and surface chemistry
GO was synthesized from graphite by a modified Hammers’
method. Three PEGylated GO-derivatives with different surface
coatings and sizes were prepared (Fig. 1a). Following our established
protocol [16e18,31], nGO-PEG were synthesized by covalently
conjugating GO with 6-armed-PEG-Amine (SunBio Inc) via carbo-
diimide catalyzed amide formation under sonciation in an aqueous
solution. Different from as-prepared GO which was in a light brown
color and rapidly aggregated in the presence of salts, nGO-PEG
showed a darker brownish color and was stable in a range of phys-
iological solutions (Fig. 1b). Strong CeH and OeH stretch peaks were
observed in the infrared (IR) spectrum of nGO-PEG sample (Fig. 1c).
To enhance the optical absorption in the NIR region, GO was
reduced into RGO by hydrazine hydrate reduction following liter-
ature protocols [40]. RGO with much less surface functional groups
became water-insoluble (Fig. 1b, Supporting Fig. S1). We thus
functionalized RGO by sonicating it in a solution of an amphiphilic
polymer, PEG grafted poly(maleic anhydride-alt-1-octadecene)

Fig. 1. Preparation of PEGylated GO-derivatives. (a) A scheme showing the preparation of nGO-PEG, RGO-PEG, and nRGO-PEG from GO. (b) Photos of GO, nGO-PEG, RGO, RGO-PEG,
nRGO and nRGO-PEG solutions in water, 9% NaCl and fetal bovine serum. (c&d) FT-IR spectra of nGO-PEG, nRGO, nRGO-PEG, RGO and RGO-PEG after purification to remove excess
polymers. The strong CeH stretch peak (w2800 cm1) and CeO stretch peaks (1100e1500 cm1) clearly indicate the presence of PEG in the PEGylated samples. The absence of PEG
signals in the nRGO sample (c) suggested that the N2H4 treatment of nGO-PEG was able remove its initial covalent PEG coating.
 K. Yang et al. / Biomaterials 33 (2012) 2206e2214 2209

(C18PMH-PEG) (Fig. 1a), which was previously employed to func-
tionalize single-walled carbon nanotubes (SWNTs) to effectively
prolong their blood circulation half-lives [8,38]. The strong hydro-
phobic interactions between numerous C18 hydrocarbon chains in
the C18PMH-PEG polymer and the hydrophobic graphene panel
allows stable PEG coating of RGO, which was also evidenced by IR
spectra (Fig. 1d). The obtained RGO-PEG was a black solution stable
in various biological buffers (Fig. 1b).
The third PEGylated GO-derivative, nRGO-PEG, was obtained via
a three step method (Fig. 1a). We first synthesized nGO-PEG, and
then treated it by hydrazine reduction, which was able to eliminate
the oxygen groups as well as to remove the PEGylation. The nRGO
sample after reduction was not water soluble anymore (Fig. 1b). IR
spectra also suggested that the PEG coating of nGO-PEG was
completely removed after hydrazine treatment (Fig. 1c). To regain
the water solubility, we further sonicated nRGO in a solution of
C18PMH-PEG, obtaining nRGO-PEG which was again stable in
physiological solutions (Fig. 1b).
As expected [16e18,31] and shown by the atomic force micro-
scope (AFM) image, nGO-PEG produced by this method exhibited
ultra-small sheet sizes (Fig. 2a). Compared to nGO-PEG synthesized
by covalent PEGylation, the non-covalently functionalized RGO-
PEG showed obviously larger sheet sizes (Fig. 2b). The third one,
nRGO-PEG, was much smaller than RGO-PEG and had similar sizes
to that of nGO-PEG (Fig. 2c). The AFM measured averaged flake
areas were 408, 3280, and 578 nm2, which were equivalent to sheet
diameters of 23, 65, and 27 nm (assuming them in round shape), for
nGO-PEG, RGO-PEG, and nRGO-PEG, respectively (Fig. 2d). The
sheet thickness of nRGO-PEG, on the other hand, was similar to that
of RGO-PEG and much higher than that of nGO-PEG (Fig. 2e), likely
owing to the non-covalent PEGylation that offered more condensed
surface polymer coating on graphene sheets [8].

Fig. 2. Characterization of PEGylated GO-derivatives. (aec) AFM images of nGO-PEG (a), RGO-PEG (b) and nRGO-PEG (c). (d&e) AFM histograms of flake areas (d) and heights (e) of
nGO-PEG, RGO-PEG and nRGO-PEG samples. The flake area was estimated as (length  width)  3.14/4. (f) UVeVISeNIR spectra of as-prepared GO, nGO-PEG, RGO-PEG and nRGO-
PEG solutions at the graphene-concentration of 0.01 mg/ml. (g) Temperature change curves of water, nGO-PEG, RGO-PEG, and nRGO-PEG solutions exposed to the 808 nm laser at
a power density of 1 W/cm2.
2210 K. Yang et al. / Biomaterials 33 (2012) 2206e2214

Consistent to their colors (Fig. 1b), UVeviseNIR spectral of as-
prepared GO, nGO-PEG, RGO-PEG and nRGO-PEG solutions at the
same weight concentration clearly revealed that reduced RGO-PEG
and nRGO-PEG exhibited dramatically enhanced NIR absorption,
with 3e4 folds of increase at 808 nm compared to that of un-
reduced nGO-PEG (Fig. 2f). Thus as expected, both RGO-PEG and
nRGO-PEG was more effective than nGO-PEG in terms of photo-
thermal heating under the NIR laser irradiation (Fig. 2g).
3.2. In vivo blood circulation and biodistribution
In order to study their in vivo behaviors, nGO-PEG, RGO-PEG,
nRGO-PEG were all labeled with 125I following our previous
protocol [31]. The radiolabeling method should be more reliable
than the fluorescence imaging method used in our earlier work [18]
for quantitative and accurate in vivo tracking. The radiolabeling
stability of three GO-derivatives was tasted in mouse plasma at
37
C (Supporting Fig. S2), showing <20% of de-iodination within 7
days. We then compared the blood circulation of three GO-
derivatives after intravenous injection of 125I-nGO-PEG, 125I-RGO-
PEG, and 125I-nRGO-PEG at the same dose (4 mg/kg, 20 mCi per
mouse) into Balb/c mice (Fig. 3a). Blood was drawn at different time
points post injection (p.i.), and measured by a gamma counter. The
blood circulation curves showed that the pharmacokinetics of GO-
derivatives followed a two-compartment model. The first and
second phase blood circulation half-lives were 0.29  0.02 and
5.8  2.8 h for nGO-PEG, 0.22  0.09 and 17.5  3.2 h for RGO-PEG,
0.51  0.2 and 16.7  1.7 h for nRGO-PEG. The areas under curves
(AUC0-N) of nGO-PEG, RGO-PEG and nRGO-PEG were 95  11,
494  20 and 461  21 mg/L$h, respectively. Compared to nGO-
PEG, RGO-PEG and nRGO-PEG functionalized by non-covalent
polymer coating exhibit remarkably prolonged blood circulation,
which allows repeated passes of nanomaterials through tumor
blood vasculatures and is favorable for passive tumor targeting via
the enhanced permeability and retention (EPR) effect.
Next, we studied the biodistribution of the three GO-derivatives.
Balb/c mice bearing 4T1 tumors were sacrificed at 2 days post
injection (p.i.) of radiolabeled 125I-nGO-PEG. 125I-RGO-PEG, and
125
I-nRGO-PEG (100 ml 20 mCi per mouse, a dose of 4 mg/kg). It was
found that RGO-PEG and nRGO-PEG showed a significant increase
of tumor uptake by 7e8 times compared to that of nGO-PEG
(Fig. 3b), owing to the improved pharmacokinetics of the former
two. However, the uptake of RGO-PEG by RES organs including liver
and spleen was also drastically higher than that of nGO-PEG, likely
attributed to the increased sheet sizes of RGO-PEG. Remarkably,
nRGO-PEG with ultra-small sizes and long blood circulation
exhibited rather efficient tumor passive accumulation which was
similar, if not higher, than that of RGO-PEG, along with the low RES
retention close to that of nGO-PEG. As a critical parameter to judge
the tumor targeting efficiency, the tumor to normal tissue (T/N)

a 40 RGO-PEG nRGO-PEG nGO-PEG

nGO-PEG
t 1/2 (h) 0.22 0.09 0.51 0.2 0.29 0.02
RGO-PEG
30 t’1/2(h) 17.5 3.2 16.7 1.7 5.8 2.8
nRGO-PEG
AUC(0- )
494 20 461 21 95 11
(mg/L·h)
%ID/g

20
c
nGO-PEG
10 RGO-PEG
nRGO-PEG
0 10
0 10 20 30 40 50
Time (h)
T/N

b 32 1
nGO-PEG
RGO-PEG
24 nRGO-PEG

0.1
%ID/g

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Fig. 3. Blood circulation and biodistribution of nGO-PEG, RGO-PEG and nRGO-PEG. (a) The blood circulation curve of 125I-labeled nGO-PEG, RGO-PEG and nRGO-PEG. The phar-
macokinetics of all samples followed the two-compartment model. Inset table: pharmacokinetic data of 125I-nGO-PEG, 125I-RGO-PEG and 125I-nRGO-PEG including circulation half-
lives and AUC0-N.(b) biodistribution of 125I-nGO-PEG, 125I-RGO-PEG and 125I-nRGO-PEG in female mice bearing 4T1 tumors. High tumor uptake of RGO-PEG and nRGO-PEG was
observed, owing to the prolonged blood circulation of these two GO-derivatives. On the other hand, nGO-PEG and nRGO-PEG with ultra-small sizes exhibited remarkably lower RES
retention compared to RGO-PEG. (c) Tumor to normal organ ratios (T/N ratios) of nGO-PEG, RGO-PEG and nRGO-PEG. The highest T/N ratios of nRGO-PEG were observed for most
organs, suggesting its superior passive cancer targeting efficiency compared to that of nGO-PEG and RGO-PEG. Error bars were based on standard deviations of 4 mice per group.
 K. Yang et al. / Biomaterials 33 (2012) 2206e2214 2211

ratios were calculated and compared among the three GO-
derivatives (Fig. 3c). The T/N ratios of nRGO-PEG were signifi-
cantly higher than that of nGO-PEG and RGO-PEG for most
measured organs, suggesting the superior passive tumor targeting
efficiency of nRGO-PEG with the optimized surface chemistry and
size. Based on our data, we propose that the tumor uptake of gra-
phene increases as the prolonging of its blood circulation, which is
closely associated to the surface coating of graphene. On the other
hand, the RES uptake of graphene may be size-dependent, with
reduced retention for those with smaller sizes.
In our previously work, we labeled nGO-PEG with a NIR fluo-
rescent dye, Cy7, to study its in vivo behaviors in tumor-bearing
mice [18]. Unfortunately, the RES uptake of GO-PEG was largely
underestimated in that study using the fluorescence imaging
method, possibly due to the quenching of fluorescent dyes in liver
and spleen. The observed low RES accumulation of GO-PEG during
fluorescence imaging leaded to the overestimation of its tumor
passive targeting efficiency. The radiolabeling method employed in
this work is clearly a more reliable approach to accurately assess the
in vivo biodistribution of nanomaterials.
3.3. In vivo photothermal therapy
Motivated by high tumor accumulation of nRGO-PEG and its
strong NIR absorbance, we then carried out an in vivo photothermal
therapy study. Ten mice bearing 4T1 tumors were intravenously
injected with 200 ml of nRGO-PEG at 2 mg/ml (a dose of 20 mg/kg)
with tumors irradiated by the 808 nm laser for 5 min at an ultra-
low power density of 0.15 W/cm2 at 48 h p.i. (nRGO-PEG þ Laser,
n ¼ 10). As the comparison, another seven 4T1 tumor-bearing mice
were injected with nGO-PEG at the same dose (20 mg/kg) and
exposed to the laser at the same conditions (nGO-PEG þ Laser,
n ¼ 7). Three other groups included saline injected mice (Control,
n ¼ 7), saline injected mice exposed to laser (Laser only, n ¼ 7), and
nRGO-PEG injected mice without laser irradiation (nRGO-PEG,
n ¼ 7), were also used as controls. The tumor sizes and mice

Fig. 4. In vivo photothermal therapy study using intravenously injected nRGO-PEG. (a) Tumor growth curves of different groups of mice after treatment. The tumor volumes were
normalized to their initial sizes. (b) Survival curves of mice bearing 4T1 tumors after various treatments indicated. nRGO-PEG injected mice after PTT treatment survived over 100
days without a single death. Seven mice were used for each group for ‘control’, ‘Laser only’, ‘nRGO-PEG only’, and ‘nGO-PEG þ Laser’ groups. Ten mice were used for the ‘nRGO-
PEG þ Laser’ group. (c) Representative photos of tumor-bearing mice after various treatments indicated. The laser irradiated tumors on the nRGO-PEG injected mice were
completely destructed. Laser wavelength ¼ 808 nm. Power density ¼ 0.15 W/cm2. Irradiation time ¼ 5 min. Error bars were based on standard deviations.
2212 K. Yang et al. / Biomaterials 33 (2012) 2206e2214

survival were monitored every the other day after treatment
(Fig. 4a and b).
While the surface temperature of tumors on nRGO-PEG treated
mice reached to w 48
C after laser irradiation, that on nGO-PEG
injected and untreated mice only showed slight increase to 41
C
and 38
C, respectively. The tumors of mice treated with nRGO-PEG
disappeared 1 day after laser irradiation, leaving the original tumor
sites black scars, which were fully recovered about 1 week after
treatment (Fig. 4c). The tumors of four control groups of mice
showed similarly rapid tumor growth (Fig. 4a and c), even for those
injected with nGO-PEG and irradiated by the laser, indicating that
nGO-PEG at this optical power was not effective in photothermal
ablation of tumors. Importantly, mice bearing 4T1 tumors treated
with nRGO-PEG and laser irradiation survived over 100 days
without tumor re-growth (Fig. 3b), in marked contrast to mice in
the four control groups which survived for only an average of w16
days post treatment (Fig. 4b), further demonstrating the excellent
efficacy of nRGO-PEG based in vivo photothermal therapy of cancer.
Gold nanomaterials are the mainstream photothermal agents
used in PTT treatment of cancer [1e5,10e13]. In 2007, Gobin et al.
[41] designed gold nanoshells that possess high NIR absorbance for
effective photothermal ablation of tumors at the laser power of
4 W/cm2. Maltzahn et al used PEG coated gold nanorods (dose:
20 mg/kg) for photothermal tumor destruction under 810 nm laser
irradiation at the power of 2 W/cm2 for 5min [1]. Effective in vivo
PTT with gold nanomaterials has been achieved at lower power
densities down to 0.75 W/cm2 (dose: 10 mg/kg), however has to be
combined with chemotherapy drugs [42]. In addition, one problem
of using gold nanomaterials for PTT is the instability or melting of
gold nanostructures under intense NIR light, resulting in the
decrease of NIR absorbance after a certain period of laser irradiation
[43e45]. In marked contrast to gold nanorods, whose absorbance
spectrum dramatically changed when exposed to the NIR light over
time, we found that nRGO-PEG was extremely stable under
continuous NIR laser irradiation for hours, without any noticeable
change in its optical absorbance (Supporting Fig. S3).

Fig. 5. No obvious toxic effect was observed for nRGO-PEG treated mice. (a) Body weight curves after various treatments indicated. Neither nanomaterials injection nor laser
irradiation resulted in significant body weight drop to the treated mice. (b) H&E stained images of major organs. Mice intravenously injected with nRGO-PEG were sacrificed 1, 7,
and 100 days after treatment. No noticeable abnormality was observed in liver, spleen, kidney, and other major organs including heart, lung, stomach, and intestine (data not
shown).
 K. Yang et al. / Biomaterials 33 (2012) 2206e2214
Surveying literature, the lowest power density that applied to
realize in vivo tumor ablation was reported to be 0.6 W/cm2 by Dai
et al. using SWNTs (dose: 3.6 mg/kg) [9]. Using a similar dose of
nRGO-PEG (4 mg/kg), we also achieved excellent PTT efficacy by
employing 0.5 W/cm2 of NIR laser irradiation (Supporting Fig. S4).
Notably, based on our own and others’ experiences, non-covalently
PEGylated SWNTs synthesized by that method has a concentration
limit and would become a sticky oil-like liquid that could hardly be
injected into mice [46]. Therefore it may not be practical to further
increase the dose and lower the laser irradiation power for SWNT-
based PTT. Our nRGO-PEG is thus unique among various light-
absorbing nanomaterials currently explored for in vivo PTT treat-
ment of cancer.
3.4. Histology examination
Our previous study revealed the minimal toxicity of nGO-PEG to
the treated animals [31]. In this work, neither death nor significant
body weight variation was noticed after nRGO-PEG treatment at
a dose of 20 mg/kg (Fig. 5a). Major organs of nRGO-PEG treated
mice whose tumors were eliminated by the photothermal therapy
were sacrificed 100 days after the treatment. Histological assess-
ment of hematoxylin and eosin (H&E) stained tissue slices showed
no appreciable normality or lesion in major organs of nRGO-PEG
treated mice (Fig. 5b). Although the systematic long-term fate
and toxicology of nRGO-PEG in animals remain to be carefully
examined, our preliminary results indicate that nRGO-PEG may not
be obviously toxic to mice.
4. Conclusions
In summary, we develop nRGO-PEG with ultra-small sizes
(average diameter ¼ 27 nm) and non-covalent PEG coating for
ultra-low power in vivo PTT treatment of cancer. How the surface
coatings and sizes of graphene affect its in vivo behaviors is studied
for the first time. It is uncovered that nRGO-PEG is able to effec-
tively target the tumor via the ERP effect with relatively low RES
retention. Owing to the high tumor passive uptake and strong NIR
absorption, nRGO-PEG is shown to be an excellent photothermal
agent that enables highly efficient in vivo tumor ablation by using
an ultra-low laser power density (0.15 W/cm2), which is an order of
magnitude lower than that usually applied for in vivo PTT treat-
ment of cancer using many other nanomaterials. The nRGO-PEG
developed in this work is obviously a powerful photothermal
agent with great potential in phototherapies of cancer. The
graphene-based PTT cancer treatment may be further combined
with other therapeutic approaches co-delivered by graphene for
synergistic cancer destruction. Our findings also suggest that the
surface chemistry and sizes of nanomaterials have unassailable
effects to their behaviors in biological systems especially in vivo,
and are critical to optimize their functionalities in biomedicine.
Acknowledgments
This work was partially supported by the National Basic
Research Program (973 Program) of China (2012CB932601,
2011CB911002), the National Natural Science Foundation of China
(51132006, 51002100), and a Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education
Institutions.
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.biomaterials.2011.11.064.
2213
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