CARBON 6 9 ( 2 0 1 4 ) 3 7 9 –3 8 9

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Hyaluronic acid conjugated graphene oxide

for targeted drug delivery

Huixia Wu a,*, Haili Shi a, Yapei Wang a, Xiaoqing Jia a, Caizhi Tang a, Jiamin Zhang b,
Shiping Yang a,*
a
The Key Laboratory of Resource Chemistry of Ministry of Education and the Shanghai Key Laboratory of the Rare Earth Functional Materials,
Department of Chemistry, College of Life and Environmental Science, Shanghai Normal University, Shanghai 200234, People’s Republic of
China
b
Shanghai (Red Cross) Blood Center, Shanghai Institute of Blood Transfusion, Shanghai 200051, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Article history: Nano-sized graphene oxide (GO) is functionalized with adipic acid dihydrazide to introduce
Received 3 July 2013 amine groups, and then hyaluronic acid (HA) is covalently conjugated to GO by the forma-
Accepted 11 December 2013 tion of amide bonds. The resulting HA-grafted GO (GO–HA) has negligible hemolytic activity
Available online 18 December 2013 and very low cytotoxicity towards HeLa and L929 cells, and it can be effectively taken up by
cancer cells through receptor-mediated endocytosis. The histological, hematological and
biochemical analysis results suggest no perceptible toxicity of GO–HA in mice at a high
exposure level of 10 mg kg1 and at an exposure time of up to 10 days. Doxorubicin
(DOX) can be efficiently loaded on the GO–HA, and the resulting GO–HA/DOX exhibits nota-
ble cytotoxicity to HeLa cells. The in vivo drug delivery capability of GO–HA is demonstrated
by following the tumor growth in mice after intravenous administration of GO–HA/DOX.
The results indicate that GO–HA can efficaciously deliver DOX to the tumors and suppress
tumor growth.
 2013 Elsevier Ltd. All rights reserved.

1. Introduction to biological applications [16]. Importantly, both sides of a GO

As a two-dimensional sp2 carbon networking material iso-
lated for the first time in 2004, graphene has attracted great
attention because of its extraordinary chemical, optical, elec-
trical and mechanical properties and potential applications
for nanoelectronic devices, transparent conductors and com-
posite materials [1–3]. Very recently, graphene has also been
used in biomedical fields, such as drug/gene delivery, biolog-
ical sensing, molecular imaging and cancer therapy [4–11].
In particular, functionalized graphene oxide (GO) and its
derivatives have emerged as promising materials for drug
delivery [4,6,10–15]. GO possesses unique features, such as
easy synthesis, high dispersibility in water as well as in phys-
iological environments, excellent biocompatibility and easily
tunable surface functionalization, which are highly propitious
sheet can be available for drug loading, which contributes to
the high drug-loading amount. Moreover, the dynamic bond-
ing interactions (for example, p–p stacking, hydrophobic,
hydrogen bonding and electrostatic interactions) between
GO and the drugs show controlled response to external stim-
uli (such as pH, temperature, chemical substances and elec-
tric fields) [17,18]. Therefore, the controlled drug release
from GO can be achieved by various routes. All these positive
attributes make GO much more efficacious than other carbo-
naceous nanomaterials as drug carriers for in vitro and in vivo
biological applications.
As a naturally occurring polysaccharide composed of alter-
nating units of N-acetyl-D-glucosamine and D-glucuronic acid,
hyaluronic acid (HA) has unique and excellent physicochem-
ical properties, such as biodegradability, biocompatibility and

* Corresponding authors.
E-mail addresses: wuhuixia@shnu.edu.cn (H. Wu), shipingy@shnu.edu.cn (S. Yang).
0008-6223/$ - see front matter  2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbon.2013.12.039
380 CARBON 6 9 ( 2 0 1 4 ) 3 7 9 –3 8 9

O H
NH2 N
N NH2
H O
OH OH
O O

HOOC (a) O
NH
HN
H
N
O NH2
O

O OH
OH
OH
O
O OH O
O O O
NH HO
NH n
OH
NH O
CH3
O
O
NH (b)
OH OH NH OH
O O
O O OH O O OH O
[ HO O O O ]n
NH HO NH
OH OH
O O
CH3 CH3

O OH O
OH

(c) OH

O O OH O OH
NH2
O

O
O OH
OH
O OH
OH

O HO O
O OH

O NH2
O
NH

NH
O
O (d)
NH
OH OH NH OH
O O
O O OH O O OH O
[ HO O O O ]n
NH HO NH
OH OH
O O
CH3 CH3

Fig. 1 – Schematic diagram of the synthesis of GO–HA and succedent DOX loading: (a) Functionalizing GO with ADH to
produce GO–ADH; (b) conjugation of HA with GO–ADH by the formation of amide bonds to produce GO–HA; (c) loading DOX
onto GO–HA and (d) intravenous administration of GO–HA/DOX by tail vein. (A colour version of this figure can be viewed
online.)

nonimmunogenicity [19,20]. HA is widely present in the extra-
cellular matrix, connective tissues and bodily fluids, and it
plays important roles in many pathophysiological processes
[21,22]. It is also known that HA has a strong affinity with
cell-specific surface markers such as cluster determinant 44
(CD44) and receptor for hyaluronate-mediated motility
[23–25]. These HA receptors, especially the CD44, show
dramatically higher levels on the surface of various tumor
cells [25]. CD44 overexpression is also associated with cancer-
ous angiogenesis and other types of tumor progression
[26,27]. Such an interesting selectivity of HA to CD44 over-
expressing cancer cells has shown great application potential
in biomedical fields such as cancer diagnosis and therapy [28–
30]. In view of the above-mentioned targeting characteristics
of HA, the conjugation of HA to GO is a promising method
for targeting tumor cells.
In this contribution, to integrate the advantages of GO and
HA, the nano-sized GO was functionalized with adipic acid
dihydrazide (ADH), and then the introduced amino groups
were used to conjugate HA. The in vitro and in vivo toxicity
studies of the resulting HA-grafted GO (GO–HA) were carried
out by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
 CARBON
tetrazolium bromide (MTT) assays, hematological and serum
biochemistry measurements, and histological assessments.
Doxorubicin hydrochloride (DOXÆHCl), a hydrophilic drug,
can be loaded onto the functionalized GO in a controlled
way (Fig. 1). The in vitro selective targeting and cytotoxic effect
of the DOX-loaded GO–HA (GO–HA/DOX) to CD44 over-
expressing HeLa cells were examined. Moreover, the chemo-
therapy effects of HeLa tumor-bearing mice intravenously in-
jected with GO–HA/DOX were studied.
2. Experimental
2.1. Chemicals and materials
ADH (P98%) was provided by TCI. Sodium hyaluronate (ex-
tracted from cockscomb) was obtained from Shanghai Haoran
Biological Technology Co., Ltd. DOXÆHCl (>99%) was acquired
from Beijing Hua Feng United Technology Co., Ltd. Other chem-
icals and reagents were purchased from Sinopharm Chemical
Reagent Co., Ltd. Deionized water with resistivity higher than
18 MX cm was used in all experiments. GO was prepared by a
modified Hummers’ method [31] using natural graphite pow-
der as a starting material (Supporting information).
2.2. Synthesis of GO–HA
GO (50 mg) was dispersed in distilled water (50 mL), and then
N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochlo-
ride (EDC, 250 mg) and N-hydroxysuccinimide (NHS, 750 mg)
were introduced to activate the carboxylic acid groups of
GO. ADH (520 mg) was added to the activated GO solution, fol-
lowed by another 24 h of stirring. The produced GO–ADH was
separated by centrifugation and rinsed repeatedly with water.
Sodium hyaluronate (15 mg), EDC (38 mg) and NHS (113 mg)
were dissolved in phosphate buffered saline (PBS, pH 5.6,
15 mL), then the above-mentioned GO–ADH was introduced,
and the mixture was stirred at room temperature for 24 h.
The resulting GO–HA was collected by centrifugation, washed
repeatedly with water, and then dispersed in PBS (pH 7.4).
2.3. Characterization
Transmission electron microscopy (TEM) images were ob-
tained on a JEOL JEM-2100 high-resolution transmission elec-
tron microscope. Morphological analyses were performed
with a Veeco Multimode IIIa atomic force microscope (AFM).
Fourier transform infrared (FT-IR) spectra were recorded on
a Nicolet Avatar 370 FT-IR spectrophotometer using KBr pel-
lets. X-ray diffraction (XRD) patterns were obtained on a Rig-
aku DMAX 2000 diffractometer using Cu-Ka radiation
(k = 0.15405 nm) (40 kV, 40 mA). Raman spectra were collected
with a super LabRam II confocal microscopic Raman spec-
trometer instrument (Dilor, France). Ultraviolet–visible
(UV–vis) absorption spectra were obtained with a BeckMan
coulter DU 730 spectrophotometer.
2.4. Cell culture
HeLa and L929 cell lines, which were provided by the Institute
of Biochemistry and Cell Biology, SIBS, CAS (China), were
6 9 (2 0 1 4) 3 7 9–38 9 381
cultured in regular growth medium consisting of Roswell Park
Memorial Institute medium (RPMI) 1640 supplemented with
10% fetal bovine serum at 37 C under a 5% CO2 atmosphere.
The cells were routinely harvested by treating with a trypsin–
ethylene diamine tetraacetic acid (EDTA) solution (0.25%).
2.5. In vitro cytotoxicity and hemolysis assay
Cytotoxicity: HeLa and L929 cell lines were used to evaluate the
in vitro cytotoxicity of GO and GO–HA. The cells, which were
seeded in a 96-well cell culture plate at a density of 1 · 104
cells per well, were cultured under 5% CO2 at 37 C for 24 h.
Then the cells were exposed to serial concentrations of the
materials, and further incubated for 12 or 24 h. Finally, the cell
viability was assessed using the MTT assays. The cytotoxicity
was expressed as the percentage of cell viability compared to
that of untreated control cells.
Hemolysis assay: Human red blood cells (HRBCs) stabilized
with EDTA were kindly provided by Shanghai Blood Center.
The in vitro hemolysis assays were carried out following the
literature method [32]. The concentration of the test material
is 50, 100, 200, 300 and 400 lg mL1.
2.6. In vivo toxicity studies
The healthy male Kunming (KM) mice (body mass 20 g) were
obtained from Shanghai Laboratory Animal Center, Chinese
Academy of Science, Shanghai, China (Warrant No. SCXK
[Shanghai] 2012-0002). All animal operations were conducted
in accordance with the guidelines of the Institutional Animal
Care and Use Committee. GO–HA dispersed in physiological
saline (0.2 mL) at a total dose of 10 mg kg1 was injected into
KM mice (n = 3 per group) by the tail vein. KM mice (n = 3) with
injection of only physiological saline were chosen as the con-
trol group.
Histology and hematology studies: Blood samples and tissues
were harvested from mice 4 h and 10 days post-injection.
Blood was collected from the orbital sinus by quickly remov-
ing the eyeball from the socket with a pair of tissue forceps.
Two indicators for kidney functions (creatinine and blood
urea nitrogen (BUN)) and three important hepatic indicators
(alanine aminotransferase (ALT), aspartate aminotransferase
(AST) and total bilirubin (TBIL)) were determined. For the
preparation of blood smear, a drop of blood was placed on
one end of a slide and dispersed along the length of the slide
by another slide. The slide was left to air dry, after which the
blood was stained with hematoxylin–eosin (HE) to distinguish
the cells from each other. Upon completion of the blood col-
lection, the mice were sacrificed. The heart, liver, spleen, lung
and kidneys were removed and fixed in 4% formaldehyde,
then dehydrated and embedded in paraffin. 5 mm transverse
sections were cut (Leica RM2235 microtome) and stained with
HE for histological analysis.
2.7. Luminescence imaging
Confocal luminescence imaging was performed with a Leica
TCS SP5 II laser scanning confocal microscope and a 63· oil-
immersion objective lens. The cells were seeded on coverslips
and incubated with 30 lg mL1 of GO–HA/DOX and GO/DOX
382 CARBON 6 9 ( 2 0 1 4 ) 3 7 9 –3 8 9
for 3 h. The cells were washed 3 times with PBS and then
stained with 4,6-diamino-2-phenyl indole (DAPI) solution be-
fore measurements.
2.8. Drug loading, release and cytotoxicity assay
Loading DOX onto GO–HA was performed by mixing DOXÆHCl
with the material (5 mg) in PBS (pH = 7.4, 10 mL) for 20 h un-
der dark conditions at a certain drug concentration. The
resulting GO–HA/DOX was collected by centrifugation, and
washed with PBS repeatedly to remove the excess drug. The
loading ratios of DOX were estimated by UV–vis spectroscopy
at 490 nm.
To study the release behavior of DOX from GO–HA, GO–HA/
DOX (5 mg) was suspended in a PBS buffer (2.0 mL, pH 7.4, 6.3
and 5.2) and sealed in dialysis membranes (Mw cutoff = 3500).
The dialysis bags were incubated in PBS buffer (8.0 mL) of the
same pH value at 37 C under shaking (200 rpm min1). The
amount of the released DOX was measured at different time
points by a UV–vis spectrophotometer (490 nm). Each experi-
ment was repeated three times.
The cytotoxicity of free DOX and GO–HA/DOX against HeLa
cells was determined using MTT assays. The determination
procedure was similar to the cytotoxicity assay of GO–HA.
2.9. In vivo tumor therapy
The HeLa tumor models were generated by subcutaneous
injection of 5 · 106 cells in RPMI 1640 (0.2 mL) into the left lat-
eral abdominal wall of the nude mice. Treatment was initi-
ated when the tumors reached an approximate size of
100 mm3. The mice were divided into three groups, and each
experimental group included 3 mice. The mice were adminis-
tered with (a) physiological saline (200 lL), (b) GO–HA (200 lL,
10 mg kg1), (c) GO–HA/DOX (200 lL, 6 mg kg1 in terms of
DOX, with the same amount of GO–HA as that of GO–HA
group) intravenously through tail veins every three days.
The tumor volume and body weight were monitored every
other day. The tumor volume was calculated using the follow-
ing formula: tumor volume = (tumor length) · (tumor width)2/
2. Relative tumor volumes were calculated as V/V0 (V0 is the
tumor volume when the treatment was initiated).
2.10. Statistical analysis
Statistical analysis was performed using the unpaired two-
tail Student’s t-test. The data are presented as means ± stan-
dard deviation. A P value of less than 0.05 was considered sta-
tistically significant.
3. Results and discussion
3.1. Synthesis and characterization of GO–HA
The water-soluble GO was prepared by the oxidation of graph-
ite according to a modified Hummers’ method [31] and the
following sonication treatment. The obtained GO was stable
in pure water for over a month and no agglomeration
occurred. TEM (Fig. 2a) and AFM (Fig. 2b) images provide the
morphological information of the as-prepared GO. The
nanosheets have a thickness of 0.8–1.3 nm (Fig. 2c), suggest-
ing the complete exfoliation of GO sheets down to individual
ones. The determined thickness of GO is larger than the the-
oretical value of a single layer graphene because of the pres-
ence of covalently bound oxo-groups [33]. The GO sheets
show good separation, and their lateral width changes from
60 to 500 nm and more than 80% of the GO sheets are smaller
than 300 nm (Fig. 2d).
The FTIR spectrum (Fig. 2e) of the GO reveals the presence
of –OH (3380 cm1), C@O (1725 cm1), C@C (1622 cm1) and C–
O (1060 cm1) [14,34]. The as-synthesized GO was further
investigated by XRD analysis. As shown in Fig. 2f, a sharp
peak is observed at 11.3 (d = 0.78 nm), which usually appears
in the XRD patterns of GO samples [14,35]. This peak is due to
the peak position of stacked GO analogs formed during the
process of preparation of XRD samples. The characteristic dif-
fraction peak of graphite at 26.5 disappears due to the com-
plete oxidation of graphite [14]. The Raman spectrum of GO
(Fig. 2g) shows two strong peaks at 1333 and 1592 cm1 that
are assigned to the D and G bands, respectively. The D/G ratio
of GO is about 1.2, which is higher than that of as-prepared
graphite oxide (0.79) (Fig. S1). The significant increase of the
D/G ratio for GO compared to that of graphite and graphite
oxide suggests the introduction of a higher level of disorder
onto the graphene layer and the decrease in the size of the
in-plane sp2 domains after oxidation and succedent exfolia-
tion [14,35]. Compared with that of graphite oxide, the 2D
band of GO is shifted from around 2700 to 2650 cm1. The
2D band is the second order band of zone-boundary phonons.
The shift of the 2D peak depends on the number of graphene
layers. Single-layer graphene shows a single sharp 2D peak
below 2700 cm1, while bilayer or more sheets have a broader
and upshifted 2D peak located at about or more than
2700 cm1. The Raman spectrum of GO exhibits a 2D peak
which is lower than 2700 cm1, suggesting the single layer
crystal structure of the GO sheets [14].
In order to introduce amine moieties for further graft of
HA, the as-prepared GO was first conjugated with ADH
through carbodiimide coupling. Fig. 3A(a) shows the FT-IR
spectrum of the obtained GO–ADH, which reveals the disap-
pearance of the C@O stretching of –COOH (1725 cm1) and
the appearance of a strong band at 1630 cm1 associated with
the amide bonds [36]. Meanwhile, the amine content present
in GO–ADH was determined to be about 5.9 · 105 mol g1
using the standard FMOC (9-fluorenylmethyl chloroformate)
quantification protocol [37], so the weight percent of conju-
gated ADH is about 1.0%. Afterwards, the primary amine
groups in GO–ADH were allowed to react with HA through
an amide linkage. The FT-IR spectrum of the resulting GO–
HA is present in Fig. 3A(b). Several new absorbance character-
istics of HA appear at 1627 cm1 (amide I band), 1405 cm1
(carboxylate symmetric stretching), 1155 cm1 (skeletal acetal
valence band) and 1045 cm1 (C–O stretching), confirming the
successful conjugation of HA on GO [38]. The weight content
of HA in GO–HA has been estimated by TGA method (Fig. S2).
The thermogram of pristine GO shows a little mass loss
(10%) below 120 C, which is associated with the adsorbed/
trapped water molecules. A significant mass loss (31%) in
the range of 120–260 C is attributed to the decomposition of
oxygen-containing groups [39]. From the TGA curve of GO–
 CARBON 6 9 (2 0 1 4) 3 7 9–38 9 383

(a) (b) 1.077 nm (c)

40

30
(d)

Percentage %
20

10

0
0 100 200 300 400 500
Size (nm)

(e) (f) 26.5

o (g)
D

G
Intensity (a.u.)
Transmittance

Intensity (a.u.)
1725 Graphite powder
1060
1622
o
11.3

2D
3380 GO

4000 3500 3000 2500 2000 1500 1000 10 20 30 40 50 60 70 80 1000 1500 2000 2500 3000
-1
Wavenumber (cm )
-1 2θ (degree) Raman shift (cm )

Fig. 2 – TEM image (a), AFM image (b) and height histogram (c), size distribution obtained from AFM images (d), FT-IR
spectrum (e), XRD pattern (f), and Raman spectrum (g) of GO. The XRD pattern of original graphite powder was also shown in
(f) for comparison. (A colour version of this figure can be viewed online.)

(A) a
(B)

1630
b
1155
Transmittance

c 1405
1627 1045

4.925 nm

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )

Fig. 3 – (A) FT-IR spectra of GO–ADH (a), GO–HA (b) and HA (c); (B) AFM image and height histogram of GO–HA. (A colour version
of this figure can be viewed online.)

HA, it is observed that about 14% weight loss occurs between GO due to the sonication treatment during the synthesis.
120 and 220 C, which is caused by the removal of remaining Meanwhile, the measured thickness of GO-HA increases to
oxygen-containing groups on GO. The notable weight loss of 2.0–5.0 nm, mainly due to the attachment of HA to the GO
49% in the range of 230–680 C mainly corresponds to the sheet [40]. The initial GO has a zeta potential of 30.0 mV,
complete degradation of grafted HA segments [30,39]. which changes to 11.4 mV when modified with ADH. After
According to the TGA data of GO and GO–HA, the weight HA grafting, the zeta potential of the resulting GO–HA is
percentage of grafted HA chains is 49% by ignoring the significantly reduced to 34.2 mV (Fig. S3). The zeta-poten-
conjugated amount of ADH. Fig. 3B shows the AFM image tial measurement results further indicate the successful
of the resulting GO–HA. The GO-HA remains well dispersed, grafting of HA on GO. The as-prepared GO–HA can be
which is critical for biological applications. The GO-HA colloidally stable in water and physiological saline at room
sheets have sizes (40–350 nm) lower than that of the original temperature (Fig. S4).
384 CARBON 6 9 ( 2 0 1 4 ) 3 7 9 –3 8 9

3.2. In vitro cytotoxicity of GO–HA 2.0 2.0

1.5 GO
Water
The cytotoxicity effects of GO-HA against the HeLa and L929 GO-HA

Hemolysis %
1.0
cell lines were investigated by conducting MTT assays. As 1.5
0.5
shown in Fig. 4a and b, these two cell lines give similar results

Absorbance
for GO-HA, and the difference in cell viabilities after 12 and 0.0
1.0 0 100 200 300 400
24 h of incubation is negligibly small. No obvious cytotoxicity -1
Concentration (μg mL )
against HeLa and L929 cells was found after 24 h of treatment
at a concentration as high as 200 lg mL1. These results indi-
0.5
cate that GO–HA has low cytotoxicity, which is an important
PBS, GO-HA
character for drug carriers. GO-HA
H2O 400 300 200 100 PBS
0.0
3.3. In vitro blood compatibility 500 550 600 650 700
Wavelength (nm)
For successful intravenous administration and transport of
anticancer drug, the carriers must have excellent blood com- Fig. 5 – UV–vis absorption spectra to detect the presence of
patibility, such as a very low hemolytic effect. Very recently, hemoglobin in the supernatant of GO–HA suspensions in
several groups have researched the hemolytic effect of GO, PBS at concentrations of 50, 100, 200, 300 and 400 lg mL1
but they get the opposite results [41–44]. Sasidharan et al. using water and PBS as the positive and negative controls,
found that HRBCs treated with carboxyl-functionalized respectively. The insets: photographic image for direct
graphene did not show any hemolysis up to 75 lg mL1 [41], observation of hemolysis by GO–HA (lower right) and
while Singh et al. reported that GO exhibited significant hemolysis percentages of HRBCs by GO and GO–HA at
hemolytic activity [42]. Herein, hemolysis assays were utilized different concentrations (upper right). (A colour version of
to evaluate the blood compatibility of GO and GO–HA at differ- this figure can be viewed online.)
ent concentrations, as presented in Fig. 5. No hemolytic effect
of GO and GO–HA can be observed visually in the experiment
over a broad concentration range of 0–400 lg mL1. The GO–HA. At the time point of 4 h and 10 days after injection,
hemolytic effect of the samples was further quantitatively blood was drawn for blood smear analysis and biochemistry
determined by measuring the absorbance of the supernatant assays. Upon completion of blood collection, the mice were
at 541 nm (hemoglobin) using UV–vis spectroscopy. Even at a sacrificed, and the main organs were harvested for histologi-
high concentration of 400 lg mL1, the mean hemolytic cal study.
activity of GO and GO–HA has been detected to be as low as Nanoscale materials, whose sizes are within the size range
1.5% and 0.7%, respectively. Consequently, both GO and GO– of viruses and large proteins, may induce inflammatory re-
HA have negligible hemolytic activity. sponses and alter the activity of the immune system and
the hematological factors [45,46]. Therefore, hematological
3.4. In vivo toxicity studies and biochemical analyses were carried out to quantify the po-
tential toxicity of GO–HA after administration. Fig. 6a and b
The GO–HA was intravenously injected into KM mice through show the photomicrographs of the blood smears from blood
tail vein with a dose of 10 mg kg1. Over the entire study per- in vivo treated with physiological saline (control) and with
iod, the observed behaviors of all the treated mice were nor- GO–HA, respectively. The blood smears reveal that the GO–
mal, and no infection, impaired mobility, or reduced food HA treatment does not alter the number and shape of red
taking was observed, indicating a negligible acute toxicity of blood cells, platelet and white blood cells in the test mice as

(a) 120
(b) 120
12 h 24 h 12 h 24 h
100 100
Cell viability %

80 80
Cell viability %

60 60

40 40

20 20

0 0
0 5 25 50 100 200 0 5 25 50 100 200
-1
-1
Concentration (μg mL ) Concentration (μg mL )

Fig. 4 – Viability of HeLa (a) and L929 (b) cells incubated with different concentrations of GO–HA for 12 and 24 h. (A colour
version of this figure can be viewed online.)

compared to the control mice. Meanwhile, more quantitative
evaluation about the influence of GO–HA on the exposed mice
was studied by using the established serum biochemistry as-
says. Three important hepatic indicators, viz. ALT, AST and
TBIL, are at similar levels for the mice exposed to GO–HA
and for the control mice (Fig. 6c). The test groups (4 h and
10 days post-injection) also show no significant difference in
the two indicators for kidney functions (creatinine and BUN)
as compared with the control group (Fig. 6c, P > 0.05). These
results suggest no toxicity of GO–HA in mice at a high expo-
sure level of 10 mg kg1 and at an exposure time of up to
10 days.
To determine whether or not the GO–HA cause tissue dam-
age, inflammation or lesions, the mouse organs including
heart, lung, liver, spleen and kidneys were harvested, sec-
tioned into thin slices, and H&E stained for histological
assessment. In comparison with an untreated control group,
no obvious abnormality can be found in various organs from
the GO–HA-exposed mice (Fig. S5). Although further long-
term studies are needed to fully illustrate the toxicology pro-
files of GO-HA, our preliminary results show that GO-HA has
good biocompatibility.
3.5. In vitro loading and release of DOX
Cancer chemotherapy agents with extended p-structures lar-
ger than one aromatic ring can be efficiently loaded on GO by
strong p–p stacking and hydrophobic interaction [4,6]. Com-
pared with their CNT analogs, graphene derivatives are more
attractive as drug carriers because both sides of a single sheet
can be accessible for drug binding. DOX is one of the most
(a) (b)
(c) Control
150
GO-HA 4 h
GO-HA 10 days
120
Concentration
90
60
30
0.3
0.0
Creatinine BUN ALT
(μM) (mM) -1
(IU L )
(IU L )
Fig. 6 – Blood smear images of the mice at 10 days post-
injection with physiological saline (a) and with GO–HA (b); (c)
serum biochemistry results obtained from mice injected
with physiological saline (10 days post-injection) and with
GO–HA (4 h and 10 days post-injection). (A colour version of
this figure can be viewed online.)
CARBON 6 9 (2 0 1 4) 3 7 9–38 9 385
widely-used chemotherapeutic anticancer drugs, but its
administered dosage is strongly limited by the severe side ef-
fects. In view of the unique structure and properties of GO,
the disadvantages of DOX can be efficaciously resolved by
using functionalized GO as drug carriers. Therefore, herein
DOX was used as a model drug to examine the drug loading
and release properties of GO–HA.
The drug loading of GO–HA was started by mixing GO–HA
with a PBS solution of DOXÆHCl. The UV–vis absorbance at
490 nm (characteristic of DOX) was measured to estimate
the amount of the loaded drug. As indicated in Fig. 7a, the
drug loading ratio (the weight ratio of the loaded drug to
GO–HA) for DOX increases with the increase in initial drug
concentration and finally reaches its saturation value when
the drug concentration is higher than 1000 lg mL1, suggest-
ing that the loading of DOX onto GO–HA can be well con-
trolled. The maximum loading ratio of DOX is up to 81.5%
(815 mg DOX per gram of GO–HA).
The release profile of DOX from the GO–HA/DOX (60.0%
loading ratio of DOX) in PBS buffer (pH 7.4, 6.3 and 5.2) at
37 C is presented in Fig. 7b. Only a little of loaded drug
(6.8% for pH 7.4 and 10.9% for pH 6.3) has been released after
65 h of immersion in PBS at pH 7.4 and 6.3. However, when
the pH of the solution is decreased to 5.2, much more DOX
is released, and the amount of released DOX within 65 h
markedly increases to 26%. This is attributed to the increased
hydrophilicity and solubility of DOX at this pH [6]. The pH-
dependent drug release from the GO–HA is important in prac-
tical bio-applications, since the microenvironments in can-
cerous tissue and intracellular lysosomes and endosomes
are acidic, which potentially facilitates active drug release
from the carriers [47,48]. In addition, it should be noted that
the minimal drug leakage from the GO–HA under normal
physiological conditions will be of great significance because
of the minimized side-effects to normal organs.
3.6. Cellular uptake and cytotoxicity of drug-loaded GO–
HA
HeLa cell line shows very high levels of CD44 protein [30],
while L929 cells are low-expressing for CD44, as indicated in
Fig. S6. Moreover, CD44 on normal cells is usually in a quies-
cent state and does not have the binding activity to HA, while
the tumor cells overexpressing CD44 are in a highly activated
state capable of binding HA [49]. Herein, these two cell lines
were selected to evaluate the targeting effect of GO–HA to tu-
mor cells. The GO–HA/DOX (60.0% loading ratio of DOX) was
incubated with HeLa and L929 cells at 37 C for 3 h, and the
cell uptake efficiency was analyzed by confocal fluorescence
microscopy. As shown in Fig. 8a, strong red fluorescence can
be seen in HeLa cells after incubation with GO–HA/DOX. In
contrast to HeLa cells, very weak fluorescence signal is de-
AST TBIL
-1
(μM) tected in L929 cells (Fig. 8b), indicating that GO–HA/DOX is
up-taken by HeLa cells more efficiently than by L929 cells.
To further confirm the dominant receptor-mediated endocy-
tosis mechanism of the GO–HA/DOX, HeLa cells were pre-
treated with excess free HA and then incubated with
GO–HA/DOX under the same conditions. As a result, HA-
pre-incubated HeLa cells no longer show appreciable fluores-
cence (Fig. 8c), demonstrating the specific uptake of GO–HA/
386 CARBON 6 9 ( 2 0 1 4 ) 3 7 9 –3 8 9

(a) 100 (b) 40

5.2
80 6.3
30
7.4

DOX loading ratio (%)

Drug-released (%)
2.0

60
1.5
20

Absorbance
Incubated
Original solution with GO-HA
40 1.0

0.5 10
20
0.0
300 400 500 600
Wavelength (nm) 0
0
0 500 1000 1500 2000 0 10 20 30 40 50 60 70
-1 Time (h)
Concentration of DOX (μg mL )

Fig. 7 – (a) Plot of loading ratio of DOX to GO–HA versus the drug concentration; (b) release profiles of DOX from GO–HA at pH
7.4, 6.3 and 5.2. The inset in (a): Typical UV–vis spectra of DOX solutions before and after interaction with GO–HA, the
solutions were diluted with PBS prior to the measurements. (A colour version of this figure can be viewed online.)

(a) (b) (c) (d) (e)

a1 b1 c1 d1 e1

a2 b2 c2 d2 e2

a3 b3 c3 d3 e3

Fig. 8 – Confocal fluorescence images of GO–HA/DOX (30 lg mL1) in HeLa (a), L929 (b), and free HA pre-treated HeLa (c) cells
incubated at 37 C for 3 h; (d) confocal fluorescence images of GO–HA/DOX in HeLa cells incubated at 4 C for 3 h and (e)
confocal fluorescence images of GO/DOX (30 lg mL1, with the same concentration of DOX) in HeLa cells incubated at 37 C for
3 h. Scale bar (in c3) corresponds to 20 lm for all images. Red fluorescence (a1–e1): DOX molecules; blue fluorescence (a2–e2):
nuclei stained with DAPI; (a3–e3): merged images of blue and red fluorescence with the bright-field image. (A colour version
of this figure can be viewed online.)

DOX could be blocked by the excess free HA. The above con- GO/DOX show a very weak fluorescence. Therefore, the up-
focal fluorescence imaging results are consistent with the take of GO–HA/DOX to the cancer cells overexpressing HA
data obtained by flow cytometric determination (Fig. S7). Fur- receptors is associated with the HA-receptor-mediated endo-
thermore, when HeLa cells were treated with GO–HA/DOX at cytosis [50]. The grafted HA segments on GO sheets controls
4 C to block the endocytosis process, negligible red the targeted drug delivery to HeLa cells by selective binding
fluorescence can be observed (Fig. 8d). Confocal fluorescence to the HA receptors. The significant selectivity of drug-loaded
images of nontargeted GO/DOX in HeLa cells incubated at GO–HA towards cancer cells with high-expressing HA recep-
37 C for 3 h are also shown in Fig. 8 for comparison tors is vital for chemotherapeutic drug delivery. The contact
(Fig. 8e). Compared to GO–HA/DOX, HeLa cells treated with of CD44 receptor with HA is dominated by hydrogen bonds
 CARBON 6 9 (2 0 1 4) 3 7 9–38 9 387

DOX HA receptor-mediated endocytosis has been blocked by free

120
GO-HA/DOX HA. In our case, a similar cytotoxicity to HeLa cells is observed
GO-HA/DOX + free HA
100 for GO–HA/DOX and free DOX in the concentration range of
* * * 0.1–10.0 lg mL1. Several papers have also reported that the
Cell viability %

80
60
40
20
0 through diffusion [54], whereas the entry route into the cells
0 0.1
Concentration (μg mL )
Fig. 9 – In vitro cytotoxicity of free DOX and GO–HA/DOX
against HeLa cells after 12 h of incubation, together with
viabilities of HA-pre-treated HeLa cells incubated with GO–
HA/DOX (marked as GO–HA/DOX + free HA) for 12 h. *P < 0.01
(GO–HA/DOX group versus GO–HA/DOX + free HA group). (A
colour version of this figure can be viewed online.)
and van der Waals forces rather than electrostatic or aromatic
stacking interactions [51]. The carboxyl groups (hexasaccha-
ride) and the N-acetyl groups in the HA molecule are related
with its binding to HA receptors [51]. The confocal microscopy
observations also suggest that the binding ability of HA with
the receptors on cancer cells has not been inhibited after its
grafting to GO and the subsequent drug loading.
The target-specific delivery of GO–HA/DOX to the cells
with high levels of HA receptors encouraged us to evaluate
the anticancer efficacy of GO–HA/DOX by determining its
in vitro cytotoxicity against HeLa cell line using MTT assays.
Fig. 9 presents the viabilities of HeLa cells against GO–HA/
DOX and against free DOX after incubation for 12 h. The
GO–HA itself has negligible cytotoxic effects on the cancer
cells (Fig. 4), while notable cytotoxicity to the HeLa cells is
observed for GO–HA/DOX. The cytotoxic effects of GO–HA/
DOX increase with the increase of drug concentration. How-
ever, when the GO–HA/DOX was incubated with HA-pre-trea-
ted HeLa cells, the cytotoxicity becomes significantly lower at
DOX concentrations of 1.0–10.0 lg mL1 (P < 0.01), because the
*
DOX-loaded nanocarriers with targeting effect show similar
or lower cytotoxicity to the cancer cells in comparison with
the same incubation concentration of free DOX [6,52,53].
The similarities in cytotoxicity might be attributed to the dif-
ferent uptake mechanisms between free DOX and GO–HA/
DOX. Free DOX molecules directly enter the HeLa cells
1.0 2.0 5.0 10.0
-1
for GO–HA/DOX is associated with HA-receptor-mediated
endocytosis. Although the conjugation of HA facilitates the
uptake of GO–HA/DOX by HeLa cells, it would take additional
time for DOX to be released from the surface of GO–HA com-
pared to free DOX [52,53]. To support this, the time-dependent
intracellular uptake and distribution of DOX for GO–HA/DOX
and free DOX were observed by confocal fluorescence analy-
sis (Figs. S8 and S9). At an incubation time of 0.5 h, HeLa cells
treated with free DOX show red fluorescence, while no red
fluorescence signal is detected for the cells treated with GO–
HA/DOX. When the incubation time is prolonged to 1.5 h,
strong red fluorescence due to the released DOX from GO–
HA is observed mainly in the cytoplasm. The released DOX
from GO–HA diffuses through the nuclear membrane and
accumulates in the nuclear, as indicated by the intense red
fluorescence located in the nucleus after 3 h of incubation.
Although the toxicity of GO–HA/DOX is comparable to that
of free DOX, the good selectivity to the cancer cells high-
expressing HA-receptors and minimal drug leakage under
normal physiological conditions will be of great significance
for practical bio-applications. Targeted drug delivery is be-
lieved to improve the therapeutic efficacy while reducing
the toxic side effects of the drugs [55].
3.7. In vivo cancer therapy
To validate the practical chemotherapy application of drug-
loaded GO–HA in cancer treatments, in vivo anticancer exper-
iments were carried out using the HeLa tumor model on nude
mice. The HeLa tumor-bearing mice were divided into 3

(a) 8 (b) 110

7
105
Body weight change (%)
Relative tumor volume (V/V0)

6
100
5

4 95

3
** 90 Control
2 * ** ** GO-HA
* Control 85
GO-HA/DOX
1 GO-HA
GO-HA/DOX
0 80
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Days Days

Fig. 10 – In vivo antitumor effects of GO–HA/DOX in tumor-bearing nude mice: (a) Tumor growth curves for mice treated with
GO–HA/DOX and GO–HA, in comparison with the blank control. The tumor volumes were normalized to their initial sizes.
*P < 0.05, **P < 0.01 (GO–HA/DOX group versus saline injection group). (b) Body weight (normalized to day 0) curves of different

groups during the experiments. The insets in (a): Representative photos of mice from control group (left) and GO–HA/DOX
group (right) taken at the end of treatment. (A colour version of this figure can be viewed online.)
388 CARBON 6 9 ( 2 0 1 4 ) 3 7 9 –3 8 9
groups. The first group which was given intravenous injection
of saline was used as the control. The second and third groups
were intravenously injected with GO–HA and GO–HA/DOX,
respectively. The mice were monitored for 16 days for their
tumor growth pattern. The change of relative tumor volume
as a function of time is plotted in Fig. 10a, and the typical pho-
tographs of mice from GO–HA/DOX and control groups are
shown in the insets of Fig. 10a. Mice treated only with the sal-
ine experience a rapid growth of tumor volume over the en-
tire experiment period. The mice treated with GO–HA
(material control) do not show tumor regression either, and
the tumor kept on growing. However, the tumor growth of
the mice treated with GO–HA/DOX is suppressed considerably
with an inhibition of 40% by day 16. Because of the enhanced
permeability and retention effect and the specific binding be-
tween the HA and the CD44 receptor expressed on the surface
of tumor cells, the GO–HA/DOX can selectively accumulate in
the malignant tumor issues and inhibit the tumor growth
[56,57]. We also measured the body weight of the mice for
all groups during the treatments (Fig. 10b). For GO–HA and
GO–HA/DOX treated groups, no noticeable weight loss is ob-
served, indicating that the toxicity of treatments is not severe.
The results of the above cancer treatments reveal that anti-
cancer drug loaded GO–HA has great application potential
for in vivo cancer chemotherapy.
4. Conclusions
HA has been covalently conjugated to GO using ADH as a linker
to produce GO–HA, and the application of GO–HA in cancer
treatment has been studied. The in vitro and in vivo toxicity
studies show that the resulting GO–HA exhibits very low cyto-
toxicity, good blood compatibility and no evident toxic effects
in mice. Cellular uptake experiments demonstrate that the
GO–HA can targetedly deliver the anticancer drugs into the
cells by receptor-mediated endocytosis. The GO–HA shows a
high loading capacity for the anticancer drug DOX, and the
resulting GO–HA/DOX exhibits high cytotoxicity to HeLa cells.
Moreover, the chemotherapeutic effects of tumor-bearing mice
intravenously injected with GO–HA/DOX have been studied.
GO–HA/DOX can selectively accumulate in the malignant
tumor issues and inhibit the tumor growth. All these positive
results suggest that the GO–HA is a promising candidate as
anticancer drug carriers for cancer treatment.
Acknowledgements
This work is supported by the National Natural Science Foun-
dation of China (Grant no. 21271130), the Shanghai Natural
Science Foundation (13ZR1430300), the Shanghai Municipal
Education Commission (14ZZ128), and the Opening Project
of State Key Laboratory of High Performance Ceramics and
Superfine Microstructure (SKL201212SIC).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.carbon.201
3.12.039.
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