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Aptamer–conjugated graphene oxide–gold

nanocomposites for targeted chemo-
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Cite this: J. Mater. Chem. B, 2015,

3, 4036 photothermal therapy of cancer cells†
Xinhuan Wang,‡a Qiusen Han,‡a Ning Yu,*b Jingying Li,a Lin Yang,a Rong Yang*a
and Chen Wanga

Received 19th January 2015,
Accepted 9th April 2015
DOI: 10.1039/c5tb00134j
www.rsc.org/MaterialsB
The current cancer therapies in clinical practice demonstrate the need for improvements such as improving
the efficiency and reducing the severe side effects. Herein, we integrated the targeted chemotherapy and
photothermal therapy in a multifunctional drug-delivery platform. The targeting DNA aptamer (Apt)–modified
graphene oxide–gold nanoparticle (GO–AuNP) composites were successfully synthesized. The doxorubicin
(DOX)–loaded GO–AuNP–Apt system showed heat-stimulative and sustained release characteristics. In vitro
cell cytotoxicity experiments showed that combined therapy had the highest rate of death of tumor cells
compared to that of single photothermal therapy or chemotherapy. Furthermore, aptamer-modification
could significantly enhance the accumulation of nanocomposites within cancer cells. Our study demonstrates
that aptamer–modified GO–Au nanocomposites may have potential in the development of targeted photo-
thermal therapy and chemotherapy against cancer cells.

1. Introduction In order to deliver drugs to tumor cells, targeting ligands are

Current cancer therapies in clinical practice consist of surgery,
chemotherapy, and radiation therapy.1 Chemo- and radiation
therapies often damage normal tissues while killing the cancer
cells, resulting in many side effects.2–4 Recently, thermotherapy
has been explored as a non-invasive adjunctive therapeutic
approach triggered by light. Photothermal therapy5,6 uses photo-
absorbing agents to transfer the absorbed light energy into heat
and damage tumor cells. A good drug-delivery system that has
combined functions of both the chemo- and photothermal
therapy would be highly desirable.
Due to its novel physical and chemical properties, graphene has
inspired extensive studies in different fields. In the biomedical
fields, graphene and graphene oxides (GO) have been utilized in
biosensing, bio-imaging probes, and drug carriers for cancer
therapy.7–10 Due to their high optical absorption in the near-
infrared (NIR) region, graphene and GO nanosheets show potential
ability for photothermal therapy in vitro and in vivo.11–17 Targeted
delivery of graphene oxides to tumor cells may further improve the
efficacy and reduce the side effects associated with thermotherapy.
a
CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety,
National Center for Nanoscience and Technology, Beijing 100190, P. R. China.
E-mail: yangr@nanoctr.cn
b
Chinese PLA General Hospial, Beijing 100853, China. E-mail: yuning12@sina.com
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5tb00134j
‡ These authors contributed equally to this work.
needed. Currently used tumor-targeting molecules include
antibodies,18 small molecules,19 short peptides,20 and apta-
mers.21–24 Antibodies and peptides may cause the immune
response of the host.
Aptamers25–30 are folded single-strand DNA or RNA oligo-
nucleotides. Similar to antibodies, aptamers can bind to targets
with high affinity and specificity. Aptamers have been shown to
have high stability, reproducibility, reduced immunogenicity
and toxicity associated with biological therapeutics commonly.
Furthermore, the adaptability and ease of modification of
aptamers offer them an advantage over other technologies.
In this study, we synthesized graphene oxide–gold nanoparticle
(GO–AuNP) composites that were modified with a MUC1 aptamer
(Apt) termed S2.2,31 for synergistically targeted chemo-photothermal
therapy. MUC1 is a large transmembrane glycoprotein,32–36 and
known as a tumor marker present in a variety of malignant tumors,
including breast, stomach, lung, prostate, colorectal and other
cancers. MUC1 may be used as a valuable therapeutic target.
In our previous studies, the GO–Au nanocomposites37 were
synthesized by the pulsed laser ablation method.38 The results
demonstrated that GO–Au nanocomposites could significantly
inhibit the aggregation of the amyloid peptides associated with
Alzheimer’s disease.
In this work, we prepared the aptamer–modified GO–Au nano-
composites, then loaded doxorubicin (DOX) onto them. DOX may
intercalate into the double-stranded regions and form a physical
complex with the aptamers through noncovalent intercalation.

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Meanwhile, GO can also adsorb DOX via p–p stacking and
hydrophobic interactions.13 The GO–Au–Apt–DOX system could
selectively attach to MUC1-overexpressing cancer cells, and greatly
enhance the targeted thermotherapy to these cancer cells when
exposed to NIR light.
2. Experimental section
2.1 Materials
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0
4 -6-Diamidino-2-phenylindole (DAPI) was purchased from Sigma
Aldrich. Dimethyl sulfoxide and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) were bought from Amresco
(USA). DMEM medium and fetal bovine serums were purchased
from HyClone (USA). All aptamers were synthesized by Sangon
Biotech (Shanghai) Co., Ltd. S2.2 aptamer: 5 0 -HS-GCA GTT GAT
CCT TTG GAT ACC CTG-FAM-3 0 .
2.2 Synthesis of graphene oxide–gold (GO–Au)
nanocomposites
GO was synthesized using a modified Hummers method.31 Then,
GO–Au nanocomposites were prepared by the laser ablation
technique. Briefly, a gold metal plate (499.99%) was placed on
the bottom of a centrifuge tube filled with 7 mL of GO solution
with the concentration of 40 mg L1. The laser ablation was
performed using a Nd:YAG pulsed laser (532 nm, 10 Hz, pulse
energy of 100 mJ and pulse duration between 6 and 9 ns). The
laser beam was focused on the gold plate through a lens with a
focal length of 250 mm. The ablation time is 10 minutes. The
gold atoms ejected from the gold plate surface by the laser beam
were decorated on GO layers immediately.
2.3 Characterization
The morphology of GO–Au nanocomposites was examined by
transmission electron microscopy (TEM). The optical absorption
spectra of the nanocomposites were recorded using a UV-Vis-NIR
spectrometer. The Zeta potential measurements of the nano-
composites were performed using a Malvern Zeta instrument.
The chemical states of GO–Au nanocomposites were studied by
X-ray photoelectron spectroscopy (XPS) using an XPS spectro-
meter (ESCALAB 250Xi).The Raman spectra of GO and GO–Au
nanocomposites were collected with 514 nm excitation wave-
length (Renishaw).
2.4 Preparation of aptamer and drug functionalized GO–Au
nanocomposites
Aptamer–functionalized GO–Au composites were prepared as
follows: prior to use, the disulfide–functionalized aptamers S2.2
were deprotected by tris(2-carboxyethyl) phosphine (TCEP). Then,
the aptamers were added to 500 mL of GO–Au colloids. The
solution was shaken at 150 rpm overnight and aged in 0.7 M
NaCl for another 12 h. Then, GO–Au–Apt composites were
purified two times by centrifugation at 20 000 rpm.
Doxorubicin (DOX) loading onto GO–Au–Apt composites was
done by mixing 0.05 mmol L1 of DOX with the GO–Au–Apt solution
for 2 h. Unbound excess DOX was removed by centrifugation
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at 20 000 rpm for 15 min. The resulting GO–Au–Apt–DOX
complexes were resuspended and stored at 4 1C.
The fluorescence spectra of the nanocomposites were collected
using a Hitachi FP-4500 fluorescence spectrophotometer. An excita-
tion wavelength of 488 nm (slit width = 5 nm) was used and data
were collected over 500–700 nm (slit width = 5 nm).
2.5 Drug release
In the in vitro drug release experiment, 0.5 mL of GO–Au–Apt–DOX
solution was transferred to dialysis vials (3.5 kDa) and dialyzed
against PBS buffer. At selected time intervals, buffer solution
outside the dialysis vials was collected for UV-Vis analysis at
480 nm and replaced with fresh buffer solution.
2.6 Cell viability assay
A549 and MCF7 cells (ATCC, USA) were cultured in F12 and
DMEM (Hyclone) with 10% (v/v) fetal bovine serum (Hyclone),
containing 100 IU mL1 penicillin and 100 mg mL1 streptomycin
(Gibco). The cells were incubated at 37 1C in a humidified
incubator (Thermo) containing 5% CO2 in 75 cm3 tissue
culture-treated flasks (Corning).
A549 and MCF7 cells (10 000 cells per well, 100 mL) were
seeded in 96-well plates (Corning) in full medium and incubated
overnight at 37 1C. Then two cell lines were treated with GO–Au,
GO–Au–Apt and GO–Au–Apt–DOX at different concentrations for
48 h, respectively. To study the effects of NIR light irradiation,
two cell lines were incubated with GO–Au, GO–Au–Apt and
GO–Au–Apt–DOX at different concentrations for 12 h. Then the
cells were irradiated with NIR light for 0, 5, 10, 15 min, respectively.
After NIR irradiation, cells were incubated for another 24 h. The
cell toxicity was studied by the MTT assay. The absorbance was
recorded at 490 nm using a microplate reader.
2.7 Cell uptake study
The fluorescence of DOX was used to detect the localization of
DOX in the cells. DOX can be excited by 488 nm light and give
out a fluorescence signal at around 530 nm. DAPI was employed
to stain the nucleus and it is excited by 340 nm light and gives
out a fluorescence signal at around 380 nm.
3. Results and discussion
3.1 Synthesis of graphene oxide–gold (GO–Au)
nanocomposites
The fabrication process of GO–Au nanocomposites is shown in
Fig. 1a. A gold plate in GO solutions is ablated by an intense
laser beam, resulting in ejection of its constituents to form the
GO–Au nanocomposites.
The morphology of the GO–Au nanocomposites was recorded
by TEM. The TEM image in Fig. 1b shows that AuNPs distribute
uniformly on the GO sheet without obvious aggregation. The size
of AuNPs is about 10 nm in diameter. (The SEM image of GO is
shown in Fig. S2, ESI†) XPS was used to study the chemical state
of GO–Au nanocomposites. Fig. 1c shows the XPS spectra of GO
and GO–AuNP nanocomposites. In the spectrum of GO–AuNP
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Fig. 1 (a) A schematic illustration showing the synthetic route to anchor
AuNPs onto GO sheets by pulsed laser ablation of a gold plate in GO
solutions. (b) TEM image of GO and GO–Au nanocomposites. The red
arrow indicates an AuNP in the nanocomposites. (c) XPS spectra and
(d) Raman spectra of GO–Au nanocomposites.
nanocomposites, one peak of Au (4f) was observed at the
binding energy of 84.5 eV, which suggests that AuNPs have
been decorated on GO layers.
Raman spectroscopy was applied to investigate the carbon
structure of GO before and after being conjugated with Au nano-
particles. As shown in Fig. 1d, the D band at 1350 cm1 can be
clearly observed, which is usually assigned to the local defects/
disorders; while the G band at 1580 cm1 is attributed to the sp2
graphitized structure.39 One can see that after AuNPs deposited on
GO sheets, the ID/IG ratio did not increase, indicating that GO was
still with a good oxidized state and did not reduce into rGO in the
GO–AuNP nanocomposites prepared using the PLA method.
3.2 Characterization of the GO–Au–Apt–DOX
In this work, aptamers S2.2 and DOX were conjugated to GO–Au
surfaces. The nanocomposites are composed of three impor-
tant parts as shown in Fig. 2a: (1) a thiol linking the aptamers to
the GO–AuNP surface by formation of thiol–Au bonds; (2) an
aptamer segment S2.2 (Fig. S1, ESI†) to specifically bind target
cancer cells; (3) DOX was introduced to intercalate within the
aptamers and adsorbed onto GO–Au surfaces.
UV-Vis-NIR spectra showed that the optical absorption peak of
the nanocomposites at 230 nm, originating from the p-plasmon
of carbon, remained essentially unchanged after aptamers and
DOX conjugation. The plasmon absorption of AuNPs at 520 nm
was also changed a little. It is also noticed that all the three
samples show strong absorption in the NIR region at around
880 nm, indicating that nanocomposites could be used as
effective photothermal agents (Fig. 2b).
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The zeta potential of the GO–AuNPs was 6.12 mV, indicat-
ing that they were negatively charged. After the modification of
aptamers, the GO–Au–Apt showed a more negative zeta-
potential (11.1 mV) than GO–AuNPs alone since DNA was
negatively charged. Furthermore, the conjugation of DOX led to
a change in the zeta potential to 2.14 mV. These results suggest
that aptamers and DOX were sequentially introduced onto the
surface of GO–AuNPs and GO–Au–Apt–DOX nanocomposites
formed (Fig. 2c). The conjugation of aptamers and DOX onto
GO–AuNPs was also confirmed by fluorescence spectra.
The physiological stability of as-prepared GO–AuNPs before
and after aptamer conjugation and DOX loading was also
studied. The GO–AuNPs and GO–Au–Apt–DOX were centrifuged
at 20 000 rpm for 15 min and then resuspended with water, PBS
and cell medium, respectively. Fig. S3 (ESI†) shows the photos
of GO–AuNPs and GO–Au–Apt–DOX (ESI†). They were very
stable without showing any agglomeration in water, PBS or
serum after centrifugation at 1000 rpm for 5 min. The loading
efficiency of DOX was determined by the UV spectrum at
594 nm. The adsorption measurements of the original solution
of DOX and the supernatant were recorded to determine the
amount of DOX that was loaded in the nanocomposites, and
the loading efficiency was calculated. It was about 11.3%.
To test the photothermal conversion efficiency of the GO–
AuNPs, the solution with different concentrations (2.7, 5.3 and
8 mg L1) was irradiated under NIR light for different times
(Fig. 2d). The photothermal heating curves were recorded using
a thermometer, which showed a dose-dependent photothermal
effect of the GO–AuNPs with the highest temperature up to
48 1C. In contrast, only B3 1C was increased for pure water after
being irradiated for 15 min.
A study of the drug release from the GO–Au–Apt–DOX over
time was conducted in phosphate buffer solution. As shown
in Fig. 2e, about 40% DOX was released from the GO–Au–Apt–
DOX complex at 25 1C after 7 h. After NIR light irradiation and
the temperature of the solution increased to 48 1C, more than
80% DOX release was observed after 2 h. The results indicate
that the heat decreases the interactions between DOX and
aptamers and more DOX molecules can be released. The NIR
irradiation time-dependent release profile of DOX also showed
that the NIR light irradiation could promote DOX release as
shown in Fig. S4 (ESI†).
3.3 Cell uptake of the GO–Au–Apt–DOX
To evaluate the feasibility of the GO–Au–Apt–DOX complexes as
a targeted drug-delivery platform, we performed in vitro binding
and uptake studies using MCF7, A549 cells (MUC1-positive
cells), and HepG2 cells (MUC1-negative cells). Before the cell
uptake study of the GO–Au–Apt–DOX, we carried out the flow
cytometric assay to study the expression level of the MUC1
protein using anti-MUC1 monoclonal antibodies for control
cells and target cells. The MUC1 expression level for both
MUC1-positive cells and MUC1-negative cells showed little
change before and after the treatment of GO–Au–Apt–DOX as
shown in Fig. S5 (ESI†).
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Fig. 2 (a) A schematic illustration of the fabrication of GO–Au–Apt–DOX composites. (b) UV-Vis absorption spectra of GO–Au, GO–Au–Apt and GO–
Au–Apt–DOX. (c) Zeta potentials of GO–Au, GO–Au–Apt and GO–Au–Apt–DOX. (d) The photothermal profile of GO–Au with different concentrations
triggered by NIR light irradiation. (e) Time-dependent release profile of DOX from the GO–Au–Apt–DOX.

Since DOX is a fluorescent molecule, it can be used to
examine whether the drug is delivered to cells. After incubating
MCF7, A549 and HepG2 cells with GO–Au–Apt–DOX nanocom-
posites, uptake of DOX by each cell line was studied by confocal
fluorescence microscopy. In the fluorescence images of the
incubated cells shown in Fig. 3, strong red fluorescence was
seen in MCF7 and A549 cells, while almost no red fluorescence
was observed in HepG2 cells. The results indicate that selective
delivery of DOX to MUC1-positive cells was attained.
3.4 Targeted chemo- and photothermal therapy of the
GO–Au–Apt–DOX
We next examined whether the targeted delivery of the GO–Au–
Apt–DOX to MUC1-positive cells can enhance cellular cytotoxicity.
The MTT assay (Fig. 4a and b) and fluorescence imaging (Fig. 4c)
were used to study the therapeutic results with and without NIR
light irradiation. Fig. 4a shows the viability of MCF7 cells in the
presence of GO–Au, GO–Au–Apt, and GO–Au–Apt–DOX at differ-
ent concentrations. GO–Au had no obvious effect on cell viability
at concentrations up to 8.0 mg L1. It is noticed that the
modification of aptamers to GO–Au surfaces increased the cell
biocompatibility of the nanocomposites, whereas the GO–Au–Apt–
DOX exhibited a notably cytotoxic effect at 8.0 mg L1.
Then we evaluated the effects of NIR light irradiation. MCF7
cells were cultured with the nanocomposites for 12 h and then
exposed to the NIR light for 15 min. One can see from Fig. 4b
that a significant cytotoxic effect at 8.0 mg L1 of the nano-
composites was observed. Such dramatically enhanced thera-
peutic efficacy of GO–Au–Apt–DOX is attributed to the targeting
effect, which greatly promoted cellular uptake, and then the
combination effects of chemo- and photothermal therapy. The
viability of MCF7 cells incubated with different concentrations of

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Fig. 3 Confocal fluorescence images of MCF-7, A549 and HepG2 cells
after incubation with GO–Au–Apt–DOX. All cells were labeled using DAPI
(blue). The red fluorescence was from DOX.
aptamers, GO–Au–Apt and GO–Au–Apt–DOX with and without NIR
light irradiation for 15 min is shown in Fig. S6 (ESI†).
Fig. 4 Cell viability of MCF7 cells incubated with different concentrations of GO–Au, GO–Au–Apt and GO–Au–Apt–DOX without (a) and with (b) NIR light
irradiation for 15 min. (c) Fluorescence images of MCF-7 cells incubated with GO–Au, GO–Au–Apt and GO–Au–Apt–DOX (concentration of 8 mg L1) with
or without NIR light irradiation for 15 min.
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Cell viability was also analyzed by fluorescence imaging
(Fig. 4c). For untreated MCF7 cells, 15 min irradiation
with the NIR light did not increase cell death. In the case of
treated cells, the GO–Au–Apt treatment increases more in cell
death than the GO–AuNPs after NIR light irradiation. For the
GO–Au–Apt–DOX-incubated cells, a significant increase in cell
death and an obvious loss of cell viability were observed after
irradiation. The results agreed well with MTT results. The
fluorescence images of MCF7 cells irradiated with NIR light
for different times with or without GO–Au–Apt–DOX at different
concentrations are shown in Fig. S7 (ESI†).
We also performed an irradiation time-dependent MTT
assay to understand whether the efficiency of the photothermal
therapy depends on the irradiation time. The viability of MCF7
cells was 90% after 5 min of irradiation, and decreased rapidly
as the irradiation time increased. Almost 80% of the cancer
cells were dead after 15 min of irradiation (Fig. S8, ESI†).
After successfully demonstrating that the targeted delivery of
drugs and of nanocomposites can greatly enhance in MCF7 cell
death, we also studied the combination efficacy of targeting,
chemical and thermal therapy of the GO–Au–Apt–DOX to A549
cells which are also MUC1-positive cells. We incubated A549
cells with the GO–Au–Apt–DOX at different concentrations and
irradiated them with NIR light for 15 minutes. Similarly, the
results of the MTT assay (Fig. 5) and the fluorescence micro-
scopy study (Fig. S9 and S10, ESI†) also showed that combining
photothermal therapy and targeted drug delivery could enhance
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Fig. 5 Cell viability of A549 cells with different concentrations of GO–Au, GO–Au–Apt and GO–Au–Apt–DOX without (a) or with (b) NIR irradiation for
15 min.
the cellular cytotoxicity of MUC1-overexpressed cancer cells.
The cell viability of A549 cells with different concentrations of
aptamer, GO–Au–Apt and GO–Au–Apt–DOX with and without
NIR irradiation for 15 min is shown in Fig. S11 (ESI†).
4. Conclusions
In summary, novel multifunctional nanocomposites were success-
fully constructed by attaching MUC1 aptamers to GO–AuNPs.
The GO–Au–Apt nanocomposites could selectively attach to
MUC1-overexpressing cancer cells, and greatly enhance the
targeted thermotherapy to these cancer cells when exposed to
NIR light. Our results suggest that GO–AuNP–Apt composites
may have great potential in biomedical applications.
Acknowledgements
This work is supported by the National Natural Science Founda-
tion of China (21261130090) and the Chinese Academy of Sciences
(XDA09030303). Financial support from CAS Key Laboratory
for Biological Effects of Nanomaterials and Nanosafety is also
gratefully acknowledged.
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