Nanoscale

View Article Online

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

PAPER

Cite this: Nanoscale, 2015, 7, 18584

View Journal | View Issue

The synergistic eect of folate and RGD dual

ligand of nanographene oxide on tumor
targeting and photothermal therapy in vivo
Cheol Jang, Jong Hyun Lee, Abhishek Sahu and Giyoong Tae*
Eective delivery of nanoparticles to the target site is necessary for successful biomedical applications.
Inecient targeting is a major concern for nanomedicines in cancer therapy. Conjugation of multiple targeting ligands to the nanoparticle surface might further enhance the targeting eciency by a co-operative eect of individual ligands. In this study, a dual ligand targeting nanographene oxide (nGO) was
developed by non-covalent interaction with folate and cRGD functionalized pluronic, which allowed
precise control of ligand number on the nGO surface and ensured stability under physiological conditions.
The tumor targeting abilities of single and dual ligand decorated nGOs were evaluated in vitro by using KB
cells, over-expressing folate and integrin v3 receptors. In vitro cellular uptake analysis by ow cytometry
and confocal laser scanning microscopy showed enhanced uptake of dual ligand modied nGO compared to any of the single ligand modied nGOs. The cellular uptake of dual targeted cRGDFAnGO
was increased by 1.9 and 2.4 folds compared to single targeted cRGDnGO or FAnGO, respectively. The
in vivo biodistribution experiment in a mouse xenograft model also conrmed the synergistic targeting
eect of cRGD and folate dual functionalized nGO. A signicantly higher tumor accumulation of cRGD

Received 29th July 2015,

Accepted 26th September 2015

FAnGO was observed compared to cRGDnGO or FAnGO. The higher tumor accumulation of dual

DOI: 10.1039/c5nr05067g

targeted nGO resulted in complete ablation of tumor tissue through an enhanced photothermal eect by
NIR laser irradiation. Therefore, co-functionalization of a nanoparticle by cRGD and folate is a potentially

www.rsc.org/nanoscale

useful way to enhance the tumor targeting ecacy.

Introduction
Nonspecificity is a major drawback in conventional cancer
chemotherapy. Nonspecific distribution of chemotherapeutic
drugs in the healthy tissues results in high systemic toxicity
and undesirable side eects. It also reduces the potency of the
treatment due to the insucient delivery of the drugs into the
tumor site.1,2 In the past decade, nanoparticles have been
widely investigated as promising drug delivery carriers because
they can passively accumulate in solid tumors through an
enhanced permeation and retention (EPR) eect, resulting
from the leaky blood vessels with a high permeability associated with the extensive angiogenesis induced for sucient
blood supply.3,4 However, the eciency of passive targeting is

School of Materials Science and Engineering, Gwangju Institute of Science and

Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea.
E-mail: gytae@gist.ac.kr
Electronic supplementary information (ESI) available: UV-Vis spectra, photograph, and FTIR spectra of GO and nGO, TGA curve of nGO and PF-nGO, mice
body weight change after photothermal therapy. See DOI: 10.1039/c5nr05067g
Both authors contributed equally.

18584 | Nanoscale, 2015, 7, 1858418594

often limited by the degree of tumor vascularization, which

varies extensively depending on the tumor type or state.2,5 To
overcome these limitations, active targeting strategies were
used, in which the nanoparticle surface was modified with targeting ligands which allow them to bind specifically to the
target cell.3,6,7 Nanoparticles have been coupled with various
tumor targeting ligands to interact with specific receptors that
are overexpressed in the tumor cells. However, the outcome of
this active targeting has not been really impressive, so the
eort and cost involved in decorating a ligand on the surface
may not be appreciated in vivo compared to the simple passive
targeting. The eciency of single ligand conjugated nanoparticles is often unsatisfactory due to a phenomenon known
as receptor saturation.8 To improve the active targeting eect,
researchers took inspiration from nature, where viruses simultaneously target more than one receptors on the cell surface
for strong attachment and ecient delivery of their genetic
material.9 So, dual ligand conjugated nanoparticles were developed, where nanoparticles are coupled with two dierent
ligands on the surface.10,11 One ligand is dedicated specifically
for tumor targeting, whereas the other ligand can be used as a
membrane penetration moiety to increase cellular uptake, an

This journal is The Royal Society of Chemistry 2015

View Article Online

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Nanoscale

intracellular targeting moiety to guide nanoparticles to specific

intracellular compartment, a bloodbrain barrier (BBB) penetrating moiety to deliver nanoparticles to the brain, or another
tumor targeting ligand to enhance tumor targeting
specificity.1114
Recently, nanographene oxide (nGO) has emerged as a very
promising nanomaterial for drug delivery to the tumor tissues
due to its unique physiochemical and photothermal
properties.1517 nGOs have been modified with dierent
ligands to selectively target the cancer cells.1821 But, no
studies have been reported with the dual ligand modified
nGOs. Previously, we investigated the tumor targeting characteristics of folate functionalized nGOs by systematically modulating the folate density on the nGO surface.22 A simple
method for fabrication of tumor targeting nGOs with controllable ligand density was established by adjusting the mixing
ratio of folate conjugated pluronic and unmodified pluronic
during the coating process. Our results suggested the existence
of a critical folate density for tumor targeting in vivo.22
In this study, folic acid (FA) and cyclic RGD (cRGD) peptide
were used as tumor targeting ligands to functionalize nGO. FA
selectively binds to the folate receptor (FR) with a high anity
and overexpression of this receptor was frequently observed in
many types of cancer cells while the expression of FR in most
normal tissues is generally low.23,24 RGD is a cell adhesion
motif that can interact more eciently with overexpressed
integrin receptors (mainly v3), which plays a major role in
tumor-induced angiogenesis, tumor neovascularization, and
tumor metastasis.2527 Although active tumor targeting of nGO
and other nanoparticles by folate or RGD peptide individually
have been studied in many reports, combined use of these two
ligands on cancer targeting has not been systematically
studied.19,22,25,28 We have prepared folate and RGD dual-ligand
functionalized nGOs and investigated its eect on cancer targeting in vitro and in vivo. Moreover, we have also compared
the near infrared (NIR) laser-mediated photothermal therapy
ecacy of single and dual-ligand targeted nGOs in a tumor
xenograft mouse model.

Materials and methods

Materials
Graphene oxide was purchased from Angstron materials Inc.
(Dayton, OH, USA) as a 2% (w/v) solution. Pluronic F127 (PEO
98 PPO 67 PEO 98, MW 12 600) was kindly donated by BASF
Corp. (Seoul, Korea). Chloroacetic acid, sodium hydroxide,
cysteamine hydrochloride, folic acid, N-hydroxysuccinimide
(NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) were purchased from Sigma Aldrich (St. Louis, MO,
USA). The mono-reactive hydroxysuccinimide ester of Cy5.5
(NHS-Cy5.5) was purchased from Amersham Bioscience (Piscataway, NJ, USA). Cyclo(RGDfC) peptide (MW 578.7, 98%
purity) was purchased from ChinaPeptides Co., Ltd (Shanghai,
China). RPMI1640, folate free RPMI1640, fetal bovine serum
(FBS), and penicillinstreptomycin were purchased from Gibco

This journal is The Royal Society of Chemistry 2015

Paper

(Grand Island, NY, USA). WST-8 reagent was purchased from

Dojindo Laboratories (Kumamoto, Japan). SlowFade Gold
Antifade Reagent with DAPI was purchased from Life Technologies (Eugene, OR, USA). KB cell (human epithelial mouth
carcinoma cell from oral cavity) was obtained from Korean Cell
Line Bank (Seoul, Korea).
Preparation of nanographene oxide (nGO)
Nanographene oxide (nGO) was prepared by ultrasonication of
carboxylated graphene oxide as reported previously with some
modifications.29 Chloroacetic acid (400 mg) and NaOH
(400 mg) were added to a graphene oxide solution (15 ml,
1 mg ml1) and the mixture was stirred overnight at 80 C.
After reaction, the mixture was sonicated for 2 h by an ultrasonic probe (750 W, 40% intensity) (Vibra cell VCX750, Sonics
& Materials Inc., Newtown, CT, USA). The sonicated solution
was centrifuged at 3000 rpm for 5 min and the supernatant
was discarded. The precipitate was resuspended in 10 ml of
water and dialyzed (MWCO 3.5 kDa, Spectrum Laboratories
Inc., Rancho Dominguez, CA, USA) against deionized water for
2 days with frequent changes of water. The dialyzed GO suspension was further sonicated and filtered with 0.1 m syringe
filter to obtain nGO stock solution.
Synthesis of cRGD peptide-conjugated pluronic (cRGDPF)
To prepare cRGD peptide-conjugated pluronic, we first
synthesized diacrylated pluronic (DAPF) as previously
reported.30,31 9.3 mg (16 mol) of cyclo(RGDfC) peptide was
reacted with 50 mg (4 mol) of DAPF in 1 ml of phosphate
buer (100 mM, pH 8.0) for 12 h at room temperature.
Unreacted peptide was removed by dialysis (MWCO 3.5 kDa)
for 24 h in deionized water. Purified cRGDPF was lyophilized
and analyzed by 1H NMR (400 MHz, JNM-ECX-400P, JEOL,
Tokyo, Japan) to calculate the conjugation eciency.
Synthesis of folate-conjugated pluronic (FAPF)
Folate-conjugated pluronic (FAPF) was synthesized as previously described with minor modification.22 Briefly, 100 mg
(7.8 mol) of amine-functionalized pluronic was reacted with
69 mg (156 mol) of folate, 24 mg (156 mol) of EDC, and
22 mg (195 mol) of NHS in 30 ml of anhydrous DMSO for
17 h at room temperature in the dark with argon purging. The
crude product was dialyzed (MWCO 3.5 kDa) against phosphate buer ( pH 8.0) at room temperature for 36 h to remove
free folate and DMSO, followed by dialysis in deionized water
for 36 h to eliminate the remaining salts. The resulting
product was lyophilized and analyzed by 1H NMR.
Synthesis of Cy5.5-conjugated pluronic (Cy5.5PF)
To conjugate NIR fluorescent dye Cy5.5 to amine-functionalized pluronic, 1 mg of Cy5.5NHS was mixed with 5 mg of
aminePF. The mixture was stirred at room temperature overnight and dialyzed (MWCO 3.5 kDa) against deionized water
for 12 h to remove the unconjugated dye. Dialyzed solution
was lyophilized and stored at 20 C for further use.

Nanoscale, 2015, 7, 1858418594 | 18585

View Article Online

Paper

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Preparation of single-ligand functionalized nGO

Single-ligand functionalized nGO samples were prepared by
mixing nGO aqueous suspension with cRGDPF (or FAPF)
and unmodified pluronic (PF), and the content of each ligand
was regulated by varying the mixing ratio between cRGDPF
(or FAPF) and unmodified pluronic (PF). The mixture was
placed in a shaking incubator at 37 C overnight to induce
coating of cRGDPF (or FAPF) and PF on nGO by hydrophobic
association between the hydrophobic PPO unit of pluronic and
nGO. After coating, the mixture was dialyzed (MWCO 50 kDa)
against deionized water for 6 h at 37 C to remove free
pluronic.
Preparation of dual-ligand functionalized nGO
The dual-ligand functionalized nGO sample was prepared by
the same method as single-ligand functionalized nGO, except
for using both cRGDPF and FAPF simultaneously. The
content of each ligand was controlled by adjusting the mixing
ratio of RGDPF, FAPF, and unmodified PF.
Characterization of functionalized nGO samples
The hydrodynamic radius and the surface charge of nGO, PF
nGO, cRGDnGO, FAnGO, and cRGDFAnGO samples were
measured by dynamic light scattering (DLS) spectrometer
(ELS-Z2, Otsuka Electronics Co., Osaka, Japan) and transmission electron microscopy (TecnaiG2, FEI Co., Hillsboro,
OR, USA). Thermal degradation profiles of nGO and PFnGO
were recorded using a thermal gravimetric analyzer (TGA4000,
PerkinElmer, Waltham, MA, USA). 5 mg of freeze dried
samples were heated from 30 C to 800 C at a scanning rate of
5 C min1 under N2 purging. The ratios of dierent pluronic
coating on the surface of cRGDnGO, FAnGO, and cRGDFA
nGO samples were analyzed by 1H NMR.
In vitro cytotoxicity assay
Cytotoxicity of pluronic coated nGO was evaluated by the
WST-8 assay. Briefly, KB cells were seeded into a 96 well cell
culture plate (10 000 cells per well) and incubated for 6 h in a
5% CO2 incubator at 37 C. Then, the media was replaced with
200 l of fresh media containing dierent concentrations of
PFnGO. After 24 h incubation, media was removed and cells
were washed with PBS. Next, WST-8 solution was added into
the wells and 100 l of solution from each well was transferred
to a new 96 well microplate after incubation for 1 h at 37 C.
Absorbance of produced formazan was measured by a microplate reader at 450 nm (SpectraMax M2e, Molecular Devices,
Sunnyvale, CA, USA).
In vitro cellular uptake by flow cytometry analysis
KB cells were maintained in normal RPMI1640 media containing 10% FBS and 1% streptomycinpenicillin. Before flow cytometry analysis, KB cells were passaged two times in folate free
RPMI1640 media (10% FBS, 1% antibiotics) and seeded in a
24 well cell culture plate with 5 104 cells per well density.
Seeded cells were incubated for 24 h and media were replaced

18586 | Nanoscale, 2015, 7, 1858418594

Nanoscale

with the media containing functionalized nGO samples (5% of

PF was substituted with Cy5.5PF as a fluorescent probe) at
20 g ml1 of nGO. After 24 h incubation, cells were detached
with 100 l of trypsin and collected by centrifugation (1500
rpm, 4 C, 5 min) and resuspended in 1 ml of ice cold PBS
containing 10% FBS. Cells were directly analyzed by a flow
cytometer (FACS caliber, BD Biosciences, San Jose, CA, USA) at
FL4 channel and 30 000 events were recorded for each sample.
In vitro cellular uptake by confocal laser scanning microscopy
KB cells were passaged two times with folate free RPMI
1640 media and seeded onto round 12 mm gelatin coated
glass coverslips in a 24 well cell culture plate at 5 104
cells per well density. Sample containing media were prepared, similar to the flow cytometry analysis, and cells were
incubated with samples for 24 h at 37 C. Cells were
washed with PBS and fixed with 4% paraformaldehyde solution for 20 min. Cells on the coverslips were counter-stained
with DAPI and mounted on a glass slide. Then, cells were
imaged with confocal microscopy (FV1000, Olympus, Center
Valley, PA, USA).
In vivo biodistribution
An in vivo tumor xenograft model was established by injecting
KB cells (1 107 cells) subcutaneously into the right flank of
athymic nude mouse (male, 6 week old, C3H/HeN, Orient Bio
Inc., Seoul, Korea). Mice were maintained on a low-fluorescence (alfalfa free) diet and handled in accordance with the
guidelines of animal care and use committee of Gwangju Institute of Science and Technology (GIST). Tumors were allowed to
grow to an average volume of 150 mm3. After tumors reached
the desired size, Cy5.5 labeled functionalized nGO samples
(7.5 mg kg1, 200 l) were intravenously (i.v.) injected through
the tail vein. Then, fluorescence signal was monitored by an
in vivo imaging system (IVIS 100, Xenogen Corp., Alameda, CA,
USA) at 6 h, 12 h, 24 h, and 48 h post-injection. Cy5.5 was
excited with 675 nm light and the emission filter was set at
700 nm. The fluorescence exposure time was 1 second and the
field of view was 12.5 cm. Emitted signals were collected by a
time-correlated single-photon counting system. Mice were
sacrificed at 48 h post-injection and major organs were harvested. NIR fluorescence images of the excised tumors and
major organs including livers, spleens, kidneys, hearts, and
lungs were recorded.
NIR laser-mediated in vivo photothermal therapy
KB tumor xenograft mice were prepared and when the average
tumor volume reached 150 mm3, functionalized nGO
samples (7.5 mg kg1, 200 l) were administered (i.v. injection)
through the tail vein. After 12 h of injection, mice were treated
with laser for photothermal therapy (PTT). The tumors of each
mice were irradiated with a continuous wave NIR laser light
(808 nm, Dragon Lasers Co., Changchun, China) for 10 min at
2 W cm2 intensity. Tumor size and body weight of mice were
frequently measured for 21 days post treatment.

This journal is The Royal Society of Chemistry 2015

View Article Online

Nanoscale

Paper

Results

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Preparation of nanographene oxide (nGO)

Nanographene oxide (nGO) was prepared by intensive ultrasonication under strong basic conditions, which reduced the size
of GO from 450 nm to 44 nm (Table 1). The surface charge
(zeta potential) of nGO also dropped from 34.9 1.3 mV to
45.3 4.4 mV due to the increase in carboxyl groups. The
absorbance of nGO increased significantly after carboxylation
and the peak shifted to over 240 nm from 230 nm of original
GO (ESI Fig. 1a). All these characteristics were similar to the
reduction of GO.32 During carboxylation the color of the GO
solution changed from brown to black (ESI Fig. 1b), due to
the released local strain and restoration of the electronic conjugation within graphene sheets by opening of epoxide groups
and hydrolysis of esters on GO under strong basic conditions.33 The restoration of electronic conjugation and
decreased polar functional groups resulted in the increased
hydrophobicity of nGO sheets, which is beneficial for their
interaction with pluronic block copolymers. The decrease in
characteristic FTIR peaks of oxygen containing groups on GO
such as OH (3423 cm1), CO (epoxy, 1223 cm1), CO
(alkoxy, 1223 cm1) after carboxylation also confirmed the
reduction of nGO (ESI Fig. 3).32,33
Preparation of single and dual ligand functionalized nGOs
Diacrylated pluronic and amine functionalized pluronic were
synthesized as previously reported.22,28 Folate was conjugated
to both ends of amine functionalized pluronic through its
-COOH group by EDC/NHS mediated conjugation (Fig. 1a).
1
H NMR analysis of the conjugate confirmed the successful
synthesis of folate conjugated pluronic (FAPF) with the
appearance of three characteristic benzyl group peaks at 6.8,
7.7, and 8.7 ppm (Fig. 2a). The conjugation eciency was over
95% as calculated by comparing the methyl protons of PPO
units (1.7 ppm) and benzyl protons. cRGD conjugated pluronic
was synthesized by the Michael type addition reaction between
the thiol (SH) group of the peptide and the vinyl group of diacrylated pluronic (Fig. 1a).31 The appearance of signature
proton peaks from phenyl groups of phenylalanine at
7.267.42 ppm in the 1H NMR spectra confirmed the successful synthesis of cRGDPF. The conjugation eciency was calculated by comparing the methyl protons from PPO units

Table 1 Mixing ratio of dierent pluronic to prepare single and dual

ligand functionalized nGOs, their average hydrodynamic radius, and
surface charge

Samples

Mixing ratio
(cRGDPF : FAPF : PF)

Size
(nm)

Zeta potential
(mV)

nGO
PFnGO
cRGDnGO
FAnGO
cRGDFAnGO

0:0:0
0 : 0 : 100
25 : 0 : 75
0 : 25 : 50
25 : 25 : 75

44 2
52 3
52 2
49 2
56 3

45.3 4.4
5.6 1.1
2.3 1.9
6.1 0.6
4.6 0.9

This journal is The Royal Society of Chemistry 2015

(1.7 ppm) and phenyl protons (7.267.42 ppm) from phenylalanine and found to be over 95%.
The design concept of single and dual ligand functionalized nGO is illustrated in Fig. 1b. The degree of functionalization of nGO by dierent ligand compositions was controlled
by mixing cRGDPF, FAPF, and unmodified pluronic (PF)
with nGO at dierent ratios as shown in Table 1. After pluronic
coating, the average size of PFnGO increased from 44 2 nm
to 52 3 nm, and the surface charge increased from
45.3 4.4 mV to 5.6 1.1 mV due to the coverage of the nGO
surface by nonionic PF (Table 1). The surface charges of single
and dual-ligand functionalized nGOs were similar to each
other and close to that of non-targeting nGO (PFnGO). By
ligand functionalization, the size of nGOs were similar to nontargeting PFnGO (Table 1), suggesting a minimal eect of targeting ligands at each distal end of PF on the size of the functionalized nGO. The final sizes of single and dual ligand
functionalized nGOs (cRGDnGO, FAnGO, and cRGDFA
nGO) were all similar (Table 1). Electron microscopy (TEM)
was also used to analyze the lateral size of nGO samples. TEM
results showed that there was no significant change in the size
and shape of nGO after pluronic coating (Fig. 2b), which
clearly supports the data obtained by DLS measurement. Thus,
there would be no eect of the size and surface charge on the
tumor targeting among ligand functionalized nGOs. The stability of nGO samples in biological solutions is essential for
better in vivo performance. The pluronic coating is known to
increase the stability of graphene oxide in electrolyte solutions
and it also increases cellular uptake and in vivo biocompatibility of graphene and graphene oxide.34,35 Previously, we have
successfully used pluronic coated nGO for in vivo applications.22,29 The long term stability of dual ligand functionalized cRGDFAnGO was characterized in a serum containing
cell culture media (DMEM + 10% FBS). Up to 24 h, the average
size of nGO increased from 50 nm to 110 nm that was still
small enough for i.v. injection and in vivo applications. Moreover, from 24 h to 15 days, there was no significant change in
the size of cRGDFAnGO (ESI Fig. 2a). The zeta potential
analysis showed no significant change in the surface charge of
the cRGDFAnGO for 15 days (ESI Fig. 2b). This confirmed
the long term stability of pluronic coated nGO samples in biological solutions.
The total amount of pluronic coated on nGO was estimated
by thermogravimetric analyzer (TGA). The TGA curve of PF
nGO showed 86% weight loss in the temperature range
between 300 C and 410 C due to the decomposition of PF
(ESI Fig. 4), implying that almost seven times more pluronic
was coated on nGO by mass.
We decided to use 25% of functionalization of each ligand
to see the combined eect of dual ligand functionalization on
tumor targeting, based on our previous study showing that the
folate conjugation at 10% was not high enough to show the
tumor targeting eect whereas 50% functionalization of folate
alone showed a very significant targeting eect.22 The total
amounts of folate and/or cRGD ligands in nGO were calculated
by 1H NMR analysis (Table 2).

Nanoscale, 2015, 7, 1858418594 | 18587

View Article Online

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Paper

Nanoscale

Fig. 1 (a) Synthesis scheme of cRGDPF and FAPF, (b) Illustration of single (FAnGO, cRGDnGO) and dual (cRGDFAnGO) ligand functionalized nGO.

Fig. 2 (a) NMR spectra of a: FolPF, b: cRGDPF, c: PF 127, d: FAnGO, e: cRGDnGO, f: cRGDFAnGO, (b) transmission electron microscopy
(TEM) images of nGO and PFnGO.

18588 | Nanoscale, 2015, 7, 1858418594

This journal is The Royal Society of Chemistry 2015

View Article Online

Nanoscale

Paper

Table 2 The amount of dierent targeting ligands (cRGD and FA) conjugated to nGO

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Samplesa
FAnGO
cRGDnGO
cRGDFAnGO
a

Final FA or RGD content

(mg mg1 of nGO)

FA or cRGD number
(per mg of nGO)

0.137 0.014
0.177 0.018
FA: 0.144 0.021
RGD: 0.2 0.008

(1.8 0.14) 1017

(1.8 0.17) 1017
FA: (1.98 0.19) 1017
cRGD: (2.05 0.09) 1017

The amount of nGO was same for all the samples (n = 3).

In vitro cellular uptake of single and dual ligand

functionalized nGOs
No cytotoxicity of pluronic functionalized nGOs (PFnGO) up
to 50 g ml1 on KB cells was confirmed by the WST-8 assay
(Fig. 3a). The cellular uptake of single and dual ligand functionalized nGOs was analyzed with KB cells by flow cytometry
and confocal microscopy. The nGO samples were fluorescently
labeled by adding a small amount (0.2 wt%) of Cy5.5 conjugated pluronic (Cy5.5PF) during the coating process. After
24 h incubation with dierent nGO samples, the fluorescence
intensity in the KB cells was quantitatively measured by flow
cytometry analysis. KB cells treated with cRGD or FA decorated
nGOs exhibited enhanced fluorescence intensities compared
to cells treated with non-targeting nGO (PFnGO) (Fig. 3b).
The cellular uptake of single ligand cRGDnGO and FAnGO
were 2.7 fold and 2.1 fold higher than that of non-targeting
PFnGO, respectively (Fig. 3c) suggesting that the single ligand
functionalization of nGOs either by cRGD or FA was eective
in targeting the cells in vitro. On the other hand, dual ligand
functionalized cRGDFAnGO showed 5.1 fold stronger fluorescence intensity than that of PFnGO. Thus dual ligand
functionalization significantly enhanced the cellular uptake of
KB cells compared to either single ligand groups (Fig. 3c). The
mean fluorescence intensity of cells treated with cRGDFA
nGO was 1.9-times and 2.4-times stronger than that of cRGD
nGO and FAnGO, respectively. This result clearly demonstrates the synergistic eect of dual ligand functionalization of
nGO on the uptake of KB cells in vitro compared to single
ligand functionalization.
Confocal laser scanning microscopy (CSLM) was used to
confirm the internalization of ligand functionalized nGOs in
KB cells. After treating the cells with Cy5.5 labeled, single or
dual ligand functionalized nGOs for 24 h, their nuclei were
stained with DAPI. Cy5.5 from internalized nGOs was visualized as red fluorescence and nuclei of the cells were visualized
as blue fluorescence. As shown in Fig. 4, red fluorescence was
clearly observed in the cytoplasm, revealing successful internalization of functionalized nGOs in KB cells. Compared to PF
nGO, an enhanced red fluorescence intensity was observed in
the cytoplasm of cRGDnGO or FAnGO treated cells, showing
the eect of single-ligand functionalization on tumor targeting. As expected, the red fluorescence intensity from cRGD
FAnGO treated cells was stronger than any other groups,
which was well correlated with the flow cytometry data.

This journal is The Royal Society of Chemistry 2015

Fig. 3 (a) Viability of KB cells treated with dierent concentrations of

PFnGO for 24 h, measured by WST-8 assay (n = 3). (b) Flow cytometry
analysis of the cellular uptake of non-targeted (PFnGO), single ligand
targeted (FAnGO, cRGDnGO) and dual ligand targeted (cRGDFA
nGO) samples in KB cells after 24 h incubation. (c) Quantitative analysis
of the cellular uptake of dierent nGO samples as measured by ow
cytometry (n = 3) (**p < 0.001, N.S. not signicant).

In vivo tumor targeting of single and dual ligand

functionalized nGOs
The synergistic tumor targeting eect by dual ligand
functionalization of nGOs was characterized in vivo using a KB
tumor xenograft model. Fluorescently labeled nGO samples
were intravenously injected into tumor-bearing mice, and
monitored by a non-invasive near-infrared optical imaging
technique. Initially at 6 h and 12 h post-injection, the fluorescence signals were spread throughout the body and no
specific localization of the nGOs was observed for all groups
(Fig. 5a). However, at 24 h, clear dierences in the fluorescence
signals at the tumor site were observed from ligand functionalized nGOs compared to the non-targeting nGO group. Among
ligand functionalized groups, the tumor site of dual ligand
functionalized nGO showed a higher level of fluorescence

Nanoscale, 2015, 7, 1858418594 | 18589

View Article Online

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Paper

Fig. 4 Confocal microscopy images of the cellular uptake of uorescently labeled nGO samples. KB cells were treated with dierent nGO
(20 g ml1) samples for 24 h. Blue color: DAPI stained cell nuclei, Red
color: Cy5.5 labeled nGO.

Nanoscale

signal than single ligand functionalized groups. At 48 h, the

fluorescence signal at the tumor site of the mice treated with
non-targeting nGO (PFnGO) was very low and the signals
from the mice treated with FAnGO or cRGDnGO also weakened. In contrast, mice treated with cRGDFAnGO still
showed a strong fluorescence signal at the tumor site. To
characterize the tumor targeting eect of various nGOs more
accurately, ex vivo fluorescence images of the excised tumor
tissues and other major organs were taken after scarifying all
the mice at 48 h post-injection. A strong fluorescence signal
was observed from the excised tumors of the mice administered with cRGDFAnGO compared to the other three groups
(Fig. 5b). Quantitative analysis of the fluorescence signals
obtained from the excised organs revealed that the tumors
exhibit significantly higher signals than any other major
organs whereas livers showed a very weak signal. It proves that
pluronic stabilized nGOs can be an ideal nanomaterial for
long circulation, low nonspecific uptake by the liver, and
superior tumor targeting. The tumor targeting characteristics
of nGOs decorated with a single ligand were similar to those
in in vitro studies. FAnGO and cRGDnGO showed slightly
higher tumor accumulation than non-targeting PFnGO, but
nGO with dual ligands (cRGDFAnGO) exhibited significantly
higher tumor accumulation than any other groups (Fig. 5c).

Fig. 5 (a) In vivo targeting of single and dual ligand functionalized nGOs in KB tumor bearing mice, (b) ex vivo uorescence images of tumor
and other major organs after 48 h post-injection, (c) biodistribution analysis of various nGO samples in tumor and other major organs (n = 3)
(**p < 0.001, *p < 0.05).

18590 | Nanoscale, 2015, 7, 1858418594

This journal is The Royal Society of Chemistry 2015

View Article Online

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Nanoscale

The fluorescence signal from the tumors of cRGDFAnGO

administered mice was 1.7, 2.0, and 2.3 times stronger than
that of cRGDnGO, FAnGO, and PFnGO, respectively
(Fig. 5c). The liver accumulation of all nGO groups was
minimal. Dual ligand functionalization increased the lung
accumulation of nGOs compared to the single ligand or no
ligand groups, although the overall accumulation in the lung
was much weaker than that in the tumor.
In vivo photothermal therapeutic ecacy
The excellent in vivo tumor targeting eect of cRGDFAnGO
encouraged us to investigate its photothermal therapeutic
ecacy on tumor xenograft mice. KB tumor bearing mice were
divided into 5 groups and were intravenously injected with
saline, non-targeted nGO (PFnGO), single ligand functionalized nGOs (FAnGO and cRGDnGO), and dual ligand functionalized nGO (cRGDFAnGO). At 24 h post-injection, the
tumors were exposed to NIR laser irradiation for photothermal
treatment. The temperature increase in the tumor area was
monitored by an IR thermal camera (Fig. 6a). The saline

Paper

injected mice showed a slight temperature increase from 36 C

to 42 C at the tumor site during laser irradiation. In the case
of PFnGO, FAnGO, and cRGDnGO, the temperature at the
tumor site reached 44 C, 47 C, and 50 C, respectively
(Fig. 6b). The temperature at the tumor site of the mice treated
with cRGDFAnGO was much higher than any other groups
and reached 57 C (Fig. 6b). The results of a temperature
increase during laser treatment were well matched with the
in vivo tumor accumulation of dierent nGO groups; increased
nGO accumulation at the tumor resulted in the higher photothermal eect. During laser irradiation, no significant temperature increase was observed in the surrounding tissue near
the tumor, showing that the eect of laser treatment was
highly focused on the tumor site and non-specific tissue
damage would be minimal.
The changes in the tumor volume and body weight were
measured for 21 d after photothermal treatment. The saline
group mice exhibited a rapid growth of tumor volume, followed by the mice treated with non-targeting PFnGO (Fig. 6c).
In comparison, mice treated with single functionalized nGOs

Fig. 6 Photothermal therapy, (a) whole body temperature image, (b) temperature of tumor site, (c) antitumor tumor activity in KB tumor bearing
mice after treatments with saline and various nGOs (d) the photographs of tumors at the end of treatment (*p < 0.05, ***p < 0.0002, N.S. not
signicant).

This journal is The Royal Society of Chemistry 2015

Nanoscale, 2015, 7, 1858418594 | 18591

View Article Online

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Paper

(FAnGO and cRGDnGO) exhibited significant inhibition of

tumor growth, resulting from the higher nGO accumulation
and a better photothermal therapeutic eect than PFnGO.
However, single ligand functionalized nGOs failed to completely eradicate the tumor. The best result was observed for mice
treated with dual ligand decorated cRGDFAnGO, where all
tumors disappeared within 5 d of laser treatment and no
regrowth occurred up to 21 d. The complete tumor ablation by
cRGDFAnGO could be attributed to its higher tumor
accumulation and better photothermal therapeutic eect.
During the photothermal treatment experiment, no mice died
and there was no loss of bodyweight (ESI Fig. 5), revealing the
nontoxicity of nGO samples.

Discussion
The main purpose of active targeting of nanoparticles is to
increase specific cell binding and enhance cellular uptake.
But, conventional active targeting of single ligand conjugated
nanoparticles often results in inadequate therapeutic response
due to the poor endocytosis eciency.36 So, dual ligand conjugated nanosystems were developed to improve the ecacy of
active targeting by simultaneously targeting two dierent
surface receptors overexpressed in tumor tissues.8,10,11 A wide
variety of ligands including proteins, peptides, nucleic acids,
sugars, and vitamins have been used for targeting purpose.6,7
Among them, small molecules and peptides are more attractive because of their small size, high stability, low immunogenicity, and easy conjugation ability.6 Folic acid (FA) and RGD
peptide are two very popular ligands used for active targeting
of dierent nanosystems.2327 In dual ligand targeting studies,
FA or RGD have been used separately with other ligands such
as transferrin, antibodies, cell penetrating peptides, etc.810
But, there is no report of using these two ligands coupled into
a single nanosystem so far. Therefore, in this study, we functionalized nGO with FA and cRGD and compared its targeting
eciency with single ligand functionalized nGOs both in vitro
and in vivo.
Our previous study suggests that the degree of ligand
functionalization on the nGO surface plays a critical role in
the tumor targeting eciency.22 Therefore, we focused on
synthesizing nGO samples with a known density of ligand
functionalization rather than just conjugating them indiscriminately. We have developed a system which allows us to
precisely control the surface functionalization of nGO.22 In
this system, a certain amount of nGO was mixed with nonmodified and functionalized pluronic at particular ratios
(Table 1) and allowed to attach to the nGO surface via
non-covalent hydrophobic interaction between the nGO and
the hydrophobic PPO group of pluronic. As expected, the
final amount of FA and cRGD functionalization onto the
nGO surface was very similar for single and dual ligand
systems (Table 2). Dual ligand functionalization also
increased the total concentration of the ligand on the nGO
surface.

18592 | Nanoscale, 2015, 7, 1858418594

Nanoscale

The cellular uptake of nGOs was analyzed in KB cells,

which are known to overexpress both the folate receptor and
integrin receptor.37,38 Flow cytometry analysis (Fig. 3b and c)
showed that the uptake of cRGDFAnGO was increased by 5.1
fold compared to 2.7 and 2.1 folds of cRGDnGO and FAnGO,
respectively. This enhancement reveals the synergistic eect of
dual ligands on the cellular uptake in vitro. The similar eect
was observed for polymer nanoparticle conjugated with transferrin and cRGD.39 Gold nanoparticles functionalized with
folic acid and glucose or folate receptor (FR) and epidermal
receptor growth factor receptor (EGFR) antibodies also showed
enhanced uptake in cancer cells compared to the single ligand
targeting.40,41 Cancer cells often overexpress multiple receptors
on their surface, which allows a nanoparticle with two
dierent ligands to interact strongly through multivalent interactions and this strong binding leads to enhanced endocytosis,
thus improved cellular uptake.39,41 Apart from the synergistic
eect of two dierent ligands, the ligand density might also
play an important role in the increased targeting eciency.
The increase in ligand density also increases the potential of
strong cellular attachment and endocytosis through multivalent interactions.22,42 However, the increase in cellular
uptake of nanoparticles by the increase in ligand concentration is limited due to the limited amount of a particular
receptor on the target cell surface.43 Therefore, two dierent
ligands on the nGO surface, which increases the target selectively, could be more beneficial than only one ligand.
Our previous study showed that the in vitro eciency of a
targeting system does not always confirm its in vivo
eciency.22 Most of the previous reports of dual ligand targeting of nanoparticles showed mainly in vitro data, and only very
few of them were tested for in vivo eciency.11,14,36,44,45 Therefore, in this study, we further verified the eciency of dual
ligands on our system in a tumor xenograft model in vivo. The
IVIS imaging clearly demonstrated the higher tumor accumulation of cRGDFAnGO compared to cRGDnGO, FAnGO, or
PFnGO (Fig. 5). Some accumulation of non-targeted PFnGO
was observed in the tumor tissue, although it was relatively
low, presumably due to the passive targeting through the EPR
eect.29 Only folate functionalized nGO (FAnGO) did not
show any significant improvement in the tumor accumulation
of nGO compared to non-targeted PFnGO at this folate
density, as we previously reported.22 cRGDnGO showed a
slightly better eect than FAnGO, but still minimal improvement was observed compared to the non-targeted PFnGO. In
contrast, the tumor accumulation of cRGDFAnGO was significantly higher than any other groups, confirming that the
dual ligand functionalization could eectively improve the targeting eciency of nGO in vivo. Since both folate and integrin
(v3) receptors are also present in the normal tissues, it might
cause the slightly enhanced accumulation of cRGDFAnGO in
the lung, too.23,25,46 But, the accumulation of modified nGOs
in any other organs was much lower than that in the tumor.
In a previous study, PEGylated liposome modified with
RGD and interleukin-13 (IL-13) peptide was used for simultaneous targeting of neovascular and the glioma cells.45 RGD

This journal is The Royal Society of Chemistry 2015

View Article Online

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Nanoscale

peptide has a high binding anity to integrin receptors (v3)

which is overexpressed on the neovascular endothelial cells,
whereas IL-13 peptide targets IL13R2, a glioma restricted
receptor.45 Also, polymeric nanoparticle conjugated with cRGD
peptide and transferrin was able to deliver chemotherapeutic
drug more eciently.39 Similarly, cRGD functionalization in
this study could help the accumulation of nGOs in the tumor
by delivering nGOs first to the angiogenic blood vessels in the
tumor and subsequently providing more chances for the
strong attachment and endocytosis to the cancer cells through
cRGD and folate dual ligands.
Functionalized nGO has been used as photothermal agents
for in vivo cancer treatment with encouraging therapeutic outcomes.22,28,29 During the reduction of size of GO by ultrasonication and chemical reaction, the reduction of GO occurs, so
the absorbance in NIR wavelengths increases. The unique physiochemical properties of nGO such as high surface area,
intrinsic high optical absorbance in NIR wavelengths, and
good heat-capacity makes it an excellent choice for photothermal treatment applications.28,47 nGO showed better photothermal anticancer properties than carbon nanotubes, and the
reduced nGO was shown to be an eective photothermal agent
even with ultra-low laser power.48,49 In this study we hypothesized that nGOs with high tumor targeting eciency should
show a better photothermal treatment eect because a higher
localized concentration of nGOs can result in better photothermal eciency. As expected, mice treated with cRGDFAnGO
showed the highest temperature increase during laser irradiation
and resulted in complete removal of the tumor tissue.

Conclusions
An ecient active tumor targeting system was designed by successful functionalization of nGO with folate and cRGD
peptide. The amount of each ligand on the nGO could be precisely controlled by non-covalent association between nGO and
pluronic conjugated with cRGD and folate. The tumor targeting eciency of the dual ligand modified nGO was evaluated
in vitro and in vivo. Dual ligand modification significantly
enhanced the nGO uptake in the cancer cells compared to
single ligand functionalized nGOs. The combination of integrin and folate receptor targeting was also successfully amplified in vivo tumor targeting of nGOs beyond the levels attained
by either ligand alone. Furthermore, the enhanced tumor
localization of nGOs resulted in better photothermal tumor
ablation in vivo. These impressive results suggest that the dual
ligand combination of folate and cRGD might be used to
increase the tumor targeting ecacy of other nanosystems in
addition to nGOs in cancer therapy.

Acknowledgements
This research was financially supported by the National
Research Foundation of Korea (NRF) funded by MEST of Korea
(2013R1A2A2A03068802).

This journal is The Royal Society of Chemistry 2015

Paper

References
1 M. Ferrari, Nat. Rev. Cancer, 2005, 5, 161.
2 D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit
and R. Langer, Nat. Nanotechnol., 2007, 2, 751.
3 N. Bertrand, J. Wu, X. Xu, N. Kamaly and O. C. Farokhzad,
Adv. Drug Delivery Rev., 2014, 66, 2.
4 H. Maeda, J. Wu, T. Sawa, Y. Matsumura and K. Hori,
J. Controlled Release, 2000, 65, 271.
5 U. Prabhakar, H. Maeda, R. K. Jain, E. M. Sevick-Muraca,
W. Zamboni, O. C. Farokhzad, S. T. Barry, A. Gabizon,
P. Grodzinski and D. C. Blakey, Cancer Res., 2013, 73, 2412.
6 T. M. Allen, Nat. Rev. Cancer, 2002, 2, 750.
7 J. Y. Yhee, S. Lee and K. Kim, Nanoscale, 2014, 6, 13383.
8 G. Kibria, H. Hatakeyama, N. Ohga, K. Hida and
H. Harashima, J. Controlled Release, 2011, 153, 141.
9 Y. Nie, D. Schaert, W. Rdl, M. Ogris, E. Wagner and
M. Gnther, J. Controlled Release, 2011, 152, 127.
10 J. M. Saul, A. V. Annapragada and R. V. Bellamkonda,
J. Controlled Release, 2006, 114, 277.
11 J. Yu, X. Xie, X. Xu, L. Zhang, X. Zhou, H. Yu, P. Wu,
T. Wang, X. Che and Z. Hu, J. Mater. Chem. B, 2014, 2,
2114.
12 L. Mei, Q. Zhang, Y. Yang, Q. He and H. Gao, Int. J. Pharm.,
2014, 474, 95.
13 Y. C. Chen, C. F. Chiang, L. F. Chen, P. C. Liang,
W. Y. Hsieh and W. L. Lin, Biomaterials, 2014, 35, 4066.
14 X. Ying, H. Wen, W. L. Lu, J. Du, J. Guo, W. Tian, Y. Men,
Y. Zhang, R. J. Li, T. Y. Yang, D. W. Shang, J. N. Lou,
L. R. Zhang and Q. Zhang, J. Controlled Release, 2010, 141,
183.
15 Y. Wang, Z. Li, J. Wang, J. Li and Y. Lin, Trends Biotechnol.,
2011, 29, 205.
16 S. Goenka, V. Sant and S. Sant, J. Controlled Release, 2014,
173, 75.
17 J. Liu, L. Cui and D. Losic, Acta Biomater., 2013, 9, 9243.
18 S. Shi, K. Yang, H. Hong, H. F. Valdovinos, T. R. Nayak,
Y. Zhang, C. P. Theuer, T. E. Barnhart, Z. Liu and W. Cai,
Biomaterials, 2013, 34, 3002.
19 X. Yang, G. Niu, X. Cao, Y. Wen, R. Xiang, H. Duan and
Y. Chen, J. Mater. Chem., 2012, 22, 6649.
20 H. S. Jung, M.-Y. Lee, W. H. Kong, I. H. Do and S. K. Hahn,
RSC Adv., 2014, 4, 14197.
21 S. Shi, K. Yang, H. Hong, F. Chen, H. F. Valdovinos,
S. Goel, T. E. Barnhart, Z. Liu and W. Cai, Biomaterials,
2015, 39, 39.
22 J. H. Lee, A. Sahu, C. Jang and G. Tae, J. Controlled Release,
2015, 209, 219.
23 J. Sudimack and R. J. Lee, Adv. Drug Delivery Rev., 2000, 41,
147.
24 X. C. Qin, Z. Y. Guo, Z. M. Liu, W. Zhang, M. M. Wan and
B. W. Yang, J. Photochem. Photobiol., B, 2013, 120, 156.
25 F. Danhier, A. L. Breton and V. Prat, Mol. Pharm., 2012, 9,
2961.
26 F. Wang, Y. Shen, W. Zhang, M. Li, Y. Wang, D. Zhou and
S. Guo, J. Controlled Release, 2014, 196, 37.

Nanoscale, 2015, 7, 1858418594 | 18593

View Article Online

Published on 07 October 2015. Downloaded by Nanyang Technological University on 27/09/2016 11:30:00.

Paper

27 F. Wang, W. Zhang, Y. Shen, Q. Huang, D. Zhou and

S. Guo, Acta Biomater., 2015, 23, 136.
28 J. T. Robinson, S. M. Tabakman, Y. Liang, H. Wang,
H. Sanchez Casalongue, D. Vinh and H. Dai, J. Am. Chem.
Soc., 2011, 133, 6825.
29 A. Sahu, W. I. Choi, J. H. Lee and G. Tae, Biomaterials,
2013, 34, 6239.
30 S. Y. Lee and G. Tae, J. Controlled Release, 2007, 119, 313.
31 J. Y. Kim, W. I. Choi, Y. H. Kim, G. Tae, S. Y. Lee, K. Kim
and I. C. Kwon, J. Controlled Release, 2010, 147, 109.
32 S. Pei and H.-M. Cheng, Carbon, 2012, 50, 3210.
33 X. Fan, W. Peng, Y. Li, X. Li, S. Wang, G. Zhang and
F. Zhang, Adv. Mater., 2008, 20, 4490.
34 B. J. Hong, O. C. Compton, Z. An, I. Eryazici and
S. T. Nguyen, ACS Nano, 2012, 6, 63.
35 M. C. Duch, G. R. S. Budinger, Y. T. Liang, S. Soberanes,
D. Urich, S. E. Chiarella, L. A. Campochiaro, A. Gonzalez,
N. S. Chandel, M. C. Hersam and G. M. Mutlu, Nano Lett.,
2011, 11, 5201.
36 J. L. Crisp, E. N. Savariar, H. L. Glasgow, L. G. Ellies,
M. A. Whitney and R. Y. Tsien, Mol. Cancer Ther., 2014, 13,
1514.
37 J. A. Reddy and P. S. Low, J. Controlled Release, 2000, 64, 27.
38 R. Shukla, T. P. Thomas, J. Peters, A. Kotlyar, A. Myc and
J. R. Baker Jr., Chem. Commun., 2005, 5739.

18594 | Nanoscale, 2015, 7, 1858418594

Nanoscale

39 Q. Xu, Y. Liu, S. Su, W. Li, C. Chen and Y. Wu, Biomaterials,

2012, 33, 1627.
40 X. Li, H. Zhou, L. Yang, G. Du, A. S. Pai-Panandiker,
X. Huang and B. Yan, Biomaterials, 2011, 32, 2540.
41 S. Bhattacharyya, J. A. Khan, G. L. Curran, J. D. Robertson,
R. Bhattacharya and P. Mukherjee, Adv. Mater., 2011, 23,
5034.
42 S. Hong, P. R. Leroueil, I. J. Majoros, B. G. Orr, J. R. Baker
Jr. and M. M. B. Holl, Chem. Biol., 2007, 14, 107.
43 N. Kamaly, Z. Xiao, P. M. Valencia, A. F. Radovic-Moreno
and O. C. Farokhzad, Chem. Soc. Rev., 2012, 41, 2971.
44 J. Q. Gao, Q. Lv, L. M. Li, X. J. Tang, F. Z. Li, Y. L. Hu and
M. Han, Biomaterials, 2013, 34, 5628.
45 H. Gao, Z. Yang, S. Cao, Y. Xiong, S. Zhang, Z. Pang and
X. Jiang, Biomaterials, 2014, 35, 2374.
46 B. Singh, C. Fu and J. Bhattacharya, Am. J. Physiol.: Lung
Cell. Mol. Physiol., 2000, 278, L217.
47 K. Yang, L. Feng, X. Shi and Z. Liu, Chem. Soc. Rev., 2013,
42, 530.
48 Z. M. Markovic, L. M. Harhaji-Trajkovic, B. M. TodorovicMarkovic, D. P. Kepi, K. M. Arsikin, S. P. Jovanovi,
A. C. Pantovic, M. D. Dramicanin and V. S. Trajkovic, Biomaterials, 2011, 32, 1121.
49 K. Yang, J. Wan, S. Zhang, B. Tian, Y. Zhang and Z. Liu, Biomaterials, 2012, 33, 2206.

This journal is The Royal Society of Chemistry 2015