View Article Online

View Journal

Nanoscale
Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: X. Wang, W. Gao,
H. Fan, D. Ding, X. Lai, Y. Zou, L. Chen, Z. Chen and W. Tan, Nanoscale, 2016, DOI:
10.1039/C6NR00369A.

This is an Accepted Manuscript, which has been through the

Royal Society of Chemistry peer review process and has been
accepted for publication.

Accepted Manuscripts are published online shortly after

acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.

You can find more information about Accepted Manuscripts in the

Information for Authors.

Please note that technical editing may introduce minor changes

to the text and/or graphics, which may alter content. The journals
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.

www.rsc.org/nanoscale
 Please do not adjust margins
Page 1 of 7 Nanoscale
View Article Online
DOI: 10.1039/C6NR00369A

Journal Name

PAPER

Simultaneous tracking of drug molecules and carriers using

aptamer- functionalized fluorescent superstable gold
Published on 09 March 2016. Downloaded by Gazi Universitesi on 09/03/2016 11:41:54.

Received 00th January 20xx, nanorod-carbon nanocapsules during thermo-chemotherapy

Accepted 00th January 20xx

Xue-Wei Wang,,a Wei Gao,,a Huanhuan Fan,a Ding Ding,a Xiao-Fang Lai,a Yu-Xiu Zou,a

Nanoscale Accepted Manuscript

DOI: 10.1039/x0xx00000x
Long Chen,b Zhuo Chen,*,a Weihong Tan*,a
www.rsc.org/

Controlling and monitoring the drug delivery process is critical to its intended therapeutic function. Many nanocarrier
systems for drug delivery have been successfully developed. However, biocompatibility, stability, and simultaneously
tracing drugs and nanocarriers present significant limitations. Herein, we have fabricated a multifunctional nanocomposite
by coating gold nanorod (AuNR) with a biocompatible, superstable and fluorescent carbon layer, obtaining the
AuNR@Carbon core-shell nanocapsule. In this system, the carbon shell, originally obtained in aqueous glucose solutions
and, therefore, biocompatible in physiological environments, could be simply loaded with cell-specific aptamers and
therapeutic molecules through - interactions a useful tool for cancer-targeted cellular imaging and therapy. Moreover,
such stable and intrinsic fluorescent effect of the AuNR@Carbon enabled simultaneous tracking of released therapeutic
molecules and nanocarriers under thermo-chemotherapy. The AuNR@Carbons had high surface areas and stable shells, as
well as unique optical and photothermal properties, making them promising nanostructures for biomedical applications.

surface area guarantees a high drug payload.8-9 Nucleic acid

Introduction
Based on their remarkable physicochemical properties, the
application of nanomaterials in biomedicine offers potential
solutions for many of the current challenges in cancer treatments.
Combining different materials into coordinated nanocomposites
provides even more creative possibilities,1 including the
development of so-called theranostic nanoplatforms, in which
multiple nanocomponents are integrated into a single
nanoassembly to realize both diagnostic and therapeutic functions
simultaneously. Another emerging direction is
thermo-chemotherapy mediated by photothermal inorganic
nanoparticles.2-3 Owing to their tunable localized surface plasmon
resonance and photothermal effects, AuNRs have proven to be
promising in a wide range of biomedical applications, such as
theranostics and thermo-chemotherapy.4-5 To reduce the clustering
and aggregating of AuNRs, the surface of AuNRs-based
nanoplatforms is typically modified with mesoporous silica or
different kinds of polymers.6-7 As an additional benefit, their large
a.
Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for
Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical
Engineering, College of Biology and Collaborative Innovation Center for
Molecular Engineering and Theranostics, Hunan University, Changsha 410082,
China.
b.
Faculty of Science and Technology, University of Macau, Av. Padre Toms Pereira
Taipa, Macau, China.
Email: zhuochen@hnu.edu.cn, tan@chem.ufl.edu
Electronic Supplementary Information (ESI) available: Experimental details and
characterization data for all new compounds. See DOI: 10.1039/x0xx00000x
These authors contributed equally.
aptamers are nucleic acid species that have been engineered
through repeated rounds of in vitro selection to bind to various
molecular targets. Based on their unique advantages, increasing
interest has been shown in the use of aptamer-conjugated
nanomaterials as target ligands for specific cancer cell recognition
and targeted cancer therapy.10 However, the performance of these
modified drug-delivery nanoparticles has not been as good as
initially envisioned because mesoporous silica or polymers will
typically separate from AuNRs, raising concerns about their stability
and potential toxicity.11-13 At the same time, monitoring the drug
delivery process is critical to its intended therapeutic function.
Therefore, fluorescence techniques have been developed to track
drug molecules and carriers through labeling with dyes or quantum
dots.14-16 However, the dynamics of drug molecules and
nanocarriers need to be treated simultaneously when tracking
intracellular drug release, and labeled dyes or particles may leak
out, making it difficult to trace the carriers with precision. Although
the fabrication of fluorescent drug carriers could be a counterplan,
the problem of combining uniformity, stability and biocompatibility
in one single nanoparticle still remains to be addressed.
Therefore, we herein report the synthesis of a novel type of
uniform, fluorescent AuNR@Carbon core-shell nanocapsule by an in
situ coating method. Monodispersed AuNR@Carbon core-shell
nanocapsules were prepared by hydrothermal treatment.17-18 A
nucleolin-specific aptamer was used to functionalize the
AuNR@Carbons for targeted cancer cell imaging.19-22 Doxorubicin
(DOX), an aromatic chemotherapy drug, was also effectively loaded
into the carbon shell of AuNR@Carbon nanocapsules. Using
DOX-loaded nanoplatforms, the intracellular uptake of

This journal is The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1

Please do not adjust margins

 Please do not adjust margins
Nanoscale
Journal Name PAPER
AuNR@Carbon-DOX complexes could be stimulated by NIR laser
irradiation, resulting in controllable drug release inside the cell.
Furthermore, the fluorescent shell of AuNR@Carbon nanocapsules
could be utilized to track the drug carrier before, or after, NIR laser
exposure. Thus, our in vitro results showed the AuNR@Carbon
nanocapsule to be a novel and promising theranostic platform not
only in positioning anticancer drugs but also in tracking drug
nanocarriers, suggesting its potential for in vivo applications.
Experimental
Published on 09 March 2016. Downloaded by Gazi Universitesi on 09/03/2016 11:41:54.
material
Anhydrous chloroauric acid (HAuCl4) and glucose were
purchased from Aladdin. Hexadecyl trimethyl ammonium bromide
(CTAB) and L-ascorbic acid (AA) were purchased from Sigma. Trypan
blue dye (0.04%) was purchased from Bio-Rad. Aptamer AS1411
with sequence 5-d (GGT GGT GGT GGT TGT GGT GGT GGT GGA AAA
AAA AA)-3 was obtained from Shanghai Biotech (China). DOX was
purchased from Hisun Pharmaceutical (Zhejiang, China).
RPMI-DMEM medium, penicillin/streptomycin solution and feta
bovine serum were obtained from Invitrogen. MTS was purchased
from Beyotime (China). The ultrapure water was from a Milli-Q
Integral System. All other chemical reagents were analytical grade
and used without further purification. Other chemicals were all
commercially available.
Synthesis of AuNRs nanoparticles.
AuNRs with a large average aspect ratio were prepared following a
seed-mediated growth method. An aqueous mixture solution of
HAuCl4 (0.01 M, 0.25 mL) and cetyltrimethylammonium bromide
(CTAB) (0.1 M, 9.75 mL) was added into a freshly prepared, ice-cold
aqueous NaBH4 solution (0.01 M, 0.6 mL) as the seed solution. The
resultant solution was mixed by rapid stirring with magnetic stirrers
and then kept at room temperature for 2 h before use. The growth
solution was prepared by mixing together HAuCl4 (0.01 M, 20 mL),
AgNO3 (0.01 M, 4 mL) and CTAB (0.1 M, 400 mL), followed by the
addition of a freshly prepared aqueous ascorbic acid solution (0.1
M, 3.2 mL) and an aqueous HCl solution (1.0 M, 8 mL). After the
resultant solution was mixed by inversion, the seed solution (1 mL)
was rapidly injected. The reaction mixture was subjected to gentle
inversion for 10 s and then left undisturbed for at least 6 h.
Synthesis of AuNR@Carbon nanocapsules.
55 mL of the produced AuNRs were centrifuged and washed with
double-distilled water to remove excess CTAB surfactant and then
dispersed in 12 mL of double-distilled water with vigorous magnetic
stirring to form a dark solution. 0.6 g of glucose was added into the
previous solution, and the mixture was stirred for 15 min. The final
solution was transferred to a Teflon-lined stainless steel autoclave
(15 mL in total volume), sealed and maintained at 160 C for 12 h.
The final product was centrifuged and washed with double-distilled
water several times until the supernatant was colorless.
Synthesis of AuNR@SiO2 nanoparticles.
15mL produced AuNRs were centrifuged and washed with
double-distilled water at 10,000 rpm for 30 minutes to remove
excess CTAB surfactant, followed by dispersal in 10 mL of
This journal is The Royal Society of Chemistry 20xx
Please do not adjust margins
Page 2 of 7
View Article Online
DOI: 10.1039/C6NR00369A
double-distilled water. 100 L of 0.1 M NaOH solution were added
upon stirring. Following this step, three 6 L injections of pure
tetraethyl orthosilicate (TEOS) were added under gentle stirring at
30 minute intervals. The mixture was reacted for 3 days.
Characterizations
Transmission electron microscope (TEM) images were obtained by
JEM-2010 (JEOL). Scanning electron microscopy (SEM) imaging of
AuNR@Carbons was obtained by field emission electron microscopy
(Tecnai G2F20 S-TWIN). UV-Vis spectra were measured on the
UV-2450 spectrophotometer (Shimadzu). Hydrodynamic diameters
were measured using a Zetasizer Nano ZS90 DLS system (Malvern
Instruments Ltd., Worcestershire, England), and -Potential
measurements were carried out at room temperature on the
Nanoscale Accepted Manuscript
Zetasizer Nano ZS90 Zeta system (Malvern Instruments Ltd.,
Worcestershire, England). All fluorescence measurements were
carried out on a Fluoromax-4 spectrofluorometer (HORIBA Jobin
Yvon, Edison, NJ).
Cell culture
Human breast cancer cells (MCF-7) and embryonic kidney cells
(HEK-293) were cultured at 37 C in DMEM medium supplemented
with 10% premium fetal bovine serum (FBS) and 1% penicillin
/streptomycin in a 5% CO2 environment.
NIR photothermal treatment of cancer cells
To determine the effect of AuNR@Carbons concentration on
photothermal heating eect, a series of solutions of
AuNR@Carbons with dierent concentrations from 0.05 to 0.6
mg/mL were irradiated by NIR laser (wavelength, 808 nm; power
2
density, 2 W/cm ; Kaisite Electronic Equipment Co., Ltd., Beijing,
China) for different times. To determine the effect of NIR power
density, the 0.3 mg/mL AuNR@Carbons solution was irradiated
using different power densities, while the temperature was
monitored by a thermometer. AuNRs and AuNR@SiO2 normalized
to AuNR@Carbons concentration at 0.3 mg/mL, together with
water, were used as controls. For photothermal cell therapy, MCF-7
cells were precultured for 24 h, followed by the addition of
-3
AuNR@Carbons with a final concentration of 3.510 mg/mL. After
incubation for 2 h at 37 C, cells were washed with DPBS to remove
excess AuNR@Carbons. Then an 808 nm NIR laser was used to
2
irradiate MCF-7 cells at a power density of 2 W/cm at different
times and with various AuNR@Carbons concentrations. After
staining with 0.4% trypan blue solution (Sigma Inc.) for 5 min,
microscopic images of the cells were obtained using an
epifluorescence microscope (Olympus IX71, 40X, 0.65 N.A.
objective).
DOX loading on AuNR@Carbon nanocapsules
DOX loading was achieved by simply mixing aqueous DOX solution
and 0.3 mg/mL AuNR@Carbons. The loading was performed with
3.3 mM DOX PBS solution, adjusting the pH to 8.5-9 by using 0.5 M
carbonate buffer. Samples were incubated for 14 hours at room
temperature with gentle agitation. After the incubation, excess DOX
was removed through centrifugation until no DOX was detected in
the supernatant. The concentration of DOX was determined by
-1 -1
UV-Vis using a DOX extinction coefficient of 10500 M cm at 490
J. Name., 2013, 00, 1-3 | 2
 Please do not adjust margins
Page 3 of 7 Nanoscale
View Article Online
DOI: 10.1039/C6NR00369A
Journal Name PAPER

nm. All UV-Vis measurements were conducted using a UV-2450

UV-Vis spectrophotometer (Shimadzu).
Cell viability test of photothermal enhanced chemotherapy
Measurement of cell viability was evaluated by the MTS assay. First,
MCF-7 cells were cultured in 96-well microplates with 100 L of
media for 18 hours. DOX, AuNR@Carbon and AuNR@Carbon-DOX
complexes were added to separate wells and cultured for 2 h,
respectively. Then the cells were washed with DPBS and added to
100 L of culture media. For thermal therapy, corresponding wells
2 2
were irradiated with an 808 nm NIR laser (1 W/cm or 2 W/cm ) for
6 min. The culture media were then replaced with fresh media and
Published on 09 March 2016. Downloaded by Gazi Universitesi on 09/03/2016 11:41:54.

cultured for a further 48 h, followed by standard MTS assay. The

Fig 1. Characterization of AuNR@Carbon nanocapsules. a) Schematic
absorbance value at 490 nm was determined by a Synergy 2
illustration of AuNR@Carbon nanocapsules fabrication; b) and c) TEM

Nanoscale Accepted Manuscript

Multi-Mode Microplate Reader (Bio-Tek, Winooski, VT). The
experiments were performed at least 3 times.
CLSM images of cells
MCF-7 cells were seeded in a 30-mm glass dish and incubated for 24
h. After removing cell medium, cells were incubated with DOX,
AuNR@Carbon and AuNR@Carbon-DOX complexes in 2 mL DPBS at
37 C for 2 h. Then cells were washed three times with DPBS,
followed by analysis using a fluorescence confocal microscope.
Targeted CLSM images with aptamer-functionalized
AuNR@Carbons
The efficiency of adsorbing aptamers on AuNR@Carbons was
also clearly demonstrated by the fluorescence spectra of
50 nM random DNA sequence
FAM-TAC GAG TTG AGA CCG TTA AGA CGA GGC AAT CAT GCA TAT A
TT GGA CGC TTT ACC GAC ATC TGG CAT CGAT-3 mixed with various
concentrations of AuNR@Carbons in Dulbecco`s phosphate
buffered saline (DPBS, Gibco) for 30 min. AuNR@Carbons were able
to quench FAM fluorescence through the fluorescence resonance
energy transfer (FRET) process once the FAM-DNA had
adsorbed on the surface. For targeted CLSM imaging, MCF-7
and HEK-293 cells were incubated with AS1411-AuNR@Carbons
solutions for 0.5 h at 4 C. After washing with DPBS three times,
cells were imaged under a fluorescence confocal microscope.
images of AuNR@Carbons; d) SEM image of the ordered cocklebur
structure of the monodispersed AuNR@Carbons.
hydrated diameter of 165 nm under dynamic light scattering
measurements. Scanning electron microscopy (SEM) images (Fig 1d,
S1) of the AuNR@Carbon samples displayed elliptic and uniform
structures. This resulted in a cocklebur-like structure, effectively
increasing the surface area of the AuNR@Carbon and, hence,
guaranteeing a higher drug payload than that of AuNR@SiO2 (Fig
S2) 25-27. AuNR@Carbons has two prominent Raman vibration bands
(Fig S3), a disordered (D) peak around 1330 cm-1 and a graphitic
carbon (G) peak around 1590 cm-1, which demonstrated carbon
shell has a certain degree of graphitization.11 Fourier transform
infrared spectroscopy (Fig S4) was used to detect the functional
groups of AuNR@Carbon nanocapsules. The peaks around
1000-1300 cm-1, which include the C-OH stretching and OH bending
vibrations, imply the existence of residual hydroxy groups
compared to those of AuNRs. The bands at 1710 cm-1 and 1620 cm-1
attributed to C=O and C=C vibrations, respectively, support the
concept of aromatization of glucose during hydrothermal
treatment. The -potential value (Fig S5) of AuNR@Carbon in water
solution was negative, further indicating the presence of OH and
C=O groups on carbon frameworks, thereby improving the
hydrophilicity and stability of the nanocapsules and greatly

Results and discussion

Synthesis and Characterization of AuNR@Carbons
A seed-mediated growth method was utilized for the fabrication of
23-24
AuNRs. AuNR@Carbons were prepared through an in situ
coating method with cetyltrimethylammonium bromide (CTAB) as
25
the template. The monodisperse AuNR@Carbons were obtained
in aqueous glucose solutions, and neither toxic reagents nor organic
solvents, both common in the preparation of polymer and silica
11,26
spheres, were used during synthesis. The hydrothermal reaction
time and ratio of the AuNR and glucose could be used to modulate
the nanocapsulate size (Fig 1a). AuNR@Carbon had a uniform size
distribution of around 120 nm in length and 80 nm in width (Fig 1b, Fig 2. The fluorescence and cytotoxicity assay of AuNR@Carbons. a)
c). Obvious core-shell structure was observed in the transmission Fluorescence spectra of AuNR, AuNR@SiO2 and AuNR@Carbon (Ex=405
electron microscopy (TEM) images, and the carbon shell was nm); b) Cytotoxicity assay of MCF-7 cells treated with different
estimated to have a homogeneous thickness of ~30 nm. concentration of AuNR@Carbons; c) and d) CLSM images of normal MCF-7
AuNR@Carbon had a monodisperse distribution with an average cells incubated without and with AuNR@Carbons (Ex=405 nm).

This journal is The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

Please do not adjust margins

 Please do not adjust margins
Nanoscale Page 4 of 7
View Article Online
DOI: 10.1039/C6NR00369A
Journal Name PAPER
20
efficient, - stacking to realize targeted imaging (Fig 3a). We
further identified the functionalization of AuNR@Carbons using
FAM-modified AS1411 aptamers. Graphitic carbon has been
35
demonstrated as a good quencher of nearby fluorescence dyes . As
shown in Fig 3b and S8, the fluorescence signal of
AuNR@Carbon-AS1411 was significantly reduced compared to
AS1411 aptamer alone. Furthermore,we investigated the stability of
AuNR@Carbon-AS1411 complex in DPBS, DMEM and cell culture,
repectively (Fig S8). The fluorescence signal of FAM still quenched in
DPBS and DMEM, while in cell culture, about 9% fluorescence
intensity recovery was observed. The result demonstrated
Published on 09 March 2016. Downloaded by Gazi Universitesi on 09/03/2016 11:41:54.

AuNR@Carbon-AS1411 complex exhibited superior stability in DPBS

Fig 1. Characterization of AuNR@Carbon nanocapsules. a) Schematic and DMEM, and relative good stability in cell culture. The
illustration of AuNR@Carbon nanocapsules fabrication; b) and c) TEM AuNR@Carbon-AS1411 complex was then utilized for targeted

Nanoscale Accepted Manuscript

images of AuNR@Carbons; d) SEM image of the ordered cocklebur
structure
Fig of the monodispersed
3. AuNR@Carbon AuNR@Carbons
loaded with .
aptamer. a) Schematic illustration of
AS1411 loaded on AuNR@Carbon; b) Fluorescence spectra of 50 nM
FAM-aptamer mixed with various concentrations of AuNR@Carbons in 20
mM Tris-HCl buffer for 30 min; c) and d) CLSM images (Ex=405nm) of
HEK-293 and MCF-7 cells incubated with AS1411-AuNR@Carbons for 0.5 h
at 4 .
widening their range of applications.
AuNR@Carbons exhibited superior stability provided by the
robust carbon shell. Such nanoparticles without any additional
modification were found to be very stable under extreme pH
environments and salt conditions (Fig S6). Such excellent stability
and biocompatibility are ideal properties for biomedical
applications.
The fluorescence imaging and cytotoxicity assay with
AuNR@Carbons
Compared to AuNRs or AuNR@SiO2 nanoparticles, the unique
carbon shells of the AuNR@Carbons allowed fluorescence of the
nanocarriers (Fig 2a,S7) when the maximum excitation wavelength
was 405 nm. To explain this property, it is possible that the
glucose-based hydrothermal method used to fabricate the carbon
imaging of MCF-7 and embryonic kidney (HEK-293) cells since they
showed different binding affinities to AS1411 aptamers (Fig 3c,d).
Only MCF-7 cells, which had higher binding affinities, showed a
positive signal. We further used AuNR@Carbon and
AuNR@Carbon-AS1411 complex to incubate MCF-7 cell for 0.5h at
4, which verified AuNR@Carbon-AS1411 complex can target
MCF-7 cell quickly (Fig S9). Thus, AuNR@Carbon-AS1411
demonstrated good targeted imaging capabilities, indicating
efficient functionalization of the AS1411 aptamer and, hence, the
potential of AuNR@Carbon-AS1411 complex in biomedical
applications.
Thermo-chemotherapy with AuNR@Carbon-Dox complexes
Based on strong absorbance in the NIR region, combined with
the unique properties of carbon and AuNR, AuNR@Carbon was also
demonstrated to be stable with significant photothermal heating
36
capability. Compared to AuNR and AuNR@SiO2, the
AuNR@Carbon may be more resistant to melting under
photoinduced local heating because of the stable carbon shell. The
photothermal heating effects were then investigated (Fig 4a). The
a) 80 b)
Temperature( C)

shells could cause the formation of large conjugated aromatic

o

60 H2O
30-31 AuNR
structures, which could be the sources of fluorescence. Such AuNR@SiO2
AuNR@Carbon
40
fluorescent AuNR@Carbons were utilized for cell imaging. The
cytotoxicity was investigated through the MTS assay. 20

AuNR@Carbons exhibited good biocompatibility. Negligible 0 100 200 300 400 500
Irradiation Time(s)
inhibition of proliferation was observed in human breast cancer c) 0.8
DOX d)1.2 Non-ir
cells (MCF-7) stained with AuNR@Carbons (Fig 2b). Nanocapsules in 0.6
AuNR@Carbon-DOX
Supernatant
Ir-1
Ir-2
Cell Viability

AuNR@Carbon
high concentration were incubated with MCF-7 cells at 37 C for 2 0.8
Abs(a.u.)

0.4
hours. As indicated by confocal laser scanning microscopy (CLSM),
0.4
the AuNR@Carbons demonstrated good cell imaging capability (Fig 0.2

2c,d). 0.0
400 500 600 700 800 900
0.0
X on OX ol
DO arb -D ntr
Wavelength(nm) @C on Co
arb
Aptamer Functionalization of AuNR@Carbons for Targeted @C

Imaging Fig 4. a) The heating curves of water, AuNR, AuNR@SiO2 and

AuNR@Carbon under 808 nm laser irradiation at a power density of 2
Nucleolin, a bcl-2 mRNA binding protein, is highly expressed by
32 W/cm2; b) Digital photo of the DOX, AuNR@Carbon and
cancer cells, both intracellularly and on the cell surface. Aptamers
AuNR@Carbon-DOX complexes in solution; c) UV-Vis characterization
with high binding affinity and specificity to nucleolin have already
33-34 of the DOX-loaded AuNR@Carbons; d) Relative cell viabilities of DOX,
been developed. In particular, aptamer AS1411 with sequence
AuNR@Carbon, and MCF-7 cells treated with AuNR@Carbon-DOX
5-d (GGT GGT GGT GGT TGT GGT GGT GGT GGA AAA AAA AA)-3
complexes with or without laser irradiation (808 nm, 1 W/cm2, 6 min-Ir-1;
has been functionalized on the AuNR@Carbon through simple, but
808 nm, 2 W/cm2, 6 min-Ir-2).

This journal is The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 4

Please do not adjust margins

 Please do not adjust margins
Page 5 of 7
Journal Name PAPER
solution temperature of AuNR@Carbons increased to around 75
2
after 4 minutes of 2 W/cm laser irradiation. This result was similar
to that of AuNRs and AuNR@SiO2, but much higher than that of
water. The thick carbon-isolated shells provided the
AuNR@Carbons with superstability under photothermal heating
without interfering with heating efficiency. In vitro photothermal
tests were further explored with MCF-7 cells. Increasing the
concentration or laser irradiation time could efficiently improve
photothermal efficiency (Fig S10). With the addition of 8 L 0.2
mg/mL of AuNR@Carbons solution and laser irradiation at 808 nm
over 6 minutes, most cells were found dead, indicating the high
Published on 09 March 2016. Downloaded by Gazi Universitesi on 09/03/2016 11:41:54.
efficiency of AuNR@Carbon as a good material for NIR
photothermal therapies.
Integrating chemotherapy with photothermal therapy will
greatly enhance therapeutic efficiency. The AuNR@Carbons
demonstrated drug loading capability superior to that of
AuNR@SiO2 nanoparticles (Fig S11a, b). The carbon shell was a good
substrate for loading DOX via simple hydrophobic interaction and
- stacking with a high loading content up to 46 % (Fig S12a, b).
From the UV-Vis spectrum, the obvious peak around 490 nm
indicated conjugation of DOX on the AuNR@Carbon (Fig 4c). Fig 4b
was the digital photo of DOX, AuNR@Carbon and
AuNR@Carbon-DOX complexes in solution. The
AuNR@Carbon-DOX complexes was very stable and could be stored
for a long time. DOX loading efficiency was also explored (Fig S13).
After DOX was loaded on the AuNR@Carbons, the fluorescence of
DOX was quenched through fluorescence resonance energy
transfer, indicating the close attachment of DOX on the
AuNR@Carbons. Also observed was a pH-dependent release
behavior with more DOX released at lower pH (Fig S14), owing to
the protonation of the amino group in the DOX molecule that
50m 50m 50m 50m
50m
50m
50m 50m
50m 50m 50m 50m
50m 50m
50m 50m
Fig 5. a) CLSM images of MCF-7 cells and HEK-293 cells incubated with
AS1411-AuNR@Carbon-DOX complexes for 0.5 h at 4 , cultured in fresh
media with 6 min NIR laser irradiation, and then incubated for another 5 h
at 37 ; b) Schematic illustration of the cell-targeted, photocontrolled
nanodrug delivery system for cancer therapy.
This journal is The Royal Society of Chemistry 20xx
Please do not adjust margins
Nanoscale
View Article Online
DOI: 10.1039/C6NR00369A
afforded DOX a positive charge, thus facilitating drug release under
acidic pH.
The cytotoxicity of AuNR@Carbon-DOX complexes was then
tested. MCF-7 cells were incubated with DOX, AuNR@Carbon and
AuNR@Carbon-DOX complexes, respectively. After 808 nm laser
2
irradiation for 6 min at the power density of 1 W/cm (Ir-1) and 2
2
W/cm (Ir-2), relative cell viabilities were investigated (Fig 4d).
Without laser irradiation, the toxicity of DOX, AuNR@Carbon-DOX
complexes and AuNR@Carbon in MCF-7 cells receded successively.
This demonstrated good biocompatibility and the relatively slow
release of DOX from the AuNR@Carbon-DOX complexes by lower
37
pH inside the endosome. When all MCF-7 cell samples were
irradiated with Ir-1 condition, no obvious differences were
observed for the DOX, AuNR@Carbon and control samples, either
Nanoscale Accepted Manuscript
with or without laser irradiation. However, for the sample treated
with AuNR@Carbon-DOX, cell death significantly increased and was
even higher than that of the DOX-only samples, demonstrating that
chemotherapeutic effect had been sustained in complex with
AuNR@Carbon and that nanoparticle carriers could improve drug
efficacy. Importantly, when increasing laser intensity to Ir-2, cell
death significantly increased and was even higher than that of the
samples treated with AuNR@Carbon-DOX under Ir-1 conditions,
indicating that the combination of photothermal treatment and
chemotherapy can exert a synergistic effect. Such controllable drug
release with NIR laser irradiation could significantly reduce the side
effects of chemotherapy, making AuNR@Carbon a promising
platform for future clinical applications.
Co-localized imaging with Aptamer-Functionalized
AuNR@Carbon-DOX
Fluorescent AuNR@Carbons were then used as NIR-responsive drug
nanocarriers for simultaneous tracking of drug molecules and
carriers under photochemotherapies. AuNR@Carbon carrying a
DOX payload in combination with NIR irradiation could effectively
colocate the nanocarrier and therapeutic molecule in cells, leading
to multifunctional monitoring. When AuNR@Carbons load DOX and
aptamer at the same time, there is an inevitable competition
between DOX and Aptamer adsorption. Whereas most of the DOX
molecules were remained on AuNR@Carbons, after
AuNR@Carbon-DOX further mixed with 50nM aptamer in DPBS or
cell culture (Fig S15). We also investigated the possible interaction
between DOX and the AS1411 aptamer molecules. As showed in Fig
S16, the interaction is very weak and no obvious complex was
obtained. Targeted cellular uptake and intracellular drug release
behavior of AS1411 aptamer-functionalized AuNR@Carbon-DOX
complexes in response to NIR laser irradiation were studied in
MCF-7 and HEK-293 cells. Neither DOX fluorescence nor
AuNR@Carbon fluorescence was observed in HEK-293 cells
incubated with AS1411-AuNR@Carbon-DOX complexes (Fig 5a)
because normal cells either lack, or have very low levels of,
nucleolin receptors on the plasma membrane. Subsequently,
cell-specific internalization of AuNR@Carbon-DOX complexes was
determined. Before NIR laser irradiation, only weak fluorescence of
DOX molecule was observed in MCF-7 cells treated with
AS1411-AuNR@Carbon-DOX complexes, essentially resulting from
the slight release of DOX by decreasing the pH (Fig 5a). On the other
hand, NIR laser irradiation led to stronger DOX fluorescence inside
J. Name., 2013, 00, 1-3 | 5
 Please do not adjust margins
Nanoscale Page 6 of 7
View Article Online
DOI: 10.1039/C6NR00369A
Journal Name PAPER

MCF-7 cells (Fig 5a), indicating efficient internalization, as well as
fast drug release, from the nanocapsule complexes. Further culture
of MCF-7 cells following NIR laser irradiation for another 5 h
showed that DOX had been efficiently transported into the nuclei of
MCF-7 cells (Fig 5a). Moreover, it is possible that the functionalized
aptamers and pre-released DOX molecules loosened the nuclear
membrane, allowing a few AuNR@Carbon nanocarriers to be
transported into the nuclei of MCF-7 cells (Fig 5a).
The schematic illustration of AS1411-AuNR@Carbon-DOX
complexes, which coordinate efficient cell-targeted delivery and
nuclear uptake of drugs for cancer therapy, was shown in Fig 5b.
Published on 09 March 2016. Downloaded by Gazi Universitesi on 09/03/2016 11:41:54.
1 R. Ghosh Chaudhuri, S. Paria, Chem. Rev., 2011, 112, 2373.
2 Y. Fang, G. Zheng, J. Yang, H. Tang, Y. Zhang, B. Kong, Y. Lv, C. Xu,
A. M. Asiri, J. Zi, Angew. Chem., 2014, 126, 5470.
3 T. Sun, Y. S. Zhang, B. Pang, D. C. Hyun, M. Yang, Y. Xia, Angew.
Chem., Int. Ed.,2014, 53, 12320.
4 K. M. Mayer, S. Lee, H. Liao, B. C. Rostro, A. Fuentes, P. T. Scully,
C. L. Nehl, J. H. Hafner, Acs Nano, 2008, 2, 687.
5 V. Juv, M. F. Cardinal, A. Lombardi, A. Crut, P. Maioli, J.
Prez-Juste, L. M. Liz-Marzn, N. Del Fatti, F. Valle, Nano
letters, 2013, 13, 2234.
6 A. Shiotani, Y. Akiyama, T. Kawano, Y. Niidome, T. Mori, Y.

The nanocapsule complexes could be directed to the target cancer Katayama and T. Niidome, Bioconjugate. Chem., 2010, 21, 2049.
cell by aptamers on the carbon shell. After nanocapsule complexes 7 Z. Zhang, J. Wang, X. Nie, T. Wen, Y. Ji, X. Wu, Y. Zhao and C.
endocytosed into the cancer cells, the DOX was slightly released Chen, J. Am. Chem. Soc., 2014, 20, 7317.

Nanoscale Accepted Manuscript

from the AuNR@Carbon surface by decreasing the pH. Upon NIR 8 Y. Zhong, C. Wang, L. Cheng, F. Meng, Z. Zhong and Z. Liu,
laser irradiation, the photothermal effect could be used to
stimulate the release of the chemotherapeutic DOX on the surface 9 T. Nakamura, F. Sugihara, H. Matsushita, Y. Yoshioka, S.
of the AuNR@Carbons. Thus, the multifunctional nanocomposite
could be used for simultaneous monitoring of DOX molecules and 10
AuNR@Carbon to achieve colocalization of released drug molecules
and drug carriers during the photochemotherapies. 11
12
Conclusions 13
In summary, a novel type of theranostic agent with good
biocompatibility and stability based on the AuNR@Carbon 14
core-shell nanoparticle was synthesized with an in situ growth
method. We successfully integrated photothermal, fluorescent and
drug delivery properties in a single nanocomposite by coating AuNR 15
with a carbon shell. The AuNR core could transform NIR laser to
heat, which could then be used for PTT, as well as external 16
laser-triggered drug release. The AuNR@Carbons were fabricated
through a green method and showed minimal cytotoxicity, good
biocompatibility and superior stability. The prepared carbon shell 17
also afforded the AuNR@Carbon with significant fluorescence and
simple chemical surface modifiability, including functionalization 18
with aptamers for targeted celluar imaging. Furthermore, owing to
the integrated features of the fluorescent AuNR@Carbon and the 19
fluorescence of DOX drug molecules, together with the core-shell 20
structure, AuNR@Carbon could be an effective tool for drug
delivery and simultaneous in vitro tracking of drug nanocarriers and 21
released therapeutic molecules. By coupling the intrinsic properties
of both carbon shell and gold nanorod core, the synthetic 22
AuNR@Carbon nanocomposite herein reported could be an 23
interesting nanoplatform showing promise for applications in
cancer therapy, optical imaging and biosensing. 24
25
Acknowledgements
This work was funded by the National Key Basic Research Program 26
of China (No. 2013CB932702), the National Natural Science 27
Foundation of China (No. 21522501), and the Science and
Technology Development Fund of Macao S.A.R (FDCT, 067/2014/A). 28
29
Notes and references
30
Biomacromolecules, 2013, 14, 2411.
Mizukami and K. Kikuchi, Chem. Sci., 2015, 3, 1007.
Q. L. Liu, C. Jin, Y. Y. Wang, X. H. Fang, X. B. Zhang, Z. Chen and
W. Tan, NPG Asia Material, 2014, 6, 95.
W. Gao, X. W. Wang, H. H. Fan, Z. L. Song, X. F. Lai, Z. Chen and
W. Tan, Sci. Bull., 2015, 60, 1101.
E. Jakab, G. Varhegyi, O. Faix, J. Anal. Appl. Pyro., 2000, 56, 273.
C. Park , J. G. Smith Jr., J. W. Connell, S. E. Lowther, D. C.
Working, E. J. Siochi, Polymer,2005, 46, 9694.
V. Bagalkot, L. f. Zhang, E. Levy-Nissenbaum, S. Jon, Philip W.
Kantoff, R. Langer and O. C. Farokhzad, Nano Letter 2007, 7,
3065.
B. Kang, J. Li, S. Q. Chang, M. Z. Dai, C. Ren, Y.-D. Dai and D.
Chen, Small 2012, 8, 777.
Y. Zhang, S. Swaminathan, S. C. Tang, J. Garcia-Amoros, M.
Boulina, B.r Captain, J. D. Baker and F. M. Raymo, J. Am. Chem.
Soc., 2015, 135, 4709.
H. S. Qian, S. H. Yu, L. B. Luo, J. Y. Gong, L. F. Fei and X. M. Liu,
Chem. Mater., 2006, 18, 2102.
T. Zhu, J. S. Chen and X. W. Lou, J. Phys. Chem. C, 2011, 115,
9814.
X. Sun and Y. Li, Angew. Chem., Int. Ed., 2004, 43, 597.
C. R. Ireson and L. R. Kelland, Mol. Cancer Ther., 2006, 5,
2957.
P. J. Bates, D. A. Laber, D. M. Miller, S. D. Thomas and J. O. Trent,
Exp. Mol. Pathol., 2009, 86, 151.
J. Ai, Y. Xu, B. Lou, D. Li and E. Wang, Talanta, 2014, 118, 54.
H. M. Meng, T. Fu, X. Z. Zhang, W. H. Tan, Natl. Sci. Rev.,
2015, 2, 71.
L. Vigderman, B. P. Khanal and E. R. Zubarev, Adv. Mater., 2012,
24, 4811.
X. Ye, Y. Gao, J. Chen, D. C. Reifsnyder, C. Zheng and C. B.
Murray, Nano letters, 2013, 13, 2163.
I. Gorelikov and N. Matsuura, Nano letters, 2008, 8, 369.
C. Wang, Z. Ma, T. Wang and Z. Su, Adv. Funct. Mater., 2006, 16,
1673.
J. R. Anema, J. F. Li, Z. L. Yang, B. Ren and Z. Q. Tian, Ann. Rev.
Anal. Chem., 2011, 4, 129.
H. Su, Y. Zhong, T. Ming, J. Wang and K. S. Wong, J. Phys. Chem.
C, 2012, 116, 9259.
S. T. Yang, L. Cao, P. G. Luo, F. Lu, X. Wang, H. Wang,

This journal is The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 6

Please do not adjust margins

 Please do not adjust margins
Page 7 of 7 Nanoscale
View Article Online
DOI: 10.1039/C6NR00369A
Journal Name PAPER

M. J. Meziani, Y. Liu, G. Qi and Y. P. Sun, J. Am. Chem. Soc.,

2009, 131, 11308.
31 S. T. Yang, X. Wang, H. Wang, F. Lu, P. G. Luo, L. Cao, M. J.
Meziani, J. H. Liu, Y. Liu, M. Chen, Y. Huang and Y. P. Sun, J.
Phys. Chem. C, 2009, 113, 18110.
32 S. Soundararajan, W. Chen, E. K. Spicer, N. Courtenay-Luck and
D. J. Fernandes, Cancer Res., 2008, 68, 2358.
33 F. Mongelard and P. Bouvet, Curr. Opin. Mol. Ther., 2010, 12,
107.
34 J. Wu, C. Song, C. Jiang, X. Shen, Q. Qiao and Y. Hu, Mol. Pharm.,
2013, 10, 3555.
35 Z. L. Song, X. H. Zhao, W. N. Liu, D. Ding, X. Bian, H. Liang, X. B.
Published on 09 March 2016. Downloaded by Gazi Universitesi on 09/03/2016 11:41:54.

Zhang and Z. Chen, W. Tan, Small, 2013, 9, 951.

36 Y. V. Kaneti, C. Y. Chen, M.S. Liu, X. C. Wang, J. L. Yang, R. A.

Nanoscale Accepted Manuscript

Taylor, X. C. Jiang, and A. B. Yu, ACS Appl. Mater. Inter.,2015, 7,
25658.
37 Y. Wang, K. Wang, J. Zhao, X. Liu, J. Bu, X. Yan and R. Huang, J.
Am. Chem. Soc., 2013, 135, 4799.

This journal is The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

Please do not adjust margins