Biomaterials 38 (2015) 10e21

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

Hyaluronic acid-modified Fe3O4@Au core/shell nanostars for

multimodal imaging and photothermal therapy of tumors
Jingchao Li a, c, 1, Yong Hu c, 1, Jia Yang b, 1, Ping Wei c, Wenjie Sun c, Mingwu Shen c, ***,
Guixiang Zhang b, **, Xiangyang Shi a, c, *
a
State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University,
Shanghai 201620, PR China
b
Department of Radiology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200080, PR China
c
College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, PR China

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

Article history: Development of multifunctional theranostic nanoplatforms for diagnosis and therapy of cancer still re-
Received 25 August 2014 mains a great challenge. In this work, we report the use of hyaluronic acid-modified Fe3O4@Au core/shell
Accepted 19 October 2014 nanostars (Fe3O4@Au-HA NSs) for tri-mode magnetic resonance (MR), computed tomography (CT), and
Available online
thermal imaging and photothermal therapy of tumors. In our approach, hydrothermally synthesized
Fe3O4@Ag nanoparticles (NPs) were used as seeds to form Fe3O4@Au NSs in the growth solution. Further
Keywords:
sequential modification of polyethyleneimine (PEI) and HA affords the NSs with excellent colloidal
Fe3O4@Au nanostars
stability, good biocompatibility, and targeting specificity to CD44 receptor-overexpressing cancer cells.
Hyaluronic acid
CT imaging
With the Fe3O4 core NPs and the star-shaped Au shell, the formed Fe3O4@Au-HA NSs are able to be used
MR imaging as a nanoprobe for efficient MR and CT imaging of cancer cells in vitro and the xenografted tumor model
Tumors in vivo. Likewise, the NIR absorption property enables the developed Fe3O4@Au-HA NSs to be used as a
Photothermal therapy nanoprobe for thermal imaging of tumors in vivo and photothermal ablation of cancer cells in vitro and
xenografted tumor model in vivo. This study demonstrates a unique multifunctional theranostic nano-
platform for multi-mode imaging and photothermal therapy of tumors, which may find applications in
theranostics of different types of cancer.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction toxicity [15e17]. Another promising nanoplatform is gold NPs

The past decade has seen a myriad of interest in using various
inorganic or organic nanoparticles (NPs) or microparticles for a
wide variety of biomedical applications because of their unique
structural features and functionalities [1e7]. In particular, for
magnetic iron oxide (Fe3O4) NPs, besides their uses in magnetic
separation [8,9], hyperthermia [10,11], catalysis [12], and drug/gene
delivery [13,14], Fe3O4 NPs have been used as negative contrast
agents for T2-weighted magnetic resonance (MR) imaging due to
their high relaxivity, excellent contrast enhancement, and low
* Corresponding author. College of Chemistry, Chemical Engineering and
Biotechnology, Donghua University, Shanghai 201620, PR China. Tel.: þ86 21
67792656; fax: þ86 21 67792306 804.
** Corresponding author. Tel.: þ86 21 63240090 4166; fax: þ86 21 63240825.
*** Corresponding author. Tel.: þ86 21 67792750; fax: þ86 21 67792306 804.
E-mail addresses: mwshen@dhu.edu.cn (M. Shen), guixiangzhang@sina.com
(G. Zhang), xshi@dhu.edu.cn (X. Shi).
1
Authors contributed equally to this work.
(AuNPs). On one hand, with a higher atomic number than that of
iodine, AuNPs have been extensively employed as a contrast agent
for CT imaging of different biological systems due to their better X-
ray attenuation property than that of Omnipaque (a conventional
iodine-based CT contrast agent) [18e23]. On the other hand, AuNPs
with particular shapes such as nanorods [24,25], nanoshells
[26,27], nanoflowers [28], nanocages [29,30], or nanostars (NSs)
[31,32] display strong surface plasmon resonance (SPR) absorption
intensity in near infrared (NIR) region [33], enabling their uses for
thermal imaging and photothermal therapy of cancer or other
biological systems.
For diagnosis and therapy of cancer, it is essential to develop a
theranostic platform that is able to integrate both diagnosis ele-
ments and therapeutic agents [34,35]. In particular, for accurate
cancer imaging applications, it is meaningful to design a multi-
functional platform affording dual or multi-mode imaging because
each imaging modality has its own limitations and advantages [36].
For instance, Cai et al. prepared Fe3O4@Au nanocomposite particles
for MR/CT dual mode imaging [37]. Tian et al. reported the

http://dx.doi.org/10.1016/j.biomaterials.2014.10.065
0142-9612/© 2014 Elsevier Ltd. All rights reserved.
 J. Li et al. / Biomaterials 38 (2015) 10e21
Fe3O4@Cu2xS core/shell NPs for both MR and thermal imaging of
tumors [38]. To exert a therapeutic effect to tumors, an anticancer
drug is usually encapsulated within a designed nanoparticulate
system [39e42] or conjugated onto the surface of NPs [43e46].
Alternatively, cancer cells can also be ablated under laser irradia-
tion in the presence of NPs with strong NIR absorption property,
such as tungsten oxide (WO2.9) nanorods [47], Cu7.2S4 nanocrystals
[48], Au nanorods [49], and Au NSs [50]. Therefore, for accurate
theranostics of cancer or imaging-guided cancer therapy, it is
essential to integrate multi-mode imaging elements and thera-
peutic agents within one nanoparticulate system. Although some of
the above NP systems have been demonstrated to be able to exert
both dual mode imaging (e.g., CT/thermal or MR/thermal imaging)
and photothermal therapeutic efficacy of cancer [35,38,47], these
NP systems are lack of targeting specificity presumably due to the
technical difficulty of surface biofunctionalization. Development of
various multifunctional nanoplatforms that enable targeted multi-
mode imaging and photothermal therapy of cancer still remains a
great challenge.
Our previous work has shown that dendrimer-entrapped AuNPs
[19e23,51,52] and Fe3O4 NPs prepared via either controlled co-
precipitation [53,54] or hydrothermal [15e17,55] approaches are
able to be used as CT and MR contrast agents, respectively for
molecular imaging applications. Dual mode MR/CT imaging func-
tionality can be easily realized by assembly of dendrimer-
entrapped AuNPs onto preformed Fe3O4 NPs [37] or by hydro-
thermal synthesis of Fe3O4/Au composite NPs [56,57]. In another
work, Liu and coworkers have shown that Au NSs having a strong
surface plasmon resonance (SPR) band at NIR region can be pre-
pared from Au seeds in the growth solution using silver ion com-
plexes as growth inhibitors [58]. Likewise, Au NSs with magnetic
Fe3O4 or Fe cores formed by exposing the core/shell Fe3O4@Au NPs
(seeds) or Fe seeds into the Au growth solution can be used for
gyromagnetic imaging of cells or fluorescence imaging/photo-
thermal destruction of cancer cells [59,60], while Fe3O4@Au
nanostars (NSs) formed by the reduction of Au(III) onto the
dextran-coated Fe3O4 NPs with hydroxylamine as a seeding agent
displayed five distinct functions (aptamer-based targeting, MR
imaging, optical imaging, photothermal therapy and chemo-
therapy) [34]. However, these studies have not completely
demonstrated the potentials to use the developed nanoplatforms
for CT/MR dual mode imaging and photothermal therapy of cancer
cells in vitro and in vivo. Our previous successes in the preparation
of Fe3O4/Au composite NPs lead us to hypothesize that the hydro-
thermally synthesized Fe3O4@Au or Fe3O4@Ag seeds may further
grow to form star-shaped Au shells onto the Fe3O4 core NPs,
thereby affording the creation of Fe3O4@Au NSs for multi-mode
imaging and photothermal therapy of cancer. Our prior work has
also shown that in the presence of branched polyethyleneimine
(PEI), hydrothermally formed Fe3O4 NPs are able to be afforded
with amine functionality [17]. Hence, Fe3O4 NPs can be easily
modified with targeting ligand folic acid (FA) or hyaluronic acid
(HA) for targeted MR imaging of FA receptor- and CD44 receptor-
overexpressing tumors, respectively [15,16]. Logically, the
Fe3O4@Au NSs to be designed in this work may also be modified
with PEI for further modification of targeting ligands, thereby
generating a multifunctional nanoplatform for targeted thera-
nostics of cancer.
In this present study, we report the formation of HA-targeted
Fe3O4@Au NSs for tri-mode (MR/CT/thermal) imaging and photo-
thermal therapy of cancer. First, Fe3O4@Ag seeds were synthesized
via a facile one-pot hydrothermal route according to our previous
work with some modifications [56]. Then the Fe3O4@Ag seeds were
added into the Au growth solution to form Fe3O4@Au NSs with the
help of silver nitrate. The formed Fe3O4@Au NSs were then surface
11
modified by partially thiolated PEI (PEI-SH) via AueS bond, fol-
lowed by modification with HA via 1-ethyl-3-[3-
dimethylaminopropyl] carbodiimide hydrochloride (EDC)
coupling reaction with the PEI amines on the surface of the NSs
(Scheme 1). The formed Fe3O4@Au-HA NSs were characterized via
different techniques. Their stability, biocompatibility including
hemocompatibility and cytocompatibility, targeting specificity to
CD44 receptor-overexpressing cancer cells, and potentials to be
used as T2-weighted MR and CT contrast agents for dual mode MR/
CT imaging of cancer cells in vitro and xenografted tumor model
in vivo were investigated in detail. Furthermore, the developed
Fe3O4@Au-HA NSs were used for photothermal ablation of cancer
cells in vitro and xenografted tumor model in vivo, as well as
thermal imaging of the tumor model in vivo.
2. Experimental section
2.1. Materials
Hyaluronic acid (HA, Mw ¼ 31,200) was purchased from Zhenjiang Dong Yuan
Biotechnology Corporation (Zhenjiang, China). EDC, N-hydroxysuccinimide (NHS),
cetyltrimethyl-ammoniumbromide (CTAB), and methyl thioglycolate (MTG) were
supplied by J&K Chemical Ltd (Shanghai, China). Branched polyethyleneimine (PEI,
Mw ¼ 25,000) and sodium borohydride (NaBH4) were purchased from Aldrich (St.
Louis, MO). HAuCl4$4H2O, ferrous chloride tetrahydrate (FeCl2$4H2O > 99%),
ammonia (25e28% NH3 in water solution), silver nitrate, ascorbic acid (AA) and all
other chemicals and solvents were from Sinopharm Chemical Reagent Co., Ltd
(Shanghai, China). All chemicals were used as received. HeLa cells (a human cervical
carcinoma cell line) and U87MG cells (a human glioblastoma carcinoma cell line)
were obtained from Institute of Biochemistry and Cell Biology, the Chinese Academy
of Sciences (Shanghai, China). Modified eagle medium (MEM), Dulbecco's modified
eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were
from Hangzhou Jinuo Biomedical Technology (Hangzhou, China). 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased
from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd
(Shanghai, China). Water used in all experiments was purified using a Milli-Q Plus
185 water purification system (Millipore, Bedford, MA) with a resistivity higher than
18.2 MU$cm. Regenerated cellulose dialysis membranes with molecular weight cut-
off (MWCO) of 14,000 were acquired from Fisher.
2.2. Synthesis of partially thiolated PEI
The partially thiolated PEI (PEI-SH) was synthesized according to protocols
described in the literature [61]. In brief, MTG (108 mL) was added to a freshly pre-
pared PEI aqueous solution (10 mL, 1.0 g), and the mixture was continuously stirred
at 60e70  C in a water bath for 24 h to complete the reaction. After that, the reaction
mixture was dialyzed against water (6 times, 2L) using a dialysis membrane with
MWCO of 14,000 for 3 days, followed by lyophilization to obtain the purified PEI-SH.
2.3. Synthesis of Fe3O4@Ag seeds
Fe3O4@Ag seeds were synthesized according to our previous work with some
modifications [56]. Firstly, PEI was used as a stabilizer to synthesize Ag NPs at the
PEI/Ag salt molar ratio of 1:20. Namely, a silver nitrate aqueous solution (68.0 mg,
2 mL) was added into a PEI aqueous solution (0.05 g/mL, 10 mL) under vigorous
magnetic stirring. After 30 min, an icy cold NaBH4 aqueous solution (75.66 mg, 1 mL)
was rapidly added into the above mixture and the mixture was continuously stirred
for 2 h to complete the reaction. The obtained PEI-Ag NPs were then purified via
dialysis as described above and finally redispersed in 5 mL water for further use.
Then, Fe3O4@Ag seed particles were synthesized using a one-pot hydrothermal
approach as described in our previous work [56]. FeCl2$4H2O (1.25 g) dissolved in
7.75 mL water was mixed with ammonium hydroxide (6.25 mL) under vigorous
magnetic stirring. The mixture was kept in air for about 10 min while stirring to
ensure iron (II) to be oxidized. Then the mixture was transferred into a 50-mL
autoclave (KH-50 Autoclave, Shanghai Yuying Instrument Co., Ltd., Shanghai,
China) and the obtained suspension of PEI-Ag NPs (5 mL) was also added into the
autoclave. The mixture was stirred thoroughly and then autoclaved in a sealed
pressure vessel at 134  C for 3 h. Subsequently, the autoclave was cooled down to
room temperature, and the product was collected via magnetic separation. The
formed Fe3O4@Ag seeds were further purified by rinsing with water for 3 times and
finally redispersed in 15 mL water.
2.4. Synthesis of Fe3O4@Au NSs
To prepare a gold growth solution, HAuCl4 (30 mg/mL, in 580 mL water) was
added to an aqueous solution of CTAB (381 mg, 10 mL), followed by successive
addition of AgNO3 (1.1 mg) and ascorbic acid (12 mg) under vigorous magnetic
stirring. Then 0.1 mL Fe3O4@Ag seeds (10 times diluted with water from the above
12 J. Li et al. / Biomaterials 38 (2015) 10e21
AgNO3
NaBH4
PEI
HA
EDC/NHS
Fe3O4@Au-HA NSs Fe3O4@Au-PEI NSs
Scheme 1. Schematic illustration of the synthesis of Fe3O4@Au-HA NSs.
suspension) were added and the mixture solution changed to blue within a few
minutes, indicating the formation of Fe3O4@Au NSs. After additional stirring for 1 h,
the product was purified by 3 cycles of centrifugation/redispersion in water to
remove CTAB, and the obtained Fe3O4@Au NSs were redispersed in 10 mL water.
2.5. Formation of Fe3O4@Au-PEI NSs
PEI-SH (0.1 g) dissolved in 1 mL water was added into the above aqueous so-
lution of Fe3O4@Au NSs (10 mL). The mixture was sonicated for 30 min, and then
stirred at room temperature for 24 h. After removing the non-absorbed PEI-SH
through 3 cycles of centrifugation (5000 rpm, 5 min)/redispersion in water, the
Fe3O4@Au-PEI NSs were obtained and redispersed in 10 mL water.
2.6. Formation of Fe3O4@Au-HA NSs
HA (520 mg, in 10 mL water) was mixed with EDC (128 mg) and NHS (80 mg)
under vigorous magnetic stirring for 3 h. Then, the activated HA solution was
dropped into the above aqueous suspension of Fe3O4@Au-PEI NSs (10 mL) under
magnetic stirring. After 3 days, the HA-modified Fe3O4@Au NSs (Fe3O4@Au-HA NSs)
were subjected to multiple cycles of centrifugation/redispersion in water to remove
the small molecular impurities and the non-reacted HA. Finally, the purified
Fe3O4@Au-HA NSs were redispersed in water and/or phosphate buffered saline (PBS)
before further use.
2.7. Characterization techniques
Fourier transform infrared (FTIR) spectra were collected using a Nicolet Nexus
670 FTIR spectrophotometer (Thermo Nicolet Corporation, USA). A Bruker AV400
nuclear magnetic resonance spectrometer was used to record the 1H NMR spectra of
PEI and PEI-SH samples. The samples were dissolved in D2O before measurements. A
Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, U.K.) equipped with a
standard 633 nm laser was used to analyze the hydrodynamic sizes and zeta po-
tentials of the samples. Thermal gravimetric analysis (TGA) was carried out using a
TG 209 F1 (NETZSCH Instruments Co., Ltd., Germany) thermal gravimetric analyzer
at a heating rate of 20  C/min under N2 atmosphere. UVevis spectroscopy was
performed using a Lambda 25 UVevis spectrophotometer (PerkinElmer, Boston,
MA) and the samples were dispersed in water before measurements. Transmission
electron microscopy (TEM, JEOL 2010F, Japan) was performed at an operating
voltage of 200 kV. TEM samples were prepared by dropping an aqueous particle
suspension (6 mL) onto a carbon-coated copper grid and air dried before measure-
ments. X-ray diffraction (XRD) measurements were performed on a D/max 2550 PC
X-ray diffractometer (Rigaku Cop., Japan) with Cu Ka radiation (l ¼ 0.154056 nm).
The Fe and Au concentrations of the samples dispersed in water or PBS were
analyzed using Leeman Prodigy inductively coupled plasma-optical emission spec-
troscopy (ICP-OES, Hudson, NH). The T2 relaxometry measurements and T2-
weighted MR imaging of the samples dispersed in water at different Fe concentra-
tions (0.005e0.08 mM) were performed using an NMI20-Analyst NMR Analyzing
Hydrothermal synthesis
Fe(II) salt
PEI-Ag NPs Fe3O4@Ag seeds
Growth
PEI-SH
Fe3O4@Au NSs
and Imaging system (Shanghai Niumag Corporation, Shanghai, China). The param-
eters were set as following: CPMG sequence, 0.5 T magnet, point
resolution ¼ 156 mm  156 mm, section thickness ¼ 0.6 mm, TR ¼ 6000 ms,
TE ¼ 80 ms, number of excitation ¼ 1. The linear fitting of the inverse T2 relaxation
times (1/T2) as a function of the Fe concentration was used to calculate the T2
relaxivity (r2). CT imaging of the samples dispersed in water with different Au
concentrations (0.01e0.08 M) was performed using a GE LightSpeed VCT imaging
system (GE Medical Systems) with 100 kV, 80 mA, and a slice thickness of 0.625 mm.
The X-ray attenuation intensity in Hounsfield units (HU) was evaluated by loading
the digital CT images in a standard display program and then selecting a uniform
round region of interest on the resultant CT image for each sample. To determine the
photothermal property of the Fe3O4@Au-HA NSs, an aqueous suspension of
Fe3O4@Au-HA NSs with different Au concentrations (0.32e24 mM, 0.3 mL),
Fe3O4@Ag seeds (with Fe concentration similar to that of the Fe3O4@Au-HA NSs with
Au concentration of 24 mM), or water was put into a quartz cuvette, and illuminated
by a 915 nm laser (Shanghai Xilong Optoelectronics Technology Co. Ltd, Shanghai,
China) with a power density of 1.2 W/cm2 for 300 s. The temperature of different
samples was recorded by an online DT-8891E thermocouple thermometer (Shenz-
hen Everbest Machinery Industry Co., Ltd., Shenzhen, China) every 5 s.
2.8. Hemolysis and cytocompatibility assay
Hemolysis assay of Fe3O4@Au-HA NSs was carried out according to protocols
described in the literature [55]. Briefly, fresh human blood (kindly provided by
Shanghai First People's Hospital with approval by the ethical committee of Shanghai
First People's Hospital) was centrifuged, purified, and 10 times diluted with PBS to
obtain human red blood cells (HRBCs). Then, 0.1 mL diluted HRBC suspension was
added to 0.9 mL water (as a positive control), 0.9 mL PBS (as a negative control), and
0.9 mL PBS containing Fe3O4@Au-HA NSs at different Au concentrations (0.25, 0.5,
1.0, 2.0, and 4.0 mM, respectively). The mixtures were gently shaken, and then kept
still at room temperature for 2 h. After that, the samples were centrifuged
(10,000 rpm, 1 min) and the absorbance of the supernatants (hemoglobin) was
measured by UVevis spectrophotometer. The hemolysis percentage was calculated
based on the absorbance at 541 nm according to the literature [56].
HeLa cells were continuously cultured and passaged in 25 cm2 plates with
DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and
100 mg/mL streptomycin under 37  C and 5% CO2. For MTT assay, HeLa cells were
seeded into 96-well plates with 200 mL fresh medium at a density of 1  104 cells/
well. After incubation for 12 h to bring the cells to confluence, the medium was
replaced with 200 mL fresh medium containing Fe3O4@Au-HA NSs with different
final Au concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.5, and 2.0 mM, respectively) and the
cells were incubated for 24 h at 37  C and 5% CO2. Thereafter, MTT solution (20 mL,
5 mg/mL in PBS buffer) was added into each well and the cells were incubated for
another 4 h. The assays were carried out according to the manufacturer's instruction.
The absorbance at 570 nm of each well was measured using a Thermo Scientific
Multiskan MK3 ELISA reader (Thermo Scientific, USA). The background subtraction
 J. Li et al. / Biomaterials 38 (2015) 10e21
at 570 nm was applied to eliminate the influence of the added materials. For each
sample, mean and standard deviation of five parallel wells were recorded.
2.9. Targeted MR and CT imaging of cancer cells in vitro
HeLa cells seeded in 6-well plates at a density of 2  106 cells/well in DMEM
were brought to confluence after overnight culture. Then the medium was replaced
with fresh medium containing Fe3O4@Au-HA NSs at different Au concentrations (0,
1.25, 2.5, 3.75, and 5.0 mM, respectively) and the cells were incubated at 37  C and 5%
CO2 for 6 h. U87MG cells cultured with MEM were used as control and were treated
in the same manner [16]. Thereafter, the cells were washed 3 times with PBS,
trypsinized, centrifuged, and resuspended in 0.5 mL PBS (containing 0.5% agarose) in
1.5-mL Eppendorf tubes before MR and CT imaging. T2-weighted MR imaging was
carried out using a 1.5 T Signa HDxt superconductor clinical MR system (GE Medical
Systems, Milwaukee, WI) under the following parameters: point
resolution ¼ 156 mm  156 mm, section thickness ¼ 0.6 mm, TR ¼ 3000 ms,
TE ¼ 90 ms, and number of excitation ¼ 1. CT scanning was performed using GE
LightSpeed VCT imaging system (GE Medical Systems) under the conditions similar
to those used to analyze the X-ray attenuation intensity of samples as described
above.
2.10. In vitro cellular uptake assay
The specific uptake of Fe3O4@Au-HA NSs by HeLa cells overexpressing CD44
receptors was investigated by ICP-OES. U87MG cells without CD44 receptor
expression [16] were used as control. Briefly, 5  105 HeLa or U87MG cells per well
were seeded in 12-well plates at 37  C and 5% CO2 the day before the experiment.
The next day, the medium was replaced with fresh medium containing Fe3O4@Au-
HA NSs at the Au concentrations of 1.0 or 2.0 mM. After 6 h incubation, the medium
was discarded and the cells were washed with PBS for 3 times, trypsinized,
centrifuged, and resuspended in 1 mL PBS. A portion of cells (100 mL cell suspension)
was counted, and the remaining cells were centrifuged, collected, and lysed using
aqua regia solution (1.0 mL, nitric acid/hydrochloric acid, v/v ¼ 1:3). Then the
samples were diluted with 1.0 mL PBS, and the Au concentration in different cells
was measured by ICP-OES.
2.11. In vivo MR and CT imaging of tumors
All animal experiments were performed according to the guidelines of the
institutional committee for animal care, and also in accordance with the policy of the
National Ministry of Health. Male 4- to 6-week-old BALB/c nude mice (15e20 g)
were purchased from Shanghai Slac Laboratory Animal Center (Shanghai, China). To
establish a xenografted tumor model, HeLa cells (2  106/mouse) were subcutane-
ously implanted into the back of the nude mouse. When the tumor nodules reached
a volume of 0.12e0.30 cm3 after 10 days, the tumor-bearing mice were used.
For MR imaging of tumors, HeLa tumor-bearing nude mice were first anes-
thetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg), then a PBS
solution of Fe3O4@Au-HA NSs ([Fe] ¼ 5.0 mM, 0.1 mL) was intratumorally injected
into the tumor site. After 10 min, the mice were placed inside a custom-built rodent
receiver coil (Chenguang Med Tech, Shanghai, China) and MR imaging was per-
formed using a 1.5 T Signa HDxt superconductor clinical MR system. T2-weighted
MR images of the mice before and after 10 min post injection were obtained using a
conventional spin-echo sequence under the parameters similar to those used for MR
imaging of cancer cells in vitro.
For tumor CT imaging, HeLa tumor-bearing nude mice were anesthetized as
mentioned above and intratumorally injected with a PBS solution of Fe3O4@Au-HA
NSs ([Au] ¼ 123.5 mM, 0.1 mL). CT scans were performed before and at 10 min post
injection using a GE LightSpeed VCT clinical imaging system with 100 kV, 80 mA, and
a slice thickness of 0.625 mm.
2.12. In vitro photothermal ablation of HeLa cells
HeLa cells were seeded into 96-well plates with 200 mL fresh DMEM at a density
of 1  104 cells/well and incubated for 12 h to allow the cells to be attached before
photothermal experiments. Then the medium was carefully removed and fresh
medium (200 mL) containing 20 mL Fe3O4@Au-HA NSs at different final Au concen-
trations (0, 0.1, 0.2, 0.3, or 0.4 mM, respectively) was added into each well. After
incubation for another 6 h, the cells were irradiated by a 915 nm laser with an output
power density of 1.2 W/cm2 for 5 and 10 min, respectively. The cell viability was then
measured via MTT assay according to the procedures described above. Mean and
standard deviation for the triplicate wells were reported. In parallel, the morphology
of cells treated with PBS or Fe3O4@Au-HA NSs at the Au concentration of 0.4 mM for
6 h, followed by irradiation with a 915 nm laser (at an output power density of 1.2 W/
cm2) for 10 min and then rinsing with PBS for 3 times was observed by Leica DM IL
LED inverted phase contrast microscope.
2.13. In vivo photothermal imaging
HeLa tumor-bearing nude mice were first anesthetized by intraperitoneal in-
jection of pentobarbital sodium (40 mg/kg), then a PBS solution of Fe3O4@Au-HA
NSs ([Au] ¼ 32 mM, 0.1 mL) was intratumorally injected into the mice. The mice
13
intratumorally injected with PBS (0.1 mL) were used as control. After 10 min, the
tumor site was exposed to a 915 nm laser with a power density of 1.2 W/cm2 for
5 min. During the process of laser radiation, a photothermal medical device (GX-300,
Shanghai Infratest Electronics Co., Ltd, Shanghai, China) with an infrared camera was
used to obtain the whole-body infrared thermal images at different time points.
2.14. In vivo photothermal ablation of HeLa tumors
HeLa tumor-bearing nude mice were randomly divided into four groups (n ¼ 4
for each group). The mice were intratumorally injected with 0.1 mL PBS without
laser irradiation (Control group), 0.1 mL PBS and then the tumor site was exposed to
a 915 nm laser with a power density of 1.2 W/cm2 for 10 min (Laser group), 0.1 mL
PBS containing Fe3O4@Au-HA NSs ([Au] ¼ 32 mM) without laser irradiation (NSs
group), and 0.1 mL PBS containing Fe3O4@Au-HA NSs ([Au] ¼ 32 mM) with laser
irradiation under similar power density and time period (NSs þ Laser group). The
similar treatments were carried out again after the next day (at day 3), but the
volume of PBS or PBS solution containing Fe3O4@Au-HA NSs at the same Au con-
centration was decreased to 0.05 mL. The tumor size and body weight of all mice
were measured and pictures of mice were taken at pre-determined time points. The
length and width of the tumors were measured by using a digital vernier caliper and
the tumor volumes were calculated according to the formula of (tumor
length  (tumor width)2)/2. The survival rate of the mice in each group was
calculated according to the formula of N1/N  100%, where N1 and N represent the
number of surviving mice and the number of total mice in each group, respectively.
2.15. H&E and TUNEL staining
Four groups of HeLa tumor-bearing nude mice were treated according to the
above protocols (n ¼ 1 for each group). After 2 h, the mice were euthanized and the
tumors were removed, fixed in 4% paraformaldehyde, and embedded in paraffin for
H&E staining and TUNEL staining according to standard protocols described in our
previous work [44]. The morphology of tumor sections after different treatments
was observed using a Leica DM IL LED inverted phase contrast microscope. The
number and percentage of TUNEL-positive cells in each sample were counted and
determined from five random selected fields.
2.16. Statistical analysis
One-way ANOVA statistical analysis was performed to evaluate the significance
of the experimental data. 0.05 was selected as the significance level, and the data
were indicated with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001,
respectively.
3. Results and discussion
3.1. Synthesis and characterization of Fe3O4@Au-HA NSs
In our previous work, we synthesized Fe3O4@Au composite
nanoparticles via a one-pot hydrothermal route for in vivo dual
mode MR/CT imaging applications [56]. By virtue of the same hy-
drothermal approach, Fe3O4@Ag seed particles were formed, fol-
lowed by exposure to Au growth solution to form the Fe3O4@Au
NSs. To render the Fe3O4@Au NSs with targeting specificity, the
Fe3O4@Au NSs were first reacted with PEI-SH via AueS bond, and
then reacted with HA via EDC chemical crosslinking of the PEI
amines onto the surfaces of Fe3O4@Au NSs (Scheme 1).
The formation of PEI-SH (Fig. S1, Supporting Information) was
first confirmed by FTIR spectroscopy (Fig. S2, Supporting
Information). By comparison with PEI, an obvious peak emerging
at 1640 cm1 in the spectrum of PEI-SH is attributed to the char-
acteristic peak of amido linkage (Fig. S1). In addition, the appear-
ance of another weak band at 2570 cm1 suggests the existence of
eSH in the formed PEI-SH, in agreement with the literature [61]. 1H
NMR was also carried out to confirm the structure of PEI-SH and to
quantify the degree of PEI thiolation (Fig. S3, Supporting
Information). We can see that the PEI and PEI-SH show similar
characteristic peaks except for the peak at 3.4 ppm, which can be
attributed to the proton signal of eCOeCH2eSe in MTG. Based on
the NMR integration, the average number of SH coupled to each PEI
was estimated to be 16.9. Both FTIR and NMR results suggest the
successful formation of PEI-SH.
To form the Fe3O4@Ag seed particles, PEI-stabilized Ag NPs (PEI-
Ag NPs) with a mean diameter of 5.3 nm (Fig. S4aeb, Supporting
14
Information) were first formed via NaBH4 reduction chemistry,
similar to our previous work related to the formation of PEI-
stabilized Au NPs [56]. In the presence of PEI-stabilized Ag NPs, a
hydrothermal approach [17,56] was used to synthesize Fe3O4@Ag
seeds with a size of 13.1 nm (Fig. S4ced, Supporting Information)
using FeCl2$4H2O as Fe precursor. Then, the formed Fe3O4@Ag
seeds were developed into Fe3O4@Au NSs by dropping them into
the Au growth solution containing silver ions (Fig. S4eef,
Supporting Information). The selected volume and concentration
of Au growth solution have been optimized to enable the formed
NSs with a desirable Fe/Au molar ratio to ensure their subsequent
effective multimode imaging and therapy applications. To render
the Fe3O4@Au NSs with good colloidal stability in aqueous solution
and amine functionality, Fe3O4@Au NSs were washed with water to
remove the capping agent CTAB in the solution, followed by
modification with PEI-SH via AueS bond. The aminated Fe3O4@Au-
PEI NSs were then modified with HA to be afforded with targeting
specificity to CD44 receptor-overexpressing cancer cells.
Zeta potential measurements were employed to confirm the
successful modification of HA onto the surface of NSs (Fig. S5a,
Supporting Information). Fe3O4@Au-PEI NSs dispersed in water
had a positive potential of þ32.7 mV because of the surface
modification of PEI with abundant amines. After modification with
HA, the zeta potential of Fe3O4@Au-HA NSs was reversed to be
negative (27.7 mV), suggesting the successful conjugation reac-
tion [16,62,63]. The hydrodynamic sizes of NSs in aqueous solution
before and after HA modification were measured to be 298.8 and
339.4 nm (Fig. S5b, Supporting Information), respectively by dy-
namic light scattering (DLS). This suggests that the HA coating
enlarged the periphery of Fe3O4@Au-PEI NSs, further confirming
the successful HA conjugation. What's more, the hydrodynamic
sizes of the Fe3O4@Au-HA NSs at different storage time periods
1.5
a Fe3O4@Ag seeds
b c
Fe3O4@Au-HA NSs
Absorbance
1.0
0.5
0.0
400 600 800 1000
Wavelength (nm)
60
d Mean diameter = 119.4 nm
σ = 19.4 nm e f
50
Frequency (%)
40
30
20
10
0 20 30
60 80 100 120 140 160 180
Diameter (nm)
Fig. 1. (a) UVevis spectra of Fe3O4@Ag seeds and Fe3O4@Au-HA NSs (inset is the photograph of Fe3O4@Ag seeds and Fe3O4@Au-HA NSs dispersed in water), (b, c) TEM image, (d) size
distribution histogram, (e) high-resolution TEM image, and (f) XRD pattern of the Fe3O4@Au-HA NSs.
J. Li et al. / Biomaterials 38 (2015) 10e21
were also recorded to evaluate their long-term colloidal stability
(Fig. S6, Supporting Information). It is clear that the hydrodynamic
size does not have any appreciable changes within a time period of
2 weeks, indicating their good colloidal stability. Furthermore, the
colloidal stability of Fe3O4@Au-HA NSs was also checked by
exposing them to water, PBS, and cell culture medium (DMEM) for
at least one month. We show that the NSs are still stable and no
precipitation occurs (Fig. S7, Supporting Information), further
confirming their excellent colloidal stability in different aqueous
media.
The grafting of PEI and subsequent conjugation of HA onto the
surface of Fe3O4@Au NSs were also confirmed by TGA (Fig. S8,
Supporting Information). Due to the fact that at 700  C, most of
the organic components have been burned off, we selected 700  C
to calculate the weight loss of NSs after each step of surface
modification. Compared with Fe3O4@Au NSs just showing a weight
loss of 0.94%, the PEI grafting via AueS bond formation rendered
the NSs with a weight loss of 14.23%. Further conjugation of HA via
EDC chemistry resulted in an increased weight loss of 24.36% for
Fe3O4@Au-HA NSs. The grafting percentages of PEI and HA were
calculated to be 13.29% and 10.13%, respectively.
UVevis spectroscopy was used to investigate the optical prop-
erty of the Fe3O4@Au-HA NSs (Fig. 1a). It is clear that Fe3O4@Au-HA
NSs exhibit an obvious surface plasmon resonance (SPR) peak at
870 nm, which is amenable for photothermal therapy applications
under NIR laser irradiation [47]. In contrast, Fe3O4@Ag seeds do not
show any obvious absorption features in the same region. It should
be noted that the solution of Fe3O4@Au-HA NSs became blue
because of the surface coating of Au shells, which is different from
that of the Fe3O4@Ag seeds (Fig. 1a, inset).
The morphology and size of the Fe3O4@Au-HA NSs were
investigated by TEM imaging (Fig. 1bee). It can be seen that star-
* (111)
* Au
Fe3O4
Intensity (a.u.)
(400)
* (311)
*(220)
(220)
* (200)
* (222)
(311)
(511)
(422)
(440)
40 50 60 70 80 90
2θ(degree)
 J. Li et al. / Biomaterials 38 (2015) 10e21 15

shaped Au shells are coated onto the surface of clustered Fe3O4 NPs,
and the Fe3O4@Au-HA NSs have a quite uniform size distribution
(Fig. 1b). A close observation of a single Fe3O4@Au-HA NS reveals
that several spike-like gold shell crystals are densely and discon-
tinuously surrounding a cluster of Fe3O4 NPs (Fig. 1c), in agreement
with the literature [34]. The mean diameter of the internal sphere
(Fig. 1c) was estimated to be 119.4 ± 19.4 nm (Fig. 1d). High-
resolution TEM image confirmed the crystal structure of the star-
shaped Au shells, as lattices of the crystals can be clearly
observed (Fig. 1e). In addition, the NSs were also found to be sur-
rounded with a transparent polymer shell on the outer surface,
which is associated with the PEI coating and HA modification
(Fig. 1e). The crystalline structure of the Fe3O4@Au-HA NSs was also
characterized by XRD (Fig. 1f). The diffraction peaks well match the
planes of Fe3O4 and Au crystals, indicating the formation of crys-
talline Fe3O4@Au composite structures. Meanwhile, due to the core/
shell structure, some peaks related to Fe3O4 are not prominent. The
elemental composition of the Fe3O4@Au-HA NSs dispersed in water
or PBS was quantitatively measured using ICP-OES, and the Fe/Au
molar ratio was estimated to be 1:24.7.
3.2. T2 MR relaxometry and X-ray attenuation property
Fe3O4 NPs have been known to be able to shorten the T2
relaxation time of water protons, resulting in MR contrast
enhancement. The transverse relaxivity (r2, the transverse relaxa-
tion rate per mM of Fe) is usually used to quantify the efficiency to
use Fe3O4 NPs as contrast agents. T2-weighted MR imaging data
show that the developed Fe3O4@Au-HA NSs are able to weaken the
signal intensity of the MR images with the Fe concentration
(Fig. 2a). By plotting T2 relaxation rate (1/T2) as a function of Fe
concentration (Fig. 2b), a linear relationship between the relaxation
rate and the Fe concentration (R2 ¼ 0.9999) can be found with a
slope of 144.39 mM1 s1, which is identified to be the r2 value of
the Fe3O4@Au-HA NSs. It seems that the star-shaped Au shell
coating and the further conjugation of PEI and HA do not appre-
ciably weaken the r2 relaxivity when compared to Fe3O4@Au
composite NPs reported in our previous work [56]. The relatively
high r2 value of Fe3O4@Au-HA NSs may be due to the fact that water
protons are accessible to the surface of clustered Fe3O4 NPs in the
core of the NSs via the interstitial spaces between the Au spikes.

a H c

Fe (mM) 0.005 0.01 0.02 0.04 0.08 Au (M) 0.01 0.02 0.04 0.06 0.08
L
600
b d
r2=144.39 mM-1s-1, R2=0.9999
12
450
Hounsfield Unit (HU)
1/T2 (s-1)

8 300

4 150

0
0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08
Fe concentration (mM) Au concentration (M)
e 90 f 70
water seeds 0.32 mM 0.8 mM
1.6 mM 3.2 mM 16 mM 24 mM
80 60
70 50
Temperature (°C)

60 40
ΔT (°C)

50
30
40
20
30
10
20
0
0 50 100 150 200 250 300 0 1 2 3 16 24
Time (s) Au concentration (mM)

Fig. 2. (a) Color T2-weighted MR images and (b) linear fitting of 1/T2 of Fe3O4@Au-HA NSs at different Fe concentrations (The color bar from red to blue indicates the gradual
decrease of MR signal intensity). (c) CT images and (d) X-ray attenuation intensity of the Fe3O4@Au-HA NSs with different Au concentrations. (e) Temperature elevation of water and
the aqueous solution of Fe3O4@Ag seeds ([Fe] ¼ 0.972 mM) or Fe3O4@Au-HA NSs at different Au concentrations (0.32, 0.8, 1.6, 3.2, 16 and 24 mM, respectively) under the irradiation of
a 915 nm laser with a power density of 1.2 W/cm2 as a function of irradiation time. (f) The temperature change (DT) of an aqueous suspension of Fe3O4@Au-HA NSs at different Au
concentrations over a period of 300 s. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
16 J. Li et al. / Biomaterials 38 (2015) 10e21
Our data suggest a great potential to use the Fe3O4@Au-HA NSs as a
T2 negative contrast agent for MR imaging applications.
On the other hand, the potential to use Fe3O4@Au-HA NSs as a
CT contrast agent was confirmed by X-ray attenuation intensity
measurement (Fig. 2c). We show that the CT image of Fe3O4@Au-
HA NSs becomes brighter with the Au concentration, correlating
well with the quantitative analysis of the attenuation intensity
change of the Fe3O4@Au-HA NSs as a function of Au concentration
(Fig. 2d). Since AuNPs are known to be a good CT contrast agent
[20], there is no doubt to conclude that the Fe3O4@Au-HA NSs can
be potentially used as a contrast agent for CT imaging applications
due to the integrated Au component, similar to our previous study
related to Fe3O4@Au composite NPs [56,57].
3.3. Photothermal property of Fe3O4@Au-HA NSs
The strong SPR absorption of Fe3O4@Au-HA NSs in the NIR re-
gion drove us to explore their photothermal property. The tem-
perature change of an aqueous suspension of the Fe3O4@Au-HA NSs
as a function of Au concentration (0.32e24 mM) under laser irra-
diation for 300 s was monitored (Fig. 2e). It is clear that the
Fe3O4@Au-HA NSs with higher Au concentration result in a more
prominent temperature increase, and the solution temperature
reaches 81.2  C at the Au concentration of 24 mM. In contrast, the
water and the aqueous suspension of Fe3O4@Ag seeds (with the
same Fe concentration as the Fe3O4@Au-HA NSs at the Au con-
centration of 24 mM) do not have obvious temperature increase
under similar experimental conditions. The plot of temperature
change (DT) over a time period of 300 s versus Au concentration
shows that the temperature only increase 5.0 and 10.1  C for water
and Fe3O4@Ag seeds, respectively. With the increase of Au con-
centration (from 0.32 to 24 mM), the aqueous suspension has a
temperature increase of 15.4, 24.8, 34.8, 47.1, 57.0, and 62.8  C,
respectively (Fig. 2f). Our results indicate that Fe3O4@Au-HA NSs
are able to generate heat rapidly and efficiently upon laser irradi-
ation. It should be noted that the developed Fe3O4@Au-HA NSs still
have a very good colloidal stability during and after the laser irra-
diation process, which is amenable for their effective photothermal
therapy applications.
3.4. Hemolytic assay and cytotoxicity assay
For biomedical applications, hemocompatibility and cyto-
compatibility of the developed Fe3O4@Au-HA NSs should be eval-
uated. Hemolytic assay was used to assess the hemocompatibility
of the Fe3O4@Au-HA NSs (Fig. S9, Supporting Information). We can
see that the Fe3O4@Au-HA NSs at the Au concentration ranging
from 0.25 to 4.0 mM do not cause any obvious hemolysis effect
when compared with the negative control (PBS). In contrast, the
positive control of water induces a significant hemolysis of HRBCs.
Based on the absorbance of the supernatant at 541 nm that is
associated with the absorption of the released hemoglobin from
HRBCs, the hemolysis percentages of HRBCs in the presence of
Fe3O4@Au-HA NSs at the Au concentrations of 0.25, 0.5, 1.0, 2.0, and
4.0 mM were calculated to be 0.26%, 0.62%, 0.79%, 1.39%, and 1.65%,
respectively (Fig. S9, inset), which are all less than the threshold
value of 5% [19]. This suggests that the developed Fe3O4@Au-HA
NSs have a good hemocompatibility in the studied concentration
range.
The cytotoxicity of the Fe3O4@Au-HA NSs was evaluated by MTT
viability assay of HeLa cells treated with the particles (Fig. S10,
Supporting Information). It can be seen that the viability of HeLa
cells does not have any appreciable changes after incubation with
the Fe3O4@Au-HA NSs at the Au concentrations of 0.2, 0.4, 0.6, 0.8,
1.0, and 1.5 mM, respectively, when compared with the PBS control.
When the Au concentration increases to 2.0 mM, the Fe3O4@Au-HA
NSs start to display cytotoxicity and the cell viability can still reach
66.5%. Taken together with the results from hemolytic assay, we can
safely conclude that the developed Fe3O4@Au-HA NSs have a good
biocompatibility in the studied concentration range, which is
essential for their further biomedical applications.
3.5. In vitro targeted MR and CT imaging of cancer cells
We next investigated the potential to use the developed
Fe3O4@Au-HA NSs as a nanoprobe for targeted dual mode MR/CT
imaging of cancer cells in vitro. In this study, we selected HA as a
targeting ligand. HA, a member of the glycosaminoglycan family
composed of repeating disaccharide units of D-glucuronic acid and
N-acetyl-D-glucosamine, has been recognized as an attractive tar-
geting ligand that can bind to CD44 receptor-overexpressing cancer
cells [16,62,64]. U87MG cells without CD44 receptor over-
expression were used as control. After incubation with the NSs at
different Fe or Au concentrations for 6 h, the cells were subjected to
T2-weighted MR imaging and CT imaging, respectively (Fig. 3aeb).
It can be seen that the MR signal intensity of both HeLa and U87MG
cells decreases with the Fe concentration, however the decreasing
trend of U87MG cells is much less than that of HeLa cells under
similar conditions (Fig. 3a). This can be further confirmed by
quantitative analysis of the signal intensity of the cells (Fig. 3c),
where the signal intensity of the treated HeLa cells was much lower
than that of the U87MG cells under a given Fe concentration
(p < 0.01).
For CT imaging, due to the fact that it is difficult to visually
differentiate the brightness of the CT images of the cells treated
with the NSs at different Au concentrations [19,65], it is essential to
perform quantitative analysis of the CT signal intensity using the
manufacturer's standard display program (Fig. 3d). It can be seen
that the CT values of both HeLa and U87MG cells treated with the
Fe3O4@Au-HA NSs increase with the Au concentration. Apparently,
at a relatively high Au concentration, the CT value of HeLa cells with
CD44 receptor-overexpression is much higher than that of U87MG
cells at the same Au concentration (p < 0.05). These results suggest
that the developed Fe3O4@Au-NSs have a high affinity to CD44
receptor-overexpressing cancer cells, thereby enabling targeted
dual mode MR/CT imaging of cancer cells via receptor-mediated
active targeting pathway.
3.6. In vitro cellular uptake assay
The targeted uptake of the Fe3O4@Au-HA NSs by HeLa cells was
further quantitatively confirmed by ICP-OES analysis of the Au
uptake (Fig. 3e). It is clear that for both HeLa and U87MG cells, the
treatment of Fe3O4@Au-HA NSs with a higher concentration leads
to a higher Au uptake within the cells. Importantly, at the same Au
concentration (1.0 and 2.0 mM), the Au uptake in HeLa cells over-
expressing CD44 receptors is significantly higher than that in
U87MG cells without CD44 receptor overexpression. This further
confirmed the role played by HA-mediated targeting that enables
specific uptake the Fe3O4@Au-HA NSs, in agreement with our
previous work [16].
3.7. In vivo MR and CT imaging of a xenografted tumor model
With the excellent targeting specificity of the Fe3O4@Au-HA NSs
for in vitro MR/CT imaging of cancer cells, we next explored the
potential to use the NSs as a contrast agent for MR/CT imaging of a
xenografted tumor model. The tumor-bearing mice intratumorally
injected with the Fe3O4@Au-HA NSs before and at 10 min post in-
jection were imaged by MR and CT, respectively (Fig. 4aeb). We can
 J. Li et al. / Biomaterials 38 (2015) 10e21 17

a [Fe]/mM 0 0.05 0.10 0.15 0.20 b [Au]/mM 0 1.25 2.50 3.75 5.00
U87MG cells U87MG cells

HeLa cells HeLa cells

c 1600 d 120
U87MG cells ***
** HeLa cells U87MG cells
*** HeLa cells
1200 90

Hounsfield unit (HU)

*** **
Signal intensity

*** *
800 60

400 30

0 0
0 0.05 0.10 0.15 0.20 0 1.25 2.50 3.75 5.00
Fe concentration (mM) Au concentration (mM)
e 420 f 120
0 min 5 min 10 min
***
Cellular Au uptake (pg/cell)

U87MG cells
*** *** *** ***
HeLa cells 100 *** *** *** ***
*** *** ***
Cell viability (%)

280 80
***
60

140 40

20
0
0
Control 1.0 2.0 0.0 0.1 0.2 0.3 0.4
Au concentration (mM) Au concentration (mM)

Fig. 3. (a) T2-weighted MR images, (b) CT images, (c) MR signal intensity, and (d) CT values of HeLa and U87MG cells after treated with Fe3O4@Au-HA NSs at different Fe or Au
concentrations for 6 h. (e) The Au uptake by HeLa and U87MG cells treated with Fe3O4@Au-HA NSs at the Au concentration of 1.0 and 2.0 mM for 6 h. (f) MTT viability assay of HeLa
cells after treatment with the Fe3O4@Au-HA NSs at different Au concentrations and different laser irradiation time periods.

clearly see that the tumor region darkens obviously in a typical T2-
weighted MR image at 10 min post injection (Fig. 4a), when
compared to before injection. The quantitative signal intensity
analysis reveals that the MR signal intensity of the tumor region
before injection (641.0) dramatically decreases to 41.1 at 10 min
post-injection. Likewise, in the CT images, the intratumoral
administration of the Fe3O4@Au-HA NSs makes the tumor region
much brighter at 10 min post injection (Fig. 4b). To quantify the CT
contrast enhancement, the CT values of the tumor region before and
at 10 min post injection were measured. The CT value of the tumor
region was estimated to be 2303.3 HU at 10 min post injection, much
higher than that before injection (157.4 HU). This suggests that the
intratumoral injection of the NSs leads to a quite uniform distribu-
tion of the particles within the tumor region, allowing for effective
MR/CT imaging of the whole tumor. These results suggest that the
formed Fe3O4@Au-HA NSs have a great potential to be used as a
contrast agent for in vivo tumor MR/CT imaging.
3.8. In vitro photothermal ablation of cancer cells
The high photothermal conversion efficiency and the targeting
specificity of the Fe3O4@Au-HA NSs drove us to investigate the
potential to use these NSs for photothermal ablation of cancer cells
in vitro. The viability of HeLa cells treated with the Fe3O4@Au-HA
NSs at different Au concentrations under laser irradiation was
assessed by MTT assay (Fig. 3f). In all cases, HeLa cells without
treatment with Fe3O4@Au-HA NSs under laser irradiation (5 or
10 min), or HeLa cells treated with Fe3O4@Au-HA NSs at different
Au concentrations (0.1e0.4 mM) without laser irradiation do not
display any appreciable viability changes when compared to the
PBS control. In contrast, when HeLa cells treated with the
Fe3O4@Au-HA NSs were irradiated by a 915 nm NIR laser for 5 min,
the cell viability started to have a significant decrease at the Au
concentration of 0.2 mM (p < 0.001). Further extension of the laser
irradiation time to 10 min led to more prominent cell death under
all the studied Au concentrations (p < 0.001). The cells treated with
Fe3O4@Au-HA NSs at a higher Au concentration under a longer time
of laser irradiation have more decreased viability. Around 62.2%
HeLa cells treated with the Fe3O4@Au-HA NSs at an Au concen-
tration of 0.4 mM were able to be killed under laser irradiation for
10 min. Our results suggest a great potential to use Fe3O4@Au-HA
NSs for photothermal ablation of cancer cells.
The photothermal therapeutic efficacy of Fe3O4@Au-HA NSs was
further evaluated by optical microscopic observation of cell
18 J. Li et al. / Biomaterials 38 (2015) 10e21

a b

0 min 10 min 0 min 10 min

c (d) 70
Region 11 Region 12

60

Temperature (°C)
50

40

30

20
0 50 100 150 200 250 300
Time (s)

Fig. 4. (a) T2-weighted MR images and (b) CT images of the tumors before injection and at 10 min post intratumoral injection of 0.1 mL PBS solution containing Fe3O4@Au-HA NSs
([Fe] ¼ 5.0 mM, [Au] ¼ 123.5 mM). (c) Photothermal images of two tumor-bearing mice injected with 0.1 mL PBS (the left mouse, indicated region 11) or 0.1 mL PBS containing
Fe3O4@Au-HA NSs ([Au] ¼ 32 mM, the right mouse, indicated region 12), respectively, followed by irradiation with a 915 nm laser (1.2 W/cm2) at a time point of 0, 1.5 min, and 5 min,
respectively. (d) The temperature profiles in Regions 11 and 12 as a function of the laser irradiation time.

morphology (Fig. S11, Supporting Information). The cells treated
with laser alone or with Fe3O4@Au-HA NSs without laser irradia-
tion have similar attachment morphology, with cell numbers
approximately similar to the PBS control (Fig. S11aec). In contrast,
when the cells were incubated with Fe3O4@Au-HA NSs and then
irradiated by the laser for 10 min, most of the cells died and only a
small portion of adherent cells left after the washing step. Our re-
sults suggest that the treatment of laser or Fe3O4@Au-HA NSs alone
does not exert any therapeutic efficacy to the cancer cells, and the
developed Fe3O4@Au-HA NSs are able to effectively ablate cancer
cells under laser irradiation, corroborating the MTT results.
3.9. In vivo photothermal imaging and ablation of a xenografted
tumor model
We next explored the feasibility to use the developed
Fe3O4@Au-HA NSs for photothermal imaging and ablation of a
xenografted tumor model in vivo. Two HeLa tumor-bearing mice
were intratumorally injected with 0.1 mL PBS or 0.1 mL PBS con-
taining Fe3O4@Au-HA NSs with Au concentration of 32 mM,
respectively (Regions 11 and 12 in Fig. 4c). After 10 min, Regions 11
and 12 were irradiated under a 915 nm laser (1.2 W/cm2) for 300 s.
The full-body thermal images of the mice were captured using an
infrared camera. It is clear that only a slight temperature change
was detected at Region 11. In contrast, Region 12 displays a sig-
nificant temperature increase due to the injection of the Fe3O4@Au-
HA NSs. The tumor temperature was also monitored as a function of
the laser irradiation time (Fig. 4d). It can be observed that Region 11
injected with PBS only has a slight temperature increase of 4.5  C
and remains below 36.8  C during the laser irradiation. In sharp
contrast, Region 12 injected with Fe3O4@Au-HA NSs has a rapid
temperature increase from 32.8 to 58.9  C after 90 s laser irradia-
tion, and then remains above 50  C for the following 205 s. Our data
suggest a great potential to use the developed Fe3O4@Au-HA NSs
for thermal imaging of tumors.
The high local temperature in the tumor region after treatment
with the Fe3O4@Au-HA NSs under laser irradiation is believed to be
able to kill the tumor cells. We then investigated the photothermal
therapeutic efficacy of Fe3O4@Au-HA NSs by measuring the vol-
umes of the tumors after different treatments (Fig. 5a). It is obvious
that the volumes of tumors treated with laser alone or Fe3O4@Au-
HA NSs alone increase with the time, similar to the control group,
suggesting that the laser irradiation alone or injection of
Fe3O4@Au-HA NSs without laser irradiation does not have any
impact on the tumor growth. In contrast, the tumors treated with
Fe3O4@Au-HA NSs under laser irradiation are able to be completely
inhibited. On day 19, the tumor tissue almost completely dis-
appeared in the mice after the photothermal therapy, which is
significantly different from other groups (Fig. S12, Supporting
Information). Furthermore, mice in different treatment groups
maintained their weights during the experimental time period
(Fig. 5b), implying that the laser irradiation alone, the injection of
the Fe3O4@Au-HA NSs alone, or the combination of the above two is
unable to generate toxicity to the mice. To further investigate the
photothermal therapeutic efficacy of the Fe3O4@Au-HA NSs, the
survival rate of the mice in the four groups was evaluated (Fig. 5c).
It is obvious that the mice treated with the Fe3O4@Au-HA NSs un-
der laser irradiation maintain a 100% survival rate after 60 days,
which is significantly higher than the mice in the other three
groups. The survival rate of the mice without treatment, treated
 J. Li et al. / Biomaterials 38 (2015) 10e21 19

a 12 b 35 c
Control Control
Laser Laser 100
Relative tumor volume

NSs NSs
9

Body weight (g)

30 NSs + Laser

Survival rate (%)

NSs + Laser
75
Control
Laser
6 25 NSs
50 NSs + Laser

3 20 25

0 15 0
0 4 8 12 16 20 0 4 8 12 16 20 0 10 20 30 40 50 60
Time (day) Time (day) Time (day)

Fig. 5. The relative tumor volume (a), body weight (b), and survival rate (c) of HeLa tumor-bearing mice as a function of time post treatment.

with Fe3O4@Au-HA NSs only, and treated with laser only is 25%, 0% Information). These results demonstrate that the developed
and 0%, respectively. Our results suggest that the developed Fe3O4@Au-HA NSs are able to be used as an efficient nanoplatform
Fe3O4@Au-HA NSs have a great potential to be used for photo- for photothermal ablation of tumors in vivo.
thermal therapy of tumors.
4. Conclusion
3.10. H&E and TUNEL staining
In summary, we developed a convenient approach to synthe-
The photothermal ablation of tumors using Fe3O4@Au-HA NSs sizing Fe3O4@Au-HA NSs with a quite uniform morphology for
was further confirmed by histological examination of tumor sec- multi-mode imaging and photothermal therapy of tumors. The
tions after different treatments (Fig. 6). H&E staining of the tumor hydrothermally synthesized Fe3O4@Ag seeds are able to be
sections shows that the tumors treated with laser alone and with deposited with star-shaped Au shells that can be modified with PEI
the Fe3O4@Au-HA NSs alone display well-shaped tumor cells via AueS bond. Likewise, the PEI-mediated reaction can be used for
without the appearance of necrosis region, similar to the control HA conjugation onto the surface of the NSs to render them with
group treated with PBS. However, obvious necrosis area can be seen targeting specificity to CD44 receptor-overexpressing cancer cells.
in the tumor tissue treated with Fe3O4@Au-HA NSs under laser The formed Fe3O4@Au-HA NSs are water dispersible, colloidally
irradiation (Fig. 6a). The photothermal ablation of tumors using stable, and biocompatible in the given concentration range.
Fe3O4@Au-HA NSs was further characterized by TUNEL staining Importantly, the Fe3O4@Au-HA NSs are able to be used as a multi-
(Fig. 6b). It is obvious that only sparse positive staining of apoptotic functional nanoplatform for MR/CT imaging of cancer cells in vitro
cells appear in the tumor sections treated with PBS, laser alone, or and xenografted tumor model in vivo. The NIR absorption property
Fe3O4@Au-HA NSs alone. In contrast, a large area of positive stained of the NSs enables them to be used as a platform for photothermal
apoptotic cells can be seen after the tumors were treated with therapy of cancer cells in vitro and the xenografted tumor model
Fe3O4@Au-HA NSs under laser irradiation. Additional quantitative in vivo, as well as additional thermal imaging of tumors. The
analysis of TUNEL-stained tumor sections reveals that the apoptosis developed Fe3O4@Au-HA NSs may be used as a multifunctional
rate of the tumors in the Control, Laser, NSs, and NSs þ Laser groups nanoplatform for efficient theranostics of different types of CD44
is 6.4%, 12.3%, 7.8%, and 88.6%, respectively (Fig. S13, Supporting receptor-overexpressing cancer.

Control Laser NSs NSs + Laser

a

Fig. 6. Representative H&E staining images (a) and TUNEL assay images (b) of xenografted HeLa tumors with different treatments. The scale bars in each panel of (a) and (b)
represent 50 and 100 mm, respectively.
20 J. Li et al. / Biomaterials 38 (2015) 10e21
Acknowledgments
This research is financially supported by the National Natural
Science Foundation of China (21273032, 81101150, and 81371623),
the Fund of the Science and Technology Commission of Shanghai
Municipality (11nm0506400 for X. S. and 12520705500 for M. S.),
the Sino-German Center for Research Promotion (GZ899), and the
Program for Professor of Special Appointment (Eastern Scholar) at
Shanghai Institutions of Higher Learning.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.biomaterials.2014.10.065.
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