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Hyaluronic acid-modied 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 Modication of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University,

Shanghai 201620, PR Chinab Department of Radiology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200080, PR Chinac College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, PR China

a r t i c l e i n f o

Article history:

Received 25 August 2014

Accepted 19 October 2014

Available online

Keywords:

Fe3O4@Au nanostars

Hyaluronic acid

CT imaging

MR imaging

Tumors

Photothermal therapy

a b s t r a c t

Development of multifunctional theranostic nanoplatforms for diagnosis and therapy of cancer still re-

mains a great challenge. In this work, we report the use of hyaluronic acid-modied Fe3O4@Au core/shell

nanostars (Fe3O4@Au-HA NSs) for tri-mode magnetic resonance (MR), computed tomography (CT), and

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

sequential modication of polyethyleneimine (PEI) and HA affords the NSs with excellent colloidal

stability, good biocompatibility, and targeting specicity to CD44 receptor-overexpressing cancer cells.

With the Fe3O4core NPs and the star-shaped Au shell, the formed Fe3O4@Au-HA NSs are able to be used

as a nanoprobe for efcient MR and CT imaging of cancer cells in vitroand the xenografted tumor model

in vivo. Likewise, the NIR absorption property enables the developed Fe 3O4@Au-HA NSs to be used as a

nanoprobe for thermal imaging of tumors in vivoand 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 nd applications intheranostics of different types of cancer.

2014 Elsevier Ltd. All rights reserved.

1. Introduction

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 contrastagents for T2-weighted magnetic resonance (MR) imaging due to

their high relaxivity, excellent contrast enhancement, and low

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

(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 CTcontrast agent) [18e23]. On the other hand, AuNPs

with particular shapes such as nanorods [24,25], nanoshells

[26,27], nanoowers [28], nanocages [29,30], or nanostars (NSs)

[31,32]display strong surface plasmon resonance (SPR) absorptionintensity 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

* Corresponding author. College of Chemistry, Chemical Engineering and

Biotechnology, Donghua University, Shanghai 201620, PR China. Tel.: 86 21

67792656; fax: 86 21 67792306 804.

** Corresponding author. Tel.: 86 21 63240090 4166; fax: 86 21 63240825.

*** Corresponding author. Tel.: 86 21 67792750; fax: 86 21 67792306 804.

E-mail addresses: [email protected] (M. Shen), [email protected]

(G. Zhang),[email protected](X. Shi).1 Authors contributed equally to this work.

Contents lists available at ScienceDirect

Biomaterials

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . co m / l o c a t e / b i o m a t e r i a l s

http://dx.doi.org/10.1016/j.biomaterials.2014.10.065

0142-9612/

2014 Elsevier Ltd. All rights reserved.

Biomaterials 38 (2015) 10e21
mailto:[email protected]:[email protected]:[email protected]://www.sciencedirect.com/science/journal/01429612http://www.elsevier.com/locate/biomaterialshttp://dx.doi.org/10.1016/j.biomaterials.2014.10.065http://dx.doi.org/10.1016/j.biomaterials.2014.10.065http://dx.doi.org/10.1016/j.biomaterials.2014.10.065http://dx.doi.org/10.1016/j.biomaterials.2014.10.065http://dx.doi.org/10.1016/j.biomaterials.2014.10.065http://dx.doi.org/10.1016/j.biomaterials.2014.10.065http://www.elsevier.com/locate/biomaterialshttp://www.sciencedirect.com/science/journal/01429612http://crossmark.crossref.org/dialog/?doi=10.1016/j.biomaterials.2014.10.065&domain=pdfmailto:[email protected]:[email protected]:[email protected]

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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], Cu7.2S4nanocrystals

[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 onenanoparticulate 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 efcacy of cancer[35,38,47], these

NP systems are lack of targeting specicity presumably due to the

technical difculty 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 formolecular 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

Fe3O4or 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 uorescence 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 agentdisplayed ve 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

cellsin vitroand 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 easilymodied 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 modied

with PEI for further modication 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

viaa facile one-pot hydrothermal route according to our previous

work with some modications [56]. Thenthe 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

modied by partially thiolated PEI (PEI-SH) via AueS bond, fol-

lowed by modication 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 specicity 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 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 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 fromInstitute of Biochemistryand Cell Biology, the Chinese Academy

of Sciences (Shanghai, China). Modied eagle medium (MEM), Dulbecco's modied

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 puried using a Milli-Q Plus

185 water purication 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 protocolsdescribed 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

at60e70 C ina waterbathfor 24h to complete thereaction. After that, thereaction

mixture was dialyzed against water (6 times, 2L) using a dialysis membrane with

MWCO of 14,000 for 3 days, followed bylyophilization to obtain the puried PEI-SH.

2.3. Synthesis of Fe3O4@Ag seeds

Fe3O4@Ag seeds were synthesized according to our previous work with some

modications[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

magneticstirring.After30 min, an icycold 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 puried via

dialysis as described above and nally 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 in7.75 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 puried by rinsing with water for 3 times and

nally 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

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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 puried 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-modied 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 puried

Fe3O4@Au-HA NSs wereredispersed inwater and/or phosphatebuffered 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

PEIand PEI-SH samples. Thesampleswere dissolved inD 2O before measurements.AMalvern 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 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 (0.005e

0.08 mM

) were performed using an NMI20-Analyst NMR Analyzing

and Imaging system (Shanghai Niumag Corporation, Shanghai, China). The param-

eters were set as following: CPMG sequence, 0.5 T magnet, point

resolution 156 mm 156 mm, section thickness 0.6 mm, TR 6000 ms,

TE 80 ms, number of excitation 1. The linear tting of the inverse T2relaxation

times (1/T2) as a function of the Fe concentration was used to calculate the T2relaxivity (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 (GEMedical Systems)with100 kV,80 mA,and a slice thicknessof 0.625 mm.

The X-ray attenuation intensity in Hounseld units (HU) was evaluated by loading

the digital CT images in a standard display program and then selecting a uniform

roundregion ofintereston the resultantCT image foreachsample. Todeterminethe

photothermal property of the Fe3O4@Au-HA NSs, an aqueous suspension ofFe3O4@Au-HA NSs with different Au concentrations (0.32e24 mM, 0.3 mL),

Fe3O4@Agseeds(with Fe concentration similar tothatof 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]. Briey, 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, puried, 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

100mg/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 con uence, the medium was

replaced with 200 mL fresh medium containing Fe3O4@Au-HA NSs with different

nal 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 werecarriedout according tothe manufacturer's instruction.

The absorbance at 570 nm of each well was measured using a Thermo Scientic

Multiskan MK3 ELISA reader (Thermo Scienti

c, USA). The background subtraction

AgNO3

NaBH4

PEI

HA

EDC/NHS

PEI-Ag NPs

Hydrothermal synthesis

Fe(II) salt

Fe3O

4@Ag seeds

Fe3O

4@Au-HA NSs Fe

3O

4@Au-PEI NSs Fe3O4@Au NSs

Growth

PEI-SH

Scheme 1. Schematic illustration of the synthesis of Fe 3O4@Au-HA NSs.

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at 570 nm was applied to eliminate the inuence of the added materials. For each

sample, mean and standard deviation ofve 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 conuence 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 5.0mM, respectively)and the cells were incubatedat 37 C and 5%

CO2for 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 (containing0.5% 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 156 mm 156 mm, section thickness 0.6 mm, TR 3000 ms,

TE 90 ms, and number of excitation 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 specic 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. Briey, 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 Fe 3O4@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 mLPBS. 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 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 foranimal care,and alsoin accordance withthe 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 intothe 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 rst anes-thetized by intraperitoneal injection of pentobarbital sodium(40 mg/kg), then a PBS

solution of Fe3O4@Au-HA NSs ([Fe] 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 minpost injection were obtained using a

conventionalspin-echo sequence underthe 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] 123.5 mM, 0.1 mL). CT scans were performed before and at 10 min post

injectionusing a GE LightSpeed VCTclinical imaging system with 100kV, 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 200mL 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 nal Au concen-

trations (0, 0.1, 0.2, 0.3, or 0.4 mM, respectively) was added into each well. After

incubation foranother6 h,the cells were irradiated bya 915 nmlaserwithan output

power density of 1.2 W/cm2 for5 and10 min, respectively.The cell viabilitywas then

measuredvia MTT assay according to the procedures described above. Mean and

standarddeviation for the triplicate wellswere reported.In parallel,the morphology

of cells treated with PBS or Fe3O4@Au-HA NSs at the Au concentration of 0.4 m Mfor

6 h,followed byirradiationwith a 915 nmlaser(at anoutput powerdensityof 1.2W/

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 rst anesthetized by intraperitoneal in-

jection of pentobarbital sodium (40 mg/kg), then a PBS solution of Fe3O4@Au-HA

NSs ([Au]

32 mM

, 0.1 mL) was intratumorally injected into the mice. The mice

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/cm 2 for

5 min.Duringthe process of laserradiation,a photothermal medical device (GX-300,

ShanghaiInfratest Electronics Co.,Ltd, Shanghai,China) withan infraredcamerawas

used to obtain the whole-body infrared thermal images at different time points.

2.14. In vivo photothermal ablation of HeLa t umors

HeLa tumor-bearing nude mice were randomly divided into four groups (n 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] 32 mM) without laser irradiation (NSs

group), and 0.1 mL PBS containing Fe 3O4@Au-HA NSs ([Au] 32 mM) with laser

irradiation under similar power density and time period (NSs 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 ofN1/N 100%, whereN1and Nrepresent 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 theabove protocols (n 1 for each group). After 2 h, the mice were euthanized and the

tumors were removed, xed in 4% paraformaldehyde, and embedded in parafn 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 ve random selected elds.

2.16. Statistical analysis

One-way ANOVA statistical analysis was performed to evaluate the signicance

of the experimental data. 0.05 was selected as the signicance 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 specicity, the

Fe3O4@Au NSs were rst 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

rst conrmed by FTIR spectroscopy (Fig. S2, Supporting

Information). By comparison with PEI, an obvious peak emergingat 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 conrm 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 ofeCOeCH2eSein 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. S4ae

b, Supporting

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Information) were rst 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

modication with PEI-SHviaAueS bond. The aminated Fe3O4@Au-

PEI NSs were then modied with HA to be afforded with targeting

specicity to CD44 receptor-overexpressing cancer cells.

Zeta potential measurements were employed to conrm the

successful modication of HA onto the surface of NSs ( Fig. S5a,

Supporting Information). Fe3O4@Au-PEI NSs dispersed in water

had a positive potential of 32.7 mV because of the surface

modication of PEI with abundant amines. After modication withHA, 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 modication 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 conrming

the successful HA conjugation. What's more, the hydrodynamic

sizes of the Fe3O4@Au-HA NSs at different storage time periods

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

conrming 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 conrmed 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

modication. 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-HANSs 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-

a b c

fd e

1.5

1.0

0.5

0.0400 600

Wavelength (nm)

Diameter (nm) 2(degree)

Frequency(%)

Absorbance

Intensity(a.u.)

20 30 40 50 60 70 80 90

* Au

800 1000

Fe3O

4@Ag seeds

Fe3O

4

(220)

(311)

(400)

(200)

(111)

(311)

(222)

(422)

(511)

(440)

(220)

*

*

* *

*

Fe3O4@Au-HA NSs

Mean diameter = 119.4 nm

= 19.4 nm

60

50

40

30

20

10

060 80 100 120 140 160 180

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 Fe 3O4@Au-HA NSs.

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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 Fe3O4NPs (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 conrmed 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 modication

(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 Fe3O4are 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. T2MR relaxometry and X-ray attenuation property

Fe3O4 NPs have been known to be able to shorten the T2relaxation 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 efciency 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 0.9999) can be found with a

slope of 144.39 mM1 s1, which is identied 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 tothe 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.

0.00 0.02 0.04 0.06 0.08

12

8

4

Fe concentration (mM)

600

450

300

150

00.00 0.02 0.04 0.06 0.08

Au concentration (M)70

60

50

40

30

20

10

00 1 2 3 16 24

Au concentration (mM)Time (s)

90

80

70

60

50

40

30

20

0 50 100 150 200 250 300

Temperatur

e(C)

1

/T2(s-1)

T(C

)

Hounsf

ieldUnit(HU)

r2=144.39 mM-1s-1, R2=0.9999

water seeds 0.32 mM 0.8 mM

24 mM16 mM3.2 mM1.6 mM

Fe (mM) Au (M)0.005 0.01 0.010.02 0.020.04 0.04 0.06 0.08

a cH

L

b d

e f

0.08

Fig. 2. (a) Color T2-weighted MR images and (b) linear tting 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] 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 Fe 3O4@Au-HA NSs at different Au

concentrations over a period of 300 s. (For interpretation of the references to color in this

gure legend, the reader is referred to the web version of this article.)


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Our data suggest a great potential to use the Fe3O4@Au-HA NSs as a

T2negative contrast agent for MR imaging applications.

On the other hand, the potential to use Fe3O4@Au-HA NSs as a

CT contrast agent was conrmed 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 temperaturereaches 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 efciently upon laser irradi-

ation. It should be noted that the developed Fe 3O4@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 signicant hemolysis of HRBCs.

Based on the absorbance of the supernatant at 541 nm that isassociated 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 mMwere 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 ofD-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 undersimilar conditions (Fig. 3a). This can be further conrmed 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 difcult 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 U87MGcells at the same Au concentration (p < 0.05). These results suggest

that the developed Fe3O4@Au-NSs have a high afnity 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 conrmed 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 signicantly higher than that in

U87MG cells without CD44 receptor overexpression. This further

conrmed the role played by HA-mediated targeting that enables

specic 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 specicity 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. 4ae

b). We can

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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 CTcontrast 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 minpostinjection, 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 vivotumor MR/CT imaging.

3.8. In vitro photothermal ablation of cancer cells

The high photothermal conversion efciency and the targeting

speci

city 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 thePBS 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 signicant 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 longertime

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 mMwere 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 efcacy of Fe3O4@Au-HA NSs was

further evaluated by optical microscopic observation of cell

1600

1200

800

400

00 0.05 0.10 0.15 0.20

420

280

140

0

Control 1.0 2.0

120

90

60

30

00 1.25 2.50 3.75 5.00

120

100

80

60

40

20

0

0.0 0.1 0.2 0.3 0.4

Fe concentration (mM)Au concentration (mM)

Au concentration (mM)Au concentration (mM)

[Fe]/mM

U87MG cells

HeLa cells

0a b

c d

fe

0.05 0.10 0.15 0.20 [Au]/mM

U87MG cells

HeLa cells

0 1.25 2.50 3.75 5.00

Signalintensity

CellularAuuptake(pg/cell)

Hounsfieldunit(HU)

Cellviability(%)

U87MG cells

HeLa cells U87MG cells

HeLa cells

U87MG cells

*****

***

***

***

0 min 5 min 10 min***

***

******

******

******

***

******

***

***

**

*

HeLa cells

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 m Mfor 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.


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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 efcacy 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 developedFe3O4@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 inFig. 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-

nicant 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 efcacy 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 completelyinhibited. On day 19, the tumor tissue almost completely dis-

appeared in the mice after the photothermal therapy, which is

signicantly 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-HANSs alone, or the combination of the above two is

unable to generate toxicity to the mice. To further investigate the

photothermal therapeutic efcacy 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 signicantly higher than the mice in the other three

groups. The survival rate of the mice without treatment, treated

a b

c

0 min10 min0 min 10 min

70

60

(d)

50

40

30

200 50 100 150 200 250 300

Time (s)

Temperature(C)

Region 11 Region 12

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 Fe 3O4@Au-HA NSs

([Fe] 5.0 mM, [Au] 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] 32 mM, the right mouse, indicated region 12), respectively, followed by irradiationwith a 915 nm laser (1.2 W/cm2) at a time point of 0, 1.5 min, and 5 min,

respectively. (d) The temperature proles in Regions 11 and 12 as a function of the laser irradiation time.

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with Fe3O4@Au-HA NSs only, and treated with laser only is 25%, 0%

and 0%, respectively. Our results suggest that the developed

Fe3O4@Au-HA NSs have a great potential to be used for photo-

thermal therapy of tumors.

3.10. H&E and TUNEL staining

The photothermal ablation of tumors using Fe3O4@Au-HA NSs

was further conrmed by histological examination of tumor sec-

tions after different treatments (Fig. 6). H&E staining of the tumor

sections shows that the tumors treated with laser alone and with

the Fe3O4@Au-HA NSs alone display well-shaped tumor cells

without the appearance of necrosis region, similar to the control

group treated with PBS. However, obvious necrosis area can be seen

in the tumor tissue treated with Fe3O4@Au-HA NSs under laser

irradiation (Fig. 6a). The photothermal ablation of tumors using

Fe3O4@Au-HA NSs was further characterized by TUNEL staining

(Fig. 6b). It is obvious that only sparse positive staining of apoptotic

cells appear in the tumor sections treated with PBS, laser alone, orFe3O4@Au-HA NSs alone. In contrast, a large area of positive stained

apoptotic cells can be seen after the tumors were treated with

Fe3O4@Au-HA NSs under laser irradiation. Additional quantitative

analysis of TUNEL-stained tumor sections reveals that the apoptosis

rate of the tumors in the Control, Laser, NSs, and NSs Laser groups

is 6.4%, 12.3%, 7.8%, and 88.6%, respectively (Fig. S13, Supporting

Information). These results demonstrate that the developed

Fe3O4@Au-HA NSs are able to be used as an efcient nanoplatform

for photothermal ablation of tumors in vivo.

4. Conclusion

In summary, we developed a convenient approach to synthe-

sizing Fe3O4@Au-HA NSs with a quite uniform morphology for

multi-mode imaging and photothermal therapy of tumors. The

hydrothermally synthesized Fe3O4@Ag seeds are able to be

deposited with star-shaped Au shells that can be modied with PEI

viaAueS bond. Likewise, the PEI-mediated reaction can be used for

HA conjugation onto the surface of the NSs to render them with

targeting specicity to CD44 receptor-overexpressing cancer cells.

The formed Fe3O4@Au-HA NSs are water dispersible, colloidally

stable, and biocompatible in the given concentration range.

Importantly, the Fe3O4@Au-HA NSs are able to be used as a multi-

functional nanoplatform for MR/CT imaging of cancer cells in vitro

and xenografted tumor model in vivo. The NIR absorption propertyof the NSs enables them to be used as a platform for photothermal

therapy of cancer cells in vitro and the xenografted tumor model

in vivo, as well as additional thermal imaging of tumors. The

developed Fe3O4@Au-HA NSs may be used as a multifunctional

nanoplatform for efcient theranostics of different types of CD44

receptor-overexpressing cancer.

a b c

00

3

6

9

12

Relativetumorvolume

Bodyweight(g)

Survivalrate(%)

ControlLaser

NSsNSs + Laser

ControlLaser

NSsNSs + Laser

ControlLaserNSsNSs + Laser

4 8 12 16 20 0 4 8 12 16 20 0 10 20 30 40 50 60

100

75

50

25

0

35

30

25

20

15

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.

Control Laser NSs NSs + Laser

a

b

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.

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Acknowledgments

This research is nancially 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 .

References

[1] Taylor-Pashow KM, Della Rocca J, Huxford RC, Lin W. Hybrid nanomaterials forbiomedical applications. Chem Commun 2010;46:5832e49.

[2] Shen M, Shi X. Dendrimer-based organic/inorganic hybrid nanoparticles inbiomedical applications. Nanoscale 2010;2:1596e610.

[3] Leung KC-F, Xuan S, Zhu X, Wang D, Chak C-P, Lee S-F, et al. Gold and ironoxide hybrid nanocomposite materials. Chem Soc Rev 2012;41:1911e28.

[4] Du C, Zhao J, Fei J, Gao L, Cui W, Yang Y, et al. Alginate-based microcapsuleswith a molecule recognition linker and photosensitizer for the combinedcancer treatment. Chem Asian J 2013;8:736e42.

[5] Qi W, Duan L, Li J. Fabrication of glucose-sensitive protein microcapsules andtheir applications. Soft Matter 2011;7:1571e6.

[6] Li D, He Q, Yang Y, Mohwald H, Li J. Two-stage pH response of poly (4-vinylpyridine) grafted gold nanoparticles. Macromolecules 2008;41:7254e6.

[7] Qi W, Wang A, Yang Y, Du M, Bouchu MN, Boullanger P, et al. The lectinbinding and targetable cellular uptake of lipid-coated polysaccharide micro-capsules. J Mater Chem 2010;20:2121e7.

[8] Liu HL, Sonn CH, Wu JH, Lee K-M, Kim YK. Synthesis of streptavidin-FITC-conjugated coreeshell Fe3O4-Au nanocrystals and their application for thepurication of CD4 lymphocytes. Biomaterials 2008;29:4003e11.

[9] Bao J, Chen W, Liu T, Zhu Y, Jin P, Wang L, et al. Bifunctional Au-Fe3O4nanoparticles for protein separation. ACS Nano 2007;1:293e8.

[10] Samanta B, Yan H, Fischer NO, Shi J, Jerry DJ, Rotello VM. Protein-passivatedFe3O4 nanoparticles: low toxicity and rapid heating for thermal therapy.

J Mater Chem 2008;18:1204e

8.[11] Shi D, Cho HS, Chen Y, Xu H, Gu H, Lian J, et al. F luorescent polystyreneeFe3O4

composite nanospheres for in vivo imaging and hyperthermia. Adv Mater2009;21:2170e3.

[12] Wang C, Daimon H, Sun S. Dumbbell-like PtFe3O4 nanoparticles and theirenhanced catalysis for oxygen reduction reaction. Nano Lett 2009;9:1493e6.

[13] Pan B, Cui D, Sheng Y, Ozkan C, Gao F, He R, et al. Dendrimer-modiedmagnetic nanoparticles enhance efciency of gene delivery system. CancerRes 2007;67:8156e63.

[14] Chen Y, Chen H, Zeng D, Tian Y, Chen F, Feng J, et al. Core/shell structuredhollow mesoporous nanocapsules: a potential platform for simultaneous cellimaging and anticancer drug delivery. ACS Nano 2010;4:6001e13.

[15] Li J, Zheng L, Cai H, Sun W, Shen M, Zhang G, et al. Polyethyleneimine-mediated synthesis of folic acid-targeted iron oxide nanoparticles for in vivotumor MR imaging. Biomaterials 2013;34:8382e92.

[16] Li J, He Y, Sun W, Luo Y, Cai H, Pan Y, et al. Hyaluronic acid-modi ed hy-drothermally synthesized iron oxide nanoparticles for targeted tumor MRimaging. Biomaterials 2014;35:3666e77.

[17] Cai H, An X, Cui J, Li J, Wen S, Li K, et al. Facile hydrothermal synthesis and

surface functionalization of polyethyleneimine-coated iron oxide nano-particles for biomedical applications. ACS Appl Mater Interfaces 2013;5:1722e31.

[18] Guo R, Wang H, Peng C, Shen M, Pan M, Cao X, et al. X-ray attenuationproperty of dendrimer-entrapped gold nanoparticles. J Phys Chem C2010;114:50e6.

[19] Peng C, Li K, Cao X, Xiao T, Hon W, Zheng L, et al. Facile formation ofdendrimer-stabilized gold nanoparticles modied with diatrizoic acid forenhanced computed tomography imaging applications. Nanoscale 2012;4:6768e78.

[20] Peng C, Zheng L, Chen Q, Shen M, Guo R, Wang H, et al. PEGylated dendrimer-entrapped gold nanoparticles for in vivo blood pool and tumor imaging bycomputed tomography. Biomaterials 2012;33:1107e19.

[21] Wang H, Zheng L, Peng C, Guo R, Shen M, Shi X, et al. Computed tomographyimaging of cancer cells using acetylated dendrimer-entrapped gold nano-particles. Biomaterials 2011;32:2979e88.

[22] Wang H, Zheng L, Peng C, Shen M, Shi X, Zhang G. Folic acid-modied den-drimer-entrapped gold nanoparticles as nanoprobes for targeted CT imaging

of human lung adenocarcinoma. Biomaterials 2013;34:470e

80.

[23] Wen S, Li K, Cai H, Chen Q, Shen M, Huang Y, et al. Multifunctional dendrimer-entrapped gold nanoparticles for dual mode CT/MR imaging applications.Biomaterials 2013;34:1570e80.

[24] Huang X, El-Sayed IH, Qian W, El-Sayed MA. Cancer cell imaging and photo-thermal therapy in the near-infrared region by using gold nanorods. J AmChem Soc 2006;128:2115e20.

[25] Seo S-H, Kim B-M, Joe A, Han H-W, Chen X, Cheng Z, et al. NIR-light-inducedsurface-enhanced Raman scattering for detection and photothermal/photo-dynamic therapy of cancer cells using methylene blue-embedded goldnanorod@SiO2 nanocomposites. Biomaterials 2014;35:3309e18.

[26] Ji X, Shao R, Elliott AM, Stafford RJ, Esparza-Coss E, Bankson JA, et al.Bifunctional gold nanoshells with a superparamagnetic iron oxide-silica coresuitable for both MR imaging and photothermal therapy. J Phys Chem C2007;111:6245e51.

[27] Ma Y, Liang X, Tong S, Bao G, Ren Q, Dai Z. Gold nanoshell nanomicelles forpotential magnetic resonance imaging, light-triggered drug release, andphotothermal therapy. Adv Funct Mater 2013;23:815e22.

[28] Han J, Li J, Jia W, Yao L, Li X, J iang L, et al. Photothermal therapy of cancer cellsusing novel hollow gold nanoowers. Int J Nanomed 2014;9:517e26.

[29] Yang J, Shen D, Zhou L, Li W, Li X, Yao C, et al. Spatially conned fabrication ofcoreeshell gold nanocages@mesoporous silica for near-infrared controlledphotothermal drug release. Chem Mater 2013;25:3030e7.

[30] Gao L, Fei J, Zhao J, Li H, Cui Y, Li J. Hypocrellin-loaded gold nanocages withhigh two-photon efciency for photothermal/photodynamic cancer therapyin vitro. ACS Nano 2012;6:8030e40.

[31] Yuan H, Fales AM, Vo-Dinh T. TAT peptide-functionalized gold nanostars:enhanced intracellular delivery and efcient NIR photothermal therapy usingultralow irradiance. J Am Chem Soc 2012;134:11358e61.

[32] Wang S, Huang P, Nie L, Xing R, Liu D, Wang Z, et al. Single continuous wavelaser induced photodynamic/plasmonic photothermal therapy usingphotosensitizer-functionalized gold nanostars. Adv Mater 2013;25:3055e61.

[33] Hwang S, Nam J, Song J, Jung S, Hur J, Im K, et al. Sub 6 nanometer plasmonicgold nanoparticle for pH-responsive near-infrared photothermal cancertherapy. New J Chem 2014:918e22.

[34] Li C, Chen T, Ocsoy I, Zhu G, Yasun E, You M, et al. Gold-coated Fe3O4 nano-roses with ve unique functions for cancer cell targeting, imaging, and ther-apy. Adv Funct Mater 2013;24:1772e80.

[35] Tian Q, Wang Q, Yao KX, Teng B, Zhang J, Yang S, et al. Multifunctional pol-ypyrrole@Fe3O4 nanoparticles for dual-modal imaging and in vivo photo-thermal cancer therapy. Small 2013;10:1063e8.

[36] Zhou T, Wu B, Xing D. Bio-modied Fe3O4 core/Au shell nanoparticles fortargeting and multimodal imaging of cancer cells. J Mater Chem 2012;22:470e7.

[37] Cai H, Li K, Shen M, Wen S, Luo Y, Peng C, et al. Facile assembly of Fe3O4@Aunanocomposite particles for dual mode magnetic resonance and computedtomography imaging applications. J Mater Chem 2012;22:15110e20.

[38] Tian Q, Hu J, Zhu Y, Zou R, Chen Z, Yang S, et al. Sub-10 nm Fe 3O4@Cu2exS

coree

shell nanoparticles for dual-modal imaging and photothermal therapy.J Am Chem Soc 2013;135:8571e7.[39] Wang Y, Guo R, Cao X, Shen M, Shi X. Encapsulation of 2-methoxyestradiol

within multifunctional poly (amidoamine) dendrimers for targeted cancertherapy. Biomaterials 2011;32:3322e9.

[40] Zhang X-y, Chen J, Zheng Y-f, Gao X-l, Kang Y, Liu J-c, et al. F ollicle-stimulatinghormone peptide can facilitate paclitaxel nanoparticles to target ovariancarcinoma in vivo. Cancer Res 2009;69:6506e14.

[41] Feng W, Zhou X, He C, Qiu K, Nie W, Chen L, et al. Polyelectrolyte multilayerfunctionalized mesoporous silica nanoparticles for pH-responsive drug de-livery: layer thickness-dependent release proles and biocompatibility.

J Mater Chem B 2013;1:5886e98.[42] Li D, Li C, Wang A, He Q, Li J. Hierarchical gold/copolymer nanostructures as

hydrophobic nano tanks for drug encapsulation. J Mater Chem 2010;20:7782e7.

[43] Zheng Y, Fu F, Zhang M, Shen M, Zhu M, Shi X. Multifunctional dendrimersmodied with alpha-tocopheryl succinate for targeted cancer therapy. MedChem Commun 2014;5:879e85.

[44] Zhu J, Zheng L, Wen S, Tang Y, Shen M, Zhang G, et al. Targeted cancer

theranostics using alpha-tocopheryl succinate-conjugated multifunctionaldendrimer-entrapped gold nanoparticles. Biomaterials 2014;35:7635e46.

[45] Zhu J, Shi X. Dendrimer-based nanodevices for targeted drug delivery appli-cations. J Mater Chem 2013;1:4199e211.

[46] Zheng L, Zhu J, Shen M, Chen X, Baker Jr JR, Wang SH, et al. Targeted cancercell inhibition using multifunctional dendrimer-entrapped gold nanoparticles.Med Chem Commun 2013;4:1001e5.

[47] Zhou Z, Kong B, Yu C, Shi X, Wang M, Liu W, et al. Tungsten oxide nanorods:an efcient nanoplatform for tumor CT imaging and photothermal therapy.Sci Rep 2014;4:3653.

[48] Li B, Wang Q, Zou R, Liu X, Xu K, Li W, et al. Cu7.2S4 nanocrystals: a novelphotothermal agent with a 56.7% photothermal conversion efciency forphotothermal therapy of cancer cells. Nanoscale 2014;6:3274e82.

[49] Mackey MA, Ali MR, Austin LA, Near RD, El-Sayed MA. The most effective goldnanorod size for plasmonic photothermal therapy: theory and in vitro ex-periments. J Phys Chem B 2014;118:1319e26.

[50] Chen H, Zhang X, Dai S, Ma Y, Cui S, Achilefu S, et al. Multifunctional goldnanostar conjugates for tumor imaging and combined photothermal andchemo-therapy. Theranostics 2013;3:633e49.

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