Biomaterials 38 (2015) 10e21

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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 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 b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 August 2014 Accepted 19 October 2014 Available online

Development of multifunctional theranostic nanoplatforms for diagnosis and therapy of cancer still remains a great challenge. In this work, we report the use of hyaluronic acid-modified 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 modification of polyethyleneimine (PEI) and HA affords the NSs with excellent colloidal stability, good biocompatibility, and targeting specificity to CD44 receptor-overexpressing cancer cells. With the Fe3O4 core NPs and the star-shaped Au shell, the formed Fe3O4@Au-HA NSs are able to be used as a nanoprobe for efficient MR and CT imaging of cancer cells in vitro and the xenografted tumor model in vivo. Likewise, the NIR absorption property enables the developed Fe3O4@Au-HA NSs to be used as a 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 nanoplatform 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.

Keywords: Fe3O4@Au nanostars Hyaluronic acid CT imaging MR imaging Tumors Photothermal therapy

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 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: [email protected] (M. Shen), [email protected] (G. Zhang), [email protected] (X. Shi). 1 Authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.10.065 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

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 Xray 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 elements and therapeutic agents [34,35]. In particular, for accurate cancer imaging applications, it is meaningful to design a multifunctional 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

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 irradiation 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 therapeutic 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 multimode 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 coprecipitation [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 functionality can be easily realized by assembly of dendrimerentrapped AuNPs onto preformed Fe3O4 NPs [37] or by hydrothermal 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 prepared from Au seeds in the growth solution using silver ion complexes 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/photothermal 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 chemotherapy) [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 hydrothermally 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 receptoroverexpressing 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 theranostics of cancer. In this present study, we report the formation of HA-targeted Fe3O4@Au NSs for tri-mode (MR/CT/thermal) imaging and photothermal 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, followed by modification with HA via 1-ethyl-3-[3dimethylaminopropyl] 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$4H2O > 99%), ammonia (25e28% NH3 in water solution), silver nitrate, ascorbic acid (AA) and all other chemicals and solvents were from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All chemicals were used as received. HeLa cells (a human cervical carcinoma cell line) and U87MG cells (a human glioblastoma carcinoma cell line) were obtained from Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). Modified eagle medium (MEM), Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were from Hangzhou Jinuo Biomedical Technology (Hangzhou, China). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with a resistivity higher than 18.2 MU$cm. Regenerated cellulose dialysis membranes with molecular weight cutoff (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 prepared 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

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J. Li et al. / Biomaterials 38 (2015) 10e21

Hydrothermal synthesis

AgNO3 NaBH4

Fe(II) salt

PEI

Fe3O4@Ag seeds

PEI-Ag NPs

Growth

PEI-SH

HA EDC/NHS

Fe3O4@Au-HA NSs

Fe3O4@Au-PEI NSs

Fe3O4@Au 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 solution 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 potentials 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 measurements. 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 spectroscopy (ICP-OES, Hudson, NH). The T2 relaxometry measurements and T2weighted MR imaging of the samples dispersed in water at different Fe concentrations (0.005e0.08 mM) were performed using an NMI20-Analyst NMR Analyzing

and Imaging system (Shanghai Niumag Corporation, Shanghai, China). The parameters 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 (Shenzhen 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

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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 concentration 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.

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@AuHA 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.

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.

2.11. In vivo MR and CT imaging of tumors

2.16. Statistical analysis

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 subcutaneously 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 anesthetized 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 performed 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.

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.

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 concentrations (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 injection 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

2.15. H&E and TUNEL staining

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 hydrothermal approach, Fe3O4@Ag seed particles were formed, followed 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 characteristic peak of amido linkage (Fig. S1). In addition, the appearance 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 (PEIAg NPs) with a mean diameter of 5.3 nm (Fig. S4aeb, Supporting

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J. Li et al. / Biomaterials 38 (2015) 10e21

Information) were first formed via NaBH4 reduction chemistry, similar to our previous work related to the formation of PEIstabilized 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@AuPEI 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 reaction [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 dynamic 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

Fe3O4@Ag seeds Fe3O4@Au-HA NSs

b

c

1.0

0.5

* (111)

Mean diameter = 119.4 nm σ = 19.4 nm

f

e

* Au

0

60

80 100 120 140 160 180 Diameter (nm)

20 30

40

* (311)

*(220)

50 60 70 2θ(degree)

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(440)

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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 property 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-

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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

3.2. T2 MR relaxometry and X-ray attenuation property

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 discontinuously 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). Highresolution TEM image confirmed the crystal structure of the starshaped Au shells, as lattices of the crystals can be clearly observed (Fig. 1e). In addition, the NSs were also found to be surrounded 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 crystalline 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.

a

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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 relaxation 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 appreciably 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.

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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.)

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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@AuHA 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 region drove us to explore their photothermal property. The temperature change of an aqueous suspension of the Fe3O4@Au-HA NSs as a function of Au concentration (0.32e24 mM) under laser irradiation 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 concentration 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 concentration (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 irradiation. It should be noted that the developed Fe3O4@Au-HA NSs still have a very good colloidal stability during and after the laser irradiation process, which is amenable for their effective photothermal therapy applications. 3.4. Hemolytic assay and cytotoxicity assay For biomedical applications, hemocompatibility and cytocompatibility of the developed Fe3O4@Au-HA NSs should be evaluated. 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 targeting ligand that can bind to CD44 receptor-overexpressing cancer cells [16,62,64]. U87MG cells without CD44 receptor overexpression 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 overexpressing 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 injection were imaged by MR and CT, respectively (Fig. 4aeb). We can

J. Li et al. / Biomaterials 38 (2015) 10e21

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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 T2weighted 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 distribution 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 concentration 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

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J. Li et al. / Biomaterials 38 (2015) 10e21

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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 irradiation 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 results 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 containing 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 significant temperature increase due to the injection of the Fe3O4@AuHA 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 irradiation, 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 volumes of the tumors after different treatments (Fig. 5a). It is obvious that the volumes of tumors treated with laser alone or Fe3O4@AuHA 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 disappeared 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 under 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

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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 photothermal therapy of tumors.

Information). These results demonstrate that the developed Fe3O4@Au-HA NSs are able to be used as an efficient nanoplatform for photothermal ablation of tumors in vivo. 4. Conclusion

3.10. H&E and TUNEL staining The photothermal ablation of tumors using Fe3O4@Au-HA NSs was further confirmed by histological examination of tumor sections 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, or Fe3O4@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 þ Laser groups is 6.4%, 12.3%, 7.8%, and 88.6%, respectively (Fig. S13, Supporting

Control

Laser

In summary, we developed a convenient approach to synthesizing 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 modified with PEI via AueS bond. Likewise, the PEI-mediated reaction can be used for HA conjugation onto the surface of the NSs to render them with targeting specificity 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 multifunctional nanoplatform for MR/CT imaging of cancer cells in vitro and xenografted tumor model in vivo. The NIR absorption property of 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 efficient theranostics of different types of CD44 receptor-overexpressing cancer.

NSs

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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 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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shell nanostars for multimodal imaging and photothermal therapy of tumors.

Development of multifunctional theranostic nanoplatforms for diagnosis and therapy of cancer still remains a great challenge. In this work, we report ...
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