Article pubs.acs.org/molecularpharmaceutics

Multifunctional Metal Rattle-Type Nanocarriers for MRI-Guided Photothermal Cancer Therapy Yuran Huang,†,‡ Tuo Wei,† Jing Yu,§ Yanglong Hou,§ Kaiyong Cai,*,‡ and Xing-Jie Liang*,† †

CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, 11 Beiyitiao, Zhongguancun, Beijing, 100190 China ‡ Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, 174 Shazhengjie, Shapingba, Chongqing, 400031 China § Department of Advanced Materials and Nanotechnology, College of Engineering, Peking University, Beijing, China S Supporting Information *

ABSTRACT: In the past decade, numerous species of nanomaterials have been developed for biomedical application, especially cancer therapy. Realizing visualized therapy is highly promising now because of the potential of accurate, localized treatment. In this work, we first synthesized metal nanorattles (MNRs), which utilized porous gold shells to carry multiple MR imaging contrast agents, superparamagnetic iron oxide nanoparticles (SPIONs), inside. A fragile wormpore-like silica layer was manipulated to encapsulate 8 nm oleylamine SPIONs and mediate the in situ growth of porous gold shell, and it was finally etched by alkaline solution to obtain the rattle-type nanostructure. As shown in the results, this nanostructure with unique morphology could absorb nearinfrared light, convert to heat to kill cells, and inhibit tumor growth. As a carrier for multiple SPIONs, it also revealed good function for T2-weighted MR imaging in tumor site. Moreover, the rest of the inner space of the gold shell could also introduce potential ability as nanocarriers for other cargos such as chemotherapeutic drugs, which is still under investigation. This metal rattle-type nanocarrier may pave the way for novel platforms for cancer therapy in the future. KEYWORDS: rattles, nanocarriers, imaging, photothermal, cancer

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researches have demonstrated the usages of nanorattles as drug carriers, imaging contrast agents, confined nanoreactors, and catalysts.7−10 Tang and co-workers reported silica nanorattles with tailored structures that could encapsulate antitumor drug for cancer therapy,11 and that could also support gold nanoshells on their surfaces for photothermal cancer therapy.12 However, pure silica nanorattles are greatly limited to be further engineered because silica itself does not possess particular functions. Introducing other multiple functionalities needs additional modifications, which results in tedious synthesis processes. Considering this fact, other kinds of rattle-type nanostructures were developed to ameliorate. Shi and coworkers developed a novel multifunctional rattle-type nanostructure composed of an upconversional core and a porous silica shell for cisplatin delivery and magnetic/luminescent dualmode imaging.10 Herein, metal nanomaterials, including

he rapid development of nanotechnology has brought various nanoscale tools to revolutionize nanomedicine and lead disease treatments into a new era.1 The parameters of nanomaterials in size, surface, shape, targeting modification, and responsive properties have been evaluated for optimal efficacy of biomedicine.2 Among them, multifunctional nanoplatforms composed of different nanoscale objects have attracted increasing attention because of their potential applications in processing treatment under visualized diagnosis.3 Diagnosis often refers to multiple modalities of imaging while treatments include different therapeutic paradigms. This integration is crucial because the location and behavior information on the nanosystems in vivo can be obtained to improve therapeutic efficiency, reduce their toxicity, and realize noninvasive treatment. Meanwhile, it will enhance the specific response to achieve controlled targeting besides achieving the same functions as mentioned above if the materials are endowed with special stimulus-responsive properties, especially external stimuli (e.g., heat, light, magnetic field, etc.).4,5 Nanorattles were popularly utilized as multifunctional platforms by taking advantage of their large surface-to-volume ratio, mesoporous structure, interstitial hollow space, and movable cores. These unique properties provide a vast ocean of parameters that can be tuned to fit different scenarios.6 Many © XXXX American Chemical Society

Special Issue: Recent Molecular Pharmaceutical Development in China Received: January 4, 2014 Revised: May 11, 2014 Accepted: May 15, 2014

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upconversion nanoparticles, gold nanoparticles, and iron oxide nanoparticles, are easier to manipulate and assemble because of their unique physical and chemical properties. Especially, gold nanoplatforms are one of the hottest enabling techniques for pushing the advances of nanomedicine, considering their good biocompatibility (biological inert surfaces), controllable size and shape, and unique surface plasmonic properties.13 Introducing magnetic nanoparticles into gold-based nanostructures is able to realize simultaneous high-resolution magnetic resonance imaging (MR imaging or MRI) and near-infrared (NIR) light-induced photothermal cancer therapy. However, normally, an intermediate layer, such as polymer,14,15 liposome,16 and silica,17,18 must be engineered between the magnetic core and the gold shell in the synthetic procedures because of the mismatching of crystal structures of iron oxide and gold. The existence of an intermediate layer means less free space in the nanoplatform that can be utilized for potential cargo loading. According to the silica nanorattle structures, removal of the intermediate layer means more possibilities of introducing versatile functions. Furthermore, both gold nanoshell structure for lung cancer therapy and iron oxide nanoparticles for enhanced MRI contrast are now under clinical trials. Particularly, Feraheme (ferumoxytol), a SPION coated with a low molecular weight semisynthetic carbohydrate, has been approved by the Food and Drug Administration (FDA) for use for labeling stem cells and visualizing by MRI. This means it is extremely promising to apply gold/iron oxide nanocomposites into real clinical applications. In this work, we intended to synthesize a type of multifunctional metal rattle-type nanostructures for simultaneous MR imaging and photothermal effect. Briefly, in this multicomponent system, gold shell acted as a vehicle to carry MRI contrast agents SPIONs on one hand and displayed photothermal effect on the other hand; also the rest of the inner space could be used for carrying other potential cargos. First, multiple 8 nm oleylamine-coated SPIONs were synthesized by incorporation as cores as MRI contrast agents. A positively charged organic/inorganic hybrid silica layer, susceptible to alkaline solvent, encapsulated multiple SPIONs to form Fe3O4@T-Si nanocomposites. Gold seeds were synthesized and in situ anchored on the silica surfaces because of the presence of amino groups with positive charge. By continuous reduction of additional chloroauric acid (HAuCl4), the gold seeds would grow and finally transform to a gold shell, enabling a photothermal effect. Meanwhile, the porous feature of the gold shell allowed convenient alkaline etching of the intermediate hybrid silica layer and finally formation into a rattle-type nanostructure. The free hollow space of the inner gold shell resulting from etching was naturally a reservoir of cargos for potential further applications (Figure 1a). To the best of our knowledge, this is the first report about synthesizing novel rattle-type metal nanostrcutures using a porous gold shell to carry MRI contrast agents. This kind of MNR has several advantages beyond other nanosystems (e.g., polyelectrolyte capsules). First, intrinsically MNRs possess both imaging and photothermal functions based on their nature without other intermediates like polyelectrolyte materials. Besides, the gold surface makes further modification much easier and reduces the possibility of bringing toxic chemical compounds. In addition, gold shell and iron oxide nanoparticles are either under clinical trials or approved by the FDA, which shows great potential for real clinical application. Finally, as nanocarriers, the extra inner spacing renders this nanostructure

Figure 1. (a) Schematic diagram for fabrication of MNRs. (b) SEM images of nanostructures for each step in scheme a: (a1−a2) formation of Fe3O4@T-Si nanocomposites; (b1−b2) in situ reduction of Au3+ on the surface of Fe3O4@T-Si nanocomposites; (c1−c2) formation of Fe3O4@T-Si@AuShells; (d1−d2) formation of MNRs after alkaline etching.

more promising to carry cargos such as other imaging contrast agents for multimodality imaging or other chemicals for chemotherapy.

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MATERIALS AND METHODS Experimental Materials. The materials used and their sources are as follows: iron(III) acetylacetonate (Fe(acac)3, Aladdin, Shanghai, China), oleylamine (Aladdin, Shanghai, China), benzyl ether (Aladdin, Shanghai, China), ethanol, hexane, NaOH, Na2CO3, cetyltrimethylammonium bromide (CTAB, Sigma-Aldrich), N-[3-(trimethoxysily)propyl]B

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HAuCl4 solution, and we adjusted its pH to 9.0 by 0.01 M NaOH solution to finally obtain a colorless transparent solution. Then, Fe3O4@T-Si nanocomposites (3.75 mL, 2 mg/mL) were added for a further 30 min of intense stirring. In this procedure, HAuCl4 will attach on the surface of Fe3O4@TSi nanocomposites based on electrostatic adsorption. After that, 1.5 mL of sodium borohydride solution (0.01 M) was slowly added in order to reduce HAuCl4 into gold seeds on the surface of Fe3O4@T-Si nanocomposites. After aging for six more hours, Fe3O4@T-Si@AuSeeds nanocomposites were separated by centrifugation, washed with H2O, and lyophilized for storage, characterized by DLS, TEM, and SEM. Synthesis of Metal Nanorattles. First, we prepared 40 mL of aqueous solution with 10 mg of potassium carbonate (Na2CO3) under stirring for 10 min. Then 0.6 mL of HAuCl4 solution (1% (w/v)) was added for another 30 min of stirring until the yellow color of the solution became colorless. Different volumes of Fe3O4@T-Si@AuSeeds nanocomposites (2 mg/mL) were slowly added under intense stirring. A 500 μL solution of L-ascorbic acid was added, and the whole solution became green-blue and was aged for another 3 h. Fe3O4@TSi@AuShells nanocomposites were separated by centrifugation and washed with H2O, and then the mixture was added into Na2CO3 solution (0.3 M) for etching for 2 h under stirring. Finally, MNRs were obtained by centrifugation and washing with H2O and measured by DLS, TEM, and SEM. The Photothermal Effect of MNRs in Vitro. First, the photothermal effect of MNRs was verified in aqueous solution. Briefly, 200 μL of aqueous solution of MNRs with different concentrations was irradiated by using an 808 nm NIR laser with 2 W/cm2 for 5 min. The temperature of solutions was measured by a digital thermometer with a thermocouple probe. Subsequently, the photothermal effect in the cell level was measured. Cell viability assay of MNRs with NIR laser irradiation was carried out. HepG2 cells (104 cells per well) were incubated in 96-well plates (Corning, New York, USA) in an atmosphere with 5% CO2 at 37 °C using DMEM with 10% fetal bovine serum, L-glutamine, penicillin, and streptomycin for 24 h. Nanoparticle suspensions (0.1 mL) with different MNR concentrations ranging from 0.01 μg to 100 μg mL−1 were added into and incubated with cells for 4 h. After washing the additional MNRs that were not taken up, the cells were irradiated by 808 nm NIR laser under 2 W/cm2 for 5 min. After incubation for another 24 h, the cell viability was determined by MTT assay. In addition, we used calcein AM and PI staining method to qualitatively analyze the photothermal effect on cancer cells. Calcein AM could only penetrate in live cells and emit green fluorescence, and PI could only penetrate in dead cells and emit red fluorescence. After incubating with MNRs for 4 h (2.0 mL, 100 μg/mL), cells were exposed to NIR laser for 5 min under 2 W/cm2. Then, we prepared a mixture solution with 1 μg/mL calcein AM and 2 μg/mL PI for staining 30 min, and used LSCM examination. The Photothermal Effect of MNRs in Vivo. The tumorbearing mice treated with intratumorally injecting saline or MNRs (100 μL,10 μg) were exposed to NIR laser (2 W/cm2), and thermal images were taken in different points within 5 min using an infrared camera. Female BALB/c nude mice (n = 16) with weight at 18−20 g were purchased from Vital River Laboratory Animal Center (Beijing, China) and kept under SPF conditions with free access to standard food and water. The performance of all animal experiments was approved by the ethics committee of Peking University. The tumor model

ethylenediamine (TSD, Sigma-Aldrich), tetraethylorthosilane (TEOS, Sigma-Aldrich), (3-aminopropyl)triethoxysilane (APTES, Alfa Aesar), chlorauric acid (HAuCl4, Sigma-Aldrich), calcein AM (calcein acetoxymethyl ester), PI (propidium iodide), sodium borohydride (Sigma-Aldrich), L-ascorbic acid, DMEM (Hyclone, Logan, UT), fetal bovine serum (ExCell, New Zealand), MTT dye ((3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, 0.5 mg/mL, Sigma-Aldrich), microplate reader (M200, Tecan, Männedorf, Switzerland), laser scanning confocal microscope (LSCM, Ultraview VOX, PerkinElmer, Waltham, MA), transmission electron microscope (TEM, Tecnai G2 20 S-TWIN, FEI, USA), transmission electron microscope (F20, Tecnai G2 F20 U-TWIN, FEI, USA), scanning electron microscope (SEM, Hitachi S4800+EDS, Japan), dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern, England), animal MRI instrument (Bruker Pharmascan 7.0 T, Bruker, Germany), UV−vis spectrum (Lambda 950, PerkinElmer, USA), and Fourier transform infrared spectroscope (FT-IR, Spectrum One, PerkinElmer, USA). Synthesis of Superparamagnetic Nanoparticles. The 8 nm oleylamine SPIONs were supported by the Hou group synthesized as they previously reported.19 Briefly, 1.065 g of Fe(acac)3 and 34 mL of organic solution of a 1/1 ratio of oleylamine and benzyl ether were mixed, reacted with nitrogen protection by slowly rising temperature until 300 °C, kept for another 1 h reaction, and cooled down under room temperature. Finally, black SPIONs were washed with ethanol, obtained by centrifugation, and stored in hexane for subsequent usage. Synthesis of Fe3O4@T-Silica Nanospheres. First, the oilsoluble surface of the SPION nanoparticle must be converted into a water-soluble surface in order to accommodate the deposition of a silica layer. 1 mL of SPION hexane solution was redispersed in chloroform with different concentrations, and then it was slowly dropped into CTAB solution (5 mL, 55 mM) under intense stirring to form a brown emulsion. The quantity of Fe3O4 nanoparticles encapsulated as cores could be adjusted by changing the concentration of SPION solution, as shown in Figure S1 in the Supporting Information. Then, the brown emulsion was heated to 60 °C and kept for 15 min, aiming to evaporate all organic solution. That the solution turned into a black transparent solution meant that the organic solution was evaporated and CTAB had assembled on the surfaces of Fe3O4 nanoparticles, making them water-soluble. After that, this black solution was added into a mixture of 45 mL of H2O and 0.3 mL of 2 M NaOH, and then heated to 70 °C. When the temperature of the whole solution reached 70 °C, a mixture of TSD and TEOS with a ratio of 1/4, 50 μL of APTES, and 3 mL of ethyl acetate were added in order. 50 μL of APTES was added after 10 min reaction two more times. The whole mixture solution was stirred for another 3 h under refluxing. Crude Fe3O4@T-Si nanocomposites were obtained by centrifugation (10000 rpm, 10 min) and washed with ethanol three times. Then they were redispersed in 40 mL of ethanol, heated to 60 °C, and refluxed overnight under stirring to remove CTAB. Finally Fe3O4@T-Si nanocomposites were obtained by centrifugation (10000 rpm, 10 min) and washed with ethanol 3 times, stored after lyophilization for further usage, and characterized by DLS, zeta-potential, TEM, and SEM. Synthesis of Fe3O4@T-Si@AuSeeds. First, we prepared 30 mL of aqueous yellow solution containing 1 mL of 0.01 M C

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Figure 2. (a) TEM images of 8 nm superparamagnetic iron oxide nanoparticles (a1−a2), Fe3O4@T-Si nanocomposites (b1−b3), Fe3O4@T-Si@ AuSeeds nanocomposites (c1−c3), procedure of gold seeds growth on Fe3O4@T-Si@AuSeeds nanocomposites (d1−d3), Fe3O4@T-Si@AuShell nanocomposites (e1−e3), and MNRs (f1−f3). (b) EDX mapping images of Fe3O4@T-Si@AuShell nanocomposites and MNRs. (c) UV−vis-NIR spectra for the Fe3O4@T-Si@AuShellnanocomposites and MNRs.

was established by inoculation of 1 × 106 HepG2 cells per mouse in the right/left hind leg of nude mice. The test could be processed after the tumor volume reached 150 mm3. To investigate the tumor ablation efficiency by photothermal effect, mice were injected with saline or MNRs intratumorally. After 24 h, mice were anesthetized and irradiated by 808 nm laser under 2 W/cm2. T2-MRI Evaluation Relaxivity Measurement. First, we measured the T2 relaxivity of MNRs and Fe3O4@T-Si nanocomposites. Solution of MNRs and Fe3O4@T-Si nanocomposites at different concentrations were prepared in pure water. Mapping experiments for measuring T2 relaxation time were performed using a Bruker Pharmascan 7.0 T MRI animal instrument (Captial Medical University, Beijing, China) with the following paremeters: TR = 3000.0 ms, TE = 45.0 ms, matrix = 256 × 256, FOV = 3 cm, nex = 2. r2 values were calculated after the curve fitting of 1/T2 (s−1) versus CFe (mM).

Briefly, MR images were taken before and after the intratumoral injection of MNRs. Then the tumor-bearing mouse was taken for the T2-weighted MRI tests. MRI was performed using a Bruker Pharmascan 7.0 T MRI animal instrument (Captial Medical University, Beijing, China) with following parameters: TR/TE = 3000.0/45.0, echo length = 1, FOV = 4 cm, slice thickness = 2 mm, nex = 3, matrix = 256 × 256. All MRI analyses were processed by the same radiologist.

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RESULTS AND DISCUSSION Synthesis of Fe3O4@T-Si Nanocomposites. To obtain this structure, we chose highly dispersed 8 nm oleylamine SPIONs as MRI contrast agents (Figure 2a1−a2) as previous reported.19 These particles were capped with hydrophobic oleylamine ligands, which was the main reason for oil solubility. Therefore, CTAB was utilized as the phase inversion surfactant to transfer them into aqueous solutions. Meanwhile, considerD

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nanocomposites, and the Fe3O4@T-Si@AuSeeds nanocomposites were still maintained well-dispersed. Also, it could be clearly distinguished that numerous gold seeds distributed on the surface of the composites in SEM images (Figure 1b1−b2). In that sense, it was further confirmed that the simple one-pot in situ synthesis we used here could simultaneously facilitate the formation of gold seeds and subsequent anchoring onto the surface of silica smoothly, avoiding agglomeration from simple mixing/assembly of small gold nanoparticles and silica-based nanoparticles, lowering complicated surface modification of gold and/or Fe 3 O 4 @T-Si nanocomposites in previous reports,22 and reducing the possibility of toxicity brought by chemicals for surface modification. Subsequently, more sources of Au3+ were introduced for second growth of gold seeds. By precisely controlling the following seed-mediated growth, 2 nm gold seeds grew bigger and finally formed into porous gold shells. Both TEM and SEM images indicated the good dispersivity and homogeneous size distribution of Fe3O4@TSi@AuShells nanocomposites (Figure 1c1−c2, Figure 2d1− d3,e1−e3). Synthesis and Characterization of MNRs. Based on the results of TEM images of Fe3O4@T-Si@Au shell nanocomposites, there were many irregular pores on the gold nanoshells (Figure 1c2) which could allow the permeation of alkaline solution. As mentioned above, the insertion of TSD made the hybrid silica layer loose and sensitive to alkaline solution. Thus, according to the protocol by Chen et al.23 and our previous experiments, we chose a 0.3 M aqueous solution of sodium carbonate (Na2CO3) for etching 2 h at room temperature and finally obtained unique rattle-type structures of MNRs. Both SEM and TEM results indicated the fact that the MNRs still maintained stable morphology and structure after etching (Figure 1d1−d2, Figure 2f1−f3) with size around 152 nm and surface charge as −6.79 mV similar to Fe3O4@TSi@Au shell nanocomposites (−8.14 mV) (Table S1 in the Supporting Information), and the magnified images of MNR demonstrated its hollow structure (Figure S3 in the Supporting Information). The structures of Fe3O4@T-Si@Au shell nanocomposites and MNRs were further confirmed by EDX spectroscopy. The EDS line profile indicated the decrease of silicon element in MNRs and the existence of three primary elements (Figure S4 in the Supporting Information). The elemental mapping analysis (Figure 2b) revealed that Au (green) distributed in the outer layers of both Fe3O4@T-Si@ Au shell nanocomposites and MNR with shell-like structures; compared with the homogeneous distribution of silicon (orange) in Fe3O4@T-Si@Au shell nanocomposites, the inner silicon of MNRs was obviously reduced and almost distributed annularly along with the gold shell rather than the inner space after etching, suggesting the successful removal of the T-Silica layer without damage to gold shells; and Fe (orange) located in the cores of both structures were surrounded by the gold shell. The ringlike distribution of silicon in MNR was probably attributable to the residual silicon elements on the gold shell during their dissolution after etching or the background noise. The extra inner space could be potentially used to carry other cargos as in the report by Tang et al.24 As Figure 2c indicated, after etching, the MNRs still had strong absorption in the NIR light region, which provided the basis for subsequent photothermal effect experiments. In Vitro Photothermal Effects. Various gold nanostructures had been widely investigated as photothermal therapy agents for selective ablation of solid tumors because localized

ing the fact that CTAB is the most common liquid crystal template of mesoporous silica nanoparticles,39 the presence of CTAB on the surface also facilitated the formation of a silica layer on the surface of magnetic cores. To obtain a more vulnerable intermediate layer that was easily etched by alkaline solvent, TSD and TEOS with the ratio 1/4 were cocondensed to encapsulate SPIONs. Along with the increment of concentration of SPIONs used, Fe3O4@T-Si nanocomposites ranging from monocore to multicore with a slight increase of sphere size were obtained (Figure S1 in the Supporting Information). To maintain higher responsiveness to magnetic field, Fe3O4@T-Si nanocomposites were chosen with multiple magnetic cores for subsequent experiments. Besides, we removed CTAB by refluxing Fe3O4@T-Si nanocomposites in ethanol, in order to avoid the influences of its toxicity on subsequent experiments. The FT-IR data indicated that the characteristic band of C−H stretching vibration in CTAB did not distinctly show in spectrum peaks at 2921 and 2850 cm−1, which meant that CTAB had been successfully removed. In Figure 1a1−a2 and Figure 2b1−b2, it was shown that Fe3O4@T-Si nanocomposites had a well-defined spherical morphology with a uniform size of ∼120 nm (Table S1 in the Supporting Information) and high dispersivity. Tens of SPIONs were found to be encapsulated into a single T-Silica nanosphere (Figure 2b 3 ). Meanwhile, organic groups −(CH2)3−NH−CH2−CH2−NH2 of TSD homogeneously distributed in the silica layer in the cocondensation procedure forming an organic−inorganic hybrid siloxane framework, which resulted in a less compact organic-silica framework than pure Si−O−Si counterpart.7 The TEM images of Fe3O4@ T-Si nanocomposites showed the wormhole-like mesopores20 in the outer layer, unlike the orderly tubelike mesopores in the Fe3O4@Silica nanocomposites without cocondensation of TSD, which means the insertion of TSD into the silica layers (Figure S2, panels a1−a2 and b1−b2, in the Supporting Information). Also, the characteristic band of the Si−O bond (around 1100 cm−1) of Fe3O4@T-Si nanocomposites became much more placid and broad compared with Fe3O4@Silica nanocomposites and pure mesoporous silica nanoparticles (MSN), which also indicated the higher disordered structure of the silica layer; while the characteristic band of Fe−O bond (600 cm−1) clearly showed in Fe3O4@Silica and Fe3O4@T-Si nanocomposites, which again confirmed the existence of SPIONs in both core−shell nanostructures (Figure S2c in the Supporting Information).21 Besides, for subsequent growth of gold seeds on the surface of nanocomposites, moderate APTES was added to form a positive surface (+33.4 mV) (Table S1 in the Supporting Information) with the existence of amino group (−NH2). Synthesis of Fe3O4@T-Si@Au Shell Nanocomposites. Then, according to the in situ Au3+ reduction reported by Shi and co-workers,3 uniform 2 nm Au seeds (Table S1 in the Supporting Information) formed and simultaneously attached to the outer surface of Fe3O4@T-Si nanocomposites via strong electrostatic adsorption between the negative Au and positive amino groups provided by APTES. During the reduction procedure, Au3+ absorbed on the surface was locally reduced to Au atom, and along with more and more Au3+ ions being reduced, gold seeds were formed and anchored on the surface of Fe3O4@T-Si nanocomposites. This method was called in situ reduction, which was attempted to avoid aggregation and nonmonodispersity. As shown in Figure 2c1−c3, numerous 2 nm gold seeds were grown on the outer surface of Fe3O4@T-Si E

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Figure 3. (a) HepG2 cell viabilities cultured with MNRs at different concentrations with or without 808 nm laser irradiation, and only exposed to 808 nm laser irradiation. (b) Confocal microscopic images of differently treated HepG2 cells stained with calcein AM and PI, including control, laser irradiation only, MNRs without laser irradiation, and MNRs with laser irradiation. (c) Infrared thermal images of saline-injected as control and MNR-injected tumor-bearing mice at different time points under 808 nm laser irradiation. (d) In vivo photothermal effect of MNRs in HepG2 tumor-bearing mice in different groups.

temperature rise of pure water was less than 3 °C even irradiated for 10 min under the same condition (Figure S4a in the Supporting Information). In the following experiments, we chose 5 min as the time for 808 nm laser irradiation because temperature would be stable after 5 min irradiation. Then the concentration dependence for increasing temperature was also tested. As indicated in Figure S5b in the Supporting Information, the higher the concentration of MNRs was, the higher the increment of temperature would be after exposure to NIR light. The photothermal effect of MNRs was further investigated in the HepG2 cell level. In the MTT assay, HepG2 cells treated with MNRs with different concentrations up to 100 μg mL−1 still had high cell viability, indicating nontoxicity of MNRs. After irradiation by 808 nm NIR laser for 5 min under 2 W/ cm2, the viability of treated HepG2 cells decreased significantly, and only nearly 20% of HepG2 cells still remained viable at the concentration of 100 μg mL−1 (Figure 3a) . The 808 nm NIR laser only also did not show toxicity to cells. To further confirm their photothermal effect, we utilized a confocal microscope to directly observe the death of MNR-treated cells caused by the

surface plasmonic resonance (LSPR) endowed gold the capability of responding to NIR light and converting NIR light to heat.25,40 It had been reported that tumor tissues were much more vulnerable and sensitive to the temperature in the range of 42−45 °C than normal tissues were, basically because of their poorer vascularization.26 Meanwhile, NIR light has been proved to be safe and able to penetrate deeply into tissues, which made the development of a new NIR light-responsive nanogold-based biomedicine more promising.27 In our nanosystems, the gold shell is thin and porous, making it a better agent for NIR light absorption rather than scattering, which is therefore a better agent for photothermal therapy compared to gold nanospheres and gold nanorods (both of them rely on the absorption cross-section of the gold).28−30In this work, based on their absorption in the NIR region (Figure 2c), the photothermal effect of MNRs was subsequently measured in aqueous solution. The temperature of the MNR solution with high concentration 100 μg mL−1 increased dramatically from ∼30 °C to ∼63 °C only after 2 min at 808 nm laser irradiation under 2 W/cm2,3,31,32 and then the temperature tended to be stable and went into a plateau of around 65 °C. However, the F

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Figure 4. (a) In vitro T2-weighted MR imaging signal of MNRs and Fe3O4@T-Si nanocomposites. (b) Plot of the spin−spin relaxation rate (T2−1) against iron concentration of MNRs and Fe3O4@T-Si nanocomposites. (c) In vivo T2-weighted MR images of a tumor site before and after injection of MNRs.

photothermal effect was examined in vivo. After the temperature rose up to 46 °C within the first 5 min, the 808 nm laser kept irradiating the MNR-treated tumor for another 5 min under 2 W/cm2 to inhibit its growth. The skin on the tumor did not show obvious empyrosis, but the death of tumor cells caused necrosis of tumor tissue inside. Finally, the solid tumor became invisible to the naked eye after 20 days without any evidence of tumor renaissance (Figure 3d). In the tumor sites, we could clearly distinguish the renascent skin after ablation of the solid tumor, which was continent to other reported results,37 whereas the solid tumors in groups of treated only with saline, saline with laser, or MNRs without laser exposure still displayed thriving growth. MR Imaging Evaluation. Furthermore, the MNR sample was also treated as T2-weighted MR imaging contrast agent by using a Bruker Pharmascan 7.0 T MRI (for animal) instrument. The T2-weighted MR images (echo time = 45 ms) of MNRs in aqueous media with Fe concentrations ranging from 0 to 0.20 mM were obtained. The Fe concentrations in both Fe3O4@TSi nanocomposites and MNRs were determined by inductively coupled plasma optical emission spectrometry (ICP-OES). The signal intensity of the MR images decreased along with the increase of Fe concentrations, as expected for T2 contrast agents (Figure 4a). On the other hand, the tube that contained only blank solution remained bright and indistinguishable. We compared the transverse relaxivity r2 and measured the change in spin−spin relaxation rate (T2−1) per unit Fe concentration between MNRs and Fe3O4@T-Si nanocomposites. Though the r2 value decreased by 30.5% after forming the nanorattle structures, it was still as high as 174.7 mM−1 s−1 (Figure 4b). As SEM results showed, a small number of gold nanoshells were broken after etching because of their heterogeneous growth, which probably provided channels for iron oxide nanoparticles to leak out (Figure S3 in the Supporting Information). Therefore, one reason for the decrease of r2 could be the loss of iron oxide nanoparticles during the process of etching.

laser irradiation. As shown in Figure 3b, a distinct demarcation line between live (green) and dead (red) cell regions could be observed in the MNR-treated group with regional laser exposure. On the contrary, treating cells with only MNRs or 808 nm laser irradiation cannot lead to cell death. This result confirmed that MNRs were nontoxic and could kill HepG2 cancer cells assisted by NIR laser irradiation. In Vivo Phothothermal Effects. Many previous works had reported that tumor could be efficiently inhibited by heating nanocomposites up to 60 °C or even higher. However, empyrosis, even subsequent necrosis, would occur in the skin or tissue under that high temperature. As mentioned above, the temperature increment of MNRs is concentration-dependent, which means that the highest temperature capable to be achieved could be tuned readily by controlling the concentration of MNRs injected in vivo. Therefore, the laser-induced temperature-increasing effects by the synthesized MNRs were investigated in vivo. The spatial distribution of intratumoral temperature was examined by using a thermal imaging camera for the nude mouse intratumorally injected with saline or MNR solution (100 μL, 10 μg) (Figure 3c).33−36 During the irradiation, the temperature of the tumor region injected with MNRs increased from ∼32 to 46 °C and became stable within 5 min. Meanwhile, other regions, which were not exposed to the NIR laser, showed an increment less than 3 °C. In contrast, the temperature of the tumor region injected only with saline showed undetectable change under exposure to NIR light for 5 min. This result revealed clearly that the MNRs could also absorb the 808 nm light and convert it into heat in vivo just as they did in vitro, which could potentially be used for photothermal tumor therapy under NIR laser irradiation. In this work, we utilized intratumoral injection to make a proof-of-principle demonstration of the utility of our system, similar to the same protocols of other research to administer different nanoscale therapeutic agents.33−36 Based on the result of thermal imaging, tumor inhibition resulting from the G

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Additionally, the loss of T-Silica of MNRs could also weaken the proton relaxation rate because the porous silica matrices could enhance the molecular motion of H2O within the pores as previously reported.38 These combined reasons could result in the decrease in specific relaxivity of MNRs. The T2-weighted MR imaging capability of MNRs was further evaluated in vivo. As presented in Figure 4c, the left tumor site without MNR injection as the control group did not exhibit apparent change before and after injection; however, a significant darkening in the T2-weighted MR image could be observed at the right tumor site after MNRs were injected. It implied that the present superparamagnetic iron oxides in MNRs still possessed the ability for T2-weighted MR imaging and MNRs could be a promising candidate as a type of contrast agent in T2-weighted MR imaging for solid tumors. Combined with its functionality of photothermal therapy, it was anticipated that the as-prepared MNRs would be a potential multifunctional agent for MR imaging-guided photothermal cancer therapy.

Article

ASSOCIATED CONTENT

S Supporting Information *

Table of Fe3O4@T-Si nanocomposites with different iron concentration during synthesis; TEM images of Fe3O4@ SiO2nanocomposites and Fe3O4@T-Si nanocomposites; FTIR result for mesoporous silica nanoparticles (MSN), Fe3O4@ SiO2 nanocomposites, and Fe3O4@T-Si nanocomposites; average size and ζ-potential changes of nanostructures in different steps to synthesize MNRs; SEM image of MNRs and its magnification; EDS profiles of Fe3O4@T-Si@AuShell nanocomposites and MNRs; and temperature rise of MNRs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected].

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Notes

The authors declare no competing financial interest.

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CONCLUSIONS In summary, we successfully prepared and characterized novel multifunctional rattle-type metal nanocarriers (MNRs) by utilizing porous gold shell to carry MRI contrast agents, 8 nm SPIONs. In the preparation process, the wormhole-like porous silica layer formed by cocondensation of TEOS and TSD not only acted as an intermediate layer for the growth of gold nanoshells but also provided much more vulnerable porous structure for subsequent easy and rapid etching by alkaline solution. Also, the modification with APTES introduced amino groups on the surfaces of Fe3O4@T-Si nanocomposites and thus facilitated the in situ growth of gold seeds on their surfaces. Moreover, this is the first report about etching the intermediate layer to fabricate a metal nanorattle consisting of SPIONs and porous gold nanoshells by using Na2CO3 alkaline solution. Based on the specific absorption in the NIR region and the ability to convert light to heat, we demonstrated that MNRs could kill cancer cells in vitro and inhibit solid tumor in vivo effectively coupled with NIR 808 nm laser irradiation. Additionally, this multifunctional nanostructure could be potentially applied as MR imaging contrast agent evidently. This self-functionalized nanoplatform possesses practical prospects as therapeutics for MR imaging-guided photothermal cancer therapy in vivo based on the current clinical trials of gold nanoshells. The rattle structure makes our system a superior reservoir for further loading application, which potentially combines traditional chemotherapy with novel cancer therapy and therefore renders more possibility to tune the release profile and therapeutic efficiency of this system. The fact that gold nanoshell is under clinical trail and some types of SPIONs have been approved by FDA also make this novel rattle-type metal nanocarrier more promising for real application. In this work, we mainly emphasized elucidating the nanostructured MNRs which possess novel properties and proceeding with primary verification on their multiple functions. We will keep improving this nanostructure in the aspects of stability in biological fluid, surface modification for antifouling, long circulation time and specific targeting,41 and so on, expanding its applicability for delivery of other cargos, such as drugs and other contrast agents, and finally executing intravascular examinations including therapeutic efficiency, dose toxicity, long toxicity, and so on, in the future.

ACKNOWLEDGMENTS This work was supported by Chinese Natural Science Foundation projects (No. 30970784, No. 81171455), a National Distinguished Young Scholars grant (31225009), the National Key Basic Research Program of China (2009CB930200), the Chinese Academy of Sciences (CAS) “Hundred Talents Program” (07165111ZX), the CAS Knowledge Innovation Program and the State High-Tech Development Plan (2012AA020804). The authors also appreciate the support by the “Strategic Priority Research Program” of the Chinese Academy of Sciences (XDA09030301).

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