Pd NanosheetCovered Hollow Mesoporous Silica Nanoparticles as

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Pd Nanosheet-Covered Hollow Mesoporous Silica Nanoparticles as a Platform for the Chemo-Photothermal Treatment of Cancer Cells Weijun Fang, Shaoheng Tang, Pengxin Liu, Xiaoliang Fang, Jiawei Gong, and Nanfeng Zheng*

A versatile system combining chemotherapy with photothermal therapy for cancer cells using Pd nanosheet-covered hollow mesoporous silica nanoparticles is reported. While the hollow mesoporous silica core can be used to load anticancer drugs (i.e., doxorubicin) for chemotherapy, the Pd nanosheets on the surface of particles can convert NIR light into heat for photothermal therapy. More importantly, the loading of Pd nanosheets on hollow mesoporous silica nanospheres can dramatically increase the amount of cellular internalization of the Pd nanosheets: almost 11 times higher than the unloaded Pd nanosheets. The as-prepared nanocomposites efficiently deliver both drugs and heat to cancer cells to improve the therapeutic efficiency with minimal side effects. Compared with chemotherapy or photothermal therapy alone, the combination of chemotherapy and phototherapy can significantly improve the therapeutic efficacy, exhibiting a synergistic effect.

1. Introduction Nanomaterials have attracted great attention recently for biomedical applications, such as drug delivery, gene transfection, cancer diagnosis, and therapy.[1–4] Among these biomedical applications, hyperthermia with NIR resonant nanoparticles for cancer therapy has developed very quickly in recent years. The benefit of those NIR resonant nanoparticles is that the particles could strongly absorb NIR light and convert it into heat to kill cancer cells. Compared to short-wavelength light, NIR light is not harmful to the human body and can penetrate tissue deeply.[5,6] Thus, a series of NIR-light-absorbing

W. J. Fang, S. H. Tang, P. X. Liu, X. L. Fang, J. W. Gong, Prof. N. F. Zheng State Key Laboratory for Physical Chemistry of Solid Surfaces and Department of Chemistry College of Chemistry and Chemical Engineering Xiamen University Xiamen 361005, China E-mail: [email protected] DOI: 10.1002/smll.201200962

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nanomaterials, including gold nanocrystals,[7–12] carbon nanotubes (CNTs),[13–16] graphene,[17,18] organic nanoparticles,[19,20] Pd nanosheets,[21] and CuS/Cu2-xSe nanocrystals[22–26] have been prepared to achieve good results in cancer photothermal therapy. Based on the unique property of the NIR resonant nanoparticles to improve the cell-killing efficiency, the combination of chemotherapy and photothermal therapy could be a more effective way to destroy cancer cells than chemotherapy or photothermal therapy alone. This synergistic effect is confirmed by numerous successive studies. For instance, silica/Au nanoshells,[27] polymer/Au nanoshells,[28,29] Au/polymer complexes and hollow gold nanoparticles[10,30–36] have been fabricated and showed remarkable cancer-cellkilling efficiency. Alternatively, carbon-based nanomaterials, especially carbon nanotubes and graphene, have also exhibited a clear synergistic effect in cancer cells killing after absorbtion of aromatic drug molecules via π–π stacking.[37–39] Therefore, the development of novel nanomaterials for combining chemo- and photothermal therapy with high efficiency is vital. Among the above NIR-light-absorbing nanomaterials, Pd nanosheets have high photothermal stability, photothermal transformation efficiency, and biocompatibility.[21]

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Unfortunately, there is a lack of studies using Pd nanosheets for the combination of chemotherapy and photothermal therapy to improve cancer-cell-killing efficiency. We here report a simple route for the preparation of Pd nanosheetcovered hollow mesoporous nanoparticles for the combined chemotherapy and photothermal therapy of cancer cells. In the synthesized nanocomposites, doxorubicin (DOX) molecules are encapsulated in the hollow mesoporous silica particles, and the Pd nanosheets are deposited on the particle surfaces. Once the composite nanoparticles are internalized by cancer cells and irradiated by NIR laser, the Pd nanosheets on their surface can absorb the NIR light and convert it into fatal heat to kill the cancer cells. Moreover, the low pH in endosomal compartments and the heat generated by NIR irradiation can induce the significant release of the DOX molecules loaded on the mesoporous particles, improving the chemotherapeutic efficacy of DOX. The combined therapy in our system yields a synergistic effect, meaning that the combined therapy efficacy is higher than the sum of individual efficacies of chemotherapy and photothermal therapy.

Figure 1. SEM (a,c) and TEM (b,d) images of HMSS (a,b) and HMSS–[email protected] nanoparticles (c,d).

2. Results and Discussion Scheme 1 depicts the stepwise preparation procedures for our controlled drug-release system integrating hollow mesoporous silica sphere (HMSS), doxorubicin (DOX) and Pd nanosheets. HMSS particles were first synthesized by using CTAB as the template via our recently reported method.[40] The scanning electron microscope (SEM) image shown in Figure 1a revealed that the obtained HMSS were spherical

in shape and about 170 nm in diameter, and the electron contrast between the cores and the shells confirms its hollow structure. As revealed by the transmission electron microscope (TEM) image in Figure 1b, the transparency of the core of the HMSSs confirms their hollow characteristics. The shell of the HMSSs with a thickness of ∼20 nm exhibits an obvious mesoporous structure. The HMSS particles were then treated with 3-aminopropyltrimethoxysilane (APTES) to yield HMSS–NH2 particles having amino groups on their surface. The mesopores of the HMSS and HMSS–NH2 were investigated by N2 adsorption–desorption measurements. Both HMSS and HMSS–NH2 samples showed a typical type IV isotherm feature (Supporting Information, Figure S1). The Brunauer–Emmett–Teller (BET) surface area, pore volume, and pore diameter of the HMSSs were measured to be 1002.1 m2/g, 1.27 m3/g, and 2.7 nm, respectively. In comparison, these parameters of HMSS–NH2 were reduced to 815.8 m2/g, 0.94 m3/g, and 2.5 nm, respectively. The slightly decreased pore diameter and surface area of HMSS–NH2 is likely due to the presence of amino Scheme 1. Schematic procedure for the preparation of HMSS–NH2/[email protected] nanoparticles. groups on their surface. small 2012, 8, No. 24, 3816–3822

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under laser irradiation (808 nm, 1 W/cm2). As illustrated in Figure 2b, the temperature of 1.0 mL water solution containing 200 μg of HMSS–[email protected] (12.2 μg Pd) was raised from 27.0 °C to 47.5 °C after 12 min NIR irradiation. In comparison, in the absence of nanoparticles, no significant temperature change was observed to a water solution irradiated by the NIR laser under the same conditions. These data confirm that the HMSS–[email protected] can effectively absorb and convert NIR light into heat. Another unique structural feature of the HMSS–[email protected] Pd particles lies in their hollow core and mesoporous shell with perpendicular pore channels. Based on these structures, the HMSS–[email protected] particles are expected to have advantages as anticancer drug delivery carriers. DOX, an anticancer drug, was thus selected as a model drug to investigate the drug-loading capacity of the HMSS–NH2 nanoparticles. DOX molecules were loaded in the HMSS–NH2 nanoparticles to obtain the HMSS–NH2/DOX composite. The HMSS– NH2/DOX nanoparticles were then capped by Pd nanosheets. The loading capacity of DOX on the HMSS–[email protected] particles was 9.2% (by weight) as determined by fluorescence spectroscopy (Figure S3). As shown in the DOX release profiles from the HMSS–NH2/[email protected] particles (Figure 3a), the lower pH value of the phosphate buffered saline (PBS)

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Figure 2. (a) UV-vis spectra of Pd and HMSS–[email protected] nanoparticles in water solution. (b) The temperature versus time plots recordedfor 1 mL aqueous dispersions of HMSS–[email protected] nanoparticles at various concentrations on irradiation by a 1 W/cm2 laser.

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After post-grafting modification with APTES to yield HMSS–NH2 particles, Pd nanosheets (∼41 nm in diameter) were deposited onto the surface of HMSS–NH2 to obtain HMSS–[email protected] particles (see the Experimental Section for details). As illustrated in Figure 1c,d, Pd nanosheets were clearly visible on the surface of HMSS– NH2 spheres, indicating that Pd nanosheets were successfully grafted onto the surface of the HMSS–NH2 nanoparticles. Based on the inductively coupled plasma (ICP) analysis and electron microscopy results, about 14 Pd nanosheets were grafted onto the surface of each HMSS– NH2 sphere. The capping mechanism of the HMSS–[email protected] Pd system involves two possible interactions between Pd nanosheets and HMSS–NH2: 1) the electrostatic interaction between the positively charged HMSS–NH2 (ζ = +29.2 mV) and negatively charged Pd nanosheets (ζ = −13.3 mV) (Figure S2); 2) the coordination between the amino groups on the surface of HMSS–NH2 and the Pd nanosheets. Importantly, the HMSS–[email protected] particles nicely inherited the strong NIR absorption property of the Pd nanosheets (Figure 2a), making them a potential photothermal therapy agent. To investigate the photothermal effect induced by NIR absorption, the temperatures of the solutions containing various concentrations of HMSS–[email protected] were measured

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Figure 3. (a) Release kinetics of DOX from HMSS–NH2/[email protected] in PBS buffer at different pH values; (b) Release kinetics of DOX from HMSS–NH2/[email protected] in PBS buffer at pH = 5.0 with or without laser irradiation for 5 min periods.

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solution, the more DOX was released. For instance, 17.3%, 35.1% 50.3%, and 60.0% of the loaded DOX were released from HMSS–NH2/[email protected] particles in PBS buffer within 8 h at the pH values of 7.4, 6.0, 5.0, and 4.0, respectively. These results indicate that the DOX release from HMSS–NH2/ [email protected] increases with decreasing pH. Such a pH-sensitive release of DOX from HMSS–NH2/[email protected] is important for cancer therapy because the pH value of tumor tissue is typically lower than that of normal tissue or biological fluids. Having Pd nanosheets grafted on their surface, HMSS– [email protected] nanoparticles exhibited a significant photothermal effect induced by NIR irradiation. To examine whether the NIR photothermal effect offered by Pd nanosheets could trigger the DOX release, the release kinetics of DOX from the HMSS–NH2/[email protected] composite under NIR light irradiation was investigated. During the DOX release process from the HMSS–NH2/[email protected] dispersion in the PBS buffer at pH = 5, 5 min NIR laser irradiations (808 nm, 1 W/cm2) were given at 0.5, 1.0, 2.0, 3.0, 4.0, and 6.0 h. As shown in Figure 3b, the first 5 min NIR irradiation at 0.5 h increased the cumulative amount of released DOX from 14.5% to 39%. When the NIR laser was switched off, the release rate was significantly reduced, suggesting that the NIR light could enhance the DOX release from the nanoparticles. The DOX release for each cycle was 9.8%, 6.4%, 3.4%, 2.2%, and 1.6% for the following 5 min NIR radiation cycles at 1.0, 2.0, 3.0, 4.0, and 6.0 h, respectively. After the six cycles of 5 min irradiation at the given time intervals, the cumulative release of DOX reached 75.4% after 8 h. In comparison, the HMSS– NH2/[email protected] particles without NIR laser irradiation had a cumulative release of only 48.9% in the same period. The enhanced drug release under NIR irradiation can be attributed to the photothermal effect of the Pd nanosheets on the surface of HMSS–NH2/[email protected] nanospheres, which induces heat generation by NIR irradiation to allow more DOX molecules from the nanospheres by weakening the coordination between the amino groups on the surface of HMSS–NH2 and Pd nanosheets. Moreover, it was found that the binding of Pd nanosheets on the surface of HMSS–NH2 nanoparticles was quite strong. Pd nanosheets were still grafted on the surface of HMSS–NH2 nanoparticles even after 30 min NIR laser irradiation in PBS solution (808 nm, 1 W/cm2, pH = 5.0; Figure S4). It is well known that the efficient internalization of nanoparticles into cells is very important in cancer therapy. Several groups have studied the efficiency of the internalization of nanoparticles by varying their size, shape, and surface.[41–44] Recently, we also found that the ultrathin nature of 2D Pd nanosheets prevents them from efficiently entering into cells for effective photothermal therapy.[45,46] When a silica coating was applied to the Pd ultrathin nanosheets to increase their thickness, the amount of internalization of the Pd nanosheets was greatly enhanced (∼5 times). In this work, we also found that more Pd nanosheets were taken up by cancer cells when the ultrathin Pd nanosheets were coated on the surface of hollow mesoporous nanoparticles. After 12 h incubation with 0.5 mL dispersion containing 5 μg/mL Pd of unloaded Pd nanosheets or HMSS–[email protected] nanospheres, Hep G2 cells took up only 0.52% (1.3 × 10−2 μg) of the unloaded Pd small 2012, 8, No. 24, 3816–3822

Figure 4. (a) Mass of Pd nanoparticles internalized in Hep G2 cells incubated with naked Pd nanosheets and HMSS–[email protected] nanoparticles measured by ICP-MS, (b) Fluorescence microscopy image of Hep G2 cells after being incubated with HMSS–NH2–[email protected] nanoparticles for 10 h at 100 μg/mL.

nanosheets, but 4.64% (0.116 μg) of the Pd nanosheets loaded on mesoporous nanospheres (Figure 4a). The uptake of the Pd nanosheets by the cancer cells was increased by almost 9 times. Loading of the Pd nanosheets onto the surface of silica spheres further increased the uptake of the nanosheets by 8 and 11 times, respectively, when the incubation concentration of the Pd nanosheets were increased to 10 and 15 μg/ mL. The increased internalization of the Pd nanosheets with the HMSS–[email protected] particles could be attributed to the larger size of HMSS–[email protected] particles that could be endocytosed by cells more easily than the ultrathin Pd nanosheets. To clearly observe the internalization of nanoparticles into cancer cells, fluorescein isothiocyanate (FITC) was applied to label the nanoparticles. The labeled particles are denoted as HMSS–NH2–[email protected] particles. Hep G2 cells were then incubated with 100 μg/mL of the HMSS–NH2– [email protected] particles for 10 h, and washed with PBS buffer to remove the non-internalized nanoparticles. As revealed by fluorescence microscopy images (Figure 4b, S5), significant fluorescence was observed inside the cells, indicating that the HMSS–NH2–[email protected] particles were efficiently internalized into Hep G2 cells. As discussed above, the HMSS–NH2/[email protected] particles displayed the following two important properties: 1) The release of DOX from the particles was pH-dependent and could be enhanced by introducing NIR laser irradiation; 2) The uptake of the ultrathin nanosheets by cells was significantly enhanced when the sheets were loaded onto silica nanospheres. Such important features of HMSS–NH2/[email protected] Pd nanoparticles motivated us to further examine their application in controlled drug release for cancer cell therapy. The cellular cytotoxicity of the HMSS–[email protected] particles was first investigated by Hep G2 cells. After incubation with the HMSS–[email protected] carriers at different concentration for 24 h, the cell viability was measured by MTT assays. As depicted in Figure 5a, the cell viability was 97.9% even the concentration of the HMSS–[email protected] carriers as high as 600 μg/mL (the total content of the particles), demonstrating that the HMSS– [email protected] particles are highly biocompatible. Moreover, the enhanced release of DOX from HMSS–NH2/[email protected] particles by introducing NIR laser was also observed inside cells by confocal fluorescence microscopy (Figure S6). After 6 h incubation with 100 μg/mL HMSS–NH2/[email protected] nanoparticles,

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measured with or without NIR irradiation. After NIR laser irradiation (1 W/cm2, 808 nm), the HMSS–NH2/[email protected] particles showed a higher cell-killing effect in Hep G2 cells at all tested concentrations than the chemotherapy (without NIR irradiation) or the photothermal therapy (without DOX) alone (Figure 5b). For instance, 90.5% cells were killed by the HMSS–NH2/[email protected] particles under 5-min NIR irradiation at an equivalent DOX concentration of 10 μg/mL. In comparison, the HMSS–NH2/[email protected] particles without NIR irradiation killed only 28.3% of the cells. And the HMSS–[email protected] Pd particles with 5-min NIR irradiation killed 47% of the cells. To identify the cell viability, the dead cells were stained with Trypan Blue (Figure S7). All the results suggested that the chemotherapy and photothermal therapy were successfully combined in the HMSS–NH2/[email protected] particles. More importantly, the combined chemotherapy and photothermal therapy achieved in our system yielded a synergistic effect that the combined therapy efficacy was higher than the sum of individual efficacies of chemotherapy and photothermal therapy.

3. Conclusion

Figure 5. (a) Viability of Hep G2 cells incubated for 24 h with different concentrations of HMSS–[email protected] [email protected]). (b) Hep G2 cell viabilities after 10 h incubation with different concentrations of free DOX, HMSS–[email protected] and HMSS–NH2/[email protected] with or without 5 min NIR irradiation (1 W/cm2, 808 nm).

Hep G2 cells upon 5 min NIR irradiation displayed a stronger fluorescence intensity of DOX than those without NIR treatment, suggesting that the photothermal effect could induce more DOX molecule release inside Hep G2 cells. To compare the cytotoxicities of DOX loaded on HMSS– NH2/[email protected] nanoparticles and free DOX, Hep G2 cells were incubated in culture medium containing free DOX or HMSS–NH2/[email protected] particles with various concentrations for 10 h, and then the MTT assay was carried out to test the cell viability. About 49% of cells were killed by the HMSS– NH2/[email protected] particles at an equivalent DOX concentration of 20 μg/mL (Figure 5b). However, free DOX exhibited a lower toxicity with 31.3% cells killed at the same condition. The higher cytotoxicity of HMSS–NH2/[email protected] nanoparticles than free DOX molecules can be explained by the high uptake of HMSS–NH2/[email protected] particles by Hep G2 cells through endocytosis, followed by the low-pH induced release of the loaded DOX inside the endosomal compartment. To further investigate the HMSS–NH2/[email protected] particles for the combined chemo-photothermal cancer therapy, Hep G2 cells were incubated with the HMSS–NH2/[email protected] particles and the HMSS–[email protected] particles. The cell viabilities were

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In conclusion, we have developed a novel drug-delivery system based on the HMSS–[email protected] particles. Based on these Pd/silica complexes nanoparticles, combined chemotherapy and photothermal therapy was successfully achieved, and the HMSS–[email protected] particles exhibit a synergistic effect for cancer-cell killing. Moreover, we have demonstrated that the HMSS–[email protected] particles show significant enhancment of the cellular internalization of Pd particles, compared to naked Pd nanosheets. Therefore, we conclude that these complex Pd/silica nanoparticles have great potential use for drug delivery and cancer therapy.

4. Experimental Section Materials: Ammonium aqueous solution (25–28%), TBAB (tetrabutylammonium bromide), PVP (poly(vinylpyrrolidone) MW = 30 000 daltons), Na2CO3 and CTAB (cetyltrimethylammonium bromide) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). DOX was purchased form HuaFeng United Technology Co. Ltd. (Beijing, China). Pd(acac)2 (Pd(II)acetylacetonate), tetraethoxysilane (TEOS), 3-aminopropyltrimethoxysilane (APTES), MTT and FITC were purchased from Alfa Aesar. Hep G2 cells were purchased from cell storeroom of Chinese Academy of Science. All reagents were used as received without further purification. Synthesis of HMSS Spheres: The whole synthesis process of hollow mesoporous silica spheres (HMSSs) consists of three steps. Firstly, solid SiO2 spheres (sSiO2) were prepared using a modified Stöber method. Typically, 74 mL of ethanol, 3.15 mL of ammonium aqueous solution (∼28%) and 10 mL of ultra-pure water were mixed and further stirred for 1 h. The mixture was then heated up to 50 °C and 6 mL of TEOS was added. After the reaction with stirring for 3 h, sSiO2 were obtained by centrifugation and washed with ethanol. Secondly, 50 mg of sSiO2 were re-dispersed in 30 mL of ethanol/water mixture 1:2 (v/v), and then 7.5 mL of ethanol/

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water mixture 1:2 (v/v) containing 75 mg of CTAB was added. After 30 min of stirring, 125 μL of TEOS and 300 μL of ammonium aqueous solution (∼28%) were added to the above mixture. The mixture was allowed to react for 12 h at room temperature. The [email protected]/SiO2 spheres were collected by centrifugation, and re-dispersed in 10 mL of water for subsequent use. Thirdly, to transform [email protected]/SiO2 spheres to HMSS spheres, 10 mL of the above solution containing 50 mg of [email protected]/SiO2 spheres was mixed with 232 mg of Na2CO3. The reaction was stirred at 50 °C for 11 h, the HMSS spheres were harvested by centrifugation. To remove surfactant template, the products were dispersed in 60 mL of NH4NO3 ethanol solution (20 mg/mL). The mixture was heated to 45°C under stirring for 6 h, and then the products were collected by centrifugation, washed with ethanol several times. The procedures were repeated three times. Synthesis of HMSS–NH2 Spheres: 50 mg of HMSS spheres, 25 mL of ethanol, 25 μL of water and 25 μL of 3-aminopropyltrimethoxysilane (APTES) were mixed and heated to 45 °C for 8 h. The product was separated by centrifugation, washed with ethanol and finally re-dispersed in ethanol for subsequent use. Synthesis of Hexagonal Pd Nanosheets: 50 mg of Pd(acac)2, 160 mg of PVP, and 161 mg of TBAB were dissolved in 10 mL of DMF (N,N-dimethylformamide) and then 2 mL of water was added to the mixture. The resulting homogeneous yellow solution was transferred to a glass pressure vessel. The vessel was then charged with CO to 1 bar and heated at 100 °C for 3 h. The obtained dark blue solution was stored at 4 °C for further use. Preparation of HMSS–[email protected]: First, 5 mg of HMSS–NH2 spheres were dispersed in 1.0 mL of dry DMF and then reacted with 0.2 mg of FITC. The reaction was allowed to stir for 12 h under dark conditions. The product HMSS-NH2-FITC nanoparticles were collected by centrifugation and washed with ethanol at least 5 times. Then, 2.0 mg of HMSS–NH2-FITC nanoparticles were added to 1.0 mL of ultra-pure water containing 175.2 μg of Pd nanosheets. After 30 min of stirring, the resultant products were collected by centrifugation, washed with water and re-dispersed in PBS solution for subsequent use. DOX Loaded and In vitro Release: 2.5 mg of HMSS–NH2 nanoparticles were mixed with 0.25 mg of DOX in 500.0 μL of ultra-pure water, and stirred under dark conditions for 24 h. Then 120 μL of water containing 175.2 μg of Pd nanosheets were added. After 30 min of stirring, the resulting HMSS–NH2/[email protected] nanoparticles were collected by centrifugation and re-dispersed in PBS solution for subsequent use. To evaluate the DOX loading capacity, the supernatant solutions containing DOX molecules were measured by the fluorescence spectrum of DOX (ex = 500 nm). The loading capacity of the nanoparticles was determined as the percentage of the weight of DOX related to the weight of HMSS–NH2 nanoparticles. In vitro DOX release from the hollow mesoporous nanoparticles were performed in PBS buffer at pH values of 7.4, 6.0, 5.0, 4.0. For each release study, 250 μg of DOX loaded nanoparticles were dispersed in 100 μL of PBS buffer and shaken at room temperature. At selected time intervals, the sample was collected, and 100 μL of solution was removed. Then, 100 μL of fresh PBS buffer was added. The solution removed was properly diluted and the amount of DOX molecules present was measured by the fluorescence spectrum. To confirm that the drug could release increased from the nanoparticles under laser irradiation, 1.0 mg of DOX

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loaded nanoparticles were dispersed in 400 μL of PBS buffer at pH values of 7.4 or 5.0, and then repeatedly illuminated by laser lamp (λ = 808 nm, 1 W/cm2) over a period of 5 min, followed by the predetermined time intervals. At various times, the sample was collected, and 400 μL of solution was removed. Then, 400 μL of fresh PBS buffer was added. The released free DOX was quantified by the fluorescence. Characterization: Transmission electron microscopy (TEM) studies were performed on a TECNAI F-30 high resolution transmission electron microscopy operating at 300 kV. Scanning electron microscopy (SEM) images were obtained on a Hitachi S4800 scanning electron microscope with a field emission electron gun. Surface areas and pore sizes were determined by a Micromeritics ASAP2020 automated sorption analyzer. Fluorescence images were recorded on a Nikon eclipse Ti-U fluorescence microscope. Cell Culture: Human hepatoblastoma cells (Hep G2) were maintained in Duibecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, 1% penicillin, 1% streptomycin in 37 °C, 5% CO2. Cell Uptake Studies: Hep G2 cells were seeded in a 24-well plate (∼0.5 × 105 cells per well) and cultured for 24 h. The cell medium was removed, and then cells were incubated with 0.5 mL of fresh cell medium containing the Pd nanosheets or HMSS–NH2– [email protected] nanoparticles for another 12 h. After removal the cells medium, the cells were washed with PBS for several times, and then were trypsinized and centrifuged. Followed, the cells were digested by aqua regia and diluted by ultrapure water at proper concentrations for the ICP analysis. Fluorescence Imaging Analysis: Hep G2 cells were seeded in a 24-well plate as described above. After incubated with the HMSS–NH2–[email protected] nanoparticles for 10 hrs, the cell medium was removed. Cell imaging was then carried out by fluorescence microscope after washing cells with PBS. Confocal Fluorescence Imaging Analysis: For the confocal microscopy studies, Hep G2 cells were cultured on glass bottom cell culture dishes (20 mm). After the cells were then incubated with HMSS–NH2/[email protected] nanoparticles (100 μg/mL) for 6 h, the medium was replaced by 200 μL of fresh medium and irradiated by a laser lamp (λ = 808 nm, 1 W/cm2) for 5 min. DAPI was then applied to the cells for 15 min. Confocal fluorescence imaging was carried out after washing cells with PBS using a Leica TCS SP5 confocal microscope. For comparison, the confocal imaging was also performed on the Hep G2 cells with the same treatment but without NIR irradiation. In Vitro Cytotoxicity Assay: Hep G2 cells were seeded in a 96-well plate at a density 104 cells/well for 24 h to allow the cells to attach onto the surface of the wells. Then, the cells were exposed to free DOX and HMSS–NH2/[email protected] nanoparticles at desired concentration. After incubation for 10 h, cell viabilities were tested by standard MTT (3-(4,5)-dimethylthiahiazo (-zyl)-3,5-di-phenytetrazoliumromide) assay. To confirm that the HMSS–NH2/DOX-Pd could efficiently kill cancer cell under laser irradiation, the cells were incubated with HMSS–[email protected] and HMSS–NH2/[email protected] nanoparticles for 10 h. After that, the medium was replaced by 200 μL of fresh medium and irradiated by a laser lamp (λ = 808 nm, 1 W/cm2) for 5 min. Cell survival was determined using the MTT assay as described before. To identify the cell viability, the dead cells were stained with Trypan Blue.

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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements We thank the MOST of China (2011CB932403), the NSFC (21131005, 21021061, 20925103), the NSF of Fujian Province (Distinguished Young Investigator Grant 2009J06005), the Fok Ying Tung Education Foundation (121011), and NFFTBS (J1030415) for the financial support.

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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: May 3, 2012 Revised: June 22, 2012 Published online: August 20, 2012

small 2012, 8, No. 24, 3816–3822

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