Biomaterials xxx (2014) 1e9

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Bi2S3-embedded mesoporous silica nanoparticles for efficient drug delivery and interstitial radiotherapy sensitization Ming Ma a, 1, Yan Huang b, 1, Hangrong Chen a, *, Xiaoqing Jia a, Shige Wang a, Zizheng Wang b, Jianlin Shi a, * a State Key Laboratory of High Performance Ceramic and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Science, Shanghai, 200050, PR China b Department of Nuclear Medicine of Nanjing First Hospital, Nanjing Medical University, Nanjing, 210006, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 June 2014 Accepted 2 October 2014 Available online xxx

A novel design of Bi2S3 nanoparticles with a coating of mesoporous silica (BMSN) is obtained by a surfactant induced condensation method. It was found that BMSNs exhibited a high doxorubicin (DOX) loading efficiency of 45 wt% and pH-responsive controlled drug release owing to the electrostatic interaction between silanol surface and DOX molecules. The cell viability results demonstrated the encapsulation of DOX into BMSNs could lead to significantly enhanced therapeutic effect against multidrug-resistance cancer cells compared to that of free DOX drug. Furthermore, the comparable study of tumor growth by different treatments demonstrated that the introduction of BMSNs in the X-ray therapy could lead to higher therapeutic effect, with just 2.10-fold increase in tumor volume through 24 days, in comparison to 4.40-fold increase for X-ray beams treatment alone. Meanwhile, the in vitro interstitial radiotherapy experiments demonstrated that the cell inhibiting effect of P-32 interstitial radiotherapy combined with BMSNs (50 mg/mL) was 1.55-fold higher than that of P-32 alone. Significantly, it is notable that the simultaneous chemo- and interstitial radiotherapy based on BMSNs could tremendously increase the therapeutic effect compared to those treatment alone. More importantly, the in vivo P-32 radiotherapy in conjunction with BMSNs was proved to present a significantly eradication of the tumor volumes by an average of 21% reduction to its initial values, in comparison to 2.01-fold increase in case of P-32 treatment alone. Thus, it is expected that the BMSNs could be applied as a highly efficient multifunctional nanosystem to realize the enhanced chemo- and radiotherapy in the further clinical applications. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Mesoporous silica Radiotherapy sensitization Drug delivery Bi2S3

1. Introduction Since cancer has become one of the greatest threats to human health, numerous therapeutic protocols have been developed in recent years, among which radiotherapy is one of most efficient protocols used for solid tumors. The ideal radiation therapy is expected to be able to damage plenty of cancer cells, while decreasing the harm to nearby healthy tissue. To selectively increase the radioactivity deposition in the tumor region, metal nanoparticles with high atomic number (Z), have recently received wide interests for their excellent radiosensitization effect [1e5]. Their strong photoelectric absorbance capacities and the numerous short-range * Corresponding authors. Tel.: þ86 21 52412712; fax: þ86 21 52413122. E-mail addresses: [email protected] (H. Chen), [email protected] (J. Shi). 1 M. Ma and Y. Huang contributed equally to this work.

secondary electrons generated on the particle surface can accelerate the DNA break and thus kill more tumor cells when performing the radiotherapy. Meanwhile, it has been observed that nanoparticle down to 100 nm in diameter could exhibit increased tumor accumulation in the intravenous administration by virtue of the enhanced permeability and retention (EPR) effect of leaky tumor vasculature. Thus, it is believed that the nanometer sized radiosensitizer could efficiently integrate the radiosensitization effect and the enhanced tumor accumulation together, showing great potential in delivering enhanced radiation dosage to the targeted tumors. Nevertheless, previous studies mainly focused on the radiosensitization effects of particles through the external beam radiation from a machine outside the body. Few studies have provided the evidences about whether the sensitization effect is also feasible in the interstitial radiotherapy. As we known, when treating the tumor using interstitial radiotherapy, the radiation source (colloids,

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M. Ma et al. / Biomaterials xxx (2014) 1e9

small pellets, seeds and containers) should be encapsulated into the tumor, which generally causes serious side-effect to the healthy organs and even radiation harm to other persons [6e8]. Thus, it is critically important and highly desired to find an efficient nanomedicine strategy towards the enhanced radiation effect while minimizing radiation dose in the interstitial radiotherapy. Bismuth based nanoparticle has received wide attention in the field of radiotherapy research based on its remarkable radiation dose enhancement under kilovoltage X-ray beams, which is significantly higher than the well-known gold radiation sensitizer [9e11]. More recently, it has been suggested the significant radiosensitization effect of Bi2S3 nanoparticle could be successfully realized on the tumor-bearing mice model in the X-ray radiotherapy research, resulting in significantly higher inhibition effect for the tumor growth [12]. Meanwhile, as we known, the lower cost of bismuth element compared to gold could make the Bi2S3 nanoparticle a better candidate to further commercial use [13,14]. Thus, it is suggested that Bi2S3 nanoparticle is an ideal alternative to evaluate the therapeutic effect of nanomedicine based radiosensitizer in the interstitial radiotherapy research. Mesoporous silica nanoparticle (MSN) has received wide attention in recent decades, due to the excellent biocompatibility and diverse applications in the field of biomedicine [15e18]. Meanwhile, the silica coating strategy is a favorable candidate for overcoming the toxicity problem mostly existed in heavy metallic nanoparticles, since the silica shell possess the advantages of chemical stability and resistance to erosion under extreme conditions [19,20]. More importantly, MSNs have also some unique advantages such as uniform mesoporosity, high surface area and large pore volume, and thus are capable of encapsulating a high payload of anticancer drug as a drug delivery system (DDS) [21e24]. Especially, various nanoparticles, such as MnOx, gold and superparamagnetic iron oxide, have been successfully designed to create coreeshell structured DDS by mesoporous silica coating, which could efficiently combine the characteristics of nanoparticle and drug delivery property of MSNs into one nanosystem [25e27]. Unfortunately, despite significant progress in the field of MSNs based DDS, no report can be found on the successful fabrication of a multifunctional MSNs that could deliver Bi2S3-type radiosensitizer and anticancer drug simultaneously. Herein, a novel design of B2S3 based nanoparticle with a coating of mesoporous silica shell (termed as BMSN) has been developed based on a surfactant induced condensation method. This BMSN is expected to exhibit obvious advantages such as high drug loading capacity, pH-responsive release of doxorubicin (DOX) drug as well as the enhanced therapeutic effect against multidrug-resistant (MDR) cancer cells. Furthermore, in vitro and in vivo experiments were carried out to demonstrate the radiosensitization effect of BMSNs in both X-ray external beam and P-32 radionuclide interstitial radiotherapy. More importantly, our developed BMSNs could serve as a multifunctional nanosystem aiming at the efficient codelivery of Bi2S3 radiosensitizers and anticancer drugs to achieve enhanced therapeutic goal. 2. Experimental section 2.1. Chemicals The bismuth neodecanoate, oleic acid (90%), octadecene, oleylamine (98%), thioacetamide and hexadecyltrimethylammonium bromide (CTAB, 99%) were obtained from SigmaeAldrich. Tetraethyl orthosilicate (TEOS), anhydrous alcohol, methanol, sodium chloride (NaCl) and dimethylsufoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd.



mixture was heated to 165 C and then maintained at this temperature for 20 min under Ar protection. After the solution was cooled down to 100  C, 0.75 g of thioacetamide in 7 mL of oleylamine solution was quickly injected under vigorous agitation. Then, after 1 min of reaction at this temperature, the mixture was cooled to temperature. The oleic acid stabilized Bi2S3 NDs were obtained after centrifugation (20,000 rpm) and washing with ethanol (total volume: 50 mL). Finally, the  collected product was dispersed in chloroform and stored at 4 C for further use. 2.3. Synthesis of BMSNs 0.5 mL of chloroform containing B2S3 NDs was added into 5 mL of deionized water dissolving 100 mg CTAB under vigorous agitation. Then, the unsealed bottle  with above mixture was heated up to 60 C under gentle agitation to evaporate the violent chloroform. After cooled to room temperature, the B2S3 NDs suspension was sequentially added 100 mL deionized water, 3 mL NH4OH, 0.5 mL TEOS and 5 mL EtOAc under vigorous agitation. After stirring for 1 min, the mixture was kept in static condition at the same temperature for 3 h. Then, the solid products were washed for three times with a 1 wt% solution of sodium chloride (NaCl) in methanol under ultrasonication of 30 min to remove the template CTAB. After centrifugation (10,000 rpm) and washing with ethanol (total volume: 50 mL), the BMSNs were redispersed in 50 mL of deionized water and stored at 4  C for further use. 2.4. Cell culture The cells in this study containing (HK, MCF-7/ADR and PC3) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS, Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou), 100 units/ mL penicillin and 100 mg/mL streptomycin. All cells were maintained in a humidified incubator at 37  C, 5% CO2. 2.5. In vitro cytotoxicity of BMSNs In vitro cytotoxicities of BMSNs against HK-2 or MCF-7/ADR cells were evaluated based on the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays. Cells were cultured with BMSNs at different concentrations from 25 mg/mL to 200 mg/mL in the 96-well plate. After incubation for 24 h, the culture media was replaced by fresh DMEM solution containing 0.7 mg/mL MTT. The 96-well plate was placed in the incubator for another 4 h before the DMEM solution was removed. Finally, 100 mL of DMSO solution was added into each well, and the absorption intensity was recorded at 490 nm using a microplate reader (BioTek). 2.6. In vitro radiosensitization effects of BMSNs PC3 cells seeded in 96 well plates were cultured with P-32 colloid at the same radiation dose of 0.03 mCi and BMSNs at different final concentration (0, 50, 100 and 200 mg/mL) separately. Then, the cell inhibition rates at different time (24 h, 48 h and 72 h) were measured using MTT assay. 2.7. Evaluation of the DOX loading capacity 10 mg of BMSNs was added in 10 mL of PBS solution including 5 mg of DOX under vigorous agitation. The above mixture was stirred for 24 h under the dark condition before the BMSNs were collected by centrifugation. Sequentially, the BMSNs were washed with PBS solution for three times, and the supernatant DOX solution was collected to evaluate the DOX loading capacity. The DOX content in the supernate was calculated by using the UV-vis absorption peak intensity at 485 nm compared with the standard curve of DOX in PBS solution. Finally, the loading capacity was calculated by the below equation: Loading capacity (%) ¼ 100  (Total DOX  DOX in supernate)/(Total BMSNs used þ Total DOX - DOX in supernate). 2.8. In vitro drug release studies 5.0 mg of DOX loaded BMSNs was added into each dialysis membrane bag (cut off molecular weight: 3000 g/mol). Then the bag was immersed in 30 mL PBS (pH ¼ 7.4 or 5.5) and shook at the speed of 130 rpm at 37  C. At each time interval, the released DOX concentration was monitored by UV-vis spectra measurement. 2.9. Cellular confocal fluorescence imaging MCF-7/ADR cells were seeded at a density of 1  105 cells/well in a cell culture dish of CLSM and incubated for 24 h at 37  C under 5% CO2. Then, the free DOX and DOX loaded BMSNs at the same drug concentration of 20 mg/mL were added into the culture dish. After incubation for different time, the cells were washed with PBS three times, followed by the nuclei staining using DAPI solution (Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou). Finally, the fluorescence imaging was collected on an Olympus FV1000 laser-scanning microscope. 2.10. Long-term in vivo toxicity studies of BMSNs

2.2. Synthesis of B2S3 NDs B2S3 NDs were synthesized based on a well-established method with a little modification [14]. Firstly, bismuth neodecanoate (7.2 g) were mixed with 40 mL oleic acid and 80 mL octadecene in a round-bottomed flask under vigorous stirring. The

All the animal procedures were in agreement with the guidelines of the institutional Animal Care and Use Committee. Balb/c nude mice with weight about 22 g were obtained from the Nanjing First Hospital. For the biosafety evaluation, 12 nude mice were randomly divided into two groups (control and BMSNs group). The

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M. Ma et al. / Biomaterials xxx (2014) 1e9 BMSNs group was intraperitoneal injected with BMSNs. On the 28th day after injection of BMSNs, the mice were sacrificed and the whole blood was collected for biochemical characterization. The blood parameters including WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PDN, ALT, AST, BUN and Cr were measured by the JRDUN Biotechnology Co., LTD. 2.11. In vivo evaluation of the radiosensitization effect PC3 cells (5  106 cell/mL, dispersed into 0.2 mL DMEM medium) were subcutaneously injected into nude mice. The administration was carried out when the major diameter of the tumor reach about 1 cm. 18 tumor-bearing nude mice were randomly divided to six groups (n ¼ 3, each group), containing saline (I), BMSNs (II), P-32 Radiotherapy (RT) (III), BMSNs þ P-32 RT (IV), X-ray RT (V) and BMSNs þ X-ray RT (VI). The injection dosage of BMSNs in II, IV and VI group is 5 mg/kg. The radiation dose of P-32 RT and X-ray RT is 0.05 mCi and 8 Gy, respectively. The tumor volume was measured every two days for 24 days. The tissue section for apoptosis, H&E, Ki 67 immunohistochemistry and bio-TEM analysis were prepared according to the manufacturer's protocols. The proliferating index (PI) and apoptotic index (AI) were measured by evaluating the percentage of nuclei staining positive cells, which can be obtained from five randomly selected high power fields (  400 magnification).

3. Results and discussion 3.1. Materials synthesis and structure The technical route of BMSNs preparation is demonstrated in Fig. 1. The oleic acid stabilized hydrophobic B2S3 NDs of 2e3 nm in diameter were synthesized using a solvent thermal method. As shown in Fig. S1, the TEM image and XRD pattern were well consistent with the results of previous report, demonstrating the successful fabrication of B2S3 NDs [14]. However, the current NDs cannot be stable in aqueous solution owing to the hydrophobic particle surface. Herein, a typical surfactant induced condensation method was used to construct a hydrophilic mesoporous silica coating on the B2S3 NDs towards much better stability in aqueous solution [11,14]. Hydrophobic Bi2S3 NDs were transferred into aqueous phase by a chloroform evaporation process using CTAB as a stabilizing agent, followed by a solegel condensation of TEOS in basic solution containing B2S3 NDs. The extraction process using NaCl/methanol solvent was carried out to remove the template CTAB molecules and thus establish the mesopores of silica shell. The sphere shape and wormlike pore structure of silica shell can be observed from the TEM images (Fig. 2A). And the average particle size was measured to be 72.7 nm using Image J 1.40 G software (Fig. S2). Meanwhile, BMSNs dispersed in aqueous media shows a

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mean hydrodynamic diameter of 340.0 nm with a unimodal distribution as measured by the dynamic light scattering (DLS) analysis (Fig. S3). The different contrasts from STEM image (Fig. 2B) between the mesoporous shell and core regions confirms the coexistence of silica and Bi2S3 NDs, which can also be further proved by the line-scan spectrum of the energy dispersive X-ray spectroscopy (EDS, Fig. 2C and Fig. S4). The result of N2 adsorption/ desorption analysis (Fig. 2D) shows the BET surface area, average pore diameter and pore volume is 639 m2/g, 3.0 nm and 0.98 cm3/g, respectively, indicating that BMSNs are highly porous, which is expected to exhibit high encapsulation of drug molecules. 3.2. Materials biosafety In addition, BMSNs show negligible cytotoxicity against normal human renal cell HK-2 after incubation for 24 h with varied concentrations from 25 to 200 mg/mL (Fig. S5). Meanwhile, the in vivo long term toxicity of BMSNs was evaluated by intraperitoneal administrating of BMSNs saline solution into mice at dosages of 7.5 mg/kg for 28 days. Both blood chemistry and complete blood panel analysis exhibited no obvious pathological difference compared to the saline solution group (Fig. S6). All above results convincingly evidence that the BMSNs have no obvious side effect to normal tissues and show safety for biomedical application. 3.3. Drug release and MDR overcoming studies MSNs possess highly porous structure and high surface area, thus leading to various advantages in drug delivery such as high drug loading capacity and controlled drug release property [28,29]. As we expected, BMSNs exhibit high drug loading efficacy of 45 wt% for DOX drug. The characteristic absorbance of DOX was significantly decreased after drug loading process (Fig. S7), indicating a large amount of DOX molecules has been encapsulated into the BMSNs. This high drug loading efficiency may be ascribed to the electrostatic interaction between negative charged silanol groups on the particle surface and positive charged DOX molecules in aqueous solution [30e32]. Furthermore, we studied the DOX release behaviors at two representative pH values (7.4 and 5.0), which could be used to simulate the actual drug release in the neutral condition of normal tissue and weak acidic environments of

Fig. 1. (A) Technical route of BMSNs preparation and the schematic demonstration of its application in interstitial therapy.

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M. Ma et al. / Biomaterials xxx (2014) 1e9

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loaded BMSNs against MDRs of cancer cells. As clearly shown in Fig. 3C, the strong red fluorescence in the MCF-7/ADR cells demonstrated that a large amount of DOX molecules have been delivered into cells by BMSNs after co-incubation for 4 h. And the DOX molecules could be stably retained in the cytoplasm after 24 h (Fig. S8). In contrast, free DOX did not exhibit visible red fluorescence even after co-incubation for 24 h (Fig. 3B and Fig. S7), due to the over-expression of multidrug efflux pumps P-glycoprotein in the cell membrane. Furthermore, the cell viability results from MTT

tumor. As shown in Fig. 3A, the release of DOX was very slow in the neutral condition, with only 4.0% DOX releasing during the first 24 h. However, an accelerated release was observed when placed in the acidic solution (pH 5.0), and about 40% of the pre-loaded DOX was released in 24 h. Such a pH-dependent release behavior of DOX is expected to maximally prevent the drug leakage in blood stream, and increase the DOX accumulation in tumor cells. Meanwhile, the MCF-7/ADR cell was used as the model to evaluate the therapeutic effects of free DOX molecules and DOX

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assay confirmed that the encapsulation of DOX in BMSNs could exhibit significantly higher toxicity against MCF-7/ADR cell than free DOX at concentrations from 6.25 to 25.0 mg/mL (Fig. 3D). The above in vitro results indicated the encapsulation of drug into BMSNs could efficiently reversing MDR and thus enhance the therapeutic effect of chemotherapy.

mechanism of radiosensitization effect based on heavy-metal nanoparticle remain largely unexplored, the most reasonable explanation could be attributed to the large amount of free radicals generated by the low-energy electrons from the particles surface, which is severely toxic towards tumor cells in the vicinity of the nanoparticles [1].

3.4. Radiosensitization effect in external radiotherapy

3.5. In vitro study of radiosensitization effect in interstitial radiotherapy

The therapeutic effect of BMSN under X-ray irradiation was investigated using a human prostate cancer cell line PC3 cell as the model. The PC3 cells were incubated with 200 mg/mL of BMSNs and then irradiated with 6 Gy X-ray beams. Meanwhile, the group without BMSNs was also tested for comparison (Fig. 4A and B and Fig. S9). It is found that the apoptosis number of PC3 cells treated with BMSNs was significantly higher than that of saline control group. To further investigate the in vivo radiosensitization effect of BMSNs, we carried out the external X-ray radiotherapy at a dose of 8 Gy upon the tumor-bearing nude mice after intratumoral injection of BMSNs (5 mg/kg, termed as BMSNs þ X-ray RT). Meanwhile, mice administrated with saline and irradiated with the same dose of X-ray were set as control. It was observed that the BMSNs þ Xray RT produced obvious therapeutic effect, with the relative tumor volume (V/Vo) of 2.10, tremendously lower than that of X-ray treatment alone (V/Vo ¼ 4.40). Therefore, both the in vitro and in vivo results indicated the excellent radiosensitization effect of BMSNs, which is well consistent with the previous reports on Au and Ag nanoparticles [33]. Though the detailed physical

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Compared to the external radiotherapy, the interstitial therapy using radionuclides inside the tumor is more sustained and efficient. The excellent performance of BMSNs in the external radiation therapy has encouraged us to investigate whether its radiosensitization effect could be also feasible in interstitial therapy. Herein, P-32eenriched gelatine chromic phosphate colloid (abbreviated as P-32) was applied as a representative radioactive substance, which is common used in clinical application and has a more energetic beta radiation (1.71 MeV) and longer half-life (14.3 days) than other ones [34,35]. To evaluate the radiosensitization effect of BMSNs in the interstitial therapy, PC3 cells were homogenously cultured with the BMSNs at different concentrations (0, 50, 100 and 200 mg/mL) and P-32 at the same irradiation dose of 0.03 mCi (termed as BMSNs þ P-32 RT). Meanwhile, the nonirradiated cells incubated with BMSNs at same concentrations as above BMSNs þ P-32 RT groups were also tested for comparison. Cell viability after incubated with various materials for different time periods (24 h, 48 h, and 72 h) was evaluated using the STD

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MTT assay. As clearly shown in the Fig. 5A, without P-32 irradiation, BMSNs exhibit no apparent toxicity against PC3 cells in different time periods. On the contrary, the cell viability distinctively decreased after receiving only P-32 treatment, with the inhibition rate of 16.1%, 20.4% and 23.6% for 24 h, 48 h and 72 h, respectively. Moreover, it is indicated that the introduction of a very small amount of BMSNs at concentration of 50 mg/mL into the P-32 radiotherapy could lead to significantly enhanced inhibition effect, with an increase of 57% (24 h), 55% (48 h) and 57% (72 h), compared to that of only P-32 treatment. Furthermore, the flow cytometry analyses of the cells after various treatments for 24 h were evaluated to investigate the possible mechanism of anti-proliferative activity. Therein, the unaffected and apoptotic cells were distinguished using Annexin V-FITC/PI staining. The apoptotic percentage of the cells treated with only P-32 RT was measured to be 8.1%, and obviously increased to 16.5% after treated with BMSNs þ P-32 RT group (BMSNs concentration: 200 mg/mL, Fig. 5B and C and Fig. S10). The apoptotic index tremendously increased in combined treatment (BMSNs þ P-32 RT) suggested that the cell apoptosis may mainly

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contribute to the anti-proliferative effects of BMSNs in conjunction with P-32 radiation. 3.6. In vitro assessment of the combined therapeutic effect of DOX drug and radiotherapy Taking account of the unique structure of BMSNs, this developed nanosystem is expected to realize the co-delivery of Bi2S3 radiosensitizers and anticancer drug to achieve enhanced therapeutic goal. To demonstrate this assumption, cytotoxicity profiles of PC3 cells after 24 h of treatment with free DOX, DOX-loaded BMSNs, BMSNs þ P-32 RT and DOX-loaded BMSNs þ P-32 RT were studied using MTT assays. As shown in Fig. 6, no significant difference of inhibiting rate was found between DOX loaded BMSNs and free DOX formulations with the same DOX concentration (5 mg/mL), indicating the negligible influence of BMSNs on the drug effect against the non-MDR cancer cells. However, it was clearly observed that the introduction of P-32 with a radiation dosage of 0.0075 mCi could tremendously add to the cytotoxicity of DOX loaded BMSNs, with about 1.4-fold increase in cell inhibiting rate (from 16.5 % to

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Fig. 5. (A) Cytotoxicity profiles of PC3 cells after incubation with 0.03 mCi P-32 and BMSNs for 24, 48 and 72 h (aed): BMSNs at the concentrations of 0, 50, 100 and 200 mg/mL; (eeh): BMSNs (0, 50, 100 and 200 mg/mL) þ P-32 RT. (B, C) The apoptosis levels of PC3 cells at 24 h after treatment of P-32 (B) and BMSNs þ P-32 RT (C).

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39.7 %). And the cytotoxicity of DOX loaded BMSNs exhibited the positive correlation with the irradiation dosage of P-32 in the range from 0.0075 mCi to 0.03 mCi. It can be considered that the DOX drug, the gamma rays irradiated by P-32 and second electron produced from BMSNs could simultaneously take effect on the DNA and thus generated accumulative therapeutic effect on the tumor cells. 3.7. In vivo study of the radiosensitization effect in interstitial radiotherapy To further demonstrate whether the combination of BMSNs and P-32 could result in enhanced antitumor effects in vivo, the radiotherapy was performed following intratumoral injection of BMSNs and P-32 in the nude mice xenografted with PC3 cells. Therein, BMSNs was fixed at 5 mg/kg for each animal, with the same concentration as the above external in vivo X-ray radiation experiment. Meanwhile, P-32 with the dose of 0.05 mCi was applied for the irradiated groups. On the 24th day post P-32 implant, the relative tumor volumes in each group was measured to be 6.77 ± 0.83 (control group), 5.84 ± 0.57 (BMSNs), 2.01 ± 0.09 (P-32 RT) and 0.79 ± 0.04 (BMSNs þ P-32 RT), respectively (Fig. 7). The quantitative analysis revealed that BMSN alone exhibited no apparent tumor growth inhibition effect compared to that of control group. However, the P-32 radiotherapy combined with BMSNs presented an obvious eradication of the tumor by an average 21% reduction in volume, in comparison to P-32 RT alone (2.01-fold increment in tumor volume). Interestingly, the results indicated that the inhibition effect of mice treated with BMSNs þ P-32 RT was obviously higher than that of BMSNs þ X-ray RT group (V/Vo ¼ 2.10). Thus, it was considered that BMSNs þ P-32 RT exhibited the best therapeutic effect among different radiotherapy strategies, owing to the efficient combination of interstitial radiotherapy and Bi2S3 based radiosensitizer. We further confirmed the biological effect of BMSNs in the interstitial therapy by both histological and immunohistochemical methods. Upon microscopic examination on the 3rd, 13th and 24th day (the end day) after P-32 implant, tumors after combined BMSNs þ P-32 RT therapy showed significantly larger region of coagulation necrosis compared to any other groups (Fig. 8A and Fig. S11). On 24th day, it can be found that the massive coagulation

Fig. 7. (A) In vivo tumor growth inhibition curves of tumor-bearing nude mice treated with saline, BMSNs, P-32 RT and BMSNs þ P-32 RT; (B) The tumor volume at 24th day after each treatment was demonstrated by the photograph.

necrosis involving both the peripheral and central regions at the tumor site appears in the BMSNs þ P-32 RT group. Comparatively, P32 RT alone group shows numerous viable tumor cells with only partial necrosis in the central region. Furthermore, TUNEL assays also presented the obviously higher cell apoptosis after treatment with BMSNs þ P-32 RT, with the apoptotic index (0.74 ± 0.019) significantly higher than control (0.094 ± 0.023), BMSNs (0.084 ± 0.017) and P-32 RT (0.51 ± 0.032) groups, (Fig. 8B). Immunohistochemical staining of tumor section for antigen Ki67 was used to further evaluate the tumor cell proliferation (Fig. 8A and C). The expression of antigen Ki67 was positive as brown granules in the cell nucleus. As expected, tumor cells in the non-RT treated groups (control and BMSNs) exhibit obviously stronger expression of antigen Ki67 compared to the P-32 RT treated groups, while the expression of Ki67 in BMSNs þ P-32 RT group is much less than that of P-32 RT alone group. In addition, as can be found in the bio-TEM images (Fig. S12), BMSNs þ P-32 RT treated tumor cells present irreversible damages both in cell membranes and mitochondria. Comparatively, almost intact cell morphology is shown in the P-32 group. All the above results soundly demonstrate the significant radiosensitization effect of BMSNs in the P-32 interstitial therapy. 4. Conclusions A novel BMSN delivery system was successfully developed by a surfactant induced condensation method. The newly developed BMSN can be used as a high reservoir for enhanced encapsulation of DOX anticancer drug with a high loading efficiency of 45 wt%. Meanwhile, the in vitro experiments indicated that the DOX-loaded BMSNs exhibited both on-demand pH-responsive release of DOX drug and the enhanced therapeutic effect against MDR cancer cells. Significantly, BMSNs could substantially augment the therapeutic effects of interstitial P-32 radionuclide radiotherapy in the solid

Please cite this article in press as: Ma M, et al., Bi2S3-embedded mesoporous silica nanoparticles for efficient drug delivery and interstitial radiotherapy sensitization, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.001

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tumor, which has been systematically demonstrated both in vitro and in vivo. More importantly, it is confirmed by in vitro study that the simultaneous chemo- and interstitial radiotherapy based on BMSNs could tremendously increase the therapeutic effect compared to those treatment alone. Thus, the present results demonstrate that the BMSNs could be developed as a highly promising synergistic agent for both chemotherapy and interstitial radiotherapy, which open up more opportunities for future clinical cancer therapy. Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, Grant No. 2011CB707905), China National Funds for Distinguished Young Scientists (51225202), National Natural Science Foundation of China (Grant No. 51402329, 51132009), and Program of Shanghai Subject Chief Scientist (Grant No. 14XD1403800). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.10.001. References [1] Zheng Y, Hunting DJ, Ayotte P, Sanche L. Radiosensitization of DNA by gold nanoparticles irradiated with high-energy electrons. Radiat Res 2008;169: 19e27.

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Please cite this article in press as: Ma M, et al., Bi2S3-embedded mesoporous silica nanoparticles for efficient drug delivery and interstitial radiotherapy sensitization, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.001

Bi2S3-embedded mesoporous silica nanoparticles for efficient drug delivery and interstitial radiotherapy sensitization.

A novel design of Bi2S3 nanoparticles with a coating of mesoporous silica (BMSN) is obtained by a surfactant induced condensation method. It was found...
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