Tumor Targeted Albumin Coated Bismuth Sulfide Nanoparticles

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Tumor Targeted Albumin Coated Bismuth Sulfide Nanoparticles (Bi2S3) as Radiosensitizers and Carriers of Curcumin for Enhanced Chemoradiation Therapy Hamed Nosrati,† Jalil Charmi,‡ Marziyeh Salehiabar,†,^ Fatemeh Abhari,§ and Hossein Danafar*,†,∥ †

Department of pharmaceutical biomaterials, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan 45139-56111, Iran Department of Physics, Faculty of Science, University of Zanjan, Zanjan 45371-38791, Iran § Department of Medical Physics, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran ∥ Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan 45139-56111, Iran ^ Department of Organic and Biochemistry, Faculty of Chemistry, University of Tabriz, Tabriz 5166614766, Iran

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ABSTRACT: Combination therapy such as radiotherapy combined with chemotherapy has attracted excessive interest in the new cancer research area. Therefore, developing nanobiomaterials for combination of radiotherapy and chemotherapy is required for more powerful and successful cures. Because of the amazing X-ray sensitization proficiency of Bi based nanoparticles, in this work, we synthesized and used Bi2S3 as an enhancer of X-ray radiation therapy, and furthermore, Bi2S3 served as carrier of curcumin (CUR), a chemotherapy drug, for the goal of combination therapy. Additionally, we selected and conjugated folic acid (FA) as a targeting molecule for the direction of the designed system to the tumor site. After characterization of drug loaded FA conjugated Bi2S3@BSA nanoparticles (Bi2S3@BSA-FA-CUR) and in vitro and in vivo safety assessment, we applied it for enhanced chemotherapy and X-ray radiation therapy in cancer cells and a tumor bearing mice model. Moreover, the CT contrast ability of synthesized nanoparticles was examined. Here, we (1) for the first time developed the novel and targeted CUR loaded Bi2S3@BSA (Bi2S3@BSA-FA-CUR) to promote chemoradiation therapy in 4T1 cells and breast tumor in mice; (2) found the synthesized nanoparticles to have good stability; (3) injected a single dose of the designed radiosensitizer for cancer therapy; and (4) used a conventional X-ray dose, 2Gy, for X-ray radiation therapy. The result of in vivo X-ray radiotherapy shows that the mice tumors vanished near 3 weeks after radiation. Interestingly, these results show that Bi2S3@BSA-FA-CUR with the aid of X-ray can clearly promote the efficacy of chemoradiation therapy. KEYWORDS: X-ray sensitization, albumin, curcumin, chemoradiation, CT imaging, bismuth sulfide

1. INTRODUCTION Surgery, chemotherapy, and radiotherapy are the treatment methods for breast cancer. In terms of a single therapy, these treatments are not each time successful. Therefore, such treatments as radiotherapy combined with chemotherapy have attracted excessive interest in new cancer research works and clinical practice.1,2 Furthermore, to solve these issues, numerous types of nanobiomaterials have been developed with multiple applications, such as probing and therapeutic.3 For chemotherapy we can use nanobiomaterials for delivery of natural and synthetic chemotherapeutic agents. Also, the scientist can add the targeting agent on the surface of © XXXX American Chemical Society

nanoparticles to direct chemotherapeutic drugs to the tumor site.4 For radiotherapy, remarkable development has been done to enhance the therapeutic effect of X-ray therapy.5,6 For enhancing the disruptive effect of radiotherapy, scientists use radiosensitizers. In this case, high atomic number (Z) elements, such as Au and bismuth, are used as radiosensitizers.7,8 Researchers encounter side effects on normal tissues and hypoxia-resistance challenges in radiotherapy.9 So, Received: April 9, 2019 Accepted: August 7, 2019

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DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. (a) Schematic illustration of the synthesis process of Bi2S3@BSA-FA-CUR, (b) TEM image of Bi2S3@BSA, (c) TEM image of Bi2S3@ BSA-FA, (d) TEM image of Bi2S3@BSA-FA-CUR, (e) size monitoring to screen stability of Bi2S3@BSA and Bi2S3@BSA-FA-CUR, (f) UV−vis spectra of FA, CUR, Bi2S3@BSA, and Bi2S3@BSA-FA-CUR, and (g) XRD pattern of Bi2S3@BSA.

the present work. Additionally, we select and conjugated folic acid (FA) as a targeting molecule for direction of the designed system to the tumor site. After characterization of drug loaded FA conjugated Bi2S3@ BSA nanoparticles (Bi2S3@BSA-FA-CUR), we applied them for enhanced chemotherapy and X-ray radiation therapy. After assessing the in vitro anticancer effect, we evaluated the antitumor effects of this combined therapy scheme with twoin-one type nanoparticles (radiosensitizing and chemotherapeutic agents in one system) on 4T1 tumor beard mice. Moreover, the CT contrast ability of as synthesized nanoparticles were examined by CT imaging.

the development of nanobiomaterials for combined radiotherapy and chemotherapy is a requirement for more powerful and successful cures. These nanobiomaterials enable both carrying the drugs to the target site and also use as a radiosensitizer, decreasing therapeutic doses, reducing side effects, and, at the same time, increasing therapy efficacy.10 Currently, design and synthesis of high-Z number containing nanobiomaterials for radiotherapy have attracted extensive interest. Among the high-Z number atoms, bismuth based nanobiomaterials have widespread appeal.11−13 Bismuth derivatives including, MnSe@Bi2Se3 nanoparticles,14 Bi2S3,15 Bi2Se3,16 and Cu3BiS3,17 recently applied to cancer therapy and imaging. Bismuth is an inexpensive and biocompatible heavy metal element. To date, bismuth subcitrate has been used for curing gastrointestinal sicknesses in clinical settings.18 Though, based on our knowledge, there are no reports about its combination therapy especially with natural products. Also, bismuth, which has a high atomic number, is used for X-ray computed tomography (CT) imaging due to its large X-ray attenuation coefficient (bismuth: 5.74 keV).19,20 In the present report, to improve the stability of bismuth nanoparticles, we used bovine serum albumin (BSA) because of its high chemical stability, nontoxicity, and biodegradability. Moreover, a long half-life is one of the other unique properties of BSA.21 In fact, BSA, acting as a stabilizer, also functioned as a sulfur precursor for forming Bi2S3 nanoparticles.15 Because of their amazing X-ray sensitization proficiency, these Bi2S3 nanoparticles can work as enhancers of X-ray radiation therapy. Furthermore, these Bi2S3 nanoparticles can be serve as carriers of chemotherapy drugs for combination therapy. Curcumin (diferuloyl methane) not only has antioxidative and anticancer properties22,23 but also has radioprotective and radiosensitizing activities.24 For this reason, CUR was used in

2. MATERIALS AND METHODS 2.1. Materials. BSA, Bi (NO3)3, FA, N-(3-(dimethylamino)propyl)-N0-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy succinimide (NHS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and CUR were purchased from SigmaAldrich Chemicals, (St. Louis, MO, USA). All other solvents were purchased from Emertat Chimi Company (Tehran, Iran). The chemicals were used without any purification. 2.2. Preparation of BSA Coated Bismuth Sulfide Nanoparticles (Bi2S3@BSA). An 8 mL portion of BSA solution with 31.25 mg mL−1 concentration was prepared. A 50 mM portion of Bi(NO3)3 was dissolved in 1 mL nitric acid with a concentration of 2 M. Next, under magnetic stirring the prepared bismuth nitrate solution was slowly added to the BSA solution. After formation of the complex with Bi and BSA, to biomineralize BSA and form Bi2S3, NaOH was added to the above solution. The stirring continued for 12 h. After the biomineralization process was complete and the prepared BSA stabilized Bi2S3, the colorless solution was changed to black. The resulting BSA coated Bi2S3 was purified by the dialysis against water for 48 h and noted as the Bi2S3@BSA. This BSA biomineralization for facile synthesize of BSA coated Bi2S3 was previously reported by Wang et al.15 A schematic illustration of BSA biomineralization for synthesis of Bi2S3@BSA is shown in Figure 1a. B

DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering 2.3. Covalent Bonding of FA on the Surface of Bi2S3@BSA (Bi2S3@BSA-FA). For covalently bonding of FA on the surface of Bi2S3@BSA, first the carboxylic group of FA was activated by EDC and NHS and, second, reacted with the amine group of BSA on the surface of Bi2S3@BSA. Third, Bi2S3@BSA-FA was purified by a dialysis process (12 kDa). A 1 mL portion of FA in alkaline DMSO with a concentration of 10 mg mL−1 was activated with 18 mg EDC and 2.4 mg NHS. This prepared solution was slowly added to the 10 mL Bi2S3@BSA solution with a 10 mg mL−1 concentration. Again, for preparation of the alkaline medium, NaOH (3 M) was added to the solution. The reaction was protected from light and stirred for 24 h and, next, purified by dialysis against water. 2.4. Loading of CUR on Bi2S3@BSA-FA (Bi2S3@BSA-FA-CUR). For physically attachment of CUR on Bi2S3@BSA-FA, 2.5 mg CUR in 200 μL acetone was slowly added to the 10 mg Bi2S3@BSA-FA in 2.8 mL deionized water. The mixture was stirred overnight by hot plate in dark conditions. In next step, after loading of CUR, the Bi2S3@BSAFA-CUR was centrifuged at 18 000 rpm and consequently washed with H2O. 2.5. Fluorescein Isothiocyanate (FITC) Labeled Nanoparticles. Bi2S3@BSA, Bi2S3@BSA-FA, and Bi2S3@BSA-FA-CUR were labeled with FITC in order to study transfection efficiency. The FITC (in ethanol) was added to each sample solution with a labeling ratio of 10:1 (sample: FITC) for 24 h. After finishing the reaction time, the reaction media were dialyzed against deionized water for 2 days to remove excess unlabeled FITC. It is noteworthy that the materials were protected from light in both the labeling and dialysis process by an aluminum foil covering. 2.6. Characterization. The Fourier transform infrared (FTIR) (Bruker, Tensor 27), UV−visible (UV−vis) (T80), transmission electron microscopy (TEM) (Cambridge 360-1990 Stereo Scan Instrument-EDS), X-ray diffraction (XRD) (Bruker AXS model D8 Advance diffractometer), and dynamic light scattering (DLS) (Malvern Instruments, Worcestershire, UK, model Nano ZS) techniques were used for structural, chemical, and physical characterization of synthesized nanoparticles and materials. 2.7. Determination of the Conjugated Folate. The covalently bonded FA was identified by a spectrophotometric study at 365 nm. A 3 mg portion of Bi2S3@BSA-FA was dispersed in phosphate buffer saline (PBS) in the presence of 3 mg proteinase K enzyme. The vials incubated 24 h in 37 °C. Then, after centrifuging the absorbance of supernatant (released FA) was read by a UV−vis spectrophotometer. 2.8. Determination of the Loaded CUR. The physically loaded CUR was determined by a spectrophotometric study at 428 nm. Three mg Bi2S3@BSA-FA-CUR was dispersed in 1.6 mL acetone. The vials incubated 24 h in 37 °C. Then, after centrifuging the absorbance of supernatant (released CUR) was read by UV−vis spectrophotometer. 2.9. CUR Release Study. The CUR release behavior was studied by dialysis process. A 3 mg Bi2S3@BSA-FA-CUR was dispersed in PBS with 2% tween 80 and infused to the dialysis bag, prior to immersing in 40 mL. The vials were incubated in 37 °C with gentle shaking. Then, in predetermined time intervals, the samples were taken from dialysate, and then the absorbance of released CUR was read by UV−vis spectrophotometer and finally returned back to the vials. 2.10. Hemolysis Assessment. Hemolysis assay was performed by previously reported protocol.25 2.11. In Vivo Safety Assessment. Female BALB/c mice with ∼18 g weight were purchased from Pasteur Institute (Tehran, Iran) from and served under protocols approved by the National Institute for Medical Research Development of Islamic Republic of Iran. BALB/c mice were intravenously injected with Bi2S3@BSA solution in various concentration range (200, 300, 400, and 500 mg/kg). Enough repeats were done for each dose. The mortality of injected mice were monitored for 1 week. 2.12. In Vitro CT Imaging. For evaluation of contrast effect of Bi2S3@BSA in CT imaging, CT signal intensities in the region of interest (ROI) were measured. CT imaging was performed with a U-

SPECT+/CT imaging system (MILABS, Netherlands) with two various parameters as follows: full angle, 3 frame averaging, 126 and 53 mA tube current, and 120 and 90 kV tube voltage. The CT contrast, Hounsfield units (HU), was calculated as follows: HU = signal intensities of Bi 2S3@BSA − signal intensities of water 2.13. Cell Cytotoxicity. The mouse breast carcinoma cell line (4T1) (1 × 105 cells per well) was seeded into 96-well plates in RPMI-1640 culture medium with 10% fetal bovine serum (FBS). For attaching, the cells were incubated for 24 h. The medium was separated, and then, the attached cells further incubated with free medium (as control) and the medium containing Bi2S3@BSA, Bi2S3@ BSA-FA, and Bi2S3@BSA-FA-CUR at different concentrations (25, 50, and 100 μg/mL). After 6 h, the nanoparticle containing medium was removed, cells were washed with PBS, and finally the fresh medium was added to the wells. These cells were then irradiated by Xray radiation at doses of 2 and 6 Gy. The irradiated cells were further incubated for 12 h. The cell medium was replaced with fresh medium containing 20 μL of MTT solution (2.5 mg/mL). The incubation was continued further for 4 h. After that, 100 μL of DMSO was added to the wells. Finally, absorbance of solubilized formazan was read at 570 by a micro plate reader. The same method was repeated without X-ray radiation for comparison by irradiated groups. 2.14. Transfection Efficiency Study. Bi2S3@BSA, Bi2S3@BSAFA, and Bi2S3@BSA-FA-CUR were labeled with FITC in order to study transfection efficiency. The 4T1 cells at a density of 1.5 × 106 cells/well were seeded in 24-well plates. After incubating within 24 h to increasing confluences to 80%, the cells were transfected using FITC labeled samples with concentrations of 50 μg mL−1. Following incubation at 37 °C in a 5% CO2 incubator for a 6 h, the transfected cells were harvested, washed, and scanned by a flow cytometer (BD Biosciences, San Jose, CA). 2.15. Chemoradiotherapy in the 4T1 Tumor-Bearing Mouse Model. Female BALB/c mice with ∼18 g weight were purchased from Pasteur Institute (Tehran, Iran) and treated under protocols approved by the National Institute for Medical Research Development of Islamic Republic of Iran. 4T1 tumors were produced by subcutaneously injecting 1 × 106 4T1 cell suspensions in the right flanks of mice. The tumor-bearing mice were randomly divided into six groups (each group included five mice) and intravenously injected via tail vein with PBS (as control), PBS with X-ray radiation, CUR, CUR with X-ray radiation, Bi2S3@BSA-FA-CUR, and Bi2S3@BSA-FA-CUR with X-ray radiation. After 14 days, when the tumor volume reached around 100 mm3, each group was injected intravenously with 125 μL PBS (as control), 125 μL PBS with X-ray radiation, 125 μL CUR, 125 μL CUR with Xray radiation, 125 μL Bi2S3@BSA-FA-CUR (6 mg/mL), and 125 μL Bi2S3@BSA-FA-CUR (6 mg/mL) with X-ray radiation. The groups with X-ray radiation were irradiated 24 h post injection. The conventional 2 Gy radiation dose was used to treat mice. During treatment, the tumor volume and body weight of mice were documented.

3. RESULTS AND DISCUSSION 3.1. Synthesis of Drug Loaded Radiosensitive Nanoparticles. Bi2S3@BSA was produced through the biomineralization process at room temperature using BSA as both a sulfur precursor and coating agent. Moreover, the BSA coating can increase the circulation half-life of in vivo administrated nanoparticles. Several residues (for example 35 cysteine residues) were denatured under alkaline conditions, to form the sulfur source.26,27 At first, FA functionalized Bi2S3@BSA (Bi2S3@BSA-FA) was prepared by activation of the carboxylic acid group of FA with EDC and NHS and next preceded by reaction of this activated FA with amine groups of Bi2S3@BSA C

DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 2. FT-IR spectra of (a) BSA, (b) Bi2S3@BSA, (c) FA, (d) Bi2S3@BSA-FA, (e) Bi2S3@BSA-FA-CUR, and (f) CUR.

Figure 3. (a) Release profile of Bi2S3@BSA-FA-CUR, (b) survival curve and mortality monitoring of injected mice in various concentration ranges, (c) in vitro CT images in full angle, three frame averaging, 53 mA tube current, and 90 kV tube voltage parameters, and (d) in vitro CT images in full angle, three frame averaging, 126 mA tube current, and 120 kV tube voltage parameters.

conjugated silica-modified gold nanorods as targeted CT imaging and also radiation therapy.30 Second, for combination chemotherapy and radio therapy the CUR was loaded on Bi2S3@BSA-FA (Bi2S3@BSA-FACUR). The amount of loaded CUR was determined by UV−

surface. The amount of conjugated FA was determined by UV−vis spectrophotometry analysis and calculated to be 0.9 ± 0.31 mg FA per 100 mg Bi2S3@BSA-FA. The FA conjugate was used to target delivery of the synthesized nanoparticles to the tumor site.28,29 For example, Huang et al. reported FA D

DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 4. (a) Transfection rates of Bi2S3@BSA, Bi2S3@BSA-FA, and Bi2S3@BSA-FA-CUR analyzed by flow cytometry, in vitro enhanced X-ray radiotherapy by the designed radiosensitizer at (b) 0, (c) 25, (d) 50, and (e) 100 μg/mL concentrations on the 4T1 cell line. * indicates significant difference with P < 0.05, ** indicates significant difference with P < 0.01, *** indicates significant difference with P < 0.001, **** indicates significant difference with P < 0.0001.

Furthermore, the hyperchromic effect at 420 nm can be related to loading of CUR. Although the X-ray diffraction peaks are closely matched with reported pattern in literature structure (JCPDS no. 431471) (Figure 1g).15 Furthermore, to confirm the modification of nanoparticles a step by step FTIR technique was used. The BSA FTIR spectrum (Figure 2a) showed characteristic peaks at 1661 cm −1 and 1535 cm −1. After denaturation of BSA and giving the −SH for preparation of Bi2S3 the capping was proven by observation of BSA absorptions in the Bi2S3@BSA FTIR spectrum (Figure 2b). Also, shifting of BSA peaks to 1639 and 1547 cm −1 prove the interaction of BSA with Bi2S3 and further formation of Bi2S3@BSA. After conjugating FA to the surface of Bi2S3@BSA, we can see the shift of 1639 and 1547 cm −1 to 1650 and 1539 cm −1 in the spectrum of Bi2S3@BSA-FA (Figure 2d), and moreover, we can see a few characteristic FA bands between 950 and 1082 cm−1. Also, after loading of CUR obviously we can see the presence of characteristic peaks of CUR in the FTIR spectrum of Bi2S3@BSA-FA-CUR (Figure 2e). For example, a new peak at 1513 cm−1 in the FTIR spectrum of Bi2S3@BSA-FA-CUR is related to the CUR peak at 1501 cm−1 that has a red shift after loading. We can see in Figure 3a that CUR can be released with sustained release behavior. This release behavior can be useful for cancer therapy. The released CUR in way to reach the tumor site may be protect the normal tissues and released CUR in tumor site can be kill the cancer cells by down regulation of TNF-α and NF-κβ.24

vis spectrophotometry analysis at 428 nm and drug loading percentage calculated about 10 ± 1.51%. 3.2. Characterization. The hydrodynamic size distributions of Bi2S3@BSA and Bi2S3@BSA-FA-CUR were documented by DLS. The DLS result demonstrates that the average hydrodynamic diameter of Bi2S3@BSA was 78.9 nm. In comparison, the average hydrodynamic diameter related to Bi2S3@BSA-FA-CUR was 170.9 nm. Also, the zeta potential Bi2S3@BSA-FA-CUR was about −23.2 mV. The synthesized nanoparticle shows good stability. The stored synthesized nanoparticles do not show any precipitate. Also, for further stability evaluation of synthesized nanoparticles the size distribution of these nanoparticles was monitored by DLS for 2 months (Figure 1e). The monitored size distribution shows that Bi2S3@BSA-FA-CUR does not exhibit any increase in the diameter size. The synthesized Bi2S3@BSA, Bi2S3@BSAFA, and Bi2S3@BSA-FA-CUR have uniform size distributions and morphologies. The TEM images of Bi2S3@BSA, Bi2S3@ BSA-FA, and Bi2S3@BSA-FA-CUR are shown in Figure 1b, c, and d. The images prove that the shape and monodispersity of particles did not change in the modification process. To confirm the presence of FA and CUR on the surface of as synthesized Bi2S3@BSA-FA-CUR, the UV−vis spectrophotometer was used. The UV−vis spectra of Bi2S3@BSA, FA, CUR, and Bi2S3@BSA-FA-CUR are shown in Figure 1f. As we can see, the FA related absorbance peaks at Bi2S3@BSA-FA have a blue shift, in comparison with the free FA spectrum. Also, the absorbance at 352 nm due to FA was expanded in the Bi2S3@BSA-FA-CUR spectrum. These changes confirmed the existence of FA in the structure of Bi2S3@BSA-FA-CUR. E

DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 5. (a) Changes of relative tumor volume, (b) body weight, and (c) percent survival of six groups after various treatments. **** indicates significant difference with P < 0.0001 compared to Bi2S3@BSA-FA-CUR + X-ray.

3.3. Safety Studies. The safety of designed radiosensitizers are important values for in vivo and clinical applications. A hemolytic activity test was used for determination of the blood safety of the designed radiosensitizer. The hemolytic value was determined to be less than 3.8%. An LD 50 test was used for the evaluation of biosafety. BALB/c mice were intravenously injected with Bi2S3@BSA solution in various concentration ranges (200, 300, 400, and 500 mg/kg). Enough repeats were done for each dose. The mortality of injected mice were monitored for 1 week. The survival curve in Figure 3b shows the mortality of injected mice in various concentration ranges. At the concentrations of 200, 300, and 400 mg/kg, we did not see any death in injected mice. Whereas, at the

concentration of 500 mg/kg, one of the mice was alive within 1 week. One of the other mice that was injected with the 500 mg/kg concentration died overnight. Also, one of the other mice in this group died 48 h after injection. This result obviously shows that intravenous injection of Bi2S3@BSA up to 400 mg/kg did not show any toxicity. Wang et al. evaluated complete blood, hematological and biochemical markers, and liver injury markers and reported that Bi2S3@BSA exhibited no long-term systemic effects in rats.15 In other studies, the authors reported the synthesis of oleic acid coated Bi2S3 nanoparticles with admirable biocompatibility. The nanoparticles exhibited long blood-circulation times and also no damage to organ tissue.31 F

DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering 3.4. CT Imaging Contrast Efficacy. The use of bismuth as a contrast agent of CT imaging recently has attracted more attention because of the high X-ray attenuation coefficient of bismuth (5.74 cm2 kg−1 at 100 keV).32 To measure the CT contrast efficacy, we compared HU at two different CT parameters. The CT images of aqueous dispersions of Bi2S3@ BSA at two different CT parameters with a series of concentrations and water as the control were shown in Figure 3c and d. The HU increased linearly with increase in the concentration of Bi.31 We found that higher tube current and tube voltage can produce higher contrast. In other words, increasing the tube current and tube voltage produced higher contrast. This result shows that these types of nanoparticles may be of use in clinical settings as CT contrast agents.33 3.5. In Vitro Enhanced X-ray Radiotherapy. Before evaluation of in vitro enhanced X-ray radiotherapy, we investigated in vitro uptake analysis. The result is shown in Figure 4a. FA-conjugated particles were uptaken via folate receptormediated endocytosis into cells.34 FA conjugation significantly increased nanoparticle uptake. For this reason, the transfection efficacy of both Bi2S3@BSA-FA and Bi2S3@BSA-FA-CUR is more than Bi2S3@BSA. This result shows that conjugation of FA can increase uptake of nanoparticles into cancer cells. To examine the in vitro enhanced X-ray radiotherapy by the designed radiosensitizer, the MTT assay was done (Figure 4). The inhibitory effect of Bi2S3@BSA on 4T1 cells showed that this designed radiosensitizer, Bi2S3@BSA, exhibited no significant toxicity. After loading of CUR, the inhibitory effect was increased, but this difference is not significant. The cells that were not treated with the designed radiosensitizer were irradiated with X-rays at 2 and 6 Gy. The result showed above 85% cell viability at the radiation dose of 2 Gy. The result showed no significant difference between not irradiated and irradiated groups. The inhibitory effect increased at the radiation dose of 6 Gy. Overall, the MTT assay result showed that the cell viability reduced after radiation. The cells irradiated with X-rays at a dose of 6 Gy showed significant difference between the cells that were not irradiated. To assess the radiosensitizer and enhanced X-ray radiotherapy ability of Bi2S3@BSA, Bi2S3@BSA-FA, and Bi2S3@ BSA-FA-CUR, the cells were irradiated with X-rays at 2 and 6 Gy. The cells after treatment with Bi2S3@BSA, Bi2S3@BSA-FA, and Bi2S3@BSA-FA-CUR were irradiated with X-rays at 2 and 6 Gy. Interestingly, the result showed that all treated groups with Bi2S3@BSA, Bi2S3@BSA-FA, and Bi2S3@BSA-FA-CUR after radiation at different doses (2 and 6 Gy) showed significant difference with the not irradiated groups. X-ray radiation of bismuth nanoparticles could generate secondary electrons and Auger electrons, which can ionize water molecules to produce reactive oxygen species (ROS) including hydroxyl radicals, resulting in DNA damage in cancer cells.35 3.6. In vivo Enhanced X-ray Radiotherapy. Recently, many efforts for optimization of radiosensitization and combinatorial therapeutic planes for increased therapeutic efficacy with a harnessing of nanotechnology power have been evaluated.36,37 After examining the in vitro enhanced X-ray radiotherapy by designed radiosensitizer, in vivo enhanced X-ray radiotherapy was evaluated in tumor bearing mice. The conventional 2 Gy

radiation dose was used to treat mice. The mice were irradiated 24 h postinjection. The tumor-bearing mice were randomly divided into six groups (each group included five mice) and intravenously injected via a tail vein with PBS (as a control), PBS with X-ray radiation, CUR, CUR with X-ray radiation, Bi2S3@BSA-FACUR, and Bi2S3@BSA-FA-CUR with X-ray radiation. The relative tumor growth is shown in Figure 5a. In vivo chemoradiotherapy results show that the X-ray conventional dose, 2 Gy, does not show any treatment effect. Also, in treatment dose the CUR and CUR with X-ray radiation groups show an increase in tumor volume. The results show that Bi2S3@BSA-FA-CUR does not show any treatment effect. Nevertheless, the treatment effect becomes obvious with the aid of X-ray. Mice that are injected with Bi2S3@BSA-FA-CUR and follow irradiation with X-ray show comparable treatment effects against other groups. The results indicated that PBS (as control), PBS with X-ray radiation, CUR, CUR with X-ray radiation, and Bi2S3@BSA-FA-CUR did not inhibit tumor growth. Interestingly, these results showed that the Bi2S3@ BSA-FA-CUR with the aid of X-ray can be clearly promote the efficacy of chemoradiation therapy. During the treatment time body weight of mice were monitored. Observations show that the body weight of all groups have an inconspicuous difference during the experiment (Figure 5b). These data reveal that Bi2S3@BSA have negligible systemic toxicity. The survival percentages were shown in Figure 5c. In outstanding contrast, all of the five mice in the Bi2S3@BSA-FACUR + X-ray groups were alive up to 60 days.

4. CONCLUSION In this study, for first time we developed novel and targeted CUR loaded Bi2S3@BSA (Bi2S3@BSA-FA-CUR) to promote chemoradiation therapy in 4T1 cells and breast tumor in mice. In the present report, to improve the stability of bismuth nanoparticles, we used BSA because of their high chemical stability, nontoxicity, and biodegradability. The synthesized nanoparticle could keep colloidal stability. The stored synthesized nanoparticles do not show any precipitate. Also, for further stability evaluation of synthesized nanoparticles, the size distribution of these nanoparticles were monitored by DLS for 2 month. Bi2S3@BSA-FA-CUR can inhibit tumor growth and enhance survived ratios in vivo. In addition, Bi2S3@BSA had excellent contrast in CT images. In summary, Bi2S3@BSAFA-CUR would be a promising theranostic agent for cancer therapy.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hamed Nosrati: 0000-0002-7487-8188 Hossein Danafar: 0000-0001-8956-7895 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Institute for Medical Research Development (Grant No. 971457). G

DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.9b00489 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX