Construction of multifunctional organic–inorganic hybrid Bi₂S₃–PLGA capsules for highly efficient ultrasound-guided radiosensitization of brachytherapy

Abstract

The development of nanobiotechnology provides an efficient strategy to improve the therapeutic outcome of various therapeutic modalities against cancer. In this work, we successfully constructed a novel multifunctional theranostic platform based on Bi₂S₃-embeded poly(lactic-co-glycolic acid) composite capsules (Bi₂S₃–PLGA) to realize the highly efficient ultrasound-guided synergistic brachytherapy. The as-synthesized hydrophobic Bi₂S₃ nanodots were encapsulated into PLGA capsules to form Bi₂S₃–PLGA composite capsules by a simple but efficient water/oil/water (W/O/W) emulsion approach. The composite Bi₂S₃–PLGA capsules exhibit excellent contrast-enhanced ultrasound imaging performance in the prostate tumor-bearing nude mice model, which could accurately indicate the location of a radiation source. More importantly, this research has demonstrated for the first time both in vitro and in vivo that Bi₂S₃–PLGA capsules could function as an efficient radiosensitizer for synergistic brachytherapy. Thus, the elaborately designed novel Bi₂S₃–PLGA capsules based on nano-biotechnology provides an efficient protocol for enhancing the therapeutic efficiency of ultrasound imaging-guided brachytherapy against cancer.

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1. Introduction

Brachytherapy has been widely used as an efficient therapeutic modality against prostate carcinoma due to its unique advantage of long-term and durable radiation effect by placing the radiation source directly inside the tumor.^1–4 The implanted radiation source, containing different formations, such as colloids, seeds, and containers, can deliver a significantly higher radiation dose to the prostate carcinoma with mitigated side-effects to the surrounding normal tissues compared to the traditional external radiation therapy. To increase the accuracy of radiation source placement and ensure that the tumor can receive efficient concentration and distribution of seeds throughout the prostate tumor, the in situ ultrasound imaging was generally applied, by which the catheter can be monitored to guide the implanting process.^5–7 However, the proper catheter position cannot sufficiently ensure the accurate delivery of radiation sources at the desired tumor site. Thus, the development and application of an efficient ultrasound-based contrast agents to highlight the location of radiation source is highly desirable for the brachytherapy.^8–12 In addition, the brachytherapeutic efficiency strongly depends on the radiation doses, which was generated by various implanted radiation sources. The potential radiation danger to normal tissues, however, substantially restricts the administration doses of radiation sources. It is now still the great challenge to realize the efficient brachytherapy under the low administration doses of radiation sources.

Inspired by the radiosensitization effect of heavy metal element-based nanoparticles (NPs) for external radiotherapy such as Au, Gd, Bi, et al.,^13–18 it is anticipated that these NPs can exhibit the similar radiosensitization effect for brachytherapy due to the same radiation mechanism for killing the cancer cells. However, no report has confirmed the sensitization effect of NPs in the brachytherapy because nanotechnology-based radiosensitization is still in its infancy. In this respect, bismuth-based NPs are the most preferred radiosensitization agent because bismuth (Bi) is the biocompatible metal element with much higher atomic number (Z) than traditional Au, Gd, Ag, et al.

Herein, a unique Bi₂S₃-embedded poly(lactic-co-glycolic acid) capsule (Bi₂S₃–PLGA) was developed, for the first time, to realize the concurrent contrast-enhanced ultrasound imaging and heavy metal element-induced radiosensitization of brachytherapy by a simple but efficient double emulsion protocol. Biocompatible PLGA capsules have been demonstrated as the excellent contrast agents for ultrasound imaging.^19–23 Additionally, PLGA capsule is considered as efficient carriers for the delivery of functional NPs, such as Gd₂O₃, iron oxide and gold NPs, which could realize multifunctional theranostic functions besides ultrasound contrast-enhanced imaging. Recently, Bi₂S₃ NPs have been employed as the contrast agents for computed tomography due to their satisfactory biocompatibility and large X-ray attenuation coefficient (Bi: 5.74, Au: 5.16, Pt: 4.99 and Ta: 4.3 cm² kg⁻¹ at 100 eV).^24–26 Especially, it is anticipated that Bi₂S₃ can be used as the radiation sensitizer because of their strong photoelectric adsorption and ejected short-range secondary electron after exposure to X-ray or gamma irradiation, which can enhance the DNA breakage and cause the death of tumor cells. The elaborate combination of organic PLGA capsules and inorganic Bi₂S₃ NPs can construction a unique organic–inorganic hybrid multifunctional capsule with the following structural and compositional characteristics for brachytherapy. On the one hand, the administration of Bi₂S₃-embeded PLGA capsules can function as the contrast agents for ultrasound imaging, by which the radiation sources of brachytherapy can be guided into the desired position within the tumor tissues. On the other hand, the integrated Bi₂S₃ NPs within PLGA capsule can be used as the radiation sensitizer to synergistically improve the efficiency of brachytherapy. The performances of Bi₂S₃–PLGA for ultrasound-guided brachytherapy have been systematically demonstrated both in vitro and in vivo in this work.

2. Experimental

2.1 Materials

Poly(lactic-co-glycolic acid) (50 : 50) with the molecular weight of 15 000 MW was purchased from Polyscience, USA. The bismuth neodecanoate, octadecene, oleic acid, thioacetamide and oleylamine were obtained from Sigma-Aldrich. Methylene chloride and poly(vinyl alcohol) was obtained from Sinopharm Chemical Reagent Co. Colloidal chromium phosphate (P-32) was purchased from Beijing Yuanzi High Tech. Company. All chemicals were received without further purification. Deionized water was used in the whole experiment.

2.2 Preparation of the Bi₂S₃–PLGA capsules

The Bi₂S₃ NPs were prepared based on the previous report.²⁴ The prepared Bi₂S₃ NPs were dispersed into methylene chloride at the concentration of 10 mg mL⁻¹. Furthermore, Bi₂S₃–PLGA capsules were prepared by a simple but efficient double emulsion protocol. Typically, 20 mL of the methylene chloride solution was prepared containing 5 mg of PLGA and 5 mg of Bi₂S₃ NPs (0.5 mL, 10 mg mL⁻¹), which was formed by dispersing Bi₂S₃ NPs and dissolving PLGA under ultrasound treatment. Then, 4 mL of deionized water was added dropwise, and the mixture was emulsified using an ultrasonic probe with the power of 180 W at room temperature. Sequentially, the above mixture solution was added into 100 mL of poly(vinyl alcohol) solution with the concentration of 5% w/v and homogenized for 10 min at room temperature. Finally, the methylene chloride was extracted from the emulsion by gently stirring overnight at room temperature. The obtained Bi₂S₃–PLGA capsules were collected by centrifugation and washed with deionized water for three times by repeated centrifugations.

2.3 In vivo contrast-enhanced ultrasound imaging

The mixture of 0.05 mCi P-32 enriched gelatin chromic phosphate colloid (P-32) and 100 μg of Bi₂S₃–PLGA capsules in saline solution was intratumoral administrated into the prostate tumor bearing nude mice. The ultrasound imaging was performed before and after the injection, respectively.

2.4 In vitro evaluation of the synergistic radiosensitization effect

A human prostate carcinomas cells line, PC3 cells were cultured in the RPMI 1640 medium with 10% FBS, and 1% penicilin–streptomycin. Cells were incubated at 37 °C in a humidified 5% CO₂ incubator. To evaluate the in vitro enhancement of Bi₂S₃–PLGA in P-32 radiation, PC3 cells were cultured in a 96-well plate with the density of 1 × 10⁵ per well. After incubation overnight, the medium was replaced by fresh medium with 0.03 mCi P-32 and different concentration of Bi₂S₃–PLGA nanocapsules. Then the cells were cultured for another 24 h and 48 h, respectively. The cell viabilities were evaluated by the typical MTT assay. In addition, the apoptosis levels of PC3 cells after each treatment were demonstrated by the flow cytometry analysis.

2.5 In vivo evaluation of the synergistic radiosensitization effect

The nude mice prostate carcinomas xenograft was firstly established for in vivo evaluations. The P-32 radiation treatment was performed when the tumor diameter reached about 1 cm. The prostate tumor-bearing nude mice (n = 12) were randomly divided to 4 groups (n = 3) and intratumorly received the treatment of saline solution, Bi₂S₃–PLGA capsules, P-32 radiation therapy (RT) and Bi₂S₃–PLGA capsules combined with P-32 radiation therapy, respectively. The amount of administrated Bi₂S₃–PLGA capsules is 100 μg per mouse and the radiation dose of P-32 is 0.05 mCi. The tumor volume was measured every two days. After the 24^th day treatment, the tissue section was dissected for Ki 67 immunohistochemisty analysis according to the manufacture's protocol.

2.6 Characterizations

Transmission electron microscopy (TEM) image was acquired on a JEM-2100F electron microscope operated at 200 kV. Scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM) were obtained on a field-emission Magellan 400 microscope (FEI Company). Ultrasound imaging was acquired on a VisualSonics Vevo2100, MS-400 with the parameter of B-mode and 30 MHz frequency. The corresponding average gray values of ultrasound imaging were determined by the analysis with SONOMATH-DICOM software. All the animal procedures were performed under the guidance approved by the University Animal Care and Use Committee of Nanjing Medial University.

3. Results and discussion

As shown in Fig. 1A, Bi₂S₃–PLGA composite capsules exhibit the typical spherical morphology. The shell of such composite capsule was composed of PLGA with embedded Bi₂Si₃ nanodots. The as-synthesized monodispersed Bi₂S₃ nanodots were hydrophobic (Fig. 2), which could be easily encapsulated into the hydrophobic tails of PLGA molecules during the typical double emulsion process. The well-defined spherical morphology of Bi₂S₃–PLGA capsules can be directly observed in SEM image (Fig. 1B), which shows that such capsules possess the smooth surface with the diameter in the range of 500–1000 nm. Bright-field TEM images exhibit the similar spherical morphology of Bi₂S₃–PLGA capsules, but the presence of Bi₂S₃ nanodots within PLGA capsule is hardly to distinguish. To give the further information of the distribution of Bi₂S₃ nanodots within PLGA capsules, dark-field TEM image of Bi₂S₃–PLGA composite capsules was acquired (Fig. 1D). Based on the contrast differences between Bi element of Bi₂S₃ nanodots and C element of PLGA capsule, dark-field TEM image shows that Bi₂S₃–PLGA capsules possess the typical “sesame ball” microstructure with the uniform distribution of Bi₂S₃ nanodots (much brighter dots in Fig. 1D) within the PLGA matrix. These electron microscopic characterizations give the strong evidence that Bi₂S₃ nanodots have been successfully encapsulated into PLGA capsules to achieve a unique organic–inorganic hybrid composite capsule with multifunctions for further imaging-guided brachytherapy.

Fig. 1 (A) Schematic illustration of the structural characteristics of Bi₂S₃–PLGA capsules; SEM (B), bright-field TEM (C) and dark-field TEM (D) images of Bi₂S₃–PLGA composite capsules. The scale bar of D is 1 μm.

Fig. 2 TEM image of as-synthesized hydrophobic Bi₂S₃ nanodots.

Imaging-guided brachytherapy is very important for the guidance of implanting process for radiation sources because such guidance can assist the determination of the accurate implanting sites to guarantee the high radiation efficiency and mitigate the side-effects of radiation sources. Ultrasound has been proved to function as an efficient imaging modality to achieve this goal due to its portable and operational features. PLGA capsules with varied particle sizes have demonstrated their excellent performance to enhance the contrast-enhanced ultrasound imaging performance for ultrasonography.¹⁹ To demonstrate the feasibility of Bi₂S₃–PLGA capsules as the contrast agents for ultrasound-guided brachytherapy, the ultrasound imaging of tumor-bearing mice before and after administration of Bi₂S₃–PLGA capsules was conducted. As shown in Fig. 3, the contrast-enhance ultrasound imaging of tumor tissues (Fig. 3B) shows the significantly enhanced positive ultrasound signals after the injection of Bi₂S₃–PLGA capsules compared to the initial tumor tissue (Fig. 3A). The corresponding grey value of Bi₂S₃–PLGA-treated tumor tissue shows a great increase from the initial 27.6 dB to 46.2 dB, which further gives a strong evidence of the contrast-enhanced ultrasound imaging performance and is expected to be potentially used for the ultrasound-guided brachytherapy.

Fig. 3 Ultrasound image of the tumor tissue before (A) and after (B) administration of Bi₂S₃–PLGA capsules.

It has been reported that heavy metal elements can be used as the sensitizer for synergistic radiotherapy because of its strong photoelectric absorption and generated secondary electron under the radiation exposure.^17,18 Different from most reported heavy metal element-based nanoparticles for radiotherapy, we evaluated the radiosensitization effect Bi₂S₃ nanodots within PLGA capsules in brachytherapy. Bi₂S₃–PLGA capsules were homogenously mixed with 0.03 mCi P-32 enriched gelatin chromic phosphate colloid (P-32). Then, Bi₂S₃–PLGA capsules with different concentrations were employed for in vitro assessments against PC3 cancer cells. The mixtures were added into the 96-well plate containing PC3 cells and co-incubated for 24 h and 48 h, respectively. As shown in the Fig. 4, no obvious cell toxicity was found for the Bi₂S₃–PLGA capsules groups without P-32 radiation. Comparatively, the obvious cell toxicity was found when performing P-32 radiation, and increased with concentration increment of capsules. In addition, it was also found the apoptotic percentage of PC3 cells after the combined treatment of Bi₂S₃–PLGA capsules and P-32 RT was 13.1% (Fig. 5), which is obviously higher than 8.8% of the P-32 RT group. Both the above in vitro MTT results (Fig. 4) and apoptosis data (Fig. 5) indicate the obvious sensitization effect of Bi₂S₃–PLGA in the brachytherapy.

Fig. 4 (A) The PC3 cell inhibiting rate after incubation with 0.03 mCi P-32 colloids and Bi₂S₃–PLGA capsules for 24 h and 48 h, respectively. The concentrations of Bi₂S₃–PLGA capsules are in the range of 0 to 200 μg mL⁻¹. The P-32 RT treated groups were highlighted in the figure.

Fig. 5 (A–D) The apoptosis profile of PC3 cells demonstrated by the flow cytometry analysis after treatment of saline solution (A), Bi₂S₃–PLGA capsules (B), P-32 RT (C) and Bi₂S₃–PLGA capsules combined with P-32 RT (D), respectively. The cells were stained by Annexin V-FITC/PI staining; (E) the apoptosis index (AI) of PC3 cells after treatment of saline solution, Bi₂S₃–PLGA capsules, P-32 RT and Bi₂S₃–PLGA capsules combined with P-32 RT, respectively.

To preliminary evaluate the radiosensitization effect of Bi₂S₃–PLGA capsules in vivo, nude mice bearing PC3 prostate carcinomas tumor xenograft was established for the assessment. The tumor-bearing mice were administrated with saline solution (group I), Bi₂S₃–PLGA capsules (group II), 0.05 mCi P-32 RT (group III) and Bi₂S₃–PLGA capsules combined with 0.05 mCi P-32 RT (group IV), respectively. It was found the tumor growth curves exhibited no obvious difference between the saline solution (I) and Bi₂S₃–PLGA capsules (II) group in the 24^th day (Fig. 6A), further demonstrating the negligible biological effect and high biocompatibility of fabricated composite capsules. Importantly, an obvious eradication of the tumor by about 30% reduction in the tumor volumes was recorded in the combined (IV) group, in comparison to the P-32 RT (III) group with nearly double growth, which strongly demonstrates the high synergistic therapeutic efficiency of Bi₂S₃–PLGA composite capsules for brachytherapy. To further reveal the synergistic mechanism, we analyzed the immunohistochemical results for Ki67 of tumor cells, which reflects the cell proliferation condition after each treatment. Obvious lowest expression of Ki67 in combined (IV) group can be found compared to P-32 RT (III) group, saline group (I) and Bi₂S₃–PLGA group (II) (Fig. 6B), indicating the significantly suppressed activity of tumor cells and improved synergistic efficiency after the introduction of Bi₂S₃–PLGA composite capsules combined with radiation therapy for brachytherapy. Based on the tumor growth curve and the analysis of Ki67 of tumor cells, it can be concluded that the fabricated Bi₂S₃–PLGA capsules can function as the efficient in vivo radiation sensitizer for enhancing the therapeutic efficiency of brachytherapy. In addition, the in situ intratumor administration was adopted because brachytherapy typically implants the radiation source into the tumor clinically,^1–4 which can maximally accumulate the Bi₂S₃–PLGA capsules and P-32 radiation source within the tumors. Thus, the administration dose can be much lower than traditional systemic administration, such as intravenous injection, causing the significantly reduced toxicities to mice.

Fig. 6 (A) The tumor growth curve of prostate tumor-bearing nude mice after treatment of saline solution, Bi₂S₃–PLGA capsules, P-32 RT and Bi₂S₃–PLGA capsules combined with P-32 RT; (B) the immunohistochemical staining analyses for Ki67 of tumor cells after treatment of saline solution, Bi₂S₃-PLGA capsules, P-32 RT and Bi₂S₃–PLGA capsules combined with P-32 RT, respectively.

4. Conclusions

In summary, a novel organic–inorganic hybrid Bi₂S₃–PLGA composite capsule was successfully developed for ultrasound-guided synergistic brachytherapy based on nano-synthetic chemistry and nano-biotechnology. The Bi₂S₃–PLGA capsules with high dispersity and spherical morphology exhibit excellent contrast-enhanced ultrasound-imaging performance confirmed by the in vivo imaging evaluation against mice xenograft. Importantly, due to the presence of heavy metal element bismuth, high radiosensitization effect of Bi₂S₃–PLGA capsules in the P-32 interstitial radiation therapy was confirmed by systematic in vitro and in vivo assessments. Thus, it is anticipated that the elaborately designed Bi₂S₃–PLGA capsules could be used as an efficient sensitizer in the ultrasound imaging-guided brachytherapy against cancer, which also provides an alternative approach to enhance the therapeutic efficiency of brachytherapy rather than the traditional increase of dangerous radiation doses.

Acknowledgements

The authors acknowledge the financial support from Natural Science Foundation of China (no. 81071185).

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