原位生成砒霜的生物杂化体的制备及其体内外抗肝细胞癌评价

Preparation of in situ arsenic formation biological hybrid and evaluation of its anti-hepatoma effect in vivo and in vitro

关键词:  生物杂化体  砒霜  希瓦氏菌  肝细胞癌  乳酸

Key words:  biological hybrid  arsenic  Shewanella oneidensis  hepatocellular carcinoma  lactic acid

摘要:

目的 为改善砒霜在肝细胞癌治疗过程中高毒副作用问题,制备一种原位生成砒霜的生物杂化体(CeAs-Bac),并评价其体内外抗肝细胞癌效果。方法 用静电吸附法和原位矿化法制备CeAs-Bac,并通过透射电镜(TEM)、扫描电镜(SEM)、马尔文粒径仪和X射线光电子能谱(XPS)评价其外观形貌、电位及元素组成；采用透析袋法和XPS考察CeAs-Bac的体外释放和释放前后As价态变化；以吲哚菁绿(ICG)为荧光标记物考察HepG2摄取情况；采用MTT、活/死细胞染色法和Annexin V-FITC凋亡试剂盒分别考察CeAs-Bac的细胞毒性和凋亡情况；建立H22肝癌小鼠模型考察CeAs-Bac的体内抗肿瘤效果。结果 成功制备了生物杂化体CeAs-Bac,TEM和SEM结果表明砷铈化合物紧密附着在希瓦氏菌表面,且修饰Ce和As后细菌表面电位由-32.2±2.05 mV变为-15.3±0.81 mV,砷装载效率为60.6%；体外释放显示CeAs-Bac具有乳酸响应性释放的特点,且能在乳酸存在条件下生成砒霜；细胞实验结果证明CeAs-Bac对HepG2具有较高细胞毒性,并可诱导HepG2细胞凋亡；体内抗肿瘤结果显示CeAs-Bac可有效靶向肿瘤部位,抑制肿瘤生长,并促进肿瘤组织坏死。结论 本研究成功制备了具有乳酸响应性释放及原位生成砒霜特点的生物杂化体CeAs-Bac,其能够有效降低砒霜在应用过程中的毒副作用,实现肝细胞癌的安全有效治疗。

Abstract:

ABSTRACT: OBJECTIVE In order to improve the high toxicity and side effects of arsenic in the treatment of hepatocellular carcinoma, a biological hybrid (CeAs-Bac) of arsenic in situ formation was prepared and its anti-hepatocellular carcinoma efficacy in vivo and in vitro was evaluated. METHODS CeAs-Bac was prepared by electrostatic adsorption and in situ mineralization, and its appearance, Zeta potential and element composition were evaluated by transmission electron microscope (TEM), scanning electron microscope (SEM), Malvern particle size analyzer and X-ray photoelectron spectroscopy (XPS). Dialysis bag method and XPS were used to investigate the valence changes of As before and after the release of CeAs-Bac in vitro. Indocyanine green (ICG) was used as a fluorescent marker to detect HepG2 uptake, and MTT, living / dead cell staining and AnnexinV-FITC apoptosis kit were used to detect the cytotoxicity and apoptosis of CeAs-Bac. Establishment of a mouse model of H22 hepatocellular carcinoma to investigate the anti-tumor effect of CeAs-Bac in vivo RESULTS The CeAs-Bac was successfully prepared, and CeAs were closely attached to the surface of Shewanella oneidensis, and the surface zeta potential of bacteria changed from-32.2 ±2.05 mV to-15.3 ±0.81 mV after modification of Ce and As. The As loading efficiency of Shewanella oneidensis was 60.6%. CeAs-Bac has the characteristics of responsive release of lactic acid and can produce arsenic in the presence of lactic acid. The results of cell experiment showed that CeAs-Bac had high cytotoxicity to HepG2 and could induce apoptosis of HepG2 cells. The anti-tumor results in vivo showed that CeAs-Bac could effectively target the tumor site, inhibit tumor growth and promote tumor tissue necrosis. CONCLUSION In this study, a biological hybrid CeAs-Bac with the characteristics of responsive release of lactic acid and formation of arsenic was successfully prepared, which can effectively reduce the toxic and side effects of arsenic in the application process and treat hepatocellular carcinoma safely and effectively.