二氧化锰纳米颗粒负载槲皮素对乳腺癌细胞的放射增敏作用研究

Radiosensitizing effect of quercetin-encapsulated manganese dioxide nanoparticles on breast cancer cells

目的探究二氧化锰纳米颗粒负载槲皮素[Mn(QU)]对乳腺癌4T1细胞及移植瘤小鼠的放射增敏作用。方法用油酸模板法合成二氧化锰(MnO_(2))纳米颗粒,采用透射电子显微镜、扫描电镜和能量色散X射线光谱仪表征合成的MnO_(2)纳米颗粒的形貌和化学组分。进一步用物理吸附法负载槲皮素(quercetin,QU),通过傅里叶变换红外光谱仪和紫外分光光度计进行成分表征,并利用溶氧仪检测Mn(QU)在不同pH与不同浓度过氧化氢反应生成氧气的能力;利用CCK-8法检测不同浓度Mn(QU)对小鼠乳腺癌4T1细胞活性的影响;克隆形成、γ-H2AX荧光染色、ROS荧光染色、活/死细胞荧光染色和流式细胞术检测Mn(QU)联合放疗对4T1细胞的放射增敏作用以及对细胞凋亡的促进作用。最后,构建小鼠乳腺癌4T1细胞移植瘤模型评价Mn(QU)联合放疗抑制小鼠乳腺癌生长的作用。结果成功合成了粒径大小约为120 nm MnO_(2)纳米颗粒并负载QU。所制备的Mn(QU)与过氧化氢反应的氧气生成能力与pH大小呈负相关而与过氧化氢浓度呈正相关;细胞实验结果显示,Mn(QU)在浓度为50μg/mL时对4T1细胞无明显毒性,但能显著增强X射线诱导的4T1细胞杀伤作用(放疗增敏比为1.61),增加DNA双链的断裂以及ROS的产生,细胞死亡比例增加,诱导4T1细胞凋亡;体内移植瘤实验结果显示不同处理组对小鼠肿瘤体积抑制能力:Mn(QU)联合放疗组>MnO_(2)联合放疗组>QU联合放疗组>放疗组>对照组。结论Mn(QU)联合放疗对乳腺癌4T1细胞表现出显著的增殖抑制和放射增敏作用,并对移植瘤的生长也表现出明显的抑制效果。

Objective To investigate the radiosensitizating effect of quercetin(QU)loaded manganese dioxide nanoparticles[Mn(QU)]on breast cancer cell line 4T1 and tumour-bearing mice.Methods Manganese dioxide(MnO_(2))nanoparticles were synthesized by oleic acid template method.The morphology and chemical composition of MnO_(2) nanoparticles were characterized by transmission electron microscopy(TEM),scanning electron microscopy(SEM)and X-ray photoelectron spectroscopy.Then QU nanomaterials were encapsulated by using physical adsorption.The composition was characterized by ultraviolet spectrophotometer,and the ability of Mn(QU)nanoparticles reacting with different doses of hydrogen peroxide to produce oxygen at different pH values was detected by dissolved oxygen analyzer.CCK-8 assay was employed to detect the effects of different concentrations of Mn(QU)nanoparticles on the viability of 4T1 cells.Colony formation,γ-H2AX fluorescence staining,ROS fluorescence staining,LIVE/DEAD cell viability assay and flow cytometry were used to evaluate the radiosensitizing and pro-apoptotic effects of Mn(QU)nanoparticles on 4T1 cells.Finally,the effect of Mn(QU)nanoparticles combined with radiotherapy on tumor growth inhibition was evaluated in mouse model of 4T1 cell transplanted tumor.Results MnO_(2) nanoparticles with particle size of about 120 nm were successfully synthesized and encapsulated with QU.The oxygen generation capacity of the prepared Mn(QU)nanoparticles reacting with hydrogen peroxide was negatively correlated with pH value and positively with hydrogen peroxide concentration.The results of cell experiments showed that Mn(QU)nanoparticles at a concentration of 50μg/mL had no obvious toxicity to 4T1 cells,but could significantly enhance the X-ray-induced killing effect on 4T1 cells,at a radiotherapy sensitization ratio of 1.61,improve DNA double-strand breaks and ROS production,and induce apoptosis of 4T1 cells.The results of tumor xenograft model experiment indicated that the inhibition of tumor volume was Mn(QU)nanoparticles combined with radiotherapy>MnO_(2) nanoparticles combined radiotherapy>QU combined radiotherapy>Radiotherapy>Control.Conclusion Mn(QU)nanoparticles combined with radiotherapy can significantly inhibit the proliferation and show radiosensitization of breast cancer 4T1 cells,and also exert a significant inhibitory effect on the growth of the transplanted tumor.