自噬调节剂雷帕霉素和羟氯喹纳米载体联合靶向耐药肿瘤细胞研究  

Combined autophagy modulators rapamycin and hydroxychloroquine-loaded 7pep-modified nanomedicines for targeting drug-resistant tumor cells

作　　者：梅冬 张晓燕 赵立波[1] 王晓玲[1] 张强[2] MEI Dong;ZHANG Xiao-yan;ZHAO Li-bol;WANG Xiao-ling;ZHANG Qiang(National Center for Children＇s Health/Beijing Children＇s Hospital,Capital Medical University,BEIJING 100045,China;Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems,School of Pharmaceutical Sciences,Peking University,BEIJING 100191,China)

机构地区：[1]国家儿童医学中心/首都医科大学附属北京儿童医院,北京100045 [2]北京大学药学院分子药剂学与新释药系统北京市重点实验室,北京100191

出　　处：《中国新药与临床杂志》2018年第10期571-580,共10页Chinese Journal of New Drugs and Clinical Remedies

基　　金：国家科技部重大专项(2018ZX09721003);首都医科大学附属北京儿童医院国家自然科学基金培育项目(GPY201711)

摘　　要：目的制备7pep修饰的雷帕霉素胶束和羟氯喹脂质体,考察两种纳米载体联合给药对耐药肿瘤细胞的协同抗肿瘤作用,并探索可能的机制。方法采用薄膜分散法和硫酸铵梯度法分别制备7pep修饰的主动靶向胶束和主动靶向脂质体,对纳米载体的粒径大小、分布、包封率以及Zeta电位进行表征,采用流式细胞术和激光共聚焦法观察7pep修饰纳米载体的靶向性, SRB法评价两种纳米药物联合给药的细胞毒性,通过MDC染色法考察胞内自噬囊泡蓄积情况, ELISA法定量测定自噬体标记物LC3Ⅱ的表达水平。结果雷帕霉素胶束粒径在20 nm左右,包封率在90%以上。羟氯喹脂质体粒径在90～100 nm之间,包封率在88%以上。主动靶向胶束和主动靶向脂质体通过转铁蛋白受体介导的途径增加胞内摄取。7pep修饰的雷帕霉素胶束和羟氯喹脂质体对耐药细胞呈剂量依赖性细胞毒性,且两种纳米载体联合给药具有协同抗肿瘤作用,并导致细胞内自噬囊泡大量蓄积和自噬标记物LC3Ⅱ表达水平升高。结论 7pep修饰的雷帕霉素胶束和羟氯喹脂质体联合给药能抑制耐药肿瘤细胞增殖,诱导细胞过度自噬可能是其作用机制之一。AIM To construct 7 pep-modified rapamycin-loaded polymer micelles（ 7 pep-PM-RAPA）and hydroxychloroquine-containing liposomes（ 7 pep-SSL-HCQ）,and investigate the synergistic anti-tumor effects of these two nanocarriers on drug-resistant tumor cells,as well as its possible mechanisms.METHODS7 pep-PM-RAPA and 7 pep-SSL-HCQ were prepared by a thin-film dispersion method and ammonium sulfate gradient method,respectively.The particle size,distribution,encapsulation efficiency and Zeta potential of two nanocarriers were characterized.The targeted efficiency of nanomedicines was characterized by flow cytometry and laser confocal microscopy.The cytotoxicity of two nanomedicines used in single or in combination was evaluated by SRB method.The intracellular autophagic vesicle accumulation was examined by monodansylcadaverine（MDC） staining.The expression level of autophagosome marker LC3Ⅱ was quantified by ELISA.RESULTS The size of 7 pep-PM-RAPA was about 20 nm and the encapsulation efficiency was above90%.The size of 7 pep-SSL-HCQ was between 90 and 100 nm,and the encapsulation efficiency was greater than 88%.The intracellular uptake of 7 pep-PM-RAPA and 7 pep-SSL-HCQ was increased via transferrin receptor-mediated pathways.Both 7 pep-PM-RAPA and 7 pep-SSL-HCQ showed dose-dependent cytotoxicity to drug-resistant tumor cells.The combination therapy had a synergistic anti-tumor effect and resulted in the intracellular accumulation of autophagic vesicles as well as the increase of LC3 Ⅱ expression.CONCLUSION The combination therapy of 7 pep-PM-RAPA and 7 pep-SSL-HCQ can inhibit the proliferation of drug-resistant tumor cells,and one of the mechanisms may be the induction of excessive cellular autophagy.

关 键 词：受体 转铁蛋白 自噬 雷帕霉素 羟氯喹 联合给药 

分 类 号：R979.1[医药卫生—药品]