氯喹脂质体的制备及其对外泌体分泌量的影响  

Preparation of chloroquine liposomes and its influence on exosome secretion

作　　者：晏兰兰 杨艾 张菜 刘杰峰 王学丽 鲁卫东 YAN Lan-lan;YANG Ai;ZHANG Cai;LIU Jie-feng;WANG Xue-li;LU Wei-dong(School of Pharmaceutical Science,Yunnan Key Laboratory of Pharmacology for Natura Products,Kunming Medical University,Kunming 650500,China)

机构地区：[1]昆明医科大学药学院/云南省天然药物药理重点实验室,昆明650500

出　　处：《中国新药杂志》2024年第20期2153-2158,共6页Chinese Journal of New Drugs

摘　　要：目的:制备氯喹脂质体(chloroquine liposomes,CQ-Lips),探究CQ-Lips对细胞外泌体分泌量的影响,建立高效促外泌体分泌技术体系。方法:以大豆卵磷脂、胆固醇为膜材,通过单因素实验筛选最佳处方,制备CQ-Lips;通过CCK-8实验探究CQ-Lips载不同浓度CQ对细胞活性的影响;将CQ-Lips载不同浓度CQ(5~100μmol·L^(-1))与细胞共孵育,以CD9和TSG101这2种外泌体表面标记物为指标,检测外泌体分泌量,确定最佳脂质体载药浓度;CQ-Lips载不同浓度CQ(5,10μmol·L^(-1))与细胞共孵育不同时间点,检测外泌体的分泌量,确定收取上清最佳时间点。结果:以最佳处方药物与磷脂质量比1∶80、磷脂与胆固醇质量比3.75∶1,20℃下孵育10 min,冻融2次,制备CQ-Lips,其粒径均一,呈正态分布,包封率为72.71%;CCK-8结果显示,单纯CQ 20μmol·L^(-1)对细胞活性无影响,CQ-Lips载不同浓度CQ,细胞活性随CQ浓度增大而逐渐降低,包载CQ(5,10μmol·L^(-1))时,细胞活性较高,但随孵育时间的延长而细胞活性逐渐降低;CQ-Lips刺激外泌体的分泌效果优于单纯CQ,综合考虑毒性,CQ-Lips载CQ 5μmol·L^(-1)效果最佳;外泌体分泌量随CQ-Lips孵育时间延长而逐渐降低,48 h为收取细胞上清最佳时间点。结论:本研究通过最佳处方CQ-Lips的制备,促使更多CQ进入溶酶体内,减少了溶酶体对外泌体的降解,提高了外泌体的分泌量,为外泌体的基础研究及临床使用提供实验基础。Objective:To establish a highly efficient exosomal secretion promoting technology system by preparing chloroquine liposomes(CQ-Lips)and exploring the effects of CQ-Lips on the secretion of exosomes.Methods:Using soybean lecithin and cholesterol as membrane materials,the best prescription of CQ-Lips was screened by single factor experiment.The effects of CQ-Lips loaded with different concentrations of CQ on cell viability were explored through CCK-8 experiments.CQ-Lips loaded with chloroquine of 5~100μmol·L^(-1)were incubated with cells,taking exosome surface markers CD9 and TSG101 as indicators to detect the secretion of exosomes to determine the optimal liposome drug loading concentration.CQ-Lips loaded with chloroquine of 5 and 10μmol·L^(-1)were incubated with cells for different time,then the secretion of exosomes was detected to determine the optimal time point for collecting supernatant.Results:CQ-Lips were prepared under the optimum condition which was as follows:phospholipid mass ratio 1∶80,phospholipid cholesterol mass ratio 3.75∶1,incubated at 20℃for 10 min,freeze-thawed for twice.The resulting product had uniformly sized particles with the encapsulation rate reaching to 72.71%;CCK-8 results showed that CQ of 20μmol·L^(-1)alone had no effect on cell activity.For different concentration groups of CQ in CQ-Lips,the cell activity decreased gradually with the increasing of CQ concentration.The cell activity was higher for 5 and 10μmol·L^(-1)CQ-loaded liposomes,but gradually decreased with the prolongation of incubation time.The effect of CQ-Lips on exosome secretion was better than that of CQ alone,CQ-Lips containing CQ of 5μmol·L^(-1)had the best effect.The secretion of exosomes decreased with the prolonging of incubation time,and 48 h was the best time point for collecting cell supernatant.Conclusion:In this study,more chloroquine was promoted to enter the lysosome through preparation of chloroquine liposomes with the best prescription,so the production of exosomes could be increased by the

关 键 词：脂质体 外泌体产量 氯喹 溶酶体抑制剂 

分 类 号：R917[医药卫生—药物分析学]