间充质干细胞膜仿生纳米药物制备及抗乳腺癌实验研究  

Preparation of mesenchymal stem cell membrane-biomimetic nano drug and its anti-tumor activity

作　　者：李威 童金莲 夏桂民[3] 黄卫[4] 袁伟 马洁 LI Wei;TONG Jin-lian;XIA Gui-min;HUANG Wei;YUAN Wei;MA Jie(National Cancer Center/National Clinical Research Center for Cancer/State Key Laboratory of Molecular Oncology,Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021;Beijing Hospital Biotherapy Center/National Gerontology Center,Beijing 100730,China;Preparation Room,Institute of Medical Biotechnology Peking Union Medical College,Chinese Academy of Medical Sciences,Beijing 100050,China;State Key Laboratory of Metal Matrix Composites,School of Chemistry and Chemical Engineering Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院/分子肿瘤学国家重点实验室,北京100021 [2]北京医院生物治疗中心/国家老年医学中心,北京100730 [3]中国医学科学院北京协和医学院医药生物技术研究所制剂室,北京100050 [4]上海交通大学化学化工学院金属基复合材料国家重点实验室,上海200240

出　　处：《中华肿瘤防治杂志》2021年第10期719-726,共8页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家重点研发计划(2016YFA0201500);国家自然科学基金(51972343);国家科技重大专项(2016ZX09101094)。

摘　　要：目的制备间充质干细胞膜(MSCm)仿生纳米药物lac-DOX/DOX@MSCm,探讨其对乳腺癌4T1的靶向能力及抑瘤效果。方法薄膜水化法制备载阿霉素(DOX)的乳糖(lac)-阿霉素纳米胶束lac-DOX/DOX,反复冻融提取MSCm、超声后挤压成囊泡并用其包被lac-DOX/DOX制得细胞膜仿生纳米药物lac-DOX/DOX@MSCm。使用透射电子显微镜、动态光散射法及聚丙烯酰胺凝胶电泳对lac-DOX/DOX@MSCm的形貌、粒径、电位及膜蛋白表达情况进行表征。使用激光共聚焦显微镜检测4T1细胞对lac-DOX/DOX和lac-DOX/DOX@MSCm摄取情况。使用CCK-8法检测MSC膜囊泡、lac-DOX/DOX及lac-DOX/DOX@MSCm对4T1细胞的杀伤能力。建立小鼠原位4T1移植瘤模型,实验分为磷酸盐缓冲液(PBS)组、lac-DOX/DOX组和lac-DOX/DOX@MSCm组,每组5只,按5mg/kg量给药,1次/3d,通过小鼠肿瘤体积及瘤重评价抑瘤效果,小鼠体质量变化及主要脏器组织学变化评价药物的安全性。结果lac-DOX/DOX@MSCm呈核-壳结构,粒径为(187.0±1.5)nm,其蛋白表达情况与MSC膜囊泡相似。相比于lac-DOX/DOX组,lacDOX/DOX@MSCm组细胞红色荧光强度明显增强,且随着药物与细胞孵育时间的延长,荧光强度不断增加。浓度为0.0025~5μg/mL的MSCm囊泡对4T1细胞存活率差异无统计学意义,F=1.643,P>0.05。lac-DOX/DOX@MSCm和lac-DOX/DOX均表现出浓度依赖性的4T1细胞毒性;其中5和10μg/mL的lac-DOX/DOX@MSCm比相同浓度的lac-DOX/DOX具有更强的细胞杀伤能力,差异有统计学意义,均P<0.05。动物抑瘤实验中PBS、lac-DOX/DOX和lacDOX/DOX@MSCm组小鼠肿瘤体积间的效应F处理=34.481,P<0.001。其中lac-DOX/DOX@MSCm组与lac-DOX/DOX组比较,肿瘤生长受到抑制,差异有统计学意义,P<0.05。3组小鼠瘤重分别为(1.244±0.236)、(0.680±0.174)和(0.434±0.081)g,3组间均值差异有统计学意义,F=27.992,P<0.001。其中lac-DOX/DOX@MSCm组与lacDOX/DOX组相比瘤重降低,差异有统计学意义,P<0.05。实验结束时PBS、lac-DOX/DOX和lac-DOX/DObjective To design a kind of Mesenchymal stem cell(MSC)membrane-camouflaged nanodrug and to evaluate its targeting ability and therapeutic effect on breast cancer.Methods The lac-DOX/DOX was prepared by filming-rehydration method.The MSC membrane(MSCm)was extracted by repeated freezing and thawing.The MSCm vesicles were prepared by sonicating and extruding the MSCm.Then lac-DOX/DOX was coated with MSCm vesicles to prepare lac-DOX/[e-mail] particle size and zeta potential were measured by dynamic light scattering and the core-shell structure was observed under transmission electron microscopy.The surface protein contents were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Laser confocal microscopy was used to detect the internalization efficiency of lac-DOX/DOX and lac-DOX/DOX@MSCm by 4 T1 breast cancer cells.The 4 T1 cytotoxicity of MSCm vesicles,lac-DOX/DOX and lac-DOX/DOX@MSCm was evaluated by cell counting kit-8(CCK-8).The 4 T1 orthotopic murine breast cancer model was established,and the models were divided into phosphate buffered saline(PBS)group,lac-DOX/DOX group and lac-DOX/DOX@MSCm group,5 mice in each group.The mice were intravenously injected with 5 mg/kg lac-DOX/DOX,lac-DOX/DOX@MSCm or PBS once every three days.The tumor volumes and weights were measured to evaluate the tumor inhibition efficacy.The changes of the body weight and the organs tissue pathological changes were used to evaluate the safety in vivo.Results The lac-DOX/DOX@MSCm exhibited a spherical coreshell structure with hydrodynamic diameter of(187.0±1.5)nm.The protein profiles of lac-DOX/DOX@MSCm and MSCm vesicles were similar.Compared to lac-DOX/DOX group,the red fluorescence intensity of cells in the lac-DOX/DOX@MSCm group was significantly increased,and the fluorescence intensity increased with the incubation time.MSCm vesicles with concentrations of0.0025-5μg/ml had no statistically significant difference in the viability of 4 T1 cells,F=1.643,P>0.05.Both lac-DOX/DOX@MSCm and lac-DOX/DOX showed

关 键 词：间充质干细胞膜 仿生 纳米药物 阿霉素 乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤]