叶酸修饰的克班宁聚乙二醇-聚乳酸羟基乙酸共聚物纳米粒的制备及其体外 抗肿瘤活性研究  

Preparation and anti-tumor activity study of folic acid modified crebanine polyethylene glycol-polylactic acid hydroxyacetic acid copolymer nanoparticles in vitro

作　　者：潘蕊 张海亮 赵笑雨 唐钧泽 吴健铭 郑永仁 程欣 PAN Rui;ZHANG Hai-liang;ZHAO Xiao-yu;TANG Jun-ze;WU Jian-ming;ZHENG Yong-ren;CHENG Xin(College of Traditional Chinese Medicine,Yunnan University of Chinese Medicine,Kunming 650500,China;Key Laboratory of External Drug Delivery System and Preparation Technology in University of Yunnan Province,Kunming 650500,China;College of Chinese Materia Medica and Yunnan Key Laboratory of Southern Medicinal Utilization,Kunming 650500,China;Yunnan Key Laboratory of Dai and Yi Medicines,Kunming 650500,China;Science and Technology Office,Yunnan University of Chinese Medicine,Kunming 650500,China)

机构地区：[1]云南中医药大学中药学院,云南昆明650500 [2]云南省高校外用给药系统与制剂技术研究重点实验室,云南昆明650500 [3]云南省南药可持续利用研究重点实验室,云南昆明650500 [4]云南省傣医药与彝医药重点实验室,云南昆明650500 [5]云南中医药大学科学技术处,云南昆明650500

出　　处：《中草药》2023年第20期6643-6656,共14页Chinese Traditional and Herbal Drugs

基　　金：云南省科技厅--云南中医药大学应用基础研究联合专项重点项目(202001AZ070001-008);云南省科技厅生物医药重大科技专项(202002AA100007);云南省科技人才平台计划(202105AG070012);云南省傣医药与彝医药重点实验室开放课题(202210SS2205);云南省傣医药与彝医药重点实验室开放课题(202210SS2206);国家中医药管理局“十二五”重点学科--傣药学。

摘　　要：目的制备叶酸修饰的克班宁聚乙二醇-聚乳酸羟基乙酸共聚物纳米粒(folic acid modified crebanine polyethylene glycol-polylactic acid hydroxyacetic acid copolymer nanoparticles,FA-Cre@PEG-PLGA NPs),并考察其体外释放及抗肿瘤活性。方法以克班宁为模型药,用PEG-PLGA共聚物、叶酸等为材料,通过超声乳化-溶剂挥发法制备FA-Cre@PEG-PLGA NPs,采用激光粒度仪、透射电子显微镜和荧光显微镜对FA-Cre@PEG-PLGA NPs的粒径、ζ电位及形态进行表征,紫外光谱法测定并计算克班宁的包封率和载药量,比较FA-Cre@PEG-PLGA NPs和克班宁原料药72 h的体外释放规律,通过溶血实验考察二者生物学特性,CCK-8实验考察两者对正常肝L02细胞存活率的影响及对肝癌Bel-7402细胞体外增殖抑制作用。细胞划痕法考察纳米粒和原料药对肝癌Bel-7402细胞迁移能力的影响。荧光显微镜观察纳米粒在不同时间点对肝癌Bel-7402细胞的摄取情况。结果FA-Cre@PEG-PLGA NPs的平均粒径为(247.67±2.49)nm,多分散指数(polydispersity index,PDI)为0.139±0.027,ζ电位为(−9.40±0.54)mV,透射电子显微镜下呈圆球状核壳结构,荧光显微镜下观察纳米粒,其气化后明场呈蓝色光点、暗场呈红色光点。FA-Cre@PEG-PLGA NPs中克班宁的包封率为83.78%,载药量为67.78%。FA-Cre@PEGPLGA NPs与克班宁原料药体外释放均符合Ritger-Peppas模型;安全性评价结果显示,FA-Cre@PEG-PLGA NPs相比于克班宁原料药在50~300μg/mL内具有良好的生物相容性。FA-Cre@PEG-PLGA NPs对人正常肝L02细胞毒性较低,相比于克班宁原料药有一定的安全性。体外增殖抑制结果显示,两者对Bel-7402肿瘤细胞的增殖抑制率均呈现出剂量依赖性和时间依赖性,且FA-Cre@PEG-PLGA NPs联合超声辐照后对Bel-7402肝癌细胞具有更强的杀伤作用。细胞划痕实验表明,克班宁原料药、FA-Cre@PEG-PLGA NPs对Bel-7402肿瘤细胞迁移均有抑制作用,呈剂量依赖性,且纳米粒联合超�Objective To prepare folic acid modified crebanine polyethylene glycol-polylactic acid hydroxyacetic acid copolymer nanoparticles(FA-Cre@PEG-PLGA NPs)and investigate their in vitro release and anti-tumor activity.Methods FA-Cre@PEGPLGA NPs were prepared by ultrasonic emulsion-solvent evaporation method with crebanine as a model drug,polyethylene glycol-polylactic acid hydroxyacetic acid(PEG-PLGA)copolymer and folic acid(FA)as materials.The particle size,potential and morphology of FA-Cre@PEG-PLGA NPs were characterized by laser particle size measurement,transmission electron microscopy and fluorescence microscope.The encapsulation rate and drug loading capacity of crebanine were determined and calculated by Ultraviolet spectroscopy.Meanwhile,the in vitro release patterns of FA-Cre@PEG-PLGA NPs and crebanine bulk drug at 72 h were compared.The biological properties were investigated by hemolysis assay.The safety evaluation on L02 cells and the proliferation inhibition on Bel-7402 cells were investigated by CCK-8 assay in vitro.The proliferation of Bel-7402 cells was quantified by cell wound scratch assay.Finally,the uptake capacity of nanoparticles to Bel-7402 cells at different time points was observed by fluorescence microscope.Results The average particle size of FA-Cre@PEG-PLGA NPs was(247.67±2.49)nm,with a polydispersity index(PDI)of 0.139±0.027 and aζpotential of(−9.40±0.54)mV.The transmission electron microscopy revealed a core-shell structure,while the fluorescence microscopy showed blue dots in the bright field and red dots in the dark area after gasification.The encapsulation rate of crebanine in nanoparticles was 83.78%and the drug loading was 67.78%.The release of FA-Cre@PEG-PLGA NPs and crebanine bulk drug conformed to the Ritger-Peppas model in vitro.The safety evaluation showed that FA-Cre@PEG-PLGA NPs had good biocompatibility within the range of 50—300μg/mL compared with crebanine bulk drug.The FA-Cre@PEG-PLGA NPs showed lower toxicity to L02 cells than crebanine bulk drug.The proliferati

关 键 词：叶酸 聚乙二醇-聚乳酸羟基乙酸 克班宁 纳米粒 抗肿瘤活性 体外释放 超声乳化-溶剂挥发法 

分 类 号：R283.6[医药卫生—中药学]