MiRNA-126硬脂胺阳离子脂质体递送系统的构建及细胞摄取考察  

Construction of miRNA-126 stearylamine cationic liposome delivery system and cellular uptake investigation

作　　者：唐永瑜 邰诗蝶 吴兴杰 陈英 冯发进 沈祥春 邹弢 陶玲 TANG Yongyu;TAI Shidie;WU Xingjie;CHEN Ying;FENG Fajin;SHEN Xiangchun;ZOU Tao;TAO Ling(Guizhou Medical University,Guiyang 550025,China;School of Pharmaceutical Sciences,Guizhou Medical University,Guizhou Province Specialized Natural Medicine Resource Efficient Utilization Engineering Center,Key Laboratory of Pharmacology and Pharmacological Evaluation of Natural Medicines in Guizhou Higher Education Institutions,Guizhou Medical University-Guiyang Joint Key Laboratory,Key Laboratory of Utilization of Natural Medicine Resources,Guiyang 550025,China;Guizhou Biotechnology Research and Development Base Co.,Ltd.,Guiyang 551400,China;Beijing Jishui Tan Hospital Guizhou Hospital,Guiyang 550007,China)

机构地区：[1]贵州医科大学,贵州贵阳550025 [2]贵州医科大学药学院,贵州省特色天然药物资源高效利用工程中心,贵州省高等学校天然药物药理与成药性评价特色重点实验室,贵州医科大学-贵阳市联合重点实验室,天然药物资源优效利用重点实验室,贵州贵阳1550025 [3]贵州省生物技术研究开发基地有限公司,贵州贵阳551400 [4]北京积水潭医院贵州医院,贵州贵阳550007

出　　处：《药物评价研究》2024年第9期2006-2016,共11页Drug Evaluation Research

基　　金：国家自然科学基金资助项目(No.82260827和U1812403-4-4);贵州省自然科学基金会([2020]1Z069);贵州医科大学国家自然科学基金培育项目(No.20NSP050);贵州省科技计划项目(黔科合支撑[2020]4Y240号);贵州省高层次创新型人才十层次人才黔科合平台人才(GCC[2023]048);黔科合中引地([2023]003)。

摘　　要：目的基于阳离子脂质体制备血管细胞黏附分子-1(VCAM-1)单克隆抗体(VCAM-1 mAb)修饰的负载微小RNA-126(miR-126)的靶向递送系统,并初步评价其稳定性、细胞毒性及细胞摄取。方法采用薄膜分散-挤出法制备硬脂胺阳离子脂质体(SCL),以脂质体粒径、电位为指标,单因素法考察大豆磷脂与胆固醇质量比、硬脂胺用量、无水乙醇的用量、成膜温度、水合介质用量、水合制备温度、水合制备时间;并考察其稀释稳定性及储存稳定性。采用琼脂糖凝胶阻滞实验考察SCL对miR-126的负载能力及对酶解的保护效果。利用1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)/N-羟基琥珀酰亚胺(NHS)交联法制备VCAM-1 mAb修饰的SCL(Va-SCL),并通过SDS-PAGE法考察VCAM-1 mAb与SCL的耦连效率。MTT法考察其对人脐静脉内皮细胞(HUVECs)的毒性,流式细胞术和荧光显微镜考察正常HUVECs及LPS(1μg·mL^(-1))刺激后HUVECs对miR-126、SCL/miR-126、Va-SCL/miR-126的摄取情况。结果精密称量大豆磷脂120mg、胆固醇40mg、硬脂胺10 mg于茄形瓶中,加入5 mL无水乙醇,50℃超声(功率300 W、频率40 kHz)使其充分溶解,肉眼观察无可见颗粒物或者不溶物,水浴减压蒸发除去有机溶剂,在瓶壁上形成均匀透明的脂质薄膜;加入12 mL DEPC水作为水合介质,水浴超声30 min,挤出器过孔径为200 nm聚碳酸酯膜7次,取续滤液,得SCL,稳定性良好。当氮磷比为10∶1时,SCL能够有效负载miR-126;miR-126通过静电吸附于SCL表面,SCL一定程度上保护miR-126不被酶解。SDS-PAGE结果显示,VCAM-1mAb与SCL成功偶联,接枝率为(53.2±7.6)%。MTT结果显示,Va-SCL、Va-SCL/miR-126对HUVECs的半数抑制浓度(IC_(50))值分别为17.38、71.61 nmol·L^(-1),SCL、SCL/miR-126的IC_(50)值分别为81.03、97.79 nmol·L^(-1)。荧光显微镜及流式结果均显示Va-SCL/miR-126的细胞摄取效果更为明显。结论成功制备了Va-SCL/miR-126,能明显增加miR-126的细胞摄取�Objective To prepare a VCAM-1 monoclonal antibody(VCAM-1 mAb)modified miR-126 targeted delivery system based on cationic liposomes,and to preliminarily evaluate its stability,cytotoxicity and cellular uptake.Methods SCL was prepared by a thin film dispersion-extrusion method.With liposome size and zeta potential as the indicators,single factor method was used to investigate the effects of soybean phospholipid to cholesterol ratio,amount of stearic amine,amount of ethanol,film-forming temperature,amount of hydration medium,hydration preparation temperature,and hydration preparation time on the liposome.The dilution stability and storage stability were also examined.The loading capacity of miR-126 by SCL and the protection effect of SCL on the enzymatic hydrolysis were investigated by agarose gel retardation assay.The coupling efficiency of VCAM-1 mAb with SCL was examined by SDS-PAGE.The toxicity of Va-SCL,Va-SCL/miR-126 to human umbilical vein endothelial cells(HUVECs)was investigated by MTT assay,and the uptake of miR-126,SCL/miR-126,and Va-SCL/miR-126 by normal HUVECs and LPSstimulated HUVECs was examined by flow cytometry and fluorescence microscopy.Results Weigh out 120 mg of soybean phospholipids,40 mg of cholesterol,and 10 mg of stearic amine into a funnel-shaped bottle,and add 5 mL of anhydrous ethanol.Ultrasonicate at 50℃(power 300 W,frequency 40 kHz)to dissolve the ingredients completely,and visually inspect for any visible particles or insoluble substances.Remove the organic solvent by rotary evaporation at 50℃under a vacuum,forming a uniform,transparent lipid film on the bottle wall.Add 12 mL of DEPC-treated water as a hydration medium,and ultrasonicate at 50℃for 30 min.Pass the mixture through a 200 nm polycarbonate membrane using a syringe pump 7 times,collect the filtrate,and obtain SCL.The stability is good.When the nitrogen-to-phosphorus ratio is 10:1,SCL can effectively load miR-126.miR-126 adheres to the surface of SCL via electrostatic attraction,and SCL to some extent protects miR-126 fr

关 键 词：阳离子脂质体 MIR-126 薄膜分散法 EDC/NHS法 血管细胞黏附分子-1单克隆抗体修饰 

分 类 号：R943.42[医药卫生—药剂学] R979.1[医药卫生—药学]