miR-126阳离子纳米乳的制备及初步药效  

Preparation of miR-126 cationic nanoemulsion and its preliminary pharmacodynamic

作　　者：张丽丹 唐永瑜 汪巳卜 陈英 吴兴杰 沈祥春 陶玲 ZHANG Lidan;TANG Yongyu;WANG Sibu;CHEN Ying;WU Xingjie;SHEN Xiangchun;TAO Ling(School of Pharmacy,Guizhou Medical University/Guizhou Engineering Center of Efficient Utilization of Natural Medicine Resources&Key Laboratory of Pharmacology and Druggability Evaluation of Natural Medicine&Key Laboratory of Optimal Utilization of Natural Medicine Resources,Guizhou Colleges and Universities,Guiyang 550025,Guizhou,China)

机构地区：[1]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,天然药物资源优效利用重点实验室,贵州贵阳550025

出　　处：《贵州医科大学学报》2024年第6期811-818,共8页Journal of Guizhou Medical University

基　　金：贵州省科技计划项目(黔科合支撑[2020]4Y240);贵州省高层次创新型人才百层次人才项目(贵州科技厅黔科合人才[2015]4029);贵州医科大学药学国际科技合作基地(黔科合平台人才[2017]5802);贵州省科技创新基地(黔科合中引地[2023]003);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)。

摘　　要：目的采用阳离子纳米乳(CNE)载miRNA-126(miR-126)形成miR-126阳离子纳米乳递送系统(CNE-miR126),并对其药剂学性能、时间稳定性、血清稳定性、细胞毒性、细胞摄取及血管黏附分子-1(VCAM-1)蛋白表达量进行评估。方法用琼脂糖凝胶电泳考察CNE和miR-126的最佳氮磷比(N/P),采用纳米粒度及Zeta电位分析测量仪测定其粒径和进行药剂学性能评价;在4℃条件下,评估CNE-miR-126在1周内粒径及电位的稳定性;用血清稳定性实验评估CNE-miR-126在48 h之内的稳定性,MTT法评估CNE-miR-126的细胞毒性;流式细胞术和荧光显微镜观察人脐静脉内皮细胞(HUVECs)对CNE-miR-126的摄取情况,免疫蛋白质印迹法(Weston blot)检测CNE-miR-126对由脂多糖(LPS)致炎的HUVECs中VCAM-1的表达水平。结果CNE与miR-126最佳N/P为16∶1,CNE-miR-126外观基本圆整、平均粒径(D_(50))为(144.79±1.85)nm、Zeta电位为(22.92±1.24)mV;CNE-miR-126在1周内粒径及电位稳定性良好,且在48 h之内血清稳定性良好;在10^(-6)~1 nmol/L浓度内对HUVECs细胞生长无明显抑制作用,能够促进HUVECs对miR-126的摄取;Weston blot结果显示,与Control组相比,LPS(1 mg/L)处理细胞8 h后VCAM-1蛋白水平显著升高(P<0.01);与Model组相比,CNE-miR-126组预处理24 h后VCAM-1蛋白水平降低(P<0.05)。结论CNE-miR-126粒径适宜,稳定性良好,可降低VCAM-1蛋白的表达,可为心血管疾病的治疗提供参考。Objective To evaluate pharmaceutical properties,time stability,serum stability,cytotoxicity and cellular uptake of miR-126 cationic nanoemulsion(CNE)delivery system(CNE-miR126)loaded by CNE and assess the effect of CNE-miR126 on protein expression of vascular adhesion molecule-1(VCAM-1).Methods Optimal nitrogen-phosphorus ratio(N/P)of CNE and miR-126 was observed by agarose gel electrophoresis.Pharmaceutical performance was evaluated using nanoparticle size and Zeta potential measured by Zetasizer Nano ZS.The stability and potential of CNE-miR-126 particle size within 1 week under 4℃were evaluated.Serum stability was used to assess the stability of CNE-miR-126 within 48 hours.MTT assay was applied to evaluate the cytotoxicity of CNE-miR-126.Flow cytometry and fluorescence microscopy were used to observe the uptake of CNE-miR-126 by human umbilical vein endothelial cells(HUVECs).Western blot was performed to detect the effect of CNE-miR-126 on VCAM-1 expression in inflammatory HUVECs induced by lipopolysaccharide(LPS).Results The optimal N/P ratio for CNE and miR-126 was 16∶1.The appearance of CNE-miR-126 was basically round,with an average particle size(D_(50),144.79±1.85)nm and a Zeta potential(22.92±1.24)mV.CNE-miR-126 had good particle size and potential stability within 1 week,and serum stability was good within 48 hours.CNE-miR-126 at the concentration ranging from 10^(-6) to 1 nmol/L had no obvious inhibitory effect on HUVEC growth and promoted the uptake of miR-126 by HUVECs.Western blot result showed that VCAM-1 protein level was significantly upregulated after LPS(1 mg/L)treatment for 8 h when compared to control(P<0.01).When compared to model group,CNE-miR-126 group showed a decrease in VCAM-1 protein level after 24 hours of pretreatment(P<0.05).Conclusion CNE-miR-126 has suitable particle size and good stability,which can reduce VCAM-1 protein expression,providing a reference for cardiovascular disease therapy.

关 键 词：阳离子纳米乳 miRNA-126 基因递送 细胞摄取 人脐静脉内皮细胞 

分 类 号：R944[医药卫生—药剂学]