初步构建与评估递送柚皮素的RGD外泌体  

Preliminary construction and evaluation of RGD exosomes for delivering naringin

作　　者：冯玘 刘洪峰[2] FENG Qi;LIU Hongfeng(Department of Pharmacy,North Anhui Health Vocational College,Suzhou Anhui 234000;Department of Pharmacy,Suzhou Municipal Hospital Affiliated to Anhui Medical University,Suzhou Anhui 234000,China)

机构地区：[1]皖北卫生职业学院药学系,安徽宿州234000 [2]安徽医科大学附属宿州市立医院药剂科,安徽宿州234000

出　　处：《江苏大学学报（医学版）》2024年第5期407-413,共7页Journal of Jiangsu University：Medicine Edition

基　　金：安徽省教育厅重点项目(2022AH053142);校级科研项目(WZK201906)。

摘　　要：目的:本研究旨在制备生物合成纳米载体递送柚皮素(naringenin,Ng),并研究其对乳腺癌的体外抑瘤效果。方法:使用精氨酸甘氨酸天冬氨酸前列腺素F2受体阴性调节因子绿色荧光蛋白(Arg-Gly-Asp-prostaglandin F2 receptor negative regulator-green fluorescent protein,RGD-P-G)质粒进行转染,构建分泌RGD-P-G外泌体(RGD-P-G-Exosomes,RGD-P-G-Exo)的293F细胞。通过超速离心收集293F细胞上清液中的RGD-P-G-Exo,加入Ng,并通过水浴超声促使RGD-P-G-Exo包封Ng,形成RGD-P-G-Exo@Ng。使用荧光显微镜对转染RGD-P-G的293F细胞进行鉴定。通过透射电子显微镜、纳米颗粒跟踪分析技术、蛋白质免疫印迹等方法对RGD-P-G-Exo@Ng的形貌、粒径、标志物和绿色荧光蛋白(GFP)表达情况进行表征。采用紫外分光光度法评估包封率和载药量。通过细胞摄取实验评估纳米药物被吞噬摄取的效果。通过CCK-8法检测Exo@Ng和RGD-P-G-Exo@Ng对乳腺癌MDA-MB-231细胞的杀伤能力。结果:电镜结果显示RGD-P-G-Exo@Ng呈茶托状结构,纳米颗粒追踪分析技术显示RGD-P-G-Exo@Ng粒径主要分布在147.0 nm。蛋白免疫印迹结果表明RGD-P-G-Exo@Ng表达CD9、CD63等外泌体标志物和GFP,证明RGD-P-G-Exo的构建成功。RGD-P-G-Exo@Ng纳米药物的包封率为(28.1±0.6)%,载药量为(6.0±0.1)%。在高浓度下,Exo及RGD-P-G-Exo对MDA-MB-231细胞的存活率无显著影响,具有良好的生物相容性。在细胞摄取实验中,与Exo@Ng相比,RGD-P-G-Exo@Ng具有更强的细胞摄取效果(P<0.05)。药效实验中,Exo@Ng和RGD-P-G-Exo@Ng均表现出浓度依赖性的MDA-MB-231细胞毒性,其中RGD-P-G-Exo@Ng比相同浓度的Exo@Ng具有更强的细胞杀伤能力(P<0.05)。结论:本研究初步合成了一种用于靶向递送Ng的纳米药物,为开发生物纳米靶向肿瘤药物递送系统提供了新的策略。Objective:To prepare biosynthetic nanocarriers to deliver naringenin(Ng)and to investigate its in vitro tumor-suppressive effect on breast cancer.Methods:293F cells secreting Arg-Gly-Asp-prostaglandin F2 receptor negative regulator-green fluorescent protein exosomes(RGD-P-G-Exo)were constructed by transfection using Arg-Gly-Asp-prostaglandin F2 receptor negative regulator-green fluorescent protein(RGD-P-G)plasmid.The nanomedicine(RGD-P-G-Exo@Ng)was prepared by collecting RGD-P-G-Exo in the supernatant by ultracentrifugation,adding Ng,and inducing the RGD-P-G-Exo to encapsulate the Ng by sonication in a water bath.293F cells transfected with RGD-P-G were characterized using fluorescence microscopy.The morphology,particle size,markers and green fluorescent protein(GFP)expression of RGD-P-GFP-Exo@Ng were characterized by transmission electron microscopy,nanoparticle tracking analysis technique and protein immunoblotting.The encapsulation rate and drug loading capacity were assessed by UV spectrophotometry.Phagocytic uptake of the nanodrugs was assessed by cellular uptake assay.The killing ability of Exo@Ng and RGD-P-G-Exo@Ng on MDA-MB-231 cells was detected by CCK-8 assay.Results:Electron microscopy revealed a cup-like structure of RGD-P-G-Exo,with a particle size of 147.0 nm according to nanoparticle tracking analysis.Immunoblotting confirmed the surface expression of exosome markers(CD9,CD63)and GFP on RGD-P-G-Exo,validating the successful construction of RGD-P-G-Exo.The encapsulation efficiency of RGD-P-G-Exo@Ng was(28.1±0.6)%,and the drug loading content was(6.0±0.1)%.At high concentrations,Exo and RGD-P-G-Exo had no significant effect on the viability of MDA-MB-231 cells with good biocompatibility.In the cellular uptake assay,the RGD-P-G-Exo@Ng group had a stronger cellular uptake effect compared with the Exo@Ng group(P<0.05).In the pharmacodynamic assay,both Exo@Ng and RGD-P-G-Exo@Ng groups exhibited concentration-dependent cytotoxicity in MDA-MB-231 cells,in which the RGD-P-G-Exo@Ng group possessed a strong

关 键 词：乳腺癌 精氨酸甘氨酸天冬氨酸 前列腺素F2受体阴性调节因子 外泌体 柚皮素 

分 类 号：R944.9[医药卫生—药剂学]