通过系统性阳离子材料筛选优化牛奶外泌体载体以实现mRNA的高效肺部靶向递送OA
Optimizing milk-derived exosome carriers through systematic cationic material screening for efficient pulmonary-targeted mRNA delivery
目的 通过系统性筛选阳离子修饰材料,旨在构建一种工程化的牛奶来源外泌体(milk-derived exosome,mExos)载体,以实现信使核糖核酸(messenger RNA,mRNA)在呼吸道内的高效递送.方法 采用超速离心法提取mExos,并通过电穿孔法将体外转录的萤火虫荧光素酶(firefly luciferase,Fluc)或增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)mRNA负载于mExos中.使用壳聚糖(chitosan,CS)、聚乙烯亚胺(polyethylenimine,PEI)、聚(β-氨基酯)[poly(β-amino ester),PBAE]、阳离子脂质(1,2-dioleoyl-3-trimethylammonium-propane chloride,DOTAP)及鱼精蛋白分别对负载mRNA的mExos进行修饰.采用动态光散射、纳米颗粒追踪分析、流式细胞术、荧光素酶报告基因检测等方法系统评价各修饰载体的的粒径、电位、胶体稳定性、细胞摄取、mRNA转染效率及细胞毒性.选用50只6～8周龄雌性BALB/c小鼠(体质量约20 g),经鼻内滴注染料标记或负载Fluc-mRNA的制剂,采用小动物活体成像系统观察制剂在其体内分布与肺部转染情况,并于给药24 h后取主要器官进行组织病理学和血清生化分析.结果 成功构建了系列阳离子修饰mExos.经筛选,壳聚糖修饰的mExos(Chitosan-modified milk exosomes,CS-mExos)在保持合适粒径和良好稳定性(48 h内PDI无显著变化)的前提下,其表面电位发生了反转.体外实验显示,CS-mExos在16HBE和A549细胞中的摄取效率分别较未修饰mExos提高2.56倍(P＜0.001)和4.56倍(P＜0.001),并显著提升Fluc-mRNA的转染效率达28.9倍(P＜0.001)和26.5倍(P＜0.001),且无明显细胞毒性(细胞活力＞95%).活体成像表明,CS-mExos经鼻给药后可特异性富集于肺部并长效滞留至少72 h,且介导的Fluc表达在给药后6 h达峰值,强度为mExos组的9.5倍.组织病理学与血清生化分析显示,CS-mExos未引起明显脏器损伤或肝功能、肾功能指标异常.结论 通过系统性筛选,确定CS为优化mExos的mRNA递送性能的最佳阳离子修饰材料.基于此构建的CS-mExos载体能高效靶向递送mRNA至呼吸道,显著提升肺部转染效率并具备良好生物安全性.
Objective To construct an engineered milk-derived exosome(mExos)vector for efficient messenger RNA(mRNA)delivery to the respiratory tract by systematically screening cationic modification materials.Methods mExos were isolated via ultracentrifugation,followed by electroporation-mediated loading of firefly luciferase(Fluc)or enhanced green fluorescent protein(EGFP)mRNA.Chitosan(CS),polyethyleneimine(PEI),poly(beta-amino ester)(PBAE),cationic lipid(DOTAP),and protamine were used to modify mRNA-loaded mExos.Dynamic light scattering,nanoparticle tracking analysis,flow cytometry and luciferase reporter assay were employed to evaluate particle size,zeta potential,colloidal stability,cellular uptake,transfection efficiency,and cytotoxicity.Fifty female BALB/c mice(6 to 8 weeks,about 20 g)received intranasal administration of dye-labeled or Fluc-mRNA-loaded formulations.In vivo distribution and pulmonary transfection kinetics were monitored by IVIS imaging,while histopathology and serum biochemistry of major organs were analyzed at 24 h post-administration.Results Cationic-modified mExos were successfully constructed.CS-modified mExos(CS-mExos)demonstrated surface charge reversal while maintaining optimal size and stability(PDI unchanged within 48 h).In vitro,CS-mExos enhanced cellular uptake in 16HBE and A549 cells by 2.56-fold(P＜0.001)and 4.56-fold(P＜0.001),respectively,and increased Fluc-mRNA transfection efficiency by 28.9-fold(P＜0.001)and 26.5-fold(P＜0.001)versus unmodified mExos,with＞95%cell viability.In vivo imaging revealed CS-mExos specifically accumulated in the lungs with sustained retention＞72 h,mediating 9.5-fold higher peak Fluc-mRNA expression at 6 h post-administration.Histopathology and serum biochemistry confirmed no significant organ damage or abnormal hepatic/renal indices.Conclusion Systematic screening identified chitosan as the optimal cationic material for engineering milk exosomes.The developed CS-mExos vector enables efficient targeted mRNA delivery to the respiratory tract with enhanced pulmonary transfection and favorable biosafety.
罗明星;廖睿;关山;张卫军;罗萍;张怡;程平;李飏;邹全明
陆军军医大学(第三军医大学)药学与检验医学系,重庆陆军军医大学(第三军医大学)药学与检验医学系,重庆陆军军医大学(第三军医大学)药学与检验医学系,重庆陆军军医大学(第三军医大学)药学与检验医学系,重庆陆军军医大学(第三军医大学)药学与检验医学系,重庆陆军军医大学(第三军医大学)药学与检验医学系,重庆陆军军医大学(第三军医大学)药学与检验医学系,重庆陆军军医大学(第三军医大学)第一附属医院药剂科,重庆陆军军医大学(第三军医大学)药学与检验医学系,重庆
医药卫生
牛奶外泌体壳聚糖信使RNA肺部给药基因治疗
milk-derived exosomeschitosanmessenger RNApulmonary administrationgenetic therapy
《陆军军医大学学报》 2026 (4)
407-419,13
国家自然科学基金面上项目(82173764,32370993)重庆市自然科学基金面上项目(CSTB2025NSCQ-GPX0624) Supported by the General Program of National Natural Science Foundation of China(82173764,32370993)and the General Program of Natural Science Foundation of Chongqing(CSTB2025NSCQ-GPX0624).
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