外泌体负载甲氨蝶呤递药体系的构建及表征

Construction and characterization of delivery system for exosome-loaded methotrexate

目的以巨噬细胞来源外泌体(exosomes,Exos)为载体,包载抗肿瘤药物甲氨蝶呤(methotrexate,MTX),构建一种纳米载药体系(MTX-Exos),并对其理化性质及在体外对肝细胞癌增殖的抑制作用进行研究。方法通过超速离心从RAW264.7细胞中提取纯化Exos,采用超声孵育法将MTX载入Exos,利用动态光散射(dynamic light scattering,DLS)、透射电镜(transmission electron microscope,TEM)和Western blot对其进行表征,并通过MTT实验研究了该递药体系抑制肝细胞癌增殖的作用。结果最优处方下的MTX-Exos的平均粒径为(174.3±3.2)nm,Zeta电位为(-10.8±0.3)mV,载药量为(2.81±0.02)%,外观形态呈茶托状双层膜结构并表达外泌体特异性蛋白Alix和TSG101。MTX-Exos对肝癌细胞的毒性作用比游离MTX更强。结论Exos成功负载MTX,制备方法简单可行,在体外试验中对肝细胞癌的增殖展示出良好的抑制作用。

Objective To construct a nano-drug delivery system with macrophage-derived exosomes(Exos)encapsulated with anticancer drug methotrexate(MTX),and to determine its physicochemical properties and inhibition of hepatocellular carcinoma proliferation in vitro.Methods Exos were purified from RAW264.7 cells by ultracentrifugation,and MTX was loaded into Exos by ultrasonic incubation,and characterized by dynamic light scattering,transmission electron microscope(TEM),and Western blot.The effect of this drug delivery system in inhibiting the proliferation of hepatocellular carcinoma was determined by MTT assay.Results The average particle size of MTX-Exos in the optimal prescription was(174.3±3.2)nm,the Zeta potential was(-10.8±0.3)mV,and the drug loading was(2.81±0.02)%.These exosomes contained known markers Alix and TSG101 with the classic saucer-like bilayer structure of exosomes observed by TEM.The toxic effect of MTX-Exos on the hepatoma cells was stronger than that of free MTX.Conclusion MTX is successfully loaded by Exos with a simple and feasible method,and the in vitro experiment shows a good inhibition on the proliferation of hepatocellular carcinoma.