前阳离子脂质体的构建及有关性质研究

 目的制备前阳离子脂质体并考察其相关性质。方法化学合成含二硫键的胆固醇衍生物(2-4-(羟甲基)二硫]-1-亚氨丁基]氨基]乙基]氨基甲酸胆固醇酯,CHETA);薄膜分散-膜挤压法制备空白前阳离子脂质体;以鱼精蛋白缩合质粒DNA再与空白前阳离子脂质体作用,形成载基因前阳离子脂质体;粒度电位测定仪测定前阳离子脂质体的粒径和Zeta电位,电镜观察形态;考察在二硫键还原酶二硫苏糖醇的作用下前阳离子脂质体Zeta电位变化情况;以LacZ为报告基因转染HepG2细胞,X-gal原位染色定性观察,并定量测定β-半乳糖苷酶活性和总蛋白含量计算转染效率。结果前阳离子脂质体形态近似于球体,平均粒径为145.8 nm(PDI=0.086),Zeta电位为-33.83 mV,载基因前阳离子脂质体转染肝癌细胞HepG2转染效率为12.18 mU·mg^-1 protein,是裸质粒的10.5倍。结论二硫键修饰的前阳离子脂质体是具有发展潜力的非病毒载体。

Preparation and Characterization of Procationic Liposomes for Gene Delivery

OBJECTIVE To develop a novel non-viral gene delivery system procationic liposome(PCL) and investigate the related characteristics.METHODS CHETA,a kind of Cholesterol derivative containing disulfide bond,was synthesized and used to prepare liposomes in combination with phospholipid and cholesterol.Blank procationic liposomes were prepared by thin-film dispersion method.PCL encapsulating DNA was prepared by mixing the plasmid DNA and protamine together,and then the polyplexes were incubated for 10 min at room temperature,followed by addition of formed free PCL.The particle size and Zeta potential were determined by Malvern Zetasizer Nano ZS90(Malvern instruments Ltd.,UK) instrument with a 50 mV laser at a scattering angle of 90°.The morphological examination was performed on a JEM-100SX electron microscope(Japan).The Zeta potential of PCL was measured by the incubation of DTT with PCL.The estimation of transfection efficiency was performed by galactosidase assay in HepG2 cells using lacZ as reporter gene.RESULTS The resulting PCL had a regular spherical surface with an average size of 145.8 nm and a Zeta potential of-33.83 mV.The transfection efficiency of the delivery systems of PCL in HepG2 cells were 12.18 mU·mg^-1 protein,10.5 times greater than that of the naked plasmid DNA.CONCLUSION The PCL modified with disulfide bond is a perspective non-viral vector for gene delivery systems.