N-(N',N'-二甲基)丙基丁二酸单胆固醇酯单酰胺的合成、性质及作为基因载体的研究

Synthesis and Characterization of N-( N',N'-Dimethyl) Propyl Succinic Mono-Cholesteryl Mono-Amide as Gene Carrier

目的合成新型的阳离子胆固醇衍生物——N-(N',N'-二甲基)丙基丁二酸单胆固醇酯单酰胺(DMAPA-CHEMS),考察其作为非病毒类基因载体的可行性。方法以N-(N',N'-二甲基)丙基丁二酸单胆固醇酯单酰胺和普通磷脂为膜材采用薄膜分散法制备阳离子脂质体,透射电镜观察脂质体的形态,激光粒度仪测定其粒径和表面电位;琼脂糖凝胶电泳考察脂质体对DNA的结合能力和保护作用;红细胞溶血实验和噻唑蓝(MTT)比色法测定其细胞毒性;流式细胞仪测定Hep G2细胞对FITC标记寡核苷酸的摄取,倒置荧光显微镜观察脂质体对绿荧光蛋白质粒DNA转染的促进作用。结果所得脂质体的形态规整,粒径均匀,平均粒径为94.0 nm,Zeta电位为24 m V。当阳离子脂质体和DNA的正负电荷比大于4∶1时,脂质体能将DNA完全结合,且能保护DNA免受DNase I酶的降解。所得脂质体具有较低的毒性,其细胞摄取效率不如DC-胆固醇脂质体,但转染效率明显好于后者。结论 N-(N',N'-二甲基)丙基丁二酸单胆固醇酯单酰胺是一种高效低毒的基因载体,具有较强的应用前景。

OBJECTIVE To synthesize a novel cationic cholesterol derivative,N-（ N＇,N＇-dimethyl） propyl succinic mono-cholesteryl mono-amide（ DMAPA-CHEMS）,and evaluate its feasibility as a none-viral gene vehicle. METHODS DMAPA-CHEMS and phospholipid were employed to produce liposomes by thin membrane dispersion method,and the morphology of liposomes was observed under transmission electron microscope. The average diameter and Zeta potential were determined by laser particle size analyzer. The combination to and protection for DNA were investigated via agarose gel electrophoresis. The uptake of FITC-labelled oligonucleotides into Hep G2 cells was determined by flow cytometry,while the transfection promotion of liposomes for GFP-plasmid DNA was observed by inverted fluorescence microscope. RESULTS The obtained liposomes showed regular shape and uniform size,with a mean diameter of 94. 0 nm and Zeta potential of 24 m V. The liposomes could combine DNA completely and protect DNA from the degradation of DNase I when the charge ratio of cationic cholesterol to DNA was more than 4. The cationic liposomes had low cytotoxicity. Although the uptake efficiency of liposomes made of DMAPA-CHEMS was lower than that of DC-Chol,the gene expression efficiency was higher. CONCLUSION DMAPACHEMS is a novel gene carrier with high transfection efficiency and low cytotoxicity,which has a great potential for application.