MT肽修饰阳离子脂质体作为新型基因载体的体外细胞学评价

The in vitro cytological evaluation of cationic liposome modified by MT peptide as a novel gene vector

目的通过体外细胞学实验对MT肽修饰的阳离子脂质体进行基因载体递送能力的评价。方法通过点击反应制备DSPE-PEG2000-MT用于修饰阳离子脂质体,并通过1H-NMR对DSPE-PEG2000-MT进行结构表征。以能够表达增强型绿色荧光蛋白的pEGFP-C1报告基因为模型基因,首先通过琼脂糖凝胶电泳实验进行复合物包封率测定,选出最佳质量比,然后应用A549细胞对其进行细胞毒性评价、报告基因转染与表达研究,以及细胞摄取实验。以PEG2000修饰阳离子脂质体和空白阳离子脂质体为对照,充分研究MT肽修饰阳离子脂质体作为基因递送系统的潜力。结果MT肽修饰阳离子脂质体(cationic liposomes modified by DSPE-PEG2000-MT,MCL)对比参照阳离子脂质体(cationic liposomes,CL;cationic liposomes by DSPE-PEG2000,PCL)具有更高的稳定性及包封率;并通过细胞毒性实验验证MT肽修饰阳离子脂质体的细胞毒性较低;通过共聚焦显微镜和流式细胞仪验证该复合物具有更好的细胞摄取能力,能够促进绿色荧光蛋白的表达;而且通过时间、温度依赖性实验表明了该摄取行为与时间和温度有一定的关系。结论MT肽修饰的阳离子脂质体拥有较好的稳定性,较低的细胞毒性以及更好的细胞摄取能力,是一个十分有潜力的基因递送载体。

Objective To evaluate the gene delivery ability of cationic liposomes modified by MT peptide in vitro cytology experiments.Methods DSPE-PEG2000-MT was prepared by click reaction to modify cationic liposomes,and the structure of DSPE-PEG2000-MT was characterized by 1H-NMR.The pEGFP-C1 reporter gene was capable of expressing enhanced green fluorescent protein and was used as a model gene.First,the agarose gel electrophoresis experiment was carried out to determine the encapsulation efficiency of the composite,and the best mass ratio was selected.Then A549 cells were used for cytotoxicity evaluation,reporter gene transfection and expression studies,and cell uptake experiments.PEG2000 modified cationic liposomes were compared with blank cationic liposomes to fully study the potential of MT peptide modified cationic liposomes as a gene delivery system.Results MCL(cationic liposomes modified by DSPE-PEG2000-MT)had higher stability and encapsulation efficiency than the reference complexes,and the cytological experiments confirmed that the MCLP could express green fluorescent protein more effectively,which was determined by confocal microscopy and flow cytometry.Conclusion MT peptide modified cationic liposomes has good stability,low cytotoxicity and higher cell uptake capacity,making them as a very potential gene delivery vehicle.