Background It is a key step to transfect the therapeutic gene into target tissue safely and effectively.Because of its potential pathogenicity,immunogenicity and mutagenicity,viral vector is limited in clinical application.Therefore,nonviral vector is becoming a study hotpot due to its security,low p rice and flexibility.However,the study of octadecyl-quaternized lysine modified chitosan/cholesterol (OQLCS/chol) as genetic vector is rare.Objective This study was to investigate the optimal conditions for gene transfection by new polymeric liposome-OQLCS/chol.Methods Human retinal pigment epithelial (RPE) cells were cultured in modified 1640 medium containing fetal bovine serum.Plasmid DNA was prepared and extracted based on the sequence of human vascular endothelial growth factor (VEGF) from GeneBank.The loading efficiency of OQLCS/chol lipidosome to plasmid DNA was clarified by electrophoresis to select the optimal tranfected conditions,including the mass ratio,action time as well as with or without blood serum.The dissolution curve of OQLCS/chol lipidosome was drafted by supercentrifuge process.The RPE cells were transfected by OQLCS/chol lipidosome and Lipo2000,respectively,and the transfected efficiency was detected by green fluorescence protein (GFP) and compared between the OQLCS/chol lipidosome group and Lipo2000 group.The in vitro survival rate of the cells was evaluated by cell counting kit-8(CCK-8).Results The average size of OQLCS/chol liposome nanoparticles was 134 nm,and the Zeta potential was ±39.64 mV.About 70％ of total encapsulated plasmid DNA was rapidly released within initial 5 days followed by a constant and sustained release for 14 days.This new polymeric liposome was safe for biological use under the 20 μg/ml,with the optimal OQLCS/chol:plasmid DNA of ≥2∶1 and incorporation time of 30-40 minutes in the free-serum medium.The transfected cell rate was 71.05％ in the OQLCS/chol liposome group,and that in the Lipo2000 group was 70.58％,showing a significant difference between the two groups (x2 =0.534,P =0.465).Conclusions OQLCS/chol loposome is synthesized successfully with uniform size,better stability,less cytotoxicity,higher Zeta potential and cellular uptake efficiency in comparison with Lipo2000.This novel polymeric liposome loaded plasmid DNA can transfect VEGF siRNA into RPE cells efficiently like a Lipo2000.%背景 将目的基因安全、有效地导入并使其适度表达是基因治疗的关键.病毒载体具有潜在的致病性、免疫原性及致突变性等缺点,而非病毒载体以其安全、价廉、灵活性成为目前研究的热点.目前,O-羧甲基壳聚糖十八烷基季铵盐/胆固醇(OQLCS/chol)脂质体作为基因载体的相关研究鲜见报道. 目的 研究OQLCS/chol脂质体的性能及其作为非病毒载体的最适转染条件. 方法 常规培养人视网膜色素上皮(RPE)细胞株;根据GeneBank人(NM-00171626)血管内皮生长因子(VEGF) mRNA的已知序列制备和抽提质粒DNA,采用电泳阻滞法研究OQLCS/chol脂质体与质粒DNA在不同质量比、不同作用时间及是否存在血清等条件下的包载效能,筛选最优转染条件;采用高速离心法进行体外释放动力学实验,绘制体外释放曲线;将培养的细胞分为OQLCS/chol脂质体组和Lipo2000组,采用共培养法分别用OQLCS/chol脂质体和Lipo2000转染R PE细胞,采用绿色荧光蛋白(GFP)染色法比较和评价其转染效率;利用细胞计数试剂盒-8(CCK-8)检测OQLCS/chol脂质体转染后RPE细胞的存活率,确定OQLCS/chol脂质体的最佳转染安全质量浓度. 结果 OQLCS/chol脂质体纳米粒平均有效粒径为134 nm,Zeta电位为±39.64 mV.体外释放实验显示,载质粒DNA的OQLCS/chol脂质体的体外快速释放相在最初5d,质粒释放量约为70.0％,至14 cl缓释量呈稳态相.OQLCS/chol的最高安全质量浓度为20 μg/ml,OQLCS/chol与质粒DNA质量比≥2∶1、混合时间为30～40 min、无血清培养条件下转染条件为最优.载质粒DNA的OQLCS/chol脂质体转染组RPE细胞转染率为71.05％,lLipo2000转染组为70.58％,差异无统计学意义(x2=0.534,P=0.465). 结论 自行制备的跨膜肽修饰的OQLCS/chol脂质体具有粒径均匀、分散性好、Zeta电位高、细胞毒性小、能在较短时间释放达到治疗质量浓度的基因且后续可缓慢平稳释放等特性.OQLCS/chol脂质体将VEGFsiRNA转染到RPE细胞的转染效率与Lipo2000接近.
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