磁性阳离子高分子脂质体结合野生型p53投递系统的构建

目的用合成的磁性阳离子高分子脂质体与野生型p53(WTp53)基因质粒结合,构建靶向纳米载体投递系统,为进一步行静脉内皮细胞转染奠定基础。方法应用大肠杆菌重组质粒扩增法扩增WTp53质粒DNA,合成的磁性阳离子高分子脂质体为羧甲基壳聚糖十八烷基季铵盐/胆固醇(OQCMC/Chol)包覆油溶性Fe3O4的载体粒子。在不同的pH值及不同的WTp53质粒DNA和载体粒子质量比的条件下,将WTp53质粒DNA与OQCMC/Chol超微磁性载体粒子进行混合,并分别进行DNA结合实验和沉淀实验。观察电泳结果,并用SPSS 13.0统计软件进行数据分析。在DNaseⅠ模仿体内酶的环境下,将其加入制备好的超微载体投递系统中,电泳后观察投递系统对DNA的保护作用。应用透析系统模拟体内环境,观察超微投递系统载体与基因分离情况,并利用紫外分光光度计测量超微载体投递系统累积缓释曲线。结果应用透射电镜观察OQCMC/Chol超微磁性载体粒子和WTp53结合后的投递粒子。在pH 7条件下,WTp53质粒DNA和载体粒子质量比为1∶0.6,p53质粒DNA与OQCMC/Chol超微磁性载体粒子经过直接静电作用几乎100%结合,并证实该投递系统对DNA具有较好的保护作用。超微载体投递系统在7～12 h左右有明显的突释波峰,到第54小时以后,超微载体释放浓度变化缓慢,并可持续释放9 d以上。结论成功构建磁性阳离子高分子脂质体结合WTp53投递系统,将为进一步行细胞转染奠定可靠的基础,并为构建血管内靶向定位基因投递新方法和新技术提供可靠理论依据。

Construction and identification of magnetic cationic polymeric liposome combined with WTp53

Objective To construct the target gene-carrier nanodelivery system through magnetic cationic polymeric liposome combined with wild type p53 ge/ne(WTp53) plasmid. Methods The WTp53 DNA was amplified by alkaline lysis-neutralization method.Magnetic cationic polymeric liposome was made from the ocradecyl quaternized carboxymethyl chitonsan/cholesterol(OQCMC/Chol).The OQCMC coated by magnetic iron oxide nanoparticles was produced by transonic emulsification and coprecipitation method.Under different pH of solutions,WTp53 DNA was mixed with nanoparticles through different quality ratios.The DNA combination tests and DNA precipitation tests were performed on the mixtures.The electrophoresis results were observed.DNase I and dialysis system were used for imitating in vivo environment.The release curve of nanodelivery system was evaluated by ultraviolet spectrophotometer. Results Under neutral pH and quality ratios of 1∶0.6 or higher,the WTp53 DNA could almost fully combine with OQCMC coated magnetic iron oxide nanoparticles and a good protection for DNA of nanodelivery system were observed.Release experiment showed that the nanodelivery system had a sudden release at 7 to 12 hours and slowed down after 54 hours.The release could last for more than nine days. Conclusions The delivery system,OQCMC/Chol coated magnetic iron oxide combined with WTp53 DNA,may be used for further investigation in the cell transfection.