核酸载体lipopolyplex 的制备及细胞毒性研究

目的：为降低聚阳离子基因载体polyplex 的正电荷和毒性，在其表面构建中性磷脂膜制备lipopolyplex，并测定lipopolyplex 对小鼠结肠癌细胞CT26 和人乳腺癌细胞MCF-7 的细胞毒性。方法：采用PEI25KDa与DNA 复合制备polyplex，在polyplex 体系 中加入中性脂质体和SADGE 制备lipopolyplex。采用琼脂糖凝胶电泳考察lipopolyplex 对质粒DNA的包裹能力；采用激光粒度 仪和zeta 电位分析仪测定lipopolyplex 的粒径与zeta 电位；采用透射电镜观察lipopolyplex 的形态；采用CCK-8 试剂盒考察 lipopolyplex 对CT26和MCF-7 的细胞毒性。结果：琼脂糖凝胶电泳显示lipopolyplex 可以完全包裹质粒DNA；lipopolyplex 的粒 径在200 nm 左右，电位在-20 mV 左右；透射电镜下为较为规则的球状颗粒；lipopolyplex 在CT26 和MCF-7 细胞中的毒性明显 低于聚阳离子基因载体polyplex。结论：在polyplex 表面成功构建中性磷脂膜制备的lipopolyplex，可以完全的包裹DNA 并且细 胞毒性明显低于polyplex，在基因输送载体领域具有潜在应用价值。

Preparation and Cytotoxicity Studies of Nucleic Acids Vectors Lipopolyplex

Objective:To prepare lipopolyplex by assembing a stable neutral lipid membrane around polyplex surface in order to reduce positive charge and cytotoxicity of polyplex. Investigate cell cytotoxicity of lipopolyplex in CT26 and MCF-7 cells.Methods:Polyplex was prepared by PEI25KDa and DNA, then neutral liposomes and SADGE solutions were added into polyplex to prepare lipopolyplex. The ability to wrap plasmid DNA of lipopolyplex was examined by agarose gel electrophoresis(AGE). Particle size and zeta potential of lipopolyplex were measured by particle sizer and zeta potential analyzer. The morphology of lipopolyplex was observed by transparent electron microscopic (TEM). The cytotoxicity of lipopolyplex in CT26 and MCF-7 cells was detected by CCK-8 method.Results:The results of AGE showed that the plasmid was fully protected by lipopolyplex. The particle size was about 200 nm and the zeta potential was around -20 mV. The results of TEM showed that the lipopolyplex could display as regular spherical particles. The cytotoxicity of lipopolyplex in CT26 and MCF-7 cells was significantly lower than polyplex, respectively.Conclusion:The lipopolyplex prepared by building a neutral lipid membrane around polyplex surface has a lower zeta potential and cytotoxicity compared with polyplex. It has potential application value in the field of gene delivery vectors.