DESIGN OF CELL SPECIFIC CARRIERS FOR GENES AND PEPTIDES

采用由质粒DNA、低密度脂蛋白和疏水聚赖氨酸组成的双体基因传递系统在A7R5小鼠平滑肌细胞（SMC）上的体外表达研究。以聚l -赖氨酸为骨架合成了一种带正电荷、疏水侧链（c18 -硬脂酰25摩尔%）的可降解聚合物。将不同重量比的DNA/H-PLL/LDL形成复合体系，并在SMC上测试其体外转染效率。与DNA与H-PLL复合物或DNA与LipofectinTM复合物相比，在血清存在的情况下，质粒DNA在A7R5细胞上的转染效率提高了2- 5倍。与LipofectinTM制剂相比，在没有血清的情况下，复合系统的转染效果也增加了1.5倍。转染前用100微米氯喹预处理细胞30分钟，可使复合物系统的转染效率提高30%。流式细胞分析表明，DNA应该与H-PLL结合，以实现有效的细胞结合和摄取，LDL通过受体介导的内吞作用帮助细胞内化。100微米EDTA或过量的游离LDL的存在抑制了DNA复合物系统进入细胞的结合和/或内化，这表明LDL通过受体介导的方式在复合物系统的内吞作用中起重要作用。当H-PLL与LDL络合时，在本实验使用的浓度范围内未观察到H-PLL引起的相当大的细胞毒性。综上所述，该结果表明，H-PLL/DNA/LDL复合体系实现了相当程度的外源基因传递及其在细胞内的表达，为开发高效、安全的体内基因治疗基因传递载体提供了有益的信息。

In vitro gene expression was studied using a teplex gene delivery system consisting of plasmid DNA,low density lipoprotein, and hydrophobized poly-L-lysine on A7R5 murine smooth muscle cells (SMC). A positively charged giodegradable polymer with hydrophobic side chain (25 mol % of C18-stearyl group) was synthesized using poly-L-lysine as a backbone. A terplex system was formed from various weight ratios of DNA/H-PLL/LDL and its in vitro trahsfection efciency was tested on SMC.The terplex system showed a 2-to 5-fold increase in transfection efficiency of plasmid DNA on A7R5 cells in the presence of serum, compared to the complex of DNA with H-PLL,or DNA with LipofectinTM.Transfection efficacy of the terplex system in the absence of serum also showed a 1.5-fold increase, comopared to LipofectinTM formulation. Pretreatment of the cells with 100 micro M chloroquine for 30 min prior to the transfection resulted in a 30% increase in transfection efficiency of the terplex system. Flow cytometric analysis indicates that DNA should be bound to H-PLL for efficient cellular binding and uptake, and LDL helps internalization of the terples via receptor-mediated endocytosis. The association and/or internalization of the DNA terplex system into cells was inhibited either by the presence of 100 micro M EDTA or excess amount of free LDL,suggesting that LDL plays an important role in the endocytosis of the terplex system by a receptor-mediated fashion. Considerable cytotoxicity due to the H-PLL was not observed at the concentration range used for this experiment when H-PLL was complexed with LDL.In conclusion, this result indicates that a significant degree of exogenous gene delivery and its intracellular expression was achieved by the H-PLL/DNA/LDL terplex system, providing useful information for the development of efficient and safe gene delivery vectors for in vivo gene therapy.