DESIGN OF CELL SPECIFIC CARRIERS FOR GENES AND PEPTIDES

在体外基因表达进行了研究，使用teplex基因传递系统组成的质粒DNA，低密度脂蛋白，和疏水化的聚-L-赖氨酸对A7 R5小鼠平滑肌细胞（SMC）。以聚L-赖氨酸为骨架，合成了一种带正电荷的疏水侧链（25mol%的C18-硬脂基）的生物可降解聚合物。以不同重量比的DNA/H-PLL/LDL组成复合体系，在SMC上进行体外转染实验，结果表明，在血清存在下，复合体系的质粒DNA对A7 R5细胞的转染效率比DNA与H-PLL复合物提高2 ~ 5倍，在不存在血清的情况下，与LipofectinTM制剂相比，terplex系统的转染效率也显示出1.5倍的增加。在转染前用100 μ M氯喹预处理细胞30分钟，导致三重系统的转染效率增加30%。流式细胞仪分析表明，DNA应结合到H-PLL有效的细胞结合和摄取，和LDL有助于通过受体介导的内吞作用的三重内化。100 μ M EDTA或过量的游离LDL均可抑制DNA三重体系统的结合和/或内化，表明LDL通过受体介导的方式在三重体系统的内吞作用中起重要作用。结果表明，H-PLL/DNA/LDL复合物系统可实现外源基因的有效传递和细胞内表达，为开发高效、安全的体内基因治疗载体提供了有用的信息。

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.