DESIGN OF CELL SPECIFIC CARRIERS FOR GENES AND PEPTIDES

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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.