In vitro gene expression on smooth muscle cells using a terplex delivery system

In vitro gene expression was studied using a terplex gene delivery system consisting of plasmid DNA (pSV-beta-gal), low density lipoprotein, and hydrophobized poly-L-lysine (H-PLL) on A7R5 murine smooth muscle cells (SMC). A positively-charged biodegradable polymer with hydrophobic side chain (25 mol % of C-18-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 transfection efficiency was tested on SMC. The terplex system showed a 2- to 5-fold increase in transfection efficiency of plasmid DNA (pSV-beta-gal) on A7R5 cells in the presence of serum, compared to the complex of DNA with H-PLL, or DNA with Lipofectin(TM). Transfection efficiency of the terplex system in the absence of serum also showed a 1.5-fold increase, compared to Lipofectin(TM) formulation. Pretreatment of the cells with 100 mu 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 terplex via receptor-mediated endocytosis. The association and/or internalization of the DNA terplex system into the cells was inhibited either by the presence of 100 mu 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.