A NEW NONVIRAL DNA DELIVERY VECTOR - THE TERPLEX SYSTEM

A new DNA delivery vector (the terplex system) based on a balanced hyd rophobicity and net surface charge between stearyl-poly(L-lysine), low density lipoprotein (LDL), and genetic material (i.e. plasmid DNA or antisense oligonucleotide) was developed. The pSV-beta-gal plasmid in terplex system showed a 2-5-fold increase in beta-galactosidase expres sion on murine smooth muscle cells (A7R5) compared to Lipofectin(TM). Delivery of unmodified c-myb antisense oligonucleotide to A7R5 cells w as also facilitated significantly by the terplex system, requiring as little as 5.4 nM of antisense oligonucleotide to achieve a 50% antipro liferative effect. Similar antiproliferative effect was observed when the c-myb antisense/terplex formulation was tested on CCD-32 Lu human lung fibroblasts. Characterization of the physical properties of the t erplex system was performed using various techniques. Plasmid DNA was condensed by addition of stearyl-PLL and LDL, resulting in the terplex system of about 100 nm in diameter as shown by atomic force microscop y. A strong hydrophobic interaction between stearyl-poly(L-lysine) and LDL was registered by H-1-NMR spectrometry, showing a significant dec rease in the epsilon-methylene signal of poly(L-lysine) backbone when stearyl-poly(L-lysine) was mixed with LDL; however, this phenomenon wa s not observed with unmodified poly(L-lysine). Agarose gel electrophor esis revealed that electrophoretic mobility of the terplex system decr eased with increasing amounts of stearyl-poly(L-lysine), indicating th at the surface charge of the terplex system became more positive by ad dition of stearyl-poly(L-lysine). Zeta-potential measurement showed th at the terplex system exerted a slightly positive charge (+2 mV) at a 1:1:1 weight ratio of plasmid DNA:LDL:stearyl-poly(L-lysine). The obta ined results will be utilized in;he design of more efficient and safer DNA delivery vectors for in vivo gene therapy. (C) 1998 Elsevier Scie nce B.V.