Peptide-mediated Gene Transfer of Cationic Lipid/Plasmid DNA Complexes to Endothelial Cells

Peptide-mediated Gene Transfer of Cationic Lipid/Plasmid DNA Complexes to Endothelial Cells
May 2004
K. Anwer1; G. Kao2; A. Rolland3; W.H.P. Driessen4; S.M. Sullivan4
1: Expression Genetics Huntsville AL USA
2: Valentis The Woodlands TX USA
3: Vical, Inc. San Diego CA USA
4: Department of Pharmaceutics, College of Pharmacy University of Florida Gainesville FL USA
Journal of Drug Targeting, May 2004, vol. 12, no. 4, pp. 215-221(7)
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Abstract:
The purpose of this research is to develop ligand-targeted plasmid based gene delivery systems for gene transfer to tumor endothelium. Cell adhesion assays were used to test the peptide inhibition of human endothelial cell adsorption to vitronectin-treated tissue culture plates. A series of RGD containing peptides were tested in linear form and with one and two disulfide bonds. The linear and two disulfide bond peptides yielded similar IC50 (ap1 ? 10-7 M). Substitution of two methionines for cysteines yielded a single disulfide bond that increased the IC50 by 10-fold. The single and double disulfide peptides were derivatized to N-succinyl-dioleoylphopsphatidylethanolamine and incorporated into 100 nm liposomes radiolabeled with 3H-cholesterylhexadecylether. Liposome uptake by human umbilical vein endothelial cells was tested as a function of lipopeptide surface density. Increase in membrane surface density from 5 to 20 mol% increased human umbilical derived endothelial cell (HUVEC) uptake of the liposomes for both the single and double disulfide peptides. Liposome uptake by HUVECs was 3-fold greater for the double disulfide compared to the single disulfide. The single and double disulfide lipopeptides were then tested for gene transfer to HUVECs using DOTMA:Cholesterol cationic liposomes. The polyplexes were formed by rapidly mixing plasmid DNA with DOTMA:CHOL liposomes at a 3:1 charge ratio in 2% ethanol, 10% lactose. The ethanol was removed by lyophilization and upon rehydration, the lipoplexes had a mean diameter of approximately 100 nm. HUVEC transfection studies showed that increasing the mol% of the single disulfide RGD lipopeptide to 20 mol% increased gene transfer by 10-fold. This increase in transfection could be reduced to that obtained in the absence of lipopeptide by co-incubating the HUVECs with a 100-fold excess of the single disulfide RGD peptide, thus demonstrating lipopeptide mediated gene transfer to endothelial cells.