Development of peptide-targeted lipoplexes to CXCR4-expressing rat glioma cells and rat proliferating endothelial cells.

A peptide analog, 4-fluorobenzoyl-RR-(L-3-(2-naphthyl)alanine)-CYEK-(L-citrulline)-PYR-(L-citrulline)-CR, covalently linked to a phospholipid, was used for targeting a lipid-based gene delivery vehicle to CXCR4(+)-cells. Characterization of transfection activity was done in vitro using a transformed rat glioma cell line (RG2) that expresses CXCR4. The substitution of the targeting lipid at increasing mole percentages in the place of helper lipids yielded a progressive increase in reporter gene expression, reaching a maximum of 2.5 times the control value at 20 mol% of ligand. The substitution of helper lipids with cysteine-derivatized phospholipid analog or phosphatidylethanolamine resulted in a progressive decrease in transfection activity, with complete inactivation of the complex occurring at 20 mol%. A DNA dose-response with 10 mol% of lipopeptide reduced the effective DNA dose at least fivefold with regard to the number of transfected cells and >20-fold with regard to the amount of gene expression. Gene transfer to rat endothelial cells was studied in the context of an arterial organ culture. Mesenteric arteries were cannulated and maintained in culture for up to 4 days. CXCR4 cell-surface expression on endothelial cells was induced after overnight incubation with vascular endothelial growth factor (VEGF). Gene transfer studies showed that only the peptide-targeted lipoplexes transfected the endothelium, and only after CXCR4 had been induced with VEGF. These results demonstrate that non-viral transfection complexes can be targeted to cells expressing CXCR4, and that gene transfer is dependent upon cell surface receptor expression levels.