Peptide-mediated gene transfer of cationic lipid/plasmid DNA complexes to endothelial cells.
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 (approximately 1 x 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 H-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 20mol% 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 100nm. HUVEC transfection studies showed that increasing the mol% of the single disulfide RGD lipopeptide to 20mol% 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.
Anwer
K
K
Expression Genetics, Huntsville, AL, USA.
Kao
G
G
Rolland
A
A
Driessen
W H P
WH
Sullivan
S M
SM
D002412:Cations; D002448:Cell Adhesion; D002784:Cholesterol; D004220:Disulfides / Q000737:chemistry; D042783:Endothelial Cells / Q000378:metabolism* / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000096:biosynthesis; D008024:Ligands; D008081:Liposomes; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids; D000644:Quaternary Ammonium Compounds; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D002412:Cations; D004220:Disulfides; D039302:Integrin alphaVbeta3; D008024:Ligands; D008081:Liposomes; D009842:Oligopeptides; D010455:Peptides; D000644:Quaternary Ammonium Compounds; C054005:N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium; C047981:arginyl-glycyl-aspartic acid; D002784:Cholesterol; D008156:Luciferases