Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes.
Unilamellar activated cationic liposomes containing 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol, dioleoyl phosphatidylethanolamine (DOPE) and the N-hydroxysuccinimide ester of cholesteryl hemisuccinate (4:5:1, molar ratio) have been prepared and their DNA-binding capacity has been assessed in a gel retardation assay. Ternary complexes composed of activated cationic liposomes, carbodiimide-cationized asialoorosomucoid (Me+AOM) and pRSVL plasmid DNA were assembled for receptor-mediated DNA delivery into cells expressing the asialoglycoprotein receptor (ASGP-R). Binding of complexes in which Me+AOM was replaced by fluoresceinated Me+AOM (FMe+AOM) to the human hepatocellular cell line HepG2 at 4 degrees C was severely reduced by co-incubation with asialoorosomucoid (AOM). Moreover, assemblies containing liposomes, pRSVL DNA and Me+AOM (8:1:4, w/w/w) promoted high levels of luciferase activity in this cell line (1.3 x 10(7) relative light units/mg soluble cell protein). Assays conducted in the presence of a hundred-fold excess of the ligand AOM afforded considerably lower levels of transfection (2.5 x 10(5) relative light units/mg soluble cell protein). In contrast, the highest level of luciferase activity achieved with liposome, pRSVL DNA, AOM complexes was only a quarter of the best levels obtained with liposome, pRSVL DNA, Me+AOM assemblies. These findings strongly support the notion that complexes gain entry into hepatocyte-derived cells by ASGP-R mediation and that they are potentially useful gene carriers to liver hepatocytes.
Biochemistry, School of Biochemistry and Microbiology, Faculty of Science, University of Durban-Westville, Private Bag X54001, Durban, South Africa.
Moganavelli
M
Singh
Mario
M
Ariatti
D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D001212:Asialoglycoproteins / Q000737:chemistry* / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002788:Cholesterol Esters / Q000737:chemistry; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry; D004337:Drug Carriers / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008081:Liposomes / Q000737:chemistry*; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008745:Methylation; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry; D014162:Transfection / Q000379:methods*
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D002788:Cholesterol Esters; D004337:Drug Carriers; D008081:Liposomes; D009961:Orosomucoid; D010714:Phosphatidylethanolamines; D013388:Succinimides; C017030:asialoorosomucoid; D004024:Dicyclohexylcarbodiimide; D004247:DNA; D008156:Luciferases; C001426:N-hydroxysuccinimide; C013440:cholesteryl succinate