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Biotechniques. 1998 Aug;25(2):282-4, 286-92.

Enhanced gene transfer into HuH-7 cells and primary rat hepatocytes using targeted liposomes and polyethylenimine.

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  • 1University of Minnesota Medical School, Minneapolis, USA.

Abstract

Different ratios of DNA phosphate to polyethylenimine amine were used for encapsulation and delivery to liver cells of chloramphenicol acetyl transferase (CAT) or luciferase expression plasmids in cationic, neutral and anionic liposomes. Positive liposomes consisted of dioleoyl phosphatidylcholine (DOPC): dioleoyl trimethylammonium propane (DOTAP) (6:1 molar ratio); neutral liposomes were composed of DOPC and dioleoyl phosphatidylethanolamine (DOPE) (1:1); and negative liposomes contained dioleoyl phosphatidylserine (DOPS) and DOPC (1:1). All formulations included 8 mol% galatocerebroside for targeting to the hepatocyte asialoglycoprotein receptor. Liposomes were prepared by film hydration followed by sequential extrusion through 0.8-0.2 mumol polycarbonate membranes. Transfection efficiency of HuH-7 human hepatoma cells and isolated rat hepatocytes was determined by CAT enzyme-linked immunosorbent assay (ELISA) or luciferase activity. Uptake of liposomal-encapsulated, fluorescently labeled 68-mer oligonucleotides was assessed by confocal microscopy. All three formulations demonstrated a twofold or greater increase in transfection efficiency and significantly lower toxicity compared to nonencapsulated polyethylenimine complexes. Negative liposomes were most effective, particularly in the rat hepatocytes. Only the cationic and anionic liposomal formulations exhibited significant thermodynamic stability. These formulations are readily characterized for size, phospholipid and DNA content, and they represent feasible systems for optimizing in vivo delivery systems to hepatocytes.

PMID:
9714889
[PubMed - indexed for MEDLINE]
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