ENHANCED GENE-TRANSFER INTO HUH-7 CELLS AND PRIMARY RAT HEPATOCYTES USING TARGETED LIPOSOMES AND POLYETHYLENIMINE

Different ratios of DNA phosphate to polyethylenimine amine were used for encapsulation and deliver?, to liver cells of chloramphenicol acet yl;vl transferase (CAT) or luciferase expression plasmids in cationic, neutral and anionic liposomes. Positive liposomes consisted of dioleo yl phosphatidylcholine (DOPC):dioleoyl trimethylammonium propane (DOTA P) (6:1 molar ratio); neutral liposomes were composed of DOPC and diol eoyl phosphatidylethanolamine (DOPE) (1:1) and negative liposomes cont ained dioleoyl phosphatidylserine (DOPS) and DOPC (1:I). All formulati ons included 8 mol% galactocerebroside for targeting to the hepatocyte asialoglycoprotein receptor Liposomes were prepared by film hydration followed by sequential extrusion through 0.8-0.2-mu m polycarbonate m embranes. Transfection efficiency of HuH-7 human hepatoma cells and is olated rat hepatocytes was determined by CAT enzyme-linked immunosorbe nt assay (ELISA) or luciferase activity Uptake of liposomal-encapsulat ed fluorescently labeled 68-mer oligonucleotides was assessed by confo cal microscopy. All three formulations demonstrated a twofold or great er increase in transfection efficiency and significantly lower toxicit y compared to nonencapsulated polyethylenimine complexes. Negative lip osomes 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 sy stems for optimizing in vivo delivery systems to hepatocytes.