ABSTRACT
To enhance gene transfection to hepatocytes by cationic liposomes, it is necessary to overcome a number of barriers existing in the process from administration to gene expression. Recently we and other group have demonstrated that the escape of plasmid DNA (pDNA)/cationic liposome complexes (lipoplexes) from the endosome to cytoplasm was rate limiting. In this study, to enhance transfection efficiency by promoting the release of lipoplexes from the endosome to cytoplasm, we proposed utilizing the "proton sponge effect". Here, we synthesized a novel pH-sensitive histidine-modified galactosylated cholesterol derivative (Gal-His-C4-Chol), for a more efficient gene delivery to hepatocytes. Liposomes containing Gal-His-C4-Chol showed much greater transfection activity than conventional Gal-C4-Chol liposomes based on a receptor-mediated mechanism in HepG2 cells. Hence, this finding should contribute to the development of gene therapy using cationic liposomes toward their clinical application.
Subject(s)
Asialoglycoprotein Receptor/metabolism , Carcinoma, Hepatocellular/metabolism , Cholesterol Esters/metabolism , DNA/metabolism , Glycopeptides/metabolism , Liposomes , Liver Neoplasms/metabolism , Transfection/methods , Active Transport, Cell Nucleus , Animals , Cations , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Survival/drug effects , Cholestenes/metabolism , Cholesterol Esters/chemistry , Cholesterol Esters/toxicity , Cytoplasm/metabolism , DNA/chemistry , Endosomes/metabolism , Genes, Reporter , Glycopeptides/chemistry , Glycopeptides/toxicity , Humans , Hydrogen-Ion Concentration , Luciferases , Mannitol/metabolism , Mice , NIH 3T3 Cells , Particle SizeABSTRACT
In this study, we evaluated the effect of blood components (whole blood and serum) on asialoglycoprotein receptor-mediated in vivo gene transfer. The hepatic transfection activity of galactosylated lipoplex preincubated with serum was approximately 10 times higher than that without incubation after intraportal injection in mice. However, preincubation with whole blood significantly reduced hepatic transfection activity. Fluorescent resonance energy transfer analysis and agarose gel electrophoresis revealed that preincubation with serum reduced the degree of destabilization of the galactosylated lipoplex in blood, partially supporting enhanced hepatic transfection activity by preincubation with serum. Inhibition of hepatic transfection activity by predosing galactosylated bovine serum albumin indicated that the galactosylated lipoplex exposed to serum is recognized by asialoglycoprotein-receptors on hepatocytes. Inactivation of serum prior to mixing with galactosylated lipoplex reduced liver accumulation and completely abolished enhancement of hepatic transfection activity by preincubation with active serum, suggesting that not only the stability of the lipoplex in blood but also the serum opsonin activity plays important roles. Alternatively, preincubation with inactivated serum reduced the lung accumulation and inflammatory cytokine production of galactosylated lipoplex. The information provided by this study will be valuable for the future use, design, and development of galactosylated lipoplex for in vivo asialoglycoprotein receptor-mediated gene transfer.
Subject(s)
Asialoglycoprotein Receptor/metabolism , Blood , Gene Transfer Techniques , Animals , Cytokines/blood , Cytomegalovirus/genetics , DNA/administration & dosage , DNA/chemistry , Drug Carriers , Female , Liposomes/chemistry , Liver/metabolism , Luciferases/genetics , Male , Mice , Mice, Inbred ICR , Plasmids/genetics , Rats , Rats, Wistar , Tissue Distribution , TransfectionABSTRACT
In this study, we demonstrated that the presence of an essential amount of sodium chloride (NaCl) during the formation of cationic liposome/plasmid DNA complexes (lipoplexes) stabilizes the lipoplexes according to the surface charge regulation (SCR) theory. Fluorescence resonance energy transfer analysis revealed that cationic liposomes in an SCR lipoplex (5 and 10 mM NaCl solution in lipoplex) increased fusion. Also, aggregation of SCR lipoplexes was significantly delayed after exposure to saline (150 mM NaCl) as a model of physiological conditions. After intraportal administration, the hepatic transfection activity of galactosylated SCR lipoplexes (5 and 10 mM NaCl solution in lipoplex) was approximately 10- to 20-fold higher than that of galactosylated conventional lipoplexes in mice. The transfection activity in hepatocytes of galactosylated SCR lipoplexes was significantly higher than that of conventional lipoplexes, and preexposure to competitive asialoglycoprotein-receptor blocker significantly reduced the hepatic gene expression, suggesting that hepatocytes are responsible for high hepatic transgene expression of the galactosylated SCR lipoplexes. Pharmacokinetic studies both in situ and in vivo demonstrated a higher tissue binding affinity and a greater expanse of intrahepatic distribution by galactosylated SCR lipoplexes. Moreover, enhanced transfection activity of galactosylated SCR lipoplexes was observed in HepG2 cells, and investigation of confocal microscopic images showed that the release of plasmid DNA in the cell was markedly accelerated. These characteristics partly explain the mechanism of enhanced in vivo transfection efficacy by galactosylated SCR lipoplexes. Hence, information in this study will be valuable for the future use, design, and development of ligand-modified lipoplexes for in vivo applications.
