Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer into hepatic cells by asialofetuin-labeled liposomes

Abstract

Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer using asialofetuin-labeled liposomes (AF-liposomes) were examined with HepG2 cells and rat hepatocytes in primary culture. AF-liposomes were sufficiently taken up by both types of cells through the asialoglycoprotein receptor-mediated endocytosis. In HepG2 cells, bacterial beta-galactosidase (beta-Gal) gene expression was observed by transfection using AF-liposomes encapsulating plasmid pCMV beta DNA (AF-liposome-pCMV beta). By addition of dioleoylphosphatidylethanolamine (DOPE) to the liposomal lipid composition (AF-liposome(DOPE)-pCMV beta), the transfection efficiency was clearly increased. The effects of DOPE were more conspicuous in the presence of chloroquine in the medium throughout the transfection. When pCMV beta complexed with gramicidin S (pCMV beta (GrS)) was encapsulated (AF-liposome(DOPE)-pCMV beta (GrS) and was transfected to HepG2 cells, an significantly high beta-Gal activity in the cells was observed as compared with that in the cells transfected with AF-liposome(DOPE)-pCMV beta. No effects of GrS were found in the transfection using AF-non-labeled control liposomes. In primary culture of rat hepatocytes, no beta-Gal gene expression was observed even though AF-liposome(DOPE)-pCMV beta was introduced into the cells prepared from adult rats. However, following the transfection with AF-liposome(DOPE)-pCMV beta, the beta-Gal activity was expressed in the cells from immature rats cultured in the medium supplemented with epidermal growth factor and insulin, and the transfection efficiency was 2-fold higher than that transfected with pCMV beta encapsulated in AF-non-labeled control liposomes. By the complex formation of pCMV beta with GrS, the transfection efficiency of AF-liposome(DOPE)-pCMV beta (GrS) increased according to the increase of GrS in the complex. It was shown that AF-liposome(DOPE)-pCMV beta (GrS) did efficiently introduce and express beta-Gal gene in both HepG2 cells and primary hepatocytes in the receptor mediated manner.

Liver Targeting Liposomes Containing .BETA.-Sitosterol Glucoside with Regard to Penetration-Enhancing Effect on HepG2 Cells.

“…AF, which is a glycoprotein having galactose residues, competes with galactose residues for the specific recognition by ASGP-R and thereby inhibits ASGP-R-mediated endocytosis. 17,18) The inhibition of the association by AF indicates the involvement of galactose-modified carriers such as liposomes modified with AF 19) or galactosylated lipid 20) with ASGP-R-mediated endocytosis. The association of SG-liposomes was significantly reduced by addition of 1 mg/ml AF (pϽ0.05), unlike non-SGliposomes.…”

Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes

“…Various liposomal formulations containing modified galactolipids have been optimized for maximum liver uptake. 26,27 Hara et al [28][29][30] reported that AF-labeled liposomes encapsulating plasmid DNA is effective in gene expression after intraportal injection with a preload of EDTA. On the other hand, Zanta et al 31 reported ASGPr-mediated in vitro gene delivery by a 5% galactose-bearing-PEIpolymer.…”

Efficient Gene Transfer to Hepatoblastoma Cells through Asialoglycoprotein Receptor and Expression under the Control of the Cyclin A Promoter.

“…17) Overexpression of cyclin A contributes to the high proliferative activity of hepatoma, 18) indicating that the cyclin A promoter is active in such cells. Therefore we examined the gene expression in the hepatoma cell line HepG2 under the regulation of the cyclin A promoter using AF-liposomes, which can deliver genes specifically to hepatic cells through the AgpR, 11,12) as a nonviral vector. Polyplex Formation and Encapsulation into AF-Liposomes To encapsulate pCyclin A-Luc into AF-liposomes, DNA condensation was carried out by means of electrostatic interaction with PLL.…”

“…10) The formation of the DNA-PLL complex (polyplex) was evaluated in the gel retardation assay: samples (10 ml) were elec- 12) Briefly, palmitoyl-AF (125 mg as AF) dissolved in 50 ml of 10 mM Tris-HCl buffer (pH 7.5) containing 2% sodium deoxycholate (DOC) was added to 1 mmol of liposomal lipid film and mixed micelles were prepared by vigorous vortexing. DOC was dialyzed against 10 mM Tris-HCl buffer (pH 7.5) for 24 h, and empty AF-liposomes were obtained.…”

“…Ligand-labeled cationic carriers have been developed to deliver plasmid DNA to specific tissues or cells. [8][9][10] We also reported that asialofetuin (AF)-and epidermal growth factor (EGF)-labeled cationic liposomes associated with plasmid DNA were efficiently taken up and showed efficient transfection in the human hepatoblastoma cell line HepG2 and EGF receptor-overexpressing cancer cells through the asialoglycoprotein receptor (AgpR) 11,12) and EGF receptor, 13) respectively. Such an approach will also lead to expression in normal cells expressing AgpR and EGF receptors when plasmid DNA constructed with a viral promoter is used.…”