Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer into hepatic cells by asialofetuin-labeled liposomes.
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.
Hara
T
T
Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan.
Kuwasawa
H
H
Aramaki
Y
Y
Takada
S
S
Koike
K
K
Ishidate
K
K
Kato
H
H
Tsuchiya
S
S
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D002788:Cholesterol Esters / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D060748:Fetuins; D017930:Genes, Reporter / Q000235:genetics; D006096:Gramicidin / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008561:Membrane Fusion; D010714:Phosphatidylethanolamines / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D014162:Transfection / Q000187:drug effects*; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D001212:Asialoglycoproteins; D002788:Cholesterol Esters; D060748:Fetuins; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000509:alpha-Fetoproteins; C017029:asialofetuin; D006096:Gramicidin; C094877:dioleoyl phosphatidylethanolamine; C014409:cholesteryl oleate; D002738:Chloroquine; D001616:beta-Galactosidase