Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro.
The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.
Singh
Ashika
A
Pfizer Molecular Biology Research Facility, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa.
Ariatti
Mario
M
Singh
Moganavelli
M
Hawtrey
Arthur
A
Naidoo
Richard
R
D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D001212:Asialoglycoproteins / Q000009:adverse effects / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000009:adverse effects / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use; D019921:Biotinylation; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000628:therapy; D049109:Cell Proliferation / Q000187:drug effects; D002784:Cholesterol / Q000009:adverse effects / Q000031:analogs & derivatives / Q000737:chemistry; D060748:Fetuins; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods*; D056945:Hep G2 Cells; D006801:Humans; D008024:Ligands; D008081:Liposomes; D008113:Liver Neoplasms / Q000378:metabolism* / Q000628:therapy; D049329:Nanostructures / Q000009:adverse effects / Q000737:chemistry* / Q000627:therapeutic use / Q000648:ultrastructure; D009961:Orosomucoid / Q000009:adverse effects / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000009:adverse effects / Q000737:chemistry; D010957:Plasmids / Q000009:adverse effects / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000009:adverse effects / Q000378:metabolism / Q000627:therapeutic use; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D000509:alpha-Fetoproteins / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D060748:Fetuins; D008024:Ligands; D008081:Liposomes; D009961:Orosomucoid; D010714:Phosphatidylethanolamines; D000509:alpha-Fetoproteins; C017029:asialofetuin; C017030:asialoorosomucoid; D001710:Biotin; C020888:1,2-dielaidoylphosphatidylethanolamine; D019809:Streptavidin; D002784:Cholesterol