Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes Moganavelli Singh, Mario Ariatti * Biochemistry, School of Biochemistry and Microbiology, Faculty of Science, University of Durban-Westville, Private Bag X54001, Durban, South Africa Received 14 May 2003; accepted 14 July 2003 Abstract Unilamellar activated cationic liposomes containing 3h[N-(NV ,NV -dimethylaminopropane)-carbamoyl] cholesterol, dioleoyl phosphatidylethanolamine (DOPE) and the N-hydroxysuccinimide ester of cholesteryl hemisuccinate (4:5:1, molar ratio) have been prepared and their DNA-binding capacity has been assessed in a gel retardation assay. Ternary complexes composed of activated cationic liposomes, carbodiimide – cationized asialoorosomucoid (Me + AOM) and pRSVL plasmid DNA were assembled for receptor-mediated DNA delivery into cells expressing the asialoglycoprotein receptor (ASGP-R). Binding of complexes in which Me + AOM was replaced by fluoresceinated Me + AOM (FMe + AOM) to the human hepatocellular cell line HepG2 at 4 jC was severely reduced by co-incubation with asialoorosomucoid (AOM). Moreover, assemblies containing liposomes, pRSVL DNA and Me + AOM (8:1:4, w/w/w) promoted high levels of luciferase activity in this cell line (1.3 Â 10 7 relative light units/mg soluble cell protein). Assays conducted in the presence of a hundred-fold excess of the ligand AOM afforded considerably lower levels of transfection (2.5 Â 10 5 relative light units/mg soluble cell protein). In contrast, the highest level of luciferase activity achieved with liposome, pRSVL DNA, AOM complexes was only a quarter of the best levels obtained with liposome, pRSVL DNA, Me + AOM assemblies. These findings strongly support the notion that complexes gain entry into hepatocyte-derived cells by ASGP-R mediation and that they are potentially useful gene carriers to liver hepatocytes. D 2003 Elsevier B.V. All rights reserved. Keywords: Cationic; Liposome; Transfection; Asialoorosomucoid; HepG2 1. Introduction The directed delivery of corrective DNA into hepatocytes and hepatocyte-derived cells by receptor mediation is of considerable importance in the devel- opment of gene therapy protocols for human diseases and disorders of the liver. These include low-density lipoprotein receptor deficiency, haemophilia and a 1 - antitrypsin deficiency [1]. The asialoglycoprotein re- ceptor (ASGP-R), which is abundantly and selectively expressed in hepatocytes [2], is found predominantly on the sinusoidal surface of parenchymal cells [3]. This lectin which has a high affinity for the galactose- terminating triantennary N-linked heteroglycans of 0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-3659(03)00360-2 * Corresponding author. Tel.: +27-31-2044981; fax: +27-31- 2044942. E-mail address: mariatti@pixie.udw.ac.za (M. Ariatti). www.elsevier.com/locate/jconrel GENE DELIVERY Journal of Controlled Release 92 (2003) 383 – 394