Active targeting of asiaglycoprotein receptor using sterically stabilized lipoplexes

The large numbers of hepatic disorders of a genetic origin continues to drive the quest for safe and efficient non-viral gene delivery systems with the design features suitable for in vivo application. Here, we examine the efficacy of hepatocyte-specific stealth lipoplexes in the human hepatoma cell line HepG2. Thus cationic liposomes were formulated with the cytofectin N,N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09), the neutral co-lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with and without the asialoglycoprotein receptor (ASGP-R)-specific ligand cholesteryl--d-galactopyranoside (Chol--Gal), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) 2000] (DSPE-PEG(2000)) in the 0-5 mol% range. Lipoplexes formed with pCMV-luc plasmid DNA were characterized by cryo-transmission electron microscopy (cryo-TEM), gel retardation, and ethidium displacement assays showed that cargo DNA was compacted and protected from serum nucleases by cationic liposomes. Hydrodynamic diameters were in the 1146.2-2245.3nm size range while particle sizes, polydispersity indices (PDI), and -potentials decreased with increasing degree of PEGylation. Cell viabilities of >75% were attained by targeted complexes in the ASGP-R-negative human embryonal kidney cell line HEK293 and in ASGP-R-positive HepG2 cells. Targeted 5% PEGylated lipoplexes at N/P ratio=3.5 achieved the highest transfection activity in the hepatocyte-derived cells while competition assays and fluorescence microscopy confirmed that all targeted lipoplexes transfected HepG2 cells largely by ASGP-R mediation. Practical applications: The findings of this study show that stealth lipoplexes bearing PEG(2000) at a density of 5 mol% may be effectively targeted to the ASGP-R, which is almost exclusively expressed on hepatocytes and hepatocyte-derived cells, by inclusion of the simple monoantennary cholesteryl--d-galactopyranoside at 10 mol% in the parent cationic liposome preparation. The remarkable efficacy of this cholesteryl glycoside ligand, in the presence of serum, shows that a -glycosidic link between the cholesteryl membrane anchor moiety and the pyranose ring is sufficient to ensure efficient ASGP-R recognition of the sugar hexose at clinically relevant PEG grafting densities, by lipoplexes into which it has been incorporated. We conclude that the targeted, PEGylated lipoplexes characterized and evaluated in this study constitute a solid platform for the development of hepatotropic nucleic acid delivery vehicles.