Galactosylated liposomes with proton sponge capacity : a novel hepatocyte-specific gene transfer system.
Hepatocyte-directed liposomal gene delivery systems have received much attention in view of the present lack of suitable treatment alternatives for several liver-associated disorders. While targeting of liposomes to the asialoglycoprotein receptor (ASGP-R), nearly-exclusive to hepatocytes, is a well-documented means of achieving cell-specificity, several intra- and extracellular barriers reduce the efficacy of liposomal gene transfer. These include the aggregation and opsonisation of lipoplexes by serum components; and endo/lysosomal degradation of internalised DNA. This study has attempted to address the individual concerns by modifying hepatotropic liposomes with a steric stabilising, polyethylene glycol (PEG) shroud, and an endosomal escape-inducing proton sponge moiety. Novel galactosylated (SH02) and imidazolylated (SH04) cholesterol derivatives were successfully synthesised with the aim of conferring the respective functions of ASGP-R-specificity and proton sponge capability upon cationic liposome formulations. The individual derivatives afforded stable, unilamellar vesicles (< 200 nm, Z-average diameter) when incorporated at 10 % on a molar basis with the cytofectin, 3β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and co-lipid, dioleoylphosphatidylethanolamine (DOPE). Modification of these formulations with 1,2-distearoyl-sn-glycero-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000] (DSPEPEG₂₀₀₀), at 5 mol %, gave smaller vesicles (< 110 nm, Z-average diameter) and moderately reduced the instability associated with the combination of both SH02 and SH04 in a single formulation. Individual preparations formed electrostatic complexes with pCMV-luc plasmid DNA, as demonstrated by gel retardation assays and electron microscopy. Furthermore, the liposomes afforded some protection to the DNA cargo against serum nuclease attack during a 4 hour-long exposure to foetal calf serum at 37 °C. However, the DNA-binding and protecting capabilities of the liposomes were reduced upon addition of the PEG coating. Growth inhibition assays showed that lipoplexes derived from individual formulations were well tolerated by human hepatocyte-derived, HepG2, and embryonic kidney, HEK293, cell lines. Expression of the luciferase transgene mediated by non-pegylated formulations containing SH02 was significantly higher in hepatocytes than in the ASGP-R-negative, kidney cells. Furthermore, receptor-mediated internalisation of non-pegylated, galactosylated carriers by hepatocytes was demonstrated by the gross inhibition of transfection in the presence of excess asialofetuin, a natural ligand to the ASGP-R. Liposome acid titration profiles highlighted the endosomal pH-buffering capacity afforded by SH04. However, the imidazolylated lipid enhanced the transfection activity of the non-sterically stabilised Chol-T/DOPE system, but not that of its targeted counterpart, and only with respect to HEK293 cells. Finally, pegylation reduced the transfection capability of liposomes by at least three orders of magnitude in both cell lines. The results suggest that further optimisation of liposome composition is necessary in order to achieve a liposomal system that simultaneously embodies hepatocyte-targeting, proton sponge and long-circulating properties.