The comparative effects of acetylated and deacetylated galactose derivatives in liposomal gene delivery.

Abstract

The use of cationic liposomes remains the most attractive non-viral approach in gene therapy as these gene carriers provide for ease and versatility in design. In targeted gene delivery, liposomes are coupled to ligands tailored to possess desired characteristics for improved cell-specificity. Carbohydrates have been established as useful targets for the asialoglycoprotein (ASGP) receptor in liver-directed delivery. The main purpose of this study was to comparatively evaluate physicochemical characteristics, DNA-binding interactions and in vitro transfection activities of hepatocyte-targeted liposomes bearing acetylated and deacetylated galactosides in ASGP receptor-mediated gene delivery. Furthermore, in silico studies were carried out to assess ligand-receptor interactions for both galactosides. Novel targeted cationic liposomes conjugated with galactosyl ligands viz. cholest-5-en-3-yl 2-[4-(β-D-galactopyranosyl-1-oxymethyl)-1H-1,2,3-triazol-1-yl]ethylcarbamate (Sc6) and cholest-5-en-3-yl 2-[4-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-1-oxymethyl)-1H-1,2,3-triazol-1-yl]ethylcarbamate (Sc9) were formulated with cytofectin 3β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and the neutral co-lipid dioleoylphosphatidyl ethanolamine (DOPE), using the thin film hydration–sonication method. Characterisation of lipoplexes by cryo-transmission electron microscopy (cryo-TEM) showed unilamellar liposomes, and lipoplexes ranging between ~80 – 140 nm. DNA was fully liposome-bound at N:P ratios 2.5:1 – 3:1. Upon inclusion of polyethylene glycol 2000 -distearoylphosphatidyl ethanolamine (DSPE-PEG₂₀₀₀) in liposome formulations, vesicles were more compacted due to steric stabilisation. UnPEGylated lipoplexes achieved better condensation of DNA as determined in band shift and ethidium bromide displacement assays. Nuclease digestion assays revealed suitable protection of cargo DNA by some formulations, with the least protection afforded by the acetylated SM3 derivatives. Cytotoxicity studies in the HEK293 and HepG2 cell lines revealed good cell viabilities under transfection conditions for all liposomes. Transfection efficiency was assessed using the luciferase reporter gene assay. Higher transfection activities were observed in the ASGP receptor-positive HepG2 cell line than the ASGP receptor-negative HEK293 cells line for all lipoplexes. While the acetylated unPEGylated derivative (SM3) demonstrated better transgene expression levels compared to other derivatives, this was not found to be significant. High transfection levels were attributed to favourable size and surface charge, as well as galactoside ligand accessibility to the receptor. In the presence of excess asialofetuin, a marked decrease in transfection efficiencies was observed for all targeted derivatives. Docking scores further confirmed good binding affinity for the deacetylated Sc6 ligand and acetylated Sc9 ligand at ˗6.7 and ˗5.5 kCal/mol, respectively. The acetylated SM3 however, achieved avidity to the binding site through hydrogen bonding via the triazine linker. Overall transfection efficiency results were corroborated by outcomes from molecular studies as both galactoside ligand-conjugated liposomes presented similar binding affinities and transfection efficiency results. It is thus concluded that both these galactosides, with further optimization could present the potential for hepatocyte-specific delivery via ASGP receptor-mediated endocytosis.