The use of cholesterol-galacto compounds in liver directed gene delivery.

Abstract

Gene therapy has to date gained immense interest as a potential method for treating genetic disorders such as Parkinson’s and cystic fibrosis. The liver being a central organ of metabolism is susceptible to several metabolic disorders which could be targeted through liver directed gene delivery. The most common diseases being viral hepatitis and hepatocellular carcinoma towards which this study is focused. Non-viral vectors have gained wide interest as the vector of choice for delivering genes to organs such as the liver, because of their large-scale production potential, easy preparation, low cost and are relatively non-immunogenic to target cells / organs. This study utilized non-viral cationic liposomes targeted to hepatocytes via ligand-receptor recognition. A total of six cationic liposomes (targeted acetylated /non-acetylated, nontargeted, pegylated, non-pegylated) were prepared according to the lipid film hydration method. The liposomes and liposome:DNA complexes were characterized using transmission electron microscope (TEM) and Zeta sizing to determine morphology, lamellarity and size. Results showed spherical, unilamellar cationic liposomes and lipoplexes in the size range of 50-200 nm in diameter. Band shift assays showed that these liposomes have strong DNA binding capabilities which was further confirmed by the ethidium bromide intercalation assays. Nuclease protection assays showed that liposomes were able to protect the integrity of the DNA cargo. From the MTT cell viability assays, low cytotoxicity was observed for all liposomes with cell survival as high as 80 % in most cases. Higher transfection activities were noted in the hepatocellular carcinoma receptor positive cell line (HepG2), for targeted non-acetylated liposomes, compared to the acetylated liposomes. The ligand competition assay and the use of the human embryonic kidney receptor negative cell line (HEK293) confirmed that the complexes entered the cells via receptor mediated endocytosis. Furthermore, it was confirmed that the acetylation of the galactose ligands hindered the process of receptor mediation. Overall, these liposomal formulations are serum tolerant, have low cytotoxicity and are able to selectively target and transfect hepatocytes in vitro. Hence, they have the potential as future non-viral gene delivery vehicles and with further optimisation can be tested in vivo.