The use of cholesterol-galacto compounds in liver directed gene delivery.
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Date
2014
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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.
Description
M. Sc. University of KwaZulu-Natal, Durban 2014.
Keywords
Gene therapy., Liver., Liposomes., Theses -- Biochemistry.