Cationic liposome mediated targeted gene delivery with and without pegylated accessories.
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Date
2009
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Abstract
As a consequence of safety issues encountered by the use of viral vectors in gene therapy,
there has been a steady increase in the development and application of non-viral vectors,
especially liposomes. Cationic liposome mediated delivery is one of the most promising nonviral
delivery methods. These liposomes are prepared from synthetic lipids, are positively
charged and interact favourably with DNA through electrostatic interactions. Cationic
liposomes have also shown immense potential in the targeting of specific cell types such as
HepG2 (hepatocellular carcinoma) cells, a model in vitro gene delivery system for the study
of hepatocyte function. However, these liposomes also have a number of limitations in vivo.
In an attempt to overcome these restrictions, a hydrophilic polymer, polyethylene glycol
(PEG) is incorporated into the cationic liposome. This covalent attachment of (PEG) to the
liposomal surface is thought to sterically stabilise liposomes, promote biological stability,
inhibit aggregation, decrease toxicity and immunogenicity, prevent interaction with serum
proteins and complement and thus prolonging the circulation time of liposomes in vivo. The
versatility and simplicity of cationic liposomes have made them vitally significant non-viral
gene delivery vehicles for human gene therapy.
In this investigation novel untargeted and targeted glycosylated liposomes with and without
PEG were synthesised to evaluate their gene transfer activities in vitro to potentially develop a
suitable gene delivery system for future in vivo applications. A constant molar quantity of the
cationic cholesterol derivative, 3 [N-(N’, N’-dimethylaminopropane)-carbamoyl]
cholesterol (CHOL-T) was mixed with dioleoylphosphatidylethanolamine (DOPE) and a
galactose/glucose derivative to produce targeted cationic liposomes. PEG liposomes were
prepared in the same way with the addition of distearoylphosphoethanolamine polyethylene
glycol 2000 (DPSE-PEG2000), 2% on a molar basis.
Supported by transmission electron microscopy characterisation, we present evidence that the
pegylation of liposomes affects the DNA binding capability and transfection efficiencies of
the cationic liposomes in addition to protecting the plasmid DNA in lipoplexes from serum
nuclease degradation. Optimal DNA : liposome binding ratios were obtained from gel
retardation studies and confirmed by ethidium bromide intercalation assays. These complexes
were then tested on the human hepatoma cell line, HepG2, to determine toxicity and assess
transfection efficiencies. From results obtained in this study, it appears that both cationic and
pegylated cationic liposomes are well tolerated by cells in vitro. The results further suggest
that targeting by use of glycolipids incorporated into the structure of the liposome increases
transfection, while pegylation of cationic liposomes marginally decreases the transfection
efficiency of the lipoplexes to HepG2 cells in vitro.
Description
Thesis (M.Sc.)-University of KwaZulu-Natal, 2009.
Keywords
Genetic engineering., Gene therapy., Theses--Biochemistry.