The high gene knockdown specificity and efficiency of RNA interference (RNAi)
provides a potentially viable avenue for the development of a new class of nucleic
acid therapeutics for gene-based disease conditions. However, serum instability,
inefficient cellular trafficking and non-specific effects of small interfering RNAs
(siRNAs), one of the functional mediators of RNAi, has necessitated the development
of carriers to facilitate targeted cell delivery. The decline of viral vectors in human
gene therapy as a consequence of safety issues has intensified the importance of
non-viral vector development. Among the non-viral vectors available for siRNA
delivery, cationic liposomes have emerged as an attractive option owing to their
simplicity, versatility, relatively low toxicity and potential for cell-specific targeting.
Although existing cationic lipids and liposomes traditionally used for DNA delivery
have also been used for siRNAs, there still exists a need to develop cationic lipids
tailored towards siRNA transfection for improved gene silencing efficiency. Among
the cell specific targets available for RNAi therapeutics, hepatocytes expressing the
asialoglycoprotein receptor (ASGP-R) are an ideal choice due to the large number of
disease targets present for treatment.
In this investigation four novel cationic liposome formulations were prepared from
equi-molar quantities of either the cationic cholesterol derivative 3β [N-(N’,N’-
dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or 3β [N-(N’, N’, N’-
trimethylammoniumpropyl)-carbamoyl] cholesterol iodide (Chol-Q) and DOPE, with
and without the hepatotropic ligand, cholesteryl-β-D-galactopyranoside.
Electrophoretic gel analysis and SYBR®green displacement assays were employed to
determine siRNA binding and condensation efficiencies for all cationic liposomes;
while liposome and lipoplex size measurements were made by cryoTEM. SiRNAlipoplex
stability in serum was determined by the nuclease protection assay. Cell
studies performed on the ASGP-R+ human hepatoma cells, HepG2 and the ASGP-Rembryonic
kidney cells, HEK293, to determine lipoplex toxicity and transfection
efficiencies were also undertaken. We show that the cationic liposomes formulated for this investigation were able to
bind synthetic siRNA optimally at a positive to negative charge ratio of ± 1 : 6. In
addition, the cationic liposomes were able to afford siRNA duplexes substantial
protection from ribonuclease digestion in serum. From the results obtained in this
study, it appears that the cationic liposomes are well tolerated by both the HEK293
and HepG2 cells in vitro. More importantly, the results obtained demonstrated
higher transfection efficiencies for the targeted lipoplexes compared with the
untargeted controls, strongly supporting the notion that incorporation of the
cholestryl-β-D-galactopyranoside into the liposome structure increases transfection
efficiency to the targeted HepG2 cells in culture via ASGP receptor mediation.
Comparative studies in the HEK293 cell line yielded low transfection effeciences in
the order of 20%, with no significant difference being recorded between
galactosylated and non-galactosylated lipoplexes. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2011.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/11134 |
| Date | January 2011 |
| Creators | Dorasamy, Shantal. |
| Contributors | Singh, Moganavelli., Ariatti, Mario. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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