Receptor targeted gene delivery using folate ligand conjugated cationic liposomes.
| dc.contributor.advisor | Singh, Moganavelli. | |
| dc.contributor.advisor | Ariatti, Mario. | |
| dc.contributor.author | Gorle, Sridevi. | |
| dc.date.accessioned | 2015-09-09T07:11:02Z | |
| dc.date.available | 2015-09-09T07:11:02Z | |
| dc.date.created | 2014 | |
| dc.date.issued | 2014 | |
| dc.description | Ph. D. University of KwaZulu-Natal, Durban 2014. | en |
| dc.description.abstract | Gene therapy has become an important strategy to treat several human diseases, including cancer, viral infections and inherited disorders. In response to this growing trend, a number of gene delivery vectors have been manufactured both to facilitate nucleic acid uptake by target cells and also to promote the transport of genetic materials into the nucleus. The success of gene therapy however depends on the efficient delivery of therapeutic genes into target cells both in vitro and in vivo. Cationic liposomes represent a class of non-viral vectors that have shown the ability to bind and deliver DNA cargo to defective cells efficiently. This study has focused on the development of a novel folate-targeted cationic liposome-mediated gene delivery system. This receptor is overexpressed on numerous cancer cell types and offers a convenient docking point for subsequent cellular uptake of folate decorated liposome-DNA complexes by receptor mediation. In this study, a total of six cationic liposome preparations comprising either cationic cholesterol cytofectin -dimethylpropylamidosuccinylcholesterylformylhydrazide (MSO9) or 3β[N(N1,N1-dimethlaminopropylsuccinamidoethane)-carbamoyl]-cholesterol (SGO4) were formulated by mixing the fusogenic neutral helper lipid, dioleoylphosphatidylethanolamine (DOPE) as a common constituent. DSPE-PEG₂₀₀₀ was also used in formulations for possible in vivo development of PEGylated, targeted liposomes. The targeting ligand folate was appended to the distal end of liposome-anchored DSPEPEG₂₀₀₀, for prominent display and optimal receptor recognition. Transmission electron micrographs revealed liposomes to be unilamellar, spherical shaped vesicles with a narrow size range (50 - 80 nm in diameter). Agarose gel retardation studies demonstrated complex formation between cationic liposomes and plasmid DNA, whilst serum nuclease protection assays showed that the liposome formulations were capable of protecting the complexed DNA in lipoplexes against serum nuclease digestion. Ethidium bromide dye displacement studies yielded information on the compaction or condensation efficacy of the liposomes with respect to the cargo plasmid. In addition, particle sizes determined by dynamic light scattering confirmed the suitability of lipoplexes for future in vivo applications in which extravasation is essential. Importantly, these liposome:DNA complexes were found to exhibit minimal growth inhibition levels in HEK293, HeLa and KB cells. Further investigations were carried out to determine the optimal transfection activity of complexes in the folate receptor-positive cell lines (HeLa and KB). The plasmid containing the transgene firefly luciferase (pCMV-luc) was used in transfection studies. Results showed that folate targeted liposomes, irrespective of cytofectins MSO9 or SGO4 achieved highest transfection activities in vitro, specifically via receptor mediation. Lower transfection activity was observed for by untargeted PEGylated and unPEGylated liposomes compared to that of the folate targeted liposomes, strongly implicating folate receptor-mediation in the uptake of ligand-displaying lipoplexes. This was further confirmed by flow cytometry analysis. Furthermore, zeta potential values obtained for targeted complexes revealed low negative surface charge, thus minimizing the possibility of electrostatic interaction between lipoplexes and target cells. The cytofectin, MSO9, achieved 10 fold greater transfection activity than the cytofectin SGO4 although they are closely related, differing only in their spacer lengths. Competition assays using free folate (200 μM) to confirm folate receptor mediated lipoplex uptake in the HeLa, and KB cells revealed a dramatic decline in transfection activity due to the excess free folate binding to and blocking access to the folate receptors on the cell membrane. The two novel PEGylated lipoplexes designed for folate receptor-mediated uptake by transformed mammalian cells display very favourable physicochemical characteristics, low cytotoxicity and promising transfection profiles in vitro. Therefore further investigation of the cationic liposome formulations examined in this study in vivo is warranted. | en |
| dc.identifier.uri | http://hdl.handle.net/10413/12434 | |
| dc.language.iso | en_ZA | en |
| dc.subject | Gene therapy. | en |
| dc.subject | Genetic vectors. | en |
| dc.subject | Liposomes. | en |
| dc.subject | Theses--Biochemistry. | en |
| dc.title | Receptor targeted gene delivery using folate ligand conjugated cationic liposomes. | en |
| dc.type | Thesis | en |