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Non-viral gene delivery with pH-sensitive gemini nanoparticles : synthesis of gemini surfactant building blocks, characterization and in vitro screening of transfection efficiency and toxicity

dc.contributor.advisorFoldvari, Mariannaen_US
dc.contributor.committeeMemberVerrall, Ronald E.en_US
dc.contributor.committeeMemberNazarali, Adil J.en_US
dc.contributor.committeeMemberAttah-Poku, Samen_US
dc.contributor.committeeMemberAlcorn, Janeen_US
dc.contributor.committeeMemberWettig, Shawnen_US
dc.creatorDonkuru, McDonalden_US
dc.date.accessioned2009-01-13T13:14:23Zen_US
dc.date.accessioned2013-01-04T04:23:57Z
dc.date.available2010-01-14T08:00:00Zen_US
dc.date.available2013-01-04T04:23:57Z
dc.date.created2008en_US
dc.date.issued2008en_US
dc.date.submitted2008en_US
dc.description.abstractResearch on self-assembling gemini surfactants and other amphiphiles for potential gene delivery applications in research as well as in clinical practice, and as alternatives to viral gene delivery vectors, is beginning to focus more on ‘structure–activity relationships’ to address the current low gene delivery efficiencies of amphiphiles. Some underlying structure–activity relations are beginning to emerge. But, as a better understanding of the factors that govern the transfection abilities of amphiphile molecules emerges, development of improved non-viral vectors with clinical potential may also emerge. The research conducted for this thesis was aimed at the design, synthesis and in vitro investigation of gemini surfactants as one of a family of novel amphiphiles being investigated for gene therapeutic applications. The properties of these compounds can be controlled as well as allowed to vary naturally. Gemini surfactant-based gene delivery systems were prepared and characterized for transfer of Luciferase plasmid (pMASIA.Luc) to both COS-7 and PAM 212 cells. Characterization was accomplished using microscopy, dynamic light scattering (DLS) and zeta (ζ) potential analysis. In vitro gene expression and toxicities were evaluated in COS-7 cell and PAM 212 keratinocyte cultures. The level of in vitro transfection in general was found to correlate strongly with the structure of the gemini surfactants. Among the 12-spacer-12 surfactants, incorporation of a pH-sensitive aza (N-CH3) group, which is also steric hindrance-imposing, in the spacer chain yielded increased transfection, particularly for the 12-7N-12 surfactant. In comparison, the incorporation of the more pH-sensitive imino (N-H) group in the 12-7NH-12 surfactant yielded the highest increase in transfection among the 12-spacer-12 surfactants. The deleterious effect of steric hindrance due to the aza group is more evident when comparing the transfection efficiency of 12-5N-12 (1 × aza, higher) vs. 12-8N-12 (2 × aza, lower transfection). Another highlighted structural feature is provided by the fact that both the 12-7NH-12 and 12-7N-12 surfactants had higher transfection efficiencies than 12-5N-12 and 12-8N-12 surfactants; the first pair has trimethylene spacing, which constitutes an optimal separation between nitrogen centres, while the second pair has shorter dimethylene spacings. After expanding the structure of surfactants, transfection efficiencies were found to increase in response to increase in hydrocarbon tail length, but were much lower for surfactants with no amino functional groups, those that lacked the optimal trimethylene spacing, or those having both of these limitations in the gemini surfactant spacer. The 18-7NH-18 surfactant had the highest overall transfection in both COS-7 and PAM 212 cells. Gemini surfactant-based gene delivery systems capable of adopting both polymorphic structural phases and which could undergo pH-induced structural transition demonstrated high transfection efficiencies. Gemini surfactants with both characteristics (e.g., 12-7NH-12-based complexes are both polymorphic and pH-sensitive) had higher transfection than gemini surfactants with only one (e.g., 12-3-12-based complexes are only polymorphic). Overall, the m-7NH-m surfactants, the most efficient surfactants studied, had transfection efficiencies similar to that of the commercial Lipofectamine Plus™ reagent and imposed no higher toxicity on cells relative to the less efficient surfactants. Thus, the design of the m-7NH-m surfactants to enhance their transfection abilities also ensured that their toxicity to cells were kept minimal. Overall, the design, synthesis and in vitro transfection screening of gemini surfactant candidates has revealed that the m-7NH-m surfactants have the highest transfection efficiencies; they have emerged as suitable candidates for non-viral gene delivery in vivo or at higher levels. Gene delivery investigations for six of the gemini surfactant candidates are being reported for the first time.en_US
dc.identifier.urihttp://hdl.handle.net/10388/etd-01132009-131423en_US
dc.language.isoen_USen_US
dc.subjectGemini Surfactant; Gene Deliveryen_US
dc.subjectNanoparticleen_US
dc.subjectpH-sensitive; Structure-Activity Relationship; DNAen_US
dc.titleNon-viral gene delivery with pH-sensitive gemini nanoparticles : synthesis of gemini surfactant building blocks, characterization and in vitro screening of transfection efficiency and toxicityen_US
dc.type.genreThesisen_US
dc.type.materialtexten_US
thesis.degree.departmentPharmacyen_US
thesis.degree.disciplinePharmacyen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Science (M.Sc.)en_US

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