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dc.contributor.advisorBadea, Ildikoen_US
dc.creatorSingh, Jagbiren_US
dc.date.accessioned2015-10-21T12:00:32Z
dc.date.available2015-10-21T12:00:32Z
dc.date.created2013-08en_US
dc.date.issued2015-10-20en_US
dc.date.submittedAugust 2013en_US
dc.identifier.urihttp://hdl.handle.net/10388/ETD-2013-08-1189en_US
dc.description.abstractGemini surfactants are versatile gene delivery agents because of their ability to bind and compact DNA and their low cellular toxicity. The aim of my dissertation work was to develop non-invasive mucosal formulations of novel amino acid-substituted gemini surfactants with the general chemical formula C12H25(CH3)2N+-(CH2)3-N(AA)-(CH2)3-N+(CH3)2-C12H25 (AA= glycine, lysine, glycyl-lysine, lysyl-lysine). These compounds were formulated with a model plasmid DNA, encoding for interferon-γ and green fluorescent protein, in the presence of helper lipid, 1,2 dioleyl-sn-glycero-phosphatidyl-ethanolamine. Formulations were assessed in Sf 1 Ep epithelial cells. Among the novel compounds, plasmid/gemini/lipid (P/G/L) nanoparticles formulated using glycine- and glycyl-lysine substituted gemini surfactants achieved significantly higher gene expression than the parent unsubstituted compound. The key physicochemical properties, e.g. size, surface charge, DNA binding, and toxicity of P/G/L complexes were correlated with transfection efficiency. The presence of amino-acid substitution did not interfere with DNA compaction and contributed to an overall low toxicity of all P/G/L complexes, comparable to the parent gemini surfactant. A cellular uptake mechanistic study revealed that both clathrin- and caveolae-mediated uptake were major uptake routes for P/G/L nanoparticles. However, amino acid substitution in the gemini surfactant imparted high buffering capacity, pH-dependent increase in particle size, and balanced DNA binding properties. These properties may enhance endosomal escape of P/12-7NGK-12/L resulting in higher gene expression. Finally, the P/G/L complexes were incorporated into an in-situ gelling dispersion containing a thermosensitive polymer, poloxamer 407, and a permeation enhancer, diethylene glycol monoethyl ether (DEGEE). A 16% w/v poloxamer concentration produced a dispersion that gelled at body temperature and exhibited sufficient yield value to prevent formulation leakage from the vaginal cavity. The formulations were prepared with a model plasmid, encoding for red fluorescent protein, and administered topically to rabbit vagina. In agreement with our in vitro results, confocal microscopy revealed that glycyl-lysine substituted gemini surfactant exhibited higher gene expression compared to the parent unsubstituted gemini surfactant. This provided proof-of-concept for use of amino acid-substituted gemini surfactant in non-invasive mucosal (vaginal) gene delivery systems with potential therapeutic applications. These formulations will be developed with therapeutically relevant genes to assess their potential as genetic vaccines. In addition, new gemini surfactants will be developed by grafting other amino acids via glycine linkage to retain conformation flexibility and enhance endosomal escape of DNA complexes for higher transfection efficiency.en_US
dc.language.isoengen_US
dc.subjectAmino acid-substituteden_US
dc.subjectgemini surfactantsen_US
dc.subjectplasmiden_US
dc.subjectnon-viral gene deliveryen_US
dc.subjectcellular uptakeen_US
dc.subjectclathrin-mediated endocytosisen_US
dc.subjectcaveolae-mediated endocytosisen_US
dc.subjectmucosalen_US
dc.subjectintravaginal administrationen_US
dc.subjectgelling temperatureen_US
dc.subjectrheologyen_US
dc.subjectgene expressionen_US
dc.titleDevelopment of Amino acid-Substituted Gemini Surfactant-Based Non-invasive Non-Viral Gene Delivery Systemsen_US
thesis.degree.departmentPharmacy and Nutritionen_US
thesis.degree.disciplinePharmacyen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US
dc.type.materialtexten_US
dc.type.genreThesisen_US
dc.contributor.committeeMemberKrol, Eden_US
dc.contributor.committeeMemberVerrall, Ronald E.en_US
dc.contributor.committeeMemberEl-Aneed, Anasen_US
dc.contributor.committeeMemberAlcorn, Janeen_US


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