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dc.contributor.authorRafiee, Amirreza
dc.date.accessioned2018-09-04 21:49:03 (GMT)
dc.date.issued2018-09-04
dc.date.submitted2018-08-30
dc.identifier.urihttp://hdl.handle.net/10012/13734
dc.description.abstractThe development of non-viral gene delivery vectors is highly challenging and aims to provide a safe while cost-effective manufacturing alternative to viral vectors. Eleven novel gemini surfactants (G4-G14) were designed and synthesized by covalent linking of 10 different functional moieties (R = R1-R10) to the spacer regions of gemini surfactants (chemical formula m-s-m; m = saturated 12, 18 carbon alkyl chains, s = R-linked-imino-substituted-7 methylene spacer group (7NR)). These R-functionalities include imidazole and thiol containing functional groups (R1 = imidazolepropionyl, R2 = thiopropionyl; for synthesis of G4 (18-7NR1-18) and G5 (18-7NR2-18)), linear RGD derivatives (R3 = RGDG, R4 = GRGDSPG; for synthesis of G6 (12-7NR3-12), G7 (18-7NR3-18), G8 (18-7NR4-18)), polyhistidine derivatives (R5 = E(H)5; for synthesis of G9 (18-7NR5-18)), bifunctional RGD-polyhistidine peptides (R6 = EGRGDSPG(H)5; for synthesis of G10 (18-7NR6-18)), and arginine-rich peptide motifs (R7 = Suc-(E)2G(R)2, R8 = Suc-(E)2G(R)3, R9 = Suc-(E)2(G)3(R)3, R10 = Suc-DE(G)3(R)3); for synthesis of G11 (18-7NR7-18), G12 (18-7NR8-18), G13 (18-7NR9-18), G14 (18-7NR10-18)]. The RGD-functionalized gemini surfactants were evaluated for targeted gene delivery. Further, the impact of non-covalent addition of designed peptide enhancers (7 types; PA-PG) [zwitterionic RGD peptide enhancer (PA), cationic peptide enhancers rich in histidine and/or arginine (PB, PD, PF), or bifunctional cationic, RGD peptide enhancers (PC = PA+ PB, PE = PA+ PD, PG = PA+ PF)] were examined for development of peptide-based lipopolyplexes, named peptide-driven tri-modal gene delivery systems (PDTMG). The PDTMG were formulated using peptide enhancers/gemini surfactants/1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) helper lipid, for in vitro delivery of green fluorescent protein (GFP)-expressing plasmid DNA. Using quantitative flow cytometry, transfection activity was investigated by both the percentage of the transfected cells and the intensity of GFP expression level determined by mean fluorescence intensities (MFI). The correlation of the transfection activity and viability to the physicochemical properties of delivery systems, size and zeta potential, were identified to advance formulation strategies for development of a potent delivery system with negligible cytotoxicity. These include optimization of cationic quaternary ammonium of gemini surfactants/anionic phosphate of DNA (N/P) mole ratios (ρ values), DOPE/gemini molar ratios (r values), and the molarity of the compositional elements in the formulation mixtures (MP, MG and ML for molar concentrations of peptide enhancers (P), gemini surfactants (G) and DOPE helper lipids (L), respectively). In vitro transfection studies demonstrated that among PDTMG delivery systems formulated using fourteen different gemini surfactants [G1-G14] differing in their headgroups and alkyl tails lengths (i.e., s = 3, 7NH, 7NR1-10; m = 12, 18), remarkably gemini surfactants with short RGD functional headgroups and C18 alkyl tails (G7 and G8) provided elevated cell-penetrating activity and endosomal rupturing functionality. The G7-based PDTMG formulation (prepared at ρ = 1.1 and r = 6.8; MP = 267 µM, MG = 17 µM, ML = 113 µM) revealed up to 120-fold increase in MFI as compared to bi-modal gemini/DOPE formulation (prepared at ρ = 10 and r = 3.3). Compared to the PDTMG systems formulated using G1-G6 and G9-G14 gemini surfactants (prepared at ρ = 1.1 and r = 6.8), G7 counterparts resulted up to 38-fold higher in MFI. Further, the G7- and G8-based PDTMG nanoparticles demonstrated comparable transfection activity with the gold-standard Lipofectamine 3000 transfection reagent. It is believed that the short RGD functional peptides (R3, R4) covalently linked to 18-series gemini surfactants provided reduced steric hindrance on the surface of the PDTMG nanoparticles when compared to other functional peptides (R5-R10), and exhibited endosomal destabilizing effects in response to cellular environment. The non-covalent addition of cationic peptide enhancers in formulation of the PDTMG delivery systems demonstrated a synergistic effect for DNA condensation, particle stability, cellular uptake, amplified endosomal release, protecting and facilitating the intracellular delivery of the pDNA. This project has demonstrated that G7- and G8-based PDTMG nanoparticles have the capability to undergo conformational changes in response to the cellular environment, disrupt the endosome, and release genetic materials into the cell cytoplasm. The development of these novel peptide-based lipopolyplexes provided a solid foundation for design of the versatile derivatives for in vivo targeted delivery of nucleotide-based therapeutics.en
dc.language.isoenen
dc.publisherUniversity of Waterlooen
dc.subjectGene Deliveryen
dc.titlePeptide-Driven Tri-Modal Gene Delivery Systems (PDTMG): Novel Versatile Peptide-Based Lipopolyplexes Incorporating Peptide-Functionalized Gemini Surfactants for Targeted Gene Therapy- Implementation of RGD Motifs as a Means for Endosomal Escapeen
dc.typeDoctoral Thesisen
dc.pendingfalse
uws-etd.degree.departmentSchool of Pharmacyen
uws-etd.degree.disciplinePharmacyen
uws-etd.degree.grantorUniversity of Waterlooen
uws-etd.degreeDoctor of Philosophyen
uws-etd.embargo.terms1 yearen
uws.contributor.advisorEdwards, David
uws.contributor.affiliation1Faculty of Scienceen
uws.published.cityWaterlooen
uws.published.countryCanadaen
uws.published.provinceOntarioen
uws-etd.embargo2019-09-04T21:49:03Z
uws.typeOfResourceTexten
uws.peerReviewStatusUnrevieweden
uws.scholarLevelGraduateen


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