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dc.contributor.advisorEl-Aneed, Anas
dc.creatorDonkuru, McDonald 1978-
dc.date.accessioned2017-09-01T21:40:07Z
dc.date.available2017-09-01T21:40:07Z
dc.date.created2017-06
dc.date.issued2017-09-01
dc.date.submittedJune 2017
dc.identifier.urihttp://hdl.handle.net/10388/8079
dc.description.abstractDicatonic gemini surfactants have, within the past two decades, demonstrated advancing non-viral gene transfection ability in cell cultures and in animal models. However, knowledge of the intracellular/subcellular fate of gemini surfactants that may further advance gemini surfactant-based gene transfection is very limited. Therefore, my Ph.D. research conducted the investigation of gemini surfactants within transfected PAM212 keratinocytes including the development of effective bioanalytical mass spectrometric (MS) methods necessary for such investigations. For effective mass spectrometric bioanalysis of the gemini surfactants within cellular matrix their fingerprint fragment ions necessary for targeted identification and quantification were first determined through single-stage (MS), tandem (MS/MS) and multi-stage (MS3) analyses. The molecular composition of gemini surfactants was confirmed. In addition, fragmentation mechanisms of novel dipyridinium and β-cyclodextrin-based diquaternary ammonium molecules (chosen as study compounds) were established in detail, allowing for their qualititive and quantitative analysis. Hydrophilic interaction liquid chromatography-based (HILIC)-MS/MS methods, alone and in conjunction with the method of standard addition, were subsequently developed/validated by adopting multiple reaction monitoring (MRM), ensuring selectivity alongside the distinctive chromatographic separation. The analytical strategy ensured selectivity/specificity for target gemini surfactants including two lead compounds, 16(Py)-S-2-S-(Py)16 and 16-3-16. The validated HILIC-MS/MS methods were more sensitive, faster and have simplified isocratic elution relative to recently reported methods. In the application to nanoparticle-based gene transfection studies, the HILIC-MS/MS and standard addition–HILIC-MS/MS methods allowed a comprehensive investigation of the cellular uptake, intracellular deposition and subcellular distribution of the 16(Py)-S-2-S-(Py)16 and 16-3-16 gemini surfactants. The results showed similar cellular uptake and intracellular depletion trends but different subcellular distribution profiles. Both gemini surfactants showed an initial spike in their concentration within cells upon addition of gemini surfactant-based DNA nanoparticles to the cells, as would be expected to achieve nanoparticle entry into cells during transfection. The intracellular gemini surfactant content, however, underwent a depletion upon removal of the added nanoparticles – a trend observed for the first time and attributable to either gemini surfactant biodegradation or exocytosis from host cells. Results of subcellular distribution showed higher distribution of 16-3-16 to the mitochondria and nucleus relative to its 16(Py)-S-2-S-(Py)16 counterpart (p < 0.05), with the two having similar distribution to the cell membrane, cytosol and the remnant subcellular residue. This differential subcellular distribution, determined for the first time, may explain a suggested higher toxicity for 16-3-16 as its increased distribution to the mitochondria and nucleus could impact their biological integrity and function. Herein, the investigations and findings will benefit further exploration of gemini surfactants through the established molecular fragmentation fingerprints of novel compounds and comprehensive LC-MRM-MS bioanalytical strategies for studying the biological fate, elucidating varying toxicity and assessing possible metabolite formation.
dc.format.mimetypeapplication/pdf
dc.subjectGemini surfactants, gene/drug delivery, gene therapy, nanoparticle-based transfection, liquid chromatography-mass spectrometry (LC-MS/MS), qualitative/quantitative analysis, biological/intracellular fate, subcellular distribution.
dc.titleMASS SPECTROMETRIC STUDIES OF NOVEL LIPID-BASED NANOMATERIALS: INVESTIGATIONS OF THE INTRACELLULAR FATE
dc.typeThesis
dc.date.updated2017-09-01T21:40:07Z
thesis.degree.departmentPharmacy and Nutrition
thesis.degree.disciplinePharmacy
thesis.degree.grantorUniversity of Saskatchewan
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)
dc.type.materialtext
dc.contributor.committeeMemberIldiko, Badea
dc.contributor.committeeMemberKatselis, George K
dc.contributor.committeeMemberHeadley, John V
dc.contributor.committeeMemberBlackburn, David
dc.contributor.committeeMemberKroll, Ed
dc.creator.orcid0000-0002-1863-7988


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