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dc.contributor.authorPaluwatte Muhandiramlage, Thusithaen_US
dc.creatorPaluwatte Muhandiramlage, Thusithaen_US
dc.date.accessioned2011-12-05T22:25:47Z
dc.date.available2011-12-05T22:25:47Z
dc.date.issued2010en_US
dc.identifier.urihttp://hdl.handle.net/10150/194267
dc.description.abstractThe dynamics of glucose-stimulated insulin secretion play a key role in normal physiological regulation, with altered temporal profiles observed in diabetic patients. Elucidation of the underlying mechanisms responsible for insulin secretion is key to understanding the development and progression of diabetes, though such studies have been limited due to a dearth of methods with sufficient sensitivity and spatiotemporal resolution to facilitate direct quantification of cellular glucose. In this work, we developed a cell-penetrating, glucose-responsive fluorescence indicator protein (FLIP) capable of detecting dynamic glucose changes in single pancreatic β-cells. Flow cytometry and confocal microscopy revealed that cell penetrating FLIP was directly loaded into single cells via simple incubation, avoiding the requirement for cellular transfection. The cell-penetrating FLIP yielded similar analytical performance in vitro compared to unmodified FLIP, and facilitated direct observation of cellular glucose levels, though with a lower in vivo response compared to FLIP-transfected cells. To minimize intracellular proteolytic degradation and/or compartmentalization that may contribute to the lowered sensor response, FLIP was encapsulated in stabilized, porous phospholipid nanoshells (PPNs). In vitro characterization showed that the FLIPPPN (K(d) = 854 ± 32 μM) yielded similar analytical performance to FLIP with < 1 min response time. FLIP-PPN was readily loaded into live cells upon modification of the PPN with TAT peptide. Intracellular delivery and localization of FLIP-PPN was confirmed using confocal microscopy. FLIP-PPNs exhibited ca. 50% more activity in the intracellular environment compared to non-encapsulated FLIP, suggesting the importance of encapsulated delivery. To obtain optimized glucose transport, the molecular weight cutoff of PPNs was examined using a novel dextran retention method with a mass resolution of 162 Da. Stabilized PPNs exhibited 90 % retention at MW ca. 1800. Kinetic control of permeability with binary lipid mixtures was investigated, and composition dependent permeability of PPNs was observed. Lipid domain formation was evident in PPNs prepared using binary lipid mixtures (30-100% bis-SorbPC), where the MW cutoff was found to be unchanged compared to 100% bis-SorbPC. Proton permeability studies of lipid mixtures below the resolution of the dextran retention assay (< 20% bis-SorbPC) confirmed composition dependent change of permeability.
dc.language.isoenen_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.subjectChemistryen_US
dc.titlePREPARATION AND CHARACTERIZATION OF GLUCOSE NANOSENSORS FOR INTRACELLULAR APPLICATIONSen_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.contributor.chairAspinwall, Craig A.en_US
thesis.degree.grantorUniversity of Arizonaen_US
thesis.degree.leveldoctoralen_US
dc.contributor.committeememberSaavedra, S. Scotten_US
dc.contributor.committeememberHeien, Michaelen_US
dc.contributor.committeememberTollin, Gordonen_US
dc.contributor.committeememberGhosh, Indraneelen_US
dc.identifier.proquest11296en_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.namePh.D.en_US
refterms.dateFOA2018-08-24T23:50:48Z
html.description.abstractThe dynamics of glucose-stimulated insulin secretion play a key role in normal physiological regulation, with altered temporal profiles observed in diabetic patients. Elucidation of the underlying mechanisms responsible for insulin secretion is key to understanding the development and progression of diabetes, though such studies have been limited due to a dearth of methods with sufficient sensitivity and spatiotemporal resolution to facilitate direct quantification of cellular glucose. In this work, we developed a cell-penetrating, glucose-responsive fluorescence indicator protein (FLIP) capable of detecting dynamic glucose changes in single pancreatic β-cells. Flow cytometry and confocal microscopy revealed that cell penetrating FLIP was directly loaded into single cells via simple incubation, avoiding the requirement for cellular transfection. The cell-penetrating FLIP yielded similar analytical performance in vitro compared to unmodified FLIP, and facilitated direct observation of cellular glucose levels, though with a lower in vivo response compared to FLIP-transfected cells. To minimize intracellular proteolytic degradation and/or compartmentalization that may contribute to the lowered sensor response, FLIP was encapsulated in stabilized, porous phospholipid nanoshells (PPNs). In vitro characterization showed that the FLIPPPN (K(d) = 854 ± 32 μM) yielded similar analytical performance to FLIP with < 1 min response time. FLIP-PPN was readily loaded into live cells upon modification of the PPN with TAT peptide. Intracellular delivery and localization of FLIP-PPN was confirmed using confocal microscopy. FLIP-PPNs exhibited ca. 50% more activity in the intracellular environment compared to non-encapsulated FLIP, suggesting the importance of encapsulated delivery. To obtain optimized glucose transport, the molecular weight cutoff of PPNs was examined using a novel dextran retention method with a mass resolution of 162 Da. Stabilized PPNs exhibited 90 % retention at MW ca. 1800. Kinetic control of permeability with binary lipid mixtures was investigated, and composition dependent permeability of PPNs was observed. Lipid domain formation was evident in PPNs prepared using binary lipid mixtures (30-100% bis-SorbPC), where the MW cutoff was found to be unchanged compared to 100% bis-SorbPC. Proton permeability studies of lipid mixtures below the resolution of the dextran retention assay (< 20% bis-SorbPC) confirmed composition dependent change of permeability.


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