PREPARATION AND CHARACTERIZATION OF GLUCOSE NANOSENSORS FOR INTRACELLULAR APPLICATIONS
Committee ChairAspinwall, Craig A.
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PublisherThe University of Arizona.
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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.