Biochemical changes of the insulin molecule due to functionalized surfaces: An investigation from a therapeutic perspective

Abstract

Insulin therapeutics is the cornerstone in current diabetes management, especially while treating Type I diabetes. However, being a protein, insulin is known to demonstrate agglomeration when subjected to alterations in its biochemical environment, such as acidity, temperature fluctuation, coming in contact with hydrophobic surfaces, and agitation. This thesis describes a detailed and systematically designed study to investigate the curious case of insulin agglomeration, understand the mechanism(s) that drive such agglomeration, and realize the therapeutic impacts of such insulin agglomeration. To begin with, a detailed literature survey was conducted on insulin agglomeration, with or without fibrillation, and converge the available knowledge into a single discourse, which is presented as the Chapter 1 (introduction) of this thesis. This literature survey was followed by four experimental chapters (Chapters 2–4) where through a range of systematic and consistent experimental approach the phenomena of acidity-, heating-, and hydrophobic surface-induced insulin agglomeration was investigated. To characterize the insulin agglomerates, a broad range of analytic platforms, such as transmission and scanning electron microscopy, circular dichroism, nanoparticle tracking analysis, and zeta potential were used. Additionally, this thesis work also exploited the promise of advanced optical microscopy platforms, such as the confocal laser scanning microscopy and fluorescence lifetime imaging microscopy to probe the agglomerates and identify the varied segments of fluctuating acidity inside them. Taken together, the data was able to provide vital information on insulin agglomeration with a mechanistic understanding of what drives such agglomeration. An in vitro study was also conducted (Chapter 3) to find out if the insulin agglomerates generated due to heating at high temperatures (50°C and 65°C) were bioactive, and the results came positive. The Chapter 5 then explains another in vitro study where fluorophore-labeled insulin uptake in human hepatocellular carcinomaderived HepG2 cells was investigated with confocal microscopy. Finally, Chapter 6 summarized the findings, provide a few additional perspectives on overall realizations made through these studies, and prioritized some lines of research for future explorations.