Synthesis, Characterization and In-vivo Testing of Photoactivatable Insulin Depots for Continuously Variable and Minimally Invasive Insulin Delivery

Title from PDF of title page viewed May 15, 2020Dissertation advisor: Simon H. FriedmanVitaIncludes bibliographical references (pages 275-290)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2019Proteins are macromolecules involved in a diverse array of functions. Mutations or abnormal levels of proteins are indicated in several diseases. Despite showing early promise, the translation of protein therapeutics into the clinics has been challenging. The stability of these macromolecules, their delivery, and penetration inside the cells have been the main hurdles limiting their true potential. In the dissertation, various strategies to overcome such protein delivery challenges are discussed. Insulin is a lifesaving peptide for millions of diabetics around the world. Despite significant progress in insulin therapies, the quality of life in diabetics is constrained by the burden of multiple daily injections, invasive nature of therapy and inability to control the blood glucose tightly. To address these concerns, we constructed a photoactivatable insulin depot (PAD). In the approach, an insoluble depot of modified insulin was created by linking insulin covalently to photolabile caging moieties. Transcutaneous irradiation breaks the bond to release insulin from the depot into the systemic circulation. Chapter 3 describes the first successful testing on our PAD technology in diabetic animal models. In Chapters 2 and 4, I describe second-generation materials incorporating more efficient photolabile groups utilizing visible light wavelengths and PAD material with greater insulin loading. These changes improved the overall performance by several folds when tested in-vivo. Chapter 5 discusses the strategies addressed to deliver siRNA inside cells for effective light-activated RNA interference (LARI). LARI can be used for studying biology and cellular processes. Once administered, proteins are prone to degradation by ubiquitous proteases, limiting their circulation time and therapeutic effect significantly. Chapter 6 discusses prodrug strategies to temporarily modify proteins to shield them against proteases. We envisioned cross-linking amino acid residues on the surface via small crosslinkers. The tight bridges would hinder proteases from binding to proteins and unwinding the helices preventing their proteolysis. We also attempted integration of this approach to achieve intracellular protein delivery which is another obstacle in protein delivery. Here, the cross linking was performed via disulfide linkages. The disulfide groups would be reduced once inside the cells, yielding native proteins.Introduction: photoactivatable insulin depot -- Synthesis of insulin macro polymer -- In-vivo testing of first-generation pad material -- Synthesis and testing of advanced second-generation material -- Light activated SiRNA nanoparticles -- Intracellular protein delivery using protein prodrug

Light Control of Insulin Release and Blood Glucose Using an Injectable Photoactivated Depot

In this work we demonstrate that blood glucose can be controlled remotely through light stimulated release of insulin from an injected cutaneous depot. Human insulin was tethered to an insoluble but injectable polymer via a linker, which was based on the light cleavable di-methoxy nitrophenyl ethyl (DMNPE) group. This material was injected into the skin of streptozotocin-treated diabetic rats. We observed insulin being released into the bloodstream after a 2 min trans-cutaneous irradiation of this site by a compact LED light source. Control animals treated with the same material, but in which light was blocked from the site, showed no release of insulin into the bloodstream. We also demonstrate that additional pulses of light from the light source result in additional pulses of insulin being absorbed into circulation. A significant reduction in blood glucose was then observed. Together, these results demonstrate the feasibility of using light to allow for the continuously variable control of insulin release. This in turn has the potential to allow for the tight control of blood glucose without the invasiveness of insulin pumps and cannulas