Development and Characterization of Novel Peptide- and Protein-Based Amyloid Depots for Therapeutic Delivery

Abstract

This dissertation investigates amyloids, which are a specific type of protein aggregate, and their potential in drug delivery focusing the research on amyloid complexes in the receptor-interacting protein kinase (RIP) family. Given their involvement in both disease pathology and normal cellular processes, the study investigates the potential of this amyloid aggregate in therapeutic drug delivery. This is done by utilizing ultrasound-responsive and thermoresponsive polymers. The key objectives include developing and characterizing RIP1/RIP3 and RIP3 amyloids, formulating tunable (ultrasound or temperature-sensitive) amyloids, formulating microvesicle-encapsulated amyloids, and developing amyloid depots for oligonucleotide delivery. For the study, amyloid-prone amino acid sequences within the RHIM region of RIP1 and RIP3 proteins are used because they form functional amyloid structures. These amyloids are then used as drug depots using cisplatin as a model drug. Furthermore, peptide/polymer-based amyloid aggregates were studied by combining the amyloidogenic RIP3-derived NIYNCSGVQVGD sequence with the stimuli-responsive polymer Pluronic F127 using doxorubicin (DOX) as a model drug. This makes a remarkable exploration in the scientific field because this is the first time amyloid structures are combined with Pluronic F127. The research continues with developing microvesicle-encapsulated amyloid aggregates (MV-DOX-BSA) to enhance the therapeutic effectiveness of DOX with the goal of producing minimal toxicity. This was done by loading microvesicles derived from RAW 264.7 macrophage cells with BSA-DOX amyloid aggregates. The evaluation processes yielded results that demonstrated improved drug delivery efficiency and increased cancer cell death compared to free drugs. The study also uses cyclic-diguanylate monophosphate (c-di-GMP) as a model drug, and amyloid depots were explored for oligonucleotide delivery by combining cationic lipid ALC-0315 with RIP3 and BSA amyloid aggregates. This system was assessed for the ability to improve intracellular delivery and immune-stimulating properties. The results showed effective means of improving the uptake and immunomodulatory effects of c-di-GMP. Overall, this dissertation presents a new way of amyloid-based drug delivery tactics with promising results of improving therapeutic effectiveness of the drug while minimizing side effects. By combining protein aggregates with certain drug delivery techniques, this research provides a new and innovative way of improving cancer treatments and immunotherapy practices.