Novel Delivery of Cyclic-Diguanylate Monophosphate Utilizing Amyloid Depots

<b>Background:</b> Recently, cyclic diguanylate monophosphate (c-di-GMP) drug delivery has garnered interest due to its potential in cancer immune modulation. In this pilot study, we developed a novel c-di-GMP formulation based on peptide amyloids. The amyloid depots were formed by combining an amyloidogenic prone 12 amino acid peptide sequence of receptor-interacting protein kinase 3 (RIP3) with cationic lipid ALC-0315, or using lysozyme proteins. Both RIP3 and lysozyme proteins have intrinsic physiological functions. This is the first time intrinsic peptides/protein-based amyloids have been explored for c-di-GMP delivery. The main goal was to evaluate how these amyloid depots could enhance c-di-GMP drug delivery and modulate responses in RAW 264.7 macrophage-like cells. <b>Methods:</b> Physicochemical characterization and cellular assays were utilized to characterize the amyloid structures and assess the efficacy. <b>Results:</b> Our results show that amyloid aggregates significantly improve the therapeutic efficacy of c-di-GMP. When RAW 264.7 cells were treated with c-di-GMP amyloids, we observed at least a 1.5-fold change in IL-6 expression, nitric oxide (NO) production, and reactive oxygen species (ROS) production compared to treatment with 5x free c-di-GMP treatment, which suggests that this system holds promise for enhanced therapeutic effects. <b>Conclusions:</b> Overall, these findings emphasize the potential of amyloid-based delivery systems as a promising approach for c-di-GMP delivery, warranting further investigations into their potential in therapeutic applications.