Amyloid aggregates play a major role in diseases as well as in normal physiological function. Receptor-interacting protein kinases 1 and 3 (RIP1/RIP3) aggregates complexes in cellular necroptosis is one example of protein aggregation in normal cellular function. Although recently there have been several studies on full kinase proteins aggregation, the aggregation potential of small peptide sequences of RIP1/RIP3, the physicochemical properties, and the potential in biomedical applications have not been explored. Hence, in this paper, we study the aggregation propensity of peptides consisting of four and twelve amino acid sequences in the RHIM region of RIP1/RIP3 proteins that are known to drive the beta-sheet formation and the subsequent aggregation. The aggregation kinetics, physicochemical characterization, mechanosensitive properties, cellular effects, and potential as a cancer drug depot have been investigated. The results show that the number and concentration of amino acids play a role in amyloid-like aggregates’ properties. Further, the aggregates when formulated with cisplatin-induced significant lung cancer cell toxicity compared to an equal amount of cisplatin with and without ultrasound. The study would serve as a platform for further investigation on RIP1/RIP3 peptide and protein aggregates, their role in multiple cellular functions and diseases, and their potential as drug depots.

Amyloid aggregates have attracted significant interest in regard to diverse biomedical applications, particularly in the field of drug delivery. Here, we report novel amyloid aggregates based on a 12-amino-acid peptide from the amyloidogenic region of the receptor-interacting kinase 3 (RIP3) protein and a thermoresponsive triblock copolymer, namely, Pluronic F127 (RIP3/F127). Physicochemical characterization was performed to determine the aggregation size, morphology, and stimuli-responsive properties. The potential of the aggregates as a drug depot was assessed in lung cancer cells, using Doxorubicin (Dox) as a model drug. The results show that RIP3 and RIP3/F127 exhibit amyloidogenic properties. Further, the RIP3/F127 amyloids exhibited significant ultrasound-responsive properties compared to amyloid aggregates without Pluronic F127. Moreover, the RIP3/F127/Dox amyloid formulations that were subjected to ultrasound treatment exhibited greater toxicity to lung cancer cells compared to that of Dox alone at equal concentrations. Overall, the results from this proof-of-concept study show that amyloidogenic peptide aggregates with stimuli-responsive properties can be utilized as efficient drug delivery depots.

Background/Objectives: Lysozyme-based amyloid aggregates offer a promising platform for RNA delivery due to their stability, cationic nature, biocompatibility, and ability to form well-defined structures. In this study, we evaluated their potential as drug carriers, focusing on the delivery of polyinosinic–polycytidylic acid (Poly(I:C)), an immunostimulatory synthetic RNA. To validate RNA delivery capability and rule out the possibility that observed effects arose from the lysozyme–Poly(I:C) complex itself, small interfering RNA (siRNA) was also used to verify that the successful delivery of intact and functional RNA was the cause of the observed effects. Methods: The aggregates were characterized by particle size, zeta potential, morphology, and RNA encapsulation efficiency. Results: In vitro studies using RAW 264.7 macrophage-like cells demonstrated that Poly(I:C)-loaded aggregates improved RNA uptake and triggered significant immune activation without inducing toxicity. To further confirm the potential of lysozyme amyloids in RNA delivery, GFP siRNA-loaded aggregates were evaluated in A549-GFP cells. A notable decrease in GFP expression, confirmed through confocal microscopy and flow cytometry, confirmed successful intracellular delivery. Conclusions: These results highlight the potential of lysozyme amyloids as non-viral vectors for RNA delivery, with promising applications in immunotherapy.

Background: 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. Methods: Physicochemical characterization and cellular assays were utilized to characterize the amyloid structures and assess the efficacy. Results: 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. Conclusions: 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.

Pulmonary arterial hypertension (PAH) is a devastating disease driven largely by oxidative stress and inflammation. The pulmonary arterial endothelium plays a key role in PAH pathogenesis. In this study, we developed novel poly(lactic-co-glycolic acid) (PLGA)-based lutein nanoparticles coated with a Vascular Cell Adhesion Molecule-1 (VCAM-1) antibody to target pulmonary arterial endothelial cells (PAECs) associated with PAH. Lutein, a potent antioxidant and anti-inflammatory carotenoid, served as the model drug. The lutein nanoparticles were produced by nanoprecipitation, followed by surface modification with VCAM-1 antibody. Physicochemical characterization and cellular efficacy studies were performed to assess the potential of the formulation. The studies revealed the production of nanoparticles with an average size of approximately 170 nm that exhibited specificity for PAH patient-derived PAECs (PAH-PAECs) and antioxidant and anti-inflammatory properties. Moreover, the nanoparticles did not exhibit significant cellular toxicity toward healthy PAECs. This proof-of-principle study demonstrates the therapeutic potential of targeted nanoparticles for the treatment of PAH.