Branched Amphiphilic Peptides Capsules for RNA Delivery: Design, Synthesis, and Optimization

Abstract

RNA is a versatile biomolecule with diverse therapeutic applications for vaccine development, cellular engineering, and other areas of biomedicine. Alone, however, RNA has little therapeutic potential, being readily degraded by environmental nucleases, and proving ineffective at cellular entry and expression. Delivery vectors are therefore needed to facilitate the protection and transfection of RNAs into cells. For this purpose, organic nanoparticles are most often used. Branched amphiphilic peptide capsules (BAPCs) are a novel class of organic nanoparticles which have previously been shown effective at RNA delivery in various in vivo systems with insect and murine models. Previously, BAPC peptides have varied significantly between batches and dependent on the synthesizer, resulting in high variation in experimental BAPC formulation impurities and results. BAPC toxicity has also received little investigation and never been directly compared with major FDA-approved or clinically used nanoparticle formulations such as lipid nanoparticles (LNPs). Here, I outline standardized synthesis, purification, and assembly protocols for branched amphiphilic peptides which compose BAPCs. Furthermore, I investigate potential mechanisms associated with BAPC toxicity in comparison to LNPs and elucidate potential avenues for the improvement of BAPC formulations moving forward. This work will allow for high-fidelity reproduction of BAPCs on any synthetic-chemistry capable lab bench. Further, this work outlines a comparable level of toxicity for BAPCs when compared to leading lipid alternatives, impressing the potential of this platform as a future clinically-relevant nanoformulation.