(414c) The Green Chemistry Synthesis of Cationic Lipids and Polymers for Nanoparticle-Based mRNA Vaccines and Therapeutics

Lipid nanoparticles (LNPs) formulated with ionizable cationic lipids are clinically advanced non-viral gene delivery platforms and hold great potential for gene therapeutics, exemplified by the two COVID-19 vaccines developed by Pfizer-BioNTech and Moderna. The FDA-approved ionizable cationic lipids such as DLin-MC3-DMA (MC3), ALC-0315, and SM-10218 are synthesized through multistep reactions and present drawbacks such as a low chemical yield especially after multiple post-reaction processes. Moreover, most published lipid-like materials are synthesized through Michael addition or epoxide ring-opening reactions, which makes the resulting lipid-like materials nonbiodegradable with potential cytotoxicity. Herein, we propose a 'Green Chemistry' approach to develop a new chemical library of ionizable cationic lipids or polymers using a one-step chemical-biological enzyme-catalyzed esterification method. Subsequently, these lipids or polymers are formulated into LNPs or polymer-lipid hybrid nanoparticles for mRNA delivery and COVID-19 vaccine development. For example, we have utilized a chemical and biological tool, the enzyme catalyst Candida antarctica Lipase B, to synthesize a chemical library of degradable ionizable cationic lipids using amino alcohols and lipid acids, resulting in 144 lipid-like materials with high chemical yield and purity. Through highthroughput screening, the top-performing ionizable lipid was determined to be AA3-DLin, which is rationally designed to be composed of two unsaturated linoleic lipid chains, two tertiary amine head groups from piperazine which can provide enough positive charge to condense the gene payloads in acidic buffer, and two biodegradable ester linkers. To further investigate the optimized AA3-DLin LNP formulation, an orthogonal design of experiment (DoE) methodology was employed to fine-tune the molar ratios of AA3-DLin, DOPE, cholesterol, and DMGPEG. The top-performing AA3-DLin LNPs show outstanding mRNA delivery efficacy and long-term storage capability. Furthermore, the AA3-DLin LNPs efficiently deliver SARS-CoV-2 full-length spike encoded mRNA in vitro and in vivo. Both fresh and 12-month stored AA3-DLin COVID-19 vaccines successfully elicit spike-specific antibodies and Th1-biased T cell immune response in immunized mice. The AA3-DLin LNPs are an excellent mRNA delivery platform, and this study provides an overall perspective of new ionizable cationic lipids from aspects of lipid design, synthesis, screening, optimization, fabrication, characterization, and application.