Choice of organic solvent affects function of mRNA-LNP; pyridine produces highly functional mRNA-LNP

Lipid nanoparticles (LNPs) are well-known nanocarriers for mRNA delivery. mRNA-encapsulated LNPs (mRNA-LNPs) are prepared by alcohol dilution (broadly defined as solvent dilution) method, in which mRNA dissolved in acidic buffer is mixed with lipid dissolved in an organic solvent. Ethanol is the most commonly used organic solvent for dissolving lipids during the preparation of mRNA-LNPs. However, no studies have systematically investigated the effects of organic solvents that dissolve lipids during the preparation of mRNA-LNPs on the properties and functions of mRNA-LNPs. In this study, we prepared mRNA-LNPs by using a series of organic solvents and evaluated their characteristics. After screening, we discovered that pyridine, an organic solvent, improved the quality of mRNA-LNPs and their function in vitro and in vivo. Pyridine was applied versatilely to some lipid-composition combinations generally used in the preparation of mRNA-LNPs and can also be adapted to microfluidic-based preparation. Furthermore, with appropriate purification, the amount of pyridine remaining in the final preparation of the mRNA-LNPs was extremely low and did not affect safety. Although further mechanism-based studies are required, we conclude that pyridine is a solvent that can be applied to the production of mRNA-LNPs as a pharmaceutical product.