(512b) Post-Loading RNA in Lipid Nanoparticles

Lipid nanoparticles (LNPs) have recently emerged as effective vehicles for the delivery of ribonucleic acid (RNA) in vaccine formulations. LNP-based RNA vaccines for COVID-19 are among the most recent and critically important examples of such advanced formulations. A major limitation of current COVID-19 vaccines is the requirement of cold chain handling (-20 °C for Moderna, -70 °C for Pfizer). While there has been extensive research on lipid structure, lipid formulations, and RNA modifications to optimize in vivo efficacy, there has been less work on how processing affects LNP structure and properties. In this work, we have used a Confined Impinging Jet (CIJ) mixers to produce LNPs that are co-loaded or post-loaded with RNA while tracking their physicochemical properties. In CIJ mixers, an organic solvent stream—containing the lipids—and an aqueous anti-solvent stream are rapidly mixed. We have tuned buffer, pH, lipid concentration, RNA concentration, and RNA payload to produce <100 nm co-loaded LNPs—an optimal size for the best in vivo practices—with an encapsulation efficiency of >90%. Moreover, we have tuned these parameters to produce <100 nm empty LNPs (eLNPs) to be used for post-loading practices. In both cases, we have found that the co-loaded LNPs and eLNPs have been stable for several days. While electrostatic interactions control the complex formation of RNA and ionizable lipid, we show that by controlling the mobility of lipids, we can reach an encapsulation efficiency of as high as 80% via post-loading eLNPs. Using small-angle x-ray scattering, we further compare the internal structure of co-loaded and post-loaded LNPs. Given the similar physicochemical properties of post-loaded LNPs to those for co-loaded LNPs, we propose alternative routes to produce and store LNP-based vaccines to address the current cold chain handling challenge.