Scalable Formulation of Lipid Nanoparticles for RNA Encapsulation by Flash NanoPrecipitation

Abstract

Lipid nanoparticles (LNPs) have recently emerged as effective vehicles for ribonucleic acid (RNA) delivery in vaccine formulations. LNP-based RNA vaccines for COVID-19 are among the most recent and critically important examples of such advanced formulations. While there has been extensive research on lipid structure, lipid formulations, and RNA modifications to optimize performance, there has been less work on how processing affects LNP structure and properties. In this work, we have used a Confined Impinging Jet (CIJ) and a Multi-Inlet Vortex Mixer (MIVM) to produce LNPs using Flash NanoPrecipitation (FNP). In these turbulent mixers (Reynolds number>5000), an organic solvent stream—containing the lipids—and an aqueous anti-solvent stream which may contain RNA are rapidly mixed. Through this scalable process, LNPs are formed only at the proper compositions and flow rates of the fluid streams. We have explored the effects of buffer pH, ionic strength, and lipid concentrations on the key properties of LNPs, including size, zeta potential, and colloidal stability. We show how these parameters can be tuned to produce 60 to 80 nm LNPs—an optimal size for the best in vivo practices. Moreover, we have maximized the loading of RNA in LNPs under controlled electrostatic and steric interactions. These findings are critical to produce RNA-loaded LNPs, and further develop therapeutic formulations.