Lipid nanoparticles (LNPs) are the leading platform for delivery of RNA-based therapeutics. There is considerable interest in optimizing the transfection potency of these systems. Standard formulation protocols involve mixing lipids in ethanol with a buffer containing mRNA at pH 4, followed by dialysis against a pH 7.4 saline buffer to remove ethanol and raise the pH. The dialysis process results in fusion of mRNA-lipid complexes and small vesicles formed at pH 4, driven by two processes: Conversion of the ionizable lipid from the positively charged bilayer form to the neutral oil form and fusion stimulated by higher ionic strengths. Here we explored the consequences of a two-step dialysis process involving formulating at pH 4, 25 mM NaOAc, and then inducing fusion at pH 4 by dialyzing against a higher ionic strength buffer while maintaining the ethanol concentration at 25% (v/v). This was followed by dialysis against PBS to raise the pH. We show that the two-step protocol results in increased mRNA loading per LNP, a decreased proportion of "empties" as compared to the standard process, and improved transfection potencies in vitro and in vivo when compared to LNP mRNA formed from the traditional one-step buffer exchange process.
