Mechanistic Insight into pH-Driven Phase Transition of Lipid Nanoparticles

Abstract

Lipid nanoparticles (LNPs) are essential delivery vehicles in mRNA-based vaccines, with their functionality largely governed by aminolipids. At low pH, protonated aminolipids facilitate efficient mRNA encapsulation, while their deprotonation at physiological pH drives LNP remodeling into a stable spherical structure, potentially aiding mRNA release during endosomal re-protonation and concomitant restructuring at low pH. Our study uncovers the pH-dependent phase transition of the Comirnaty lipid formulation at the molecular scale, highlighting the intricate interplay between lipid reorganization and aminolipid protonation. We observe a substantial shift in the aminolipid pKa, from 7-8 at the LNP surface to below 4 within the hydrophobic LNP core, resulting in an overall apparent pKa of the lipid nanoparticle of 6.3. The pH-dependent protonation significantly affects the aminolipid's localization, with protonated aminolipids enriched in vicinity of the mRNA and at the LNP surface at neutral pH. The lipid monolayer covering LNPs at neutral pH comprises cholesterol (60-65%), helper phospholipids (20-25%), PEGylated lipids (5%), and a mix of protonated and deprotonated aminolipids (5-10%). These findings provide mechanistic insights into aminolipids behavior in LNPs, offering a foundation for optimizing LNP design to enhance stability, tunable pH responses, and therapeutic efficacy.