Structuring of lipid nanoparticle mRNA formulations at acidic and neutral pH: X-ray scattering and molecular dynamics studies

mRNA lipid nanoparticles (LNPs) effectively deliver mRNA into cells. However, the structure of these particles remains poorly understood. LNP formulations are manufactured by initially forming nanoparticles at pH 4, to incorporate the RNA, then adjusting the pH 7.4 to trap the RNA within. This work investigates the structural rearrangements that occur during this process. We used the Martini 2 coarse-grained force field to model LNPs with varying water content and placement of PEGylated lipids. New parameters were developed for the lipids Dlin-MC3-DMA, its ionized form and the PEGylated lipid DMG-PEG 2000. At pH 4, the simulations suggest that bilayers are formed at the aqueous interface of the LNPs, with a largely amorphous core, whereas at pH 7.4, the bulk of the LNP is amorphous. Based on our models, we estimate that LNPs contain less than 1% w/w water, with the RNA cargo residing at the interface between water clusters and lipids. RNA entrapment is enhanced when water clusters form inside LNPs. Small-angle X-ray scattering (SAXS) measurements of LNPs at the two modeled pH values are consistent with the MD simulations. This study provides insight into water cluster formation, lipid arrangement, and RNA distribution inside LNPs, advancing our understanding of mRNA-LNP formulations.