Molecular Insight into Lipid Nanoparticle Assembly from NMR Spectroscopy and Molecular Dynamics Simulation.

Lipid nanoparticles (LNPs) have emerged as the premier drug delivery system for oligonucleotide vaccines and therapeutics in recent years. Despite their prosperous advancement in research and clinical applications, there is a significant lack of mechanistic understanding of the assembly of lipid particles at the molecular level. In our study, we utilized a combination of solution and solid-state NMR, together with molecular dynamics simulations, to elucidate local structures and interactions of chemical components across multiple motional regimes. Our results comprehensively evaluated the impact of formulation components and engineering process factors on the particle formation and identified the interplay of phospholipids (DSPC), poly(ethylene glycol) (PEG) lipid conjugates, and cholesterol in governing the particle size and lipid dynamics from a structural perspective, using static 31P NMR techniques. These studies provide novel insights into the impact of particle engineering on the molecular properties of the LNP envelope membrane. Additionally, molecular interactions and compositional distribution play a critical role in particle engineering and the consequent stability and potency. In this study, we have identified intermolecular contacts among the lipid components using one-dimensional 1H-13C cross-polarization magic angle spinning experiments, 1H relaxation measurements, and two-dimensional 1H-1H correlation methods, providing a structural basis for the lipid assembly. Interestingly, the cationic and ionizable lipids, conventionally regarded as stabilizing agents primarily located within the core of LNPs, were found to interact with PEG lipids and coexist in the outer layer of the particles. We suggest that LNPs examined here are comprised of an outer layer rich in lipid components surrounding a core region. Our high-resolution findings offer insightful structural and dynamic details pertaining to the individual chemical components in the lipid particles and their interactions influence lipid complex structure and stability in particle engineering.