Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by H-1 NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties

H-1 NMR relaxometry was applied for molecular-level structuralanalysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify theimpact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC) and cholesterol, on the physicochemical properties of LNP.Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreasedthe molecular mobility of ionizable lipids. DSPC reduced the overallmolecular mobility of ionizable lipids, while cholesterol specificallydecreased the mobility of the hydrophobic tails of ionizable lipids,suggesting that cholesterol filled the gap between the hydrophobictails of ionizable lipids. The decrease in molecular mobility andchange in orientation of lipid mixtures contributed to the maintenanceof the stacked bilayer structure of siRNA and ionizable lipids, therebyincreasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometryrevealed that incorporating those neutral lipids enhanced PEG chainflexibility at the LNP interface. Notably, a small amount of DSPCeffectively increased PEG chain flexibility, possibly contributingto the improved dispersion stability and narrower size distributionof LNPs. However, cryogenic transmission electron microscopy representedthat adding excess amounts of DSPC and cholesterol into LNP resultedin the formation of deformed particles and demixing cholesterol withinthe LNP, respectively. The optimal lipid composition of ionizablelipid-based LNPs in terms of siRNA encapsulation efficiency and PEGchain flexibility was rationalized based on the molecular-level characterizationof LNPs. Moreover, the NMR relaxation rate of tertiary amine protonsof ionizable lipids, which are the interaction site with siRNA, canbe a valuable indicator of the encapsulated amount of siRNA withinLNPs. Thus, NMR-based analysis can be a powerful tool for efficientlydesigning LNP formulations and their quality control based on themolecular-level elucidation of the physicochemical properties of LNPs.