Molecular Dynamics of Lipid Nanoparticles Explored through Solution and Solid-State NMR Spectroscopy and MD Simulations

Lipid nanoparticles (LNPs) are a critical platform for nucleic acid delivery, characterized by their kinetic assemblies and structural complexity. In this study, we integrated solution and solid-state NMR with molecular dynamics (MD) simulations to probe lipid dynamics in therapeutic siRNA-encapsulated LNP formulations over a temperature range of 30 °C to -50 °C. Using ¹H-¹³C cross-polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) solid-state NMR experiments, we probed mobile and rigid components by analyzing ¹³C signal attenuation due to molecular motions spanning nanoseconds to seconds. Our findings demonstrate that the cationic lipid, Lipid X, exhibits significantly higher dynamics at nanosecond time scales than other lipid components, with siRNA encapsulation reducing its mobility, thereby supporting a dense core model for siRNA-loaded LNPs. In contrast, DSPC and cholesterol, which constitute the outer envelope membrane of LNP particles, exhibit slower motion compared to the cationic lipid. PEGylated lipid content strongly influences LNP membrane dynamics, displaying a broad dynamic distribution of its polyethylene glycol chains on the particle surface, as shown by relaxation-filtered Diffusion-Ordered NMR Spectroscopy (DOSY). Phase transition studies indicate that the siRNA-cationic-lipid core shifts to a slower motional state at -50 °C, evidenced by the disappearance of Lipid X ¹³C INEPT signals, whereas the DSPC/cholesterol/PEG membrane undergoes a phase change at -20 °C, marked by an increase in ¹³C CP intensity. Interestingly, the freezing of the bulk solution at -15 °C to -20 °C and the water domain within the interior core region at -35 °C to -50 °C appear to couple with the slowing of motions in the outer membrane and the siRNA-cationic-lipid complex, respectively. Complementary MD simulations provide detailed insights into lipid organization and dynamics across the examined temperature range. Collectively, these spectroscopic and computational findings deepen our molecular-level understanding of the LNP core and surface dynamics and offer valuable guidance for optimizing stable LNP formulations for therapeutic applications.