NMR-Based Molecular-Level Characterization: A Review of Its Application in Unveiling Lipid Nanoparticle Formulations

Lipid nanoparticles (LNPs) are crucial for delivering nucleic acid therapeutics, yet their complex and heterogeneous nature poses significant challenges for comprehensive quality assessment. Traditional characterization methods provide macroscopic data, failing to offer the molecular-level insights necessary for understanding LNP quality, stability, and in vivo performance. NMR spectroscopy emerges as a powerful, non-destructive tool to bridge this analytical gap. This review highlights the diverse applications of NMR in characterizing LNP formulations. NMR techniques, including solution-state and solid-state NMR, provide atomic-level information on chemical composition, molecular structure, dynamics, and interactions. Specifically, NMR can probe lipid dynamics and molecular organization, revealing how components like cholesterol and helper lipids influence the mobility and arrangement of ionizable lipids. It elucidates the impact of cargo encapsulation on LNP structure, showing how nucleic acids like small interfering RNA (siRNA) interact with and suppress the mobility of LNP constituents. Furthermore, NMR helps understand intermolecular interactions and spatial organization within LNPs, mapping the distribution of lipids and cargo. Finally, NMR can assess the influence of preparation methods on LNP heterogeneity and monitor kinetic processes such as shedding of polyethylene glycol-conjugated lipids. NMR spectroscopy is indispensable for rational LNP design, robust quality control, and ultimately, enhancing the therapeutic efficacy of LNP-based medicines.