Thermodynamic Model and Experiments Indicate Kinetically Trapped States in Ionisable Lipid Nanoparticles

Lipid nanoparticles (LNPs) are the leading delivery vehicles for RNA-based therapies, with pH-dependent mesophase transitions of their ionizable lipids proposed to play a crucial role in LNP function and endosomal escape. However, a consistent quantitative framework linking molecular lipid properties to the thermodynamics of these transitions is still lacking. Here, we establish such a framework by combining X-ray scattering, fluorescence spectroscopy, all-atom molecular dynamics simulations, and continuum modeling. Using small-angle X-ray scattering (SAXS) and Laurdan fluorescence spectroscopy, we characterize the pH- and temperature-dependent phase behavior of both bulk lipid systems and LNPs containing the ionisable lipids MC3 or ALC-0315. Laurdan fluorescence measurements reveal the same pH- and temperature-dependent transitions in bulk and LNP systems, which closely follow the inverse micellar (LII) - inverse hexagonal (HII) transitions identified by SAXS, establishing Laurdan as a reporter of these mesophase transitions in LNPs. We combine experimental and simulation data to parameterize a continuum model of the LII - HII transition, providing a quantitative description of mesophase stability. The model reveals that geometric frustration arising from packing constraints is a key determinant of mesophase stability. By combining experimental and simulation data with theoretical analysis, we determine the relevant membrane parameters, including mean and Gaussian curvature bending rigidities, and identify the parameter ranges that govern phase stability. Together, these results establish a quantitative continuum theory for pH-dependent mesophase transitions of ionisable lipid and suggest metastable states in response to pH changes as an inherent feature of LNP core phase.