Combining SAXS analysis and MD simulation to determine structure and hydration of ionizable lipid hexagonal phases

Cationic ionizable lipids (CILs) are fundamental components of inverse hexagonal (H_II) lipid assemblies, which mediate the encapsulation and release of negatively charged mRNA through a pH-dependent mechanism. Since variations in the structure and composition of the H_II phases can significantly impact the biological efficacy of the mRNA-carrying lipid nanoparticles (LNP), a comprehensive understanding of the ionizable lipid H_II phases is necessary. We present an integrated approach combining small-angle X-ray scattering (SAXS) experiments, molecular dynamics (MD) simulations and a continuum model to elucidate lipid distribution and water content within H_II phases. Our results indicate strong agreement between structures derived from MD simulations and SAXS data. To this end, we introduce a method to correct for periodic boundary artifacts when computing scattering profiles from MD simulations. This enables direct, model-free comparisons between experimental and simulated data, enhancing the reliability of structural interpretations, specifically the water content of the H_II phases. Next, we developed a continuum model to extend structural analysis to CIL H_II phases for which MD data is unavailable. This integrative framework not only provides molecular-level insights into the ionizable lipid H_II mesophase but also enables the prediction of hydration properties across different CIL compositions. The different approaches consistently yield water contents that seem to correlate with the lipids' transfection efficiencies. By bridging experimental and simulation data, our approach offers a powerful tool for the rational design and optimization of lipid nanoparticles, potentially linking a lower water content with an increased therapeutic performance.