Messenger RNA (mRNA)-loaded lipid nanoparticles (LNPs) represent a transformative platform for nucleic acid delivery. Among various ionizable cationic lipids, [7-[4-(dipropylamino)butyl]-7-hydroxy-13-[()-octadec-9-enoyl]oxytridecyl] ()-octadec-9-enoate (CL4H6) has recently emerged as a promising alternative to DLin-MC3-DMA. In this study, we investigate the physicochemical properties and internal structures of mRNA-loaded CL4H6-LNPs using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), asymmetric flow field-flow fractionation coupled with multiangle light scattering and differential refractive index (AF4-MALS/dRI), and cryogenic transmission electron microscopy. SAXS and DLS analyses reveal that certain lipid compositions yield mRNA-loaded CL4H6-LNPs with remarkably narrow size distributions. The formation of monodisperse LNPs achieved even in a multicomponent system without optimized molecular design suggests that CL4H6 is structurally suited for forming the LNP with a specific curvature of the surface. The combined results of SAXS and AF4-MALS/dRI indicate that compositional control─particularly the CL4H6-to-DSPC and cholesterol ratios─critically governs the uniformity of the internal structure and dispersity of mRNA-loaded CL4H6-LNPs. Z Z
