Mapping Hydration and Nanoarchitecture in mRNA‐Loaded Lipid Nanoparticles Through Small‐Angle Neutron Scattering

mRNA‐loaded lipid nanoparticles (LNPs) offer significant therapeutic potential for various diseases, yet their structural characteristics and component distribution remain incompletely understood. This study utilizes small‐angle neutron scattering (SANS) to explore the internal architecture of KC2 LNPs, employing deuterated lipid substitutions in contrast‐varied media. Our analyses reveal that KC2 LNPs have diameters of 50–60 nm, consistent with cryogenic transmission electron microscopy (cryoTEM) and dynamic light scattering. Core–shell modeling indicates a core radius of 16–17 nm and a shell thickness of 6–8 nm, details often overlooked by cryogenic electron microscopy. Interestingly, results demonstrated that LNPs contain 48%–55% water (total particle volume), which is highly exchangeable with the dispersing medium. This water exchange is crucial during structural monitoring under conditions simulating endosomal environments, as acidification leads to an internal structural reorganization, involving solvent influx into the LNPs and potentially reorganization of components within and across the core and shell phases. Invariant analysis further confirms the high water content of LNPs, validating the model fitting results, affirming KC2 LNPs’ compositional complexity, which are pivotal for understanding LNP stability and responsiveness. These insights can guide the future design of mRNA nanotherapeutics, enhancing their therapeutic efficacy.