Lipid nanoparticles (LNPs), a promising drug delivery system, face key limitations in terms of a poor understanding toward their assembling mechanism and intracellular dynamics. Conventional characterization methods like dynamic light scattering and spectroscopy fail to capture the dynamic mesoscopic structural changes of LNPs under pH gradient. To address this issue, here we employed synchrotron small-angle X-ray scattering (SR-SAXS) to ex-situ monitor the dynamic self-assembly process of mRNA-encapsulated LNPs at different physiologically-relevant pH. By performing time-resolved SAXS sampling during the dialysis process, we observed the dynamic structural evolution of LNPs, particularly their critical transitions from neutral (pH 7) to the more acidic environment (pH 4). These observations were cross-validated via cryo-transmission electron microscopy. The scattering profiles of mRNA-LNPs formed by three independent ionizable lipids approved by the FDA (ALC-0315, DLin-MC3-DMA, and SM-102) were comparably fitted based on the core-triple shell model to obtain the corresponding critical structural parameters. Cell transfection experiments showed that the MC3-mRNA-LNPs achieved the highest cellular transfection efficiency, which strongly correlates with the capacity of MC3 to form inverted hexagonal phases (H) that facilitate LNPs membrane perturbation. This study suggests SAXS as a practical tool to monitor the structural evolution of mRNA-LNPs under physiologically-relevant pH gradient, thus shedding light on the rational design of nucleic acid-based therapeutics, as well as extending the application of SAXS to the field of nanomedicine discovery. II
