A Time-Resolved In Situ SAXS Method for Real-Time Monitoring of Lipid Nanoparticles Assembly

Lipid nanoparticles (LNPs) have emerged as popular nucleic acid delivery systems, yet the dynamic mechanisms related to their self-assembly and structural maturation remain insufficiently understood due to the limitations of traditional offline characterization tools. This study establishes a time-resolved (TR) in situ small-angle X-ray scattering (SAXS) methodology to monitor the structural evolution of LNPs during microfluidic formulation and subsequent maturation. By integrating a dual-channel microfluidic mixing system with a SAXS measurement platform, we successfully captured the real-time scattering profiles of both empty and messenger RNA-loaded nanoparticles (mRNA-LNPs). The results demonstrate distinct assembly pathways for empty-LNPs and those encapsulated with mRNA. The empty-LNPs undergo a gradual transition toward periodic nanostructures, whereas mRNA-LNPs exhibit rapid complexation into stable subunits followed by hierarchical assembly. Furthermore, the platform effectively tracked nanoscale structural rearrangements during a microfluidic dilution process, revealed by subtle shifts in scattering peaks and internal periodicity. Overall, this time-resolved approach provides a robust experimental framework for capturing transient intermediate states, offering a valuable tool to elucidate molecular assembly mechanisms and facilitate the rational design of next-generation nanomedicines.