From varied initiation strategies to converged ends: key factors in mRNA-LNPs formation and functional attributes.

The particle properties and transfection potency of mRNA lipid nanoparticles (mRNA-LNPs) are largely determined by the preparation process. We investigated the role of initiation methods by comparing distinct strategies, such as Conventional preparation method, Post-encapsulation method, and Tertiary butanol-water monophase method, using a standardized Two-step tangential-flow filtration (TFF) process. Our findings indicate that the pH-driven fusion process during TFF-mediated solvent exchange plays a predominant role in shaping mRNA-LNPs properties by facilitating the redistribution of components and the reorganization of nanoparticles. In contrast, variations in the initiation method-including the temporal sequence, spatial organization, and ethanol content during loading-appear to have minimal impact. mRNA-LNPs with similar properties and potency can be produced using different initiation methods, provided the solvent exchange parameters are consistent. Importantly, this work reveals the key determinants of particle formation and mRNA encapsulation across the initial preparation and solution exchange phases. These findings not only deepen the fundamental understanding of mRNA-LNP self-assembly and offer flexible preparation strategies, but also highlight the critical role of solvent exchange in defining nanoparticle attributes, thereby providing theoretical and practical guidance for controlled preparation, process scale-up, and diverse clinical applications.