Storage Buffer Composition Impacts Internal Structure, Freeze–Thaw Stability, and Transfection Efficiency of mRNA-Lipid Nanoparticles

High Resolution Image Download MS PowerPoint Slide Messenger RNA (mRNA) lipid nanoparticles (mRNA-LNPs) are central to emerging vaccines and therapeutics, but their wide implementation is constrained by limited endosomal escape and instability during long-term storage and freezing. While buffers are routinely optimized to prevent instability, the impact of buffer on the internal structural organization of LNPs and, consequently, their delivery efficiency remain unresolved. Here, we study the impact of storage in Tris, histidine, and citrate buffers for mRNA-LNPs formulated with LP-01, MC3, and SM-102 ionizable lipids. We demonstrate that storage buffer identity and concentration govern mRNA-LNP internal ordering before and after freeze–thaw and are thus critical parameters for engineering high-performance formulations. Deconvoluting ordered phases into an mRNA-lipid region and excess lipid region reveals the importance of excess ionizable lipid behavior in enhancing endosomal escape. Prior to freezing, citrate buffer enhances transfection efficiency by promoting a transition to the fusogenic inverse hexagonal (H II ) phase earlier during acidification, facilitated by a greater amount of ordered excess ionizable lipid. In contrast, Tris buffer provides the highest transfection efficiency after freeze–thaw by preventing aggregation and cargo loss while promoting favorable internal structure. Increasing Tris concentration from 10 to 50–150 mM leads to mRNA-rich bleb formation in freeze–thawed mRNA-LNPs, which improves freeze–thaw stability and thus transfection efficiency by mitigating mRNA-lipid adduct formation and accommodating a larger excess ionizable lipid region to facilitate H II phase formation. These findings establish a direct structural link between buffer conditions, particle size, internal morphology, and transfection efficiency, highlighting the importance of buffer composition in modulating mRNA-LNP performance.