Buffer Specificity of Ionizable Lipid Nanoparticle Transfection Efficiency and Bulk Phase Transition

: Lipid nanoparticles (LNPs) are efficient and safe carriers for mRNA vaccines based on advanced ionizable lipids. It is understood that the pH-dependent structural transition of the mesoscopic LNP core phase plays a key role in mRNA transfer. However, buffer-specific variations in transfection efficiency remain obscure. Here we analyze the effect of the buffer type on the transfection efficiency of LNPs. We find that LNPs formulated with the cationic ionizable lipids DLin-MC3-DMA (MC3), SM-102, and ALC-315 in citrate compared to phosphate and acetate buffers exhibit earlier onset and stronger mRNA-GFP expression in vitro. Using synchrotron small-angle X-ray scattering (SAXS) we determine the buffer specificity of the pH-dependent structure of ionizable lipid/cholesterol/water mesophases that serve as model systems for the LNP core phase. The results show that the phase transition from inverse micellar to inverse hexagonal with decreasing pH is shifted to a lower transition pH for acetate and phosphate compared with citrate buffer. Based on continuum theory and ion-specific adsorption obtained from all-atom MD simulations, we propose a mechanism for buffer specificity. Citrate stabilizes the inverse hexagonal phase thus shifting the formation of HII to a higher pH. By contrast, phosphate and acetate stabilize LII. It stands to reason that the inverse micellar to inverse hexagonal transition, which is facilitated in citrate buffer, enables a sensitized pH response of the LNP core phase. This, in turn, enhances endosomal release efficiency and accounts for the earlier onset of gene expression observed in LNPs prepared with citrate buffer.