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 buffer type on the structure and transfection efficiency of LNPs. We find that LNPs formulated with the cationic ionizable lipid DLin-MC3-DMA (MC3) 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 pH dependent mesophases of ionizable lipid MC3/cholesterol/water dialyzed against the various buffers. The results show that the phase transition with decreasing pH from inverse micellar cubic to inverse hexagonal (Fd3m-H _II ) is shifted by one unit to a lower transition pH for acetate and phosphate compared to 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 H _II to a higher pH. By contrast, phosphate and acetate stabilize L _II . We propose that the Fd3m-to-H _II transition, which is facilitated in citrate buffer, is responsible for 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.