Lastly, while simulations performed in more basic environments (pH > pKa) are not directly physiologically relevant, there are still meaningful mRNA-lipid interactions, which demonstrate the environment-dependent nature of lipid protonation. Notably, all structures that formed in basic environments (pH > pKa) heavily favored the formation of bilayers as ionizable lipids were almost entirely deprotonated, similar to the structure observed for the pre-packed DLin-MC3-DMA membrane. However, the ionizable lipids adjacent to the mRNA payloads remained protonated, even at these elevated pH values, as the negatively charged nucleobases produced favorable interactions with a cationic lipid. Such interactions likely contribute to the strong acidic shifts in apparent LNP pKa values observed (Table ) between water-soluble lipid analogs and their LNP formulations.