Membrane environment sets the functional pK_a of ionizable lipids

Ionizable aminolipids enable lipid nanoparticles (LNPs) to encapsulate nucleic acids at neutral pH and to release their cargo upon endosomal acidification. The discrepancy between this effective, acidic LNP pK_a and the basic intrinsic pK_a of aminolipids, however, remains poorly understood. Here, we performed microsecond constant-pH molecular dynamics simulations of five widely used aminolipids (DODAP, DLin-MC3-DMA, DLin-KC2-DMA, ALC-0315, and SM-102) embedded in different LNP-relevant ternary DOPC/DSPC-cholesterol membranes to quantify how aminolipid structure and membrane composition jointly govern aminolipid protonation and the associated pH-dependent membrane remodeling. Across all systems, membrane embedding lowers the apparent aminolipid pK_a, yielding physiologically relevant values of 6-7.5 corresponding to shifts by up to 3.5 pK_a units or approx. 20 kJ mol^-1 with respect to the intrinsic pK_a. Strikingly, pH-driven membrane remodeling upon deprotonation differs between aminolipids: polyunsaturated aminolipids undergo surface-to-core translocation, branched aminolipids preferentially form laterally segregated surface domains, and DODAP remains interfacially anchored through sustained hydration and hydrogen bonding. Saturated helper lipids (DSPC) systematically enhance segregation and amplify pK_a shifts relative to DOPC. Together, these results identify membrane phase behavior as a primary regulator of aminolipid protonation equilibria and provide insights into how formulation may influence LNP pK_a and thereby could control membrane remodeling and delivery performance.