Membrane Environment Sets the Functional pKa 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 pKa and the basic intrinsic pKa 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/D-SPC-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 pKa, yielding physiologically relevant values of 6-7.5 corresponding to shifts by up to 3.5 pKa units or approx. 20 kJ mol-1 with respect to the intrinsic pKa. Strikingly, the magnitude of the pKa shift correlates with pH-driven membrane remodeling upon deprotonation: 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 pKa shifts relative to DOPC. Together, these results identify membrane phase behavior as a primary regulator of aminolipid protonation equilibria and establish quantitative design principles for tuning LNP composition toward desired pKa, membrane remodeling, and delivery performance.