Parameterization of Ionizable Groups. Atomic charges for charged ( = 0) and neutral ( = 1) states of each ionizable lipid were adopted from the CHARMM36 forcefield. The pKa values of soluble versions of the ionizable lipids investigated in this study were previously reported. We used these experimental pKa values to parametrize the dV/dλ values for the ionizable lipids based on an optimized parametrization protocol described below. Notably, the experimental pKa values for individual (soluble) small molecule analogs of the ionizable lipids in water differ by ~ 2-3 pKa units from the apparent pKa of their LNP formulation (Table ), which clearly indicates the large effects of the LNP environment on the pKa values of the ionizable lipids. This procedure corrects for changes in the potential energy arising from deprotonation as detailed elsewhere. To increase the conformational sampling during the fixed -state calculations, Hamiltonian replica exchange (HREX) with the replica exchange solute tempering using REST2 as implemented in PLUMED (v. 2.9.0) was used. For the REST2 calculations, the force field parameters and atomic charges of the ionizable lipid were scaled according to well-established protocols to afford 10 replicas with different effective solute temperatures, ranging from 310 K to 400 K. As reported in a recent publication discussing best practices for CpHMD parameterization, buffer particles and the titratable site had their positions restrained during these parametrization rungs to ensure that there is no interaction between the buffers and the ionizable groups during parameterization. Each fixed -state calculation was run for 10 ns, with  values ranging -0.1 to 1.1 Finally, the CpHMD parameters obtained for each ionizable lipid were validated by performing 100 ns (n = 10) simulations with simulation pH set to the pKa at which the lipids were parameterized, and the energy barrier between the different protonation states set to 0. The results of all validation runs are provided in Table and Figures S1-S10 in the Supplementary Information. Membrane Simulations. Modeled membrane systems were packed using the membrane builder web tool afforded by CHARMMGUI. Systems were packed utilizing 100 ionizable lipids (50 per leaflet, 50 mol%), 78 cholesterol (CHL1 -39 per leaflet, 39 mol%), 20 distearoylphosphatidylcholines (DSPC -10 per leaflet, 10 mol%), and 2 DMG-PEG2000 (1 per leaflet, 1%) in a 7nm x 7nm x 15 nm periodic box. Ratios of LNP components were determined as previously reported in Ref. 59; however, the mRNA payload was not included in simulated membrane systems.