Implementing Hamiltonian replica exchange via the REST-2 approach during the CpHMD parametrization step greatly improved sampling and enhanced (Figure ) the quality of the dV/dλ parameters for the correction potential for deprotonation of ionizable lipids. Specifically, without replica exchange, intramolecular hydrogen bonding between the titratable proton of the ionizable site and nearby ester or ether oxygens resulted in biased conformations during parameterization, which act as a conformational trap and can lead to biased CpHMD parameters, as the environment around the titratable sites can strongly influence the outcome of the parameterization. Overall, the application of HREX for the CpHMD parametrization improves conformational sampling while retaining the accuracy of the standard forcefield. Specifically, this approach is relatively simple to implement, as it does not require adjusting or revalidating the torsional potential terms of the force fieldan alternative method that has been previously employed to enhance the sampling of ionizable carboxylic acids functions. With validated parameters for all the ionizable lipids in hand, initial simulations were performed on packed membrane systems (without the mRNA payload) on the DLin-MC3-DMA LNP formulation (pKa TNS = 6.57) 59 as shown in Figure . The apparent pKa of the membrane quickly converges after ~5 ns to a much more acidic predicted apparent pKa (pKa theo = 3.89 ± 0.04) than what is observed experimentally. The discrepancy between experimental and simulated apparent pKa values for pre-packed membrane systems is attributed to the preference for ionizable lipid headgroups to neutralize and tuck into the membrane. Once neutralized, ionizable groups remain deprotonated throughout the entirety of the simulation as the local environment of the lipid bilayers greatly favors the uncharged species. The resulting preference in charge distribution produces a significantly lower apparent pKa (~3 pKa units lower than the experimental pKa) than what is observed from experimental TNS titration assays. Therefore, the influence of the local environment and the overall structure on the protonation state of an ionizable lipid indicates that performing theoretical titrations on systems with predetermined structures might not be an accurate method for predicting the apparent pKa for the LNP formulations being modeled in this work. To meet this challenge and avoid this charge distribution bias, which we observed for packed membrane systems, we subjected the LNP formulations to 100 ns self-assembly simulations (each run in triplicate, starting with a different random seed) in different pH environments. The models for each formulation are meant to replicate the interior of the LNP, hence, no PEG-lipid was incorporated into these structures. The large size of the LNP suggests that most of the ionizable lipids are located in the interior of the nanoparticles, and as a result, the protonation state of exterior lipids is expected to exert a much weaker influence on the apparent pKa values of the LNP. With this assumption, systems containing 100 ionizable lipids were created to sufficiently sample the influence of pH on the interior environment of the LNPs. After self-assembly, the apparent pKa values were determined for each formulation using the ratio of protonated and deprotonated lipids to generate titration curves. Theoretical apparent pKa values were determined by fitting the distributions of -states with the pH of the simulation, as detailed in the supplementary information, and compared to experimental values (Figure and Table ). Notably, as shown in Figure , Supplementary Figures , both the experimental and simulated titration curves deviate significantly from classical Henderson-Hasselbalch fits, as is expected for structure-dependent distributions of ionizable lipids. Significant structural rearrangements are observed upon changing the solution pH (Figure ). Notably, our CpHMD simulations accurately reproduce (Table ) these significant, environment-dependent pKa shifts for all five ionizable lipids investigated.