Self-Assembly Simulations. Systems were randomly assembled using GROMACS 2021 using 100 ionizable lipids (50 mol%), 80 CHL1 (40 mol%), 20 DSPC (10 mol%), and mRNA (20 nucleotides) in a 7 nm x 7nm x 11 nm periodic box. Ratios of LNP components were chosen according to the previously reported formulations with known apparent pKa values. Theoretical titrations were performed using LNP formulations that included DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, DODMA, or DODAP ionizable lipids shown Figure . Randomly assembled systems were allowed to self-assemble in triplicate in simulated pH environments ranging from pH 3 to 10. The distributions of lipid protonation states for the self-assembly simulation are stable after ~50 ns of simulation time as shown in Figures . Finally, computational titration curves were generated by calculating the ratio of -states (Sprot = 1/total) for all the ionizable lipids of each system over the last 50 ns of each simulation. Error bars represent standard deviations obtained from the three independent replicas run for each simulation pH.. Representative snapshots are shown in Figures and Movies S1a -S40c.