Overall, the unbiased structures obtained with the first reported CpHMD-based, all-atom self-assembly model of an LNP interior under both acidic and neutral conditions provide structural support for hypotheses supporting the benefits of LNP formulation techniques that produce bleb structures. The mechanisms of how pH-dependent structures during the manufacturing process can increase loading efficiency and improve payload stability are illustrated in Figure . Mixing mRNA with LNP components under buffered acidic conditions promotes the formation of polar-mesoporous features in the bleb that increases the loading efficiency of mRNA through ionic interactions. Dialysis of the highly loaded mRNA/LNP vehicle using PBS buffer (pH = 7.4 ≈ LNP pKa) transitions the formulation from an acidic to a neutral environment. Upon neutralization of the reaction mixture, the mRNA payload can better incorporate into the ionizable lipids, forming more stable structures, as observed in our simulations, that are likely to help protect the payload from degradation in vivo. Therefore, these simulations offer a plausible mechanism for how mRNA-lipid adducts can form inside bleb structures of LNPs, which are known to help improve the mRNA transfection efficiency after administration. 59 Representative images of the final self-assembly structures for DODAP in different pH environments (protonated lipidred, deprotonated lipidblue, mRNApink, cholesterolgreen, DSPCorange). Self-assembly simulations (A) show that in acidic conditions mesoporous structures that harbor mRNA (pH < pKa) in water pores in acidic conditions. Neutral conditions see the incorporation of mRNA into the ionizable lipids (pH ≈ pKa) into the lipid membrane or woven between tubular lipid structures. Basic conditions, while not representative of physiological or manufacturing conditions, form bilayers (pH > pKa) in the mRNA sits on the surface and demonstrates the importance of the local environment on lipid pKa, as the only remaining protonated lipids are interacting with the mRNA nucleobases. Dialysis of the DODAP system was simulated (B) and was performed by modeling the acidic structure (that we associate with the high loading efficiency of blebs) at the pH in which they are dialyzed. The system quickly rearranges to incorporate mRNA into the lipids after ~200 ns of simulation time (Figures S142, Movies S41 -S43). These results correspond well with the increased loading and transfection efficiency that are observed with bleb structures, which are highly polar macrostructures known to efficiently incorporate mRNA. Under acidic conditions, in which blebs are formed, the protonated mesoporous lipid structures strongly interact with mRNA to increase loading efficiency. Upon dialysis to a more neutral pH the mRNA incorporates with the lipids to increase stability and improve the efficiency of transfection.