These fusion PMFs (Figure3D) revealed a substantial decrease in the free energy of stalk formation for Lipid 10 compared to MC3, and a slight increase for Lipid 2 compared to MC3, consistent with the protein expression trend reported by Sabnis et al. . Other studies have also shown that replacing alkenes with alkanes and introducing esters and branching of the lipids tails enhances delivery, since it helps in lipid protrusion, enabling the lipid to more easily overcome the dehydration barrier between the two membranes, and thereby forming the stalk . This in turn helps to explain why Lipid 10 outperforms MC3 in the PMF assays, due to the increased branching and introduction of an additional ester. These fusion assays were conducted between a membrane mimicking the LNP formulations and a simplified plasma membrane, with negatively charged lipids predominantly present in the inner leaflet. However, considering the possibility that in the endosome, negatively charged lipids might migrate to the LNP-interacting leaflet, we sought to evaluate the impact of this potential redistribution. To this end, we performed additional PMF fusion assays against a membrane model enriched with 10 mol % PAPS lipids in the LNP-interacting leaflet to better mimic the compositional differences observed in the endosome. PS was used as the negative lipid for simplicity and comparison to an earlier similar study .