Incorporating negatively charged lipids into the interacting leaflet of the lipid vesicle may accelerate the initiation of fusion between positively charged LNPs and the endosomal membrane . To test this hypothesis, we modified the vesicles to contain 10 mol% of negatively charged PAPS in the inner leaflet and performed three 10 µs LNP-vesicle simulations for Lipid 10, Lipid 2, and MC3. For each IL system, LNP-vesicle fusion was observed in one replica (Figure ), while in the other three replicas, the LNP remained inside the vesicle without any vesicle-LNP contacts. Although the unbiased approach did not clearly distinguish between the three IL types, it confirmed that LNP and vesicle lipid fusion is not a barrierless process, even with a fraction of negatively charged lipid in the vesicle. However, this barrier is reduced when negatively charged lipids are present in the target membrane.