We used a plasma membrane model called A8 , which has reduced complexity, but captures reasonably well the properties of more complex compositions. The LNP interacting leaflet of this membrane is composed of (in mol%) 24 % POPC, 12 % PAPC, 2 % POPE, 6 % DIPE, 24 % DPSM and 31 % CHOL while the non-interacting leaflet contains 14 % POPC, 8 % PAPC, 5 % POPE, 16 % DIPE, 11 % DPSM, 28 % CHOL, 25 % PAPS, 2 % PAP6. An additional membrane model, enriched with 10 mol % PAPS lipids in the LNP-interacting leaflet, was also considered to better model the differences in composition in the endosome. All bilayer fusion systems were built with an automated protocol written in python, and the same protocol was used for all studies of these systems, differing only in the composition of the membranes. Initially, two independent membrane bilayers corresponding to the target LNP and endosomal target membrane are built in a 10 × 10 × 8 nm 3 box using the insane software . Each system was then hydrated and neutralized using sodium ions. This was followed by energy minimization and equilibration for 20 ns to relax the box dimensions completely.