The initial barrier to membrane fusion is the formation of a hemifusion stalk, which should be followed by the formation of a fusion pore , process not covered by our fusion assays. Here we use the PMF method to calculate the free energy landscape of the fusion intermediates, providing insights into the energetic barriers and stability of the stalk. Figure presents a schematic overview of the process by which the LNP enters the cell and leads to protein expression, with stalk formation highlighted in Figure as the proposed initial barrier for endosomal escape.