The results for these fusion assays demonstrated a slight reduction in both the initial energetic barrier for stalk formation and in the actual stalk across all three lipids (Figure ), though for Lipid 10 most of the PMF curves overlap the difference in free energy for the stalk formation at x=1 show a statistically meaningful difference, confirming the trend. This reduction in the barrier reinforces the idea that anionic lipids, such as PS, may play an important role in the endosomal membrane during the transfection mechanism. Morover, the overall findings align with experimental data indicating that LNPs containing Lipid 10 lead to higher protein expression , thereby enhancing transfection efficiency.