The PMF fusion assays described in Section 2.3 have proven extremely valuable for quantifying the efficacy of stalk formation, the initial step in fusion, across different LNP formulations and target membrane compositions. However, this approach has limitations: it is performed in the absence of curvature in both the LNP and target membrane, and it does not account for cargo. Additionally, the fusion protocol only quantifies stalk formation and does not encompass other steps involved in complete transfection. While curvature may be incorporated by new approaches and additional collective variables could be applied to explore subsequent steps in transfection, such as pore formation , PMF fusion assays rely on one fundamental assumption: it assumes that the fusion process occurs in an equilibrium regime, where lipids of LNP and endosomal membrane fully mix, which may not reflect the real endosomal escape process.