Unbiased fusion simulations presented in Section 2.4.3 provide a natural alternative to PMF fusion assays, as they naturally account for curvature and cargo and reached similar conclusions regarding the impact of negatively charged lipids on fusion. However, unbiased simulations require significantly more sampling to distinguish between formulations and to fully capture the transfection process , which remains challenging. Previous work has demonstrated that observing the entire process is possible with unbiased simulations , although not observed in our work. The lack of observed phenomena may be related to limitations in Martini, even in its latest version, particularly in forming pores with potentially still show too larger barriers .