Proton sponge or membrane fusion? Endosomal escape of siRNA polyplexes illuminated by molecular dynamics simulations

To achieve a therapeutic effect, nanoparticles delivering nucleic acids must facilitate endosomal escape (EE) of their cargo. Despite extensive research, the mechanisms that lead to an effective EE are not sufficiently understood. Herein, we utilized Molecular Dynamics (MD) simulations in All Atom (AA) and Coarse Grained (CG) resolutions to differentiate the interaction of four polymeric formulations (polyplexes) and one lipid nanoparticle (LNP) with endosomal membranes. On the one hand, the results emphasize the benefit of hydrophobic residues in the nanoparticles. On the other hand, the role of anionic lipids in the biological membranes is demonstrated. Furthermore, the identified interaction patterns were successfully correlated to the in vitro performance of the formulations. For the first time, different EE mechanisms of polyplex formulations are visualized in simulation and therefore distinguishable from one another. Hence, this work highlights the power of MD simulations for taking a big step towards better understanding EE efficiency.