The box was enlarged (35 nm side), hydrated, neutralized with Cl -ions and then Cl -and Na + ions were added in physiological conditions. The system was equilibrated and simulated for 10 𝜇s with annealing, as in the case of pre-assembled LNPs and in previous work . The structures of the LNPs were then characterized in terms of their size, shape, density distribution and hydration level using the same protocols provided in ref , with average values calculated for the last 2 𝜇s of the simulation.