Assembly of polyplexes for RNA delivery

Messenger RNA-based pharmaceuticals have recently demonstrated their po- tential with the successful COVID-19 vaccination campaign. They are now undergoing clinical trials for a broad range of therapeutic indications, including cancer and genetic diseases. While in the COVID-19 vaccines selected lipid-based nanoparticles (LNPs), have proven to be successful, there is a need of alternative delivery systems matching efficacy and safety requirements in a broad range of applications. Polyplexes, formed by self-assembly of cationic polymers with the anionic nucleic acids, constitute a valuable extension to the technological space represented by lipid-based systems. For medical applications, it is crucial to pre- cisely control both the number of encapsulated RNA molecules in the polyplexes and their shape. Here, we use molecular dynamics simulations of a coarse-grained model of the system to show that the single most important factor characterizing it is the total ratio of charges on polyelectrolytes and RNA during the prepara- tion of the nanoparticles. Close to the isoelectric point, the polyplexes are large, whereas further away, their size decreases and the preparation of polyplexes con- taining only one RNA copy becomes possible. Our results are consistent with recent experimental work on poly-ethylenimine (PEI) and self-amplifying RNA (saRNA).