Polyanion Chemistry Engineers Ternary RNA Nanoparticle Structure/Function from the Inside-Out

Formulating cationic polyplexes (PP) with polyanions as ternary polyelectrolyte nanoparticles (TNP) offers a polymeric alternative to lipid nanoparticles (LNP) for targetable nucleic acid delivery. Although TNP in vivo transport is credited to their anionic surface charge, the relationships between polyanion chemistry and TNP structural stability, protein binding, and transfection are poorly understood compared to lipid-based systems. We hypothesized that carefully engineered hydrophobic polyanions could simultaneously endow TNPs with negative surface charge and enhanced extracellular stability critical to the future development of actively targeted formulations. We synthesized chemically diverse PEGylated polyanions to coat self-amplifying RNA (saRNA) PP, systematically studying how PEG architecture and polyanion chemistry modulate TNP structure and function. In both high-throughput stability assays and Small Angle Neutron Scattering structural studies, we found that PEG5k-bl-polyanion5k yields remarkably small particles with a pH-responsive core–shell structure. We identify a lead formulation (TNP5) with moderate hydrophobicity and charge density that balances extracellular stability and intracellular unpackaging for transfection. In agreement with spectroscopic characterization and in vitro cell studies, Molecular Dynamics simulations support the hypothesis that polyanions dictate TNP function from the inside-out by excluding water from the RNA core and by exposing functional groups that modulate protein binding. Our work correlates high throughput assays and detailed neutron scattering analysis to uncover mesoscale structural differences between two- and three-component polyelectrolyte delivery systems. These screening methods and the critical balances between polymer properties they uncover establish a framework for high throughput engineering of pH-responsive nanoparticle structure/function to navigate biological barriers to RNA delivery.