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

## TL;DR

Researchers engineered RNA nanoparticles using polyanions to improve stability and delivery of nucleic acids, offering an alternative to lipid-based systems.

## Contribution

The study introduces a framework for engineering pH-responsive RNA nanoparticle structure and function using polyanion chemistry.

## Key findings

- PEG5k-bl-polyanion5k creates small, pH-responsive core-shell nanoparticles.
- TNP5 balances extracellular stability and intracellular unpackaging for efficient transfection.
- Polyanions influence nanoparticle function by modulating water exclusion and protein binding.

## Abstract

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.