Structure-function relationships in polyelectrolyte nanoparticles for saRNA delivery

Thesis

Formulating cationic polyplexes (PP) with polyanions as ternary nanoparticles (TN) offers a promising alternative platform to lipid nanoparticles (LNP) for targetable systemic nucleic acid delivery. Beyond their role in conveying negative charge, the relationship between polyanion chemistry and TN structure, transport, and transfection is poorly understood. Given established correlations between cationic lipid chemistry and LNP structure/function, it was hypothesized that varying polyanion chemistry and architecture could simultaneously engineer RNA packaging, colloidal stability, and transfection.

Chemically diverse PEGylated polyanions was designed to coat self-amplifying mRNA (saRNA) poly(cystamine bisacrylamide-co-4-amino-1-butanol) (pABOL) PP, enabling systemic evaluation of how PEG architecture and polyanion chemistry modulate TN structure and function. High-throughput stability screening assays identified that PEG5k-bl-polyanion5k with moderate charge density and hydrophobicity were essential for colloidally stable, charge-neutralized TN. Small angle neutron scattering (SANS) studies and molecular dynamics (MD) simulations revealed that polyanion hydrophobicity governed tight core packaging while creating solvent-accessible apolar surfaces that influenced protein binding. These structural characteristics directly influenced cellular interactions, where more hydrophobic particles showed reduced uptake in protein-free conditions but exhibited serum-enhanced uptake and serum-compromised RNA release. It was revealed that PEG-bl-polyanion must balance hydrophobicity and charge density to yield stable TN capable of shielding PP from non-specific uptake mechanisms.

Finally, it was demonstrated that the reduced cellular internalization of optimized PEG-bl-polyanion TN formulations could be restored through mannose ligand targeting, which enabled receptor-mediated endocytosis. The developed screening methods and identified critical balances between polymer properties laid the foundation for the high-throughput engineering of physiologically stable nanoparticles capable of transport without sacrificing intracellular responsiveness.

Authors: Hu, L

• DOI: 10.5287/ora-gpakkxmrj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: PET-RAFT; self-amplifying mRNA; high throughput; structure/function; SANS; molecular dynamics
• Subjects: Nucleic acid delivery; Polyelectrolytes