Protein-polyelectrolyte complexation: effects of sterically repulsive groups, macromolecular architecture and hierarchical assembly.

Self-assembly of proteins and polyelectrolytes in aqueous solutions is a promising approach for the development of advanced biotherapeutics and engineering efficient biotechnological processes. Synthetic polyions containing sterically repulsive ethylene oxide moieties are especially attractive as protein modifying agents, as they can potentially induce a PEGylation-like stabilizing effect without the need for complex covalent binding reactions. In this study, we investigated the protein-binding properties of anionic polyelectrolytes based on an inorganic polyphosphazene backbone, with ethylene oxide groups incorporated into both grafted and linear macromolecular topologies. The study was conducted in aqueous solutions using isothermal titration calorimetry, dynamic light scattering, and cryogenic electron microscopy to analyze the samples in their vitrified state. Our findings revealed that the stability of the resulting protein-polyion complexes and the thermodynamic profiles of these interactions were influenced by the molecular architecture of the polyions. Furthermore, the formation of hierarchical assemblies of polyions, through ionic crosslinking into nanogels, rapidly reduced or eliminated the ability of the polyelectrolyte to bind proteins. The comprehensive analysis, combining thermodynamic, spectroscopy and direct visualization techniques, provides valuable insights into the multivalent charge-charge interactions that are critical for the development of successful non-covalent protein modification methods.