Direct structure characterization of polyelectrolytes provide new insights on a pathway to protein binding mechanisms

Well-characterized polyelectrolytes are key strategic materials for developing new vaccine formulations by offering a simplified approach to associate with cargo, such as protein antigens, via charge complexation and other binding mechanisms. Understanding the relationship between their structure, formulation properties, and preservation of protein function is critical. Here, we report the direct structural characterization of a clinically-relevant polyphosphazene, poly[di(carboxylatophenoxy)phosphazene] (PCPP). This highly charged water-soluble polyelectrolyte was characterized by cryogenic electron microscopy (cryo-EM) to visualize individual macromolecular chains in a vitrified aqueous solution, complemented by atomic force microscopy (AFM). Cryo-EM and AFM visualize both linear and cyclic chain topologies. This direct observation of the cyclic noncatenated macrocycles provides conclusive evidence for a ring expansion polymerization mechanism in the chain-growth synthesis of polyphosphazenes. Furthermore, PCPP binds to both net cationic and net anionic proteins while preserving their antigen function. This work established a direct link between the macromolecular topology of polyphosphazenes and their material properties, opening avenues to rationally enhance their performance by correlating macromolecular structure with mechanisms of biological activity.