Cryo-EM and AFM visualize linear polyorganophosphazene: individual chains and single-chain assemblies with proteins

Polyorganophosphazenes are biodegradable macromolecules with potent immunoadjuvant activity that self-assemble with protein antigens to provide biological activity. Direct imaging by cryogenic electron microscopy reveals the coil structure of the highly-charged high molecular mass synthetic polyorganophosphazenes within the vitrified state without any additives for contrast enhancement for the first time. Upon mixing with protein antigens under a controlled stoichiometric ratio, multiple proteins bind at the single chain level revealing a structural change reminiscent of compact spherical complexes or stiffened coils depending on the bound protein antigen. The structural outcome depends on the protein charge density that cannot be deduced by methods, such as dynamic light scattering, thus revealing direct morphological insight necessary to understand in vivo biological activity. Complementary atomic force microscopy supports the binding morphology outcomes as well as additional analytical techniques that indicate binding. These observations open opportunities to understand supramolecular assembly of proteins and other biomacromolecules at the single chain level with highly charged polyelectrolytes for vaccines as well as important to developing fields such as polyelectrolyte complex coacervation.