Molecular-Level Interactions of Polyphosphazene Immunoadjuvants and Their Potential Role in Antigen Presentation and Cell Stimulation.

Two macromolecular immunoadjuvants, poly[di(carboxylatophenoxy)phosphazene], PCPP, and poly[di(carboxylatoethylphenoxy)phosphazene], PCEP, have been investigated for their molecular interactions with model and biopharmaceutically important proteins in solutions, as well as for their TLR stimulatory effects and pH-dependent membrane disruptive activity in cellular assays. Solution interactions between polyphosphazenes and proteins, including antigens and soluble immune receptor proteins, have been studied using Asymmetric Flow Field Flow Fractionation (AF4) and Dynamic Light Scattering (DLS) at near physiological conditions: phosphate buffered saline, pH 7.4. Polyphosphazenes demonstrated selectivity in their molecular interactions with various proteins, but displayed strong binding with all vaccine antigens tested in the present study. It was found that both PCPP and PCEP showed strong avidity to soluble immune receptor proteins, such as Mannose Receptor (MR) and certain Toll-Like Receptor (TLR) proteins. Studies on TLR stimulation in vitro using HEK293 cells with overexpressed human TLRs revealed activation of TLR7, TLR8, and TLR9 signaling pathways, albeit with some nonspecific stimulation, for PCPP and the same pathways plus TLR3 for PCEP. Finally, PCEP, but not PCPP, demonstrated pH-dependent membrane disruptive activity in the pH range corresponding to the pH environment of early endosomes, which may play a role in a cross-presentation of antigenic proteins.