Supramolecular Protein-Polyelectrolyte Assembly at Near Physiological Conditions—Water Proton NMR, ITC, and DLS Study

Abstract

The in vivo potency of polyphosphazene immunoadjuvants is inherently linked to the ability of these ionic macromolecules to assemble with antigenic proteins in aqueous solutions and form physiologically stable supramolecular complexes. Therefore, in-depth knowledge of interactions in this biologically relevant system is a prerequisite for a better understanding of mechanism of immunoadjuvant activity. Present study explores a self-assembly of polyphosphazene immunoadjuvant—PCPP and a model antigen—lysozyme in a physiologically relevant environment—saline solution and neutral pH. Three analytical techniques were employed to characterize reaction thermodynamics, water-solute structural organization, and supramolecular dimensions: isothermal titration calorimetry (ITC), water proton nuclear magnetic resonance (wNMR), and dynamic light scattering (DLS). The formation of lysozyme–PCPP complexes at near physiological conditions was detected by all methods and the avidity was modulated by a physical state and dimensions of the assemblies. Thermodynamic analysis revealed the dissociation constant in micromolar range and the dominance of enthalpy factor in interactions, which is in line with previously suggested model of protein charge anisotropy and small persistence length of the polymer favoring the formation of high affinity complexes. The paper reports advantageous use of wNMR method for studying protein-polymer interactions, especially for low protein-load complexes.

Immunopotentiating Polyphosphazene Delivery Systems: Supramolecular Self-Assembly and Stability in the Presence of Plasma Proteins

“…The selection of proteins for the study was based on the following considerations. Hen egg lysozyme is an established model antigen, − which was studied for its avidity to PCPP , and found to show binding characteristics similar to other vaccine antigens, such as Hepatitis C E2 protein . Moreover, the ability of PCPP to augment antibody responses to lysozyme was proven in vivo .…”

Nano-Assembled Polyphosphazene Delivery System Enables Effective Intranasal Immunization with Nipah Virus Subunit Vaccine

“…The DLS analysis of the mixture reveals an antigen dose-dependent shift in the hydrodynamic diameter of the macromolecular peak, which independently confirms protein–polymer binding in the system (Figure d). The self-assembly process in protein–polyphosphazene systems has been recently studied in detail and is typically driven by both entropic (counterion release) and enthalpic (ionic bonds and hydrophobic interactions) factors. − …”

Fluorine-Functionalized Polyphosphazene Immunoadjuvant: Synthesis, Solution Behavior and In Vivo Potency

“…The immunoadjuvant potency of PCPP is closely associated with its ability to spontaneously self-assemble with vaccine antigens in aqueous solutions [ 21 , 25 , 26 ]. Protein-binding characteristics of PCPP-F were evaluated using a model antigenic protein—hen egg lysozyme, which has been intensively studied for its interactions with PCPP [ 28 , 32 ]. Non-covalent association of lysozyme with PCPP is typically manifested in a rise of DLS-detected hydrodynamic dimensions of the complex, with an increase in protein load above a certain threshold.…”

“…Poly[di(carboxylatophenoxy)phosphazene] (PCPP) is an important representative of this class of immunoadjuvants, which has demonstrated its safety and potency in clinical trials [ 16 , 17 , 18 , 19 ] and continues to show significant potential with newly emerging antigens or with alternative administration routes, such as microneedle-based intradermal vaccination [ 20 , 21 , 22 , 23 , 24 , 25 ]. The ability of PCPP to promote immune responses of enhanced magnitude, breadth and durability is closely associated with its exceptional capability to spontaneously self-assemble with antigenic proteins under physiological conditions [ 21 , 25 , 26 , 27 , 28 ]. However, mechanistic aspects of its immunoadjuvant activity are yet to be thoroughly investigated.…”