Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay

Published

April 21, 2023

Author(s)

Michael Lueckheide, Alexander Marin, Harichandra Tagad, Nicholas Posey, Vivek Prabhu, Alexander Andrianov

Abstract

Polyphosphazenes represent a class of intrinsically flexible polyelectrolytes with potent immunoadjuvant activity, which is enabled through non-covalent self-assembly with antigenic proteins by charge complexation. The formation of supramolecular complexes between polyphosphazene adjuvant, poly[di(carboxylatophenoxy)phosphazene] (PCPP), and a model vaccine antigen, hen egg lysozyme, was studied under physiological conditions using automated dynamic light scattering titration, asymmetric flow field flow fractionation (AF4), enzyme-linked immunosorbent assay (ELISA), and fluorescent quenching methods. Three regimes of self-assembly were observed covering complexation of PCPP with lysozyme in the nano-scale range, multi-chain complexes, and larger aggregates with complexes characterized by a maximum loading of over six hundred protein molecules per PCPP chain and dissociation constant in the micromolar range (Kd = 7 × 10–6 mol/L). The antigenicity of PCPP bound lysozyme, when compared to equivalent lysozyme solutions, was largely retained for all complexes, but observed a dramatic reduction for heavily aggregated systems. Routes to control the complexation regimes with elevated NaCl or KCl salt concentrations indicate ion-specific effects, such that more smaller-size complexes are present at higher NaCl, counterintuitive with respect to PCPP solubility arguments. While the order of mixing shows a prominent effect at lower stoichiometries of mixing, higher NaCl salt reduces the effect all together.

Citation

ACS Polymers Au

Pub Type

Journals

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https://doi.org/10.1021/acspolymersau.3c00006

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Keywords

polyphosphazenes, immunoadjuvants, protein−polymer complexes, self-assembly, dynamic light scattering, immunoassay

Citation

Lueckheide, M. , Marin, A. , Tagad, H. , Posey, N. , Prabhu, V. and Andrianov, A. (2023), Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay, ACS Polymers Au, [online], https://doi.org/10.1021/acspolymersau.3c00006, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=935450 (Accessed May 11, 2026)

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