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

[Image: see text] 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 a...

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Autores principales: Lueckheide, Michael, Marin, Alexander, Tagad, Harichandra D., Posey, Nicholas D., Prabhu, Vivek M., Andrianov, Alexander K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10571102/
https://www.ncbi.nlm.nih.gov/pubmed/37841951
http://dx.doi.org/10.1021/acspolymersau.3c00006
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author Lueckheide, Michael
Marin, Alexander
Tagad, Harichandra D.
Posey, Nicholas D.
Prabhu, Vivek M.
Andrianov, Alexander K.
author_facet Lueckheide, Michael
Marin, Alexander
Tagad, Harichandra D.
Posey, Nicholas D.
Prabhu, Vivek M.
Andrianov, Alexander K.
author_sort Lueckheide, Michael
collection PubMed
description [Image: see text] 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 (K(d) = 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.
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spelling pubmed-105711022023-10-14 Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay Lueckheide, Michael Marin, Alexander Tagad, Harichandra D. Posey, Nicholas D. Prabhu, Vivek M. Andrianov, Alexander K. ACS Polym Au [Image: see text] 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 (K(d) = 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. American Chemical Society 2023-04-21 /pmc/articles/PMC10571102/ /pubmed/37841951 http://dx.doi.org/10.1021/acspolymersau.3c00006 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Lueckheide, Michael
Marin, Alexander
Tagad, Harichandra D.
Posey, Nicholas D.
Prabhu, Vivek M.
Andrianov, Alexander K.
Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay
title Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay
title_full Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay
title_fullStr Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay
title_full_unstemmed Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay
title_short Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay
title_sort monitoring protein complexation with polyphosphazene polyelectrolyte using automated dynamic light scattering titration and asymmetric flow field flow fractionation and protein recognition immunoassay
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10571102/
https://www.ncbi.nlm.nih.gov/pubmed/37841951
http://dx.doi.org/10.1021/acspolymersau.3c00006
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