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. 2020 Apr 13;21(4):1604-1612.
doi: 10.1021/acs.biomac.0c00179. Epub 2020 Mar 31.

Polymer-Stabilized Sialylated Nanoparticles: Synthesis, Optimization, and Differential Binding to Influenza Hemagglutinins

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Free PMC article

Polymer-Stabilized Sialylated Nanoparticles: Synthesis, Optimization, and Differential Binding to Influenza Hemagglutinins

Sarah-Jane Richards et al. Biomacromolecules. .
Free PMC article

Abstract

During influenza infection, hemagglutinins (HAs) on the viral surface bind to sialic acids on the host cell's surface. While all HAs bind sialic acids, human influenza targets terminal α2,6 sialic acids and avian influenza targets α2,3 sialic acids. For interspecies transmission (zoonosis), HA must mutate to adapt to these differences. Here, multivalent gold nanoparticles bearing either α2,6- or α2,3-sialyllactosamine have been developed to interrogate a panel of HAs from pathogenic human, low pathogenic avian, and other species' influenza. This method exploits the benefits of multivalent glycan presentation compared to monovalent presentation to increase affinity and investigate how multivalency affects selectivity. Using a library-orientated approach, parameters including polymer coating and core diameter were optimized for maximal binding and specificity were probed using galactosylated particles and a panel of biophysical techniques [ultraviolet-visible spectroscopy, dynamic light scattering, and biolayer interferometry]. The optimized particles were then functionalized with sialyllactosamine and their binding analyzed against a panel of HAs derived from pathogenic influenza strains including low pathogenic avian strains. This showed significant specificity crossover, which is not observed in monovalent formats, with binding of avian HAs to human sialic acids and vice versa in agreement with alternate assay formats. These results demonstrate that precise multivalent presentation is essential to dissect the interactions of HAs and may aid the discovery of tools for disease and zoonosis transmission.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Synthetic strategy and characterization of polymers and particles. (A) Synthesis of galactose terminal PHEA; (B) step growth process for the synthesis of gold nanoparticles; (C) SEC of Gal-PHEAs; (D) 19F NMR showing displacement of the pentafluorophenyl unit; (E) UV–vis spectroscopy of uncoated AuNPs; (F) DLS of uncoated AuNPs; (G–I) TEM of uncoated AuNPs. Scale bar = 100 nm.
Figure 2
Figure 2
Galactosylated particles and SBA binding. (A) Aggregation of gold particles leading to red-blue color shift due to SPR band coupling. Change in Abs700 using 500 nM SBA as a function of coating and core size. Full binding curves from 500 to 0 nM are in the Supporting Information, Figure S7; (B) BLI schematic, showing capture of SBA and subsequent binding to AuNPs to generate signal. Total binding (Δmax) as a function of coating and core size. Full binding curves are in the Supporting Information, Figure S8.
Figure 3
Figure 3
Synthesis of sialylated gold nanoparticles. (A) Amination of 2,3- and 2,6- sialyllactoses using Kochetkov method; (B) reaction of DP25 PFP-PHEA with sialyllactoamines and subsequent immobilization of the polymers onto 70 nm gold nanoparticles.
Figure 4
Figure 4
Interaction of SL-functionalized particles: 2,3-SL-PHEA@AuNP (black ■), 2,6-SL-PHEA@AuNP (red ●), and Gal-PHEA@AuNP (blue ▲) with WGA by (A) UV–vis; (B) DLS; and (C) BLI.
Figure 5
Figure 5
Interaction of SL-functionalized nanoparticles with a panel of HAs using BLI. (A) 2,3-SL-PHEA@AuNP and (B) 2,6-SL-PHEA@AuNP BLI binding curves to HA panel (H1 from A/California/07/2009 (H1N1)pdm09, H2 from A/Singapore/1/1957 (H2N2), H3 from A/Uruguay/716/2007 (H3N2), H4 from A/mallard/Alberta/455/2015 (H4N6), H5 from A/duck/Hunan/795/2002 (H5N1), H7 from, A/Canada/rv444/2004 (H7N3), NR-43740, H9 from A/Hong Kong/33982/2009 (H9N2), and H10 from A/harbor seal/Germany/1/2014 (H10N7)); (C) comparison of the maximal binding of 2,3-SL-PHEA@AuNP and 2,6-SL-PHEA@AuNP to the HA panel; (D) difference (subtraction) between maximal binding to 2,6-SL-PHEA@AuNP and 2,3-SL-PHEA@AuNP to visualize relative binding preferences.

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