Polymer-tethered glycosylated gold nanoparticles recruit sialylated glycoproteins into their protein corona, leading to off-target lectin binding

Upon exposure to biological fluids, the fouling of nanomaterial surfaces results in non-specific capture of proteins, which is particularly important when in contact with blood for in vivo and ex vivo applications. It is crucial to evaluate not just the protein components but also the glycans attached to those proteins. Polymer-tethered glycosylated gold nanoparticles have shown promise for use in biosensing/diagnostics, but the impact of the glycoprotein corona has not been established. Here we investigate how polymer-tethered glycosylated gold nanoparticles interact with serum proteins and demonstrate that the protein corona introduces new glycans and hence off-specific targeting capability. Using a panel of RAFT-derived polymers grafted to the gold surface, we show that the extent of corona formation is not dependent on the type of polymer. In lectin-binding assays, a glycan (galactose) installed on the chain-end of the polymer was available for binding even after protein corona formation. However, using sialic-acid binding lectins, it was found that there was significant off-target binding due to the large density of sialic acids introduced in the corona, confirmed by western blotting. To demonstrate the importance, we show that the nanoparticles can bind Siglec-2, an immune-relevant lectin post-corona formation. Pre-coating with (non-glycosylated) bovine serum albumin led to a significant reduction in the total glycoprotein corona. However, sufficient sialic acids were still present in the residual corona to lead to off-target binding. These results demonstrate the importance of the glycans when considering the protein corona and how ‘retention of the desired function’ does not rule out ‘installation of undesired function’ when considering the performance of glyco-nanomaterials.


Analytical and Physical Methods
NMR Spectroscopy. 1 H-NMR and 19 F-NMR spectra were recorded at 300 MHz or 400 MHz on a Bruker DPX-300 or DPX-400 spectrometer respectively, with methanol-d4 as the solvent.
Chemical shifts of protons are reported as δ in parts per million (ppm) and are relative to solvent residual peak (CH3OH, δ = 3.31 ppm/ DMSO, δ = 2.50 ppm).
FT-IR Spectroscopy. Fourier Transform-Infrared (FT-IR) spectroscopy measurements were carried out using an Agilent Cary 630 FT-IR spectrometer, in the range of 650 to 4000 cm -1 .

Size Exclusion Chromatography in DMF.
Size exclusion chromatography (SEC) analysis was performed on an Agilent Infinity II MDS instrument equipped with differential refractive index (DRI), viscometry (VS), dual angle light scatter (LS) and variable wavelength UV detectors.
The system was equipped with 2 x PLgel Mixed D columns (300 x 7.5 mm) and a PLgel 5 µm guard column. The mobile phase used was DMF (HPLC grade) containing 5 mM NH4BF4 at 50 o C at flow rate of 1.0 mL.min -1 . Poly(methyl methacrylate) (PMMA) standards (Agilent EasyVials) were used for calibration between 955,000 -550 g.mol -1 . Analyte samples were filtered through a nylon membrane with 0.22 μm pore size before injection. Number average molecular weights (Mn), weight average molecular weights (Mw) and dispersities (ĐM = Mw/Mn) were determined by conventional calibration and universal calibration using Agilent GPC/SEC software.
Column oven and detector temperatures were regulated to 40°C, flow rate 1 mL/min. Poly(ethyleneoxide) standards (Agilent EasyVials) were used for calibration between 1,368,000 -106 g.mol -1 . Analyte samples were filtered through a hydrophilic GVWP membrane with 0.22 μm pore size before injection. Number average molecular weights (Mn), weight average molecular weights (Mw) and dispersities (ĐM = Mw/Mn) were determined by conventional calibration and universal calibration using Agilent GPC/SEC software.

S3
Dynamic Light Scattering (DLS). Hydrodynamic diameters (Dh) and size distributions of particles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS with a 4 mW He-Ne 633 nm laser module operating at 25 o C. Measurements were carried out at an angle of 173° (back scattering), and results were analysed using Malvern DTS 7.03 software.
All determinations were repeated 5 times with at least 10 measurements recorded for each run.
Dh values were calculated using the Stokes-Einstein equation where particles are assumed to be spherical.
Zeta Potential Analysis. Zeta potential was measured by the technique of microelectrophoresis, using a Malvern Zetasizer Nano ZS instrument, at room temperature at 633 nm. All reported measurements were the average of at least five runs. Zeta potential was calculated from the corresponding electrophoretic mobilities (μE) by using the Henry's correction of the Smoluchowski equation (μE = 4π ε0 εr ζ (1+κr)/6π μ).

Differential centrifugal sedimentation (DCS). Differential centrifugal sedimentation (DCS)
was performed using a CPS DC24000 disc centrifuge with 8-24% (w/w) sucrose gradient and a rotation speed of 24000 RPM. A fresh sucrose gradient was prepared after each condition (plasma, BSA, BSA + plasma). Before each run, polyvinyl chloride latex beads (483 nm) with narrow size distribution were used as calibration standard to ensure accuracy of the measurements. All measurements were performed in triplicate. The binding of biomolecules onto the gold nanoparticles' surface increases the particles' size but lowers their overall density. The CPS analysis assumes a constant particle density, so over-estimating the particle density means an under-estimate of the particle size. 4,5 For this reason, the binding of polymers or biomolecules to the gold nanoparticles results in an apparent decrease in the particle size reported by CPS. A core-shell mathematical model was used to analyse the coating thickness of the gold nanoparticles as previously described after the glycopolymers functionalisation and for each sample (Gal-PHEA25@AuNP40; Gal-PHEA50@AuNP40; Gal-PHEA75@AuNP40) in the different conditions. [6][7][8] Transmission Electron Microscopy. Dry-state TEM imaging was performed on a JEOL JEM-2100Plus microscope operating at an acceleration voltage of 200 kV. All dry-state samples were diluted with deionised water and then deposited onto formvar-coated copper grids.
The sealed vial was incubated at 37°C with magnetic stirring under 460 nm light irradiation for 12h. The polymerisation was quenched by exposing the vial to air. An aliquot was withdrawn for determination of monomer conversion by 1 H NMR spectroscopy. The polymer was precipitated into acetone from methanol to yield a sticky yellow polymer product and dialysed against dionised water for 72h (MWCO = 1 kDa) and subsequently freeze-dried to yield a paleyellow solid. Mn,NMR by end-group analysis could not be calculated due to overlapping signals of the -CH3 of methyl end-group with those of the corresponding polymer signals. 1         BSA blocking. The globulins free BSA for blocking functionalised gold nanoparticles, prior to incubation in bovine plasma, was used at two different concentrations i.e., 2.5% and 5% (v/v).
Briefly, 250L of gold nanoparticles solution was incubated with an equal volume (1:1) of either 2.5% or 5% BSA for 1 hour at 37 o C, and 300 rpm. The Gal-PHEA@AuNPs-BSA S14 complex was centrifuged for removing the unbound BSA proteins at 12000 g, 20°C for 20 min.
After centrifugation, the supernatant was discarded, and pellet was resuspended in 400 L of PBS and centrifuged again. The washing step was repeated at least three times to completely remove the loosely bound BSA molecules. After final wash, the Gal-PHEA@AuNPs-BSA complex was resuspended in 250 L of PBS and incubated with an equal volume (1:1) of the 80% bovine plasma for 1 hour at 37 o C, and 300 rpm. After incubation, the samples were processed using a similar protocol as described earlier in the section "Formation of biomolecular corona".

Characterisation of biomolecular corona
Sodium  nanoparticles (25 μL, ∼1,0 final OD) in assay buffer were added to each well and the plate was S16 gently agitated at room temperature for 30 min. The absorbance spectra were recorded from 400 to 700 nm with 1 nm interval using a Biotek Synergy HT micro-plate reader.