Externally controllable glycan presentation on nanoparticle surfaces to modulate lectin recognition

Response polymer gates are employed to enable external control of glycan expression on the surface of multivalent nanoparticles.


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Vector 22 FTIR spectrometer with a Golden Gate diamond attenuated total reflection cell. A total 64 (or 128) scans with resolution of 4 cm -1 were collected. Samples were pre-dried as a thin film for FTIR analysis. SEC analysis was conducted on Varian 390-LC MDS system equipped with a column, two PL-AS RT/MT auto sampler, a PL-gel 3 mm (50 × 7.5 mm) guard column, two PL-gel 5 mm (300 × 7.5 mm) mixed-D columns using dimethylformamide (DMF) with 1 mg mL -1 LiBr at 50 °C as the eluent at a flow rate of 1.0 mL min -1 . The GPC system was equipped with ultraviolet (UV) (set at 280 nm) and differential refractive index (DRI) detections. Narrow molecular weight poly(methyl methacrylate) (PMMA) standards (200-1.0 × 106 g mol -1 ) were used for calibration using a second order polynomial fit. Polymer solutions at 1 mg mL -1 were prepared in the eluent and filtered through 0.45 mm filters prior to injection.
UV-vis spectra were recorded in a disposable cuvette using a Cary 60 UV-vis spectrometer from Agilent at 25 °C. Lower critical solution temperatures of free pNIPAM and pNIPAM nanoparticles were also analyzed using an Agilent Cary 60 UV-vis spectrometer equipped with a temperature controller at 700 nm with a heating/cooling rate of 1 °C min -1 . The cloud point of pNIPAM and pNIPAM nanoparticles were determined by normalising the turbidimetry curve such that the values were in the range of 0 to 1, and the transition temperature was defined as being the temperature corresponding to a normalised absorbance of 0.5. A polymer concentration of 1.0 mg mL -1 was used in all experiments. DLS measurements were performed using a Nano-Zs from Malvern Instruments, UK running DTS software (4 mW, He-Ne laser, l = 633 nm) and an avalanche photodiode (APD) detector. The scattered light was measured at an angle of 173° for DLS measurement. The temperature was stabilized to ± 0.1 °C of the set temperature. All samples were prepared at the concentration of 0.057 mg mL -1 gold nanoparticles. Hydrodynamic radii were determined using the manufacturer's software.
Absorbance measurements of the nanoparticles incubated with lectin were recorded on a BioTek SynergyTM HT multi-detection microplate reader obtained using Gen5 1.11 multiple S4 data collection and analysis software. The size and morphology of the synthesized gold nanoparticles and polymer coated gold nanoparticles were estimated by JEOL 2100FX transmission electron microscopy (TEM) at an accelerating voltage 200 kV. A drop of sample solution was deposited onto a copper grid and the water was evaporated under air. No staining was applied. The x-ray photoemission spectroscopy (XPS) data were collected at the Warwick Photoemission Facility, University of Warwick. The samples investigated in this study were deposited on to Cu foil, mounted on to a sample bar and loaded in to a Kratos Axis Ultra DLD spectrometer which possesses a base pressure of ~ 5 × 10 -10 mbar. XPS measurements were performed in the main analysis chamber, with the sample being illuminated using an Al kα xray source. The measurements were conducted at room temperature and at a take-off angle of (30°) with respect to the surface parallel. The core level spectra were recorded using a pass energy of 20 eV (resolution approx. 0.4 eV). The spectrometer work function and binding energy scale were calibrated using the Fermi edge and 3d 5/2 peak recorded from a polycrystalline Ag sample immediately prior to the commencement of the experiments. The data were analysed in the CasaXPS package, using Shirley backgrounds, mixed Gaussian-Lorentzian (Voigt) lineshapes. For compositional analysis, the analyser transmission function has been determined using Ag, Au and Cu foils to determine the detection efficiency across the full binding energy range.
After stirring for 16 hour, the solvent was removed under reduced pressure and the residue was extracted into CH 2 Cl 2 (2 × 200 mL) from 1M HCl (200 mL). The organic extracts were washed with water (200 mL) and brine (200 mL) and further dried over MgSO 4 . The solvent was removed under reduced pressure and the residue was purified by recrystallization in hexane.

Polymerisation of N-isopropylacrylamide using 2-(dodecylthiocarbonothioylthio)-2methylpropanoic acid (DMP)
Polymers with three different molecular weights were synthesised in typical procedure. as an internal reference and the mixture was stirred (5 mins). An aliquot of this starting mixture was removed for 1 H NMR analysis. The vial was fitted with a rubber septum and degassed by bubbling with nitrogen gas (30 mins). The vial was then placed in an oil bath thermostated at 70 °C. After 35 minutes, the reaction mixture was opened to air and quenched in liquid nitrogen.
An aliquot was removed and conversion determined by 1 H NMR. The remainder was precipitated into diethyl ether (45 mL). The polymer was re-precipitated and purified from THF to diethyl ether three times. The product was purified three times by precipitation from toluene into diethyl ether, isolated centrifugation, and dried under vacuum overnight to give a yellow solid. The overall monomer conversion was determined from the 1 H NMR spectrum by measuring the decrease in intensity of the vinyl peaks associated with the monomer relative to mesitylene. Conversion (NMR): 86 %; M n (theoretical), 5200 g mol -1 ; M n (SEC), 7100 g mol -1 ; M w /M n (SEC), 1.10.

General procedure for the synthesis of polymer-coated gold nanoparticles
Approximately 1 mg of the desired thiol-terminated polymer (pNIPAM or pHEA-Gal) was added to a microcentrifuge tube, and dissolved in 100 µL of high-purity water. 900 µL of the citrated-stabilized gold nanoparticle solution was added to this tube (40 nm: 0.296 mmol L -1 , 60 nm: 0.288 mmol L -1 total gold concentration), which was then agitated 30 mins in the absence of light. To remove excess polymer, the particles were centrifuged and following careful decantation of the supernatant, the particles were then re-dispersed in 1 mL of highquality water and the centrifugation-resuspension process repeated for a total of 3 cycles. After the final cycle the particles were dispersed in 1 mL of high-quality water for future use.

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To test the specificity of the glyco-particle interactions, and rule out non-specific protein binding, control experiments were conducted using BSA as a non-carbohydrate binding protein ( Figure S8). Non responsive, galactosylated pHEA coated particles were incubated with a serial dilution of BSA and the UV-Vis spectra recorded, showing no change. Accordingly, the same experiment was undertaken with the pNIPAM containing particles at both low and high temperature (i.e where we see activation towards lectin binding). No change was seen indicating there was no aggregation induced by non-specific effects.