Site-specific conjugation of antifreeze proteins onto polymer-stabilized nanoparticles

Antifreeze proteins are site-specifically conjugated onto polymer-stabilised gold nanoparticles, resulting in hybrid materials capable of modulating ice growth processes.

The eluent was DMF with 5 mmol NH4BF4 additive. Samples were run at 1ml/min at 50. Poly(methyl methacrylate) standards (Agilent EasyVials) were used for calibration between 955,000 -550 gmol -1 . Analyte samples were filtered through a nylon membrane with 0.22 μm pore size before injection. Molar mass (Mn,SEC) and dispersity (Đ) values of synthesized polymers were determined by conventional calibration against PMMA standards using Agilent GPC/SEC software. Nanoparticle size was determined using Dynamic Light Scattering (DLS), performed on a Malvern Instruments Zetasizer Nano-ZS with 4mW HeNe laser 632.8 nm. UV/Vis spectroscopy, optical density and fluorescence plate readings were performed on a BioTek Synergy HT Microplate Reader. UV-vis spectroscopy was used to determine nanoparticle size according to a method developed by Haiss et al. 1 Transmission electron microscopy (TEM) was performed on a JEOL 2100 LaB6 high-resolution microscope. X-ray photoelectron spectroscopy (XPS) was carried out on the Kratos Axis Ultra with a delay-line detector. Bacterial cell lysis was performed using a STANSTED 'Pressure Cell' FPG12800 homogeniser. WAXS experiments were performed using a Xenocs Xeuss 2.0 equipped with a micro-focus Cu Kα source collimated with Scatterless slits. The scattering was measured using a Pilatus 100k detector with a pixel size of 0.172 μm x 0.172 μm. The distance between the detector and the sample was calibrated using silver behenate (AgC22H43O2), giving a value of 0.161(3) m. The detector was fixed at an angle of 36° giving a 2θ range of 18.5 to 47.5°, WAXS experiments used 1.0 mm borosilicate capillaries (Capillary Tube Supplies Ltd) and recorded patterns from 10 to 45 ° at temperatures between -35 and 10 °C. Thermal hysteresis measurements were performed using a Clifton nanolitre osmometer by Prof Peter Davies, Queens University Canada and using standard procedures 2 .

Synthesis of 4 nm gold nanoparticles
AuNPs were synthesized based on a method by Ieong et al. 3 All glassware was washed with aqua regia [CAUTION. Extreme care must be taken when handling this reagent] 4 prior to commencing the experiment, to reduce unwanted nucleation from impurities on the surface. 240 mL of a 0.21 mmol.L -1 , (0.08 mg.mL -1 ) aqueous solution of HAuCl4 was prepared at room temperature. To this was added 13.8 mg (0.05 mmol) of trisodium citrate to give a final citrate concentration of 0.21 mmol.L -1 . 5 mL of an ice-cold 0.1 M (0.5 mmol, 18.5 mg) solution of NaBH4 was added to the gold/citrate solution and stirred at room temperature overnight. Assuming complete reduction of the HAuCl4 to gold particles, the total gold concentration in the final solution was 0.21 mmol.L -1 (0.04 mg/mL). As these small AuNPs did not pellet upon centrifugation, small AuNPs were isolated by taking the supernatant after centrifugation at 13.2 k RPM for 10 mins at 25 °C. Nanoparticle size and dispersity was measured by TEM, UV-Vis spectroscopy and size and zeta potential measurements by DLS.

Synthesis of Maleimide-pHEA47@Au4
NOTE. These particles were found not to be stable in buffer and were not taken forward, but included for completeness.
PFP-pHEA47@Au4 solution (500 μL) and N-(2-Aminoethyl)maleimide trifluoroacetate salt (0.00088 g, 3.4 μmol) were dissolved in DMF (500 μL) in a 1.5 mL polypropylene microcentrifuge tube and vortexed 30 secs to dissolve. To the stirred mixture, triethylamine (3.5 μL, for 0.5 M solution) was added, and the reaction was moved to a 50 oil bath for 16 hours. The solutions were added to 4.5 mL of H2O before being concentrated by centrifugation in Amicon Ultra-0.5 centrifugal filter units with an Ultracel-30 membrane. The AuNPs were re-dispersed in 500 μL of PBS buffer, pH 7.5.
The nanoparticles were washed by concentration and re-dispersion a further 2 times and stored in the fridge until required. Nanoparticle size and dispersity was measured by TEM, UV-Vis spectroscopy and size and zeta potential measurements by DLS.
These quickly aggregated making them non-usable.

Synthesis of Ni-NTA-pHEA17@Au4
DMF, TEA, The solutions were concentrated by centrifugation in Amicon Ultra-0.5 centrifugal filter units with an Ultracel-30 membrane, before being re-dispersed in the same volume of pH 7.5 HEPES/NaCl buffer. The nanoparticles were washed in this manner a further 3 times before being re-dispersed in the same final volume of buffer and stored in the fridge until required. Nanoparticle size and dispersity was measured by TEM, UV-Vis spectroscopy and size and zeta potential measurements by DLS.

S13
AFP was conjugated to Mal-pHEA47@Au4; and His-AFP was conjugated to Ni-NTA-pHEA17@Au4. To this concentrate was added the same volume of protein solution as was originally concentrated to retain the same concentration of AuNPs. This was left, agitating, for 120 minutes at room temperature. The solutions were concentrated by centrifugation in Amicon Ultra-0.5 centrifugal filter units with an Ultracel-30 membrane, before being re-dispersed in the same volume of pH 7.5 PBS buffer. The nanoparticles were washed in this manner a further 2 times before being re-dispersed in the same final volume of PBS and stored in the fridge until required. Nanoparticle size and dispersity was measured by TEM, UV-Vis spectroscopy and size and zeta potential measurements by DLS.

Recombinant expression of AFPIII-Cys
The terminal Ala in the pET20b-AFPIII plasmid was mutated to Cys by site-directed mutagenesis using the primer sequence 5'-GTTAAAGGTTACGCTTGTCTCGAGCACCAC-3' and employing Phusion® High-Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer's protocol. The expression and purification was performed as detailed for AFPIII with the exception that 2 mM β-mercaptoethanol was added to all buffers when performing IMAC and gel filtration to prevent the formation of disulfide bonds. The βmercaptoethanol was removed prior to use by buffer exchanging using PD10 desalting columns (GE Healthcare).
The subsequent expression and purification was performed as detailed for AFPIII.

Ice Recrystallisation Inhibitory activity "splat" assay
The sample was prepared in PBS solution, and a 10 µL was dropped onto a glass microscope coverslip atop an aluminium plate cooled to -78 with dry ice. The thin wafer of ice that forms upon contact with the coverslip was transferred to a cryostage held at -8 ºC using liquid nitrogen, and left to anneal for 30 minutes at this temperature. Three images were taken at different locations on this wafer at 20 x zoom under cross polarizers. Using ImageJ, the number of crystals in the images were counted and the average crystal size per wafer was calculated as mean grain area (MGS) as well as the mean grain length size (MLGS).

Modified Sucrose Sandwich Ice Shaping Assay
Samples dissolved in PBS buffer containing 45% sucrose were sandwiched between two glass coverslips and sealed with immersion oil. Samples were cooled to −50 °C on a Linkam Biological Cryostage BCS196 with T95-Linkpad system controller equipped with a LNP95-Liquid nitrogen cooling pump, using liquid nitrogen as the coolant (Linkam Scientific Instruments UK, Surrey, U.K.). The temperature was then increased to −8 °C and held for 1 hour to anneal. The samples were then heated at 0.5 °C.min -1 until few ice crystals remained and then cooled at 0.05 °C.min -1 and the shape of ice crystals observed. Micrographs were obtained every 0.1 °C using an Olympus CX41 microscope equipped with a UIS-2 20x/0.45/∞/0−2/FN22 lens (Olympus Ltd., Southend on sea, U.K.) and a Canon EOS 500D SLR digital. Image processing was conducted using ImageJ. S16 Figure S1: pHEA polymer before and after functionalisation with primary amines: A) PFP-pHEA45 modified to BG-pHEA45, B) PFP-pHEA6 modified to NTA-pHEA6.
In each case the depletion of the three fluorine peaks is observed as evidence of the successful removal of the PFP-group. It should be noted that the elution behaviour of the PFP-pHEA polymers changed drastically after end-group modification -the hydrophobic end-group led to tailing in the SEC, and hence the non-symmetric trace was obtained. S19 Figure S4: Nanodrop measurements showing depletion of AFP in washes. Protein concentration is on the y-axis. Error bars are standard deviation from n = 3. Figure S4 shows protein residual protein concentration in the supernatant following washing of the AuNPs, determined using a Nanodrop. There was no detectable protein remaining after 4 washes, confirming the washing method success. S20 Figure S5: AuNP samples before and after conjugation with polymer and subsequent post-conjugation modification: A) Conjugation of BG-pHEA45 followed by SNAP-AFP, B) Conjugation of NTA-pHEA17 followed by reaction with nickel chloride (to form Ni-NTA complex), followed by His-AFP, C) Conjugation of PFP-pHEA47 followed by reaction to form Maleimide-pHEA47, followed by Cys-AFP.
For the SNAP-and His-AFP samples, in each case an increase in size is observed. For the Cys-AFP sample, the size of the maleimide-and Cys-AFP@Au4 showed significant increases in size that resulted in aggregation and sedimentation of the nanoparticles. In each sample no significant change in size or uniformity was observed by TEM. A shift in the mean zeta potential and conductivity confirmed successive surface modification on the AuNP samples. This was most pronounced in the steps involving the initial conjugation of polymer, the reaction with nickel, and upon adding protein to the maleimide-/BG-coated gold.  These raw data survey scans showed a difference in the x-ray photoelectron spectra as coatings were applied to the gold nanoparticles. Although the survey plots for citrate and NTA-pHEA17-coated AuNPs were superficially the same in these survey scans, a difference in several peaks was found upon scanning for specific elements such as sulfur and gold. The above values were calculated taking into account buffers present (such as PBS present in the protein samples) and against the control of citrate coated particles. These assays show the shape and directionality of ice growth as temperature is gradually decreased. Both recombinant AFP conjugated to the AuNP scaffold showed a branching, feather-like ice growth. The different monomeric recombinant AFPs showed a difference in shape: SNAP-AFP showing straight shard-like shapes and His-