Ice recrystallisation inhibiting polymer nano-objects via saline-tolerant polymerisation-induced self-assembly

Chemical tools to modulate ice formation/growth have great (bio)-technological value, with ice binding/antifreeze proteins being exciting targets for biomimetic materials. Here we introduce polymer nanomaterials that are potent inhibitors of ice recrystallisation using polymerisation-induced self-assembly (PISA), employing a poly(vinyl alcohol) graft macromolecular chain transfer agent (macro-CTA). Crucially, engineering the core-forming block with diacetone acrylamide enabled PISA to be conducted in saline, whereas poly(2-hydroxypropyl methacrylate) cores led to coagulation. The most active particles inhibited ice growth as low as 0.5 mg mL–1, and were more active than the PVA stabiliser block alone, showing that the dense packing of this nanoparticle format enhanced activity. This provides a unique route towards colloids capable of modulating ice growth.


Size Exclusion Chromatography. 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. Transmission Electron Microscopy. Dry-state stained TEM imaging was performed on either a JEOL JEM-2100 or a JEOL JEM-2100Plus microscope operating at an acceleration voltage of 200 kV. All dry-state samples were diluted with MilliQ water and then deposited onto formvar-coated copper grids.
After roughly 1 min, excess sample was blotted from the grid and the grid was stained with an aqueous 1 wt% uranyl acetate (UA) solution for 1 min prior to blotting, drying and microscopic analysis.
Cryogenic transmission electron microscopy (cryo-TEM) imaging was performed on a JEOL JEM-2100Plus microscope operating at an acceleration voltage of 200 kV. Samples for cryo-TEM imaging were prepared at 0.5% w/w solids content in milliQ water by depositing 8 µL sample onto plasmatreated lacey-carbon coated grids followed by blotting for approximately 5 s and plunging into a pool of liquid ethane, cooled using liquid nitrogen, to vitrify the samples. Transfer into a pre-cooled cryo-TEM holder was performed under liquid nitrogen temperatures prior to microscopic analysis. For the determination of average size of spherical assemblies at least 50 particles were analysed in each case.

Experimental Methods
Splat Ice Recrystallisation Inhibition Assay. Splat cooling assays were performed as previously described by Tomczak et al. 1 Briefly, a 10 μL sample was dropped 1.40 m onto a chilled glass coverslip, resting on a thin aluminium block placed on dry ice. Upon hitting the coverslip, a wafer with diameter of approximately 10 mm and thickness 10 μm was formed instantaneously. The glass S 4 coverslip was transferred onto the Linkam cryostage and held at -8°C using liquid nitrogen for 30 minutes. Photographs were obtained using an Olympus CX 41 microscope with a UIS-2 20x/0.45/∞/0-2/FN22 lens and crossed polarisers (Olympus Ltd), equipped with a Canon DSLR 500D digital camera.
Images were taken of the initial wafer (to ensure that a polycrystalline sample had been obtained) and again after 30 minutes. Image processing was conducted using ImageJ. In brief, the number of ice crystals in the field of view was measured for each photograph. The average (mean) of these three measurements was then calculated to find the mean grain area (MGS). The average value and error were compared to that of [NaCl] = 0.01 M solution, as appropriate, as a negative control. Nanoparticle samples solutions were first prepared upon dilution in [NaCl] = 0.01M on the range of PVA concentrations 5 -0.05 mg.mL -1 .
Sucrose Sandwich Ice Recrystallisation Inhibition Assay. Sucrose sandwich IRI assays were performed as described by Smallwood et al. 2,3 Briefly, nano-object samples dispersed in NaCl ([NaCl] = 0.01M) containing 45 wt % sucrose were sandwiched between two coverslips, and the edges were sealed with grease. Samples were cooled to and held at −50°C for 2 min 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). The temperature was then elevated to −8°C and held for 2 h. During this time, images were recorded every 10 mins using an Olympus CX41 microscope equipped with a UIS-2 20x/0.45/∞/0−2/FN22 lens (Olympus Ltd.) and a Canon EOS 500D SLR digital. Image processing was conducted using ImageJ.
Modified Sucrose Sandwich Ice Shaping Assay. Nano-object samples dispersed in NaCl ([NaCl] = 0.01M) containing 45 wt % 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). The temperature was then increased to −8 °C and held for 1 h 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.) and a Canon EOS 500D SLR digital. Image processing was conducted using ImageJ.

Synthesis of PVA181-g 7 -PHPMAn based nano-objects by aqueous RAFT-mediated dispersion thermally-initiated polymerisation-induced self-assembly (PISA).
A typical synthetic procedure to achieve PVA181-g 7 -PHPMA200 nano-objects at 10 wt% solids content by aqueous RAFT-mediated PISA is described. To a 6 mL vial containing a stirring bar were added PVA181 macro-CTA (40 mg, 3.7×10 -6 mol, 1 eq), HPMA (106 mg, 7.4×10 -4 mol, 200 eq), VAZO-50 (0.05 mg, 1.84×10 -7 mol, 0.05 eq) and MiliQ H2O (1.32 mL) such that the final concentration of monomer was 10 % w/w. The vial was sealed with a rubber septum and the solution was then purged with N2(g) for 20 min. The vial was placed into an aluminium heating block which had been pre-heated to 60 °C. After 4 h, the polymerisation was quenched by cooling the reaction mixture to room temperature and exposing it to air. An aliquot was removed for 1 H NMR in methanol-d4 and DMF SEC analyses. The resulting solution of particles was purified by three centrifugation/resuspension cycles in milliQ water at 14000 rpm. TEM, DLS and zeta potential analyses were performed on samples after dilution to an appropriate analysis concentration.
Saline (0.01 M NaCl) was used in place of PBS in this work, due to the colloidal stability challenges (as discussed in the text). This low salt condition has been previously used to study colloids 7 and antifreeze proteins 8 and is sufficient to prevent false positives which occur when pure water is used alone. 9 Example control (no additive) ice wafers after 30 minutes annealing for PBS and NaCl are show below. Salts can have a significant impact on IRI and the magnitude of effect of any additive may be impacted by this which should always be considered. 10