Potent Ice Recrystallization Inhibition Research Articles

Development of Macromolecular Cryoprotectants for Cryopreservation of Cells.

Cryopreservation is a common way for long-term storage of therapeutical proteins, erythrocytes, and mammalian cells. For cryoprotection of these biosamples to keep their structural integrity and biological activities, it is essential to incorporate highly efficient cryoprotectants. Currently, permeable small molecular cryoprotectants such as glycerol and dimethyl sulfoxide dominate in cryostorage applications, but they are harmful to cells and human health. As acting in the extracellular space, membrane-impermeable macromolecular cryoprotectants, which exert remarkable membrane stabilization against cryo-injury and are easily removed post-thaw, are promising candidates with biocompatibility and feasibility. Water-soluble hydroxyl-containing polymers such as poly(vinyl alcohol) and polyol-based polymers are potent ice recrystallization inhibitors, while polyampholytes, polyzwitterions, and bio-inspired (glyco)polypeptides can significantly increase post-thaw recovery with reduced membrane damages. In this review, the synthetic macromolecular cryoprotectants are systematically summarized based on their synthesis routes, practical utilities, and cryoprotective mechanisms. It provides a valuable insight in development of highly efficient macromolecular cryoprotectants with valid ice recrystallization inhibition activity for highly efficient and safe cryopreservation of cells.

Glycosylated Polyhydroxyproline Is a Potent Antifreeze Molecule.

Molecules that inhibit the growth of ice crystals are highly desirable for applications in building materials, foods, and agriculture. Antifreezes are particularly essential in biomedicine for tissue banking, yet molecules currently in use have known toxic effects. Antifreeze glycoproteins have evolved naturally in polar fish species living in subzero climates, but practical issues with collection and purification have limited their commercial use. Here, we present a synthetic strategy using polymerization of amino acid N-carboxyanhydrides to produce polypeptide mimics of these potent natural antifreeze proteins. We investigated a set of mimics with varied structural properties and identified a glycopolypeptide with potent ice recrystallization inhibition properties. We optimized for molecular weight, characterized their conformations, and verified their cytocompatibility in a human cell line. Overall, we present a material that will have broad applications as a biocompatible antifreeze.

Understanding the role of polymers on the nucleating behavior of water in dilute supercooled solutions.

Understanding the nucleation behavior of water in dilute polymeric solutions is quintessential for the development of suitable artificial ice recrystallization inhibition (IRI) agents. Although poly(vinyl alcohol) (PVA) is found to be one of the most potent biomimetic IRI agents, the molecular understanding of the nucleation behavior of water in the presence of PVA is still lacking. Here, we use molecular dynamics to elucidate the role of concentration, degree of supercooling, degree of polymerization, and amphiphilicity of PVA and PVA-like polymers on the homogeneous nucleation of water in dilute polymeric solutions using the seeding method. Using classical nucleation theory (CNT), our simulations indicate an increase in the chemical potential difference between ice and melt that favors ice nucleation. However, it also predicts a significant increase in the ice-melt interfacial energy that impedes nucleation. The relative increase in the interfacial energy dominates the increase in the chemical potential difference, which results in a decrease in the nucleation rate of water with an increase in the solute concentration. This study contradicts the previous simulation study that suggested the promotion of homogeneous ice nucleation by PVA and supports the experimental observations of the heterogeneous origins of ice nucleation. Our results also suggest the non-classical origins of ice nucleation in polymeric solutions and the limitation of the CNT in predicting heterogeneous ice nucleation in polymeric solutions.

The Atomistic Understanding of the Ice Recrystallization Inhibition Activity of Antifreeze Glycoproteins

As the most potent ice recrystallization inhibitors, antifreeze glycoproteins (AFGPs) have been extensively studied since their discovery. However, the molecular mechanism of how they inhibit ice growth remains controversial—notably, which group directly contributes to the binding of AFGPs to ice is hotly debated. Here, we use molecular dynamics simulations to investigate the atomistic details of the binding of AFGP8 to ice. We show that the binding of AFGP8 to ice can be divided into three cases: backbone dominant binding (BDB), disaccharide dominant binding (DDB) and weak binding (WB). Hydrogen-bonding and hydrophobic groups contribute equally to the binding of AFGP8 to ice and synergistically promote the binding. The –CH3 groups promote the contacting of AFGP8 to ice via hydrophobic effect, and the hydrogen-bonding groups anchor AFGP8 to ice surfaces through direct hydrogen bonding with ice. Specially, we verify that the -CONH- groups anchor the backbone of AFGP8 to ice by forming hydrogen bonds with ice surfaces while the –OH groups not only anchor the disaccharide to ice but also slow down the dynamics of the surrounding water. In addition, we reveal that both the backbone and the disaccharide can bind to ice surfaces while the latter is more flexible, which also perturbs the hydrogen bond network of potential ice-like water molecules by swaying in the solution to further enhance its antifreeze activity. This work provides the atomistic details of the ice growth inhibition mechanism of AFGP8, which is helpful for the design of high-efficacy cryoprotectants.

Bioinspired Threonine-Based Polymers with Potent Ice Recrystallization Inhibition Activity.

Ice growth mitigation is a pervasive challenge for multiple industries. In nature, ice-binding proteins (IBPs) demonstrate potent ice growth prevention through ice recrystallization inhibition (IRI). However, IBPs are expensive, difficult to produce in large quantities, and exhibit minimal resilience to nonphysiological environmental stressors, such as pH. For these reasons, researchers have turned to bioinspired polymeric materials that mimic IBP behavior. To date, however, no synthetic polymer has rivaled the ability of native IBPs to display IRI activity at ultralow nanomolar concentrations. In this work, we study the IRI activity of peptides and polypeptides inspired by common ice-binding residues of IBPs to inform the synthesis and characterization of a potent bioinspired polymer that mimics IBP behavior. We show first that the threonine polypeptide (pThr) displays the best IRI activity in phosphate-buffered saline (PBS). Second, we use pThr as a molecular model to synthesize and test a bioinspired polymer, poly(2-hydroxypropyl methacrylamide) (pHPMA). We show that pHPMA exhibits potent IRI activity in neutral PBS at ultralow concentrations (0.01 mg/mL). pHPMA demonstrates potent IRI activity at low molecular weights (2.3 kDa), with improved activity at higher molecular weights (32.8 kDa). These results substantiate that pHPMA is a robust molecule that mitigates ice crystal growth at concentrations similar to native IBPs.

Ice recrystallization inhibition activity in bile salts

Ice recrystallization inhibitors are novel cryoprotective agents that can reduce the freezing damage of cells, tissues, and organs in cryopreservation. To date, potent ice recrystallization inhibition (IRI) activity has been found on antifreeze (glyco)proteins, polymers, nanomaterials, and a limited number of chemically synthesized small molecules. This paper reports a relatively potent IRI activity on a group of small biological molecules - bile salts. The IRI activity increased as the number of hydroxyl groups decreased in bile salts. Among sodium cholate (NaC), sodium deoxycholate (NaDC), sodium chenodeoxycholate (NaCC), and sodium lithocholate (NaLC), the least hydrophilic NaLC at a concentration of 25.0 mM entirely blocked the ice growth in phosphate-buffered saline (PBS) under test conditions. The IRI activity of bile salts was not related to viscosity or gelation. No IRI activity was found below the critical micelle concentration. The IRI activity was independent of liquid crystal formation. No ice shaping and thermal hysteresis were observed on any bile salts, but NaC and NaLC could increase the ice nucleation temperature. The findings add bile salts to the existing material list of ice recrystallization inhibitors.

Disaccharide Residues are Required for Native Antifreeze Glycoprotein Activity.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP’s unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.

Insights into Design of Biomimetic Glycerol-Grafted Polyol-Based Polymers for Ice Nucleation/Recrystallization Inhibition and Thermal Hysteresis Activity.

Many species living in colder regions of the world have adapted to the extreme climate by producing antifreeze (glycol) proteins (AF(G)P) which exhibit ice recrystallization inhibition (IRI), thermal hysteresis activity (THA), as well as other interactions with the freezing process of water. Although several synthetic approaches for the exploitation of these proteins have been investigated, challenges remain in the synthetic design of biomimetic polymers. Similar to biological antifreezes, poly(vinyl alcohol) (PVA) has potent IRI activity; however, by comparison, PVA has very little THA. In this study, we explored structural variations to polyol-based polymers to contrast with PVA as a control and identified several key structural elements for performance in IRI, THA, as well as in ice nucleation inhibition (INI). These structural features are bioinspired by the typical ice-binding plane of AFPs yet are surprisingly simple to produce with potency approaching that of typical AFPs. Key to the performance is positioning small organic functionalities with known antifreeze properties (such as ethylene glycol) pendent to a host polymer chain with consideration of their conformational freedom. To build systematic variations into both the backbone and side-chain structures, we used poly(vinyl alcohol), poly(isopropenyl acetate), poly(acrylic acid), and poly(methacrylic acid) parent polymers for such pendent modifications. One structure in particular, glycerol-grafted-PVA (G-g-PVA), shows potency rivaling that of AFPs at similar micromolar concentration. The findings in this study help guide the rational design of synthetic antifreeze polymers useful for applications such as anti-icing coatings through to cryopreservation methods for organ transport and cell preservation.

Slow Propagation of Ice Binding Limits the Ice-Recrystallization Inhibition Efficiency of PVA and Other Flexible Polymers.

Ice recrystallization inhibitors (IRI) are of critical importance in biology, cryopreservation of cells and organs, and frozen foods. Antifreeze glycoproteins (AFGPs) are the most potent IRI. Their cost and cytotoxicity drive the design of synthetic flexible polymers that mimic their function. Poly(vinyl alcohol) (PVA) is the most potent biomimetic found to date, although it is orders of magnitude less potent than AFGPs. A lack of molecular understanding of the factors that limit the IRI efficiency of PVA and other flexible ice-binding polymers hinders the design of more potent IRI. Here, we use molecular and numerical simulations to elucidate how the degree of polymerization (DP) and functionalization of PVA impact its IRI. Our simulations indicate that the onset of IRI activity of PVA occurs for 15 < DP < 20, in agreement with experiments. We predict that polymers with stronger binding to ice per monomer attain IRI activity at lower DP and identify this as a contributor to the higher IRI potency of AFGPs. The simulations reveal that the limiting step for binding of flexible molecules to ice is not the alignment of the molecule to the surface or the initiation of the binding but the propagation to reach its full binding potential. This distinguishes AFGPs and PVA from rigid antifreeze proteins and, we argue, is responsible for their different scaling of efficiencies with molecular size. We use the analysis of PVA to identify the factors that control the IRI activity of flexible polymers and assess the molecular characteristics that endow AFGPs with their exceptional IRI potency.

Enhancement of Macromolecular Ice Recrystallization Inhibition Activity by Exploiting Depletion Forces.

Antifreeze (glyco) proteins (AF(G)Ps) are potent inhibitors of ice recrystallization and may have biotechnological applications. The most potent AF(G)Ps function at concentrations a thousand times lower than synthetic mimics such as poly(vinyl alcohol), PVA. Here, we demonstrate that PVA’s ice recrystallization activity can be rescued at concentrations where it does not normally function, by the addition of noninteracting polymeric depletants, due to PVA forming colloids in the concentrated saline environment present between ice crystals. These depletants shift the equilibrium toward ice binding and, hence, enable PVA to inhibit ice growth at lower concentrations. Using theory and experiments, we show this effect requires polymeric depletants, not small molecules, to enhance activity. These results increase our understanding of how to design new ice growth inhibitors, but also offer opportunities to enhance activity by exploiting depletion forces, without re-engineering ice-binding materials. It also shows that when screening for IRI activity that polymer contaminants in buffers may give rise to false positive results.

Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Microorganisms.

All modern molecular biology and microbiology is underpinned by not only the tools to handle and manipulate microorganisms but also those to store, bank, and transport them. Glycerol is the current gold-standard cryoprotectant, but it is intrinsically toxic to most microorganisms: only a fraction of cells survive freezing and the presence of glycerol can impact downstream applications and assays. Extremophile organisms survive repeated freeze/thaw cycles by producing antifreeze proteins which are potent ice recrystallization inhibitors. Here we introduce a new concept for the storage/transport of microorganisms by using ice recrystallization inhibiting poly(vinyl alcohol) in tandem with poly(ethylene glycol). This cryopreserving formulation is shown to result in a 4-fold increase in E. coli yield post-thaw, compared to glycerol, utilizing lower concentrations, and successful cryopreservation shown as low as 1.1 wt % of additive. The mechanism of protection is demonstrated to be linked not only to inhibiting ice recrystallization (by comparison to a recombinant antifreeze protein) but also to the significantly lower toxicity of the polymers compared to glycerol. Optimized formulations are presented and shown to be broadly applicable to the cryopreservation of a panel of Gram-negative, Gram-positive, and mycobacteria strains. This represents a step-change in how microorganisms will be stored by the design of new macromolecular ice growth inhibitors; it should enable a transition from traditional solvent-based to macromolecular microbiology storage methods.

Antifreeze Glycoproteins Bind Reversibly to Ice via Hydrophobic Groups.

Antifreeze molecules allow organisms to survive in subzero environments. Antifreeze glycoproteins (AFGPs), produced by polar fish, are the most potent inhibitors of ice recrystallization. To date, the molecular mechanism by which AFGPs bind to ice has not yet been elucidated. Mutation experiments cannot resolve whether the binding occurs through the peptide, the saccharides, or both. Here, we use molecular simulations to determine the mechanism and driving forces for binding of AFGP8 to ice, its selectivity for the primary prismatic plane, and the molecular origin of its exceptional ice recrystallization activity. Consistent with experiments, AFGP8 in simulations preferentially adopts the PPII helix secondary structure in solution. We show that the segregation of hydrophilic and hydrophobic groups in the PPII helix is vital for ice binding. Binding occurs through adsorption of methyl groups of the peptide and disaccharides to ice, driven by the entropy of dehydration of the hydrophobic groups as they nest in the cavities at the ice surface. The selectivity to the primary prismatic plane originates in the deeper cavities it has compared to the basal plane. We estimate the free energy of binding of AFGP8 and the longer AFGPs4-6, and find them to be consistent with the reversible binding demonstrated in experiments. The simulations reveal that AFGP8 binds to ice through a myriad of conformations that it uses to diffuse through the ice surface and find ice steps, to which it strongly adsorbs. We interpret that the existence of multiple, weak binding sites is the key for the exceptional ice recrystallization inhibition activity of AFGPs.

Synthesis of Degradable Poly(vinyl alcohol) by Radical Ring-Opening Copolymerization and Ice Recrystallization Inhibition Activity.

Poly(vinyl alcohol) (PVA) is the most active synthetic mimic of antifreeze proteins and has extremely high ice recrystallization inhibition (IRI) activity. Addition of PVA to cellular cryopreservation solutions increases the number of recovered viable cells due to its potent IRI, but it is intrinsically nondegradable in vivo. Here we report the synthesis, characterization, and IRI activity of PVA containing degradable ester linkages. Vinyl chloroacetate (VClAc) was copolymerized with 2-methylene-1,3-dioxepane (MDO) which undergoes radical ring-opening polymerization to install main-chain ester units. The use of the chloroacetate monomer enabled selective deacetylation with retention of esters within the polymer backbone. Quantitative IRI assays revealed that the MDO content had to be finely tuned to retain IRI activity, with higher loadings (24 mol %) resulting in complete loss of IRI activity. These degradable materials will help translate PVA, which is nontoxic and biocompatible, into a range of biomedical applications.

Antifreeze Protein Mimetic Metallohelices with Potent Ice Recrystallization Inhibition Activity.

Antifreeze proteins are produced by extremophile species to control ice formation and growth, and they have potential applications in many fields. There are few examples of synthetic materials which can reproduce their potent ice recrystallization inhibition property. We report that self-assembled enantiomerically pure, amphipathic metallohelicies inhibited ice growth at just 20 μM. Structure–property relationships and calculations support the hypothesis that amphipathicity is the key motif for activity. This opens up a new field of metallo-organic antifreeze protein mimetics and provides insight into the origins of ice-growth inhibition.

Regioregular Alternating Polyampholytes Have Enhanced Biomimetic Ice Recrystallization Activity Compared to Random Copolymers and the Role of Side Chain versus Main Chain Hydrophobicity.

Antifreeze proteins from polar fish species are potent ice recrystallization inhibitors (IRIs) effectively stopping all ice growth. Additives that have IRI activity have been shown to enhance cellular cryopreservation with potential to improve the distribution of donor cells and tissue. Polyampholytes, polymers with both anionic and cationic side chains, are a rapidly emerging class of polymer cryoprotectants, but their mode of action and the structural features essential for activity are not clear. Here regioregular polyampholytes are synthesized from maleic anhydride copolymers to enable stoichiometric installation of the charged groups, ensuring regioregularity, which is not possible using conventional random copolymerization. A modular synthetic strategy is employed to enable the backbone and side chain hydrophobicity to be varied, with side chain hydrophobicity found to have a profound effect on the IRI activity. The activity of the regioregular polymers was found to be superior to those derived from a standard random copolymerization with statistical incorporation of monomers, demonstrating that sequence composition is crucial to the activity of IRI active polyampholytes.

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing.

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.

25. Ice recrystallization inhibitors – Mitigating cellular damage during freezing, transient warming and thawing

Cryopreservation is a common strategy for the preservation of various cells, tissues and biomaterials. Unfortunately, in the absence of a cryoprotective agent (CPA) significant cellular damage occurs. While the use of CPAs such as dimethyl sulfoxide (DMSO) and glycerol mitigate this damage, their toxic nature necessitates removal after thawing and this can adversely affect the functionality and integrity of the recovered cells, tissues or material. During the past decade, our laboratory has studied various classes of naturally occurring biological antifreezes (BAs) found in organisms that inhabit sub-zero environments. BAs control the growth of ice and protect organisms against the cellular damage associated with freezing. Our early work has lead to the development of carbon-linked antifreeze glycoprotein (C-AFGP) analogues that exhibit “custom-tailored” antifreeze activity. In particular, these C-AFGPs are potent inhibitors of ice recrystallization and have been shown to effectively cryopreserve human liver cells in the presence of reduced concentrations of DMSO. Extensive structure-function studies with the C-AFGPs have led to the discovery that small carbohydrate-based molecules (molecular masses 300 AMU) are very effective inhibitors of ice recrystallization at concentrations in the millimolar to sub-millimolar range and that these molecules my have unrealized potential as novel cryoprotectants. Freezing of Human red blood cells (RBCs) requires a cell permeating cryoprotectant to ensure adequate post-thaw recoveries. Typically in North America 40% glycerol is utilized and the sample is frozen slowly, while in many European countries smaller quantities of glycerol are utilized in conjunction with very fast freezing rates. After thawing, the glycerol must be removed prior to transfusion in order to avoid intravascular hemolysis. The automated process of removing the 40% glycerol solution from RBC units can take up to 1 h or more. We have examined a number of small molecule ice recrystallization inhibitors (IRIs) for their ability to freeze human RBCs using only 10–15% glycerol and slow freezing rates. Three IRIs, pBrPhGlc, PMPGlc and 4ClA have proven to be very effective cryoprotectants as evidenced by the amount of post-thaw hemolysis. In addition, some of these molecules are able to protect RBCs from cryoinjury associated with transient warming events (TWEs). This presentation will describe the rational design of small molecule IRIs, assessment of their ability to function as novel cryoprotectants for human RBCs and preliminary investigations into their mechanism of action.

C-1007: The design of small molecule ice recrystallization inhibitors for use as cryopreservatives

Ice recrystallization inhibition (IRI) activity is a desirable property for a cryoprotectant. Recently, small molecules having potent IRI activity have been reported by our laboratory ( Chem. Sci. , 2012, 3, 1408). These small molecules are amphiphilic and can form micelles due to their polar carbohydrate-based head and aliphatic tail ( Chem. Sci. , 2012, 3, 1408). We have previously described a correlation between monosaccharide hydrate and IRI activity ( J. Am. Chem. Soc., 2008, 130 (10), 2928) and therefore the effect of adding hydrophobic atoms or functional groups to small molecules on IRI activity was investigated. Fluorine is of particular interest because it possesses what’s known as the ‘hydrophobic hydration effect’, in which water molecules tend to order around fluorine atoms in solution ( ChemBioChem , 2004, 5, 622). Interestingly, the location of a fluorine atom or other hydrophobic functional groups on the aliphatic tail greatly affects the IRI activity of the molecule. During the course of this investigation, several very potent inhibitors of ice recrystallization were discovered and their use as cryoprotectants for Tf-1 α cells, used as a model for stem cells derived from human umbilical cord blood, was explored. Post-thaw cell viability and apoptosis of Tf-1 α cells cryopreserved with these IRI active molecules will be presented.

C-2000: Inhibition of ice recrystallization and gas hydrate formation using small molecules

Cellular damage as a result of ice recrystallization during thawing after cryopreservation is a contributing factor to cell death ( Cryobiology 1998,37, 171–186). Our laboratory has previously shown that simple monosaccharides can inhibit ice recrystallization at a concentration of 22 mM ( J. Am. Chem. Soc. , 2008, 130, 17494–17501). Recently, we have discovered that other classes of small molecules also exhibit the ability to inhibit ice recrystallization. One class of these molecules have the pyranose ring oxygen replaced with a nitrogen atom resulting in a compound with different electronic properties. These aza-sugars are also capable of inhibiting the formation of methane gas hydrates. Gas hydrates are problematic in high-pressure environments where methane is present, such as gas pipelines ( Chem. Eur. J. , 2010, 16, 10409–10417). Several different aza-sugars have been assessed for ice recrystallization inhibition (IRI) activity, thermal hysteresis (TH) and the ability to inhibit gas hydrate formation. Structure–function studies of these small molecules have elucidated the structural features necessary for potent IRI activity and the ability to inhibit formation of gas hydrates.

Quantitative study on the antifreeze protein mimetic ice growth inhibition properties of poly(ampholytes) derived from vinyl-based polymers.

Antifreeze (glyco) proteins (AF(G)Ps) from the blood of polar fish species are extremely potent ice recrystallization inhibitors (IRI), but are difficult to synthesise or extract from natural sources. Despite this challenge, materials which display IRI are appealing due to their ability to enhance cellular cryopreservation, for applications including regenerative and transplantation medicine. Here, poly(ampholytes), which contain a mixture of cationic and anionic side chains are quantitatively evaluated for their IRI activity. Poly(aminoethyl methacrylate), obtained by RAFT polymerization, is functionalised with succinic anhydride to generate the poly(ampholytes). The charge balance of the side chains is shown to be crucial, with only 50 : 50 mixtures having strong IRI activity, which also scales with molecular weight. This is the first example of a non-hydroxylated synthetic polymer with quantifiable IRI activity and raises questions about the mechanism of IRI, as the polymers have no obvious ice-binding motif. The ampholytic structure is shown to be transferable to carbohydrate-centred polymers with activity retained, but poly(betaines) are shown to be inactive.