Inhibit Ice Growth Research Articles

Antifreeze Protein Activity: From Ice Binding to Ice Growth Inhibition

Antifreeze Protein Activity: From Ice Binding to Ice Growth Inhibition

Effect of different freezing conditions on ice crystal formation behavior and ice-growth inhibition by cryoprotectants.

The formation of ice crystals will have adverse effects on aquatic products, and the key to ensure the long-term preservation and better quality preservations of the product is to evaluate the intercellular ice crystal formation to find suitable refrigeration conditions and cryoprotectants. The ice crystal formation was successfully captured by using an inverted microscope cryomicroscopic system equipped with a low-temperature stage, the ice crystals formed under different freezing methods between tuna muscle cells were observed directly, the deformation degree of muscle tissue pores during crystallization was evaluated, and the effect of freeze-thaw times on tuna samples was analyzed. The effects of the use of cryoprotectant such as cellobiose and carboxylated cellulose nanofibers on ice-growth inhibition were investigated, and the reliability of the ice crystal observation results was further verified by the determination of physical properties. The results showed that carboxylated cellulose nanofibers had the best ice-growth inhibition effect, they prevented about 50% cell deformation compared with the control group, and also reduced the minimum size of ice crystal formation. In addition, the addition of cellobiose and sodium tripolyphosphate gave the ice crystals a more uniform size and roundness. The experiment proposed a stable and clear observation method for the process of intercellular ice crystal formation, and the accuracy of the observation method was further verified by some physical indicators. This may help in the selection of suitable measurement methods to directly observe ice crystal formation behavior and screen cryoprotectants. © 2024 Society of Chemical Industry.

Plunge-Freezing Cryopreservation of Tendons.

Allograft transplantation is an important method for tendon reconstruction after injury, and its clinical success highly relies on the storage and transportation of the grafts. Cryopreservation is a promising strategy for tendon storage. In this study, we report a novel cryopreservation agent (CPA) formulation with a high biocompatibility for tendon cryopreservation. Mainly composed of natural zwitterionic betaine and the biocompatible polymer poly(vinylpyrrolidone) (PVP), it exhibited ideal abilities to depress the freezing point and inhibit ice growth and recrystallization. Notably, after cryopreservation via plunge-freezing for 1 month, Young's modulus (144 MPa, 98% of fresh tendons) and ultimate stress (46.7 MPa, 99% of fresh tendons) remained stable, and the cross-linking of collagen microfibers, protein structures, and glycosaminoglycan (GAG) contents changed slightly. These results indicate that the formulation (5 wt % betaine and 5 wt % PVP in phosphate-buffered saline, PBS solution) effectively maintains the biomechanical properties and tissue structure. This work offers a novel cryopreservation method for tendons and may also provide insights into the long-term preservation of various other tissues.

Probing the Size Effect of Graphene Oxide Nanosheets on Ice Crystal Regulation and Laser-Assisted Rapid Rewarming.

Two-dimensional (2D) nanomaterials have attracted many researchers to explore the effect of ice control and rapid deicing due to their functional groups, large specific surface area, and excellent photothermal properties. However, the impact of size effects on ice crystal formation, growth, and photothermal performance has been rarely explored. Here, graphene oxide nanosheets (GO NSs) with controllable sizes were used as a representative of 2D nanomaterials to probe the effect of size on ice crystal regulation and rapid rewarming in cell cryopreservation. All sizes of GO NSs exhibited notable inhibitory effects on ice crystal size during the recrystallization process. Significantly, when the size of GO NSs was smaller than a certain size (<150 nm), they showed a more significant ice recrystallization suppression effects, which could reduce the ice crystal size to about 17% of that of pure water. Meanwhile, the photothermal experiments also indicated that smaller-sized GO NSs exhibited better photothermal behavior, with 90 nm GO NSs (GO-90) heating to 70 °C in just 1 min induced by an 808 nm laser (2 W/cm2). Furthermore, applying GO-90 (200 μg/mL) to cell cryopreservation, cell viability could reach 95.2% and 93% with a low amount of traditional cryoprotectant (2% v/v DMSO) for A549 cells and HeLa cells after recovery, respectively. With the assistance of a 808 nm laser, the rewarming time was also shortened to 20 s, greatly improving the rewarming rate. Our work associated specific sizes of 2D nanomaterials with their ice growth inhibition behaviors during recrystallization and photothermal properties to synergistically improve cell cryopreservation efficiency, providing guidance for effectively designing novel 2D nanomaterials for collaborative control of ice crystals in cell cryopreservation.

On the engulfment of antifreeze proteins by ice

Antifreeze proteins (AFPs) are remarkable biomolecules that suppress ice formation at trace concentrations. To inhibit ice growth, AFPs must not only bind to ice crystals, but also resist engulfment by ice. The highest supercooling, [Formula: see text], for which AFPs are able to resist engulfment is widely believed to scale as the inverse of the separation, [Formula: see text], between bound AFPs, whereas its dependence on the molecular characteristics of the AFP remains poorly understood. By using specialized molecular simulations and interfacial thermodynamics, here, we show that in contrast with conventional wisdom, [Formula: see text] scales as [Formula: see text] and not as [Formula: see text]. We further show that [Formula: see text] is proportional to AFP size and that diverse naturally occurring AFPs are optimal at resisting engulfment by ice. By facilitating the development of AFP structure-function relationships, we hope that our findings will pave the way for the rational design of AFPs.

Ice growth inhibition by poly(vinyl alcohol): Insights from near-infrared spectroscopy and molecular dynamics simulation

Poly(vinyl alcohol) (PVA) is a well-known ice recrystallization inhibitor, yet its antifreeze mechanism remains debated. Here, the mechanism of PVA is elucidated by means of near-infrared spectroscopy and molecular dynamics (MD) simulations. Analysis of the spectra of PVA solutions during the ice growth process reveals a dynamic interaction conversion from PVA-water to PVA-ice, indicating that PVA inhibits ice growth by directly binding to ice. Moreover, we observed OH binding to the ice surface through one or two hydrogen bonds but did not witness the previously reported formation of three hydrogen bonds when the OH group perfectly embeds into the ice crystal lattice. In addition, the spectral feature of CH2 groups of PVA indirectly indicates their significant contribution to inhibiting ice growth through hydrophobic interactions. The MD simulations further confirm the effective roles of both OH and CH2 groups for inhibiting ice growth.

Elevated fucose content enhances the cryoprotective performance of anionic polysaccharides

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18–35.7 mol%), uronic acids (UA) (13.5–26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. Our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection.

Investigating the Interaction Between Ice-Binding Proteins and Ice Surfaces Using Microfluidic Devices and Cold Stages.

Ice-binding proteins (IBPs) protect organisms living in sub-freezing conditions by inhibiting ice growth in fish and insects, limiting ice recrystallization in plants, and assisting bacteria to adhere to ice. The mechanisms by which these proteins bind to ice and inhibit its growth have been studied both experimentally and using molecular dynamic simulations. A unique experimental technique developed to test and characterize the interactions between IBPs and ice using a combination of a microfluidic device, cold stages with millikelvin temperature resolution, fluorescence-labeled IBPs, and fluorescence microscopy is described herein. The main advantage of this technique is the ability to exchange the solution around micron-sized ice crystals and characterize their binding to and inhibition of ice.

Functional aggregation of cell-free proteins enables fungal ice nucleation.

Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above -10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 °C. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here, we combine ice nucleation measurements, physicochemical characterization, numerical modeling, and nucleation theory to shed light on the size and nature of the INs from the fungus Fusarium acuminatum. We find ice-binding and ice-shaping activity of Fusarium IN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits that assemble into ice-nucleating complexes that can contain more than 100 subunits. Fusarium INs retain high ice-nucleation activity even when only the ~12 kDa fraction of size-excluded proteins are initially present, suggesting robust pathways for their functional aggregation in cell-free aqueous environments. We conclude that the use of small proteins to build large assemblies is a common strategy among organisms to create potent biological INs.

Self-Assembled Nanostructures of Homo-Oligopeptide as a Potent Ice Growth Inhibitor.

This study reports the formation of self-assembled nanostructures with homo-oligopeptides consisting of amino acids (i.e., alanine, threonine, valine, and tyrosine), the resulting morphologies (i.e., spherical shape, layered structure, and wire structure) in aqueous solution, and their potential as ice growth inhibitors. Among the homo-oligopeptides investigated, an alanine homo-oligopeptide (n = 5) with a spherical nanostructure showed the highest ice recrystallization inhibition (IRI) activity without showing a burst ice growth property and with low ice nucleation activity. The presence of nanoscale self-assembled structures in the solution showed superior IRI activity compared to an amino acid monomer because of the higher binding affinity of structures on the growing ice crystal plane. Simulation results revealed that the presence of nanostructures induced a significant inhibition of ice growth and increased lifetime of hydrogen bonding compared with unassembled homo-oligopeptide. These results envision extraordinary performance for self-assembled nanostructures as a desirable and potent ice growth inhibitor.

Strong Inhibition of Ice Growth by Biomimetic Crowding Coacervates

AbstractThe freezing of biological fluids is intensively studied but remains elusive as it is affected not only by the various components but also by the crowding nature of the biological fluids. Herein, we constructed spherical crowders, fibrous crowders, and coacervates by various components ranging from surfactants to polymers and proteins, to mimic three typical crowders in biological fluids, i.e., globular proteins, fibrous networks, and condensates of biomolecules. It is elucidated that the three crowders exhibit low, moderate, and strong ice growth inhibition activity, respectively, resulting from their different abilities in slowing down water dynamics. Intriguingly, the coacervate consisting of molecules without obvious ice growth inhibition activity strongly inhibits ice growth, which is firstly employed as a highly‐potent cryoprotectant. This work provides new insights into the survival of freezing‐tolerant organisms and opens an avenue for the design of ice‐controlling materials.

Strong Inhibition of Ice Growth by Biomimetic Crowding Coacervates.

The freezing of biological fluids is intensively studied but remains elusive as it is affected not only by the various components but also by the crowding nature of the biological fluids. Herein, we constructed spherical crowders, fibrous crowders, and coacervates by various components ranging from surfactants to polymer and proteins to mimic three typical crowders in biological fluids, i.e., globular proteins, fibrous networks, and condensates of biomolecules. It is elucidated that the three crowders exhibit low, moderate, and strong ice growth inhibition activity, respectively, resulting from their different abilities in slowing down water dynamics. Intriguingly, the coacervate consisting of molecules without obvious ice growth inhibition activity strongly inhibits ice growth, which is firstly employed as a highly-potent cryoprotectant. This work provides new insights into the survival of freezing-tolerant organisms and opens an avenue for the design of ice-controlling materials.

Mechanism of Binding of Polyproline to Ice via Interfacial Water: An Experimental and Theoretical Study.

The molecular mechanism underlying inhibition of ice growth by polyproline (PPro), a minimal antifreeze glycoprotein mimic, remains unclear. In this work, the change in the structure of water during the growth of ice in PPro solutions was investigated using a combination of near-infrared spectroscopy and molecular dynamics (MD) simulations. The results show that only high concentrations of PPro solutions can effectively inhibit ice growth, as indicated by the variation in the spectral intensity of ice with time. When PPro exhibits an antifreeze effect, the spectral intensity of hydrated water associated with PPro in a solution is weakened. The experiments and MD simulations reveal that the quantity of the interfacial water between the ice crystal and the hydrophobic groups of PPro progressively reaches a plateau. Most significantly, we present clear evidence that the stable existence of this interfacial water is critical for the antifreeze activity of PPro.

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.

Microcurvature Controllable Metal-Organic Framework Nanoagents Capable of Ice-Lattice Matching for Cellular Cryopreservation.

Ice-binding proteins (IBPs) produced by psychrophilic organisms to adapt for the survival of psychrophiles in subzero conditions have received illustrious interest as a cryopreservation agent required for cells and tissues to completely recover after freezing/thawing. Depressing water-freezing point and avoiding ice-crystal growth affect their activities which are closely related to the presence of ice crystal well-matched binding moiety. The interaction of IBPs with ice and water is critical in enhancing their freeze avoidance against cell or tissue damage. Metal-organic frameworks (MOFs) with a controllable lattice at the molecular level and a size at the nanometer scale can offer periodically ordered ice-binding sites by modifying organic linkers and controlling microcurvature at the ice surface. Herein, zirconium (Zr)-based MOF-801 nanoparticles (NPs) with good biocompatibility were used as a cryoprotectant that is well dispersed and colloidal-stable in an aqueous solution. The MOF NP size was precisely controlled, and 10, 35, 100, and 250 nm NPs were prepared. The specific IBPs-mimicking pendants (valine and threonine) were simply introduced into the MOF NP-surface through the acrylate-based functionalization to endow with hydrophilic and hydrophobic dualities. When small-sized MOF-801 NPs were attached to ice, they confined ice growth in high curvature between the adsorption sites because of the decreased radius of the convex area of the growth region, leading to highly enhanced ice recrystallization inhibition (IRI). Surface-functionalized MOF NPs could increase the number of anchored clathrate water molecules with hydrophilic/hydrophobic balance of the ice-binding moiety, effectively inhibiting ice growth. The MOF-801 NPs were biocompatible with various cell lines regardless of concentration or NP surface-functionalization, whereas the smaller-sized surface-functionalized NPs showed a good cell recovery rate after freezing/thawing by induction of IRI. This study provides a strategy for the fabrication of low-cost, high-volume antifreeze nanoagents that can extend useful applications to organ transplantation, cord blood storage, and vaccines/drugs.

Revealing the Mechanism of Irreversible Binding of Antifreeze Glycoproteins to Ice.

Antifreeze glycoproteins (AFGPs) are a special kind of antifreeze proteins with strong flexibility. Whether their antifreeze activity is achieved by reversibly or irreversibly binding to ice is widely debated, and the molecular mechanism of irreversible binding remains unclear. In this work, the antifreeze mechanism of the smallest AFGP isoform, AFGP8, is investigated at the atomic level. The results indicate that AFGP8 can bind to ice both reversibly through its hydrophobic methyl groups (peptide binding) and irreversibly through its hydrophilic disaccharide moieties (saccharide binding). Although peptide binding occurs faster than saccharide binding, free-energy calculations indicate that the latter is energetically more favorable. In saccharide binding, at least one disaccharide moiety is frozen in the grown ice, resulting in irreversible binding, while the other moieties significantly perturb the water hydrogen-bonding network, thus inhibiting ice growth more effectively. The present study reveals the coexistence of reversible and irreversible bindings of AFGP8, both contributing to the inhibition of ice growth and further provides molecular mechanism of irreversible binding.

Molecular Factors of Ice Growth Inhibition for Hyperactive and Globular Antifreeze Proteins: Insights from Molecular Dynamics Simulation.

The molecular mechanism behind the ice growth inhibition by antifreeze proteins (AFPs) is yet to be understood completely. Also, what physical parameters differentiate between the AFP and non-AFP are largely unknown. Thus, to get an atomistic overview of the differential antifreeze activities of different classes of AFPs, we have studied ice growth from different ice surfaces in the presence of a moderately active globular type III AFP and a hyperactive spruce budworm (sbw) AFP. Results are compared with the observations of ice growth simulations in the presence of topologically similar non-AFPs using all-atom molecular dynamics simulations. Simulation data suggest that the ice surface coverage is a critical factor in ice growth inhibition. Due to the presence of an ice binding surface (IBS), AFPs form a high affinity complex with ice, accompanied by a transition of hydration water around the IBS from clathrate-like to ice-like. Several residues around the periphery of the IBS anchor the AFP to the curved ice surface mediated by multiple strong hydrogen bonds, stabilizing the complex immensely. In the high surface coverage regime, the slow unbinding kinetics dominates over the ice growth kinetics and thus facilitates the ice growth inhibition. Due to the non-availability of a proper IBS, non-AFPs form a low-affinity complex with the growing ice surface. As a result, the non-AFPs are continuously repelled by the surface. If the concentration of AFPs is low, then the effective surface coverage is reduced significantly. In this low surface coverage regime, AFPs can also behave like impurities and are engulfed by the growing ice crystal.

Janus regulation of ice growth by hyperbranched polyglycerols generating dynamic hydrogen bonding

In this study, a new phenomenon describing the Janus effect on ice growth by hyperbranched polyglycerols, which can align the surrounding water molecules, has been identified. Even with an identical polyglycerol, we not only induced to inhibit ice growth and recrystallization, but also to promote the growth rate of ice that is more than twice that of pure water. By investigating the polymer architecture and population, we found that the stark difference in the generation of quasi-structured H2O molecules at the ice/water interface played a crucial role in the outcome of these opposite effects. Inhibition activity was induced when polymers at nearly fixed loci formed steady hydrogen bonding with the ice surface. However, the formation-and-dissociation dynamics of the interfacial hydrogen bonds, originating from and maintained by migrating polymers, resulted in an enhanced quasi-liquid layer that facilitated ice growth. Such ice growth activity is a unique property unseen in natural antifreeze proteins or their mimetic materials.

Atomic force microscopy imaging of ice crystal surfaces formed in aqueous solutions containing ice-binding proteins

Ice-binding proteins (IBPs) allow organisms to survive below the freezing point by modulating ice crystal growth. These proteins act by binding to ice surfaces, thus inhibiting ice growth. Until now, high-resolution imaging of ice growing in the presence of IBPs has not been possible. We developed a unique in-situ technique that enables atomic force microscopy (AFM) imaging of ice formation and growth in the ice-IBP system. The new technique enables controlling the growth of ice crystals under a strong and focused thermal gradient. We present images of ice crystals with sub-ten nanometer resolution. Ice was grown in the presence of two different IBPs that exhibit specific and unique structures. This development opens the path for fine elucidation of the interaction of IBPs with growing ice surfaces as well as with other frozen systems at unprecedented high resolution. Furthermore, with the exception of crystals growing in thin films, this is the first demonstration for imaging a growing crystal immersed in its own melt with AFM.

Cell‐friendly regulation of ice crystals by antifreeze organism‐inspired materials

AbstractThere is a dilemma between ice growth inhibition and cell‐threatening sharp‐edged ice formation, dominating an unfavorable efficiency of cell cryoprotection using ice control materials (ICMs). Herein, we design different‐type antifreeze organism‐inspired ICMs and show the cell‐friendly regulation of ice crystals by a synergistic effect for cell cryoprotection. Preferred rapid adsorption of “high‐diffusivity ICM” on the curved ice crystal surface induced by “ice‐binding ICM,” owing to the Gibbs–Curie–Wulff theorem, promotes smooth‐shaped and small ice crystal formation during the freeze–thaw cycle. Meanwhile, their intracellular and extracellular osmotic balance well protects the cryopreserved cells. All‐round protection benefits cell survival to achieve a cryopreservation efficiency of 97%. After cryopreservation, ice‐binding ICM exhibits double thermosensitive behaviors to directly encapsulate post‐thaw cells by gels, to further support cell‐based therapies. This work provides an in‐depth understanding of cell cryoprotection, and paves the road to the development of even better ICMs for biospecimen cryopreservation.