Ice-binding proteins-mimicking nanocryoprotectants for ice-water interface control

Abstract

The key to cryopreservation techniques to store and keep cells in their original state is to minimize damage from ice crystal growth inside and outside the cell. Recently, with the increasing demand for personalized healthcare, there has been a growing interest in cryopreservation of stem cells, germ cells, cord blood, cellular therapeutics, as well as organs or whole organisms, so that they can be recovered without damage. Existing chemical cryoprotectants such as dimethyl sulfoxide, sodium phosphate, and glycerol have the advantage of being mass-producible and cost-effective. However, at high concentrations, they exhibit cytotoxicity, causing damage to cell membranes and inducing genetic modifications during the freezing and thawing. There has been growing interest in exploring ice-binding proteins (IBPs) that are found in organisms such as winter flounders, ocean pouts, yellow mealworm beetles, or spruce budworm moths inhabiting polar regions, as potential alternatives. Nonetheless, the extraction process of IBPs from their natural sources is challenging and can elicit immune responses, limiting their practical use as long-term cryopreservatives. These limitations require the development of cryoprotectants with low toxicity and excellent biocompatibility while being easy to synthesize and cost-effective. IBPs have sites that can bind irreversibly to ice crystals, and the ice-interactable functional groups are spaced at regular intervals on specific crystal planes of ice to form strong chemical bonds, which inhibit the recrystallization of ice. The binding of IBPs to ice can control the interfacial energy at the ice-water interface, lowering the water-to-ice phase transition temperature. Therefore, new cryoprotectants can be developed by mimicking the structure of IBPs with excellent ice growth inhibition. In this study, I synthesized metal-organic framework (MOF) nanoparticles (NPs) that are easy to be modified with functional groups derived from IBPs, have a regular arrangement of ice-interactable functional groups similar to the ice crystal lattice, are relatively easy to mass produce, and are biocompatible. The thermodynamic behavior of the water-ice interface as a function of NPs size and functional group type and their performance as cryoprotectants for different cell types were investigated. The biocompatible zirconium-based MOF NP size was controlled by varying the precursor concentration, and the introduction of acrylate functional groups on the NP surface facilitated the introduction of any peptide functional groups through the Aza-Michael addition reaction. The MOF NPs without functional groups showed the best ice recrystallization inhibition (IRI) with the smallest NPs due to the control of interfacial energy by curvature at the ice-water interface, and the IRI was further improved after the introduction of peptide functional groups with both hydrophilic and hydrophobic groups. The curvature of the ice-water interface increased during freezing/thawing, which lowered the phase transition temperature. In particular, the developed MOF NPs did not show toxicity to kidney cells, cancer cells, and stem cells, and when a very small amout (1 in 2200) of the existing artificial compound cryopreservative was used, they showed corresponding post-cryopreservation cell recovery (70% on average) and cell proliferation efficacy (4-fold in 48 hours). Second, inspired by the fact that IBPs in nature have regular secondary structures and show excellent IRI effects depending on the functional groups, peptide molecules with short amino acid sequences were synthesized and they could self-assemble to form nanofibrils. The developed nanofibrils with alpha-helix or beta-sheet secondary structures exhibited excellent IRI effects depending on the functional groups. When applied to stem cells, they showed great potential for utilization as cryopreservatives with biocompatibility. In addition, by directly introducing gold NPs into self-assembled peptide nanostructures, the overestimation of the existing colorimetric assay by ligands attached to gold NPs could be solved, leading to the development of an optical screening used for facile and more accurate visualization of the cryopreservation performance of peptides with different sequences. The results confirmed that nanocryoprotectants developed by mimicking the structural characteristics of IBPs in nature could show excellent cryopreservation effects by affecting the ice-water interfacial energy during freezing/thawing. The development of cost-effective, mass-producible, and biocompatible cryoprotectants will emerge as a critical technology for the cryopreservation of cells, organ transplantation, stem cell storage, and advancements in the fields of vaccines and pharmaceuticals. Furthermore, these cryoprotectants hold immense potential for preserving physiological activities in extreme environments.