Bioinspired Crowding Inhibits Explosive Ice Growth in Antifreeze Protein Solutions

Antifreeze (glyco)proteins (AF(G)Ps) are naturally evolved ice inhibitors incomparable to any man-made materials, thus, they are gaining intensive interest for cryopreservation and beyond. AF(G)Ps depress the freezing temperature (T_f) noncolligatively below the melting temperature (T_m), generating a thermal hysteresis (TH) gap, within which the ice growth is arrested. However, the ice crystals have been reported to undergo a retaliatory and explosive growth beyond the TH gap, which is lethal to living organisms. Although intensive research has been carried to inhibit such an explosive ice growth, no satisfactory strategy has been discovered until now. Here, we report that crowded solutions mimicking an extracellular matrix (ECM), in which AF(G)Ps are located, can completely inhibit the explosive ice growth. The crowded solutions are the condensates of liquid-liquid phase separation consisting of polyethylene glycol (PEG) and sodium citrate (SC), which possess a nanoscale network and strong hydrogen bond (HB) forming ability, completely different to crowded solutions made of single components, that is, PEG or SC. Due to these unique features, the dynamics of the water is significantly slowed down, and the energy needed for breaking the HB between water molecules is distinctly increased; consequently, ice growth is inhibited as the rate of water molecules joining the ice is substantially reduced. The present work not only opens a new avenue for cryopreservation, but also suggests that the ECM of cold-hardy organisms, which also exhibit great water confining properties, may have a positive effect in protecting the living organisms from freezing damage.