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

Abstract

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.

Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives

“…This study also emphasized the significance of taking into account the molarity of hydrogen bond donating sites on the polymeric chain properly aligned with the ice planes, than the molarity of the polymeric solution, while establishing potency of the macromolecule relative to AF(G)Ps. 55 In addition, the ability of surface-bound polymers to retain IRI activity was demonstrated by Stubbs et al using PVA-coated gold nanoparticles. Low grafting densities of PVA on the nanoparticle surface favored the interaction with the ice surface, as compared to the sterically hindered star-branched conformation.…”

“…However, these effects of PVA were observed only in the case of heterogeneous nucleation. 55 Anti-freeze proteins (AFPs) control ice formation by adsorbing to the surface of ice crystals. Hydrogen bonding and hydrophobic effects possibly contribute to the interaction between AFPs and ice domains.…”

“…Glycerol-grafted PVA (G-g-PVA), on the other hand, showed higher ice nucleation inhibition (INI), IRI and THA activities, as compared to the parent PVA, and also demonstrated potency similar to that of AF(G)Ps. 55,75 Even trehalose-based synthetic polyether demonstrated IRI activity. 29 In addition, the synthetic glycopeptide (Tre) molecule demonstrated IRI activity of B45-50% (1 mg mL À1 , pH 7.4), as determined from the mean largest grain size (MLGS), owing to the presence of trehalose.…”

Poly(l-Ala-co-l-Lys) Exhibits Excellent Ice Recrystallization Inhibition Activity

“…Ice recrystallization inhibition (IRI) activity is usually assayed according to mean largest grain size (MLGS) of ice crystals formed in phosphate buffered saline (PBS) in the presence of candidate polymers. 4,5 In most cases, the results of MLGS in PBS in the absence of any polymers are used as a negative control. A wide range of synthetic polymers have been developed to control ice crystal formation, including biomimetic polypeptides from polar fish that contain special antifreezing proteins (AFPs) to protect their body from low temperatures, facially amphiphilic polymers, polyampholytes, poly(vinyl alcohol) derivatives, and block copolymers.…”

“…In particular, the material-based approach is promising in that the addition of small amounts of biocompatible compounds or polymers might solve the problem without the need to change the established procedures of cryopreservation. Ice recrystallization inhibition (IRI) activity is usually assayed according to mean largest grain size (MLGS) of ice crystals formed in phosphate buffered saline (PBS) in the presence of candidate polymers. , In most cases, the results of MLGS in PBS in the absence of any polymers are used as a negative control. A wide range of synthetic polymers have been developed to control ice crystal formation, including biomimetic polypeptides from polar fish that contain special antifreezing proteins (AFPs) to protect their body from low temperatures, facially amphiphilic polymers, polyampholytes, poly­(vinyl alcohol) derivatives, and block copolymers. , AFPs exhibit an excellent IRI activity of 10% MLGS of PBS at 0.014 mg/mL, and various AFP-mimicking analogs were synthesized for the preparation of IRI active compounds. − Facially amphiphilic glycopolymers consisting of hydrophobic rigid dimethyl fulvene with hydrophilic galactose and polyampholytes consisting of cationic and anionic characters in a molecule such as poly­(2-(dimethyl amino)­ethyl methacrylate- co -methacrylic acid) were reported. ,− These molecules interfere with the growth of ice crystals by affecting the interface between the water and ice.…”