Mimicking the Ice Recrystallization Activity of Biological Antifreezes. When is a New Polymer “Active”?

Abstract

Antifreeze proteins and ice‐binding proteins have been discovered in a diverse range of extremophiles and have the ability to modulate the growth and formation of ice crystals. Considering the importance of cryoscience across transport, biomedicine, and climate science, there is significant interest in developing synthetic macromolecular mimics of antifreeze proteins, in particular to reproduce their property of ice recrystallization inhibition (IRI). This activity is a continuum rather than an “on/off” property and there may be multiple molecular mechanisms which give rise to differences in this observable property; the limiting concentrations for ice growth vary by more than a thousand between an antifreeze glycoprotein and poly(vinyl alcohol), for example. The aim of this article is to provide a concise comparison of a range of natural and synthetic materials that are known to have IRI, thus providing a guide to see if a new synthetic mimic is active or not, including emerging materials which are comparatively weak compared to antifreeze proteins, but may have technological importance. The link between activity and the mechanisms involving either ice binding or amphiphilicity is discussed and known materials assigned into classes based on this.

Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants

“… 32 This polymer is only weakly IRI active, with its mechanism of action suggested to be due to membrane stabilization. 32 Poly(ethylene glycol) (PEG) (8 and 22 kDa) was chosen as it is a negative control in ice growth assays 19 , 20 , 44 and cryoprotective assays, but in our initial work (explored below) it was capable of showing false positive results.…”

“… 13 , 14 These organisms produce antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP), 15 , 16 which demonstrate potent ice recrystallization inhibition (IRI) activity, a key cause of cell death during thawing in vitro. 17 , 18 Biomaterials that mimic the IRI properties of AFPs, 19 , 20 such as poly(vinyl alcohol) (PVA), have been shown to improve post-thaw cell recoveries. 21 − 23 Other IRI active examples include polyproline, 24 , 25 small molecules, 26 and graphene oxide.…”

X-ray diffraction to probe the kinetics of ice recrystallization inhibition

“…[Note, it is crucial to highlight the conditions for these experiments to avoid false positives. 15,49 ] Fig. 1A is an example micrograph from a 'splat' assay.…”

“…Fig. 4E, shows a comparison of AFGP8 (antifreeze glycoprotein from Dissostichus mawsoni, 2.6 kDa) which is one of the most potent IRI's known, 15 and lysozyme as a negative protein control. Again, the MGLS and XRD methods show broad agreement, demonstrating that this is a valid analysis method for IRI activity and is complementary to the traditional optical based methods.…”

“…[12][13][14] However, questions remain if these function by the same mechanisms. 12,15 These include C-linked glycopeptides, [16][17][18] a range of small molecules, 3,19,20 polyampholytes, 21,22 oxidized quasi-carbon nitride quantum dots (OQCNs), 23 safranine-O, 24 graphene oxide, 25 zirconium acetate (ZrAc), 26 nanocelluloses, 27 and poly (vinyl alcohol) (PVA); which is one of the most active mimics reported to date. [28][29][30][31] The development and use of appropriate assays of activity is crucial to develop this field and understand the mode of action.…”