Quantitative Analysis of the Efficacy and Potency of Novel Small Molecule Ice Recrystallization Inhibitors

Abstract

The effects of ice recrystallization are well-recognized throughout the literature. This phenomenon is the major cause for cellular damage during thawing of cells, ultimately reducing post-thaw viability and function. In this paper, we describe a method for quantifying the inhibitory effect on ice recrystallization of novel small molecules that are cryoprotectants for red blood cells. The method is ideally suited to the splat-cooling assay, where high-ice volume fractions are present. Using our method, we have derived first-order rate constants for the increase in the average crystal size based upon a "binning" approach of ice crystals as a function of size and time. Using this reliable metric, dose−response curves were constructed to obtain IC 50 values. Two very effective inhibitors of ice recrystallization, p-methoxyphenyl β-D-glucopyranoside (PMP-Glc) and pbromophenyl β-D-glucopyranoside (pBrPh-Glc), had IC 50 values of 16.3 and 14.8 mM, respectively. Interestingly, the Hill slopes from these dose−response curves were 5.12 ± 0.81 for PMP-Glc and 3.12 ± 0.62 for pBrPh-Glc, suggesting that an element of cooperativity may be involved in the mechanism by which these compounds inhibit ice recrystallization. This is particularly interesting, as unlike antifreeze (glyco)proteins, these small molecules do not bind to the ice surface. ■ INTRODUCTIONRecrystallization is an extensively studied phenomenon, and its importance in the field of geology and metallurgy has been thoroughly documented. 1,2 The phenomenon is defined as the nucleation and growth of large nondeformed grains in the presence of smaller ones. In deformed grains, the atom arrangement is not ideal, increasing the overall energy of the system. Thus, reorganization to nondeformed grains and an overall decrease in the energy of the system occur. 3−5 As such, recrystallization is a thermodynamically driven kinetic process resulting in an overall reduction in the free energy of the system.In ice, this growth of larger ice crystals at the expense of smaller ones is thought to occur through either grain boundary migration or Ostwald ripening. 6−12 Grain boundary migration occurs through the direct transfer of individual water molecules from smaller unstable ice grains or crystals to larger more favorable ones, resulting in an overall decrease in curvature in an ice crystal and a subsequent reduction in energy. However, grain boundary migration neglects the presence of bulk water or the quasi-liquid layer (QLL) between individual ice crystals. 8 In the Ostwald ripening model, water molecules transfer from the surface of small ice crystals through bulk water and the QLL to larger ice crystals. Smaller ice crystals have a higher surface area to volume ratio, which gives them a higher surface free energy; thus, the Ostwald ripening process results in an overall increase in average ice crystal size and also a reduction in free energy. 10−12 Ice recrystallization has been identified as a problem in the frozen food industry as well as during cryopreservation of bio...

From ice-binding proteins to bio-inspired antifreeze materials

“…Similarly, these are used to measure the effectiveness of IBP analogues. Quantitative assays and image analysis routines have been developed to accurately measure thermal hysteresis (TH) activity, 63 – 66 ice recrystallization inhibition (IRI) activity, 56 , 67 , 68 dynamic ice-shaping (DIS), 69 and ice nucleation (IN) activity. 70 Furthermore, ice-binding can be detected directly, albeit in a qualitative fashion, by ice-etching 33 and fluorescently based ice-plane affinity (FIPA) 28 if IBPs are tagged with fluorescent proteins or dyes.…”

“…Ice recrystallization inhibition (IRI) activity is determined by polarized optical microscopy (POM) in a so-called ‘splat-assay’ 89 or ‘sandwich assay’, 67 which probe the rate and extent of ice recrystallization in thin wafers of ice. Splat assays are typically performed in the presence of >2 mM NaCl 90 or 1–100 mM phosphate-buffered saline (PBS) buffer 68 and sandwich assays in the presence of 18–45% sucrose. 56 , 91 In a splat assay quantification of efficacy is based on measurements of the (time-evolution of the) mean (largest) grain size (M(L)GS).…”

Interaction of ice binding proteins with ice, water and ions

“…9,84 To quantitatively compare the IRI activity of ice-binding compounds, automated image analysis routines have been developed to monitor the time-evolution of the mean ice grain size, r, and mean largest grain size. 80,86,87 The Koop laboratory demonstrated that r 3 increases linearly with time in 45 wt. % sucrose solutions as predicted by Lifshitz-SlyosovWagner (LSW) theory [ Fig.…”

“…71 Monte Carlo simulations on entire 3D ice crystals in the presence of TmAFP and wfAFP-I are also in line with this theory of antifreeze activity since ordered step growth appears inhibited. 91 The prevailing adsorption-inhibition model has been extended and revisited by various groups to account for nonstepwise growth on rough prism planes 86,103,104 and dynamic exchange between adsorbed and free AFPs in a two-step mechanism. 105 Knight et al pointed out that IBPs block normal growth preventing layer growth instead of lateral growth impeding step passage (Fig.…”

“…11), since all ice crystal planes except the basal plane are molecularly rough. 86,103,104 An insightful study by Kristiansen and Zachariassen builds upon the original adsorption-inhibition model, but argues that it is a pressure build-up due to convex ice growth that underlies the Gibbs-Thomson effect. 71 Alternative models consider the anisotropic surface energy of ice, 106 reversible adsorption, [107][108][109][110][111] the polymeric nature of IBPs, 112,113 and lowering of the interfacial tension or step energy.…”