Understanding Poly(vinyl alcohol)-Mediated Ice

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Understanding Poly(vinyl alcohol)-Mediated Ice Recrystallization Inhibition through Ice Adsorption Measurement and pH Effects Aaron Burkey, Christopher L Riley, Lyndsey K Wang, Taylor A Hatridge, and Nathaniel A. Lynd Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01502 • Publication Date (Web): 29 Nov 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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Understanding Poly(vinyl alcohol)-Mediated Ice Recrystallization Inhibition through Ice Adsorption Measurement and pH Effects Aaron A. Burkey,1 Christopher L. Riley,2 Lyndsey K. Wang,2 Taylor A. Hatridge,1 Nathaniel A. Lynd1,* 1

McKetta Department of Chemical Engineering, 2Department of Molecular Biosciences, The

University of Texas at Austin, Austin, TX, 78712, USA

ABSTRACT. The development of improved cryopreservative materials is necessary to enable complete recovery of living cells and tissue after frozen storage. Remarkably, poly(vinyl alcohol) (PVA) displays some of the same cryoprotective properties as many antifreeze proteins found in cold tolerant organisms. In particular, PVA is very effective at halting the Ostwald ripening of ice, a process that mechanically damages cells and tissue. Despite the large practical importance of such a property, the mechanism by which PVA interacts with ice is poorly understood, hindering the development of improved cryoprotective materials. Herein, we quantitatively evaluated ice growth kinetics in the presence of PVA at different pH conditions and in the presence of a range of neutral salts. We demonstrated that pH, but not salt identity, alters the ability of PVA to halt ice grain coarsening. These observations are consistent with hydrogenbonding playing a crucial role in PVA-mediated ice recrystallization inhibition. The evolution of

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the size distribution of ice crystals with annealing was consistent with incomplete surface coverage of ice with PVA. Binding assay measurements of dissolved fluorescently-labelled PVA in an ice slurry showed that PVA interacts with ice through weak adsorption (99%), potassium (Aldrich, 98%), trifluoroacetic acid (Millipore), allyl glycidyl ether (TCI) poly(ethylene oxide) (Aldrich, 35000 g/mol), fluorescein isothiocyanate Isomer I (Acros, 90%), and [(R,R)-N,N-Bis(3,5-di-tert-butylsalicylidene)-1,2-


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cyclohexanediaminato(2-)]cobalt(II) ((R,R)-1) (Aldrich) were used as received. Vinyl acetate (Acros, >99%) was filtered through a plug of basic alumina prior to use. Equipment. An Agilent size exclusion chromatograph (SEC) with a 1260 Infinity isocratic pump, degasser, and thermostated column chamber held at 30 °C containing Agilent PLgel 10 µm MIXED-B and 5 µm MIXED-C columns with a combined operating range of 200−10,000,000 g/mol relative to polystyrene standards was used to fractionate poly(vinyl acetate) samples for molecular weight measurement. Tetrahydrofuran and dimethylformamide were used as mobile phases at 0.5 mL/min. Three detectors in series from Wyatt Technologies provided measurement of polymer concentration, molecular weight, and viscosity. Static light scattering was measured using a DAWN HELEOS II Peltier system, differential refractive index measured with an Optilab TrEX, and differential viscosity measured using a Viscostar II. 1H NMR spectroscopy was performed on a 400 MHz Agilent MR spectrometer at room temperature and referenced to the residual solvent signal of CDCl3 or D2O (7.26 and 4.79 ppm, respectively). Ice recrystallization kinetic assays were performed using a Zeiss Axio Scope.A1 with a 10x NAchroplan objective and a Linkam LTS420 temperature controlled stage. FITC-labeled polymer concentrations were determined using a NanoDrop 2000c with polystyrene cuvettes. Poly(vinyl alcohol) synthesis. AIBN (176 mg, 0.002 eq) was dissolved in ethyl acetate (200 mL) in a round bottom flask. Vinyl acetate (50 mL, 1 eq) was filtered through a plug of basic alumina then added to the reaction flask. The mixture was bubbled with nitrogen for 20 minutes, brought to 60 °C, and allowed to react overnight. The resulting poly(vinyl acetate) (PVAc) was isolated by precipitating in hexanes and drying in vacuo. PVAc was separated into several monodisperse fractions by dissolving in acetone and fractionally precipitating with hexanes.20 Each fraction was hydrolyzed in methanol with sodium hydroxide. The resulting poly(vinyl


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alcohol) was isolated by vacuum filtration, washed with methanol, and dried in vacuo. All IRI assays were performed using a fraction with Mn = 26 kg/mol, PDI = 1.12. Unfractionated polydisperse PVA (Mn = 49 kg/mol, PDI = 1.26) was used for FITC-labeling. Molecular weights were determined using reacetylated PVA because fully hydrolyzed PVA is difficult to characterize with light scattering. Reacetylation of poly(vinyl alcohol). Poly(vinyl alcohol) was reacetylated using a previously reported procedure.21 100 mg of PVA were added to a mixture of 1 mL of acetic anhydride and 1 mL of pyridine. The mixture was stirred at 95 °C until fully dissolved. The resulting poly(vinyl acetate) was purified by precipitating in cold water and drying in vacuo. Hydrolytic kinetic resolution of allyl glycidyl ether. Enantiopure allyl glycidyl ether was prepared using a method adapted from a previously reported procedure.22 Briefly, 180 mL of allyl

glycidyl

ether,

4.62

g

of

[(R,R)-N,N-Bis(3,5-di-tert-butylsalicylidene)-1,2-

cyclohexanediaminato(2-)]cobalt(II) ((R,R)-1), and 1.75 mL of acetic acid were combined in a round bottom flask. The flask was placed in an ice bath, then 15 mL of water were added. The reaction was allowed to react at room temperature for 19 hours. Enantiopure allyl glycidyl ether was recovered by vacuum distillation. Linear polyglycerol synthesis. Allyl glycidyl ether was isomerized according to the procedure of Crivello et al.23 The resulting propenyl glycidyl ether was degassed by three freeze-pumpthaw cycles, then distilled over calcium hydride prior to polymerization. Polymerization of propenyl glycidyl ether was initiated with benzyl alcohol and potassium napthalenide as described previously.24 The resulting polymer was precipitated in hexanes and dried in vacuo. Poly(propenyl glycidyl ether) was hydrolyzed to polyglycerol by suspending 2 g of polymer in a mixture of 10 mL methanol, 1 mL water, and 0.2 mL trifluoroacetic acid. The polyglycerol was


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precipitated in THF and dried in vacuo. Isotactic polyglycerol was prepared by using enantiopure allyl glycidyl ether as a starting material. Splat ice recrystallization assay. A “splat” ice recrystallization assay was adapted from that of Knight et al.25 A 20 µL droplet was expelled from a height of 1.5 m onto a microscope slide chilled to –78 °C with dry ice, then quickly transferred to a microscope stage held at –6 °C. Images were collected after annealing for 1 hr to allow ample time for ice recrystallization to occur. Ice samples were illuminated with cross-polarized light. Glycerol ice recrystallization assay. PVA was dissolved in various 0.1 M salt solutions, then glycerol was added such that the final concentration was 17 wt% glycerol. A 2 µL droplet of each solution was placed on a glass slide and a coverslip was placed on top. On a temperaturecontrolled microscope stage, the sample was quickly dropped to –50 °C, then raised to –6 °C at a rate of 20 °C/min and annealed for 1 hr. Images were collected every 2 min and ice crystal sizes were determined using the Zeiss Zen software particle analysis tool. Ice growth rate constants were determined by calculating the slope of the cubic average radius versus time between t = 30 min and t = 60 min with a linear regression. Fluorescent-labeling of PVA and polyglycerol. Unfractionated PVA was labeled with fluorescein isothiocyanate (FITC) Isomer I using the procedure of Kaneo et al.26 After labeling, PVA was dialyzed for one week against deionized water and lyophilized. Polyglycerol was labeled according to an identical procedure. Type-III AFP Purification. The plasmid containing GFP tagged type-III AFP was a kind gift from Dr. Peter Davies. Plasmids were transformed in BL21 cells and a seed culture was grown overnight. A larger culture (3 L) was inoculated and grown at 37°C until an optical density of 0.6 was reached. Cultures were cooled to room temperature and induced with 1mM IPTG overnight


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at 18°C. Cultures were centrifuged at 4000 x g and lysed via sonication in a buffer containing 40mM HEPES, 400mM NaCl, and 10% glycerol at a pH of 7.5. The supernatant was then flowed through a His60 Ni SuperflowTM Resin column (Clonetech) and was washed with buffer containing 10mM imidazole. Protein was eluted using 100mM imidazole and the concentration of GFP-tagged protein was quantified using a NanoDrop1000 spectrophotometer, absorbance at 488nm with an extinction coefficient of 61,000 M-1cm-1. Purified protein was diluted 25 fold prior to use in ice binding experiments. Determination of fraction of adsorbed polymer in an ice slurry. 10g aqueous solutions of FITC-labeled PVA, FITC-labeled polyglycerol, and GFP-labeled type-III AFP were prepared. 2g of glycerol were added to each solution in order to allow for a controllable solid/liquid ratio in the ice slurry. Each solution was cooled to –6 °C with a cooling bath, then ice growth was seeded with a chilled pipet tip. The slurry was magnetically stirred for 1 hr, then a portion of the liquid fraction was removed by syringe from the bottom of the slurry so as not to withdraw any ice crystals. The polymer concentration in the liquid aliquot was measured by diluting with water such that the glycerol concentration was less than 1 wt% and measuring extinction at 495nm with a UV-vis spectrometer. Type-III AFP concentration was quantified by the band intensity in gel electrophoresis. The fraction of adsorbed polymer was determined by comparing the concentration in the liquid aliquot to that of the initial solution. Glycerol concentration measurements in the liquid aliquot showed that around 20% of the water in the slurry was frozen. The glycerol concentration in the liquid was measured by comparing the glycerol peak area in a 1

H NMR spectrum to that of a known mass of DMSO. Spectra were collected in D2O with a

relaxation delay of 10 sec and pulse angle of 90°. Several solutions of known glycerol concentration were measured to confirm the accuracy of the measurement technique.


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Modeling of ice crystal volume dispersity. First we assumed that the initial degree of PVA adsorption was equal to the equilibrium adsorption and that the equilibrium ice surface coverage remained unchanged. Next, we assumed that no new PVA would adsorb to a growing crystal, and that the degree of PVA adsorption would not exceed the equilibrium adsorption for a melting crystal:

(1)

θi(t) and θeq represent the fraction of surface area covered by PVA for each ice crystal, i, at time, t, and at equilibrium binding, respectively. Ai(t) and Ai0, and Vi(t) and Vi0, represent the ice crystal surface area at time, t, and at t=0 respectively. Next we assumed that the rate of ice size change would be the same as that predicted for diffusion-driven coarsening multiplied by the fraction of the ice surface uncovered by PVA. !

" #

$

(2)

The critical ice volume, Vcrit, is unknown so we must introduce another constraint in order to reach a solution. In this case, we assumed that the total volume of ice is constant: ∑

&

(3)

Here Vi is the volume of ice crystal, i, and K is a diffusive coarsening rate constant. Equations 2 and 3 were solved numerically for a collection of several hundred ice crystals using an ice crystal distribution from experimental data as initial conditions. The MATLAB code used for numerical analysis was included in the Supporting Information.

RESULTS AND DISCUSSION


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A splat assay is frequently used to assess IRI activity of a polymer or protein. Typically, splat assays are performed using phosphate-buffered saline (PBS) solution to mimic biological salinity. Here we used solutions of 0.1 M NaCl, NaOH, and HCl instead of PBS to demonstrate the strong influence of pH on PVA-induced IRI. Images shown in Figure 1 were collected after annealing samples at –6 °C for one hour. PVA was much more effective at blocking ice growth at high pH.

Figure 1. Splat assay shows PVA inhibited ice recrystallization more strongly at high pH. Scalebar is 200 µm. Samples were annealed for 1 hour at –6 °C. A key disadvantage of the splat assay is that closely packed ice crystals are difficult to analyze using automated image analysis software, and ice size quantification is often limited to measuring the few largest crystals within the field of view. Several authors have circumvented this problem through the addition of high concentrations of sucrose, which gives well-separated ice crystals whose growth kinetics can be analyzed using classical Ostwald ripening kinetic theory.27–30 In the present study, 17 wt% glycerol was used in place of sucrose because glycerol is a common cryoprotectant and more stable at acidic pH than sucrose. To quantify ice growth kinetics, partially frozen samples were annealed at –6 °C, and ice growth rate constants were


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calculated from the slope of a plot of cubic mean radius vs. time. Figure 2 shows the ice growth rate at a range of PVA concentrations in the presence of glycerol and different pH salts. A critical PVA concentration (c*), or the concentration at which PVA starts being IRI active, was defined as the inflection point of a sigmoidal fit of ice growth rate vs. concentration. Significantly, the critical PVA concentration decreases as pH increases even though pH by itself has little effect on ice growth kinetics. Qualitatively, the results of the glycerol assay match the results of the splat assay well. The critical PVA concentration observed in the presence of 45 wt% sucrose was ca. 10× lower than that observed in the presence of 17 wt% glycerol at neutral pH, likely because carbohydrates have some IRI activity on their own18,28,31,32 or because sucrose solutions are more viscous. Experimental error was somewhat large near the critical concentration, likely caused in part by large dispersity in crystal size at intermediate PVA concentrations. This trend is discussed more deeply below. Inconsistency in ice growth rate was especially large in acidic conditions, but the reason for this is unclear.


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Figure 2. Glycerol assay shows that the critical PVA concentration (c*) needed to restrict ice recrystallization decreased as pH was increased. Ice Growth Rate = d'r()/dt between t = 30 and 60 min. Error bars represent standard deviation between three trials. Increasing pH also imparted IRI activity to materials that have not been previously reported to be IRI-active. Significantly, poly(ethylene oxide) (PEO) and linear polyglycerol (PG) were both


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observed to have some IRI activity at high concentration and high pH. Ice growth kinetics in the presence of 50 mg/mL polymer at two different pH conditions are plotted in Figure 3. Linear polyglycerol, a polyol structurally analogous to PVA, was considered as a candidate material for IRI activity. Surprisingly, polyglycerol had weaker IRI activity than PEO, which is frequently used as a negative control in ice growth assays. Both atactic and isotactic linear polyglycerols were synthesized and evaluated to assess if stereoregularity would promote an epitaxial match with an ice crystal face. Polyglycerol tacticity had no significant effect on IRI, which was consistent with the large configurational disorder of a polymer chain in solution that is likely to overshadow any structural order imparted by stereoregularity. As with PVA, each polymer restricted ice growth more strongly under basic conditions. High polymer concentrations and high pH conditions are unsuitable for the purpose of cryopreserving living cells, but ice growth assays under these conditions are useful for inducing IRI activity in otherwise inactive polymers. The reason that high pH improves polymer IRI remains unclear. A key possibility is that pH affects the nature of the solution hydrogen bonding environment.33 Additionally, pH may change the surface characteristics of the ice.34


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Figure 3. PEO and PG also display stronger IRI at basic pH. Polymer concentration is 50 mg/mL. Samples were annealed at –8 °C. Error bars represent standard deviation among three trials.


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Anion and cation identity had a much weaker influence on IRI than pH, as shown in Figure 4. Measurements of ice growth kinetics in the presence of neutral salts NaCl, LiCl, and NaSCN show nearly the same critical PVA concentration in all cases. Insensitivity of IRI to ion charge density also suggests that PVA solubility does not contribute to IRI, as charge-diffuse NaSCN has been reported to enhance PVA solubility in water.35

Figure 4. Glycerol assay shows that other neutral pH salts influence PVA’s IRI activity similarly to NaCl. Salt identity has negligible influence on IRI relative to pH. Error bars represent standard deviation between 3 trials. One of the advantages of analyzing ice crystal growth using a glycerol or sucrose IRI assay is that mechanistic details of polymer/ice interactions can be suggested by the way the ice crystal


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size distribution evolves. Budke et al. observed that Ostwald ripening of well-separated ice crystals in the absence of polymer can be modeled well using Lifshitz-Slyozov-Wagner (LSW) theory.27 In the following discussion, we show that LSW behavior was not maintained near the critical PVA concentration, and this departure from LSW behavior is consistent with adsorbed PVA on facets of ice being responsible for IRI activity.

Figure 5. Polydispersity of ice crystal volumes increases dramatically near the critical PVA concentration (0.05 mg/mL). PDI = 1 + [(avg. volume)/(st.dev.)]2. Displayed microscope images were collected after annealing at –6 °C for 1 hr. Scalebar is 100 µm. Inset shows average PDI at t = 1 hr. Data shown here are collected in presence of 0.1 M NaOH. LSW theory predicts self-similarity of the crystal size distribution, i.e. crystal size dispersity is predicted to remain constant even as average crystal size increases.36 Figure 5 shows how ice crystal dispersity changed over time when ice crystals were annealed at –6 °C in the presence of 0.1 M NaOH and 17 wt% glycerol. Crystal dispersity data for other pH conditions are included


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in the Supporting Information. The assumption of self-similar crystal size distribution fails when the PVA concentration is near the critical concentration (0.05 mg/mL in this case). In order for such a large dispersity to arise, there must be a mechanism through which some crystals can grow very large while many crystals remain small. This behavior was more consistent with a situation in which adsorbed PVA inhibits ice recrystallization than one in which dissolved PVA inhibits recrystallization. To elaborate, if PVA is adsorbed to ice, then a growing ice crystal will have a thinning layer of adsorbed PVA as its surface area increases, whereas a shrinking crystal will have a thickening layer. As a result, there would be less inhibition to ice growth than melting, as illustrated in Figure 6. Conversely, if dissolved PVA near the ice interface were responsible for IRI, then a growing crystal would face greater inhibition to size change than a shrinking one because the local solution concentration of PVA would increase as nearby water is converted to ice.

Figure 6. Proposed mechanism for PDI increase at intermediate PVA concentrations. Adsorption of new polymer chains must be slower than the rate of crystal growth for this mechanism to occur. We speculate that PVA’s steric bulk slows adsorption of new chains. Based on this hypothesis, we developed a simple model to simulate how ice crystal volume dispersity would change over time at various equilibrium PVA adsorption values, the results of


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which are depicted in Figure 7. The key assumption of this model was that a growing ice crystal will have no new PVA adsorb to it, i.e. ice growth is much faster than PVA adsorption. For a shrinking crystal, we assumed that surface coverage will not exceed a predefined equilibrium level. Lastly, we assumed that the rate at which an ice crystal grows or shrinks will be the same as that predicted by classical LSW kinetic theory multiplied by the amount of surface area not covered by PVA. In this simulation, a high degree of initial ice surface coverage (>99%) was needed to give high PDI values comparable to those observed in experiment. At low surface coverage, PDI remained low as is predicted by LSW Ostwald ripening theory in the absence of adsorbed polymer. At very high surface coverage, PDI also remained low because very little ice size change was able to occur. The simulation results were in good agreement with experimental findings and were consistent with PVA being adsorbed to ice.


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Figure 7. Simulated ice crystal volume PDI evolution agrees well with experimental results. θeq represents the fraction of the ice surface covered with PVA at equilibrium. A small but measurable amount of fluorescently-labeled PVA appeared to be adsorbed to ice in an ice/water/glycerol slurry, as shown in Figure 8. Only neutral pH conditions were investigated due to high pH sensitivity of dye-labels. The existence of adsorbed PVA is consistent with ice crystal shaping at high PVA concentrations and recent molecular dynamics simulation results.37,38 The measured PVA adsorption was significantly greater than the degree of PG adsorption at 0.2 mg/mL (p = 0.065) and 0.5 mg/mL (p = 0.027) as determined by a one-tailed t-


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test, albeit with a low degree of statistical confidence. PG adsorption was measured to be 0% at all concentrations tested, indicating that it is unlikely that a significant amount of polymer was trapped in the spaces between ice crystals. An additional control was performed without seeding ice growth to determine if PVA was adsorbing to the tube walls. We determined that at 0.2 mg/mL, an insignificant amount of PVA adsorbed to the glass ([0.3±1.5]%). At 1 mg/mL, the measured fraction of adsorbed PVA was not significantly different from zero. A possible explanation for this is that the ice surface is likely to be nearly saturated at PVA concentrations well above the critical concentration (~0.3 mg/mL at neutral pH). At 0.2 mg/mL, it is unlikely that the ice surface is completely saturated because substantial ice growth is observed under the microscope at these concentrations, as shown in Figure S6. The low degree of PVA adsorption suggests that adsorption is weak and reversible, which has been previously suggested by others.39 By contrast, type-III AFP, which is known to bind ice irreversibly,40 was shown to have 65% adsorption to ice in an identical assay. Reversibility of PVA adsorption was also consistent with the negligible freezing hysteresis imparted by PVA,41 a property held by many to require irreversible ice binding.42–45 A key limitation of the ice slurry experiment is that it is difficult to estimate and control the total ice surface area in the slurry. Ice surface area is expected to be much higher for materials with higher IRI activity. IRI activity of each sample was compared by annealing samples in the temperature-controlled microscope. Ice crystals are seen to be much smaller in the presence of type-III AFP. Images are shown in the Supporting Information.


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Figure 8. (a) Schematic of experimental setup to remove an aliquot of unfrozen liquid from an ice slurry containing PVA and 0.2 [g glycerol / g water]. (b) A sparingly small percentage of PVA was measured to be adsorbed to ice. CONCLUSION PVA concentration measurements in an ice slurry suggest that PVA weakly and reversibly adsorbs to ice, in contrast to several irreversibly-binding antifreeze proteins. Large ice crystal size dispersity at intermediate PVA concentrations is also consistent with reversible and


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incomplete PVA adsorption. Additionally, the IRI efficacy of PVA was shown to be strongly pH dependent using two different ice growth assays, which suggests the importance of hydrogen bonding to the IRI activity of PVA. The identity of the anions and cations in solution, which modulate the aqueous solubility of PVA, were found to have negligible influence on IRI compared to pH. The same dependence of IRI on pH was observed for other polymers with weak IRI activity. High pH conditions imparted IRI activity upon otherwise inactive polymers. The pH dependence of IRI activity, observation of ice adsorption, and modeling of ice crystal size distribution provided a molecular picture of the causes for, and limitations of, PVA-induced ice recrystallization inhibition. Future studies of the hydrogen-bonding interaction of PVA with liquid water may be vital to understanding the mechanism of PVA-mediated ice recrystallization inhibition, and to ultimately enable the rational design of new materials for cryopreservation.

ASSOCIATED CONTENT Supporting Information. Ice growth kinetic data for all cosolutes tested. GPC and NMR characterization of all polymers. MATLAB code used for ice crystal dispersity modeling.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] ORCID Nathaniel A. Lynd: 0000-0003-3010-5068


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NOTES The authors claim no competing financial interest.

ACKNOWLEDGMENTS This is a Plan II SAWIAGOS project. The authors especially thank the laboratory of Prof. B. Keith Keitz for kindly allowing the use of their equipment and Shallaco McDonald for technical expertise in assembling experimental setups. N.A.L. is grateful to the Welch Foundation (F1904) for partial support of this research.

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For Table of Contents Use Only

Understanding Poly(vinyl alcohol)-Mediated Ice Recrystallization Inhibition through Ice Adsorption Measurement and pH Effects Aaron A. Burkey,1 Christopher L. Riley,2 Lyndsey K. Wang,2 Taylor A. Hatridge,1 Nathaniel A. Lynd1,* 1

McKetta Department of Chemical Engineering, 2Department of Molecular Biosciences, The

University of Texas at Austin, Austin, TX 78712


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