Article pubs.acs.org/Biomac
Antifreeze (Glyco)protein Mimetic Behavior of Poly(vinyl alcohol): Detailed Structure Ice Recrystallization Inhibition Activity Study Thomas Congdon, Rebecca Notman, and Matthew I. Gibson* Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom, CV4 7AL S Supporting Information *
ABSTRACT: This manuscript reports a detailed study on the ability of poly(vinyl alcohol) to act as a biomimetic surrogate for antifreeze(glyco)proteins, with a focus on the specific property of ice-recrystallization inhibition (IRI). Despite over 40 years of study, the underlying mechanisms that govern the action of biological antifreezes are still poorly understood, which is in part due to their limited availability and challenging synthesis. Poly(vinyl alcohol) (PVA) has been shown to display remarkable ice recrystallization inhibition activity despite its major structural differences to native antifreeze proteins. Here, controlled radical polymerization is used to synthesize well-defined PVA, which has enabled us to obtain the first quantitative structure−activity relationships, to probe the role of molecular weight and comonomers on IRI activity. Crucially, it was found that IRI activity is “switched on” when the polymer chain length increases from 10 and 20 repeat units. Substitution of the polymer side chains with hydrophilic or hydrophobic units was found to diminish activity. Hydrophobic modifications to the backbone were slightly more tolerated than side chain modifications, which implies an unbroken sequence of hydroxyl units is necessary for activity. These results highlight that, although hydrophobic domains are key components of IRI activity, the random inclusion of addition hydrophobic units does not guarantee an increase in activity and that the actual polymer conformation is important.
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plantation medicine.11,12 To highlight in the UK in 2011 there were almost 4000 organ transplants13 that all required appropriate logistics and delivery of the intact tissue, which could be supplemented and improved by novel cryopreservation techniques.14 Despite their obvious potential, there are several major hurdles that have limited the application of AF(G)Ps in biological cryopreservation. The first is their low availability from native sources and the huge synthetic burden associated with them: there are only three reports of the total synthesis of AFGPs, to the best of our knowledge, which are not ideally suited to scale-up.15−17 The second challenge is the mixed reports of both the success and failure of native AFGP in cryopreservation. Carpenter et al. observed that addition of AFPs to blood cryopreservation solutions enhanced recovery to a point, but increased concentrations of AFPs actually reduced the number of viable cells.18 Similar observations of the negative impact of AF(G)Ps have been made with cryopreserved sperm19 and in failed attempts to cryopreserve rat hearts.20 Conversely, improved cryopreservation of oocytes upon addition of AFGP and pancreatic islet cells21 have been reported.22 There is also the potential for cytotoxicity/
INTRODUCTION The formation and growth of ice presents a serious problem to many technological processes ranging from freeze-fracture damage, icing on surfaces such as aeroplane and turbine wings,1,2 freezer-burn in frozen foods,3 pharmaceutical storage,4 and its impact on agriculture. Nature has evolved several defense mechanisms to enable life to flourish in low temperature, ice-rich environments including the replacement of water with trehelose5 to enable desiccation or whole-body freezing, production of lipopolysaccharides,6 or alternatively by the production of antifreeze (glyco)proteins (AF(G)Ps), which enable fish to survive in subzero (ant)arctic waters.7,8 AF(G)Ps have three major effects on ice/water: (i) thermal hysteresis (TH), this is the noncolligative depression of the freezing point; (ii) dynamic ice shaping (DIS), which results in nonhexagonal ice crystal morphologies; and (iii) ice recrystallization inhibition (IRI) whereby the growth of already-formed ice crystals (i.e., Otswald ripening) is inhibited. IRI activity is a particularly desirable effect as uncontrolled ice crystal growth (recrystallization) during the thawing of cryopreserved biological tissue has been indicated to be a major source of coldinduced damage9 and presents a challenge to the vitrification (cryopreservation method) of whole organs.10 Consequently, there is a real need to develop technologies which reduce ice recrystallization, particularly to improve the storage and distribution of human tissue for regenerative and trans© 2013 American Chemical Society
Received: February 8, 2013 Revised: March 26, 2013 Published: March 28, 2013 1578
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immunogenicity associated with these proteins.23 These conflicting cryopreservation results have been speculated to be due to the macroscopic effects of TH and DIS; these two properties have been shown to cause the formation of spicular (needle-like) ice crystals during the cryopreservation of cells, leading to increased cell death due to physical piercing of cell membranes. Considering this, the design and application of materials that specifically display IRI is especially desirable but is limited by the aforementioned synthetic challenges. Most previous studies on structure−activity relationships in AFGPs have focused only on TH as a marker of activity, revealing that essentially all modifications (apart from exchange of the disaccharide with a monosaccharide) are not tolerated.16 Ben et al. have developed a series of peptide-based IRI active compounds, which specifically maintain IRI activity, but no appreciable TH or DIS activity.24,25 The activity of these compounds is surprising and suggests that IRI activity can be reproduced by compounds that are far more synthetically accessible than the native proteins. These short peptides have been shown to have some beneficial effect in cryopreservation when compared to the common (and potentially toxic) dimethyl sulfoxide.26 A key challenge in designing new, more active IRI active compounds lies in the lack of mechanistic understanding of the IRI process. In particular, the disconnect between the mechanism of TH and DIS, which have been studied and understood on a molecular recognition basis with specific binding to the basal planes of ice.27 Microfluidic studies on AFPs have also revealed that the binding to ice is essentially irreversible.28 The importance of hydrophobic domains, potentially to repel additional water molecules appears to be crucial with most AF(G)Ps having amphipathic character16,29,30 and small molecules with defined hydrophobic domains have some IRI activity.31−33 However, the mechanism of IRI and its relationship to TH/DIS are poorly understood. In an effort to both probe this mechanism of IRI and to develop more synthetically accessible inhibitors, Gibson et al. have investigated polymeric-mimics of AF(G)Ps. Polymers have the advantage of scalability, huge monomer and functional group chemical space, and the acceptability of synthetic polymers in biomaterials applications.4,34,35 Glycopolymers36 and poly(ampholytes)37 have been shown to have some degree of IRI activity, but by far the most active polymer reported to date is poly(vinyl alcohol), PVA.30 The reasons for the activity of PVA are still unknown with several groups hypothesizing that it is capable of directly binding to growing ice crystal through a match with the underlying crystal lattice.38 However, the distribution of hydroxyl groups in PVA is different to that of AFGP, which raises questions as to how they can both be binding to the same ice lattice and hence implying a different and additional mechanism. Mono/oligo saccharides also show negligible IRI activity, as do other polyols, suggesting there are unique features about the structure of PVA which endows its IRI activity.31 All previous studies of the IRI activity of PVA have focused on commercially available polydisperse samples that are partially acetylated (due to synthetic procedure via poly(vinyl acetate)).31,36,39,40 The minimum consensus sequence required for activity is unknown, along with the precise role of molecular weight and the role and tolerance of structural modifications. The aim of this manuscript is to take advantage of modern controlled radical polymerization techniques that allow for the reproducible synthesis of well-defined polymers. In particular, RAFT polymerization has been shown to be particularly useful
for deactivated monomers, such as vinyl acetate, and has advantages over other controlled radical processes in that no external catalysts are required, which themselves may prevent their biological application or modify the IRI process.
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EXPERIMENTAL SECTION
Materials. Methyl(ethoxycarbonothioyl)sulfanyl benzene (CTA 1) was prepared according to literature methods as detailed in the Supporting Information.41 Phosphate-buffered saline (PBS) solutions were prepared using preformulated tablets (Sigma-Aldrich) in 200 mL of Milli-Q water (>18.2 Ω mean resistivity) to give [NaCl] = 0.138 M, [KCl] = 0.0027 M, and pH 7.4. Vinyl acetate (VAc) and N-vinylpyrrolidone were purchased from Sigma-Aldrich and were filtered through a plug of basic alumina to remove inhibitors prior to their use. 4,4′-Azobis(4-cyanovaleric acid) was recrystallized from methanol and stored at −8 °C in the dark. Acetic acid (glacial, 99.5%) was purchased from Fischer Scientific. Hydrazine hydrate solution (approximately 80%) was purchased from Sigma Aldrich. All solvents were purchased from VWR or Sigma-Aldrich and used without further purification. Physical and Analytical Methods. 1H and 13C NMR spectra were recorded on Bruker DPX-300 and DPX-400 spectrometers using deuterated solvents obtained from Sigma-Aldrich. Chemical shifts are reported relative to residual nondeuterated solvent. Mass spectral analyses were obtained using Bruker MicroTOF or Bruker MaXis electrospray instruments using positive or negative electrospray mode. The molecular ion and mass fragments are quoted and assigned. Gel permeation chromatography (GPC) was used to determine the molecular weights and polydispersities of the synthesized polymers. The THF GPC system comprised of a Varian 390-LC-Multi detector suite fitted with differential refractive index (DRI), light scattering (LS) and ultraviolet (UV) detectors equipped with a guard column (Varian Polymer Laboaratories PLGel 5 μm, 50 × 7.5 mm) and two mixed D columns of the same type. The mobile phase was THF with 5% triethylamine (TEA) eluent at a flow of 1.0 mL/min, and samples were calibrated against Varian Polymer Laboratories Easi-Vials linear poly(styrene) and poly(methylmethacrylate) standards (162−2.4 × 105 g·mol−1) using Cirrus v3.3. The DMF GPC system comprised of a Varian 390-LC-Multi detector suite fitted with a differential refractive index (DRI) detector equipped with a guard column (Varian Polymer Laboaratories PLGel 5 μm, 50 × 7.5 mm) and two mixed D columns of the same type. The mobile phase was DMF with 5 nM NH3BF4 eluent at a flow of 1.0 mL·min−1, and samples were calibrated against Varian Polymer Laboratories Easi-Vials poly(methylmethacrylate) standards (162−2.4 × 105 g·mol−1) using Cirrus v3.3. Ice wafers were annealed on a Linkam Biological Cryostage BCS196 with T95Linkpad system controller equipped with a LNP95-Liquid nitrogen cooling pump, using liquid nitrogen as the coolant (Linkam Scientific Instruments UK, Surrey, U.K.). An Olympus CX41 microscope equipped with a UIS-2 20x/0.45/∞/0−2/FN22 lens (Olympus Ltd., Southend on sea, U.K.) and a Canon EOS 500D SLR digital camera were used to obtain all images. Image processing was conducted using Image J, which is freely available from http://imagej.nih.gov/ij/. Ice Recrystallization Inhibition (Splat) Assay. Ice recrystallization inhibition was measured using a modified splay assay.42 A 10 μL sample of polymer dissolved in PBS buffer (pH 7.4) was dropped 1.40 m onto a chilled glass coverslip sat on a piece of polished aluminum placed on dry ice. Upon hitting the chilled glass coverslip, a wafer with diameter of approximately 10 mm and thickness 10 μm was formed instantaneously. The glass coverslip was transferred onto the Linkam cryostage and held at −8 °C under N2 for 30 min. Photographs were obtained using an Olympus CX 41 microscope with a UIS-2 20x/0.45/ ∞/0−2/FN22 lens and crossed polarizers (Olympus Ltd., Southend on sea, UK), equipped with a Canon DSLR 500D digital camera. Images were taken of the initial wafer (to ensure that a polycrystalline sample had been obtained) and after 30 min. Image processing was conducted using Image J,43 which is freely available. In brief, the four largest ice crystals in the field of view were measured and the single largest length in any axis recorded. This was repeated for at least three wafers and the average (mean) value was calculated to find the largest 1579
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Scheme 1. RAFT/MADIX Mediated Bulk Polymerization of Vinyl Acetate and Subsequent Hydrolysis to PVA
grain dimension along any axis. The average of this value from three individual wafers was calculated to give the mean largest grain size (MLGS). This method has been shown to be highly reproducible, although it does not contain the distribution data of, for example, domain recognition software, but has the advantage that it shows the worst case (largest ice crystals) and may underestimate, but not overestimate, activity. Polymerization of Vinyl Acetate Using CTA 1. As a representative example, CTA 1 (0.21 g, 0.99 mmol), vinyl acetate (4.67 g, 2.64 mmol), and ACVA (4,4′-azobis(4-cyanovaleric acid); 0.013 g) were added to a stoppered vial. The solution was thoroughly degassed under a flow of N2 for 20 min, and the reaction mixture was then allowed to polymerize at 68 °C for typically 15 h. The yellow solutions were then cooled to room temperature. Poly(vinyl acetate) was then recovered as a yellow sticky solid after precipitation into hexane. The hexane was then decanted and the poly(vinyl acetate) was redissolved in THF, which was then concentrated in vacuo and thoroughly dried under vacuum at 40 °C for 24 h, forming a white crystalline solid. Representative characterization data for PVAc56: 1H NMR (400 MHz, CDCl3) δ 4.61 (−CHO−CH2, br, 1H), 1.74 (−CO−CH3, br, 3H), 1.53 (−CH2−, br, 2H); MnSEC(THF) = 5100 Da, Mw/Mn = 1.23. Hydrolysis of Poly(vinyl acetate) to Poly(vinyl alcohol). As a representative example, poly(vinyl acetate) (1.5 g, 3300 Da, Mn/Mw = 1.22) was dissolved in a methanol (20 mL) and hydrazine hydrate solution (15 mL, 80% in water) in a round-bottom flask. The reaction mixture was stirred at 30 °C for 2 h. The reaction mixture was then dialyzed using distilled water and poly(vinyl alcohol) was recovered as a spongy white solid by freeze-drying the dialysis solution. Deacetylation was determined by 1H NMR. Representative characterization data for PVA56: 1H NMR (400 MHz, CDCl3) δ 4.00 (−CHOH−, br, 1H), 1.68−1.60 (−CH2−, br, 2H). Acetylation of Poly(vinyl alcohol). As a representative example, poly(vinyl alcohol) (0.5 g, 29 kDa, Mn/Mw = 1.28) was dissolved in water (2.4 mL), acetic acid (7.6 mL), and HCl (0.1 mL, 3 M solution in water) in a vial equipped with a stir bar. The reaction mixture was stirred at 40 °C for 4 days. The reaction mixture was then dialyzed, and partially acetylated poly(vinyl alcohol) was recovered by freezedrying the dialysis solution. Conversion was determined by 1H NMR integration of the acetate methyl protons (δ = 2.08) and the −CH2− backbone protons (δ = 1.93−1.50) and IR by examining the −OH stretch at 3340 cm−1 and the −CO stretch at 1738 cm−1. Representative characterization data: 1H NMR (400 MHz, D2O) δ 4.00 (−CHOH−, br, 1H), 3.82 (−CHO−CH2, br, 1H), 2.08 (−CO− CH3, br, 3H), 1.93−1.50 (−CH2−, br, 2H); MnSEC (DMF) = 2700 Da; Mw/Mn = 1.22. Copolymerisation of Vinyl Acetate and N-Vinyl-pyrrolidone using CTA 1. As a representative example, vinyl acetate (0.68 g, 7.98 mmol), N-vinyl-pyrrolidone (0.944 g, 8.83 mmol), CTA 1 (0.035 g, 0.17 mmol), and ACVA (4.7 mg, 0.02 mmol) were dissolved in dioxane (8.41 mL) in a stoppered vial equipped with a stir bar. The reaction mixture was thoroughly degassed under a flow of N2 for 20 min, and the reaction mixture was allowed to polymerize at 70 °C for 7 h. The dark yellow solutions were then cooled to room temperature and the random copolymer was recovered as a sticky yellow solid by several precipitations into diethyl ether or hexane, depending on the
composition of the polymer. The solvent was carefully decanted and the product thoroughly dried under vacuum at 40 °C for 24 h, forming a yellow solid. Representative characterization data for PVA-(PVP)3: 1 H NMR (400 MHz, D2O) δ 3.52 (−CHN−, br, 1H), 3.26 (−CHO−, br, 1H), 3.12 (−NCH2CH2−, br, 2H), 2.49 (−NCOCH2−, br, 2H), 2.13 (−NCH2CH2CH2−, br, 2H), 1.97 (−COCH3−, br, 3H), 1.80− 1.50 (−CH2−, br, 2H); MnNMR(D2O) = 8400 Da; MnSEC(DMF) = 2700 Da; Mw/Mn = 1.30. Polymerization of N-Vinyl-pyrrolidone Using CTA 1. As a representative example, N-vinyl-pyrrolidone (2.09 g, 18.78 mmol), CTA 1 (0.037 g, 0.17 mmol), and ACVA (0.005 g, 0.02 mmol) were dissolved in dioxane (4 mL) in a stoppered vial equipped with a stir bar. The reaction mixture was thoroughly degassed under a flow of N2 for 20 min and the reaction mixture was allowed to polymerize at 70 °C for, typically, 5 h. The dark yellow solutions were then cooled to room temperature and the random copolymer was recovered as white flakes by several precipitations into diethyl ether. The bulk of the solvent was carefully decanted and the solid collected by centrifugation. The product was thoroughly dried under vacuum at 40 °C for 24 h, forming a yellow solid. Representative characterization data for PVP62: 1H NMR (400 MHz, CDCl3) δ 3.74 (−CHN−, br, 1H), 3.19 (−NCH2CH2−, br, 2H), 2.38 (−NCOCH2−, br, 2H), 2.21(-NCH2CH2CH2−, br, 2H), 1.81−1.53 (−CH2−, br, 2H); MnSEC (DMF) = 2700 Da; Mw/Mn = 1.59. Copolymerisation of Vinyl Acetate and Isopropenyl Acetate Using CTA 1. As a representative example, CTA 1 (0.032 g, 0.99 mmol), vinyl acetate (7.96 g, 92.45 mmol), isopropenyl acetate (0.83 g, 8.26 mmol), and ACVA (4,4′-azobis(4-cyanovaleric acid); 0.003 g, 0.012 mmol) were added to a stoppered vial. The solution was thoroughly degassed under a flow of N2 for 20 min and the reaction mixture was then allowed to polymerize at 75 °C for 14 h. The yellow solutions were then cooled to room temperature. The polymer was then recovered as a yellow sticky solid after precipitation into hexane. The hexane was then decanted and the polymer was redissolved in DCM, which was then concentrated in vacuo, forming a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 4.85 (−CHO−CH2, br, 1H), 2.59 (−C(CH3)−CH2−, br, 2H), 2.02 (−CO−CH3, br, 6H), 1.75 (−CH2−, br, 2H), 1.56 (−C(CH3)−CH2−, br, 3H); MnSEC(THF) = 4200 Da; Mw/Mn = 1.28. Deprotection of Poly(vinyl acetate)-co-poly(isopropenyl acetate). Poly(vinyl acetate)-co-poly(isopropenyl acetate) (1.50 g, MnSEC(THF) = 4200 Da, Mw/Mn = 1.28) was dissolved in ethanol (40 mL) and heated to 60 °C. Hydrazine hydrate (80% solution, 30 mL) was then added carefully with stirring. The solution was stirred at 60 °C for 6 h. The solution was then concentrated in vacuo leaving a clear solution. The solution was dialyzed using 1000 MWCO dialyzed tubing and distilled water. The dialyzed solution was then freeze-dried and the polymer recovered as white powder. 1H NMR (400 MHz, D2O) δ 3.95 (−CHOH, br, 1H), 1.75−1.40 (−CH2−, br, 4H), 1.28 (−C(CH3), br, 3H).
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RESULTS AND DISCUSSION The aim of this study was to investigate the influence of the structural features of PVA on its IRI activity. To obtain quantitative structure−activity relationships it was necessary to 1580
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Table 1. PVAc Precursors and Respective PVA Polymers entry
[M]/[CTA]a
conv.b (%)
Mn,theoc (g·mol−1)
Mn,NMRd (g·mol−1)
Mn,SECe (g·mol−1)
Mw/Mne (−)
DPNf (−)
PVAg
PVAc10 PVAc19 PVAc30 PVAc56 PVAc154 PVAc246 PVAc351
10 20 50 100 200 300 500
83.2 95.3 60.0 54.7 73.8 80.1 68.8
860 1720 4300 8600 17200 25800 43000
900 1640 2580 4700 12700 20660 29600
870 1700 2700 5100 13800 21700 30900
1.18 1.18 1.45 1.23 1.45 1.39 1.28
10 19 30 56 154 246 351
PVA10 PVA19 PVA30 PVA56 PVA154 PVA246 PVA351
a
Monomer to RAFT agent ratio. bDetermined by 1H NMR spectroscopy. cTheoretical Mn determined from monomer to RAFT agent ratio. Determined by 1H NMR. eDetermined by SEC in THF using PMMA standards. fNumber-average degree of polymerization. gCorresponding PVA prepared by hydrolysis of the respective PVAc. d
measured. A modified splat assay, as developed by Knight et al. was employed.42 Briefly, a polynucleated ice wafer comprised of ice crystals with diameters
