Recrystallization inhibitor

Abstract

The present invention relates to methods for preventing or inhibiting ice recrystallisation in substances (e.g. biological materials and food products) which are susceptible to ice crystal growth upon cryopreservation and/or thawing therefrom. The methods relate to the use of compositions comprising poly(proline) or a variant or derivative thereof. Also provided are kits and compositions comprising poly(proline) which can be used in the methods of the invention.

Claims

1. A method of preventing or inhibiting ice recrystallization in a substance which is susceptible to ice crystal growth upon cryopreservation and/or warming or thawing therefrom, the method comprising the step: (i) treating the substance with a composition comprising poly(proline) or a variant or derivative thereof, wherein the poly(proline) or a variant or derivative thereof is a homogeneous or heterogeneous mixture of polymers which consist substantially or exclusively of linear chains of proline residues, the polymers having the general structure: ##STR00002## wherein n=3-200 (SEQ ID NO: 1); or 3-150 (SEQ ID NO: 2) or 5-100 (SEQ ID NO: 3); or 10-100 (SEQ ID NO: 4), 10-50 (SEQ ID NO: 5) or 10-25 (SEQ ID NO: 6); or 10-20 (SEQ ID NO: 7) or 11-15 (SEQ ID NO: 8).

2. A method of cryopreserving a substance which is susceptible to ice crystal growth upon cryopreservation and/or thawing or warming therefrom, the method comprising the steps: (i) treating the substance with a composition comprising poly(proline) or a variant or derivative thereof, wherein the poly(proline) or a variant or derivative thereof is as defined in claim 1; (ii) reducing the temperature of the treated substance to a cryopreserving temperature; and optionally (iii) storing the treated substance at the cryopreserving temperature.

3. A method of reducing cell damage during the warming or thawing of a cryopreserved substance comprising biological material, the method comprising the steps: (i) warming or thawing the cryopreserved substance comprising biological material, wherein the cryopreserved substance is one which has been treated with poly(proline) or a variant or derivative thereof and wherein the poly(proline) or a variant or derivative thereof is as defined in claim 1.

4. The method of claim 1, wherein the substance is treated with poly(proline), or a variant or derivative thereof, before or during cryopreservation.

5. The method of claim 1, wherein the concentration of the poly(proline) or a variant or derivative thereof in the composition is 1-50 mg/mL, or 10-40, 10-30 or 10-20 mg/mL.

6. The method of claim 1, wherein the composition is a cryopreserving composition comprising DMSO.

7. The method of claim 1, wherein the substance is a biological material or a food product.

8. The method of claim 7, wherein the biological material comprises one or more of cells, tissues, whole organs and parts of organs.

9. The method of claim 8, wherein the cells are bacterial cells, fungal cells, plant cells, animal cells, mammalian cells, or human cells.

10. The method of claim 8, wherein the cells are monolayers of cells.

11. The method of claim 7, wherein the biological material is pretreated with proline prior to treatment with poly(proline) or a variant or derivative thereof.

12. The method of claim 7, wherein the food product comprises ice cream, sorbet, animal meat, a vegetable or a fruit.

13. The method of claim 2, wherein the concentration of the poly(proline) or a variant or derivative thereof in the composition is 1-50 mg/mL, or 10-40, 10-30 or 10-20 mg/mL.

14. The method of claim 3, wherein the concentration of the poly(proline) or a variant or derivative thereof in the composition is 1-50 mg/mL, or 10-40, 10-30 or 10-20 mg/mL.

15. The method of claim 2, wherein the composition is a cryopreserving composition comprising DMSO.

16. The method of claim 3, wherein the composition is a cryopreserving composition comprising DMSO.

17. The method of claim 2, wherein the substance is a biological material or a food product.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Condensation polymerization of proline.

(2) FIG. 2: ESI Mass Spec. A) PPro.sub.10 (SEQ ID NO: 9). B) PPro.sub.20 (SEQ ID NO: 13).

(3) FIG. 3: Polyproline structure and activity. A) Circular dichroism spectra; B) IRI activity of polyproline series; C) IRI activity compared to other homo-polypeptides; D) Cryomicrograph of a PBS negative control; E) Cryomicrograph of 20 mg.Math.mL.sup.−1 polyproline. Photos taken after 30 mins at −8° C. Error bars represent ±standard deviation from minimum of 3 replicates. Images shown are 1.2 mm across. MLGS (mean largest grain size; F) Circular dichroism spectra. Synthesised proline polypeptides compared to a polyproline II helical reference (PPro (II) Helix) [37] not corrected for concentration to enable comparison against reference standard; G) SPLAT assay PBS control (× 200); H) SPLAT assay L-proline, 20 mg.Math.mL.sup.−1 (×200).

(4) FIG. 4: Hydrophobic surface mapping of A) Recombinant Type I Sculpin AFP; B) PPro.sub.10 (SEQ ID NO: 9); C) PGlu.sub.10 (SEQ ID NO: 18) showing charged hydrophilic surface.

(5) FIG. 5: A549 Cryopreservation. A) Schematic of procedures used; B) Cell recovery by trypan blue assay. Cells were incubated either in media alone or with 200 mM proline for 24 hours, then cryopreserved by addition of 10% DMSO with or without PPro.sub.11 (SEQ ID NO: 10). Error bars±S.E.M. from n=3 with two nested replicates. (#P<0.05 compared to 10% DMSO treatment; * P<0.05 compared to 200 mM proline exposure with 10% DMSO treatment.

EXAMPLES

(6) The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

(7) Data were analysed with a one-way analysis of variance (ANOVA) on ranks followed by comparison of experimental groups with the appropriate control group (Holm-Sidak method) followed by Tukey's post hoc test. Excel 2013 (Microsoft, Redmond, Wash.) and R (R Foundation for Statistical Computing, Vienna, Austria) were used for the analyses. Data sets are presented as mean±(SEM).

Example 1: Synthesis and Characterization of Poly(Proline)

(8) To obtain polyproline of different molecular weights, a range of synthetic methods were employed. Oligo-proline of DP 10 (PPro.sub.10 (SEQ ID NO: 9)) and DP (PPro.sub.20 (SEQ ID NO: 13)) were prepared by solid-phase peptide synthesis, alongside a high molecular weight commercial sample. L, D, and D/L (racemic) polyproline were synthesized by condensation polymerization using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, FIG. 1). Following dialysis to remove excess amino acids, coupling reagents and low molecular weight fractions, the polymers were characterized by SEC (size exclusion chromatography) and the results shown in Table 1. This indicated molecular weights in the region of ˜3000 g.Math.mol.sup.−1 and less disperse than expected due to fractionation during dialysis, and the rigid-rod like nature of the PPII helix which affects its SEC behaviour. Table 1 also contains polymers from previous work, which are included for later critical IRI activity analysis (vide infra).

(9) TABLE-US-00001 TABLE 1 Poly(proline) characterization. M.sub.n, ,.sup.SECa DP (g .Math. mol.sup.−1) (−) (−) Secondary Structure PPro.sub.11 1300.sup.a 1.03 11 (SEQ ID NO: 10) PPro.sub.15 1700.sup.a 2.12 15 PPII (SEQ ID NO: 11) PPro.sub.19 2100.sup.a 1.50 19 (SEQ ID NO: 12) P(D)Pro.sub.15 1700.sup.a 1.01 15 Enantiomeric PPII (SEQ ID NO: 11) P(DL)Pro.sub.21 2400.sup.a 1.01 21 — (SEQ ID NO: 14) PPro.sub.10-100 1-10k.sup.b — 10-100 PPII.sup.e (SEQ ID NO: 4) PPro.sub.10  900.sup.c .sup.d 10 PPII.sup.e (SEQ ID NO: 9) PPro.sub.20 2000.sup.c .sup.d 20 PPII.sup.e (SEQ ID NO: 13) .sup.aDetermined by SEC; .sup.bValue from supplier; .sup.cMass Spectrometry; .sup.dSingle species .sup.eFrom Literature [34, 35 36].

(10) Circular dichroism spectroscopy (CD) confirmed that PPro.sub.15 (SEQ ID NO: 11) adopted a PPII helix (FIG. 3A), compared to a standard (ESI, FIG. 3F) [37]. PPII (in CD) can be confused with a random coil. However, the characteristic signals associated with a PPII helix are present at 207 and 228 nm, whilst a random conformation exhibits slight peak shifting, with signals absent in the 220 nm region [38]. P(D)Pro.sub.15 (SEQ ID NO: 11) gave the mirror spectrum as expected for D-amino acids, whilst the D/L racemic mixture showed essentially no secondary structure.

(11) L- and D-proline, poly-L-proline mol wt 1,000-10,000 (PPro.sub.10-100 (SEQ ID NO: 4)), ethyl (hydroxyimino) cyanoacetate (OxymaPure™), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI), dichloromethane (DCM), phosphate-buffered saline preformulated tablets, and hydrochloric acid (37%) were purchased from Sigma Aldrich Co. Ltd. (Gillingham, UK) and used without further purification. Dialysis Membrane Spectra/Por 7 Flexible 38 mm FWT 1000 MWCO 4.6 mL/cm was purchased from Fischer Scientific (Loughborough, UK) and used directly. Phosphate-buffered saline (PBS) solution was prepared using preformulated tablets in 200 mL of Milli-Q water (>18.2Ω mean resistivity) to give [NaCl]=0.138 M, [KCl]=0.0027 M, and pH 7.4. PPro.sub.10 (SEQ ID NO: 9) and PPro.sub.20 (SEQ ID NO: 13) (>90%) were purchased bespoke from Peptide Protein Research Ltd (Fareham, UK) and were used without further purification. PPro.sub.10 (SEQ ID NO: 9): m/z (ESI) 988.0 (100%, −1); PPro.sub.20 (SEQ ID NO: 13): m/z (ESI) 491.0 (20%, +4), 654.3 (100%, +3), 981.0 (30%, +2).

(12) SEC (size exclusion chromatography) was acquired a DMF Agilent 390-LC MDS instrument equipped with differential refractive index (DRI), viscometry (VS), dual angle light scatter (LS) and dual wavelength UV detectors. The system was equipped with 2×PLgel Mixed D columns (300×7.5 mm) and a PLgel 5 μm guard column. The eluent was DMF with 5 mmol NH4BF4 additive. Samples were run at 1 mL/min at 50° C. Poly(methyl methacrylate) standards (Agilent EasyVials) were used for calibration. Analyte samples were filtered through a nylon membrane with 0.22 μm pore size before injection. Respectively, experimental molar mass (M.sub.n,SEC) and dispersity (D) values of synthesized polymers were determined by conventional calibration (relative to poly(methyl methacrylate) standards) using Agilent GPC/SEC software. Refractive index was recorded.

(13) EDCI (0.50 g, 2.60 mmol) was dissolved in dry DCM (20 mL) and stirred at room temperature under a flow of nitrogen for 20 minutes, followed by cooling to 0° C. Within 5 minutes of cooling, L-proline (0.30 g, 2.60 mmol, 1 eqv) and OxymaPure™ (0.37 g, 2.60 mmol, 1 eqv) were added together to the reaction mixture, resulting in an instantaneous colour change to yellow. The mixture was stirred on ice under nitrogen for 1 further hour, and then warmed to RT with stirring overnight. The dark yellow solution was condensed in vacuo, dissolved in Milli-Q water (10 mL) acidified to pH 3-4 with 3M HCl, and a minimum volume of methanol added until residual solids dissolved. Dialysis (>1 kDa) for 48 hours was subsequently performed with regular water changes. The resulting solution was freeze dried, yielding an off-white solid. 31.4 mg (10.4%). The DL racemate, P(DL)Pron (SEQ ID NO: 19), utilised a 1:1 ratio of L- and D-proline (2.60 mmol prolines).

Example 2: Ice Recrystallization Inhibition (IRI) Activity of Poly(Proline)

(14) A series of peptides were tested for IRI activity using a SPLAT assay [39]. Briefly, this involved seeding a large number of small ice crystals, which were annealed for 30 minutes at −8° C., before being photographed. The average crystal size was then measured, relative to a PBS control, with smaller values indicating more IRI activity, FIGS. 3B/1C.

(15) All peptides displayed a dose-dependent activity relationship with grain size reducing as concentration increased. Only weak molecular weight dependence was observed in the range tested. Example micrographs of a PPro.sub.10-100 (SEQ ID NO: 4) ice wafer compared to a PBS control are shown in FIGS. 3D/1E, demonstrating potent inhibition. This activity was unexpected as most synthetic macromolecules show little or no IRI [15, 22, 40]. The shortest peptides (PPro.sub.10 (SEQ ID NO: 9)) lost activity below 10 mg.Math.mL.sup.−1, but the longer polymers retained activity down to 5 mg.Math.mL.sup.−1. The magnitude of this activity is significantly weaker than AF(G)Ps which function at concentrations as low as 0.14 μg.Math.mL.sup.−1[41], but comparable to polyampholytes which have found application in cellular cryopreservation [22-23].

(16) Ice recrystallisation inhibition (IRI) activity was measured using a modified splat assay [50]. A 10 μL sample of polymer dissolved in PBS buffer (pH 7.4) was dropped 1.40 m onto a chilled glass coverslip, resting on a thin aluminium block placed on dry ice. Upon hitting the 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 N.sub.2 for 30 minutes. Photographs were obtained using an Olympus CX 41 microscope with a UIS-2 20 ×/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 again after 30 minutes. Image processing was conducting using Image J, which is freely available. In brief, five of the largest ice crystals in the field of view were measured and the single largest length in any axis recorded. The average (mean) of these five measurements was then calculated to find the largest grain dimension along any axis. This was repeated for three individual wafers, and the average (mean) of these three values was calculated to give the mean largest grain size (MLGS). The average value was compared to that of a PBS buffer negative control.

Example 3: Effect of the PPII Helix on Ice Recrystallization Inhibition Activity

(17) Earlier work by Knight [42] observed that poly(hydroxyproline) had potent IRI activity, which was assumed to be due, in part, to the regularly spaced hydroxyl groups along the backbone. However, the observations made here suggest that it is the specific helical structure of poly(proline), rather than hydroxyl groups, which gives rise to the observed activity.

(18) FIG. 3C shows a comparison of the IRI activity of poly(hydroxyproline) versus PPro.sub.15 (SEQ ID NO: 11) and, two other alpha-helical poly(amino acids) [40]. These alpha-helical controls, poly(lysine) (PLys.sub.50 (SEQ ID NO: 20)) and poly(glutamic acid) (PGlu.sub.110 (SEQ ID NO: 21)), showed no IRI, similar to PEG, which was used as negative control.

(19) P(D)Pro.sub.15 (SEQ ID NO: 11) and P(DL)Pro.sub.21 (SEQ ID NO: 14) had statistically identical activity to PPro.sub.15 (SEQ ID NO: 11), ruling out any stereospecific effects. This may suggest that the local structure around the amide bond, and not the stereochemistry or folding, is crucial as opposed to long-range order (which may still have a contribution, however).

Example 4: Mapping of Hydrophobic/Hydrophilic Domains

(20) It is hypothesised that IRI activity requires a balance between hydrophilic and hydrophobic domains for activity (amphipathy) [25, 27]. PPro.sub.10 (SEQ ID NO: 9) was compared to that of a non-glycosylated Type I sculpin antifreeze protein (AFP) [43] and also against PGlu.sub.10 (SEQ ID NO: 18), by mapping their hydrophobic/hydrophilic domains (FIG. 4).

(21) NMR solution phase (AFP Sculpin) and X-ray crystal structures of proteins and peptides of interest were acquired from the Protein Data Bank and other publically accessible sources, or computationally modelled in-house (PPro.sub.10 (SEQ ID NO: 9) and PGlu.sub.10 (SEQ ID NO: 18)). Structures were rendered in PyMOL (Schrödinger LLC, Cambridge, Mass.), which is freely available for educational use, and surfaces on the structures were displayed. An open source script “color_h” was used to colour the protein surface according to the Eisenberg hydrophobicity scale of its constituent amino acids, from red (hydrophobic) to white (hydrophilic). For the homo-polypeptides where scaling is not possible, aliphatic hydrogen and carbon were defined as hydrophobic whilst oxygen, hydrogen and nitrogen as hydrophilic, utilising the same colour scheme. Due to the lack of hydrogen bond donors in a PPro.sub.10 (SEQ ID NO: 9) PPII helix, this was considered representative.

(22) Type I sculpin AFP (FIG. 4A) clearly possesses a segregated domain structure with regular ‘patches’ of hydrophobic/hydrophilic groups. PPro.sub.10 (SEQ ID NO: 9) (FIG. 4B) also possesses this facial amphiphilicity, with ‘hydrophilic pockets’ visible between the mostly hydrophobic polypeptide. In comparison, PGlu.sub.10 (SEQ ID NO: 18) (FIG. 4C, no IRI activity) has charged hydrophilic groups protruding from around the core of the helix, which prevents the presentation of core hydrophobic domains. This agrees with our previous study on Nisin A, which has pH-dependent IRI associated with segregated domains [27] and also of amphiphiles developed by Capicciotti et al. [25], which only function below the CMC (critical micelle concentration) [25].

Example 5: Effect of Poly(Proline) on Cryopreservation of Cells

(23) A549 (human Caucasian lung carcinoma) cells were employed as prototypical adherent cell monolayer which are challenging to cryopreserve by traditional methods [46]. Rather than traditional DMSO-only cryopreservation, the protective osmolyte proline (which has no IRI activity unlike the polymer—see ESI) was also added; proline accumulates under water stress in some organisms, and aids the cryopreservation process [47-48]. A549 cells were incubated with 200 mM (23 mg.Math.mL.sup.−1) proline or media alone for 24 hours. The solution was then removed and replaced with 10% DMSO with PPro.sub.11 (SEQ ID NO: 10) (1250 g.Math.mol.sup.−1, D=1.03). After 10 minutes exposure, all excess solvent was removed, before controlled freezing at 1° C..Math.min.sup.−1 to −80° C., FIG. 5A. Following storage at −80° C., cells were thawed by addition of warm media and the total number of viable cells assessed via trypan blue 24 hours post-thaw.

(24) FIG. 5B shows that using DMSO, the current ‘gold’ standard for cryopreservation, lead to just 27% of the frozen cells being recovered. It was also observed that addition of poly(proline) to 10% DMSO also failed to give any additional protection. However, cells which had been pre-conditioned with 200 mM proline for 24 h then treated with 5 mg.Math.mL.sup.−1 PPro.sub.11 (SEQ ID NO: 10) and 10% DMSO dramatically increased recovery of viable cells to 53%. Increasing the concentration of poly(proline) beyond 10 mg.Math.mL.sup.−1 did not increase recovery further, as reported for other IRI's [16]. This is an unprecedented improvement in recovery for a macromolecular antifreeze and demonstrated the successful, rational, design, characterisation and application of a simplistic antifreeze protein mimic.

(25) Human Caucasian lung carcinoma cells (A549) were obtained from the European Collection of Authenticated Cell Cultures (Salisbury, UK) and grown in 175 cm.sup.2 cell culture Nunc flasks (Corning Incorporated, Corning, N.Y., USA). Standard cell culture medium was composed of Ham's F-12K (Kaighn's) Medium (F-12K) (Gibco, Paisley, UK) supplemented with 10% USA-origin foetal bovine serum (FBS) purchased from Sigma Aldrich Co Ltd (Gillingham, UK), 100 units/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B (PSA) (HyClone, Cramlington, UK). A549 cells were maintained in a humidified atmosphere of 5% CO.sub.2 and 95% air at 37° C. and the culture medium was renewed every 3-4 days. The cells were subcultured every 7 days or before reaching 90% confluency. To subculture, cells were dissociated using 0.25% trypsin plus 1 mM EDTA in balanced salt solution (Gibco) and reseeded at 1.87×10.sup.5 cells per 175 cm.sup.2 cell culture flasks.

(26) Solutions for cell incubation experiments were prepared by dissolving the individual compounds in F-12K supplemented with 10% FBS and 1× PSA (solutions used as freezing buffers did not contain PSA) and sterile filtered prior to use.

(27) Cells to be frozen in the monolayer format were seeded at 0.4×10.sup.6 cells per well in 500 μL of cell culture medium in 24-well plates (Corning Incorporated, Corning, N.Y.). Plates had a total available volume of 3.4 mL with an approximate growth area of 1.9 cm.sup.2, no coverslips were used and plates were used with the accompanying lid. Cells were allowed to attach to the entire free surface of the bottom of the well and formed a confluent layer not greater in height than one cell. Before experimental treatments, cells were allowed to attach for 2 h to the plates in a humidified atmosphere of 5% CO.sub.2 and 95% air at 37° C. The medium was exchanged against medium that was or was not supplemented with solutes as indicated in the figure. Control cells received no additional solutes and experimental cells were incubated with 23.1 mg/mL L-proline for 24 h in a humidified atmosphere of 5% CO.sub.2 and 95% air at 37° C. Following the incubation period, the culture medium was removed and cells were exposed for 10 min at room temperature to different concentrations of solutes dissolved in F-12K supplemented with 10% FBS and 10% DMSO. After 10 min, the freezing solutions were removed and the plates placed inside a CoolCell® MP plate (BioCision, LLC, Larkspur, Calif., USA), transferred to a −80° C. freezer and frozen at a rate of 1° C./min. After 24 h at −80° C., cells were rapidly thawed by addition of 500 μL cell culture medium warmed to 37° C. Cells were placed in a humidified atmosphere for 24 h and then dissociated using 0.25% trypsin plus 1 mM EDTA in balanced salt solution. The number of viable cells was then determined by counting with a haemocytometer (Sigma Aldrich Co. Ltd, UK) at room temperature after 1:1 dilution of the sample with 0.4% trypan blue solution (Sigma Aldrich Co. Ltd, UK). The initial cell medium was discarded such that any non-attached cells were not included in the assessment. The percentage of recovered cells was calculated by dividing the number of cells with intact membranes after freezing and thawing by the number of cells present prior to freezing (i.e. after application of pre-treatments), multiplied by 100.

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