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Biological and Environmental Phenomena at the Interface
Synthetic Polyampholytes as Macromolecular Cryoprotective Agents Jing Zhao, mitchell johnson, Robert B Fisher, Nicholas A. D. Burke, and Harald D.H. Stöver Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01602 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018
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Synthetic Polyampholytes as Macromolecular Cryoprotective Agents J. Zhao, M. Johnson, R. Fisher, N.A.D. Burke, H.D.H. Stöver* Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON L8S 4M1, Canada
Keywords: polyampholytes, cryoprotective agent, cell viability
Abstract A series of polyampholytes based on different molar ratios on N,N-dimethylaminopropyl methacrylamide (DMAPMA), acrylic acid (AA) and optionally, N-tert-butylacrylamide (t-BuAAm), were prepared by free radical copolymerization, and tested as DMSO-free cryoprotective agents for 3T3 fibroblast cells by using a standard freeze-rethaw protocol. Polybetaines prepared by reaction of DMAPMA homo and copolymers with 1,3propanesultone were used as additional controls. Results showed strong effects of copolymer composition, molecular weight, polymer and NaCl concentrations, on postthaw cell viability. Binary (DMAPMA/AA) copolymers showed best post-thaw cell viability of 70% at a 30/70 mol% ratio of DMAPMA/AA, which increased to 90% upon introduction of 9 mol% t-BuAAm while maintaining the 30/70 mol% cation/anion ratio. The use of acrylamide linkages in DMAPMA ensures absence of hydrolytic loss of cationic side chains. These polyampholytes were found to decrease ice crystal size and to form a polymer-rich, ice-free layer around cells, reducing damage from intercellular ice 1
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crystals during both freezing and thawing steps. These polyampholytes also dehydrate cells during freezing which helps protects cells from intracellular ice damage. While cell viability immediately after thawing was high, subsequent culturing revealed poor attachment and long-term viability, which is attributed to residual cell damage from intracellular ice formation. Addition of 2 wt% DMSO or 1 % BSA to the polymer-based freeze medium was found to mitigate this damage, and result in post-thaw viabilities matching those achieved with 10 wt% DMSO.
Introduction Successful storage of cells and tissues at cryogenic temperatures requires cryoprotective agents to protect cells from ice crystal damage during freezing as well as thawing steps, ensuring good post-thaw cell viabilities. Natural cryoprotective agents include antifreeze proteins found in certain vertebrates, plants, fungi and bacteria, which can inhibit ice crystal growth and recrystallization.1 Small polar molecules such as glycerol and DMSO are also commonly used as cryoprotective agents: glycerol at 10-20 wt% was shown to protect cock spermatozoa at -80 oC,2 and is now used at up to 40 wt% for freezing red blood cells,3 while DMSO at 10% is used as key component in cryoprotective media used for many different cell lines. Anti-freeze proteins are not easily accessed in larger amounts, and have immunological issues where cell transplantation is anticipated.4,5 Both glycerol and DMSO are cellpenetrating cryoprotectants,6 and need to be removed immediately after thawing in order to minimize toxicity including changes in differentiation ability in, e.g., cardiac myocytes, 2
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neuron-like cells, and granulocytes.7-9 Removal of glycerol from thawed red blood cells requires approximately an hour to permit out-diffusion from within cells.3 Recent research into new cryoprotective agents has focused on both small molecule recrystallization inhibitors based on modified sugars,10-12 and on polymers that mimic anti-freeze proteins,13-17 such as polyampholytes.14,15,18,19 These new cryoprotective agents have low cytotoxicity, are active in lower concentration of up to 10 wt%, and do not require animal-derived components such as fetal bovine serum (FBS). Polyampholyte cryoprotective agents in particular can protect red blood cells,15 HT-1080,18 L929,19 certain stem cells,20 mouse oocytes21 and mouse embryos,22 with cell viabilities close to or better than those found with traditional methods. Polyampholytes are copolymers that carry cationic and anionic groups on different monomer units, and that show an anti-polyelectrolyte effect and protein-like isoelectric points, pH(I). Their properties can be tuned by incorporating different types of charged23 as well as hydrophilic or hydrophobic comonomers,24 and there is a large body of knowledge from their use in water purification,25 paper strength enhancement,26 oil recovery,27 non-fouling surfaces,28 drug delivery and cell adhesion.29-31 Their use as cryoprotectants is quite recent, and the mechanisms involved are still being explored. Gibson et al. suggested polyampholytes act as ice recrystallization inhibitors, and that a 1:1 charge ratio is crucial for their anti-freezing activity.14,15 Matsumura et al. found polyampholytes to coat the surface of cells, which can help dehydrate cells during freezing, and protect the cell membrane from damage by interstitial ice crystals.18,20,32
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Polyampholytes can serve as single component cryoprotective agent, requiring high ionic strength with slow freezing at 1 oC/min in a -80 oC freezer.18,20,32-34 Elsewhere, polyampholytes have been combined with hydroxyethyl starch15 and ethylene glycol21,22,35 in processes based on rapid freezing in liquid nitrogen. Some studies claim that cryoprotective polyampholytes require excess anionic charges,20,21,33,35 while others reported best results for stoichiometric polyampholytes having 1:1 charge ratios.15,18,34 It is clear that polyampholytes have the potential to complement or replace DMSO and other small molecule cryoprotectants, serving as non-penetrating agents. At the same time, key questions remain regarding the effects of net charge and molecular weight, and the actual mechanism of their cryoprotective action. Much of the work described to date focus on succinic anhydride-modified 4 kDa ε-poly-L-lysine20,21,33,35 and on ethanolamine-modified poly(methyl vinyl ether-alt-maleic anhydride),15 with only model studies on ice recrystallization inhibition reported for acrylic polymers,36 and none for more hydrolysis-resistant acrylamide-based polyampholytes. In this work, we investigate the cryo-protective potential of acrylamide-based polyampholytes prepared by free radical copolymerization. This approach permits combination of different monomer types into one chain, provided their reactivity ratios are known and managed, and such copolymers are being increasingly used in biomaterials research.29,30 Here we describe binary and ternary polyampholytes prepared by free radical copolymerization of N,N-dimethylaminopropyl methacrylamide (DMAPMA), acrylic acid (AA) and optionally, t-butyl acrylamide (t-BuAAm), having cationic/anionic (DMAPMA / AA) ratios of 30/70 and 50/50, low compositional drift and three different molecular weights. In contrast to controlled radical copolymerizations 4
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such as ATRP and RAFT, the conventional free radical polymerization used here avoids introducing low molecular weight ligands, or RAFT end groups, that might need to be carefully removed after polymerization, because the polymer made by RAFT would toxic depended on the types of RAFT agent and cells.37,38 We tested the ability of the polyampholytes to act as cryoprotective agents for 3T3 mus musculus fibroblast cells with a standard freezing protocol of about 1 oC/min to -80 oC, using 10 wt% DMSO as control.39,40 Results were compared to those obtained for a series of polybetaines prepared by reaction of the tertiary amines in PDMAPMA homopolymer and P(DMAPMA-co-AA) (60:40) with 1,3-propanesultone. After initial screening, we explored the effects of polymer concentration, ionic strength and hydrophobic comonomer t-BuAAm on post-thaw cell viability. Differential scanning calorimetry (DSC) and optical cryomicroscopy were used to study the polymer phase separation behavior and the interactions between polymer, ice crystals and cells during the freezing and thawing processes. Finally, we compared immediate post-thaw viability with longer-term viability, and explored the need for small amounts of DMSO or other additives to mitigate residual internal cell damage. This work significantly broadens the pool of potential cryoprotective agents and should stimulate the exploration of other well-defined, hydrolytically stable acrylamide-based polyampholytes prepared by radical copolymerization as cryoprotective agents. It explores the mechanism of standard slow cooling cryoprotection including the thawing process, which is important but not well studied. Acrylamide-based polyampholytes are easily prepared by free radical copolymerization from widely available, inexpensive
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monomers, without need for RAFT agents, ATRP ligands, and associated purification steps.
Materials and Experiments Materials: Acrylic acid (AA, 99%) was purchased from Sigma-Aldrich, and distilled before use. N,N-Dimethylaminopropyl methacrylamide (DMAPMA, 99%) from Sigma-Aldrich was neutralized using concentrated HCl, with stirring in an ice bath, and the concentration of the
final
DMAPMA.HCl
solution
was
measured
by
1
H-NMR.
N-(3-
Aminopropyl)methacrylamide hydrochloride (APM), ethylene carbonate (98%), N-tertbutylacrylamide
(t-BuAAm,
97%),
methylpropionamidine)dihydrochloride
1,3-propanesultone (Vazo-56),
(98%)
L-cysteine
2,2’-azobis(2hydrochloride
monohydrate (98%), 2-(cyclohexylamino)ethanesulfonic acid (CHES, 99%), fluorescein isothiocyanate isomer 1 (FITC, ≥90%), N,N-dimethylformamide (DMF, reagent grade) from Sigma-Aldrich were used as received. Deuterium oxide (D2O) was purchased from Cambridge Isotope Laboratories (Andover, MA). Sodium hydroxide and hydrochloric acid solutions (0.10 and 1.0 M each) were obtained from LabChem Inc. Dulbecco's Modified Eagle Medium (DMEM) (high glucose), phosphate-buffered saline (PBS), bovine calf serum (BCS), Trypsin-EDTA (0.05%) and Trypan Blue (0.4 wt%) were obtained from ThermoFisher Scientific. Polypropylene cryo tubes with external thread screw caps (2 ml) were obtained from Sarstedt AG & Co (Germany).
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Batch Free Radical Solution Copolymerization Synthesis of Polyampholytes Solution copolymerizations were carried out at 55 °C in 60 mL Nalgene bottles on a set of horizontal steel rollers fitted with an enclosure and a thermostatted heater pad, with 10% (w/v) total monomer loading, 1 mol% initiator (Vazo-56, relative to total monomer), in distilled water. Batch comonomer feed ratios for DMAPMA: AA of 45:55 and 30:70 were used to form the binary copolymers. Chain transfer agent (CTA, L-cysteine hydrochloride monohydrate) was added at 0, 1 or 5 mol% to control the molecular weight. For the DMAPMA/AA/t-BuAAm ternary copolymers, 5, 10 and 30 mol% t-BuAAm were used, with the DMAPMA/AA ratio held at 30:70, using a 1:1 (v:v) water/methanol mixture with 1 mol% CTA. Fluorescently labelled polyampholytes were synthesized as above, with incorporation of 1 mol% of fluorescein-modified APM prepared as described in the supporting information. The copolymerizations were monitored by removing small aliquots by glass pipette, diluting these with D2O, and determining conversion by 1H-NMR on a Bruker Avance 600. The areas of the vinyl signals of AA at 5.5 ppm and of DMAPMA at 5.3 ppm relative to an internal standard (ethylene carbonate) were used to determine the conversion of the two monomers, and polymerizations were stopped at 60-80% conversion. After polymerization, the cooled reaction mixtures were added into a 4.5-fold excess of acetone in order to precipitate the polymers, then the mixture was centrifuged at 3080 g for 10 min, and the supernatant removed. The polymers were re-dissolved in distilled water and the precipitation repeated a total of four times. The polymers were
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then dialyzed against distilled water in cellulose tubing (3.5 kDa MW cutoff) for 4 days with daily water changes. Finally, the purified polymers were isolated by freeze drying. The nomenclature used to describe the binary copolymers is PDAx, where D, A, and x stand for DMAPMA, AA, and mol% DMAPMA in the copolymer. Analogously, PDAtx is used for the ternary copolymers, where D, A, t and x stand for DMAPMA, AA, tBuAAm and mol% t-BuAAm in the terpolymer, with the ratio of DMAPMA and AA are kept at 3:7. The experimentally determined compositions are shown in Table 1.
Synthesis of Polybetaines PolyDMAPMA and poly(DMAPMA-co-AA) (60:40), prepared in presence of 1% CTA and purified as described above, were dissolved at 5 wt% in distilled water and the solution adjusted to pH 10 by addition of NaOH. Subsequently, 1.1 equivalents of 1,3propanesultone relative to tertiary amine groups were added and the reaction mixture stirred for 24 hrs at room temperature.41 The resulting polybetaines were purified by quadruple precipitation into excess acetone as described above. The
polybetaines
sulfonate]
and
poly[3-((3-methacrylamidopropyl)dimethylammonio)propane-1poly[3-((3-methacrylamidopropyl)dimethylammonio)propane-1-
sulfonate]-co-acrylic acid are abbreviated as PSPB and PSPBA60, where 60 is the mol% betaine in the copolymer.
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1
H-NMR Analysis of Copolymers
Copolymer (10 mg) was dissolved in 1 mL D2O, and the 1H-NMR spectra measured using a Bruker AV 600 NMR spectrometer, to detect any residual monomer left after purification. In addition, the comonomer ratios in PDAx were calculated from the areas of the DMAPMA methylene signals between 2.5 and 3.5 ppm (4H) compared to the combined signals of DMAPMA and AA between 0.5 to 2.5 ppm (13H of DMAPMA and 3H of AA). For other copolymers, the cumulative comonomer ratios were calculated by tracking the incremental monomer consumption during polymerization, as illustrated in Figure S1.
GPC Analysis The molecular weights (MWs) of the polymers were measured using an aqueous gel permeation chromatography (GPC) system consisting of a Waters 515 HPLC pump, Waters 717plus auto-sampler, three columns (Waters Ultrahydrogel-120, -250, -500; 30 cm, 7.8 mm dia.; 6 µm particles; 0-3, 0-50, 2-300 kDa MW ranges), and a Waters 2414 refractive index detector calibrated with narrow disperse poly(ethylene glycol) standards (Waters). The mobile phase was 1 M sodium acetate/acetic acid buffer adjusted to pH 4.7 for PDMAPMA and polybetaine, and 0.5 M NaNO3 buffered to pH 10 with 0.025 M CHES for other polyampholytes.
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Cell Culture, Cryoprotection and Proliferation NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% BCS. Cells were cultured at 37 °C in a 5% CO2 incubator. When the cells were 70-80% confluent they were washed 3 times with PBS and then treated with
trypsin
solution
(0.25%
[w/v]
trypsin
containing
0.02%
[w/v]
ethylenediaminetetraacetic acid in PBS), and incubated for 5 mins to detach the cells. The cells were centrifuged at 500 g for 5 min, then the pellet was resuspended with fresh DMEM without BCS. Polymer solutions with different polymer compositions, polymer concentrations, and NaCl concentrations were prepared in DMEM without BCS and adjusted to pH 7.3 - pH 7.6. All polymer solutions were syringe-filtered through 0.22 µm filters and stored at 4 oC. Approximately one million 3T3 cells were mixed in 1 mL cryoprotection solution in a 2 ml polypropylene cryo tube, the cryo tubes were placed in a Mr. FrostyTM plastic container filled with 2-propanol, which was then transferred to a -76 °C freezer without controlling the cooling rate (approximate 1 oC/min) for 24 h. For long term cryostorage, the vials were subsequently transferred into liquid nitrogen. Frozen vials were thawed in a 37 °C water bath for approximately 3 to 5 min, immediately diluted 10-fold with DMEM containing 10% BCS, and mixed well at room temperature. The resulting cell suspensions were centrifuged at 500 g for 5 min, and the pellets resuspended in 1 mL fresh DMEM with BCS. To count cells, 200 µl of cell suspension were stained with 20 µl trypan blue solution and counted on a hemocytometer. Cell viability was calculated as the ratio of viable cells to the total number of cells.
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After thawing, the cells were seeded at a cell density of 1 × 104/cm2 per well in 12 wellplates with 2 ml of DMEM with 10% BCS. Cells were monitored after 1 and 3 days incubation on a Zeiss Axiovert 200 optical microscope fitted with a camera and AxioVs40 V 4.8.2.0 software.
Statistical Analysis All cell data are expressed as the mean ± standard deviation (SD). A two-tailed TwoSample T-test Assuming Unequal Variances was used. Differences were considered statistically significant at a P value of
