Synthesis of Highly Biocompatible and Temperature Responsive

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Synthesis of Highly Biocompatible and Temperature Responsive Physical Gels for Cryopreservation and 3D Cell Culture Masanori Nagao, Jayeeta Sengupta, Diana Diaz-Dussan, Madeleine K. Adam, Meng Wu, Jason P Acker, Robert Ben, Kazuhiko Ishihara, Hongbo Zeng, Yoshiko Miura, and Ravin Narain ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00096 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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ACS Applied Bio Materials

Synthesis of Highly Biocompatible and Temperature Responsive Physical Gels for Cryopreservation and 3D Cell Culture

Masanori Nagao,1,2 Jayeeta Sengupta1, Diana Diaz-Dussan1, Madeleine Adam3, Meng Wu1, Jason Acker4,5, Robert Ben3, Kazuhiko Ishihara6, Hongbo Zeng1, Yoshiko Miura2 and Ravin Narain1* 1

Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada 2

3

Department of Chemical Engineering, Kyushu University, Fukuoka, Japan

Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa,

Canada 4

Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada 5

Centre for Innovation, Canadian Blood Services, Edmonton, Alberta, Canada

6

Department of Materials Engineering, The University of Tokyo, Tokyo, Japan

KEYWORDS: Atom transfer radical polymerization (ATRP), polymers, biocompatible, physical gels, thermo-responsive, cell culture, cryopreservation, ice recrystallization inhibitor

ACS Paragon Plus Environment

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ABSTRACT

There is considerable interest in the cryopreservation in 3D cell-culture, as structurally preserving intact cells and tissues is critical in utilizing these systems to promote cell differentiation and tissue organization. Temperature-responsive physical gels and zwitterionic polymers are useful materials as 3D scaffolds for cell culture which may also provide cryoprotection to the composite cells. Nevertheless, there has been a lack of relevant data for polymer systems that have both of these properties. In this study, highly biocompatible triblock copolymers were examined for their effectiveness both as gelators and as cryoprotectants. The triblock copolymers were synthesized with 2-methacryloyloxyethyl phosphorylcholine (MPC) and di(ethylene glycol) methyl ether methacrylate (DEGMA) via atom transfer radical polymerization (PDEGMA113–b–PMPC243–b–PDEGMA113). ABA triblock copolymers composed of hydrophilic “B” block and temperature responsive “A” block could form physical gels above their lower critical solution temperatures (LCST). PDEGMA113–b−PMPC243–b–PDEGMA113 triblock copolymer exhibited the LCST derived from DEGMA and assembled in micellar structures forming physical gels above the LCST. The mechanical properties of the physical gels were evaluated by rheological tests, and the low toxicity of PDEGMA113–b–PMPC243–b–PDEGMA113 was confirmed by MTT assay. Interestingly, the triblock copolymer showed ice recrystallization inhibition (IRI) activity which was determined to be suitable for the cryopreservation of several cell lines. In vitro studies were conducted to demonstrate the cryo-protectant properties and the formation of two and three-dimensional (2/3D) cell culture scaffolds with high biocompatibility. This stimuli-responsive gelator polymers can therefore be useful for cryopreservation of different cells

models,

and

a

promising

material

for

3D

cell-culture.


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INTRODUCTION Cell engineering has gained importance in the area of biomedical science owing to its significant role in assembling functional constructs to maintain, recover and improve cells, tissues and organs. In particular, three-dimensional (3D) cell culture techniques has attracted significant interest as they were found to promote cellular differentiation and tissue organization that is not feasible in conventional 2D cell culture systems.1, 2 3D scaffolds allow cells to grow and also maintain the original cell morphology and functionality, while the cells on twodimensional scaffolds differ structurally and functionally as compared to their native counterparts.1,

2

It has been experimentally shown that cell shape and the surrounding

environment affect the metabolism and behavior of cells.3, 4 Hence, from this perspective, it is preferable to use a 3D scaffold for cell culture which is analogous to the in vivo environment. Cryopreservation of different cells (normal and cancer cell lines) including sensitive cells such as stem cells, fibroblasts and red blood cells (RBCs) is a crucial technique in cell engineering;5–7 as it is essential to keep a constant supply of cells for use in transfusion, transplantation, and for research purposes. Dimethyl sulfoxide (DMSO) is commonly used as a cryo-protectant due to its high membrane permeability and ability to colligatively suppress ice formation.5–7 Nevertheless, DMSO has limitations such as toxicity to cells and the need for its post-thaw removal to avoid damage to the mitochondria, disruption to intracellular cell signaling, and/or alteration of epigenetic profiles8. If transfused into patients, DMSO leads to a vast range of clinically undesirable symptoms8. Thus, biocompatible cryo-protectants substitutes are required with low or no toxicity. Extremophile organisms has been reported to produce certain natural compounds that promote ice-growth inhibition and regulate ice formation. These compounds can be divided into the antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs). The addition of these compounds to aqueous solutions regulates three key factors associated with ice growth: dynamic ice shaping (DIS, the modification of the morphology of a growing ice crystal; ice recrystallization inhibition (IRI, the ability to suppress ice growth); and thermal hysteresis (TH, a non-equilibrium depression of the freezing point).9 Recently, it has been demonstrated that certain kinds of polymers such as poly(vinyl alcohol) and zwitterionic polymers exhibited macromolecular antifreezes properties, modulating ice formation and growth.10–16 Although the detailed mechanism is still under investigation, polymer-based cryo-protectants like copolymers


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of 2-(dimethylamino)ethyl methacrylate and methacrylic acid and carboxylated polysaccharides (dextran) act as IRI-active polymers.9 We are interested in developing polymeric materials that have IRI-active properties and could function as both cryo-protectants and 3D scaffolds which could be useful for cell and tissue engineering applications. Hydrogels have emerged as a potential novel cryo-protectant and cell scaffold candidate for use in cell engineering. Hydrogels have intrinsic properties such as network-structures, high water content and flexible matrix, which are suitable for 3D cell culture.17–21 Stimuli-responsive hydrogels that respond to environmental stimuli such as temperature, solution pH, or light irradiation, are preferred, as their properties can be tuned as a function of one or more of these parameters for practical uses.22–30 In particular, temperature responsive hydrogels are promising in the biomedical field as they can be designed to mimic the function of natural tissues at body temperature and support tissue growth.31–34 Furthermore, hydrogels composed of zwitterionic residues are expected to exhibit ice-growth inhibition properties and to work as cryoprotectants.16 However, such uses of hydrogels have not been studied so far. Here, we propose the fabrication of a highly biocompatible ABA triblock copolymer and its possible practical application in the cryopreservation of cells. ABA triblock copolymers which contain the central hydrophilic “B” block and the outer temperature-responsive “A” blocks form “flower” like micelles in water at or above the lower critical solution temperature (LCST).30–38 The physical gels are formed at certain polymer concentrations resulting from the cross-linking of the micellar structures, and the gel structure can be used for 3D cell culture. To provide icegrowth inhibition properties for the physical gel, 2-methacryloyloxyethyl phosphorylcholine (MPC) is used. MPC has a zwitterionic structure, and MPC-based polymers are known to be highly biocompatible and non-toxic.39 The ABA triblock copolymer was synthesized by atom transfer radical polymerization (ATRP) process with MPC for the hydrophilic middle block and di(ethylene glycol) methyl ether methacrylate (DEGMA) for temperature responsive outer blocks. As expected, the triblock copolymers exhibited ice modification properties that were confirmed by ice recrystallization inhibition (IRI) experiments. The triblock copolymers were then tested for the cryopreservation of different cell lines. It is useful that the same polymers behave both as cell culture scaffold and as a cryo-protectant in terms of practical handling. This study reports a


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promising highly biocompatible and temperature responsive hydrogel for cryopreservation and opens

up

new

avenues

and

possibilities

in

the

cell

engineering

domain.


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EXPERIMENTAL SECTION Materials 2-methacryloyloxyethyl phosphorylcholine (MPC) was synthesized according to a previous protocol.38 Di(ethylene glycol) methyl ether methacrylate (DEGMA, 95%), diethyl-meso-2,5dibromoadipate (DEDBA, 98%), copper(I) bromide (CuBr, 99.999%), 2,2’-bipyridine (bpy, 99%) and thiazolyl blue tetrazolium bromide (MTT, 98%) were purchased from Sigma-Aldrich Chemicals (Oakville, ON, Canada). Streptomycin (10 mg mL−1), penicillin (10 000 U mL−1), DMEM/F12 media, Opti-MEM (OMEM), 0.25% trypsin−EDTA, Dulbecco's modified Phosphate Buffer Saline (PBS) and fetal bovine serum (FBS) were purchased from Gibco. Life/Dead cell imaging kit was purchase from ThermoFisher (Burlington, ON, Canada). The solvents were purchased from Caledon Laboratories Ltd. (Georgetown, Canada), and were used without further purification. Synthesis of ABA triblock copolymers (PDEGMA113–b–PMPC243–b–PDEGMA113). MPC (1.86 g, 6.3 mmol) and DEDBA (9 mg, 2.5 µmol) were dissolved in 2.5 mL methanol. The solution was degassed for 20 min under nitrogen at 0 °C. CuBr (7.2 mg, 50 µmol) and bpy (16 mg, 100 µmol) were dissolved in 400 µL acetonitrile, and added into the above solution with syringe. The mixture was degassed for 10 min under nitrogen at 0 °C, and was kept stirring for 4 h at 30 °C. DEGMA (1.13 g, 6 mmol) was mixed along with 2 mL methanol, and degassed by freeze-thawing cycles (3 times). The degassed DEGMA solution was injected to the polymer solution by using a syringe. The reaction was kept stirring at 30 °C for 19 hours. The color remained brown. The polymer solution was diluted with excess amount of methanol and then passed through a short silica column to remove the residual copper salt. The solution turned colorless. The product was dialyzed against DI water for 24 h and then freeze-dried to obtain a white solid (2.46 g, 82% yield). NMR Spectroscopy Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian 500 MHz spectrometer using D2O as the solvent. Gel Permeation Chromatography (GPC)


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Average molecular weights and polydispersity were determined by conventional gel permeation chromatography (GPC) system using two WAT011545 water columns at a flow rate of 1.0 mL/min using a 0.5 M sodium acetate/0.5 M acetic acid buffer as eluent. Monodisperse Pullulan standards (Mw = 5900−404 000 g mol-1) were used for calibration. Dynamic Light Scattering Measurements The copolymer sizes at different temperatures were determined using Zeta Plus-Zeta Potential Analyzer (Brookhaven Instruments Corporation) at a scattering angle θ = 90°.PDEGMA113–b– PMPC243–b–PDEGMA113 was dissolved in PBS (0.025 wt%). The data were recorded with Omni size software. Gel formation The synthesized triblock copolymers were dissolved in 10 mM PBS at 10, 15, 20, 25 wt%. The polymer solutions were kept at 40 °C for 15 min, and observed. Rheological Measurements AR-G2 Rheometer (TA instruments) was used to conduct the rheology test with a 20mm 2.008º cone plate geometry. The plate is set at 20 ºC before the samples were dropped. Temperature sweep is conducted within the range of 20–50 ºC, 15–50 ºC, 0–50 ºC for 15, 20 and 25 wt% samples respectively with a heating rate of 2 ºC/min at strain of 1% and angular frequency of 10 rad/s. Frequency sweep was done at each temperature with strain of 1%. Cell Culture Cell lines [HeLa (ATCC®CCL-2TM), CHO-K1 (ATCC ® CCL-61™), PC-3 (ATCC ® CRL1435™), FaDu (ATCC ® HTB-43™), WI-38 (ATCC ® CCL-75™), K-562 (ATCC ® CCL243™), Skin fibroblasts (BJ ATCC ® CRL-2522™)] were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic antimycotic (100 units of penicillin, 100 µg streptomycin, 0.0085% fungizone) in a humidified atmosphere at 37 °C and 5% CO2. For K562 cells RPMI 1640 medium was used keeping the same other conditions. At about 80% confluency, the cells were sub-cultured by dissociating with 0.25% trypsin in versene twice per week.


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MTT Assay HeLa cells were seeded in 96-well tissue culture plates at the density of 8 × 104 cells per well. The cells were allowed to adhere overnight, and fresh media with varied polymer concentrations (prepared along with media) were added in duplicate. The cells were allowed to grow for 24 hours in the presence of PDEGMA113–b−PMPC243–b−PDEGMA113. The untreated cells and media alone were used as positive and negative controls, respectively. 100 µL MTT dye solution (1 g/L in sterilized PBS) was then added per well, and the plate was incubated for 4 h, followed by the addition of 100 µL of lysis buffer (DMSO: 2-propanol = 1: 1). The plate was then read at 570 nm and percent cell viability was determined by the following formula: % metabolic activity = 100 × (treated cells – negative control) / (positive control – negative control) Cryopreservation studies PDEGMA113–b–PMPC243–b–PDEGMA113 was dissolved in sterilized DMEM with 10% FBS at concentrations ranging from 0.3 to 15 wt % overnight and used for the cells (HeLa, CHO, PC3, FaDu, WI-38, Skin fibroblasts). For K562 cells, RPMI-1640 medium with 10% FBS was used to dissolve the polymers of different concentrations. Each cell line suspension of HeLa, CHO, PC3, FaDu, WI-38, Skin fibroblasts and K562 (106 cells/mL) were centrifuged. The supernatant was removed and the cells were re-suspended in 1 mL of each polymer solution. The polymer/cell suspension was divided into each microtube (per 100 µL) in triplicates and stored at −80 °C freezer. After a week, the microtubes were thawed immediately in a water bath maintained at 37 °C with gentle shaking. Membrane integrity was determined using tryphan blue by counting cells on a haemocytometer. Cell membrane integrity was calculated by the following formula: membrane integrity (%) = 100 × (number of intact cells) / (number of intact cells + number of damaged cells) The cell recovery percent after thawing was also calculated by counting the cells pre- and postthawing. Post thawing cell recovery was calculated by the following formula: post thawing cell recovery (%) = 100 × (post-thaw cell count)/ (pre-thaw cell count)


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HeLa, CHO, PC3, FaDu cells were cryopreserved using polymer concentrations ranging from 0.3 to 15 wt% and stored frozen for 7 days. The WI-38, Skin fibroblasts and K562 cells were cryopreserved using polymer concentrations ranging from 3 to 15 wt% and stored frozen for 24 h. Cryopreservation Studies using Temperature Controlled Freezing Method. The cryopreservation solution consists of 15 wt% of poly(DEGMA113–b–PMPC243–b– PDEGMA113 ). 1x106 cell suspensions (FaDu, PC3 and Skin Fibroblast) were mixed with freezing medium (DMEM + 10% DMSO) and DMEM + 3 wt% polymer by slowly adding the freezing medium to the cell suspension. The cells and freezing medium were dispensed into cryovials (Biologix), and incubated in an ice bath for 5 min prior to placement on an electronic, controlled-rate freezer (Asymptote Via Freeze Research) that was precooled to−8 °C. Samples were held for 10 min at −8 °C before ice nucleation was induced by touching the outside of the plastic cryotubes with pre-cooled (in liquid nitrogen) forceps. Controlled nucleation is performed to ensure that ice nucleation occurs at the same sub-zero temperature of −8 °C in each vial. All samples were then held at −8°C for 10 min before being cooled at a rate of 1 °C/min to − 80 °C and then transferred to a -80 °C freezer for overnight storage. For the thawing procedure, the cryovials were rapidly warmed in a water bath at 37 °C until all ice crystals disappeared, which took approximately 1 min. The cells aliquots were resuspended in warm DMEM + 10% FBS medium and the cells were collected by centrifugation (200 × g for 5 min). The supernatant solution was removed, and cell pellets resuspended in 5 mL fresh culture medium. Cells were plated in a 60 mm plate and cultured for 24 h in the conditions described above to evaluate the proliferation rate according to the following formula: cell proliferation (%) = 100 × (cell count after 24 h of incubation)/ (post-thaw cell count) 2D/3D Cell Scaffold Evaluation A polymer solution of 15 wt% was prepared by dissolving the polymers in serum-free highglucose DMEM containing 100 U mL 1 penicillin, 0.1 mg/mL 1 streptomycin, 10% FBS and 2 mM L-glutamine. The hydrogel was formed two ways: PC3 cells (1x105) were suspended in a poly(DEGMA113–b–MPC243–b–DEGMA113 )solution to form a cell/hydrogel (3D cell culture) at 37°C and the hydrogel was form alone (2D cell culture) and then cells were grown on top. Cell/hydrogel constructs were formed after incubation for 24 h cultured at 37 °C in a humidified


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atmosphere incubator containing 5% CO2. Live/Dead Assay was performed to evaluate membrane integrity. Two controls were used: Live Control (DMEM + 10% FBS) and Dead Control (70% ethanol exposure for 30 min). A 2X Stock Solution of Live/Dead reagent was added to the cells (300 ߤL) and incubated for 15 min at room temperature. Cells were image by Confocal Microscopy. The live cell component produces an intense, uniform green fluorescence in live cells at ex/em 488 nm/515 nm. The dead cells produce a predominantly nuclear red fluorescence at 570 nm in cells with compromised cell membranes; a strong indicator of cell death and cytotoxicity. Quantification and image processing was done using Imaris Image Analysis software. Ice Re-Crystallization Inhibition (IRI) Activity Sample analysis for IRI activity was performed using a cryo-microscope consisting of a Nikon 80i fluorescent microscope with a long working distance condenser and objectives, CCD cameras (Hammamatsu ORCA) interfaced to a personal computer and a convection cryomicroscope stage (Linkam FDCS196). A sample of 2 µL was inserted into a 15 mm inner diameter quartz sample holder and covered with a circular glass coverslip. The quartz dish was then placed on a temperature-controlled silver block inside the cryo-microscope stage. A flow of dry nitrogen was applied to prevent humidity in the ambient air from condensing on the upper window. The experimental procedure was as follows: there were two groups of samples, polymers (3 and 15 wt%) which were dissolved in DMEM and 10% FBS solution. For control group, only DMEM and 10% FBS solution was used. All the samples were rapidly cooled from room temperature to −40 °C at 50 °C /min with no controlled nucleation. Samples were held for 2 min at −40 °C and then warmed at 5 °C /min to −5 °C where the samples were held again for 2 min. The splat-cooling assay48,49 was used to obtain the IC50 for the ice recrystallization inhibition (IRI) activity of the sample. 10 µL of the polymer dissolved in phosphate-buffered saline (PBS) was dropped two meters above a polished aluminum block cooled to -80 °C with dry ice. The sample droplet immediately froze upon contact with the block and created a wafer roughly 1 cm in diameter and 20 mm thick. Using pre-cooled tools, the wafer was transferred to a cryostage maintained at -6.4 °C (Alpha Omega Instruments, Series 800 temperature controller). After


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annealing for 5 minutes at -6.4 °C, the wafer was photographed between crossed-polarizing filters using a Nikon CoolPix 5000 digital camera fitted to a microscope. Upon selection of one image per run, ice crystals with well-defined boundaries were circled in ImageJ and the area of each circled ice crystal was calculated. Ice crystal areas obtained were sorted into discrete bins based on size (bins increase in increments of 0.001 mm2) for each image analyzed. Summing the areas of each crystal within a bin and dividing by the sum of the areas of all crystals in the image provided the proportionate area of each bin. Rate constants were then determined and normalized based on the average rate constant determined for the PBS control. A dose-response curve was generated based on the normalized rate constants, vnorm, for each test concentration and the corresponding log values of the concentration. A two-parameter sigmoidal curve was fit to the data to obtain the half maximal inhibition concentration (IC50). Concentrations were tested in triplicate as well as the PBS control. Error is reported as the standard error of the mean (SEM).

RESULTS AND DISCUSSION Synthesis of temperature responsive triblock copolymers The ABA-type triblock copolymers were synthesized by a “one-pot” ATRP process according to the previous reports (Scheme 1).37 The MPC hydrophilic block in the center was first synthesized using the ATRP bifunctional initiator (DEDBA). The feed ratio of [MPC]: [DEDBA] was set to 250:1. After 4 h, the MPC conversion was found to be 97% as determined by 1H NMR (Figure S1). After MPC polymerization, degassed DEGMA was subsequently added into the solution. The feed ratio of [DEGMA]: [Br-PMPC-Br] was 240:1. DEGMA conversion was 94% as determined by 1H NMR (Figure S2). Based on these almost complete monomer consumption, the structure of the triblock copolymer was defined as PDEGMA113–b–PMPC243–b–PDEGMA113. After purification, the structure of PDEGMA113–b–PMPC243–b–PDEGMA113 was confirmed by 1

H NMR (Figure S3). While the molecular weight of the MPC block was determined by GPC

analysis (Mn = 70 kDa, Mw/Mn = 1.51), the molecular weight of PDEGMA113–b–PMPC243–b– PDEGMA113 could not be determined with our standard aqueous and organic GPC systems.


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Scheme 1. Synthesis of the triblock copolymer (PDEGMA113–b–PMPC243–b–PDEGMA113) by “one-pot” ATRP process. Characterization of PDEGMA113–b–PMPC243–b–PDEGMA113 The LCST and hydrodynamic diameter of PDEGMA113–b–PMPC243–b–PDEGMA113 was determined by dynamic light scattering (DLS) measurements. Scattered light intensity was 20 kcps below 30°C, and increased to 140 kcps along with the increase in temperature (Figure 1a). The increase of the scattered light intensity indicates that PDEGMA113–b–PMPC243–b– PDEGMA113 started to form assemblies above 32 °C which was determined as the lower critical solution temperature (LCST) of PDEGMA113–b–PMPC243–b–PDEGMA113. The stability of the intensity above 42 °C showed that assembling of the polymers was settled. The hydrodynamic diameter of PDEGMA113–b–PMPC243–b–PDEGMA113 at lower temperature was 50 nm, and started to increase above 30°C (Figure 1b). The diameter increased until 40°C, and the diameter was 95 nm above 40°C. The diameter below 30°C suggested that PDEGMA113– b–PMPC243–b–PDEGMA113 formed small aggregates at low temperature. PDEGMA113–b– PMPC243–b–PDEGMA113 was expected to dissolve in water below 30°C because DEGMA blocks of PDEGMA113–b–PMPC243–b–PDEGMA113 are relatively hydrophilic below the LCST.41 However, it is considered that DEGMA blocks were not completely hydrophilic even below LCST and PDEGMA113–b–PMPC243–b–PDEGMA113 did not dissolve molecularly


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forming aggregation.34 Conversely, the DEGMA blocks dehydrates above the LCST and PDEGMA113–b–PMPC243–b–PDEGMA113 bridged the neighboring aggregations at or above 40°C.

(c)

(d)

Figure 1. Scattered light intensity (a) and hydrodynamic diameter (b) of PDEGMA113–b– PMPC243–b–PDEGMA113 as a function of temperature (0.025 wt% in PBS buffer). Digital photographs of (c) the solution at 25°C and (d) the free-standing gel at 40°C formed with PDEGMA113–b–PMPC243–b–PDEGMA113 solution (20 wt% in PBS buffer). Physical gel formation The synthesized triblock copolymer in water was expected to form a physical gel at a certain critical concentration as a result of the crosslinking of the aggregation. PDEGMA113–b– PMPC243–b–PDEGMA113 was dissolved in PBS buffer at 10, 15, 20, 25 wt%. The polymer did not dissolve completely above a concentration of 30 wt%. PDEGMA113–b–PMPC243–b– PDEGMA113 formed a free standing gel above 15 wt% at 40°C (Figure 1c-d). It shows that the neighboring micelles were bridged, and formed a physical gel. Irrespective of the temperature,


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the minimum concentration of PDEGMA113–b–PMPC243–b–PDEGMA113 required for forming a free-standing gel was 15 wt%. The minimum polymer concentration for forming the physical gel was relatively higher than other ABA triblock copolymers.30–33, 35, however, this result corresponds to previously reported studies about MPC triblock copolymers.36, 37 It is suggested that hydrophobicity of DEGMA block at high temperature was not enough or the steric hindrance of diethylene glycol structure of DEGMA was disadvantageous for bridging the neighboring aggregation. Mechanical properties of the physical gel The mechanical properties of the gels formed with PDEGMA113–b–PMPC243–b–PDEGMA113 were studied by rheological tests. The measurement proceeded with swept angular frequency (ω) and temperature. Figure 2(a) shows the results of oscillatory shear measurements with frequency sweeping that was performed on the 20 wt% polymer concentrations solution at 25, 29 and 40°C. At 25°C, the storage modulus (G’) was smaller than the loss modulus (G”), which indicates that the sample was a liquid. At 40°C, the sample formed a free-standing gel. G’ was larger than G” and it was a typical solid-like behavior. Both G’ and G” increased with increasing frequency. At 29°C, the sample showed intermediate behavior, and G’ and G” had similar magnitudes of value. This indicates the sol-gel transition of the sample and approximates the gelation point. The polymer solutions at of different concentration (15, 25 wt %) showed similar rheological behavior at three typical temperatures (Figures S4).

(a)

(b)

(c)


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Figure 2. (a) Frequency dependences of the dynamic shear moduli (G’ and G”) for the 20 wt% PDEGMA113–b–PMPC243–b–PDEGMA113 solution in PBS measured at strain of 1% and three indicated temperatures. (b) Temperature-dependent dynamic shear moduli (G’ and G”) for the 20 wt% PDEGMA113–b–PMPC243–b–PDEGMA113 solution in PBS buffer measured at a heating rate of 2ºC/min, at strain of 1% and with angular frequency of 10 rad/s. Red circles and blue circles indicate heating curve and cooling curve, respectively. (c) Temperature-dependent dynamic shear moduli (G’ and G”) for the three PDEGMA113–b–PMPC243–b–PDEGMA113 gels with different polymer concentrations measured at a heating rate of 2 ºC/min, at strain of 1% and with angular frequency of 10 rad/s (heating).

The dynamic shear moduli of 20 wt% PDEGMA113–b–PMPC243–b–PDEGMA113 solution were measured with sweeping temperature from 15 to 50°C (Figure 2(b)). Transitions for both G’ and G” were observed as a function of temperature. Below 29°C, the sample was liquid and G’ was smaller than G”. G’ was larger than G” above 29°C, and the sample showed solid-like behavior indicating that the critical gelation point of 20 wt% PDEGMA113–b–PMPC243–b– PDEGMA113 solution was 29°C. Similar behavior was observed in both heating and cooling process of the samples. It shows the sol-gel transition was reversible and no hysteresis was observed. PDEGMA113–b–PMPC243–b–PDEGMA113 solution at 15 and 25 wt% showed similar behavior, while a hysteresis was observed at low temperature with 25 wt% solution (Figures S5, S6). In Figure 2(c), temperature-dependent dynamic shear moduli of PDEGMA113–b–PMPC243–b– PDEGMA113 solution at the three concentrations were compared (15, 20, 25 wt%). The critical gelation temperatures were 32, 29, and 19°C for 15, 20, and 25 wt% gel, respectively. Both G’ and G” of 25 wt% gel were the largest and those of 15 wt% gel were the smallest. As expected, the robustness of the gels was dependent on the polymer concentration. The G’ increased from 25 to 271 Pa at 37°C with increasing polymer concentration from 15 to 25 wt%. A similar behavior was observed in cooling the samples from high temperature (Figure S7). In all conditions, the values of G’ was below 103 Pa, and this result corresponds to the results of other triblock copolymers composed of central MPC block.35-36,42 Considering the G’ of other


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triblock copolymers is over 103 Pa,32-33,43 it is considered the physical gels formed with the MPC triblock copolymers are relatively weak than other physical gels formed with ABA triblock copolymers. Cell viability studies The biocompatibility of PDEGMA113–b–PMPC243–b–PDEGMA113 was evaluated by MTT assay with HeLa cells (Figure S8). The HeLa cells were incubated for 24 h in PDEGMA113–b– PMPC243–b–PDEGMA113 solution at 37 °C. The polymer concentrations were varied from 0.01 to 10 mg/mL. The cell viabilities were almost 100% at each concentration. It indicates that PDEGMA113–b–PMPC243–b–PDEGMA113 was highly biocompatible for HeLa cells. Cryopreservation studies Cryoprotective efficacy of PDEGMA113–b–PMPC243–b–PDEGMA113 was evaluated in different cell lines. It has been reported that zwitterionic polymers can work as cryoprotective agents.15-16 PDEGMA113–b–PMPC243–b–PDEGMA113 has MPC as the middle block, therefore this triblock copolymer is expected to work as a cryo-protectant. Conventional cryopreservation conditions for preserving cell lines involves addition of ~10 wt% DMSO in cell media. So, to understand the influence of PDEGMA113–b–PMPC243–b–PDEGMA113 as a cryo-protectant, a range of trial cryopreservation tests were conducted using 10 wt% DMSO as a control group and different concentrations (0.3, 1, 3 and 15 wt%) of the polymer. Cryopreservation solutions were prepared by dissolving the triblock copolymer in DMEM with 10%FBS, and the polymer concentration was varied to 0.3, 1, 3 and 15 wt%. HeLa cells were then re-suspended in the cryopreservation solution (106 cells/mL). The cell suspensions were evaluated using two different freezing procedures: 1) cells were directly stored in −80 °C; 2) cells were placed on an electronic, controlled-rate freezer. Cells in DMEM with 10% DMSO and 10% FBS were used as positive control. For the first freezing protocol, cells were counted after one week of storage at -80 °C. The 3 and 15 wt% polymer solutions showed high cell viability similar to the 10% DMSO solution and the post-thaw membrane integrity were more than 95% (Figure 3a). Conversely, 0.3 and 1 wt% polymer solutions showed low post thaw membrane integrity (below 30%). Similar


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results were obtained with other immortalized cell lines such as CHO, PC3, FaDu cells (Figure 3a). These results indicate that PDEGMA113–b–PMPC243–b–PDEGMA113 work as a cryoprotectant at certain polymer concentrations and requires it to be present in sufficient amount to retain its activity. Furthermore, it is hypothesized that hydrophobicity of DEGMA blocks contributed to the high cell viability according to previous studies.15, 44

Figure 3. Viability of cancer cell lines (a) cryopreserved for 7 days in PDEGMA113–b–PMPC243– b–PDEGMA113 solution and 10%DMSO solution in DMEM + 10% FBS. (b) cryopreserved for 24 hr in PDEGMA113–b–PMPC243–b–PDEGMA113 solution and 10% DMSO solution in media (DMEM/RPMI) with 10% FBS. Post thawing cell recovery percent of cancer cell lines cryopreserved for (c)7 days in PDEGMA113–b–PMPC243–b–PDEGMA113 solution and 10% DMSO solution in DMEM + 10% FBS and (d) for 24 h in PDEGMA113–b–PMPC243–b– PDEGMA113 solution and 10% DMSO solution in media (DMEM/RPMI) with 10% FBS. From the results obtained, it was confirmed that PDEGMA113–b–PMPC243–b–PDEGMA113 is a potential cryopreservation agent, hence the polymer was tested further in other cell lines


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(W138, Skin fibroblasts and K562) at a concentration range of 3 and 15 wt% with the first freezing protocol and cell viability was assessed after frozen storage for 24 h (Figure 3b). As expected, the W138, skin fibroblasts and K562 cells showed high post-thaw membrane integrity (over 90%) with PDEGMA113–b–PMPC243–b–PDEGMA113. Although the WI38, skin fibroblast cells are more sensitive to stimuli compared with the immortalized cell lines, the cell membrane integrity was high. The results also demonstrate that PDEGMA113–b–PMPC243–b–PDEGMA113 is equally potent for non-adherent cells (K562). The cell recovery percent was also calculated from the pre- and post-thaw cell counts. More than 50% cell recovery post-thawing was seen in cell lines cryo-preserved with 10% DMSO except in the CHO cell line. For the polymer hydrogel, variable results were obtained with different cell lines (Figure 3c-d). The lowest cell recovery ratio was obtained for the CHO cell line for both the control and polymer groups compared to the other cell lines. Polymer solutions at concentrations of 3 and 15 wt% showed high post-thawing cell recovery in the cell lines HeLa, FaDu, W138, K562 and Skin Fibroblasts. For PC3, FaDu and HeLa cell lines, the post-thawing recovery percentage was more with the 3 wt% polymer compared to the 15 wt % polymer solution. Conversely, the 0.3 and 1 wt% polymer solutions showed very low post-thaw cell recovery (below 20%) in HeLa, CHO and PC3 cells, while FaDu cells showed a good percentage of recovery at 0.3, 1 and 3 wt% polymer solutions (more than 40%). High cell recovery (more than 40%) comparable to the 10% DMSO control group was obtained in the cell lines W138, K562 and Skin Fibroblasts. The 10% DMSO control groups of HeLa, PC3, FaDu and K562 cells showed the highest recovery percentage (more than 65%). Polymer solution containing 3 and 15 wt% PDEGMA113–b–PMPC243–b–PDEGMA113 showed the same post-thawing recovery percentage of cells as DMSO, while 0.3 and 1 wt% PDEGMA113–b–PMPC243–b–PDEGMA113 polymer solutions showed the lower recovery percentages. The results indicate that PDEGMA113–b–PMPC243–b–PDEGMA113 has cryopreserving properties though, the post-thawing cell recovery percentage varies from cell line to cell line and also for different polymer concentrations.

For the second freezing method, cells were placed on an electronic, controlled-rate freezer. Controlled nucleation was performed to ensure that ice nucleation occurs at the same sub-zero temperature of −8 °C in each vial. All samples were then cooled at a rate of 1 °C/min to – 80 °C


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and then transferred to a -80 °C freezer for overnight storage. The rapid freezing method used on the first sets of experiments, can introduce cell osmotic damage and recrystallization phenomena that can cause mechanical damage to cells and also introduce lethal intracellular ice formation45. It is generally necessary to have a second step in which the samples are cooled from −80 °C to cryogenic temperatures to prevent these damages. Peptide and glycopeptide analogs of biological antifreeze compounds, like synthetic polymers and small molecules, have the potential to act as cryo-protectants by promoting thermal hysteresis, inhibiting ice recrystallization, and possibly improving membrane structural integrity45. PDEGMA113–b–PMPC243–b–PDEGMA113 have shown great cell viability (more than 80 %) after post-thawing in PC3, Skin Fibroblast and FaDu cells and a post-thaw cell recovery of more than 35%; being even more efficient than DMSO for Skin Fibroblast and FaDu cells (Figure 4 a-b). Post cryopreservation apoptosis and necrosis are normally observed within 6 to 24 h after postthaw culture. As a result, massive loss of cell viability and cellular function occur due to cryopreservation46. To evaluate the post-thaw cell proliferation, cells were incubated for 24 h after rapid thawing as describe earlier. In Figure 4c a higher proliferation rate was exhibited by the DMSO control than the 3 wt% polymer concentration (more than 40% for PC3 and FaDu compared to 20-25% with the polymer solution) but in the case of skin fibroblast the polymer solution showed better cell proliferation efficiency in comparison to the DMSO control. Still the polymer (PDEGMA113–b–PMPC243–b–PDEGMA113) mechanism of ice inhibition needs to be evaluated to further improve the cryo-protectant properties to understand and modulate cellular pathways involved in cryopreservation apoptosis.


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Figure 4. Post-Thaw a) cell viability and b) cell recovery percent of different cell lines (PC3, FaDu and Skin Fibroblast) with a temperature-controlled cryopreservation method for 24 h in PDEGMA113–b–PMPC243–b–PDEGMA113 (3 wt%) solution and 10% DMSO solution in media (DMEM) with 10% FBS. c) Post-Thaw cell proliferation percent after 24 h at 37 °C and 5% CO2 in DMEM + 10% FBS media. Hydrogels can also be used as 2D/3D scaffolds for cell growth. Mimicking living tissues, such as skin, bones, and muscle tissues is of great interest in the biomedical field. Polymer scaffolds of appropriate chemical, physical, and mechanical/structural properties to guide cell and tissue organization and cell/scaffold integration have a significant impact in the area of regenerative medicine, but great challenges in designing polymeric biocompatible scaffolds are still faced for its medical application including favoring angiogenesis within the hydrogel network50. Here we evaluated the hydrogel biocompatibility and cytotoxicity, using a live/dead imaging assay. The first evaluation was done growing the cells on top of the pre-formed hydrogel (2D) and letting them grow after 24 and 48 h of incubation in humidified atmosphere at 37°C. Two controls were


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used to normalized the results: a non-manipulated, unfrozen (live) control of cells incubated with DMEM media + 10% FBS and a negative control were cells where exposed to 70% ethanol for 30 min. Live cells are distinguished by the presence of ubiquitous intracellular esterase activity determined by the enzymatic conversion of cell-permeant calcein AM to the fluorescent calcein, which is well-retained within live cells. The red component propidium iodide, is cell-impermeant and therefore only enters cells with damaged membranes. In dying and dead cells a bright red fluorescence is generated upon binding to DNA. In Figure 5 confocal images were taken after 24 and 48 h of incubation with the pre-formed hydrogels and cell viability was quantify using Imaris Imaging Software (Figure 6). According to the results the hydrogel is a highly biocompatible scaffold that can be used for cell growth and integration (more than 98% viability). Furthermore, cells were grown inside the hydrogel by encapsulating PC3 cells within the hydrogel network. Cells cultured in the hydrogel maintained a high viability and proliferative capacity, revealing that the hydrogels could function as a suitable platform for 3D cell culture (Figure 7). Further evaluation of the angiogenic properties of this scaffold should be addressed by establishing in vitro models using endothelial and stromal cells regulating key signaling pathways for angiogenesis promotion51.

Figure 5. 2D Cell culture evaluation after 24 and 48 h of incubation. Cell viability of cancer PC3 cells was assessed using Live/Dead assay. PC3 were grown on top of PDEGMA113–b–PMPC243– b–PDEGMA113 (15 wt%) hydrogels. Live: green, Dead: red. Live control: DMEM + 10% FBS. Dead control: 70% ethanol.


21


3D Cell Culture Live/Dead Assay 100

Cell Viability (%)

80 60 40 20

Po ly m 15 er w (2 t% 4h ) Po ly m er (4 8h )

on tr ol

15

w

t%

D

ea d

C

C

on tr ol

0

Li ve

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 6. 3D Cell culture quantification after 24 and 48 h of incubation. Cell viability of cancer PC3 cells was assessed using Live/Dead assay. PC3 were grown on top of PDEGMA113–b– PMPC243–b–PDEGMA113 (15 wt%) hydrogels. Quantification was done using Imaris Imaging Software.

Figure 7. 3D Cell viability of cancer PC3 cells encapsulated in PDEGMA113–b–PMPC243–b– PDEGMA113 (15 wt%) hydrogels. Live/Dead staining of the encapsulated PC3 cells after injection for 24 and 48 h. Live: green, Dead: red. Live control: DMEM + 10% FBS. Dead control: 70% ethanol.


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Finally, inhibition of ice recrystallization in PDEGMA113–b–PMPC243–b–PDEGMA113 solution was studied using a cryomicroscope to determine if the triblock copolymer was able to modify ice crystal characteristics.47 Figure 8 (a-c) shows images of the ice crystals observed for the positive control used (DMEM+10%FBS) in the IRI assay and it was noted that the ice crystals are larger in size as compared to the polymer solution samples (Figure 8 (d-f) and (g-i)). (a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Figure 8. Ice crystal and cryomicroscopy images. Photographs illustrating the IRI activity of PDEGMA113–b–PMPC243–b–PDEGMA113 at different time-points and the control group. (a–c) Ice crystal grains from control group (a) at −40 °C, (b) at −15 °C, (c) at −5 °C (d–f) Ice crystal grains from 3 wt% polymer group (d) at −40 °C, (e) at −15 °C, (f) at −5 °C (g–i) Ice crystal grains from 15 wt% polymer group (g) at −40 °C, (h) at −15 °C, (i) at −5 °C


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Cryomicroscopy experiments have confirmed that the structure and size of ice during the freezing process is significantly different when PDEGMA113–b–PMPC243–b–PDEGMA113 is present as compared to the control group. In fact, depending on the percent of polymer solution used, the structure of ice in the frozen sample differs dramatically. From the images obtained, it is clearly visible that at −5 °C the size of the ice crystal in the control group (40 µm) is bigger than the size of the ice crystal in 3 wt% (20 µm) and 15 wt% (10 µm) polymer groups (Figure 9). (a)

(b)

(c)

Figure 9. Ice crystal grain sizes at −5 °C from (a) control group (b) 3 wt% polymer group (c) 15 wt% polymer group Ideally, the size of the ice crystals should be smaller in the presence of an ice recrystallization inhibitor. The images obtained from the experiment, therefore, indicate that the presence of MPC block in PDEGMA113–b–PMPC243–b–PDEGMA113 in the central part successfully work as a cryo-protectant. According to the results shown in Figure 8(g-i), solution with 15 wt% of PDEGMA113–b–PMPC243–b–PDEGMA113 has better IRI activity and anti-freezing properties. Additionally, the half-maximal concentration for the ice recrystallization inhibition (IRI) activity of PDEGMA113–b–PMPC243–b–PDEGMA113 was determined using a modified splat-cooling assay with phosphate-buffered saline as the positive control for ice recrystallization.49 As demonstrated in figure 10, the IC50 of the polymer is 12 mg/mL thus confirming that this triblock copolymer is an active IRI. These results are consistent to the previous report where at a certain concentration of the zwitterionic polymer, inhibition of large ice crystals occur making it a potential candidate for cryopreservation applications.15,16 These results suggest that PDEGMA113–b–PMPC243–b–PDEGMA113 is an IRI active compound and can be used as alternative to DMSO without any significant changes in the technique for cryopreserving cells.


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Figure 10. Dose-response curve for the ice recrystallization inhibition (IRI) activity of the triblock copolymer.

Conclusions The temperature responsive and zwitterionic triblock copolymer, PDEGMA113–b–PMPC243–b– PDEGMA113, has been synthesized by ATRP and it was shown that at a concentration of 15, 20 and 25 wt% physical gels were formed above the LCST. The rheological tests revealed that the polymer solutions showed sol-gel transition. MTT assay with HeLa cells confirmed that the triblock copolymer has low toxicity at the studied concentrations. PDEGMA113–b–PMPC243–b– PDEGMA113 in DMEM with 10% FBS worked as a cryo-protective agent for HeLa, CHO, PC3, FaDu, W138, Skin fibroblasts and K562 cell lines at 3 and 15 wt% polymer concentrations. 2D/3D culture studies were conducted to confirm the promising use of this hydrogel as a scaffold for mimicking in vivo cell culture. High biocompatibility and low cytotoxicity evidenced the great potential for this triblock copolymer to be used for cryopreservation, as well as for cell culture and tissue engineering applications. Further testing with other primary cell lines will be carried out in the future to determine the effectiveness of the temperature responsive and


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zwitterionic triblock copolymer as a cryo-protectant and elucidate important complex interactions with the biological entities to further improve its biomedical applications. ASSOCIATED CONTENT Supporting Information The supporting information is available. 1H NMR spectrum of PDEGMA113–b–PMPC243–b– PDEGMA113 (Figure S1, S2, S3), mechanical properties of PDEGMA113–b–PMPC243–b– PDEGMA113 (Figure S4–S7) and cell viability (Figure S8). AUTHOR INFORMATION Corresponding Author *Professor Ravin Narain; E-mail: [email protected]; tel. (780) 492-1736. ORCID Jayeeta Sengupta - 0000-0001-6622-8735 Diana Diaz-Dussan: 0000-0003-1778-3964 Ravin Narain: 0000-0003-0947-9719 Jason Acker: 000-0002-1445-827X CONFLICT OF INTEREST None declared

ACKNOWLEDGMENTS The authors would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and Canadian Foundation for Innovation (CFI) for funding this work. Leslie V. Sanchez Castillo is also thanked for her help with the cytotoxicity assays.

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GRAPHICAL ABSTRACT


Synthesis of Highly Biocompatible and Temperature Responsive

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