Supporting Information. Bioinspired Self-Healing Hydrogel Based on Benzoxaborole-Catechol Dynamic Covalent Chemistry for 3D Cell Encapsulation

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1 Supporting Information Bioinspired Self-Healing Hydrogel Based on Benzoxaborole-Catechol Dynamic Covalent Chemistry for 3D Cell Encapsulation Yangjun Chen, Diana Diaz-Dussan, Di Wu, Wenda Wang, Yi-Yang Peng, Anika Benozir Asha, Dennis G. Hall, Kazuhiko Ishihara #, and Ravin Narain* Department of Chemical and Materials Engineering, Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G6, Canada # Department of Materials Engineering, School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan *Corresponding author, narain@ualberta.ca S1

2 Experimental Section Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was synthesized by Prof. Ishihara s lab (Tokyo University, Japan). 5-Methacrylamido-1,2-benzoxaborole (MAABO) was synthesized according to our published papers. 1 Dopamine hydrochloride, methacrylic anhydride, sodium borate, 4,4 -azobis (4-cyanovaleric acid) (ACVA), rhodamine B (RhB), methylene blue (MB), fructose, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Live/dead assay was purchased from Thermo Fisher Scientific. All cell culture products, including DMEM medium, antibiotics, fetal bovine serum (FBS), and trypsin with EDTA, were obtained from Gibco. All organic solvents, including methanol, ethyl acetate, tetrahydrofuran (THF), and dimethyl formamide (DMF), were purchased from Caledon Laboratories Ltd. (Canada) and used without further purification. Characterization. 1 H NMR spectra of the monomers and polymers were recorded on a Varian 500 MHz spectrometer. The number (M n ) and weight (M w ) average molecular weights and polydispersity index (PDI = M w /M n ) of the zwitterionic copolymers were determined by Viscotek conventional gel permeation chromatography (GPC) system equipped with two WAT Waters Ultrahydrogel linear columns using 0.5 M sodium acetate/0.5 M acetic acid buffer as eluent at a flow rate of 1.0 ml/min. The GPC was calibrated by monodisperse pullulan standards (M w = ,000 g/mol). 1 S2

3 Synthesis of Dopamine Methacrylamide (DMA). DMA was synthesized as previously reported. 2 Briefly, 10.0 g of sodium borate (26.22 mmol) and 4.0 g of NaHCO 3 (47.62 mmol) were dissolved in 100 ml of DI water. The solution was degassed for 30 min with nitrogen. Then, 5.0 g (32.64 mmol) of dopamine hydrochloride was added to the mixture and allowed to stir under nitrogen atmosphere for another 15 min. After that, 4.7 ml (31.71 mmol) of methacrylic anhydride in 25 ml of THF was added dropwise to the above solution in ice-water bath. The ph of the reaction mixture was monitored periodically with ph paper and maintained slightly basic (ph 8-9) by adding 1 M NaOH solution. Once all the methacrylic anhydride solution was added, the mixture was allowed to stir overnight at room temperature. Then, the solution was washed with ethyl acetate for two times and the resulting aqueous layer was filtered. The filtrate was acidified to ph 1-2 with 5 M HCl and extracted three times with large amount of ethyl acetate. The organic layer was collected, dried with MgSO 4, and condensed down to approximately 150 ml under reduced pressure. The product was recrystallized in the fridge overnight, filtered, and vacuum dried to get 4.3 g of DMA monomer. 1 H NMR (DMSO-d 6 ) δ (1H, ArH), 6.55 (1H, ArH), (1H, ArH), 5.58 (1H, -CO-C(-CH 3 )=CH 2 ), 5.27 (1H, -CO-C(-CH 3 )=CH 2 ), (2H, C 6 H 3 (OH) 2 -CH 2 -CH 2 -NH-), (2H, C 6 H 3 (OH) 2 -CH 2 -CH 2 -NH-), 1.81 (3H, -CO-C(-CH 3 )=CH 2 ). Synthesis of MPC-Based Copolymers with Benzoxaborole/Catechol Groups. The zwitterionic random copolymers were synthesized via free radical polymerization. To S3

4 synthesize benzoxaborole-containing copolymer poly(mpc-st-maabo), MPC (1.062 g, 3.6 mmol), MAABO (86.8 mg, 0.4 mmol), and ACVA (5.6 mg, 20 µmol) were placed in a 50 ml polymerization tube and dissolved with a mixture of methanol (3.5 ml), DMF (1.5 ml), and DI water (1 ml). After being degassed with nitrogen for 30 min, the polymerization was carried out at 70 C for 18 h. The reaction was terminated by rapid cooling in liquid nitrogen and the resultant copolymer was isolated after precipitation in acetone. The product was further dialyzed against DI water for 24 h and lyophilized to get poly(mpc-st-maabo)with a high yield of 85.2%. For the synthesis of catechol decorated copolymer poly(mpc-st-dma), MPC (1.062 g, 3.6 mmol), DMA (88.4 mg, 0.4 mmol), and ACVA (5.6 mg, 20 µmol) were dissolved in a mixture of methanol (5 ml) and DMF (1 ml). The mixed solution was placed in a 50 ml polymerization tube and degassed using nitrogen for 30 min. After reacting at 70 C for 18 h, the polymerization was stopped by rapid cooling in liquid nitrogen, and the resultant polymer was isolated by precipitation in acetone. The product poly(mpc-st-dma) (Yield: 86.7%) could be obtained after dialyzing against DI water for 24 h and freeze-drying. Fabrication and Characterization of Hydrogels. Prior to hydrogel formation, poly(mpc-st-maabo) and poly(mpc-st-dma) were dissolved in ph 7.4 PBS solution at different concentrations (7.5 wt%, 10 wt%, and 12.5 wt%) and sonicated for 15 min. To form hydrogels, the same volume of these two polymer solutions were mixed via pipetting. The hydrogels with three different solid contents were S4

5 accordingly denoted as Gel-7.5%, Gel-10%, and Gel-12.5%, respectively. The porous morphology of these three hydrogels were observed by field emission scanning electron microscopy (FESEM, Zeiss Sigma 300/VP). The hydrogels were freeze-dried and sputter-coated with gold to provide a conductive environment prior to SEM characterization. The mechanical and self-healing properties were studied by an AR-G2 rheometer (TA instruments) with a 20 mm 2.008º cone plate geometry at 25 C. 3 To compare the mechanical properties, frequency sweeps of hydrogels with different solid contents were tested from 0.1 rad/s to 100 rad/s with a constant strain of 1%. Gel-10% was used as an example to study the self-healing ability. The hydrogel was first tested by oscillatory strain amplitude sweep from 0.1% to 1000% at a constant frequency of 1 Hz to determine the critical stain required for gel failure. Then, step-stain test was performed by repeating large strain (550%, 30 s) for network disruption and small stain (1%, 90 s) for mechanical recovery. The self-healing ability was also visually evaluated by putting four dyed hydrogel cubes together for 1 min before lifting on its own weight and stretching. ph and Sugar Responsiveness. Gel-10% with RhB dye staining was used to study the ph and sugar responsiveness. To test the ph-responsiveness, a little amount of 0.1 M HCl solution was added to the hydrogel, followed by vigorous shaking. After the hydrogel was totally dissociated into sol state, proper amount of 0.1 M NaOH solution was added to neutralize the acid and re-form the hydrogel. The cyclic addition of HCl-NaOH was repeated for 4 more times and the reversible gel-sol-gel transitions S5

6 were recorded. To investigate the sugar responsiveness, two pieces of hydrogel were immersed in 1.5 ml of PBS (ph 7.4) solutions with or without 50 mm of fructose, respectively. The volume changes of hydrogels were recorded at different time intervals. Cytotoxicity Assay. The cell cytotoxicity of both the hydrogel precursor polymers and gel extracts were evaluated by standard MTT assay. The gel extracts were prepared by immersing the hydrogel in low glucose DMEM medium at 10-, 20- and 40-times the volume of the origin hydrogel for 24 h. HeLa cells were seeded in 96-well microplates at a density of cells per well in 200 µl of DMEM culture medium with 10% FBS and 1% penicillin/streptomycin. After 24 h incubation at 37 C in a balanced air humidified incubator with an atmosphere of 5% CO 2, the culture medium was replaced with 200 µl of fresh culture medium or culture medium containing gel extracts or culture medium containing different concentrations of poly(mpc-st-maabo) and poly(mpc-st-dma). The cells were allowed to be incubated for another 24 h before the addition of MTT solution (20 µl, 5 mg/ml). After 4 h incubation, the culture medium was carefully removed and DMSO was added to dissolve the crystal formazan. Absorbance at 570 nm was measured by TECAN Genios pro microplate reader and cell viability was calculated by comparing O.D. values of cells treated with gel extracts or polymers with that of control. 3D Cell Encapsulation. HeLa cells were taken as an example for 3D encapsulation in Gel-10% under sterile conditions. HeLa cells were suspended in 200 µl of low glucose DMEM medium with 10 wt% of poly(mpc-st-dma) at a density of 2.5 S6

7 10 6 cells/ml and transferred to a glass-bottomed petri-dish. Then, 200 µl of 10 wt% poly(mpc-st-maabo) PBS solution was added and gently mixed with the cell suspension to form cell-loaded hydrogel. The gelation happened quite fast within 1 min. Then, the petri-dish was placed in the cell incubator for 10 min, followed by addition of 0.5 ml of low glucose DMEM medium. After 24 h incubation at 37 C, the cell-encapsulated hydrogel was stained with live/dead assay, and visualized by CLSM 710 Meta confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Quantification and image processing were done using Imaris Image Analysis software. 4 Table S1. Synthetic Results of poly(mpc-st-dma) and poly(mpc-st-maabo) Copolymers composition (mol%) a molecular weight b polymers MPC DMA MAABO M n (10 3 ) M w (10 3 ) PDI (M w /M n ) poly(mpc-st-dma) poly(mpc-st-maabo) S7

8 a Calculated from 1 H NMR results using D 2 O/DMSO-d 6 mixture as solvent. b Obtained from aqueous GPC using 0.5 M sodium acetate/acetic acid buffer as eluent. Figure S1. 1 H NMR spectrum of dopamine methacrylamide (DMA) in DMSO-d 6. S8

9 Figure S2. Step-strain sweep of Gel-10% at a constant frequency of 1 Hz. Figure S3. Cytotoxicity of poly(mpc-st-dma) and poly(mpc-st-maabo) copolymers after 24 h co-incubation with HeLa cells. Cell viabilities were obtained by standard MTT assay. S9

10 Figure S4. Cytotoxicity of gel extracts after 24 h co-incubation with HeLa cells. Gel extracts were obtained with three different culture medium/hydrogel weight ratios. Cell viabilities were obtained by standard MTT assay. References 1. Y. Kotsuchibashi, R. V. C. Agustin, J.-Y. Lu, D. G. Hall and R. Narain, Temperature, ph, and Glucose Responsive Gels via Simple Mixing of Boroxole- and Glyco-Based Polymers. ACS Macro Lett., 2013, 2, E. M. White, J. E. Seppala, P. M. Rushworth, B. W. Ritchie, S. Sharma and J. Locklin, Switching the Adhesive State of Catecholic Hydrogels using Phototitration. Macromolecules, 2013, 46, Y. Chen, W. Wang, D. Wu, M. Nagao, D. G. Hall, T. Thundat and R. Narain, Injectable Self-Healing Zwitterionic Hydrogels Based on Dynamic Benzoxaborole Sugar Interactions with Tunable Mechanical Properties. Biomacromolecules, 2018, 19, W. Huang, Y. Wang, Y. Chen, Y. Zhao, Q. Zhang, X. Zheng, L. Chen and L. Zhang, Strong and Rapidly Self-Healing Hydrogels: Potential Hemostatic Materials. Adv. Healthcare Mater., 2016, 5, S10