Supporting Information. Photo-Dimerization Induced Dynamic Viscoelastic Changes in ABA. Triblock Copolymer-Based Hydrogels for 3D Cell Culture

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1 Supporting Information Photo-Dimerization Induced Dynamic Viscoelastic Changes in ABA Triblock Copolymer-Based Hydrogels for 3D Cell Culture Ryota Tamate, Takeshi Ueki,*, Yuzo Kitazawa, Morinobu Kuzunuki, Masayoshi Watanabe, Aya Mizutani Akimoto, and Ryo Yoshida*, Department of Materials Engineering School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki , Japan Department of Chemistry and Biotechnology Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama , Japan * T. U.: * R. Y.: S1

2 Synthesis of 7-(2-acryloyloxyethoxy)-4-methylcoumarin (Coumarin acrylate, CA) First, 7-(2-hydroxyethoxy)-4-methylcoumarin was synthesized according to a previous report. S1 Next, a dry, three-necked round-bottle flask was charged with 7-(2- hydroxyethoxy)-4-methylcoumarin (2.00 g, 9.08 mmol) in dry dichloromethane (CH2Cl2, 200 ml). Triethylamine (3.80 ml, 27.2 mmol) was added and the flask was cooled in an ice bath. Then, acryloyl chloride (1.84 ml, 22.7 mmol) in 20 ml of dry CH2Cl2 was added dropwise under argon (Ar) atmosphere. The reaction was stirred at room temperature overnight. After the reaction, 200 ml of CH2Cl2 was added to the reaction mixture and washed with water, 5 w/v% NaHCO3 aqueous solution, 0.1 M hydrochloric acid, and saturated NaCl. Then, the organic phase was collected by filtration and evaporated. The oily liquid was dissolved in acetone and vacuum filtered to remove any insoluble impurities. Then, the solution was evaporated and dissolved in ethanol. After vacuum filtration, excess water was added to precipitate the product. The precipitate was collected by vacuum filtration and then dried under vacuum. Recrystallization from an ethanol/hexane mixture (1:1) gave 7-(2-acryloyloxyethoxy)-4- methylcoumarin (1.43 g, 57 % yield). 1 H-NMR (CDCl3) (ppm): = 7.50 (d, 1H, aromatic-h), = 6.87 (dd, 1H, aromatic-h), = 6.81 (d, 1H, aromatic-h), = 6.45 (d, 1H, Aromatic-OCH2CH2OC(=O)CH=CH 2 ), = (m, 2H, Aromatic- S2

3 OC(=O)CH=C(CH3), Aromatic-OCH2CH2OC(=O)CH=CH2), = 5.86 (d, 1H, Aromatic- OCH2CH2OC(=O)CH=CH 2 ), = 4.53 (t, 2H, Aromatic-OCH 2 CH2OC(=O)CH=CH2), = 4.26 (t, 2H, Aromatic-OCH2CH 2 OC(=O)CH=CH2), = 2.38 (s, 3H, Aromatic- OC(=O)CH=C(CH 3 )). S3

4 S4 Figure S1. 1 H NMR spectrum of the ABA triblock copolymer TMS CDCl 3 a a a b b b b b c c c d d d e e e f f g g g h, i, j, k l l l l l h i h i j k j k (ppm)

5 Elution time (min) Figure S2. GPC traces for the CTA-PEO-CTA precursor (blue line) and the ABA triblock copolymer (red line). S5

6 100 Dimerization degree (%) Storage time (day) Figure S3. Variation in the degree of dimerization of the dilute aqueous solution of the ABA triblock copolymer after UV irradiation. After 120 min of UV irradiation, the solution was stored at 37 C in dark. Time variation in the degree of dimerization during the storage was monitored using a UV-vis spectrophotometer. S6

7 Figure S4. Reversibility in the dimerization degree of the dilute aqueous solution of the ABA triblock copolymer (0.1 wt%) over five cycles of UV irradiation at 365 nm (1.1 mw/cm 2, 30 min) and 254 nm (1.3 mw/cm 2, 15 min). Handy UV lamp (SLUV- 6, AS ONE Co., Japan) was used as a light source. UV irradiations were conducted at 37 C. S7

8 10,000 1,000 G', G'' (Pa) : G before UV : G before UV : G after UV : G after UV Temperature ( C) Figure S5. Temperature dependence of storage (G, closed) and loss (G, open) moduli for the ABA triblock copolymer (10 wt% in culture medium) before (circles) and after (squares) UV illumination ( = 365 nm, 5 mw/cm 2, 10 min) at 37 C. S8

9 10,000 G', G'' (Pa) 1, : G before UV : G before UV : G after UV : G after UV Strain (%) Figure S6. Strain dependence of storage (G, closed) and loss (G, open) moduli for the ABA triblock copolymer (10 wt% in culture medium) before (circles) and after (squares) UV illumination ( = 365 nm, 5 mw/cm 2, 10 min) at a frequency of 1 rad/s, 37 C. Dotted lines are guides to the eye. Under large strain, some polymer chains are pulled out from the micellar core and results in G drops. S2 The critical strain value, c, is defined as a strain below which G remains invariant. The hydrogel before UV irradiation has a c 10 %, whereas the hydrogel after UV irradiation has a c 70 %. S9

10 Figure S7. Cell viability in ABA triblock copolymer hydrogels. Live cells were stained with calcein-am (green), whereas dead cells were stained with ethidium homodimer-1 (red). Fluorescent images were taken soon after encapsulation (a), after 24 h of incubation (b), and after 48 h of incubation (c). S10

11 Relative cell number : Without UV : With UV N.S. N.S. N.S Incubation time (day) Figure S8. Relative cell number of cells on tissue culture polystyrene dishes with (closed rectangles) and without (open rectangles) UV illumination ( = 365 nm, 5 mw/cm 2, 10 min) (N = 4). N.S., not significant. S11

12 Relative cell number : Without UV : UV-0day : UV-1day N.S. N.S. N.S Incubation time (day) Figure S9. Relative cell number for each cell culture during 2 days of incubation. Three different types of cell cultures were performed the same as in Figure 7, except that the ABA triblock copolymer was replaced by the Mebiol gel. N.S., not significant. S12

13 Relative cell number : Without UV : UV-0day : UV-1day N.S. * * ** ** Incubation time (day) Figure S10. Relative cell number for each cell culture during 2 days of incubation. Three different types of cell cultures were performed the same as in Figure 7, except that cells were not encapsulated in the ABA triblock copolymer hydrogel, but were cultured on the hydrogel (2D cell culture). N.S., not significant; *, P < 0.05; **, P < The cell proliferation of each cell culture was lower than that in 3D cell culture. It was previously reported that HeLa cells exhibited higher proliferation in 3D cell culture than that in 2D cell culture. S3 When comparing between the Without UV and the UV-0day cell cultures, the proliferation of the Without UV cell culture was higher than that of the UV-0day cell culture, which was the same tendency obtained in 3D cell culture. However, it is noticeable that the UV-1day cell culture showed higher cell proliferation than the Without UV cell culture, which was the opposite tendency obtained in 3D cell culture. Therefore, in 2D cell culture, the dynamic viscoelastic change at proper timing might promote HeLa cell proliferation. The reason is still unclear, but this result indicates that the situation is quite different between 3D and 2D cell cultures. S13

14 Figure S11. (a, b) Representative phase contrast images soon after cell encapsulation for Without UV cell cultures (a) and UV-0day cell cultures (b). It was confirmed that no cell aggregation was formed for each cell culture at this initial stage. S14

15 Supporting references S1. Sinkel, C.; Greiner, A; Agarwal, S. A. A Polymeric Drug Depot Based on 7-(2(- Methacryloyloxyethoxy)-4-methylcoumarin Copolymers for Photoinduced Release of 5-Fluorouracil Designed for the Treatment of Secondary Cataracts. Macromol. Chem. Phys. 2010, 211, S2. He, Y.; Boswell, P. G.; Bühlmann, P.; Lodge, T. P. Ion Gels by Self-Assembly of a Triblock Copolymer in an Ionic Liquid. J. Phys. Chem. B 2007, 111, S3. Zhao, Y.; Yao, R.; Ouyang, L.; Ding, H.; Zhang, T.; Zhang, K.; Cheng, S.; Sun, W. Three-Dimensional Printing of Hela Cells for Cervical Tumor Model in vitro. Biofabrication 2014, 6, S15