Supporting Information. In Situ Supramolecular Assembly and Modular Modification of Hyaluronic Acid Hydrogels for 3D Cellular Engineering

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1 Supporting Informtion In Situ Suprmoleculr Assemly nd Modulr Modifiction of Hyluronic Acid Hydrogels for 3D Cellulr Engineering Kyeng Min Prk,, Jeong-A Yng,, Hyunte Jung, c Junseok Yeom, Ji Sun Prk, d Keun-Hong Prk, d Alln S. Hoffmn, e Sei Kwng Hhn,,c, * nd Kimoon Kim,c, * Ntionl Cretive Reserch Inititive Center for Smrt Suprmolecules, Deprtment of Chemistry, Division of Advnced Mterils Science, Pohng University of Science nd Technology (PSTECH), Sn 31, Hyoj-dong, Nm-gu, Pohng, Kyunguk , Kore, Deprtment of Mterils Science nd Engineering, PSTECH, Kore c School of Interdisciplinry Bioscience nd Bioengineering, PSTECH, Kore d CHA Stem Cell Institute, College of Medicine, Pochon CHA University, , Yeoksm 1-dong, Kngnm-gu, Seoul, , Kore e Deprtment of Bioengineering, University of Wshington, Box , Settle, WA 98195, USA These uthors contriuted eqully to this work. Generl Methods. Fluorescence imges were oserved with DMI6000B (Leik) fluorescence microscope nd Eclipse TE 2000 (Nikon) confocl scnning microscope. 1 H NMR spectrometry ws performed on DRX500 (Bruker) spectrometer. Fluorescence mesurements were performed with 10

2 mm qurtz cells on RF-5301PC (Shimdzu) spectrofluorometer. Rheologicl nlysis ws performed on TA ARES rheometer with prllel-plte geometry (20 mm dimeter) t 25 C. MALDI/TF mss spectrometry ws performed with Reflex III (Bruker) mss spectrometer. H 2 N NH 2 NH 3 H 2 N NH 2 NH 3 CB[6] HN H H H H NH n CB[6] HN H H H H NH n Figure S1. 1 H NMR spectr of SPM-HA () efore nd () fter ddition of CB[6]. () Successful conjugtion of SPM (52 ± 2 mol% on HA units) on HA (230 kd) ws confirmed y the ppernce of the peks t 3.0 ppm nd etween 1.6 nd 2.1 ppm on 1 H NMR corresponding to SPM groups. () Hostguest interction of DAH-HA with CB[6] ws confirmed y the ppernce of chrcteristic inclusion pek t 0.6 ppm shifted from 1.7 ppm on 1 H NMR spectrum fter tretment with CB[6].

3 Figure S2. A centimetre scle suprmoleculr hydrogel nd its modulr modifiction with RBITC- CB[6]. () CB[6]/DAH-HA hydrogel ws formed in cylindricl shpe y mixing CB[6]-HA (300 µl, 2.0 wt%) nd DAH-HA (300 µl, 2.0 wt%) solutions. () CB[6]/DAH-HA hydrogel ws treted with solution of RBITC-CB[6] (100 µl, 60 µm) nd kept in humid chmer for 6 h to prepre RBITC- CB[6]@CB[6]/DAH-HA hydrogel. The red color remined even fter immersion in PBS for dy Moduli (P) 10 2 G' (Storge) G" (Loss) Moduli (P) 10 2 G' (Storge) G" (Loss) Frequency (rd s -1 ) Frequency (rd s -1 ) Figure S3. Frequency sweep rheologicl nlysis for storge (G ) nd loss (G ) moduli of () CB[6]/SPM-HA hydrogel nd () CB[6]/DAH-HA hydrogel treted with (H) 12 CB[6] (0.1 eq. to DAH in CB[6]/DAH-HA hydrogel). The verge storge modulus (G ) of the CB[6]/SPM-HA hydrogel (2.0 wt%) ws mesured to e 3.4 ± 0.5 kp, which ws 1.4 fold higher thn tht of the CB[6]/DAH-HA hydrogel (2.4 ± 0.2 kp). Storge modulus (G ) of CB[6]/DAH-HA hydrogel treted with (H) 12 CB[6] (0.1 eq. to DAH in CB[6]/DAH-HA hydrogel) ws mesured to e 2.2 ± 0.3 kp.

4 Figure S4. Synthesis of RBITC-CB[6] nd FITC-CB[6]. () RBITC (6.0 mg, 2.0 eq.) or FITC (4.0 mg, 2.0 eq.) ws dded to solution of mine-cb[6] (20.0 mg) in 50 vol% DMF in distilled wter (3.0 ml) nd then triethylmine ws dded until the solution ecme cler. After stirring t room temperture for dy, the rection mixture ws extensively wshed with cetonitrile. The purified product ws dried under reduced pressure to give RBITC-CB[6] (10.0 mg, 40%) or FITC-CB[6] (9.0 mg 37%). 1 H NMR dt suggested tht the verge sustitution numer of RBITC or FITC moiety to mine-cb[6] ws one. The MALDI-TF mss spectr reveled species with 1 RBITC or FITC conjugted mine-cb[6] which hd 8 ~ 12 cystemines (2-minoehnethiol) t the periphery of CB[6]. RBITC-CB[6]; 1 H NMR (500 MHz, D 2 ): δ (r, 3H), (r, 6H), 5.68 (r, 12H), 4.45 (r, 12H), (r, 32H), 3.26 (r, 24H), 2.90 (r, 12H), 2.79 (r, 12H), 2.09 (r, 24H), (r, 12H). MS (MALDI-TF): m/z [M + H] + clcd for C 125 H 200 N S 13 (m = C 2 H 7 N 1 S 1, 2- minoethnethiol), ; found , [M m + H] + clcd for C 123 H 193 N S 12, ; found , [M 2m + H] + clcd for C 121 H 186 N S 11, ; found , [M 3m + H] + clcd for C 119 H 179 N S 10, ; found , [M 3m + H] + clcd for C 117 H 172 N S 9, ; found , [M 4m + H] + clcd for C 115 H 165 N S 8, ; found

5 FITC-CB[6]; 1 H NMR (500 MHz, D 2 ): δ (r, 2H), (r, 5H), 5.68 (r, 12H), 4.45 (r, 12H), 3.77 (r, 24H), 3.26 (r, 24H), 2.90 (r, 12H), (r, 12H), 2.09 (r, 24H); MS (MALDI-TF): m/z [M + H] + clcd for C 117 H 180 N S 13 (m = C 2 H 7 N 1 S 1 ), ; found , [M m + H] + clcd for C 115 H 173 N S 12, ; found , [M - 2m + H] + clcd for C 113 H 166 N S 11, ; found , [M - 3m + H] + clcd for C 111 H 159 N S 10, ; found , [M - 4m + H] + clcd for C 109 H 152 N S 9, ; found Figure S5. Schemtics for the synthesis of c(rgdyk)-cb[6]. ) Synthesis of (HC)-CB[6]: 3-Mercptopropionic cid (48 eq., mg) ws dded to solution of (llyloxy) 12 CB[6] (50.0 mg) in methnol (5.0 ml) nd then UV (254 nm nd 300 nm) ws irrdited to the mixture for 2 dys. After evportion of methnol, the rection mixture ws purified y wshing with n excess mount of diethyl ether. The purified product ws dried under reduced pressure to give (HC)-CB[6] (110.5 mg, 81.8%). The N/S rtio in elementl nlysis suggested tht the verge sustitution numer of (HC) moiety to (llyloxy) 12 CB[6] ws 11.7 which ws consistent with the 1 H NMR dt. The MALDI-TF mss spectrum reveled species with 8 ~ 12 3-mercptopropionic cids conjugted (llyloxy) 12 CB[6]. 1 H NMR (500 MHz, D 2 ): δ 5.68 (r, 12H), 4.45 (r, 12H), 3.77 (r, 24H), 2.79 (r, 24H), 2.49 (r, 12H), 2.09 (r, 12H); MS (MALDI-TF): m/z [M + K] + clcd for C 108 H 156 N S 12 K, ; found , [M m + K] + (m = C 21 H 12 N 5 S, 3-mercptopropionic cid)

6 clcd for C 105 H 150 N S 11 K, ; found , [M 2m + K] + clcd for C 102 H 144 N S 10 K, ; found , [M 3m + K] + clcd for C 99 H 138 N S 9 K, ; found , [M 4m + K] + clcd for C 96 H 132 N S 8 K, ; found Anlysis (Clcd, found for (C 72 H 84 N )(C 3 H 6 S 1 2 ) 11.7 (H 2 ) 5 ) C (42.86, 42.82), H (5.51, 5.54), N (11.20, 11.17), S (12.50, 12.48). ) Synthesis of c(rgdyk)-cb[6]: c(rgdyk) (1.5 eq. 2.2 mg) ws dded to solution of (HC)- CB[6] (6.0 mg), N,N'-dicyclohexylcrodiimide (DCC, 0.8 mg), N-hydroxysuccinimide (NHS, 0.5 mg) nd triethylmine (5.0 µl) in DMS (2.0 ml), which stirred t room temperture for 2 dys. After the rection mixture ws purified y dilysis ginst wter using memrne with moleculr weight cutoff of 1,000, the smple ws lyophilized to give c(rgdyk)-cb[6] (6.0 mg, 82%). 1 H NMR dt suggested tht the verge sustitution numer of c(rgdyk) moiety to (HC)-CB[6] ws one. The MALDI-TF mss spectrum reveled species with 1 c(rgdyk) conjugted (HC)-CB[6] which hd 8 ~ 12 3-mercptopropionic cids t the periphery of CB[6]. 1 H NMR (500 MHz, D 2 ): δ 6.93 (r, 2H), 6.56 (r, 2H), 5.68 (r, 12H), 4.42 (r, 12H), 3.78 (r, 24H), 3.19 (r, 6H), 2.91 (r, 4H), 2.77 (r, 24H), 2.53 (r, 4H), 2.46 (r, 12H), 2.35 (r. 2H),2.05 (r, 12H), 1.60 (r, 4H), 1.45 (r, 2H); MS (MALDI- TF): m/z [M + K] + clcd for C 135 H 197 KN S ; found , [M m + K] + (m = C 3 H 6 2 S) C 132 H 191 N S 11 K, ; found , [M - 2m + K] + clcd for C 129 H 185 N S 10 K, ; found , [M - 3m + K] + clcd for C 126 H 179 N S 9 K, ; found , [M - 4m + K] + clcd for C 123 H 173 N S 8 K, ; found

7 0 d 3 d Enlrged Figure S6. The prolifertion of firolst (NIH3T3) cells in CB[6]/DAH-HA hydrogel with or without c(rgdyk)-cb[6]. NIH3T3 cells entrpped in CB[6]/DAH-HA hydrogels () with or () without c(rgdyk)-cb[6] were stined y clcein AM nd imged under fluorescence microscope.

8 Reltive prolifertive ctivity 5 * c Control c(rgdyk)-cb[6] c(rgdyk) Figure S7. BrdU ssy for the prolifertion of NIH3T3 cells fter culture for 10 dys in CB[6]/DAHHA hydrogel without tretment, nd with c(rgdyk)-cb[6] or free c(rgdyk) (* P < 0.05 versus the control group). Gel Figure S8. Histologicl nlysis of Bl/c mouse skin fter stining with hemtoxylin nd eosin (H&E). () Norml mice without tretment s control nd () mice fter tretment with CB[6]/DAH-HA hydrogel for week (scle r = 200 µm). The CB[6]-HA solution (100 µl, 5 wt%) nd the DAH-HA

9 solution (100 µl, 5 wt%) were sequentilly injected into the sucutis of the right side ck of Bl/c mice (six-week old, femle, n = 3). After 1 week post-injection, the implnttion sites were completely excised for the histologicl nlysis. The iopsy smples were fixed in 4% formldehyde solution, emedded in prffin, sectioned t thickness of 3 µm, nd stined with H&E. Movie Legend Movie S1. Rel time movie for in situ formtion of CB[6]/DAH-HA hydrogel y simple mixing of equl volume of CB[6]-HA (300 µl, 2.0 wt%) nd DAH-HA (300 µl, 2.0 wt%) solutions.