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 790-784, 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, 606-16, Yeoksm 1-dong, Kngnm-gu, Seoul, 135-081, Kore e Deprtment of Bioengineering, University of Wshington, Box 351720, 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
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].
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. 10 3 10 3 Moduli (P) 10 2 G' (Storge) G" (Loss) Moduli (P) 10 2 G' (Storge) G" (Loss) 10 1 1 10 100 Frequency (rd s -1 ) 10 1 1 10 100 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.
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 8.73-7.80 (r, 3H), 7.78 6.61 (r, 6H), 5.68 (r, 12H), 4.45 (r, 12H), 4.27 3.43 (r, 32H), 3.26 (r, 24H), 2.90 (r, 12H), 2.79 (r, 12H), 2.09 (r, 24H), 1.51 1.03 (r, 12H). MS (MALDI-TF): m/z [M + H] + clcd for C 125 H 200 N 39 27 S 13 (m = C 2 H 7 N 1 S 1, 2- minoethnethiol), 3095.2; found 3095.6, [M m + H] + clcd for C 123 H 193 N 38 27 S 12, 3018.2; found 3018.5, [M 2m + H] + clcd for C 121 H 186 N 37 27 S 11, 2941.1; found 2941.5, [M 3m + H] + clcd for C 119 H 179 N 36 27 S 10, 2864.1; found 2864.5, [M 3m + H] + clcd for C 117 H 172 N 35 27 S 9, 2787.1; found 2787.5, [M 4m + H] + clcd for C 115 H 165 N 34 27 S 8, 2710.1; found 2710.4.
FITC-CB[6]; 1 H NMR (500 MHz, D 2 ): δ 8.47-7.52 (r, 2H), 7.50 6.50 (r, 5H), 5.68 (r, 12H), 4.45 (r, 12H), 3.77 (r, 24H), 3.26 (r, 24H), 2.90 (r, 12H), 2. 79 (r, 12H), 2.09 (r, 24H); MS (MALDI-TF): m/z [M + H] + clcd for C 117 H 180 N 37 29 S 13 (m = C 2 H 7 N 1 S 1 ), 2983.0; found 2983.1, [M m + H] + clcd for C 115 H 173 N 36 29 S 12, 2906.0; found 2906.2, [M - 2m + H] + clcd for C 113 H 166 N 35 29 S 11, 2829.0; found 2829.2, [M - 3m + H] + clcd for C 111 H 159 N 34 29 S 10, 2751.9; found 2752.0, [M - 4m + H] + clcd for C 109 H 152 N 33 29 S 9, 2674.9; found 2675.1. Figure S5. Schemtics for the synthesis of c(rgdyk)-cb[6]. ) Synthesis of (HC)-CB[6]: 3-Mercptopropionic cid (48 eq., 218.9 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 24 48 S 12 K, 2979.7; found 2980.0, [M m + K] + (m = C 21 H 12 N 5 S, 3-mercptopropionic cid)
clcd for C 105 H 150 N 24 46 S 11 K, 2873.7; found 2873.9, [M 2m + K] + clcd for C 102 H 144 N 24 44 S 10 K, 2767.7; found 2767.9, [M 3m + K] + clcd for C 99 H 138 N 24 42 S 9 K, 2661.7; found 2662.0, [M 4m + K] + clcd for C 96 H 132 N 24 40 S 8 K, 2555.6; found 2555.8. Anlysis (Clcd, found for (C 72 H 84 N 24 24 )(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 33 55 S 12 3581.0; found 3581.5, [M m + K] + (m = C 3 H 6 2 S) C 132 H 191 N 33 53 S 11 K, 3477.0; found 3477.6, [M - 2m + K] + clcd for C 129 H 185 N 33 51 S 10 K, 3371.0; found 3371.5, [M - 3m + K] + clcd for C 126 H 179 N 33 49 S 9 K, 3265.0; found 3265.5, [M - 4m + K] + clcd for C 123 H 173 N 33 47 S 8 K, 3159.0; found 3159.5.
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.
Reltive prolifertive ctivity 5 * 4 3 2 1 0 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
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.