Supporting information

S1 Supporting information Biodegradable Injectable Polymer Systems Exhibiting Temperature-Responsive Irreversible Sol-to-Gel Transition by Covalent Bond Formation Yasuyuki YOSHIDA 1,2, Keisuke KAWAHARA 1, Kenta INAMOTO 1, Shintaro MITSUMUNE 1, Shinya ICHIKAWA 1, Akinori KUZUYA 1,3,4, Yuichi OHYA* 1,3 1 Department of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and Bioengineering, 3-3-35 Yamate, Suita, Osaka 564-8680, Japan 2 Research Fellow of Japan Society for the promotion of Science 3 Organization for Research and Development of Innovative Science and Technology (ORDIST), Kansai University, Suita, Osaka 564-8680, Japan 4 PREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

S2 INDEX Supporting texts NMR measurement of tri-pcg/tri-pcg-sa-osu micelles in water IR measurement of tri-pcg-1 and tri-pcg-sa-osu DLS measurements Preparation of 4-arm PEG-NH 2 Table S1 Characterization of tri-pcg-2 Table S2. Z-average of diameters and size distributions for diluted F(P1/S+PLys5k), F(P1/S) and F(P1) in PBS. Table S3. Results of SEC after 400 min incubation at 37 C for F(P1/S+PLys5k) and F(P1) Figure S1. 1 H-NMR spectra for tri-pcg, tri-pcg-sa-oh and tri-pcg-sa-osu. Figure S2. SEC elution curves for tri-pcg-1and tri-pcg-sa-osu. Figure S3. FT-IR spectra for tri-pcg, tri-pcg-sa-oh and tri-pcg-sa-osu. Figure S4. Photographs of tri-pcg-1 and tri-pcg-sa-osu in dry state. Figure S5. Phase diagram of tri-pcg-sa-osu. Figure S6. Photographs of F(P1/S+PLys5k). Figure S7. (a) Photographs for tri-pcg-2 solution (b) Photographs of F(P2/S+PLys5k) after heating at 37 C for 1 min and 1day. Figure S8. The amount of insoluble fraction as a function of incubation time. Figure S9. 1 H-NMR spectra of tri-pcg/tri-pcg-sa-osu in D 2 O at 25 and 37 C. Figure S10. Size distribution for F(P1/S+PLys5k), F(P1/S) and F(P1) at 25 and 37 C. Figure S11. Time course of storage and loss moduli. Figure S12. SEC elution curves for soluble part of F(P1/S+PLys5k) and F(P1) after 400 min incubation at 37 C. Figure S12. Degradation profiles of tri-pcg-1 in PBS at 37 C. Figure S13. Swelling and erosion profiles of F(P1/S+oligoamine) hydrogel. Figure S14. Cell viability of L929 fibroblast cells in the presence of F(P1/S+PLys5k) and PLys, and in the presence of hydrolysate of F(P1/S+PLys5k), F(P1/S) and F(P1). Figure S15. Storage and loss moduli of F(P1/S+PLys5k) and F(P1) hydrogels removed from rats.

S3 NMR measurement of tri-pcg-1/tri-pcg-sa-osu micelles in water To investigate environmental changes of PCGA segments and OSu groups of tri-pcg-1/tri-pcg-sa- OSu mixture micelles upon increasing temperature, 1 H-NMR spectra of tri-pcg-1/tri-pcg-sa-osu were measured. 1 H-NMR spectra of the tri-pcg-1/tri-pcg-sa-osu (1:1) solution in D 2 O (10 wt%) at 25 and 37 C are shown in Figure S8. In CDCl 3, methylene protons of PEG (around 3.6 ppm) and PCGA (1.0-2.0 ppm) segments in CP-OSu exhibited sharp peaks (Figure S1), because CDCl 3 is a good solvent for both PEG and PCGA segments. On the other hand, in D 2 O, methylene protons of PEG were observed as sharp peaks, but the methylene protons of the PCGA segment and OSu group were broad peaks at 25 C, because D 2 O is a good solvent for PEG, but a poor solvent for the PCGA segment and OSu group. These results suggest that tri-pcg-1/tri-pcg-sa-osu was dissolved in water as a core-shell type micelle, where PCGA segments containing OSu groups form hydrophobic cores and PEG segments form hydrophilic shells. The OSu groups on the termini of the PCGA segment existed in the micelle core and were covered by the PEG shell. Upon increasing temperature to 37 C, peaks of PEG, PCGA segment and OSu groups were slightly shifted downfield, and the peak of the OSu group became sharp. Furthermore, the integral ratio of PCGA segments slightly increased, and the relative integral ratio (a)/(b) for PCGA (a) to PEG (b) increased from 1.08 to 1.19. These results suggest that the PCGA segment and OSu group were exposed to the aqueous environment upon increasing temperature, which induced micelle aggregation and consequent sol-to-gel transition. These phenomena must occur in the F(P1/S+PLys) system, which allows the OSu groups to make contact with the NH 2 groups of PLys in the aqueous phase to form covalent bonds. IR measurement of tri-pcg-1 and tri-pcg-sa-osu IR spectra were recorded on a spectrometer FT/IR-4200 Type A (JASCO) for tri-pcg-1 and tri-pcg- SA-OSu by KBr methods. The results are shown in Figure S3. The difference was not clear in the original spectra of tri-pcg-1 and tri-pcg-sa-osu. But in the differential spectra (Figure S3(b)) the absorbance of succinimide ester group was clearly observed at 1740, 1780, and 1820 cm -1. DLS measurements To confirm the occurrence of micelle aggregation, the hydrodynamic diameters (D h ) and size distributions of F(P1/S+PLys), F(P1/S) and F(P1) micelles in water under diluted condition (0.5 wt%) at 25 C and 37 C were investigated by dynamic light scattering (DLS) measurements. The D h and polydispersity index (PDI) of the polymer aggregates or micelles in aqueous solution were measured by dynamic light scattering (DLS) (Malvern Instruments, Ltd., Zetasizer nano Z ZEN2600) at 25 or 37 C using a detection angle of 173 with a He-Ne laser as the incident beam. The copolymer was dissolved in a small amount of acetone and added dropwise to phosphate buffered saline (PBS, ph = 7.4). After stirring for 20 min, acetone was removed by evaporation. The final polymer concentration was 0.5 wt%. Before the DLS measurements, sample solutions were filtered with a filter (pore size: 0.2 µm). The results are summarized in Figure S9. As the temperature increased from 25 C to 37 C, an increase in D h value was observed for all samples. These results support that the sol-to-gel transition at high concentration was induced by temperature-induced micelle aggregation. The D h values for F(P1/S+PLys), F(P1/S) and F(P1) at 25 C were almost the same, but at 37 C the D h value of F(P1/S+PLys) was larger than those of F(P1/S) and F(P1), suggesting the aggregation of micelles by covalent bonds in addition to hydrophobic interactions. Preparation of 4-arm PEG-NH 2 After drying in vacuo at 110 C for 2 h, 4-arm PEG-OH (MW = 5,000) (1.25 g, 243 µmol) was dissolved in anhydrous CH 2 Cl 2 (10 ml), and Et 3 N (1.1 ml, 7.8 mmol) was added to the solution. MeSO 2 Cl (300 µl, 3.9 mmol) was added to the solution with cooling to 0 C. The solution was allowed to react for 2 h at 0 C and an additional 10 h at r.t. After removal of CH 2 Cl 2 by evaporation, the products were dissolved in CH 3 Cl (5 ml) and reprecipitated by diethylether (100 ml) as a poor solvent. The obtained white powder was dried in vacuo, then dissolved in aqueous ammonia (50 ml), and stirred for 2 days at r.t. The product was extracted by dichloromethane (50 ml), and reprecipitated using CH 2 Cl 2 as the good solvent and diethylether as the poor solvent. The obtained solid was dried in vacuo to give 4-arm PEG-NH 2 (0.38 g, yield 30%).

S4 Table S1. Characterization of tri-pcg-1 and tri-pcg-sa-osu Code M n (Da) a) M w (Da) b) M w /M n b) DP of CL c) DP of GA c) CL/GA d) L C f) L G f) Tri-PCG-2 4,100 5,700 1.4 8.0 2.2 3.7 3.6 1.0 a) Estimated by 1 H-NMR (solvent: CDCl 3 ). b) Estimated by SEC (eluent: DMF, standard: PEG). c) Degree of polymerization (DP) of CL and GA unit in a PCGA segment estimated from 1 H-NMR. d) Molar ratio of CL/GA in a PCGA segment estimated by 1 H-NMR. e) Average continuous sequence lengths of CL and GA units (L c and L g ) calculated from 1 H-NMR. Table S2. Z-average of hydrodynamic diameter (D h ) and polydispersity index (PDI) for diluted F(P1/S+PLys5k), F(P1/S) and F(P1) in PBS at 25 and 37 C. Sample Temperature ( C) D h (nm) PDI F(P1/S+PLys5k) 25 25.6 0.22 37 106 0.07 F(P1/S) 25 26.6 0.29 37 96.4 0.14 F(P1) 25 27.5 0.26 37 64.0 0.25 Polymer concentration was 0.5wt%. Table S3. Results of SEC after 400 min incubation at 37 C for F(P1/S+PLys5k) and F(P1) Code M n M w M w /M n Soluble part of F(P1/S+PLys5k) (after 400 min incubation at 37 C ) 5,500 10,000 1.8 F(P1) (Tri-PCG-1) (after 400 min incubation at 37 C ) 4,300 6,000 1.4 Tri-PCG-1 4,000 5,800 1.5

S5 Figure S1. 1 H-NMR spectra for (a) tri-pcg, (b) tri-pcg-sa-oh and (c) tri-pcg-sa-osu in CDCl 3. Left: 0-8.0 ppm, right: 2.5-3.2 ppm Figure S2. SEC elution curves for tri-pcg-1 (dotted line) and tri-pcg-sa-osu (solid line) (eluent: DMF, standard: PEG).

S6 (a) (b) 4000 3400 2800 2200 1600 1000 400 1850 1825 1800 1775 1750 1725 1700 Figure S3. Top: IR spectra for (a) tri-pcg and (b) tri-pcg-sa-osu. Bottom: Differential IR spectra of tri-pcg-sa-osu and tri-pcg in 1700-1850 cm -1.

S7 Figure S4. Photographs of (a) tri-pcg-1 and (b) tri-pcg-sa-osu in dry state at r.t. Figure S5. Phase diagram of tri-pcg-sa-osu in PBS. : Sol, : Gel, : Precipitate. Insets are photographs of the polymer solutions (25wt%) at 25 C and 42 C.

S8 Figure S6. (a) Photographs of F(P1/S+PLys5k) just after mixing with PLys5k and 1 day for at 25 C. (b) subsequent heating at 37 C and cooling 4 C. Figure S7. (a) Photographs for tri-pcg-2 solution in PBS (25wt%) at 25, 37, 65 C. (b) Photographs of F(P2/S+PLys5k) after heating at 37 C for 1 min and 1day.

S9 Figure S8. The amount of insoluble fraction as a function of incubation time after temperature raising to 37 C. Insoluble fraction ratio (%) = (weight of insoluble fraction) / (weight of tri-pcg- SA-OSu in the initial sample) 100 Figure S9. 1 H-NMR spectra of tri-pcg/tri-pcg-sa-osu in D 2 O (10wt%) at 25 and 37 C. (a) CH 2 of PEG and (b) CH 2 of caployl unit and (c) OSu group of CP-OSu. Left: 0-6 ppm, right: 2.5-3.1 ppm.

S10 Figure S10. Size distribution for (a) F(P1/S+PLys5k) ( ), (b) F(P1/S)( ) and (c) F(P1) ( ) in PBS at 25 and 37 C.

S11 Figure S11. Time course of storage (G, solid line) and loss (G, dotted line) moduli for F(P1/S+PLys) (red), F(P1/S) (green), F(P1+PLys) (yellow) and F(P1) (blue) at 37 C. Figure S12. SEC elution curves for (a) soluble part of F(P1/S+PLys5k) and (b) F(P1) after 400 min incubation at 37 C, and (c) tri-pcg-sa-osu, and (d) tri-pcg-1 (eluent: DMF, standard: PEG).

S12 Figure S13. Degradation profiles of tri-pcg-1 in PBS at 37 C. Figure S14. Swelling and erosion profiles of F(P1/S+oligoamine) hydrogel using Lys ( ), Lys-Lys ( ) and Lys-Lys-Lys ( ) soaked in PBS at 37 C.

S13 Figure S15. Cell viability of L929 fibroblast cells incubtated in E-MEM containing 10% FCS for 24 h in the presence of (a) F(P1/S+PLys5k) ( ) and PLys ( ) as a function of concentration of PLys, (b) and in the presence of hydrolysate of F(P1/S+PLys5k) ( ), F(P1/S) ( ) and F(P1) ( ) as a function of polymer concentration. Figure S16. Storage (G, closed symbols) and loss (G, open symbols) moduli of F(P1/S+PLys5k) (, ) and F(P1) (, ) hydrogels removed from rats on (a) day 1 and (b) day 8 after subcutaneous injection.