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1 Eletroni supporting informtion Dully Ctioni n Anioni p/temperture-sensitive Injetle yrogels n Potentil Applition s Protein Crrier Cong Tru uynh, Minh Khnh guyen n Doo Sung Lee* 1. Experimentl 1.1. Mterils Poly(ethylene glyol) (PEG, M n 1,750) ws provie y ID Biohem In. (Seoul, Kore). PEG (M n 2,000), sulfmethzine (SM), ryloyl hlorie (AC), nhyrous etone, iethnolmine (DEA), nhyrous,-imethylformmie (DMF), D,L-ltie (LA), ε-proltone (CL), stnnous 2-ethylhexnote (Sn(t) 2 ), iutyltin ilurte (DBTL), 1,6-iisoynto hexmethylene (DI), n phosphte uffer sline (PBS) were otine from Sigm-Alrih (St. Louis, M, USA) n use s reeive. umn growth hormone (hg) ws provie from LG hemils (Seoul, Kore). Soium hyroxie (), hyrohlori i (Cl), iethyl ether n n-hexne were ll prouts of Smhun Co. (Seoul, Kore). All other regents were of nlytil gre n use without further purifition Synthesis of ioegrle [PCLA-PEG-PCLA-PASM] x lok opolymers (PUASM opolymers) The PUASM lok opolymers were synthesize y the polyition of the isoynte groups of DI with the hyroxyl groups of synthesize ihyroxyl mino sulfmethzine monomer (DASM), n of trilok poly(ε-proltone-ltie)-poly(ethylene glyol)-poly(ε-proltoneltie) (PCLA-PEG-PCLA) in DMF, in the presene of DBTL s tlyst (Sheme S1). The PCLA-PEG-PCLA trilok opolymers were synthesize n hrterize s in the previous report. 1

2 2 R SM Cl R SM-A R DASM n + + Sn(t) 2 -PCLA-PEG-PCLA- PEG CL LA C C 6 DBTL PCLA-PEG-PCLA [PCLA-PEG-PCLA-PASM] x 6 m 6 x (PUASM opolymer) R R : 3 C 3 C S Sheme S1: Syntheti sheme of PUASM lok opolymers Synthesis of monomer DASM. The DASM ws synthesize y Mihel-ition retion etween the seonry mine groups of DEA n the vinyl groups of sulfmethzine-rylte (SM-A), whih ws synthesize similrly the previous proeure. The etil retion to synthesize the monomer DASM is s follows: SM-A n DEA were ple in 250 ml two-nek roun-ottom flsk t molr rtio of 1/1. Anhyrous DMF ws e to issolve the retnts t onentrtion of 10 wt%. The flsk ws then ple in 50 o C oil-th with onstnt stir rte for 12 h. After 12 h, the retion solution ws onentrte uner reue vuum n preipitte in n exess of n-hexne. The preipittion ws repete two times n DASM ws filtere n rie uner vuum t room temperture for 48 h prior to use. 2

3 Synthesis of opolymer PUASM. The synthesis of PUASM lok opolymers is s follows (PUASM-2): trilok PCLA-PEG- PCLA (M n 5860) (1.00 mmol), DASM (5.00 mmol) n DBTL (0.002 g) were rie in 250 ml two-nek roun-ottom flsk uner vuum t 80 o C for 2 h. The temperture ws then erese to 70 o C n the vuum ws reple y rie nitrogen followe y the ition of 90 ml nhyrous DMF. After the retnts ompletely issolve, DI (6.00 mmol) ws e n the retion ws rrie out for 3 h. Finlly, the retion solution ws onentrte uner reue vuum n preipitte in n exess of iethyl ether. The preipitte opolymer ws filtere n rie uner vuum t room temperture for 48 h. The finl yiel ws pproximtely 90%. The synthesize PUASM lok opolymers were hrterize using 1 MR n gel permetion hromtogrphy (GPC) Chrteriztion A Vrin Unity-500 Mz spetrometer (Vrin In., Plo Alto, CA, USA) ws use to reor the MR spetr using D 2 or CDCl 3 s the solvents. The moleulr weights of the opolymers n their istriutions were mesure using GPC (Futes S2001, Futes Co., Lt., De Jeon, Kore) with refrtive inex etetor (Shoex RI-101, Show Denko K.K., Kngw, Jpn) n three Styrgel (KF-803, KF n KF-802, Show Denko K.K., Kngw, Jpn) olumns in series, t flow rte of 1.0 ml/min (eluent: CCl 3 ; 35 C). PEG stnrs (Wters Corp., Minz, Germny) were use for lirtion Sol-gel phse trnsition mesurement The sol (flow)-gel (non-flow) phse trnsition of the opolymer in queous solution ws etermine using the tue inversion metho. Briefly, the opolymer ws issolve in phosphte uffere sline solution (PBS) t p~9 in 4 ml vil (10 mm imeter) t the given onentrtions for 4 h. The p ws juste to esigne vlues (IQ240 p meter, h Co., C, USA) using 5 n 5 Cl solutions t 0 o C n the smples were stilize t 2 o C overnight. The smple vils ontining pproximtely 0.5 ml of the opolymer solution were ple in wter-th n 3

4 hete slowly from 0 C to 65 C. The smples were equilirte for 10 min t 2 o C intervls. The sol-gel trnsition ws etermine y inverting the vils Rheologil mesurement A ynmi mehnil nlyzer (Bohlin Rottionl Rheometer, Mlvern Instruments Lt., WR14, UK) ws use to etermine the hnge in the visosity of the opolymer queous solutions. silltion moe with ontrolle stress of 0.4 P n frequeny of 1 r/s ws performe. The heting rte ws 1 o C/min. A opolymer solution in PBS ws ple etween 20 mm imeter plte n 100 mm imeter plte with gp of 250 μm n the test ws performe Zet potentil mesurement To onfirm the ul ioni properties, the zet potentils of synthesize mphoteri PUASM, tioni PAE-se (PAE-PCL-PEG-PCL-PAE: ), nioni SM-se (SM-PCLA-PEG-PCLA-SM: ) opolymers in eionize wter were ompre. The zet potentil of the opolymer solutions ws mesure using Zetsizer no ZS instrument (Mlvern Instruments Lt., WR14, UK) t room temperture. The opolymer solutions were prepre in eionize wter t onentrtion of 2.0 mg/ml with ifferent p vlues n performe the tests within n hour fter preprtion In vitro egrtion The in vitro egrtion of the PUASM hyrogels ws exmine. Briefly, 4 ml vils ontining 0.5 ml hyrogels t p 7.4 were inute t 37 o C for 15 min to form gels. Susequently, 1 ml of PBS t p 7.4 ws e n the smple vils were inute t 4 or 37 o C. At preetermine times, the smples were ollete, frozen n lyophilize. The GPC ws performe to etermine the moleulr weight of egre opolymer In vitro ytotoxiity of PUASM opolymer hyrogels In vitro ytotoxiity of opolymer hyrogels ws exmine oring to the IS/E Prt 5 Guielines. These guielines presrie the use of the Duleo s moifie Egle s meium (DMEM) extrtion test to ssess the possile toxi effets of the omponents relese from the 4

5 meil polymers uring extrtion. Different mounts ( mg/ml) of opolymer were extrte t 37 o C for 24 h using the DMEM (Invitrogen Corportion, CA, USA) ulture meium s the extrtion flui. After inution, the extrts were filtere (0.2 μm pore size; Avnte MFS, In. CA, USA) n 1mL of eh extrt ws e to the L929 firolst ells (Koren Cell Line Bnk, Seoul, Kore), whih h een seee in 24-well pltes. Fresh DMEM ws use s negtive ontrol. After 48 h inution, the ell viility n prolifertion were etermine using n MTT ssy. Briefly, 100 μl of fresh growth meium ontining 50 μg MTT (Sigm-Alrih, St. Louis, M, USA) ws e to eh well n the ells were inute t 37 o C for 4 h. The sorne t 570 nm mesure using SpetrMx M5 Miroplte Reer (Moleulr Devies, In., CA, USA) ws iretly proportionl to the numer of living ells. The perentge survivl reltive to the mok-trete ells (100 % survivl) ws lulte In vivo experiments Mle Sprgue-Dwley (SD) rts (nlim Experimentl Animl Lortory, Seoul, Kore) were use for the in vivo experiments. The rts (5-6 weeks ol, verge oy weight 200 g) were hnle in orne with the tionl Institutes of elth (I) guielines for the re n use of lortory nimls (I pulition 85-23, revise 1985). To exmine the injetility n in vivo geltion of PUASM hyrogel, n queous solution (200 µl, 25 wt%) t p 6.8 or 8.0 ws suutneously injete into the k of the SD rts. After 5 min, the rts were srifie n the gels morphology ws oserve. For in vivo hg relese experiments, 200 µl hg-loe opolymer solution (hg 10 mg/ml, 25 wt% PUASM-2) or 200 µl hg solution (10 mg/ml) ws injete into the SD rts (5 rts/group). At preetermine time points, loo smples were tken from the til vein of the rts n entrifuge to otin the ser, whih were store t -21 o C until ssye. The hg onentrtion in the ser ws nlyze using ommeril immunoenzymetri ssy kit (hg-easia, DIAsoure ImmunoAssys S.A, Belgium). 5

6 2. Results 2.1. Synthesis n hrteriztion mphoteri PUSAM opolymers The PUASM lok opolymers were synthesize y the polyition of the isoynte groups of 1,6-iisoynto hexmethylene (DI) with the hyroxyl groups of synthesize ihyroxyl mino sulfmethzine monomer (DASM) n of trilok poly(ε-proltone-ltie)- poly(ethylene glyol)-poly(ε-proltone-ltie) (PCLA-PEG-PCLA) opolymers in,imethylformmie (DMF) in the presene of iutyltin ilurte (DBTL) s tlyst (Sheme S1). Fig. S1 shows 1 MR spetr with lele protons of the synthesize trilok PCLA-PEG- PCLA (II), sulfmethzine-rylte (SM-A), DASM n PUASM opolymers. The signls t 3.65 (), 5.20 () n 2.30 () ppm in Fig. S1 were ssigne to the typil protons of PEG (-C 2 - C 2 --), LA (-C(C 3 )-) n CL (-C-C 2 -C 2 -) of the PCLA-PEG-PCLA trilok opolymer. The segment rtio of PEG, LA n CL loks in trilok PCLA-PEG-PCLA opolymer were lulte using the pek re of, n. Signls t 2.01 () n (e, e1, e2) ppm were respetively ssigne to the methylene protons of sulfmethzine (SM) (C 3 -) n protons of vinyl group (-C=C 2 ) (Fig. S1), initing the suessful onjugtion of ryloyl hlorie (AC) into SM. Signls t 2.45 (e) n 2.82 (f) ppm in Fig. S1 were ssigne to the protons of forme methylene groups ( C-C 2 -C 2 -=) n the signls t 2.60 (g) n 3.58 (h) ppm were ssigne to the protons of methylene groups (=-C 2 -C 2 -) of ouple iethnolmine (DEA) in the DASM. Fig. S1 shows 1 MR spetrum of the PUASM-2 opolymer. The presenes of PEG, LA n CL were respetively onfirme y the signls t 3.65, 5.20 n 2.30 ppm. The signl t 3.15 (i) ppm ws the first methylene protons of DI (--C 2 -). The signls t 2.55 (e), 6.58 (), 7.72 () n 8.08 () ppm onfirme the presene of DASM in the finl opolymer. The frtion of poly(mino sulfmethzine) (PASM) loks in the finl opolymer were lulte using the pek re of e (PASM) n PEG in Fig. S1. The 1 MR results onfirme the suessful synthesis of the PCLA-PEG-PCLA trilok, SM-A, DASM n PUASM opolymer. The etile hrteristis of the synthesize PUASM opolymers re liste in Tle S1. 6

7 ) 6 CDCl 3 h g SMA g h m i j j i j LA j h CL PCLA-PEG-PCLA PEG i gf e CL x LA CL j ) S e f g 2 h SMA h f e g S e e1 e2 ) e e1 e2 D 2 ppm n f g x e e y m e f ) CDCl ppm Fig. S1: 1 -MR spetr of () PCLA-PEG-PCLA (II) trilok opolymer (in CDCl 3 ), () SM-A (in D 2 ), () DASM (in D 2 ) n () PUASM-2 (in CDCl 3 ). Tle S1. Chrteristis of the synthesize PUASM opolymers. o. PCLA -PEG -PCLA PUASM [Tri-PASM ] x M n PDI PUASM (I) [ ] , PUASM (II) [ ] , provie y Sigm-Alrih n ID Biohem In; lulte from 1 MR (CL/LA 1.7, PASM=30.6 wt%); mesure n lulte y GPC. 7

8 The lose-loop gel regions of the PUASM hyrogels n e juste y vrying the moleulr weight of the PEG lok or opolymer onentrtion, s showe in Fig. S2. At fixe lok lengths of the PCLA n PASM, the gel region shifte to higher tempertures, s the moleulr weight of the PEG lok inrese from 1,750 to 2,000 (PUASM-1 to PUASM-2) (Fig. S2). This is similr to the shift of the prent temperture-sensitive PCLA-PEG-PCLA hyrogels s the inrese moleulr weight of PEG. In ition, the gel regions oul e esily expne y inresing the opolymer onentrtion (Fig. S2). ) Temperture/ o C Sol PUASM-2 PUASM-1 Gel Sol (ggregtion) C (37 o C, p 7.4) Gel p Sol ) Temperture/ ( o C C) PUASM-2 30 wt% Sol (Aggregtion) 25 wt% 20 wt% Boy onition Gel Sol p Fig. S2. The lose-loop gel regions of the mphoteri PUASM hyrogels. () The gel region shifte to higher tempertures with inresing the moleulr weight of PEG (25 wt%). () The gel regions oul e tilore y hnge in the opolymer onentrtion (PUASM-2) In vivo geltion Previous reports showe tht p/temperture-sensitive hyrogel systems oul e injete into the oy only t milly si (i.e., nioni hyrogels) or ii (i.e., tioni hyrogels) p. Interestingly, this novel mphoteri PUASM opolymer queous solution existe in the free flowing solution t oth ii n si p, whih woul filitte for simply suutneous ministrtion t the oth p onitions. Fig. S3 shows photogrphs of the gels t five minutes n one week fter injetion of opolymer solutions t ifferent p vlues (e.g., p 6.8 n 8.0) into the SD rts, presenting injetility of the PUASM solutions t oth milly ii n si p. 8

9 Eletroni Supplementry Mteril (ESI) for Chemil Communitions 5 min fter injetion ) Injete t p 8.0 ) Injete t p week fter injetion Fig. S3. Photogrphs of in vivo morphology of the 25 wt% PUASM-2 hyrogels t 5 min n one week fter the opolymer solutions were injete into SD rts. The injetions were performe t room temperture n t p 8.0 () n p 6.8 () In vitro egrtion In vitro egrtion of the PUASM opolymer hyrogels ws rrie out to onfirm their ioegrility. Fig. S4 shows ereses in moleulr weights of the prent trilok n PUASM opolymer hyrogels t p 7.4 n ifferent tempertures. At 4 oc, oth opolymers showe slow egrtion rtes up to one month. owever, t physiologil onitions (37 oc, p 7.4), oth opolymers showe fster egrtion rtes with grul ereses in their moleulr weights for over one month. The moleulr weight of the PUASM opolymer erese ontinuously from ~11.0 to ~5.0 kd y one month. This result onfirms the ioegrility of the PUASM opolymers. PUASM, 4 oc PUASM, 37 oc Trilok, 4 oc Trilok, 37 oc Mn/ g/mol Time/ y Fig. S4. In vitro egrtion of the PUASM hyrogels (25 wt% PUASM-2) n its prent PCLAPEG-PCLA (II) trilok hyrogels (20 wt%) t p 7.4 n ifferent tempertures. 9

10 2.4. In vitro ytotoxiity In vitro ytotoxiity of PUASM hyrogels ws exmine oring to the IS/E Prt 5 Guielines. This test ws esigne to etermine the possile ytotoxi effets of the mterils extrte from iomterils implnte in the oy. Vrious mounts ( mg/ml) of opolymer were inute in Duleo s moifie Egle s meium (DMEM) for 24 h t 37 o C to otin the extrts. The firolst ells L929 were expose to the extrts for 48 h n the ytotoxi effets were evlute. Fresh DMEM ws use s negtive ontrol. As shown in Fig. S5, the PUASM hyrogel exhiite low ytotoxiity t high opolymer onentrtions. The ell viility in the presene of the hyrogel ws greter 90 % up to opolymer onentrtion of 300 mg/ml. This result inites tht the PUASM hyrogels n e use s low ytotoxi mteril. 100 Cell viility/ % Control PUASM onentrtion/ mg/ml Fig. S5. Viility of L929 firolst ells expose with the PUASM-2 hyrogel (±SD, n = 3). 10