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1 <Supplementry Informtion> Disulfide-Cross-linked PEG-Poly(mino cid)s Copolymer Micelles for Glutthione-Medited Intrcellulr Drug Delivery Ahn N Koo, Hong Je Lee, Sung Eun Kim, Jeong Ho Chng, Chiyoung Prk, b Chulhee Kim, b Je Hyung Prk c nd Sng Cheon Lee* Nnomterils Appliction Division, Kore Institute of Cermic Engineering nd Technology, Seoul , Kore b Deprtment of Polymer Science nd Engineering, Hyperstructured rgnic Mterils Reserch Center, Inh University, Incheon , Kore c Deprtments of Advnced Polymer nd Nnophrmceuticl Sciences, Kyung Hee University, Gyeonggi-do , Kore *To whom correspondence should be ddressed: Sng Cheon Lee, Ph.D. Nnomterils Appliction Division Kore Institute of Cermic Engineering nd Technology Gsn-Dong, Guemcheon-Gu, Seoul , Kore Tel: Fx E-mil: sclee@kicet.re.kr

2 Experimentl Mterils nd Equipment. α-methoxy-ω-mino-poly(ethylene glycol) (CH 3 -PEG- NH 2 ) of M n of 5000 g/mol ws purchsed from SunBio Inc. (Seoul, Kore) nd used s received. N 6 -Crbobenzyloxy-L-lysine (H-Lys(Z)-H)), L-phenyllnine (Ph), nd methotrexte were purchsed from Sigm Co. (St. Louis, M) nd used without further purifiction. Triphosgene nd pyrene were purchsed from Aldrich Co. (Milwukee, WI) nd used s received. 3,3 -Dithiobis(sulfosuccinimidylpropionte) (DTSSP) ws purchsed from Pierce (Perbio Science Deutschlnd GmbH, Bonn, Germny). Tetrhydrofurn (THF) ws distilled from N/benzophenone under N 2, prior to use. N,N- Dimethylformmide (DMF) ws dried nd distilled over clcium hydride. The 1 H NMR spectr were recorded t 400 MHz on Vrin INVA400 NMR spectrometer with smple spinning rte of 5 khz t 25 o C. Moleculr weight distributions were determined using GPC equipped with Wters 2414 refrctive index detector, 515 HPLC pump, nd three consecutive Styrgel columns (HR1, HR2, nd HR4). The eluent ws THF with flow rte of 1 ml/min. The moleculr weights were clibrted with polystyrene stndrds. The zet potentil (ζ) ws mesured using 90 PLUS (BrookHAVEN Instruments Coopertion, New York, USA) prticle size nlyzer. N 6 -Crbobenzyloxy-L-lysine N-crboxynhydride (Lys(Z)-NCA) nd L- phenyllnine N-crboxynhydride (Ph-NCA). Synthesis of N-crboxynhydride of L-lysine nd L-phenyllnine (Lys(Z)-NCA nd Ph-NCA) ws crried out by the Fuchs- Frthing method using triphosgene [1]. In brief, for Lys(Z)-NCA, Lys(Z) (30g, 107 mmol) ws suspended in THF (300 ml) t 50 o C nd triphosgene (10.58 g, 35.68mmol) ws then dded into the Lys(Z)-suspended solution. After 3 h, the crude product ws 2

3 precipitted into n-hexne, nd Lys(Z)-NCA ws recrystllized from THF/n-hexne. Yield 92%. For Ph-NCA, Ph (5 g, 30.3 mmol) ws suspended in THF (50 ml) nd heted to 50 o C in nitrogen tmosphere. A solution of triphosgene (3 g, 12.1 mmol) in THF ws then dded dropwise to the stirred rection mixture. After 3 h, the rection mixture ws filtered to remove insoluble mterils, nd the filtrte ws poured into n- hexne (300 ml). The resulting suspension stored t -20 o C overnight to ssure complete crystlliztion. Ph-NCA ws recrystllized t -20 o C from mixture of THF/n-hexne. Yield 84 %. Anl. Clcd for Lys(Z)-NCA (C 15 H 18 N 2 5 ): C, 58.82; H, 5.92; N, Found: C, 58.69; H, 5.99; N, Clcd for Ph-NCA (C 10 H 9 N 3 ): C, 62.82; H, 4.74; N, Found: C, 62.24; H, 4.91; N, [1] W. H. Dly, D. Poche, Tetrhedron Lett., 1988, 29, Synthesis of PEG-b-PLys-b-PPh Triblock Copolymer. PEG 113 -b-plys 11 -b- PPh 24 tht hve EG units of 113, Lys of 11, nd Phe units of 24 ws synthesized s follows: To stirred solution of CH 3 -PEG-NH 2 (3 g, 0.6 mmol) in dry DMF (30 ml) ws dded Lys(Z)-NCA (2.02 g, 6.6 mmol) t 35 o C under nitrogen. After 24 h, Ph- NCA (3.44 g, 18 mmol) nd dry DMF (100 ml) were dded to the rection mixture, nd the rection ws mintined for further 24 h. PEG 45 -b-plys(z) 11 -b-pph 24 ws isolted by repeted precipittion from DMF into diethyl ether. Yield 92 %. Finlly, the deprotection of PEG 113 -b-plys(z) 11 -b-pph 24 ws performed by treting the block copolymer (0.5 g) with trifluorocetic cid (TFA) (5 ml) nd HBr/HAc (0.2 ml) to remove Z groups. The product, PEG 113 -b-plys 11 -b-pph 24, ws isolted by dilysis using membrne (moleculr weight cut-off: 1000) for 24 h, followed by freeze-drying. 3

4 Shell Cross-Linking of Core-Shell-Coron Micelles. Polymer micelles of PEG 113 -b-plys 11 -b-pph 24, consisting of PEG corons, PLys shells, nd PPh cores, were prepred by dilyzing the polymer solution in DMF ginst doubly distilled wter. Shell cross-linking ws crried out by dding n queous solution of DTSSP to micellr solution of PEG 113 -b-plys 11 -b-pph 24 (1 g/l) t ph 9.0. The rection mixture ws llowed to stir for 8 h t room temperture nd then dilyzed ginst doubly distilled wter for 3 h to remove unrected DTSSP, nd the dilyzte ws lyophilized to obtin the shell cross-linked polymer micelles. To control the degree of cross-linking, the feed molr rtio of DTSSP to Lys repeting units of PLys middle shells ws vried from 1:1 to 2:1. Consumption of primry mines of Lys units during the cross-linking rection ws quntified using fluorescmine ssy. Stbility of Shell Cross-Linked Polymer Micelles. Kinetic stbility of non-cross-linked micelles nd shell cross-linked micelles ws investigted by interction with the sodium dodecyl sulfte (SDS), which cts s destbilizing gent in queous medi. The effect of SDS on micelles in queous medi ws estimted by dynmic light scttering nlysis. A SDS solution (1 ml, 7.5 g/l) ws dded to queous solutions of non-cross-linked micelles or shell cross-linked polymer micelles (2 ml, 0.75 g/l) nd the solution ws stirred t 400 rpm. The finl SDS concentrtion ws 2.5 g/l. At predetermined time intervls, scttered light intensity of the micellr solutions contining SDS ws monitored. Fluorescence Mesurements. Pyrene excittion spectr were recorded on JASC FP-6500 spectrofluorometer. Excittion nd emission bnd widths were set t 1 4

5 nm nd 5 nm, respectively. The emission wvelength ws 390 nm, nd the pyrene excittion spectr were recorded in the wvelength rnge of nm. Light Scttering Mesurements. Dynmic light scttering mesurements were performed using 90 Plus prticle size nlyzer (Brookhven Instruments Corportion). All the mesurements were crried out t 25 o C. The smple solutions were purified by pssing through Millipore 0.45 μm filter. The scttered light of verticlly polrized He-Ne lser (632.8 nm) ws mesured t n ngle of 90 nd ws collected on n utocorreltor. The hydrodynmic dimeters (d) of micelles were clculted by using the Stokes-Einstein eqution d = k B T/3πηD where k B is the Boltzmnn constnt, T is the bsolute temperture, η is the solvent viscosity, nd D is the diffusion coefficient. The polydispersity fctor, represented s μ 2 /Γ 2, where μ 2 is the second cumulnt of the decy function nd Γ is the verge chrcteristic line width, ws clculted from the cumulnt method [2]. [2] A. Hrd nd K. Ktok, Mcromolecules, 1998, 31, 288. Trnsmission Electron Microscopy. Trnsmission electron microscopy (TEM) ws performed on JEM-2000EX (JEL Tokyo, Jpn), operting t n ccelertion voltge of 200 kv. To observe the size nd distribution of micellr prticles, drop of smple solution (concentrtion = 2 g/l) ws plced onto 200-mesh copper grid coted with crbon. About 1 min fter deposition, surfce wter ws removed using filter pper, followed by ir-drying. A drop of urnyl cette solution (5 wt%) ws used for negtive stining. 5

6 Preprtion of MTX-Loded Shell Cross-Linked Micelles. MTX-loded PEG 113 -b-plys 11 -b-pph 24 micelles were prepred by the dilysis method nd subsequent shell cross-linking rection. The block copolymer (20 mg) ws dissolved in 0.5 ml of DMF t 70 o C, nd MTX (2 mg) ws subsequently dded. The solution ws stirred for 6 h t room temperture nd then dilyzed ginst doubly distilled wter using membrne (Spectrpor, MWC: 1000). After 12 h dilysis, the solution of MTX-loded micelles ws collected. DTSSP s shell cross-linking gent ws then dded to this solution by vrying the feed molr rtio of [DTSSP]:[Lys] (1:2 or 1:1). The rection ws mintined for 8 h t ph 9.0, nd the solution ws dilyzed for 3 h to remove residul DTSSP, unloded MTX, nd rection by-products. The dilyzte ws lyophilized to obtin MTXloded shell cross-linked polymer micelles. For preprtion of non-cross-linked MTXloded micelles, n identicl process nd time period were employed, except for the DTSSP ddition. To determine the drug loding content nd loding efficiency, MTXloded polymer micelles were dissolved in DMF, nd the bsorbnce of MTX ws then mesured using UV-VIS spectrophotometer t 303 nm. Glutthione-Medited Controlled Drug Relese. MTX-loded micelles were precisely weighed, redispersed in the phosphte-buffered sline (1 ml PBS, ph 7.4, 1 g/l) solution, nd introduced into dilysis membrne bg (MWC=1,000). The relese experiment ws initited by plcing the dilysis bg in relese medi of vrious GSH concentrtions (0, 2 μm, 0.1 mm, 1 mm, nd 10 mm). The relese medium ws shken t speed of 100 rpm t 37 o C. At predetermined time intervls, smples (10 ml) were withdrwn nd replced with n equl volume of the fresh medium. The concentrtion of MTX in the smples ws mesured by UV-VIS spectrophotometer t 303 nm. The 6

7 ssy for MTX ws bsed on liner stndrd curve obtined using the concentrtion rnge of mg/ml. Cell Culture. A549 humn lung crcinom cells ws obtined from the Koren Cell Line Bnk (KCLB, Seoul). Cells were propgted in RPMI-1640 medium (Gibco BRL) supplemented with 10% (v/v) het-inctivted fetl bovine serum (FBS, Gibco BRL), nd 1% (v/v) penicillin streptomycin (Gibco BRL). Cells were cultured in humidified incubtor t 37 with 5% C 2. Cellulr Uptke of SCM. To monitor time dependent cellulr uptke of SCM, fluorescence isothiocynte (FITC) ws conjugted to the PLys middle lyers of SCM. A549 cells ( cells/well) incubted with FITC-lbeled SCM (100 μg/ml) were imged by IX71 fluorescence microscope (lympus, Jpn). Cytotoxicity of Polymer Micelles nd SCMs. A549 cells were mintined in RPMI-1640 medium contining 10% FBS nd 1% ntibiotic-ntimycotic. The cells were seeded into 96-well flt-bottomed tissue-culture plte t 1000 cells/well, nd incubted for 24 h in humidified tmosphere of 5 % C 2 t 37 o C. The concentrtion of SCM ws diluted with culture medium to obtin concentrtion rnge from 50 to 300 μg/ml. After the incubtion for 24 h, 50 μl of 10 mg/ml 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyl tetrzolium bromide (MTT) solution in PBS ws dded to ech well, nd the plte ws incubted for 4 h t 37 o C, llowing vible cells to reduce the MTT into purple formzn crystl. The formzn crystl ws dissolved by dding 200 μl of dimethyl sulfoxide (DMS) nd 25 μl of Sörensen s glycine buffer. The bsorbnce of individul wells ws mesured t 570 nm by microplte reder (SFTmx PR, Moleculr Devices Corportion, CA). 7

8 GSH-Controlled Intrcellulr MTX Delivery. To estimte the dependency of the intrcellulr MTX relese on GSH concentrtions, the intrcellulr level of GSH ws mnipulted by dding glutthione ethyl ester (GSH-Et) into the cell culture medi. A549 cells were seeded evenly into 96-well flt-bottomed tissue-culture plte (Corning Glss Works, Corning, NY) t 5000 cells/well concentrtion, nd incubted for 24 h in humidified tmosphere of 5 % C 2 t 37 o C. The cells were firstly treted with vried concentrtions of GSH-Et (0, 5, nd 10 mm) for 2 h nd wshed. MTX-loded SCM olutions (100 μl, concentrtion= 50 μg/ml) were then dded nd, the pltes were incubted. The MTX concentrtion ws fixed t 4.3 μg/ml. After the incubtion for 5 nd 10 h, the cytotoxicity of SCMs ws evluted by n MTT ssy. Imging of Cell Vibility. To obtin cellulr vitl/ded imges, the stock solutions of fluorescein dicette (5 mg/ml in cetone) nd ethidium bromide (1.25 mg/ml in PBS) were prepred. Shortly before use, 5 μl of the fluorescein dicette solution nd 5 μl of the ethidium bromide solution were mixed in 1 ml of PBS nd stored t 5 o C. The cells were stined with the solution mixture (50 μl) nd incubted for 10 min t 37 o C, before being subjected to fluorescence microscopic observtion. 8

9 Tble S1. Chrcteristics of the PEG 113 -PLys 11 -PPh 24 copolymer copolymer PEG 113 -PLys 11 - PPh 24 feed rtio ([EG]:[Lys(Z)]:[Ph]) composition rtio ([EG]:[Lys]:[Ph]) conversion of Lys(Z) (%) conversion of Ph (%) M n M w /M n b cmc c (mg/l) 113 : 12 : : 11 : , *The copolymers were synthesized using the mcroinititor (CH 3 -PEG-NH 2 ) with M n of 5,000 nd polydispersity of Clculted by 1 H NMR spectr. b M w /M n of PEG 113 -Plys(Z) 11 -PPh 24 estimted by GPC. c Criticl micelle concentrtion t 25 o C. Fig. S1 Synthetic route to the PEG 113 -PLys 11 -PPh 24 copolymer HN HC C C (CH 2 ) 4 NHCCH 2 CH 3 CH 2 CH 2 NH CH 3 CH 2 CH 2 NH CCHNH H DMF, 35 o C (CH 2 ) 4 PEG 113 -PLys(Z) 11 N H C HN C CH 2 HC C CH 2 DMF, 35 o C CH 3 CH 2 CH 2 NH CCHNH CCHNH H (CH 2 ) 4 PEG 113 -PLys(Z) 11 -PPh N H 24 C CH 2 CH 2 TFA HBr/AcH CH 3 CH 2 CH 2 NH CCHNH CCHNH H (CH 2 ) 4 CH 2 PEG 113 -PLys 11 -PPh 24 NH 2 9

10 Fig. S2 1 H NMR spectr of CH 3 -PEG-NH 2 (), PEG 113 -PLys(Z) 11 (b), PEG PLys(Z) 11 -PPh 24 (c), nd PEG 113 -PLys 11 -PPh 24 (d) in DMS-d 6. () CH 3 CH 2 CH 2 NH (b) CH 3 CH 2 CH 2 NH CCHNH H (CH 2 ) 4 N H C CH 2 c b (c) d d c CH 3 CH 2CH 2 NH CCHNH CCHNH H (CH 2) 4 CH 2 d + e d N H C CH 2 c b e c b b (d) CH 3 CH 2CH 2 NH CCHNH CCHNH H (CH 2) 4 CH 2 NH2 b c c b δ (PPM) Fig. S3 Gel permetion chromtogrms of CH 3 -PEG-NH 2 (), PEG 113 -PLys(Z) 11 (b), nd PEG 113 -PLys(Z) 11 -PPh 24 (c). (c) (b) () Retention Time (min) 10

11 Fig. S4 () Excittion spectr of pyrene s function of the PEG 113 -PLys 11 -PPh 24 concentrtion in wter. (b) Plot of I 336 /I 332 (from pyrene excittion spectr) vs. Log C for PEG 113 -PLys 11 -PPh 24. Intensity (.u.) g/l g/l g/l 0.25 g/l 2.5 g/l () Wvelength (nm) (b) I 336 / I cmc Log C (g/l) 11

12 Fig, S5 TEM imges of micelle nnoprticles before nd fter the shell cross-linking rection (: NCM, b: SCM1, c: SCM2) b c 50 nm 50 nm 50 nm Fig. S6 () Time-dependent chnges in light scttering intensities of queous NCM, SCM1, nd SCM2 in the presence of SDS (2.5 g/l). (b) Time-dependent chnges of micelle size distribution (polydispersity fctor, μ 2 /Γ 2 ) in the presence of SDS (2.5 g/l). Reltive Scttering Intensity (%) NCM SCM1 SCM Time (min) () μ 2 /Γ NCM SCM1 SCM Time (min) (b) 12

13 Fig. S7 () MTX relese profiles from NCM, SCM1, nd SCM2 t PBS (ph 7.4, 37 o C). Ech point represents the men vlue±s.d. (n=3). Cumultive MTX Relese (%) NCM SCM1 SCM Time (h) Fig. S8. Cellulr uptke of FITC-lbeled SCM2 s function of incubtion time. 30 min 1 h 2 h 3 h 13

14 Fig. S9 Vibility of A549 cells t vrious concentrtions of NCM nd SCMs Cell Vibility (%) 100 NCM SCM1 SCM Concentrtion (μg/ml) 14