Supplementary Information. ph-responsive robust polymer micelles with metal-ligand coordinated core cross-links

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1 Electronic Supplementry Mteril (ESI) for Chemicl Communictions. This journl is The Royl Society of Chemistry 2014 Supplementry Informtion ph-responsive roust polymer micelles with metl-lignd coordinted core cross-links Gyu H Hwng, Kyung Hyun Min, Hong Je Lee, Hye Young Nm, Gi Hyun Choi, Byung Joo Kim, Seo Young Jeong, nd Sng Cheon Lee* 1. Mterils -Methoxy- -mino-poly(ethylene glycol) (CH 3 O-PEG-NH 2 ) with numer verge moleculr weight (M n ) of 5000 g/mol ws purchsed from IDBIOCHEM Inc. (Ulsn, Kore) nd used s received. 3,4-Dihydroxy-L-phenyllnine (L-DOPA), docetxel (DTX), cetic nhydride, triphosgene, piperidine, iron (III) chloride (FeCl 3 ), nd Cremophor EL were purchsed from Sigm Co. (St. Louis, MO) nd used without further purifiction. Glcil cetic cid ws purchsed from Biosesng Co. (Kore) nd used s received. Tetrhydrofurn (THF) ws distilled from N/enzophenone under N 2, prior to use. N,N-Dimethylformmide (DMF) nd dimethyl sulfoxide (DMSO) were dried nd vcuum distilled over clcium hydride. Clcium chloride (CCl 2 ), 37 % hydrogen chloride (HCl), nd sodium hydroxide (NOH) were of the regent grde. 2. Instrumenttion Nucler Mgnetic Resonnce Spectroscopy (NMR). 1 H NMR nd 13 C NMR spectr were recorded t 400 MHz nd 100 MHz, respectively, on Vrin INOVA400 NMR spectrometer with smple spinning rte of 5 khz t 25 o C. Smples for NMR mesurement were prepred y dissolving 10 mg of smples in 1 ml of DMSO-d 6 or D 2 O. Gel Permetion Chromtogrphy (GPC). Moleculr weight distriutions 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 DMF with flow rte of 1 ml/min. The moleculr weights were clirted with polystyrene stndrds. 1

2 3. Synthesis of poly(ethylene glycol)--poly(l-3,4-dihydroxy-l-phenyllnine) (PEG-PDOPA) copolymer 3.1. Synthesis of di-o,o'-cetyl-l-dopa-n-croxynhydride ((AC 2 )-DOPA- NCA) (AC 2 )-DOPA-NCA ws synthesized y two-step literture process [1]. First, di-o,o'-cetyl-l-dopa hydrochloride ((AC 2 )-DOPA) ws synthesized. The stirred suspension of L-DOPA (10 g, 50.7 mmol) in glcil cetic cid (100 ml) ws purged with dry HCl gs for 2 h t room temperture. Under continuous purging with HCl gs, cetic nhydride (10 ml, mmol) ws dded to the suspension, nd the rection mixture ws stirred t room temperture. After 1.5 h, dditionl cetic nhydride (10 ml, mmol) ws dded, nd the rection temperture ws rised to 54 C. After 1 h, the rection mixture ecme cler. The rection mixture ws concentrted on rotry evportor to smller volume, nd ethnol (30 ml) ws dded to destroy ny remining cetic nhydride. (AC 2 )-DOPA ws isolted y repeted precipittion into diethyl ether. Yield: 71.2 % 1 H NMR (DMSO-d 6 ): 2.2 (s, 6H), 3.1 (m, 2H), 4.5 (m, 1H), (m, 3H). Second, for (AC 2 )-DOPA-NCA, to stirred suspension of the rection product (3 g, 9.4 mmol) in dry THF (15 ml) ws dded triphosgene (1.1 g, 3.7 mmol) in dry THF (5 ml) t room temperture under nitrogen. After the rection temperture ws rised to 60 C, nd the rection ws mintined for 3 h. (AC 2 )-DOPA-NCA ws isolted y repeted precipittion from THF into n-hexne. Yield : 93.5 % 3.2. Synthesis of PEG--PDOPA copolymer (PEG-PDOPA) PEG-PDOPA tht hs EG units of 113 nd DOPA units of 6 ws synthesized y ring-opening polymeriztion of (AC 2 )-DOPA-NCA in the presence of CH 3 O-PEG-NH 2 mcroinititor nd susequent deprotection process. First, to stirred solution of CH 3 O- PEG-NH 2 (2 g, 0.4 mmol) in dry DMF (10 ml) ws dded (AC 2 )-DOPA-NCA (1.23 g, 4 mmol) t room temperture under nitrogen. The rection ws mintined for 24 h. PEG- P((AC 2 )-DOPA) ws isolted y repeted precipittion from DMF into diethyl ether. Second, in order to remove AC groups, stirred solution of PEG-P((AC 2 )-DOPA) (2 g, 0.3 mmol) in dry DMF (20 ml) ws uled with nitrogen. After 5 min, piperidine (0.27 2

3 ml) ws dded, nd the rection ws stirred for 15 min. The rection mixture ws isolted y repeted precipittion from DMF into diethyl ether. A solution of the otined precipitte in 0.1 N HCl ws dilyzed ginst n cidic wter (HCl, ph 4.5) using memrne (moleculr weight cut-off (MWCO): 1000), followed y lyophiliztion. Yield : 52.5 %. 4. Preprtion of DTX-loded polymer micelles with ctechol-fe 3+ cross-linked cores DTX-loded PEG-PDOPA micelles with core-specific ctechol-fe 3+ coordinted cross-linking were performed y the dilysis method nd susequent core-cross-linking process. DTX (10 mg) in DMF (1 ml) ws dded to stirred solution of PEG-PDOPA (100 mg) in DMF (20 ml). The solution ws dilyzed using memrne (Spectrpor, MWCO: 1,000 g/mol) ginst douly distilled de-ionized wter. After 10 h, the solution of DTX-loded micelles ws collected. To form ctechol-fe 3+ cross-linked cores on the polymer micelles, n queous solution of FeCl 3 (40 mm, ph 1.73) ws firstly dded to stirred solution of polymer micelles (ph 6.0) t the feed molr rtio of [DOPA] : [Fe 3+ ] = 2 : 1. The ph of the solution mixture ws For Fe 3+ -ctechol is-complex formtion, the solution ph ws djusted to ph 7.4. After 1 h, the solution ws dilyzed to remove unrected ionic species. Unloded DTX ws eliminted y centrifugtion (3000 rpm) of the dilyzed solution, nd the superntnt solution ws freeze-dried for isoltion of DTX- CLPMs. The loding contents nd loding efficiency of DTX within the polymer micelles were mesured y high-performnce liquid chromtogrphy (HPLC). The concentrtion of DTX ws determined y HPLC fter soluiliztion of DTX-loded micelles in DMF/cetonitrile (50:50). The HPLC system consisted of reverse-phse silic column (ZORBAX Eclipse XDB-C18, mm, 5-Micron, Agilent, USA), moile phse of cetonitrile, nd wter (60:40 v/v) pumped (LC-20AT, Shimdzu) t flow rte of 1.0 ml/min t 25 o C. A 50 μl liquot of smple ws injected, nd detected t 230 nm with UV detector (CBM-20A, Shimdzu). The DTX loding mount nd efficiency ws determined using clirtion curve of vrious DTX concentrtions ( μg/ml). 3

4 5. Chrcteriztion of DTX-loded core-cross-linked polymer micelles (DTX- CLPMs) Dynmic light scttering (DLS) mesurements were crried out using 90 Plus prticle size nlyzer (Brookhven Instruments Corportion). As light source, verticlly polrized He-Ne lser (632.8 nm) ws used. The scttered light ws mesured t n ngle of 90 nd ws collected on n utocorreltor. The hydrodynmic dimeters of DTX-loded non-cross-linked polymer micelles (DTX-NPMs) nd DTX-loded corecross-linked polymer micelles (DTX-CLPMs) were clculted y using the Stokes- Einstein eqution [2]. The polydispersity fctor of vrious polymer micelles, represented s 2 / 2, ws clculted from the cumulnt method [2]. The morphology of DTX-loded core-cross-linked polymer micelles ws exmined y trnsmission electron microscopy (TEM) (CM30, Philips, CA, USA). For visuliztion of polymer micelles, negtive stining ws performed using urnyl cette solution (3 wt %). 6. Spectrophotometric nlysis of ph-dependent coordintion etween ctechols nd Fe 3+ ions in PEG-PDOPA micelle cores UV-Visile sorption spectr were otined using UV-1650PC (Shimdzu, Jpn). The queous mixtures (1 g/l) of DTX-loded PEG-PDOPA micelles nd FeCl 3 (the molr rtio of [DOPA] : [Fe 3+ ] = 2 : 1) t vrious ph conditions (ph 5.0, 7.4, 12.0) were prepred y incresing the ph (1.73) of initil cidic solution mixture of FeCl 3 nd DTX-loded micelles. ph-dependent vrition in the sorption spectr ws monitored t 400 ~ 650 nm. Photo imges of the queous mixture t vrious ph were otined in 4 cler window cuvettes. 7. Stility of DTX-CLPMs in Serums For the stility evlution in serum conditions, scttered light intensity (SLI) of the DTX-CLPMs (1.25 g/l) in serum-contining solution (ph. 7.4, 50 % fetl ovine serum (FBS)) ws mesured y dynmic light scttering. At predetermined time intervls, SLI ws monitored nd compred to the initil scttered light intensity (SLI 0 ). The rtio of scttered light intensity (SLI/SLI 0 ) ws monitored. As control experiment, the stility of the DTX-NPMs ws exmined under the identicl conditions. 4

5 8. ph-controlled DTX relese from DTX-CLPMs In vitro relese profiles of DTX from DTX-CLPMs were investigted in the queous uffer solutions (ph 7.4 nd 5.0 phosphte uffer). DTX-CLPM solutions (1 ml, 1 g/l) in dilysis memrne g (MWCO: 1,000 g/mol) were prepred. The relese experiment ws initited y plcing the dilysis g in 10 ml of relese medi. The relese medium ws shken t speed of 90 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 DTX in the smples ws mesured y HPLC t 230 nm. The ssy for DTX ws sed on clirtion curve of vrious DTX concentrtions ( μg/ml). 9. Visuliztion of cellulr uptke of DTX-CLPMs For visuliztion of cellulr uptke of DTX-CLPMs, fluorescein isothiocynte (FITC)-leled DTX-CLPMs were prepred s follows: First, for synthesis of FITCleled PEG--PDOPA, FITC (0.014 g, 0.03 mmol) ws dded to stirred solution (2 ml) of PEG--PDOPA (0.2 g, 0.03 mmol) in DMF (2 ml). After 24 h, the rection mixture ws dilyzed ginst distilled de-ionized wter using memrne (moleculr weight cut-off (MWCO): 3,500), followed y lyophiliztion. The leling efficiency of FITC to PEG--PDOPA ws clculted to e 90.2%, which ws determined using clirtion curve of vrious FITC concentrtions in DMF. Second, FITC-leled DTX- CLPMs were prepred sed on n identicl procedure formerly estlished for DTX- CLPMs, except the use of FITC-leled PEG--PDOPA insted of PEG--PDOPA. To oserve the cellulr uptke, MCF-7 cells were seeded in six-well plte t density of 1x10 5 cells/well in 2 ml of RPMI 1640 medium supplemented with 10 % (v/v) FBS, 1 %(v/v) penicillin-streptomycin. After 24 h incution t 37 o C with 5 % CO 2, the medium ws removed nd replced with 2 ml of medium contining FITC-leled DTX-CLPMs (100 g/ml). The MCF-7 cells treted with FITC-leled DTX-CLPMs were incuted t vrious incution times (10 min, 30 min, 1 h, 2 h). At the designted time, the cells were wshed three times with PBS nd then fixed with 3.7% formldehyde. 5

6 Cellulr uptke imges were confirmed y IX71 fluorescence microscope (Olympus Opticl Co., Tokyo, Jpn). 10. Biocomptiility of NPMs, CLPMs nd Cremophor EL MCF-7 humn rest cncer cells were otined from the Koren Cell Line Bnk (KCLB, Seoul) nd cultured in Dulecco s Modified Egle s Medium (DMEM, Gico BRL, Githersurg, MD) supplemented with 10 % (v/v) het-inctivted fetl ovine serum (FBS, Gico BRL), nd 1 % (v/v) penicillin-streptomycin (Gico BRL). Cells were cultured in humidified incutor t 37 o C with 5 % CO 2. The culture medium ws replced every two dys. Cells were seeded t numer of cells per well in 96- well flt-ottomed pltes for 1 dy. The cells were wshed with PBS nd incuted with 200 μl of fresh medium contining NPMs, CLPMs nd Cremophor EL t 37 o C with 5 % CO 2. The concentrtion of NPMs, CLPMs nd Cremophor EL ws diluted with culture medium to otin concentrtion rnge from 10 to 500 g/ml. After 24 h, the cells were wshed with PBS, followed y the cell counting kit (CCK) ssy. In rief, the medium ws replced with CCK-8 solutions (Dojindo Lortories, Kummoto). The sornce of ech wells ws mesured t 450 nm y microplte reder (Biord Elizer, PA). Cell viility ws evluted y reltive to the untreted control group. 11. Cytotoxicity of DTX-CLPMs MCF-7 cells were seeded t numer of cells per well in 96-well plte nd incuted for 24 h t 37 o C with 5 % CO 2. The cells were wshed with PBS nd incuted with 200 μl of fresh medium contining vrious concentrtion of DTX-NPMs, DTX- CLPMs nd DTX-Cremophor EL t 37 o C with 5 % CO 2. For preprtion of the DTX- Cremophor EL solution, DTX (5 mg) dissolved in Cremophor EL (1 ml), fterwrds mixed with fresh cell culture medi. After 24 h incution, the cells were wshed with PBS, followed y the CCK ssy. The sornce of individul wells ws mesured t 450 nm y microplte reder (Biord Elizer, PA). The dt re expressed s the percentges of vile cells compred to the survivl of control group. The IC 50 vlue presents the concentrtion of DTX yielding 50 % inhiition of cell prolifertion, compred to the untreted control. 6

7 References [1] W. D. Fuller, M. S. Verlnder, M. Goodmn, Biopolymers, 1978, 17, [2] A. Hrd nd K. Ktok, Mcromolecules, 1998, 31, 288. Tle S1 Chrcteristics of the PEG--PDOPA copolymer. copolymer composition rtio ([EG] : [DOPA]) M n M w /M n PEG-PDOPA 113 : 6 6, *The copolymers were synthesized using the mcroinititor (CH 3 O-PEG-NH 2 ) with M n of 5,000 nd polydispersity index (M w /M n ) of Clculted y 1 H NMR spectr Estimted y GPC. Tle S2 The micelle sizes, zet potentils, nd polydispersity fctors of vrious micelles micelles d (nm) 2 / 2 NPMs CLPMs DTX-NPMs DTX-CLPMs Men hydrodynmic dimeters t ph 7.4, 25 o C. Polydispersity fctor estimted y dynmic light scttering. Fig. S1 Synthetic route to PEG-PDOPA. H 3 C O CH 2 CH 2 NH O HN C + O HC C CH O 2 DMF, RT, 24 h O H H 3 C O CH 2 CH 2 NH C CH N H CH 2 OCOCH 3 OCOCH 3 H N OCOCH 3 OCOCH 3 O H H 3 C O CH 2 CH 2 NH C CH N H CH 2 DMF, RT, 24 h OH OH 7

8 Fig. S2 1 H NMR spectr of () PEG, () PEG-P((AC 2 )-DOPA), nd (c) PEG-PDOPA in DMSO-d 6. () () (ppm) (c) (ppm) c (ppm) c c (ppm) 8

9 Fig. S3 13 C NMR spectr of () PEG, () PEG-P((AC 2 )-DOPA), nd (c) PEG-PDOPA in DMSO-d 6. () (ppm) () ppm d e g g d e f g c g e c 20.2 ppm (c) (ppm) (ppm) (ppm) ppm 20.2 ppm (ppm) ppm ( ) 9

10 Intensity (% Numer) Intensity (% Numer) Fig. S4 Gel permetion chromtogrms of () PEG nd () PEG-P((AC 2 )-DOPA). () () Retention Time (min) Retention Time (min) Fig. S5 Size distriution nd morphology of DTX-NPMs ( nd c) nd DTX-CLPMs ( nd d) estimted y dynmic light scttering nd TEM ) ) Dimeter (nm) Dimeter (nm) c) d) 100 nm 100 nm 10

11 SLI / SLI 0 (%) Fig. S6 Time-dependent chnge of the rtio of scttered light intensities (SLI/SLI 0 (%)) of DTX-CLPMs nd DTX-NPMs in the FBS-contining solution (ph 7.4). Ech point represents the men vlue of n experiments ± S.D. (n = 3) Time (min) DTX-CLPMs DTX-NPMs Fig. S7 Time-dependent visuliztion of cellulr uptke of FITC-leled DTX-CLPMs s function of incution time. 10 min 30 min 1 h 2 h 11