Supplementry Mteril (ESI) for Chemicl Communictions This journl is (c) The Royl Society of Chemistry 21 Electronic Supplementry Informtion High Intensity Focused Ultrsound nd Redox Dul Responsive Polymer Micelles 1. Experiment Section Yongwen Li, Rui Tong, Hesheng Xi, Hongji Zhng, Jun Xun Mterils 3,3 -dithiodipropionic cid (DTPA), Thiodiglycolic cid (TDA), dicyclohexylcrodiimide (DCC), 4-dimethylminopyridine (DMAP), Dichloromethne (DCM), L-Glutthione reduced (GSH) nd pyrene were purchsed from Sigm-Aldrich Chemicl Compny 1 nd used s received. Polyethylene glycol (Mn=2) were otined from Tinjin Chemicl Regents Institute. 2 Synthesis of DTPA-PEG-DTPA As shown in Scheme 2, 1 mmol of PEG nd mmol of DTPA were dded into 1 ml of tetrhydrofurn (THF). The rection mixture ws stirred to otin cler solution. To this, 4. mmol of DCC nd 3 mg of DMAP in ml of THF were dded. After stirring minutes, the white precipitte strted to pper. The rection mixture ws stirred t room temperture for 2 dys nd then filtered to remove the precipitted dicyclohexylure (DCU). The cler solution ws concentrted y vcuum distilling. Then 1 ml of DCM ws dded nd llowed to stnd for 1 hour to precipitte the remining trces of DCU. ml of cold diethyl ether ws dded into the solution to precipitte the DTPA-PEG-DTPA. The otined products were dried in vcuum for 4 hours t 4 o C. DTPA-PEG-DTPA (1.9 g, 77%). The TDA-PEG-TDA ws otined y the sme procedure y using TDA insted of DTPA. TMS e,f 3 c,d ' ' 6 4 3 2 1 δ /ppm 4 Fig. 1S 1 H NMR spectr of PEG () nd DTPA-PEG-DTPA () in CDCl 3. Synthesis of PEG-S-S-PLA copolymer DCM ws dried over CH 2 nd distilled. The rection of DTPA-PEG-DTPA nd PLA ws crried out using the ove method. DCC (. g) s the dehydrolyzing gent, DMAP (.2 g) s the ctlyst nd DTPA-PEG-DTPA (1. g) were dissolved in 2 ml DCM nd the mixture ws stirred t room temperture for minutes. To this, the poly (L-lctic cid) (. g) in 1 ml DCM were dded (The PLA ws synthesized ccording to our previous pper [3] 4. The solution ws dded into the flsk nd stirred for nother 48 hours. After tht, the rection mixture ws filtered to remove the DCU. Then, ml of cold diethyl ether ws dded into the concentrted solution to precipitte the PEG-S-S-PLA. At lst, the product ws dissolved into wter nd centrifugted (1 rpm 1 min) to remove the excess DTPA-PEG-DTPA. The precipitte ws dried in vcuum for 4 hours t room temperture. The white wx solid ws otined. The synthesis process nd structure of the copolymer re shown in Scheme 2. PEG-S-S-PLA (.632 g, 84%). The PEG-S-PLA copolymer ws otined y the sme procedure y using TDA-PEG-TDA insted of DTPA-PEG-DTPA. To whom correspondence should e ddressed.
Supplementry Mteril (ESI) for Chemicl Communictions This journl is (c) The Royl Society of Chemistry 21 Scheme 1S Synthetic route to dilock copolymer of the PEG-S-S-PLA. 1 j e,f i TMS c,d,h,m '' ' '' ' k 2 6 4 3 2 1 δ /ppm Fig. 2S 1 H NMR spectr of PEG--PLA () nd PEG-S-S-PLA () in CDCl 3. d 3 c 4 3 2 1 ν /cm -1 Fig. 3S FTIR spectr of DTPA (), PEG (), DTPA-PEG-DTPA (c) nd PEG-S-S-PLA (d). 4
Supplementry Mteril (ESI) for Chemicl Communictions This journl is (c) The Royl Society of Chemistry 21 1 c 1 12 14 16 Retention time/min Fig. 4S GPC trces of PEG-S-S-PLA (), PLA () nd DTPA-PEG-DTPA (c). 2 Tle 1 The moleculr weight nd the moleculr weight distriution (D). Polymer Mn Mw D DTPA-PEG-DTPA 26 37 1.41 PLA 1 7 1.37 PEG-S-S-PLA 8 11 1.39 Tle 2 The element nlysis result of PEG-S-S-PLA copolymer. Element C H S Prcticl content (%) 46.4 6.2 1.62 Theoretic content (%).3 6.44 1.732 3 Polymer micelle preprtion Typiclly, PEG-S-S-PLA (s llowed to evporte y heting to 4 o C for 24 h. The solution ws then removed y filtrtion through.4 μm memrne. The initil mg) ws introduced to THF solution (1 ml), nd then wter (4 ml) ws dded to induce the formtion of micelles, while THF w polymer concentrtion ws. mg/ml. To lod fluorescent pyrene, which is model hydrophoic compound, the sme procedure ws used except tht the THF solution contined oth polymer nd pyrene. Upon ddition of wter, the ggregtion of the hydrophoic lock llowed some pyrene molecules to e soluilized y polymer chins forming the core of micelle. 4 HIFU irrdition of polymer micelles HIFU pprtus is shown in the supporting informtion (Fig. S). Typiclly, 8 ml pyrene/peg-s-s-pla micelle solution ws plced into cuvette rector, which ws seled y ltex memrne nd immersed in wter tnk. The focused ems of ultrsound penetrte through ltex memrne nd ct on the PEG-S-S-PLA micelle solution. In ll HIFU irrdition experiments, unless otherwise stted, the focl spot of the ems ws set t the center of the micelle solution. The HIFU power output ws set t - W. After certin time of HIFU irrdition, the cuvette rector ws removed from the wter tnk nd the micelle solution ws used for chrcteriztions t room temperture. 4 Glutthione tretment of polymer micelles Glutthione (1 mg) were dded into the pyrene/peg-s-s-pla micelle solution (1 ml) nd stirred for few seconds nd then kept in n oven t 37 o C for certin time. The micelle solution t different time ws used for chrcteriztions t room temperture.
Supplementry Mteril (ESI) for Chemicl Communictions This journl is (c) The Royl Society of Chemistry 21 Apprtus 1 2 3 Fig. S The schemtic illustrtion for experimentl setup (left) nd high intensity focused ultrsound pprtus (right). 1: Aritrry wveform genertor, 2: RF power mplifier, 3: Wter th, 4: Ultrsonic em, : Micelles, 6: Ltex memrne, 7: Acoustic lens trnsducer, 8: Cuvette rector. 4 4 Chrcteriztions FTIR nlysis of the smples ws performed on Nicolet 6 Fourier trnsform infrred (FTIR) spectrometer. Proton nucler mgnetic resonnce 1 H NMR spectr were recorded t room temperture with Bruker spectrometer operting t 4 MHz y using CDCl 3 s solvent nd tetrmethylsilne s n internl reference. The moleculr weight ws mesured with gel permetion chromtogrphy (GPC, Agilent 11 Series) with tetrhydrofurn (THF) s the eluent t flow rte of 1 ml min -1 t o C. The moleculr weight ws clirted with polystyrene stndrd. The content of ech element ws mesured y elementl nlyzer (EA; Euro EA 3 Series). Dynmic light scttering (DLS) ws performed on Brookhven BI-2 goniometer with verticlly polrized incident light of wvelength λ = 32 nm supplied y n rgon lser operting t 2 mw nd Brookhven BI-9 AT digitl utocorreltor. Mesurements were mde t. o C nd t n ngle of 9. The utocorreltion functions from DLS were nlyzed y using the nonnegtively constrined lest squre lgorithm (NNLS) method to otin the dimeter distriutions. The micellr morphology ws oserved with scnning electron microscopy (SEM, Inspect F, Philips). The specimens for SEM oservtions were prepred y depositing severl drops of micellr solutions onto silicon wfers nd were dried in vcuum t room temperture. Stedy-stte fluorescence emission spectr of pyrene/peg-s-s-pla micelle solution were recorded on n 97CRT spectrophotometer (Shnghi Precision & Scientific Instrument Co. Ltd). The excittion wvelength ws 337 nm. The trnsmittnce of pyrene/peg-s-s-pla solution in the presence of GSH ws monitored y UV-vis spectrum (UV-vis31, Hitchi, Jpn).
Supplementry Mteril (ESI) for Chemicl Communictions This journl is (c) The Royl Society of Chemistry 21 2. Pyrene/PEG-S-S-PLA micelle stility 1 1 Fluorescence Intensity 7 Before centrifugtion After centrifugtion 2 4 4 Fig. 6S The fluorescence emission spectr of pyrene/peg-s-s-pla micelle solution efore nd fter centrifugtion for 1 min t 1 rpm. No decrese in the fluorescence emission intensity ws oserved, suggesting tht the pyrene/peg-s-s-pla micelle is stle. λ/nm 3. HIFU induced pyrene/peg-s-s-pla micelle disruption c 3 1 7 Intensity/% 4 42 43 44 1 Dimeter/nm d 7 Intensity/% 4 1 2 3 Dimeter/nm Fig. 7S (1) The chnge in the size distriution of pyrene/peg-s-s-pla micelles in solution efore () nd fter () HIFU irrdition for 1 min, determined y DLS; (2) SEM imges of pyrene/peg-s-s-pla micelles efore (c) nd fter (d) HIFU irrdition for 1 min
Supplementry Mteril (ESI) for Chemicl Communictions This journl is (c) The Royl Society of Chemistry 21 4. More evidences for redox-responsive ehvior of polymer micelles H S S n S S CH 3 CH 3 H CH m 3 Glutthione 1 HS HS SH n CH 3 CH 3 H CH m 3 H SH 2 6 4 3 2 1 δ /ppm 3 Scheme 2S The schemtic illustrtion for the decomposition of PEG-S-S-PLA in the presence of GSH nd 1 H NMR spectr of solid smples recovered from the PEG-S-S-PLA micelle solution in the presence of glutthione: () min; () 48 min. The rtio of PEG protons to methylidyne protons in PLA decresed significntly. The lower rtio of pek re is indictive of the decresing mount of PEG lock in the smple. The decresing of pek t 2.9 nd 2.77 ppm confirmed tht the glutthione induced the clevge of disulfide. 4 1 2 GSH 37 Trnsmittnce/% 1 7 GSH 1 mg/ml Pyrene/PEG-S-S-PLA Pyrene/PEG-S-PLA 4 1 2 t/min Fig. 8S () Color chnge of the pyrene/peg-s-s-pla micelle solution: efore (1) nd fter (2) treting with GSH for 24 minutes; () The UV-Vis trnsmittnce of PEG-S-S-PLA nd PEG-S-PLA micelle solution t nm with time in the presence of GSH. Compred to significnt reduction for the trnsmittnce of PEG-S-S-PLA micelle solution, there is no chnge for tht of PEG-S-PLA without disulfide ond.
Supplementry Mteril (ESI) for Chemicl Communictions This journl is (c) The Royl Society of Chemistry 21 1 Intensity/% 1 7 PEG-S-PLA GSH tretment 48 min Relesed pyrene/ % 1 7 Pyrene/PEG-S-PLA GSH tretment (1 mg/ml) 33. 34. 34... Dimeter/nm 2 4 6 8 1 2 3 4 t/min 2 Fig. 9S () Prticle size distriution of PEG-S-PLA fter 48 min in the presence of GSH. The control experiment suggests tht there is no effect of GSH on the morphology of PEG-S-PLA micelle without the disulfide ond; () The relesed percentge of pyrene from PEG-S-PLA micelle with time in the presence of GSH. Compred to tht for PEG-S-S-PLA micelle, much lower relesed percentge of pyrene for PEG-S-PLA micelle under GSH tretment cn e noted.. A control experiment: the relese of pyrene from pyrene/peg-s-pla micelle 3 1 PEG-S-PLA/pyrene Relesed pyrene/% 7 HIFU/GSH tretment 4. 2.. 7. 1. t/min Fig. 1S The clculted relesed percentge of the pyrene from pyrene/peg-s-pla micelle solutions with the time (λex=337 nm). The dt shows slower relese rte for pyrene/peg-s-pla micelle compred to tht of pyrene/peg-s-s-pla, suggesting the PEG-S-S- 4 PLA hs quick response to HIFU ecuse the lile disulfide ond is more esily to e roken y ultrsonic cvittion.
Supplementry Mteril (ESI) for Chemicl Communictions This journl is (c) The Royl Society of Chemistry 21 6. A control experiment: the exmintion of pyrene degrdtion 7 utput: 8 w 1 Fluorescence Intensity HIFU min HIFU 1 min HIFU 2 min Pyrene/THF 4 4 λ/nm 2 Fig. 11S The fluorescence emission spectr of pyrene/thf solutions (λex=337 nm) efore nd fter HIFU. This control experiment shows tht pyrene cn not e degrded y HIFU, suggesting tht the decrese in the fluorescence emission intensity is not ttriuted to the degrdtion of pyrene, ut the relese of pyrene from the copolymer micelle. 7. Chnge in the fluorescence emission of PEG-S-S-PLA micelle solutions with time 3 88 4 Fluorescence Intensity 66 44 22 HIFU time min 1 min 2 min 4 min 8 min 1 min 4 4 λ/nm 4 Fig. 12S Vrition of fluorescence emission spectr of pyrene/peg-s-s-pla micelle solutions (λex=337 nm) with time t power output of 8 W.