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1 Electronic Supplementry Mteril (ESI) for ChemComm. This journl is The Royl Society of Chemistry 214 Electronic Supplementry Informtion for: Gold nnoprticles functionlized with cresyl violet nd porphyrin vi hyluronic cid for trgeted cell imge nd phototherpy Yncho Song,, Zhe Wng, Lihong Li, Wen Shi, Xiohu Li nd Huimin M* Beijing Ntionl Lortory for Moleculr Sciences, Key Lortory of Anlyticl Chemistry for Living Biosystems, Institute of Chemistry, Chinese Acdemy of Sciences, Beijing 119, Chin. E-mil: Key Lortory of Rdiologicl Protection nd Nucler Emergency, Ntionl Institute for Rdiologicl Protection, Chinese Center for Disese Control nd Prevention, Beijing 188, Chin. 1. Regents nd pprtus Hyluronic cid (HA; M w = 35 D) ws otined from Yngzhou Zhongfu New Mterils Co., Ltd. Hyluronidse, L-scoric cid plmitte, cresyl violet (CV), cystmine dihydrochloride, 1-ethyl-3-(3-(dimethylmino)propyl)crodiimide (EDC), L-cysteine, glutthione, tetr(4-minophenyl)porphyrin (referred to porphyrin), ovine serum lumin, nd humn serum lumin were otined from Sigm-Aldrich. HAuCl 4, trisodium citrte, CCl 2, NCl, KCl, MgCl 2, vitmin C, nd glutmine were purchsed from Beijing Chemicls, Ltd. Dimethylsulfoxide, N-hydroxysuccinimide (S), dithiothreitol nd morpholinoethnesulfonic cid (MES) were purchsed from J & K Chemicl Ltd. Dulecco s modified Egle s medi (DMEM), fetl ovine serum, penicillin (1 μg/ml), streptomycin (1 μg/ml), nd phosphte uffered sline (PBS) solution were otined from Invitrogen corportion. 3-(4,5-Dimethylthizol-2-yl)- 2,5-diphenyltetrzolium romide (MTT) ws purchsed from Serv Electrophoresis GmH (Germny), nd 2',7'-dichlorofluorescin dicette (DCFH-DA) from Biyuntin Co. (Chin). Ultrpure wter (over 18 M cm) from Milli-Q reference system (Millipore) ws used throughout. UV-vis spectr were recorded in 1-cm qurtz cells with TU-19 spectrophotometer (Beijing, Chin). Fluorescence mesurements were mde on Hitchi F-25 fluorescence spectrophotometer (Tokyo, Jpn). The incution ws crried out in Shker incutor (SKY-1C, Shnghi Sukun Industry & Commerce Co., Ltd). Trnsmission electron microscopy (TEM) imges were tken on JEM-111 instrument. The sornce for MTT nlysis ws recorded on microplte reder 1

2 (BI-TEK Synergy HT, USA) t 49 nm. Fluorescence imging experiments were performed on FV 1-IX81 confocl lser scnning microscope (lympus, Jpn). 2. Preprtion of nnoproe The nnoproe ws prepred vi the following three steps. Firstly, gold nnoprticles (AuNPs) were prepred s descried previously (Song et l, Tlnt, 213, 116, 237), nd their solution ws stored in refrigertor t 4 C for future use. Secondly, HA contining free thiol groups s well s CV nd porphyrin moieties, i.e., HA-SH-CV-porphyrin, ws prepred (Scheme S1). In rief, HA ws dissolved in wter nd dilyzed ( memrne with moleculr weight cutoff of pproximtely 1) for 1 dy to remove the sodium slt nd HA oligomers. The purified HA ws prepred s solid form vi lyophiliztion. Then, in 2 ml of MES uffer solution (ph 5.5), HA H 2 N 2 A N N HN N H 2 N S S 2 H 2 N 2 H 2 N 2 cystmine cresyl violet porphyrin B H H H H H H H H H H H H H H H H H H H H n S 2 S cresyl violet cystmine cresyl violet porphyrin porphyrin H H H H H H H H H H H H H H H H H n dithiothreitol SH cresyl violet porphyrin H H H H H H H H H H H H H H H H H n HA-SH-CV-porphyrin Scheme S1 (A) Structures of components. (B) Synthesis of HA-SH-CV-porphyrin. 2

3 (1 mg), EDC (2 mg), nd S (4.8 mg) were dded. After the resulting mixture ws stirred for 2 h, CV (.24 mg), porphyrin (.28 mg) nd cystmine dihydrochloride (12 mg) were dded, followed y further stirring for 5 h. Then, the rection mixture ws treted with dithiothreitol (1 mg) for 12 h to generte HA tht contins free thiol groups, followed y dilysis ( memrne with moleculr weight cutoff of pproximtely 1) ginst wter for 1 dy to remove unrected chemicls. The purified solution ws lyophilized, yielding yellow power product of HA-SH-CV-porphyrin, which ws stored in refrigertor t -2 C for future use. Finlly, the nnoproe (AuNPs-HA-CV-porphyrin) ws prepred y treting HA-SH-CV-porphyrin with AuNPs. Briefly, HA-SH-CV-porphyrin (12 mg) ws dissolved in 3 ml of wter, followed y ddition of 1 ml AuNPs solution (3 nm). The mixture ws stirred for 24 h to form stle Au-S ond. Then, the mixture ws centrifuged nd wshed three times with wter to remove the unound HA-SH-CV-porphyrin. 3. TEM imge of the nnoproe The size nd monodispersity of the nnoproe were determined y TEM nlysis. Smples for TEM nlysis were prepred y plcing out 1 μl of the nnoproe solution on the cron-coted copper grid nd then drying t room temperture. Frequency (%) 2 1 () Dimeter (nm) Fig. S1 TEM imge () nd dimeter distriution () of nnoproe. Scle r, 1 nm. 4. UV-vis sorption spectr of different sustnces The mounts of porphyrin nd CV conjugted to the AuNPs were clculted sed on the following eqution set nd the dditivity of sornce of porphyrin, CV nd AuNPs, which show the chrcteristic sorption nds t 42, 58 nd 52 nm (Fig. S2), respectively. A 42 nm = ε porphyrin42 C porphyrin + ε CV42 C CV + ε AuNPs42 C AuNPs A 52 nm = ε porphyrin52 C porphyrin + ε CV52 C CV + ε AuNPs52 C AuNPs A 58 nm = ε porphyrin58 C porphyrin + ε CV58 C CV + ε AuNPs58 C AuNPs 3

4 where A is the sornce of the nnoproe t different wvelengths; nd C re the molr sorptivity nd the concentrtion of relted sustnce, respectively. All the sornce mesurements were performed in qurtz cell with n opticl length of 1 cm. At 42, 52 nd 58 nm, the molr sorptivities of porphyrin re , nd M -1 cm -1, those of CV re , nd M -1 cm -1, nd those of AuNPs re , nd M -1 cm -1, respectively. Similrly, the mounts of porphyrin nd CV conjugted to per grm of the HA ckone cn e clculted to e.5 nd.2 mol, respectively, sed on the spectrum of HA-SH-CV-porphyrin (curve e). Asornce e c d porphyrin CV AuNPs nnoproe HA-SH-CV-porphyrin HA f Wvelength (nm) Fig. S2 UV-vis sorption spectr of different sustnces in wter. () Porphyrin (16 μm); () CV (15 μm); (c) AuNPs (3 nm); (d) nnoproe (25 μg/ml); (e) HA-SH-CV-porphyrin (2.6 mg/ml); (f) HA (1 mg/ml) 5. The colour of re AuNPs nd nnoproe in different solutions Fig. S3 The color chnges of re AuNPs nd nnoproe in different medi: wter (control), ph 12 (sic medium djusted y dding dilute NH to wter),.1 M NCl (out ph 7), nd ph 2 (cidic medium djusted y dding dilute HCl to wter). As is 4

5 seen, the color of re AuNPs is esier to chnge with the medi thn tht of the nnoproe, indicting tht the nnoproe hs higher stility. 6. ptimiztion of rection time Fluorescence Intensity (.u.) Time (min) Fig. S4 Effect of rection time on the fluorescence of the nnoproe (5 μg/ml) in 5 mm PBS of ph 7.4 in the sence () nd presence () of hyluronidse (2 μg/ml) t 37 C. λ ex/em = 55/62 nm. 7. UV-vis sorption spectr of the rection system.4 Asornce g/ml Wvelength (nm) Fig. S5 Asorption spectr of the nnoproe (5 μg/ml) with hyluronidse t different concentrtions (, 1, 2, 4, 8, 1, 12, 14, 16, 18, 2 μg/ml). The spectr were recorded ginst the corresponding regent lnk (see M et l, Mikrochim. Act, 1998, 128, 181) without hyluronidse. The inset (from left to right) shows the corresponding color chnge. 5

6 8. Inhiitor effect Fluorescence Intensity (.u.) 6 d 4 c Wvelength (nm) Fig. S6 Fluorescence spectr of different rection systems. () The nnoproe (5 μg/ml) in ph 7.4 PBS (control); () system () + hyluronidse (6 μg/ml) + inhiitor (4 μg/ml); (c) system () + hyluronidse (6 μg/ml) + inhiitor (2 μg/ml); (d) system () + hyluronidse (6 μg/ml). λ ex = 55 nm. 9. Selectivity study 6 F Species Fig. S7 Effects of common cellulr species on the fluorescence of the nnoproe (5 μg/ml): 1) 1 nm ovine serum lumin; 2) 1 nm humn serum lumin; 3) 1 mm L-cysteine; 4) 5 mm glutthione; 5) 2.5 mm MgCl 2 ; 6) 1 μm CuCl 2 ; 7) 1 mm CCl 2 ; 8) 1 mm vitmin B1; 9) 1 mm glucose; 1) 5 μm mtrix metlloproteinse 2; 11) 1 μm reduced nicotinmide denine dinucleotide; 12) 1 μm croxylesterse; 13) 1 μm cytochrome c; 14) 4 μg/ml hyluronidse. ΔF = F F, where F nd F re the fluorescence intensity efore nd fter the species is dded to the nnoproe solution, respectively. λ ex/em = 55/62 nm. 6

7 1. Fluorescence of the nnoproe under ultrviolet irrdition Fluorescence Intensity (.u.) Time (min) Fig. S8 Chnge of fluorescence intensity of the nnoproe (5 μg/ml) () without nd () with ultrviolet irrdition of 365 nm for different periods of time. λ ex/em = 55/62 nm. 11. The singlet oxygen detection 2 F Time (min) Fig. S9 Fluorescence chnge of DCFH-DA (5 μm) in the solution of the nnoproe (5 μg/ml) under ultrviolet irrdition of 365 nm for different periods of time.δf = F F, where F nd F re the fluorescence intensity efore nd fter ultrviolet irrdition, respectively. As is seen, the fluorescence of DCFH-DA is grdully incresed under ultrviolet irrdition, suggesting the genertion of singlet oxygen. λ ex/em = 488/525 nm. 12. Cell imging Unless otherwise stted, U-87, HeL, nd NIH-3T3 cells used in this study were cultured in DMEM contining 1% (v/v) fetl ovine serum nd 1% (v/v) penicillin-streptomycin t 37 C in 5% C 2 incutor. For fluorescence imging, the dherent cells grown on glss-ottom culture dishes (MtTek Co.) contining 1 ml of culture medi were first incuted with the nnoproe (5 µg/ml) or CV (5 µm) t 37 C, nd then wshed thoroughly with.1 M PBS (ph 7.4). Fluorescence imging 7

8 experiments were performed on FV 1-IX81 confocl lser scnning microscope (lympus, Jpn) with FV5-LAMAR for excittion t 559 nm nd vrile ndpss emission filter set to nm through NA ojective. pticl sections were cquired t.8 μm. min 15 min 3 min 4 min Reltive Pixel Intensity min Fig. S1 The fluorescence imges of U-87 cells treted with the nnoproe (5 μg/ml) for different periods of time. The differentil interference contrst (DIC) imges of the corresponding smples re shown t the ottom. Scle r, 1 µm. The reltive pixel intensities of the corresponding fluorescence imges re shown on the right (the intensity t min is defined s 1.). C c d e f Fig. S11 Confocl fluorescence imges of U-87 cells. () U-87 cells were incuted with 5 μg/ml nnoproe for 3 min; () U-87 cells were pre-treted with 1 μl of sturted HA solution (1 g/ml) for 1 h, nd then incuted with nnoproe (5 μg/ml) for 3 min; (c) U-87 cells were pre-treted with 1 μl of 6--plmitoyl-L-scoric cid (5 μg/ml) for 1 h, nd then incuted with nnoproe (5 μg/ml) for 3 min. The DIC imges of the corresponding smples re shown elow (pnels d-f). Scle r, 1 µm. 8

9 A) c d e f Reltive Pixel Intensity B) U-87 HeL c NIH-3T3 Fig. S12 A) Confocl fluorescence imges of different cells: () U-87, () HeL, nd (c) NIH-3T3 cells. The cells were incuted with CV (5 μm) t 37 C for 3 min (interestingly, less CV enters the nucleus of NIH-3T3 cells; the reson for this is uncler). The DIC imges of the corresponding smples re shown elow (pnels d-f). Scle r, 1 µm. B) Reltive pixel intensity otined from the corresponding fluorescence imges with ImgeJ softwre (the pixel intensity from NIH-3T3 cells is defined s 1.). c d e f Fig. S13 Fluorescence imges of different cell smples. () The cell mixture of NIH-3T3 nd U-87 cells fter incution with the nnoproe (5 µg/ml) t 37 C for 3 min. () The cell mixture of NIH-3T3 nd U-87 cells in the sence of the nnoproe (control). (c) nly NIH-3T3 cells incuted with the nnoproe (5 µg/ml) t 37 C for 3 min (nother control). The DIC imges of the corresponding smples re shown elow (pnels d-f). In imge d, the white rrows indicte the NIH-3T3 cells, which scrcely show fluorescence in imge. Scle r, 1 μm. 9

10 13. Cell viility ssys The toxic effects of the nnoproe itself on the three kinds of cells (U-87, HeL, nd NIH-3T3) were exmined in the sence of ultrviolet rdition. In rief, U-87, HeL, or NIH-3T3 cells were seeded onto 96-well pltes t density of 7 cells/well. After incution for 24 h, the culture medium ws replced with 1 μl of serum-free medium contining different concentrtions of the nnoproe (-4. mg/ml), followed y incution for 24 h t 37 C. The cells were then wshed twice with serum-free culture medium, nd the cell viility ws evluted y the MTT ssy (Song et l, J. Mter. Chem., 212, 22, 12568). To determine the cell viility fter ultrviolet irrdition, cells (U-87, HeL, or NIH-3T3) were seeded in 96-well U-ottom pltes t density of 7 cells/well, nd then treted s descried ove with serum-free medium contining different concentrtions of the nnoproe (-4. mg/ml) or free porphyrin (-25 µm). After the cells were wshed twice with serum-free medium, they were irrdited under UV lmp (8 W) of 365 nm for 5 min. Then the cells were incuted in serum-free DMEM t 37 C for 6 h, nd the viility of the irrdited cells ws evluted y the MTT ssy. 15 Cell Viility (%) 1 5 U-87 HeL NIH-3T Nnoproe Concentrtion (mg ml) Fig. S14 Cell viility chnges fter 24 h of incution with the nnoproe t vried concentrtions of -4. mg/ml. The cell survivl without tretment y the nnoproe is defined s 1%; the results re the men ± SD of 5 seprte mesurements. 1