Supporting Information for A Single fluorescent probe for Dual-imaging Viscosity and H 2 O 2 in Mitochondria with Different Fluorescence Signals in Living Cells Mingguang Ren, Beibei Deng, Kai Zhou, Xiuqi Kong, Jian-Yong Wang and Weiying Lin * Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P.R. Email: weiyinglin2013@163.com *Correspondence to: Weiying Lin, Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China. Email: weiyinglin2013@163.com. S1
Table of contents Page Materials and instruments..... S3 Viscosity determination and spectral measurement...... S3 Culture and preparation of HeLa and Raw 264.7 cells....s4 Cytotoxicity assay....s4 Imaging of viscosity in living cells..s5 Imaging of H 2 O 2 in living cells.......s5 Synthesis...S6 Figure S1...S7 Figure S2...S7 Figure S3....S8 Figure S4.......S8 Figure S5.......S9 Figure S6.......S9 Figure S7......S10 Figure S8......S10 Figure S9......S11 Figure S10......S11 Figure S11......S12 Spectral characterization... S12-S14 S2
Materials and instruments Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents used were purified by standard methods prior to use. Twice-distilled water was used throughout all experiments; Mass spectrometric analyses were measured on a Finnigan MAT 95 XP spectrometer; High resolution mass spectrometric (HRMS) analyses were measured on an Agilent 1100 HPLC/MSD spectrometer; NMR spectra were recorded on an AVANCE III 400 MHz Digital NMR Spectrometer, using TMS as an internal standard; Electronic absorption spectra were obtained on a Shimadzu UV-2700 power spectrometer; Photoluminescent spectra were recorded with a HITACHI F4600 fluorescence spectrophotometer with a 1 cm standard quartz cell; The fluorescence imaging of cells was performed with a Nikon A1MP confocal microscope; The ph measurements were carried out on a Mettler-Toledo Delta 320 ph meter; TLC analysis was performed on silica gel plates and column chromatography was conducted over silica gel (mesh 200 300), both of which were obtained from the Qingdao Ocean Chemicals. Viscosity determination and spectral measurement The solvents were obtained by mixing ethanol-glycerol systems in different proportions. Measurements were carried out with a NDJ-8S rotational viscometer, and each viscosity value was recorded. The solutions of Mito-VH of different viscosity were prepared by adding the stock solution (1.0 mm) to 10 ml of solvent mixture (ethanol-glycerol solvent systems) to obtain the final concentration of the dye (5.0 µm). These solutions were sonicated for 5 minutes to eliminate air bubbles. After standing for 1 hour at a constant temperature, the solutions were measured in a UV spectrophotometer and a fluorescence spectrophotometer. For measure the response to H 2 O 2, all the measurements were made according to the following procedure. A stock solution (0.5 mm) of Mito-VH was prepared by dissolving the requisite amount of it in DMF. In a 10 ml tube the test solution of compounds Mito-VH was prepared by placing 0.1 ml of stock solution, 2.9 ml of DMF, 3 ml of PBS buffer and an appropriate volume of H 2 O 2 sample solution. After adjusting the final volume to 10 ml with distilled-deionized water, standing at room temperature 1 h, 3 ml portion of it was S3
transferred to a 1 cm quartz cell to measure absorbance or fluorescence. The stock solutions of ions for selectivity experiments were prepared respectively by dissolving GSH, Cys, S 2-, F -, Br -, I -, Fe 3+, NO 2-, NO 3 -, NO, ClO -, OH ( OH radicals were supplied via Fenton reaction between FeSO 4 and H 2 O 2 ), DTBP (Di-t-butyl peroxide), TBHP (tert -Butyl hydroperoxide) and H 2 O 2 in twice-distilled water. Culture and preparation of HeLa cells and RAW 264.7 HeLa cells and RAW 264.7 were cultured in DMEM (Dulbecco s modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum) in an atmosphere of 5% CO 2 and 95% air at 37 C. Before the experiments, seed the HeLa cells and RAW 264.7 in 35-mm glass-bottomed dishes at a density of 2 10 5 cells per dish in 2 ml of culture medium and incubate them inside an incubator containing 5% CO 2 and 95% air at 37 C. Incubate the cells for 24 h. Cells will attach to the glass surface during this time. Cytotoxicity assay In vitro cytotoxicity was measured using the colorimetric methyl thiazolyl tetrazolium (MTT) assay on HeLa cells. Cells were seeded into the 24-well tissue culture plate in the presence of 500 µl Dulbecco's modifed eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 C and 5% CO 2 atmosphere for overnight and then incubated for 24 h in the presence of Mito-VH at different concentrations (0, 1, 5, 10, 20, 30 µm). Then cells were washed with PBS buffer and 100 µl supplemented DMEM medium was added. Subsequently, 10 µl MTT (5 mg/ml) was added to each well and incubated for 4 h. Violet formazan was dissolved in 100 µl sodium dodecyl sulfate solution in the water-dmso mixture. Absorbance of the solution was measured at 570 nm using a microplate reader. The cell viability was determined by assuming 100% cell viability for cells without Mito-VH. S4
Imaging of viscosity in living cells Before the experiments, the well prepared cells were washed with PBS (ph=7.4) buffer three times. We use nystatin stimulated HeLa cells. Firstly, HeLa cells were incubated with probe Mito-VH (5 µm) (containing 0.1 % DMSO as a cosolvent) for 30 min at 37 C. Wash cells twice with 1mL PBS at room temperature, and then add 1 ml PBS and observe under a confocal microscopy. Secondly, HeLa cells were coincubated with nystatin (10 µm) for 30 min at 37 C and then washed with PBS twice, and the cells were incubated with Mito-VH (5 µm) for 20 min at 37 C, and then washed with PBS three times, and the fluorescence images were acquired through a Nikon A1MP confocal microscopy inverted fluorescence microscopy equipped with a cooled CCD camera. Imaging of H 2 O 2 in living cells 1) Imaging of exogenous H 2 O 2 in HeLa cells Before the experiments, the well prepared cells were washed with PBS (ph=7.4) buffer three times. Subsequently, incubating with probe Mito-VH (5 µm) (containing 0.1 % DMSO as a cosolvent) for another 30 min at 37 C, the HeLa cells were rinsed with PBS three times, and the cells were incubated with H 2 O 2 (30 µm) for 30 min at 37 C, and then washed with PBS three times, and the fluorescence images were acquired through a Nikon A1MP confocal microscopy inverted fluorescence microscopy equipped with a cooled CCD camera. 2) Imaging of exogenous H 2 O 2 in RAW 264.7 cells For the detection of endogenously produced H 2 O 2, the RAW 264.7 macrophages were coincubated with 3 µg/ml PMA and 5 µm probe Mito-VH for 6 h. Prior to the imaging, the cells were washed three times with PBS, and the fluorescence images were acquired through Nikon A1R confocal microscope with a 40 objective lens S5
Synthesis Scheme S1. Synthesis of H 2 O 2 fluorescent probe of Mito-VH. Synthesis of compound Mito-VH Compound 1 (100 mg, 0.446 mmol, 1.0 eq) and compound 2 (132 mg, 0.446 mmol, 1.0 eq) were dissolved in acetonitrile (2 ml), The reaction mixture was reacted refluxing for 7 h with an inert atmosphere of nitrogen. Then the mixture evaporated under reduced pressure. The resulting residue was purified by column chromatography on silica gel (MeOH / CH 2 Cl 2 = 1:20, v/v) to afford the compound Mito-VH as a violet powder (158 mg, yield: 68%). 1 H-NMR (400 MHz, CDCl 3 ) δ 9.03 (d, J = 6.4 Hz, 2H), 7.84 (d, J = 7.9 Hz, 2H), 7.79 (d, J = 6.0 Hz, 2H), 7.59 (d, J = 16.1 Hz, 1H), 7.53 (d, J = 7.9 Hz, 4H), 6.85 (d, J = 15.7 Hz, 1H), 6.03 (s, 2H), 3.10 (s, 6H), 1.34 (s, 12H); 13 C NMR (101 MHz, CDCl 3 ) δ 154.25, 143.36, 143.05, 136.32, 135.80, 130.76, 128.44, 122.64, 84.02, 62.60, 40.26, 29.69, 26.89, 24.83; HRMS (ESI) m/z calcd for C 28 H 34 BN 2 O + 2 [M + ]: 441.2718; found 441.2720. S6
3.2 3.0 Log (I 607 nm ) 2.8 2.6 2.4 2.2 0. 5 1.0 1.5 2.0 2.5 Log(viscosity) Figure S1 The linear response between the log (I 607nm ) of the probe Mito-VH (5 µm) and the log (viscosity) in the EtOH/glycerol solvent (excited at 500 nm). Figure S2. DFT optimized structure of Mito-VH (A,B) and compound 1 (C,D) in ground state and excited state. In the ball-and-stick representation, carbon, nitrogen, oxygen and boron atoms are colored in gray, blue, red and pink, respectively. H atoms were omitted for clarity. S7
2000 Intensity of 510 nm 1000 0 Compound 1 Mito-VH 2 4 6 8 10 12 ph Figure S3 The emission intensity changes (at 510 nm) of compound 1 and probe Mito-VH at different ph PBS buffer, containing 30 % DMF as a cosolvent (λ ex = 400 nm). 0.3 Absorption 0.2 0.1 H 2 O 2 H 2 O 2 0.0 300 400 500 600 Wavelength /nm Figure S4 Absorption spectra of the probe Mito-VH (5 µm) with the addition of H 2 O 2 (0-50 µm) in PBS buffer solution (ph 8.4 containing 30 % DMF as a co-solvent). S8
1000 Intercept = 15.59242, Slope = 26.94165 Intensity at 510 nm 800 600 400 200 0 0 10 20 30 40 50 H 2 O 2 /µm Figure S5 Relationship between fluorescence intensity at 510 nm of Mito-VH (5 µm) and the amount of H 2 O 2 (0-30 µm). Figure S6 (A) The fluorescence spectra changes of probe Mito-VH (10 µm) in the presence of various analytes (100 µm). (B) Fluorescence intensities of Mito-VH (5 µm) at 510 nm treated with various species: 1. Blank; 2. GSH; 3. Cys ; 4.S 2- ;5. F - ; 6. Br - ; 7. I - ; 8. Fe 3+ ; 9.NO - 2 ; 10.NO - 3 ; 11. NO; 12. ClO - ; 13. OH; 14. DTBP; 15. TBHP; 16. H 2 O 2 in PBS buffer (ph 8.4, containing 30 % DMF as a cosolvent) S9
Figure S7 Cytotoxicity assays of Mito-VH at different concentrations for HeLa cells Figure S8 Brightfield and fluorescence images of HeLa cells stained with the probe Mito-VH (5 µm) and mitochondrial dye. a) brightfield image; b) from TRITC channel (fluorescence image with Mito-VH); c) from the Cy5 channel (mitochondria staining); d) overlay of brighfield, TRITC and Cy5 channels; e) overlay of TRITC and Cy5 channels ; f) Intensity profile of linear region of interest across the HeLa cell costained with TRITC channel of Mito-VH imaging and Cy5 channel of Mito Tracker Deep Red; g) Intensity scatter plot of TRITC and Cy5 channels. S10
Intensity (a.u.) 600 300 Probe only Probe + nystatin 0 450 500 550 600 650 700 Wavelength /nm Figure S9 Fluorescence spectra of probe Mito-VH (5 µm) in the absence and presence 10 µm of nystatin in PBS (30 % DMF) at 37 o C for 30 min. Figure S10 Imaging of exogenous H 2 O 2 in HeLa cells stained with the probe Mito-VH (a) Brightfield image of HeLa cells costained only with Mito-VH; (b) Fluorescence images of (a) from FITC channel; (c) overlay of (a) and (b); (d) Brightfield image of HeLa cells costained with Mito-VH and treated with H 2 O 2 ; (e) Fluorescence images of (d) from FITC channel; (f) overlay of the brightfield image (d) and FITC channels (e) S11
Figure S11 Brightfield and fluorescence images of HeLa cells stained with the probe Mito-VH (5 µm) treated with H 2 O 2 (30 µm) and mitochondrial dye. a) brightfield image; b) from FITC channel (fluorescence image H 2 O 2 with Mito-VH); c) from the Cy5 channel (mitochondria staining); d) overlay of brighfield, FITC and Cy5 channels; e) overlay of FITC and Cy5 channels ; f) Intensity profile of linear region of interest across the HeLa cell costained with FITC channel of Mito-VH imaging and Cy5 channel of Mito Tracker Deep Red; g) Intensity scatter plot of FITC and Cy5 channels. Figure S12 1 H-NMR (DMSO-d 6 ) spectrum of the product of Mtio-VH reacted with H 2 O 2. S12
Figure S13 1 H-NMR (CDCl 3 ) spectrum of compound 1. Figure S14 1 H-NMR (CDCl 3 ) spectrum of Mito-VH. S13
Figure S15 13 C-NMR (DMSO-d 6 ) spectrum of Mito-VH. Figure S16 HRMS (ESI) spectrum of Mito-VH. S14