A Photostable AIE Luminogen for Specific Mitochondrial

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1 SUPPORTING INFORMATION A Photostable AIE Luminogen for Specific Mitochondrial Imaging and Tracking Chris Wai Tung Leung,,# Yuning Hong,,# Sijie Chen, Engui Zhao, Jacky Wing Yip Lam, and Ben Zhong Tang *,, Department of Chemistry, State Key Laboratory of Molecular Neuroscience, Institute of Molecular Functional Materials, Institute for Advanced Study and Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China Guangdong Innovative Research Team, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou , China Table of Contents Experimental Section Scheme S1. Synthetic route to TPE-TPP Figure S1. 1 H-NMR spectrum of TPE-TPP in DMSO-d 6 Figure S2. 13 C-NMR spectrum of TPE-TPP in DMSO-d 6 Figure S3. Mass spectrum (MALDI-TOF) of TPE-TPP Figure S4. Particle size analysis of TPE-TPP nanoaggregate in PBS Figure S5. Cytotoxicity of TPE-TPP on HeLa cells determined by MTT assay. S2 S5 S6 S7 S8 S9 S10 Figure S6. (A C) Fluorescence images of the HeLa cells stained with TPE-TPP for (A) 30 min, (B) 1 h and (C) 2 h. (D) Signal to background ratio of TPE-TPP dyed HeLa cell with different incubation time. Concentration of TPE-TPP: 5 M; excitation wavelength: nm; scale bar: 15 m. S11 Figure S7. Fluorescent images of living HeLa cells stained with MitoTracker Red FM with increasing number of scans (1 6 times). Concentration of MitoTracker Red FM: 50 nm; excitation wavelength = 560 nm; emission filter: nm; irradiation time: 7.75 sec/scan. S12 Figure S8. Molecular structure of MitoTracker Red FM (MT) Reference S13 S13 S1

2 Experimental Section Materials and Method THF (Labscan) was purified by simple distillation from sodium benzophenone ketyl under nitrogen immediately before use. Zinc dust, titanium(iv) chloride, N-bromosuccinimide (NBS), benzoyl peroxide (BPO), triphenylphosphine (PPh 3 ), benzaldehyde, 4-cyanobenzaldehyde, carbon tetrachloride, 4-nitrobenzaldehyde, triphenylamine, N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethylsulfoxide (DMSO), and other reagents were all purchased from Aldrich and used as received. 1 H and 13 C NMR spectra were measured on a Bruker ARX 300 spectrometer in CDCl 3 and Bruker AV spectrometer in DMSO-d 6. Mass spectra were recorded on a Finnigan TSQ 7000 triple quadrupole spectrometer operating in fast-atom bombardment (FAB) mode or a GCT Premier CAB 048 mass spectrometer operated in MALDI-TOF mode. Photoluminescence (PL) spectra were recorded on a Perkin Elmer LS 55 spectrofluorometer with a xenon discharge lamp excitation. Particle size analysis was determined at room temperature by a ZetaPlus Potential Analyzer (Brookhaven Instruments Corporation, USA). Minimum essential medium (MEM), fetal bovine serum (FBS), penicillin and streptomycin and MitoTracker Red FM were purchased from Invitrogen. Carbonyl cyanide m-chlorophenylhydrazone (Sigma) was used as received. Synthesis Preparartion of 1,2-Bis(4-methylphenyl)-1,2-diphenylethene (2). A suspension of 4-methylbenzophenone (1, 3.6 g, 10.0 mmol), TiCl 4 (1.9 g, 10.0 mmol), and Zn dust (1.3 g, 20.0 mmol) in dry THF (100 ml) was refluxed for 20 h. Afterward, the reaction mixture was cooled to room temperature and filtered. The filtrate was evaporated and the crude product was purified on a silica-gel column using DCM as eluent. Compound 2 was isolated as white solid in 94% yield. 1 H NMR (300 MHz, CDCl 3 ), (TMS, ppm): (m, 10H), 6.91 (d, 8H), 2.26 (d, 6H). 13 C NMR (75 MHz, CDCl 3 ), (TMS, ppm): 144.8, 141.6, 141.1, 136.6, 132.0, 131.9, 129.0, 128.2, 126.9, m/z (FAB) [M + ]; calc Preparartion of 1,2-Bis[4-(bromomethyl)phenyl]-1,2-diphenylethene (3). To a mixture of 2 (1.8 g, 5.0 mmol) and NBS (1.7 g, 10.0 mmol) in CCl 4 was added catalytic amount of BPO at room temperature. The mixture was stirred and heated to reflux for 8 h. After filtration and solvent evaporation, the product was purified by silica gel chromatography using DCM/hexane (1:4 v/v) as eluent. Compound 3 was isolated as pale yellow solid in 43% yield. 1 H NMR (300 MHz, CDCl 3 ), (TMS, ppm): (m, 10H), (m, 8H), 4.41 (d, 4H). 13 C NMR (75 MHz, CDCl 3 ), (TMS, ppm): 144.4, 143.9, 141.5, 136.6, 132.3, 132.0, 129.2, 128.5, 127.4, m/z (FAB) [M + ]; calc S2

3 Preparartion of Bis(Triphenylphosphonium) Tetraphenylethene (TPE-TPP). Triphenylphosphonium salt, TPE-TPP, was prepared from 3 (0.5 g, 1.0 mmol) and triphenylphosphine (1.0 g, 4.0 mmol) in DMF at 100 o C. After stirring for 24 h, the solution was poured into large amount of toluene. The white precipitate was collected in 80% yield. 1 H NMR (400 MHz, DMSO-d 6 ), (TMS, ppm): (m, 30H), (m, 18H), (d, 4H). 13 C NMR (100 MHz, DMSO-d 6 ), (TMS, ppm): 143.4, , 135.2, 134.0, 131.1, 130.6, 130.3, 130.1, 128.0, 127.0, 126.1, 118.1, 117.2, m/z (MALDI-TOF) [(M 2Br) + ]; calc Anal. Calcd. C 64 H 52 Br 2 P 2 : C, 73.7; H, 5.0. Found: C, 73.7; H, 4.9. Cell culture and imaging Cell Culture. HeLa cells were cultured in the MEM containing 10% FBS and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin) in a 5% CO 2 humidity incubator at 37 o C. 1 Cell Imaging. HeLa cells were grown overnight on a 35 mm petri dish with a cover slip or a plasma-treated 25 mm round cover slip mounted to the bottom of a 35 mm petri dish with an observation window. The live cells were stained with 5 M of TPE-TPP for 15 min, 30 min, or 2 h (by adding 2 L of a 5 mm stock solution of TPE-TPP in DMSO to 2 ml culture medium) or 50 nm MitoTracker Red FM (MT) for 15 min (by adding 0.5 L of a 200 M stock solution of MT in DMSO to 2 ml culture medium). The cells were imaged under an FL microscope (BX41 Microscope) using different combination of excitation and emission filters for each dye: for TPE-TPP, excitation filter = nm, dichroic mirror = 400 nm, and emission filter = 420 nm long pass; for MT, excitation filter = nm, dichroic mirror = 600 nm, and emission filter = 610 nm long pass 1. For photo-stability test, the cells were imaged by confocal microscope (Zeiss Laser Scanning Confocal Microscope; LSM7 DUO) using ZEN 2009 software (Carl Zeiss). TPE-TPP was excited at 405 nm (6% laser power) and the fluorescence was collected at nm. MT was excited at 560 nm (18% laser power) and fluorescence was collected at nm. Cell Imaging with Carbonyl Cyanide m-chlorophenylhydrazone (CCCP) Treatment. HeLa cells were grown overnight on a 35 mm petri dish with a cover slip. The cells were incubation with 10 M CCCP (by adding 1 L of a 200 mm stock solution of CCCP in DMSO to 2 ml culture medium) for 30 min. The CCCP treated cells were then stained by either 5 M of TPE-TPP for 30 min or 50 nm MT (by adding 0.5 L of a 200 M stock solution of MT in DMSO to 2 ml culture medium) for 15 min. Cell Viability Evaluated by MTT Assay. Viability of the cells was assayed using cell proliferation Kit I with the absorbance of 595 nm being detected using a Perkin-Elmer Victor plate reader. Five thousand cells were seeded per well in a 96-well plate. After overnight culture, various concentrations of TPE-TPP were added into the 96-well plate. After 2 h S3

4 treatment, 10 μl of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in phosphate buffer solution) was added into the each well. After 4 h incubation at 37 C, 100 μl of solubilization solution containing 10% SDS and 0.01 M HCl was added to dissolve the purple crystals. After 12 h incubation, the optical density readings at 595 nm were taken using a plate reader. Each of the experiments was performed at least 3 times. S4

5 Scheme S1. Synthetic route to TPE-TPP. S5

6 a c Br P + b Br P + TPE-TPP a, 30H c, 18H b, 4H Chemical shift (ppm) Figure S1. 1 H-NMR spectrum of TPE-TPP in DMSO-d 6. S6

7 Chemical shift (ppm) Chemical shift (ppm) Figure S2. 13 C-NMR spectrum of TPE-TPP in DMSO-d 6. S7

8 Figure S3. Mass spectrum (MALDI-TOF) of TPE-TPP S8

9 Intensity (au) Particle size (nm) Figure S4. Particle size analysis of TPE-TPP (5 M) nano-aggregate in PBS. S9

10 Cell viability (%) Control [TPE-TPP] ( M) Figure S5. Cytotoxicity of TPE-TPP on HeLa cells determined by MTT assay. S10

11 Figure S6. (A C) Fluorescence images of the HeLa cells stained with TPE-TPP for (A) 30 min, (B) 1 h and (C) 2 h. (D) Signal to background ratio of TPE-TPP dyed HeLa cells with different incubation time. Concentration of TPE-TPP: 5 M; excitation wavelength: nm; scale bar: 15 m. S11

12 Figure S7. Fluorescent images of living HeLa cells stained with MT with increasing number of scans (1 6 times). Concentration of MT: 50 nm; excitation wavelength = 560 nm; emission filter: nm; irradiation time: 7.75 sec/scan. S12

13 Figure S8. Molecular structure of MitoTracker Red FM. Reference (1) Yu, Y.; Feng, C.; Hong, Y.; Liu, J.; Chen, S.; Ng, K. M.; Luo, K. Q.; Tang, B. Z. Adv. Mater. 2011, 23, S13