A Two-Photon Fluorescent Probe for Lysosomal Thiols in Live Cells and Tissues

Lysosome-specific fluorescent probes are exclusive to elucidate the functions of lysosomal thiols. Moreover, two-photon microscopy offers advantages of less phototoxicity, better three dimensional spatial localization, deeper penetration depth and lower self-absorption. However, such fluorescent probes for thiols are still rare. In this work, an efficient two-photon fluorophore 1,8-naphthalimide-based probe conjugating a 2,4-dinitrobenzenesulfonyl chloride and morpholine was designed and synthesized, which exhibited high selectivity and sensitivity towards lysosomal thiols by turn-on fluorescence method quantitatively and was successfully applied to the imaging of thiols in live cells and tissues by two-photon microscopy.


Materials and general methods
All of the solvents used were of analytic grade. 1 was dissolved in dimethyl sulphoxide (DMSO) to produce 5 mM stock solutions. Aliquots were then diluted to 10 µM with a HEPES buffer solution containing 40 mM acetic acid, phosphoric acid, and boric acid. Slight variations in the pH of the solutions were achieved by adding minimal volumes of NaOH or HCl. 1 H-NMR and 13 C-NMR spectra were recorded on a VARIAN INOVA-400 spectrometer. Chemical shifts (δ) were reported as ppm in CDCl 3 , with TMS as the internal standard. Mass spectrometric (MS) data were obtained with HP1100LC/MSD MS and an LC/Q-TOF-MS instruments. Fluorescence measurements were performed on a VAEIAN CARY Eclipse fluorescence spectrophotometer (Serial No. FL0812-M018). Excitation and emission slit widths were modified to adjust the fluorescence intensity to a suitable range.
Absorbance spectra were measured on a Perkin Elmer Lambda 35 UV/VIS spectrophotometer. All pH measurements were performed using a Model PHS-3C meter calibrated at room temperature (23 ± 2 °C) with standard buffers of pH 7.4.

Synthesis of 1
Synthesis of compound 5. 4-(2-aminoethyl)-morpholine (1.30 g, 0.01 mol) was added to 4-bromo-1,8-naphthalic anhydride (2.77 g, 0.01 mol) in boiling 1,4-dioxane (50 mL) under magnetic stirring for 4h. After cooling to room temperature, the yellowish sediments were collected by filtration and then dried overnight at room temperature in a vacuum oven to give 5 as a pale yellow solid (2.     in dichloromethane (5 mL) was added dropwise at 0 °C. Stirring for another 4 h at room temperature, the solvent was removed under reduced pressure and purified by silica gel column chromatography with dichloromethane/methanol (75:1) as the eluent to produce 1 as a bright yellow solid (142 mg), yield: 75.0%. 1

Determination of quantum yields
The fluorescence quantum yield of 1 was determined according to the method below 1 .
Where φ is fluorescence quantum yield; FA is integrated area under the corrected emission spectra; A is the absorbance at the excitation wavelength; λ ex is the excitation wavelength; η is the refractive index of the solution; the subscripts u and s refer to the unknown and the standard, respectively. We choose rhodamine B as standard, which has a fluorescence quantum yield of 0.49 in ethanol 2 .

Determination of the detection limit
Calculation of detection limit was based on the fluorescence titration curve (Fig 3, S2 and 3) of 1 in the presence of thiols. The fluorescence intensity of 1 was measured by three times and the standard deviation of blank measurement was achieved. The detection limit was calculated with the following equation: Where σ is the standard deviation of blank measurement, k is the slop between the fluorescence intensity versus thiols concentrations.

Cytotoxicity experiments
Measurement of cell viability was evaluated by reducing of MTT (3-(4,5)-dimethylthiahiazo (-2-yl) -3,5-diphenytetrazoliumromide) to formazan crystals using mitochondrial dehydrogenases (Mosmann, 1983). Human breast cancer cells (MCF-7) cells were seeded in 96-well microplates (Nunc, Denmark) at a density of 1×10 5 cells / mL in 100 μL medium containing 10 % FBS. After 24 h of cell attachment, the plates were then washed with 100 μL / well PBS. The cells were then cultured in medium with 2.5 μM of 1 for 24 h. Cells in culture medium without 1 were used as the control. Six replicate wells were used for each control and test concentration. 10 μL of MTT (5 mg / mL) prepared in PBS was added to each well and the plates were incubated at 37 °C for another 4 h in a 5 % CO 2 humidified incubator.
The medium was then carefully removed, and the purple crystals were lysed in 200 μL DMSO. Optical density was determined on a microplate reader (Thermo Fisher Scientific) at 570 nm with subtraction of the absorbance of the cell-free blank volume at 630 nm. Cell viability was expressed as a percent of the control culture value, and it was calculated using the following equation: Cells viability (%) = (OD dye -ODK dye) / (OD control· ODK control) × 100.

Tissue imaging experiments
Two-photon fluorescence imaging of 1 in tissues were obtained with a spectral confocal multiphoton microscope (Olympus, FV1000) with a high-performance mode-locked titanium sapphire laser source (MaiTai, Spectra-Physice, USA). Numerical aperture was 1.42 (oil) and 1.30 (sil), respectively. The excitation wavelength was 805 nm and output power was 1230 mW, which corresponded to approximately 10 mW average power in the focal plane. Images were collected at 520-560 nm.