Near-infrared fluorescent probe for hydrogen sulfide: high-fidelity ferroptosis evaluation in vivo during stroke

Ferroptosis is closely associated with cancer, neurodegenerative diseases and ischemia-reperfusion injury and the detection of its pathological process is very important for early disease diagnosis. Fluorescence based sensing technologies have become excellent tools due to the real-time detection of cellular physiological or pathological processes. However, to date the detection of ferroptosis using reducing substances as markers has not been achieved since the reducing substances are not only present at extremely low concentrations during ferroptosis but also play a key role in the further development of ferroptosis. Significantly, sensors for reducing substances usually consume reducing substances, instigating a redox imbalance, which further aggravates the progression of ferroptosis. In this work, a H2S triggered and H2S releasing near-infrared fluorescent probe (HL-H2S) was developed for the high-fidelity in situ imaging of ferroptosis. In the imaging process, HL-H2S consumes H2S and releases carbonyl sulfide, which is then catalyzed by carbonic anhydrase to produce H2S. Importantly, this strategy does not intensify ferroptosis since it avoids disruption of the redox homeostasis. Furthermore, using erastin as an inducer for ferroptosis, the observed trends for Fe2+, MDA, and GSH, indicate that the introduction of the HL-H2S probe does not exacerbate ferroptosis. In contrast, ferroptosis progression was significantly promoted when the release of H2S from HL-H2S was inhibited using AZ. These results indicate that the H2S triggered and H2S releasing fluorescent probe did not interfere with the progression of ferroptosis, thus enabling high-fidelity in situ imaging of ferroptosis.


Instruments and materials
Unless otherwise stated, all solvents and reagents were purchased from commercial suppliers and were used as received without further purification. The reactions were performed in standard glassware. All aqueous solutions were prepared in ultrapure water with a resistivity of 18.25 MΩ·cm (purified by Milli-Q system, Millipore). Column chromatography was performed using silica gel 60 (230 ± 400 mesh, 0.040 ±0.063 mm) from Dynamic Adsorbents. NMR spectra were recorded on a Bruker-400 spectrometer, using TMS as an internal standard. High-resolution mass spectrometry was performed with LTQ FT Ultra (Thermo Fisher Scientific, America) in MALDI-DHB mode. Absorption spectra were recorded with a UV-vis spectrophotometer (Shimadzu UV-2550, Japan), and fluorescence spectra were obtained with a fluorimeter (Shimadzu RF-6000, Japan). Fluorescence imaging of mice was performed on an IVIS Lumina LT Series III small animal optical in vivo imaging system (U.S.A.) with an excitation filter of 500 nm and an emission filter of 650 nm.
Experimental mice were anesthetized on an R500IE anesthesia machine. Living Image 4.5 software (PerkinElmer) was used for data analysis.

Spectroscopic measurements
Small amount of probe HL-H 2 S was dissolved in DMSO to prepare the stock solutions (5.0  10 -2 M). Unless otherwise mentioned, all the measurements for probe HL-H 2 S target reaction were tested in PBS buffer (10 mM, pH 7.4, containing 2% DMSO and 80% glycerol). After adding NaHS and incubating at 37 °C for 40 min in a thermostat, a 500 μL aliquot of the reaction solution was transferred to a quartz cell with an optical length of 1 cm for the measurement of absorbance or fluorescence. The excitation wavelength was 450 nm. For the selectivity assay, superoxide anion (O 2 •− ), ·OH, ONOO − , NO, H 2 O 2 , and NO 2  were generated according to previous report. 1

Determination of the detection limit
The limit of detection (LOD) for hydrogen sulfide was calculated based on the following equation: LOD = 3σ/k Supporting Information S4 Where σ represents the standard deviation and k represents the slope of the titration spectra curve among the limited range.

Quantum yield measurements
The measurement of the fluorescence quantum yield was measured by using an ethanol solution of rhodamine B as a standard (10 μM, = 0.71) and using Φ the following equation 2 .
Where s and r represent the sample to be tested and the reference dye, respectively. A represents the absorbance at the maximum absorption wavelength, F represents the fluorescence spectrum integral at the maximum absorption wavelength excitation, and n represents the refractive index of the sample to be tested or the reference dye solvent.

Viscosity determination and fluorescence measurements
The solvents were obtained by mixing a water-glycerol system in different proportions. Measurements were carried out with an NDJ-8S rotational viscometer, and each viscosity value was recorded. The relationship between the fluorescence emission intensity of the probe HL-H 2 S and the viscosity of the solvent is well expressed by the Förster-Hoffmann equation as follows: log I f = C+ xlog η, Where η is the value of viscosity, I is the emission intensity, C is a constant, and x represents the sensitivity of the probe HL-H 2 S to viscosity 3 .

Methylene blue method (MB) assay of H 2 S
The methylene blue method was carried out as previously described. Briefly, the vials were evaluated in PBS buffer (10 mM, pH 7.4, containing 2% DMSO and 80% glycerol) containing CA (100 μg/mL) and AZ (50 μM). After adding probe HL-H 2 S and NaHS and then incubating at 37 °C for 60 min in a thermostated bath equal volumes of MB solution were added. After reaction for 30 min, the absorbance of the mixture was determined at 670 nm. Methylene blue (MB) solution: 20% of Zn (OAc) 2  labelled group and the control group, respectively).

Cell culture and imaging
PC12 cells were cultured with DMEM supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere with 5/95 (v/v) of CO 2 /air at 37 °C. One day before imaging, cells were detached with a treatment of 0.2% (w/v) trypsin-EDTA solution (Gibco) and suspended in culture media. The cell suspension was then transferred to confocal dishes to grow with adherence. For imaging, PC12 cells at 80% confluence were harvested by scraping and transferred to confocal dishes to grow with adherence.

Ferroptosis model
Cells were treated with erastin (10 μM) for appropriate time to induce ferroptosis.
After that the culture media were removed, and the cells were washed with serum-free media and then incubated with HL-H 2 S (10 μM) for different treated. Imaging was performed with confocal microscope.

Measurement of the biomarkers of ferroptosis
Cellular Fe 2+ level was measured by using an iron assay kit (Sigma-Aldrich) according to the manufacturer's instructions. 5 Malondialdehyde (MDA, Sigma-Aldrich) level was measured by using an MDA assay kit (Sigma-Aldrich) according to the manufacturer's instructions and glutathione peroxidase 4 (GPX4, Sigma-Aldrich) level was measured by using an GSH assay kit (Sigma-Aldrich) according to the Supporting Information S6 manufacturer's instructions 6 . The results were normalized to total protein concentrations.

Western blotting assay
Western blotting was carried out as previously described. 7

Calculation of mean fluorescence intensity
The mean fluorescence density was measured by Image-Pro Plus (v. 6.0) and calculated via the equation (mean density = IOD sum /area sum ), where IOD and area were integral optical density and area of the fluorescent region.

OGD/R model of cells was performed by oxygen and glucose
deprivation/reperfusion. PC12 cells at 80% confluence were harvested by scraping and transferred to confocal dishes to grow with adherence. When the cells are adherent, the culture medium is changed to sugar-free DMEM and cultured in a three-gas incubator for 5 h without oxygen. Afterwards, these cells were incubated with high-glucose DMEM in a 5 % CO 2 and 95% O 2 atmosphere for 5 h. Then, the cells were incubated Supporting Information S7 with HL-H 2 S (10 µM) for 30 minutes. Wash cells three times with PBS for confocal imaging.

Middle cerebral artery occlusion (MCAO) model
MCAO was induced using a previously described method with slight modifications. 8 In brief, C57BL/6J wild-type mice were anesthetized with 5% isoflurane in O 2 by facemask, followed by ligation of the left middle cerebral artery with 6-0 monofilament (Doccol Corp., Redlands, CA, USA). After 1 h of occlusion, the monofilament was removed to initiate reperfusion. A homeothermic heating pad was employed to monitor and stabilize the mice body temperature at 37 ± 0.5 °C. The same procedure, but without monofilament ligation, was performed on sham-operated mice.

Measurement of infarct volume and neurological deficit
Mice were deeply anesthetized and euthanized with an overdose of isoflurane and decapitated MCAO. The brains were collected after transcranial perfusion by saline followed with 4% paraformaldehyde. Brain tissues were cut into 1-mm coronal sections, and then dipped in 2% 2,3,5-triphenyltetrazolium chloride (TTC) (17779, Sigma-Aldrich, United States) for staining. The infarct volume was measured and analyzed by a blinded observer using ImageJ v1.37 (NIH, Bethesda, MA, United States), as described previously, 9 then was normalized and presented as a percentage of the non-ischemic hemisphere to correct for edema. 9 Neurological deficit scores were evaluated MCAO as described previously. 10 The score ranged from 0 (without observable neurological deficit) to 4 (no spontaneous motor activity and loss of consciousness).

Histological staining of the tissue slices
After imaging, the mice were killed, and the brains and other tissues (brain, heart, liver, spleen, lung and kidney) were collected for tissue analysis. Through a series of standard procedures, including fixation in 10% neutral buffered formalin, embedding into paraffin and sectioning at 3 µm thickness, the tissues were stained with hematoxylin-eosin (H&E). Thereafter, the prepared slices were examined by a digital  Synthesis of probe HL-H 2 S: Compound 1 (70 mg, 0.16 mmol) and compound 7 (30 mg, 0.18 mmol) were dissolved in ice-cold anhydrous THF (10 mL), followed by the addition of sodium hydride (60% in mineral oil, 12 mg, 0.24 mmol). The mixture was stirred at 0 o C for 40 min, and then removed from ice bath, followed by stirring for another 3 h at room temperature. The solvent was evaporated under reduced pressure and the crude product was purified by column chromatography (1:50 v/v MeOH/dichloromethane), to generate the product as red solid. Yield: 30 mg (32.0%). Fig. S1. The 1 H NMR spectra of probe HL-H 2 S in the absent and present of 100 equiv NaHS.          Table   Table S1. Photophysical properties of HL-NH 2