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Reaction Based Color-Convertible Fluorescent Probe for Ferroptosis Identification Leilei Shi, Qinghua Guan, Xihui Gao, Xin Jin, Li Xu, Jian Shen, Chenwei Wu, Xinyuan Zhu, and Chuan Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01721 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Analytical Chemistry

Reaction Based Color-Convertible Fluorescent Probe for Ferroptosis Identification Leilei Shi,† Qinghua Guan,† Xihui Gao,† Xin Jin,† Li Xu,† Jian Shen,*,§ Chenwei Wu,† Xinyuan Zhu,† and Chuan Zhang*,† †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China

§

Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Nanjing Normal University, Nanjing 210046, China *email: [email protected]; [email protected]

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Ferroptosis is an iron-mediated, caspase-independent pathway of cell death that accompanies with the accumulations of reactive oxygen species (ROS) and oxygenases, as well as involves in many other pathophysiological procedures. However, specific and rapid monitoring of ferroptosis in living cells or tissues has not been achieved so far. Herein, a quinoxalinone-based fluorescent probe (termed as Quinos-4, or QS-4) with a reactive aromatic thioether moiety was designed for ferroptosis identification. Upon exposing to the high levels of ROS and hemeoxygenase-1 (HO-1) which are considered as the biochemical characteristics of ferroptosis, QS-4 could be oxidized into a sulfoxide derivative (QSO-4) and its original aggregation-induced enhanced red fluorescence emission could be converted to green fluorescent emission sharply. Based on this unique reaction-induced color conversion, this molecular probe can be employed for identifying the occurrence of ferroptosis both in vitro and in vivo.


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Ferroptosis is a recently identified caspase-independent cell death pathway that is highly related to iron.1-3 It is known as an iron-dependent form of programmed cell death, which is different from other cell death forms.4,5 The characteristic of ferroptosis involves the production of both soluble and lipid ROS through iron-dependent hemeoxygenase.6,7 Currently, two small molecular agents (erastin and RSL3) have been identified to induce ferroptosis with different mechanisms. For instance, erastin can inhibit the activity of cystine-glutamate antiporter (Xct), which results in the depletion of glutathione (GSH) and eventually inactivates the glutathione peroxidase 4 (GPX4).2 RSL3, on the other hand, induces the ferroptosis through covalently modifying the GPX4 and inhibiting its activity. As the GPX4 is a key enzyme to reduce the phospholipid hydroperoxides, the inhibition of GPX4 using erastin or RSL3 would cause the lipid peroxidation process,8 resulting in the generation of diffusible electrophilic species that modify the intracellular proteins and inhibit their functions.9 Therefore, misregulation of ferroptosis is highly related to many diseases, including neurodegenerative disorders and a variety of cancers, such as Huntington’s disease, cervical cancer and breast cancer.10-12 Theoretically, real-time monitoring of ferroptosis can provide us essential information for the diagnosis of disease. To date, the most commonly used method to identify ferroptosis relies on the determination of the expression level of ferroptosis-related biomarkers, for example, the Xct.1 However, this method is usually time-consuming and aggressive, which only deals with dead cells. As a result, there is a great demand for developing non-invasive method for monitoring ferroptosis. Herein, we report a fluorescent molecular probe that can specifically engage in an oxidation reaction catalyzed by the ferroptosis-associated intracellular environment and change its fluorescent emission. Previous studies have revealed that intracellular ROS and hemeoxygenase were significantly enhanced when a cellular ferroptosis was induced.7 Interestingly, Simonneaux



and coworkers once reported that iron-porphyrin could oxidize the aromatic thioether to sulfoxide in the existence of hydrogen peroxide in aqueous solution.13 Note that, iron-porphyrin is an important coenzyme of hemeoxygenase. With high levels of ROS and hemeoxygenase accompanying with the ferroptosis process, we hypothesized that ROS can also oxidize the aromatic thioether into sulfoxide form in the presence of high level of intracellular hemeoxygenase. Therefore, a fluorescent molecular probe containing an aromatic thioether group can be specifically designed for the ferroptosis identification based on the unique hemeoxygenase catalyzed reaction, in which the transformation from aromatic thioether into aromatic sulfoxide can cause the rearrangement of the electronic density of conjugated molecule, resulting in the change of fluorescent characteristic. In general, electron-donating groups, for instance, thioether group, methoxyl group, and amino-group, could enhance the electronic density of conjugated system and result in bathochromic-shift of fluorescent molecules. On the contrary, electron-withdrawing group, such as sulfoxide group and nitro-group, would result in hypochromatic shift of fluorescent molecules.


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Figure 1. Schematic representation of transformation of QS-4 and the color change of fluorescent emissions in ferroptotic cells. Aromatic thioether could be oxidized by ROS under the catalyzation of hemeoxygenase to form sulfoxide derivative (QSO-4), then resulting in a large blue shift of fluorescence emission.

EXPERIMENTAL SECTION Synthesis of 3-methylquinoxalin-2(1H)-one (1a) and 7-methoxy-3-methylquinoxalin2(1H)-one (1b).14 o-Phenylenediamine (0.1 mol, 10.8 g) was suspended in anhydrous ethanol (150 mL). The reaction mixture was cooled in an ice bath. Then, ethyl pyruvate (0.12 mol, 13.92 g) in anhydrous ethanol (10 mL) was added into the reaction mixture under stirring. The resulting solution was allowed to stir at the room temperature for 12 h. Finally, the obtained precipitate was filtered and washed with ethanol and dried in vacuum to give the product 1a (13.6 g, yield: 85%) as a white solid which was pure without any purification.



Synthesis of 1-allyl-3-methylquinoxalin-2 (1H)-one (2a) and 1-allyl-7-methoxy-3methylquinoxalin-2 (1H)-one (2b).15 Compound 1a (20 mmol, 3.2 g) and K2CO3 (24 mmol, 3.31 g) were dispersed in acetone (100 mL). Then a solution of 3-bromoprop-1-ene (24 mmol, 2.88 g) was added dropwise into the reaction mixture and followed with stirring in oil bath at 62oC for 12 h. After the reaction was completed, the solvent was evacuated and the residue was extracted with water (20 mL) and ethyl acetate (EA) (40 mL) mixture solution. Then, the organic layer was separated and dried over anhydrous MgSO4 and the concentrated crude sample was further purified via silica gel column chromatography (hexane: EA = 20:1, v:v) to obtain 2.21 g of 2a, (white solid, yield 55%). Synthesis of methyl-2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetate (3a), methyl-2-(7methoxyl-3-methyl-2-oxoquinoxalin-1(2H)-yl)acetate (3b). Compound 1a (20 mmol, 3.2 g) and K2CO3 (24 mmol, 3.31 g) were suspended in acetone (100 mL). Then a solution of methyl-2bromoacetate (24 mmol, 3.67 g) was added dropwise into the mixture. The reaction mixture was stirred in oil bath at 62oC for 12 h. After the reaction was completed, acetone was evacuated via rotary evaporators. Then, the crude was extracted with water (20 mL) and ethyl acetate (EA) (40 mL) mixture solution. The organic layer was then separated and dried by anhydrous MgSO4. Finally, the crude was purified by silica gel column chromatography (hexane: EA = 10:1) to obtain 3.0 g of 3a, (white solid, yield 54%). Synthesis of final product (QS-1, QS-2, QS-3 and QS-4). 5-(Methylthio)thiophene-2carbaldehyde (3 mmol, 570 mg) and catalytic concentrated sulfuric acid were added into a solution of compound 3a (2.5 mmol, 500 mg) in acetic acid (10 mL). The resulting solution was heated to 50oC and reacted for 8 h. The solvent was evacuated by rotary evaporators and then water (20 mL) and ethyl acetate (50 mL) mixture solution were added for extraction. After being


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basified with solid K2CO3, the extracted organic layer was collected and dried through anhydrous MgSO4. Finally, the crude sample was purified by silica column chromatography (PE: EA = 20 :1 to 10:1) to obtain 322 mg of QS-1 (orange solid, yield 38%). Preparation and stability study of QS-4 nanoparticles. QS-4 (40 mg) was dissolved in 2 mL DMSO, then the solution was added into 20 mL PBS buffer via syringe pump. The mixture was stirred overnight under room temperature, eventually the DMSO was removed through ultrafiltration. The obtained nanoparticles were stored at dark environment. When it was used for cellular study, the stock solution was diluted by PBS at the concentration of 20 µM. The stability of QS-4 nanoparticles was investigated by monitoring the size variations using dynamic light scattering (DLS) at different time points. Cell Culture. Cell culture process was followed our previous protocol.16 Briefly, the cervical cancer line (HeLa cells) was cultured in Dulbecco’s Modified Eagle Medium (DMEM). The culture medium contains 10% fetal bovine serum (FBS) and antibiotics (50 units mL-1 and 50 units mL-1 streptomycin) at 37oC under a humidified atmosphere containing 5% CO2. In vitro cytotoxicity studies. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based cytotoxicity assay was conducted following our previous procedure.16 In detail, HeLa cells were seeded in the 96-well plates. 12 hours later, cells were treated by QS-4 with different concentrations and incubated for another 72 h. After that, 20 µL MTT solution with a concentration of 5 mg/mL was added into each 96-well plate. After incubating at 37oC for 4 h, the medium was removed and 200 µL DMSO was added into each well. Finally, the absorbance of the solution was quantified by a Bio Tech Synergy H4 plate reader under 570 nm.



Ferroptosis models. Ferroptosis models were induced by erastin. Briefly, HeLa cells were seeded to culture dish, after being adhered, erastin was added into culture medium at the concentration (1 µM) for incubation with cells for 24 h before usage. The fluorescence intensity study of QS-4 in cells with ferroptosis. The fluorescence intensity of QS-4 in cells was measured by flow cytometry in HeLa cells. HeLa cells (6×105) were seeded in 6-well plates. After 12 h, erastin (1 µM) was added to the medium for incubation another 24 h, then QS-4 nanodots (20 µM) was added into the well for incubation 1 h. Cells without erastin treatment were used as negative control. After removing the culture medium, cells were washed three times with PBS and treated with trypsin to collect cells for flow cytometry analysis. The flow cytometry was performed on a BD LSRFFortessa flow cytometer to monitor the fluorescence intensity of QS-4 in cells by recording 1×104 gated events. Confocal imaging of cells stained QS-4. HeLa cells (1×105) were seeded in confocal dishes and cultured in DMEM culture medium for 24 h. Then, erastin (1 µM) was added to the culture dish and incubated for 24h to induce the ferroptosis. As a control, anti HO-1 siRNA was transfected via lipofectamine 2000 in Opti-MEM serum free medium for 12 h to knockdown the HO-1 expression. After that, QS-4 nanodots (20 µM) was incubated with HeLa cells for another 1 h. Cells without erastin treatment were used as the control. Finally, cells were washed with PBS three times, and 1 mL of PBS was finally added before imaging. Cells were imaged with a 20× objective lens. The fluorescence of QS-4 was excited with a 488 nm laser with emission collected at 510-550 nm and 595-650 nm. Western blot and ELISA analysis. HeLa cells were cultured in Patri dish (10 cm diameter) with 10 mL of complete DMEM medium at a density of 3.0×106 cells. After cells were adhered,


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1 µM of erastin was added into the culture medium to induce the cell ferroptosis. HeLa cells without erastin treatment were used as negative control. Then, the total proteins were extracted by Laemmli buffer and quantified through bicinchoninic acid (BCA) protein assay kit. The total proteins (20 µL/lane) were added and separated by sodium dodecyl sulfate-poly-acrylamide gels (SDS-PAGE) and then transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes. Eventually, the PVDF membranes were blocked by 5% nonfat milk in Tris buffered saline and measured with monoclonal antibodies against anti-GADPH (1:1000 dilution), anti-Hemeoxygenase (1:1000 dilution), followed by HRP-conjugated (HRP = horseradish peroxidase) antirabbit immunoglobulin-G (IgG, 1:5000 dilution) staining. The bands were monitored by chemiluminescent HRP Substrate S13 according to the manufacture’s method and quantified by ChemiDoc MP imaging System (Bio-Rad, USA). Expressions of HO-1 in HeLa cells were determined by a human HO-1 ELISA kit (abcam, ab207621) according to the manufacture’s method. Briefly, after incubation, the culture medium was removed. Samples were added into each well of microplate, which was precoated with antihuman HO-1 monoclonal antibody. Each well was then washed and incubated with the enzymelinked polyclonal antibody specific for human HO-1 for another 2 hours. The wells were washed to remove unbound antibody-enzyme, and substrate solutions were added to the each well. After being incubated for 20 min at room temperature, the enzyme reaction was blocked. HO-1 concentrations were detected by comparison of the optical density results via the standard curve. Cellular ROS measurement and fluorescence spectra analysis of QS-4 with ferroptotic cell lysate. HeLa cells (4×105) were seeded in a Patri dish (10 cm diameter) and cultured in DMEM culture medium. After 12 h, cells were treated with erastin (2 µM), CDDP (10 µM) and PTX (1 µM) for 24 h. Then, cells were washed by cold PBS and incubated with 100 µL DCFH-



DA probe at 37oC for 30 min. DCFH-DA probe is a fluorogenic probe designed to reliably measure reactive oxygen species (ROS) in live cells. The cell-permeable reagents are nonfluorescent or very weakly fluorescent while in a reduced state and upon oxidation exhibit strong green fluorogenic signal. After washing the cells with cold PBS, fluorescence intensity was measured by laser confocal fluorescence microscopy (Leica) and flow cytometry with excitation wavelength 488 nm. To verify the possibility of ferroptosis detection using the QS-4 molecular probe, HeLa cells (3×106) were added into a culture Patri dish. After 12 h, erastin (1 µM) was added to the medium for incubation another 24 h. After the removal of culture medium, cells were washed with PBS for three times and then the cells were disrupted through a cell scraper to obtain the supernatant (ferroptotic cell lysate). After that, the as-synthesized QS-4 nanodots (20 µM) was incubated with the supernatant at 37oC for 60 min, cells without any treatment were used as control. Eventually, the samples were used for steady-state fluorescence measurements. For cellular QSO-4 determination, ferroptotic cells were disrupted by cell scraper, and being extracted by ethyl acetate to afford the supernatant. Eventually, supernatant was dried through N2 and then detected via HPLC-MS with the eluent CH3CN and H2O. Animals and tumor models. Study protocols involving animals were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. Balb/c female nude mice (5 weeks of age) were purchased from Chinese Academy of Science (Shanghai). The nude mice were injected subcutaneously with 200 µL of cell suspension containing 5×106 HeLa cells. The tumors were allowed to grow to about 200 mm3 before experiment. In vivo imaging study and ex vivo fluorescence analysis. HeLa bearing nude female mice were randomly divided into two groups, and intravenously injected via tail vein with 200 µL of erastin (10 mg/kg) daily for one week, mice without any treatment were used as negative control.


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Then, 200 µL of QS-4 nanodots (10 mg/kg) were injected to the mice, briefly, QS-4 (40 mg) was dissolved in 1 mL DMSO, then the solution was added into 4 mL PBS buffer via syringe pump. The mixture was stirred overnight under room temperature, eventually the DMSO was removed through ultrafiltration. The obtained colloid nanoparticles (1 mg/mL) was used for in vivo detection immediately, after injection of 1 h, the fluorescence change of QS-4 was monitored using an in vivo imaging system with appropriate wavelength (λ ex = 460 nm, λ em = 510-550 nm and λ em = 595-650 nm). After that, the mice were anesthetized and intracardially perfused with PBS followed 4% PFA. The tumors were excised, fixed in 4% PFA and transferred to 30% sucrose aqueous solution for 12 h. Then the tumors were embedded in optimum temperature compound (OTC), frozen and sectioned with a thickness of 10 µm. Finally, the sections were observed by laser confocal fluorescence microscopy (Leica). Statistical analysis. The results were illustrated as the mean standard deviation (SD). The difference between two groups was evaluated by Student’s t test. The difference was considered as statistical significance (*p < 0.05) and very significant (**p < 0.01, ***p

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