Reaction-Based Color-Convertible Fluorescent Probe for Ferroptosis Identification

Article abstract of DOI:10.1021/acs.analchem.8b01721

Ferroptosis is an iron-mediated, caspase-independent pathway of cell death that is accompanied with the accumulations of reactive oxygen species (ROS) and oxygenases, as well as being involved 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 it to 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 fluorescence emission sharply. On the basis of this unique reaction-induced color conversion, this molecular probe can be employed for identifying the occurrence of ferroptosis both in vitro and in vivo.

Full text of DOI:10.1021/acs.analchem.8b01721

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Article
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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Reaction Based Color-Convertible Fluorescent Probe for
Ferroptosis Identification
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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: chuanzhang@sjtu.edu.cn; shenjianbio@hotmail.com
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ABSTRACT: Ferroptosis is an ironꢀmediated, caspaseꢀindependent 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
quinoxalinoneꢀbased fluorescent probe (termed as Quinosꢀ4, or QSꢀ4) with a reactive aromatic
thioether moiety was designed for ferroptosis identification. Upon exposing to the high levels of
ROS and hemeoxygenaseꢀ1 (HOꢀ1) which are considered as the biochemical characteristics of
ferroptosis, QSꢀ4 could be oxidized into a sulfoxide derivative (QSOꢀ4) and its original
aggregationꢀinduced enhanced red fluorescence emission could be converted to green fluorescent
emission sharply. Based on this unique reactionꢀinduced 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 caspaseꢀindependent cell death pathway that is highly related
to iron.1ꢀ3 It is known as an ironꢀdependent 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 ironꢀdependent 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 cystineꢀglutamate 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.10ꢀ12 Theoretically, realꢀtime
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 ferroptosisꢀrelated biomarkers, for example, the Xct.1 However, this method
is usually timeꢀconsuming and aggressive, which only deals with dead cells. As a result, there is
a great demand for developing nonꢀinvasive method for monitoring ferroptosis.
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Herein, we report a fluorescent molecular probe that can specifically engage in an oxidation
reaction catalyzed by the ferroptosisꢀassociated 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
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and coworkers once reported that ironꢀporphyrin could oxidize the aromatic thioether to
sulfoxide in the existence of hydrogen peroxide in aqueous solution.13 Note that, ironꢀporphyrin
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, electronꢀdonating groups, for
instance, thioether group, methoxyl group, and aminoꢀgroup, could enhance the electronic
density of conjugated system and result in bathochromicꢀshift of fluorescent molecules. On the
contrary, electronꢀwithdrawing group, such as sulfoxide group and nitroꢀgroup, would result in
hypochromatic shift of fluorescent molecules.
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Figure 1. Schematic representation of transformation of QSꢀ4 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 (QSOꢀ4), then resulting in a
large blue shift of fluorescence emission.
EXPERIMENTAL SECTION
Synthesis of 3-methylquinoxalin-2(1H)-one (1a) and 7-methoxy-3-methylquinoxalin-
2(1H)-one (1b).14 oꢀPhenylenediamine (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.
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Synthesis of 1-allyl-3-methylquinoxalin-2 (1H)-one (2a) and 1-allyl-7-methoxy-3-
methylquinoxalin-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 3ꢀbromopropꢀ1ꢀene (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%).
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Synthesis of methyl-2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetate (3a), methyl-2-(7-
methoxyl-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 methylꢀ2ꢀ
bromoacetate (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). 5ꢀ(Methylthio)thiopheneꢀ2ꢀ
carbaldehyde (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 QSꢀ1 (orange solid, yield 38%).
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Preparation and stability study of QS-4 nanoparticles. QSꢀ4 (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 QSꢀ4 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 mLꢀ1 and 50
units mLꢀ1 streptomycin) at 37oC under a humidified atmosphere containing 5% CO2.
In vitro cytotoxicity studies. 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide
(MTT) based cytotoxicity assay was conducted following our previous procedure.16 In detail,
HeLa cells were seeded in the 96ꢀwell plates. 12 hours later, cells were treated by QSꢀ4 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 96ꢀwell 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.
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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.
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The fluorescence intensity study of QS-4 in cells with ferroptosis. The fluorescence
intensity of QSꢀ4 in cells was measured by flow cytometry in HeLa cells. HeLa cells (6×105)
were seeded in 6ꢀwell plates. After 12 h, erastin (1 µM) was added to the medium for incubation
another 24 h, then QSꢀ4 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 QSꢀ4 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 HOꢀ1 siRNA was
transfected via lipofectamine 2000 in OptiꢀMEM serum free medium for 12 h to knockdown the
HOꢀ1 expression. After that, QSꢀ4 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 QSꢀ4 was excited with a 488 nm laser with emission
collected at 510ꢀ550 nm and 595ꢀ650 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 sulfateꢀpolyꢀacrylamide gels
(SDSꢀPAGE) 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 antiꢀGADPH (1:1000 dilution), antiꢀHemeꢀ
oxygenase (1:1000 dilution), followed by HRPꢀconjugated (HRP = horseradish peroxidase) antiꢀ
rabbit immunoglobulinꢀG (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 (BioꢀRad, USA).
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Expressions of HOꢀ1 in HeLa cells were determined by a human HOꢀ1 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 antiꢀ
human HOꢀ1 monoclonal antibody. Each well was then washed and incubated with the enzymeꢀ
linked polyclonal antibody specific for human HOꢀ1 for another 2 hours. The wells were washed
to remove unbound antibodyꢀenzyme, and substrate solutions were added to the each well. After
being incubated for 20 min at room temperature, the enzyme reaction was blocked. HOꢀ1
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 DCFHꢀ
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DA probe at 37oC for 30 min. DCFHꢀDA probe is a fluorogenic probe designed to reliably
measure reactive oxygen species (ROS) in live cells. The cellꢀpermeable reagents are nonꢀ
fluorescent 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 QSꢀ4 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 asꢀsynthesized QSꢀ4 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 steadyꢀstate fluorescence measurements.
For cellular QSOꢀ4 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 HPLCꢀMS with the eluent CH3CN and H2O.
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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 QSꢀ4 nanodots (10 mg/kg) were injected to the mice, briefly, QSꢀ4 (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 QSꢀ4 was monitored
using an in vivo imaging system with appropriate wavelength (λ ex = 460 nm, λ em = 510ꢀ550
nm and λ em = 595ꢀ650 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 ꢁm. Finally, the sections were
observed by laser confocal fluorescence microscopy (Leica).
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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<0.001).
RESULTS AND DISCUSSION
Design and synthesis of fluorescent probes. Based on above assumption, we designed a
series of small molecular quinoxalinoneꢀbased fluorescent probes, in which the quinoxalinone
backbone was modified and conjugated with a reactive aromatic thioether moiety (Figure 1). In
detail, the fluorescent probes were synthesized starting from benzeneꢀ1ꢀdiamine and 4ꢀ
methoxybenzeneꢀ1,2ꢀdiamine. After annulation and aldol condensation, four quinoxalinoneꢀ
based aromatic thioether derivatives (QSꢀ1, QSꢀ2, QSꢀ3, QSꢀ4) were obtained (Schemes S1ꢀS4).
The optical characteristics of these four molecular probes were analyzed in different dimethyl
sulfoxide/water (DMSO/water) mixtures. It could be noticed that when water content increased
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in the mixed solution, the absorption peak waveꢀlengths of four asꢀsynthesized compounds
underwent a trivial change but their intensities significantly decreased (Figure S1), which could
be attributed to the formation of nanodots after water addition. The nanodot formation was also
verified by dynamic light scattering (DLS) with hydrodynamic diameter ~52 ± 3 nm in
phosphate buffer solution (PBS, Figure 2b and S2) and transmission electron microscope (TEM)
imaging, in which spherical nanodots with diameter ~50 nm could be clearly observed (Figure
S2). Interestingly, the nanodots assembled by the molecular probe are quite stable in aqueous
solution, as evidenced by the negligible increase of the particle size in the stock solution for 7
days (Figure S2). In addition, all synthesized molecular probes exhibited enhanced fluorescent
emission with bathochromic shift of the maximum fluorescence peak when water/DMSO ratio
increased owing to the restricted intramolecular rotations in their aggregate states. These results
demonstrated that the asꢀsynthesized molecular probes possessed the property of aggregationꢀ
induced enhanced emission (AIEE, Figure 2a, b, Figures S3ꢀS5). Moreover, the absolute
quantum yields for QSꢀ1, QSꢀ2, QSꢀ3, QSꢀ4 was determined in their aggregated states with the
value of 6.06%, 7.46%, 6.25%, 8.47%, respectively (Table S1). It is wellꢀknown that AIE
fluorophores exhibit highly bright fluorescence in the aggregated state, but very weak
fluorescence in good solvent, making them to be ideal probes for biological imaging and
analysis.17ꢀ24 Based on their optical properties, QSꢀ4 showed the best AIEEꢀactive fluorescence
with the highest quantum yield among all of compounds and was employed for ferroptosis
identification (vide infra).
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Besides their AIEE feature, responsiveness of the molecular probe to the ferroptosisꢀ
associated cellular environment is a key factor for ferroptosis identification. Due to the existence
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the thiophene ring in the designed molecular probe is difficult to be oxidized into sulfoxide by
ROS alone. In contrast, when the quinoxalinoneꢀbased probes were exposed to hemeoxygenaseꢀ
1, a representative and commercially available hemeoxygenase used for this study, it could be
found that the aromatic thioether group was rapidly oxidized into sulfoxide form owing to the
catalytic activity of the enzyme, as evidenced by fluorescence measurements (Figure 2c). Then,
the selectivity of QSꢀ4 toward various catalysts including HOꢀ1, iron porphyrin, Fe2+ and Fe3+
was evaluated by monitoring the emission of sulfoxide form at 550 nm. As shown in Figure 2d,
the QSꢀ4 fluorescent emission was highly sensitive to HOꢀ1 and iron porphyrin, indicating that
QSꢀ4 could be served as an activatable probe with converted fluorescent color for detection of
high level of ROS and HO inside the cells. Before applying them to identify the ferroptotic cells,
we first verified the expression level of hemeoxygenase and ROS concentration inside the cells
when ferroptosis occurred. After incubating the HeLa cells with erastin (a typical ferroptosis
inducer) for 24 h, the intracellular ROS level was enhanced gradually with the increased
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concentration of erastin, as determined by
a
fluorometric assay using 2,7ꢀ
dihydrochlorofluorescin diacetate (DCFHꢀDA) as a molecular indicator (Figure S6). Besides,
western blot result revealed that the expression level of HOꢀ1 indeed significantly increased
when ferroptosis occurred (Figure S7). In contrast, the concentration of HOꢀ1 and ROS in typical
apoptosis cells induced by conventional chemotherapeutics (e.g. paclitaxel (PTX) and cisꢀ
platinum (CDDP)) were significantly lower than those in ferroptotic cells, as quantified by
enzyme linked immunosorbent (ELISA) and DCFHꢀDA assay (Figure S8 and S9). As such, the
significantly high levels of both HOꢀ1 and ROS are likely to enable the catalyzation of the
molecular probe to its sulfoxide form and generate a hypochromatic shift of the fluorescence
maximum for ferroptotic cell identification.
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Figure 2. Fluorescent characteristics of QSꢀ4, size of QSꢀ4 aggregates, and the change of
fluorescence emission when incubating QSꢀ4 with HOꢀ1 and H2O2. (a) The fluorescence spectra
of QSꢀ4 (5 µg/mL) in the mixed solvents with different DMSO/H2O ratios. (b) Representative
DLS measurements of QSꢀ4 (5 µg/mL) in the mixed solvents with different DMSO/H2O ratios.
(c) Fluorescent spectra of QSꢀ4 (20 µM) when it was incubated with HOꢀ1 (1% molar) and
different concentrations of H2O2 (from 0 to 500 µM) in PBS buffer for 1 h. (d) Fluorescence
intensities of QSꢀ4 (20 µM) at 550 nm at various catalysts and H2O2 (50 µM), Ipo is indicated as
aqueous iron porphyrin.
In vitro cellular ferroptosis identification. Then, we further verify the possibility of
ferroptosis detection using the QSꢀ4 molecular probe. In the experiment, asꢀsynthesized QSꢀ4
nanodots (20 µM) was incubated with ferroptotic cell lysate at 37oC for 60 min. As we can see,
the absorption spectrum of QSꢀ4 exhibited a hypochromatic shift after incubating the QSꢀ4 with
the cell lysate (Figure 3a). Meanwhile, the fluorescence maximum of the probe also
hypochromaticly shifted from 595 nm to 550 nm (Figure 3b). In a kinetic analysis, the
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fluorescence intensity of QSꢀ4 at 550 nm gradually increased over the time (Figure 3c),
indicating a rapid and continuous formation of QSOꢀ4. To confirm HOꢀ1 is a prerequisite for
catalyzing the molecular probe into its sulfoxide form, the fluorescence was also monitored
while incubating the QSꢀ4 (20 µM) with H2O2 (50 µM) alone for 60 min as the cancer cell used
in this study usually contained a relatively high concentration of intracellular H2O2 ranging from
10 µM to 50 µM.25 It could be observed that both UVꢀVis absorbance and fluorescence spectra
of the QSꢀ4 showed almost no change, indicating that H2O2 alone inside the cells could not
oxidize the QSꢀ4 rapidly (Figure 3d). Meanwhile, other ROS, for example, HClO, NaClO, •OH,
and OONOꢀ could not oxidize QSꢀ4 to form QSOꢀ4 (Figure S10). As a new compound used for
biodetection, its intrinsic cytotoxicity should be taken into consideration at first place. Therefore,
potential cytotoxicity of the probe was evaluated via standard methyl thiazolyl tetrazolium
(MTT) assay on HeLa cells. The MTT results showed that even the QSꢀ4 concentration
increased up to 100 µM and incubating with the HeLa cells for 72 h, a relatively high cell
viability remains, indicating QSꢀ4 has no obvious toxicity (Figure S11).
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Figure 3. The absorption and fluorescence spectra of QSꢀ4 before and after the incubation with
the lysate of ferroptotic cells. (a) UVꢀVis absorbance of QSꢀ4 (20 µM) of QSꢀ4 before and after
the incubation with cell lysate. (b) Fluorescent spectra of QSꢀ4 (20 µM) when it was incubated
with cell lysate extracted from HeLa cells that were induced for ferroptosis. QSꢀ4 incubated with
normal HeLa cells’ lysate was used as control. (c) Kinetic profile of fluorescence intensity at 550
nm when incubating QSꢀ4 (20 µM) with cell lysate. (d) Fluorescent spectra of QSꢀ4 (5 µg/mL)
when it was incubated with H2O2 (50 µM) only in PBS buffer for 60 min or without any
treatment.
Based on above results, the ferroptosis identification was further tested in living cells. After
incubating the molecular probe (20 µM QSꢀ4) with ferroptosis induced HeLa cells for 60 min,
flow cytometry was explored to detect the fluorescence emission of QSꢀ4 inside the cells.
Notably, a significant fluorescence enhancement was observed in cells within one hour,
indicating that QSꢀ4 nanodots could been rapidly uptaken by cells (Figure S12). At the
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meantime, HeLa cells without erastin treatment were used as negative control. In nonꢀferroptotic
condition, QSꢀ4 nanodots were found to emit obvious red fluorescence and weak green
fluorescence with a F550/F595 value of 0.65 for the control group after 1 h incubation (Figure
4a, b). However, when erastin (1 µM) was used to induce the ferroptosis first, the green
fluorescence increased dramatically and the red fluorescence was almost completely vanished,
with an F550/F595 value of 3.76. This indicated that QSꢀ4 molecular probe was indeed
converted to sulfoxide derivative when ferroptosis occurred. To further validate this assertion,
ferroptosisꢀinduced HeLa cells were harvested and lysated, and the corresponding sulfoxide
derivate of QSꢀ4 (QSOꢀ4) in the cell lysate was identified by high performance liquidꢀ
chromatographyꢀtandem mass spectrometry (HPLCꢀMS) analysis. As shown in Figure S13, the
major peak appeared at a retention time of 5.1 min exhibited a mass to charge ratio (m/z) of
419.07, which was consistent with the molecular weight of QSOꢀ4. However, when H2O2
scavenger glutathione (GSH) was added into the culture medium, ferroptosis inducer erastin (1
µM) could not initiate the change of fluorescence, which demonstrated that ROS was
indispensable for converting QSꢀ4 into its sulfoxide derivatives (Figure 4a, b). In addition,
traditional chemotherapeutics, for example, PTX and CDDP, could not trigger the change of QSꢀ
4 fluorescent emission, demonstrating the specificity of QSꢀ4 as a ferroptosis probe (Figure S14).
Moreover, when HeLa cells were pretreated with the anti HOꢀ1 siRNA to downꢀregulate the
expression of HOꢀ1, the fluorescent probe emitted strong red fluorescence instead of green
fluorescence even in the erastinꢀinduced ferroptotic cells (Figure S15), again demonstrating that
the hemeoxygenase was a key catalyst for the oxidation reaction. After that flow cytometry
analysis showed similar results, in which HeLa cells without any treatment emitted obvious red
fluorescence after being incubated with QSꢀ4 (20 µM). Comparatively, when ferroptosis was
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induced by erastin (1 µM), QSꢀ4 emitted bright green fluorescence and the cells could be sorted
to green channel (Figure S16). In all, both confocal imaging and flow cytometry analysis
demonstrated that QSꢀ4 could be used as a fluorescent probe with high specificity for ferroptosis
identification. Moreover, QSꢀ4 can also be used to monitor the level of ferroptosis. When
ferroptosis were induced by erastin with varied concentrations, the cellular fluorescence intensity
changed correspondingly. Notably, a linear profile of the fluorescence intensity versus erastin
concentrations could be observed (Figure S17), showing that mild level of ferroptosis induced by
erastin as low as 500 nM can be detected by our fluorescent probe.
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Figure 4. Confocal imaging of HeLa cells treated with fluorescent nanodots. The green channel
signal was collected in the range of 510ꢀ550 nm with laser excitation at 488 nm. The red channel
signal was collected in the range of 595ꢀ650 nm with laser excitation at 488 nm. (a) Top: HeLa
cells were cultured in normal medium without any treatment. Middle: HeLa cells were incubated
with erastin (1 µM) for 24 h, then cells were treated with QSꢀ4 nanodots (20 µM) for 1 h.
Bottom: HeLa cells were incubated with GSH (1 mM) and erastin (1 µM) for 24 h, then cells
were treated with QSꢀ4 nanodots (20 µM) for 1 h. The scale bars are 10 µm. (b) Fluorescence
intensity ratios of different fluorescent channels. Data are presented as average ±standard error (n
= 3), and the statistical significance level is **p<0.01, ***p<0.001.
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In vivo cellular ferroptosis identification. Having performed ferroptosis imaging in living
cells, we also applied the QSꢀ4 nanodots to probe the ferroptosis in the tumorꢀbearing nude mice
for in vivo imaging. After the injection of erastin (10 mg/kg) every two days for one week, then
QSꢀ4 (10 mg/kg) was injected for in vivo optical imaging. The results showed that the mice with
cellular ferroptosis exhibited an enhanced green fluorescence and weak red fluorescence (Figure
5a). In contrast, mice without erastin treatment emitted strong red fluorescence and weak green
fluorescence (Figure 5a). After that, the tumors were collected, sectioned and then imaged by
confocal microscope. As shown in Figure 5b, tumor slices with cellular ferroptosis emitted bright
green fluorescence. As a comparison, tumor slices without any treatment emitted red
fluorescence and faint green fluorescence, which is in accordance with the wholeꢀbody optical
imaging (Figure 5b, Figure S18). As such, in vivo fluorescent imaging analysis indicated that
QSꢀ4 was also suitable for monitoring cellular ferroptosis in living animals.
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Figure 5. Optical imaging of tumorꢀxenografted nude mice. (a) Wholeꢀbody imaging of female
nude mice bearing HeLa cells after injection of QSꢀ4 (10 mg/kg). The signals were collected
from the green channel (510ꢀ550 nm, left panel) and red channel (595ꢀ650 nm, right panel)
simultaneously with excitation at 470 nm. (b) Confocal imaging of tumor sections. The blue
channel indicated nucleus staining by DAPI. The green channel signal was collected in the 510ꢀ
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550 nm with laser excitation at 488 nm. The red channel signal was collected in the 595ꢀ650 nm
with laser excitation at 488 nm. The scale bar is 25 µm.
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CONCLUSIONS
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In summary, four novel quinoxalinoneꢀbased derivatives were designed and synthesized with
preferable AIEEꢀactive properties and equipped with a reactive aromatic thioether moiety.
Among these compounds, QSꢀ4 that performs the best AIEEꢀactive fluorescence with the highest
quantum yield was employed as a fluorescent molecular probe for ferroptosis identification.
When ferroptosis occurs, the simultaneous upregulation of ROS and hemeoxygenase will
catalyze a specific chemical reaction and convert the QSꢀ4 into its oxide formation rapidly,
which induces a hypochromatic shift of the emission maximum and consequently results in color
change of the fluorescence. Both in vitro and in vivo fluorescent imaging show that the red
fluorescence of QSꢀ4 can be converted into green fluorescence sharply when ferroptosis occurs.
As a result, QSꢀ4 can serve as a potential AIEEꢀactive probe for ferroptosis tracking in real time.
Compare to the most of molecular fluorescent probes that report the biomarkers via fluorescence
“onꢀoff” changes at a certain wavelength, these colorꢀconvertible probes could overcome the
drawbacks existing in traditional onꢀoff probes, such as the lacking of internal reference,
fluctuation of instrumental parameters, photo bleaching, and the variations of microꢀenvironment
around the probe.26ꢀ32 In future, molecular structures of the fluorescent probes can be further
engineered to improve their optical properties, such as promoting the quantum yield, tuning the
fluorescence emission to longer wavelength, etc., making them more sensitively to monitor
important biological process in real time.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website.
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Detailed synthetic route and characterization of QS1, QS2, QS3 and QS4. The relative
expression level of HOꢀ1 in HeLa cells induced by erastion. Cellular concentration of ROS in
HeLa cell induced by erastin. Figures S1ꢀS24. Table S1.
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AUTHOR INFORMATION
Corresponding Authors
Eꢀmail: chuanzhang@sjtu.edu.cn; shenjianbio@hotmail.com
NOTES
The authors declare no competing interest exists.
ACKNOWLEDGMENTS
This work was financially supported by the National Basic Research Program of China
(2015CB931801), the National Natural Science Foundation of China (21504053, 21661162001,
21673139, 91527304, 51690151, 51473093), the Innovation Fund from Joint Research Center
for Precision Medicine set up by Shanghai Jiao Tong University & Affiliated Sixth People's
Hospital South Campus (IFPM 2016B001), and the special program for collaborative innovation
in Shanghai University of Medicine & Health Sciences (SPCIꢀ17ꢀ15ꢀ001).
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ACS Paragon Plus Environment