Ratiometric fluorescent probe with AIE property for monitoring endogenous hydrogen peroxide in macrophages and cancer cells

Hydrogen peroxide (H2O2) plays a key role in the progression of human illnesses, such as autoimmune and auto-inflammatory diseases, infectious diseases, diabetes, and cancer, etc. In this work, we have discribed a novel probe, TPE-TLE, which remarkably displayed AIE property and ratiometric fluorescence emission profiles in the presence of H2O2. This ratiometric fluorescent probe with AIE property exhibits outstanding features such as the well-resolved emission peaks, high sensitivity, high selectivity, low cytotoxicity, and good cell-membrane permeability. These excellent attributes enable us to demonstrate the ratiometric imaging of endogenously produced H2O2 in macrophages and cancer cells based on the novel ratiometric probe with AIE property for the first time. By comparing two kinds of cells, it is firstly found that cancer cells should contain much more endogenous H2O2 than macrophages. We expect that TPE-TLE will be useful fluorescent platform for the development of a variety of ratiometric fluorescent probes with AIE property to achieve unique biological applications.

In this work, we described a novel ratiometric H 2 O 2 fluorescent probe with AIE property for the first time. This ratiometric fluorescent probe with AIE property exhibits outstanding features such as the well-resolved emission peaks, high sensitivity, high selectivity, low cytotoxicity, and good cell-membrane permeability. These features were promising this novel AIE material can be succesfully applied for ratiometric imaging endogenous H 2 O 2 in living RAW 264.7 macrophages and cancer cells HepG2. Compared to RAW 264.7 macrophages, we firstly found that cancer cells HepG2 should contain much more endogenous H 2 O 2 .

Results and Discussion
Preparation of probe. As we all know, tetraphenylethene (TPE) is an archetypal AIE luminogen 27 . On the basis of our interest on AIE material 28 , we further exploited the unique application of this class of dyes by rational sturctural modifications. As shown in Fig. 1, we introduced a thiazole group on the TPE-core to afford the novel AIE material TPE-TLE-O. On the other hand, modification of a H 2 O 2 site such as a borate moiety on the TPE-TLE-O gave the new compound TPE-TLE, which exhibited AIE character distinguishing from TPE-TLE-O. We envision that both materials exhibit distinct aggregation fluorescence signals.
For the AIE material TPE-TLE, in the presence of H 2 O 2 , the oxidation reaction of H 2 O 2 to the borate moiety will provide TPE-TLE-O, which will decrease aggregation fluorescence signal of TPE-TLE to induce aggregation fluorescence signal of TPE-TLE-O. Therefore, we envisioned that AIE material TPE-TLE may be suitable for ratiometric imaging endogenous H 2 O 2 in living systems.
Chemical synthesis of the compound TPE-TLE was accomplished in a total of two steps (Fig. 2). The compound TPE-TLE-O was prepared by the ring-closing reaction between compound 1 and 2-aminothiphenol. TPE-TLE was synthesized by a substitution reaction. The 1 H NMR, 13 C NMR, and H RMS were given in the Supporting Information.
Optical properties in various solvents. Optical properties of probes were basic properties of compounds.
These properties provided clues for speculating its optical applications. Thus, with TPE-TLE in hand, we first  evaluated its absorption and fluorescence profile in aqueous solutions and organic solutions. As shown in Fig. 3 and Table S2, our studies revealed that TPE-TLE had absorption and emission peak at 305 nm and 475 nm in aqueous solutions; In organic solutions, the absorption and emission peak of probe emerged at 305 nm and 420 nm. The results indicated that this probe had larger Stokes shift in aqueous solutions than organic solvents. Moreover, the probe exhibited higher fluorescence quantum yield (Φ = 0.61) in pure water system than organic solvents (Φ = 0.02 in EtOH). As anticipated, the aggregation of the compound TPE-TLE may affect optical physical properties of compound in pure water system. Introduction of a thiazole group on the TPE-core will obtain a novel AIE material TPE-TLE.

AIE property.
To verify the compound TPE-TLE whether was a fluorescent material with AIE property, we investigated optical properties of TPE-TLE in the distinct polar environments. We set out to investigate the fluorescence behavior of TPE-TLE in water/DMF (f w ) mixtures. Firstly, we prepared eleven organic solutions of TPE-TLE with different water fraction (f w ); Photographs of TPE-TLE in water/DMF mixtures were taken under UV illumination (Fig. 4a). The above result demonstrated that the blue fluorescence signal of TPE-TLE gradually became strong when f w increased from 0% (Φ = 0.11) to 100% (Φ = 0.61). Secondly, we systematically changed f w value, and further measured the emission spectra of TPE-TLE in water/DMF mixtures (Fig. 4b).
Fluorescence trend present gradually strong with increasing of f w values (Fig. 4c). Fluorescence characteristics of TPE-TLE were consistent with photographs of TPE-TLE in the distinct polar environments. Thus, we predicted that the compound TPE-TLE should be a fluorescent material with AIE property. The optical studies of TPE-TLE revealed that rational structural modifications on the TPE-core could exhibit AIE character.
In preparation of probe, we have envisioned that the oxidation reaction of H 2 O 2 to the borate moiety will provide TPE-TLE-O in the presence of H 2 O 2 , which will decrease aggregation fluorescence signal of TPE-TLE to induce aggregation fluorescence signal of TPE-TLE-O. The TPE-TLE-O should be an AIE material distinguishing from TPE-TLE. To prove the above assumption, we investigated optical properties of TPE-TLE-O in the distinct polar environments. As shown in Fig. 5, photographs and fluorescence characteristics of TPE-TLE-O in water/ DMF mixtures indicated that the compound TPE-TLE-O was an AIE material distinguishing from TPE-TLE.
The AIE material TPE-TLE should be suitable for ratiometric detecting H 2 O 2 based on an aggregation emission method.

Ratiometric responses of probe TPE-TLE to H 2 O 2 .
To examine whether probe TPE-TLE could ratiometric detect H 2 O 2 , TPE-TLE (5 μM) was treated with H 2 O 2 in PBS buffer solution (containing 50% DMF), and further studying the progress of the reaction by fluorescence titration experiment. As shown in Fig. 6a, in the absence of H 2 O 2 , the probe TPE-TLE exhibited no visible variations in the ratios of emission intensities at 435 nm, suggesting that TPE-TLE was stable in the assay conditions. When increasing concentrations of H 2 O 2 were introduced, the fluorescence spectra of probe TPE-TLE exhibited significant changes. The intensity of the emission maximum at 435 nm was gradually decreased with the simultaneous appearance of a new red-shifted emission band centered at 550 nm. The red-shift in the excitation spectra of the TPE with the increase H 2 O 2 is due to intramolecular charge-transfer (ICT) 29,30 . Moreover, the ratios of emission intensities at 550 and 435 nm (I 550 /I 435 ) displayed a large increase from 0 to 4.25 (Fig. 6a, inset). Furthermore, a new fluorescence emission peak was observed at 550 nm. This result suggests that a new compound was formed during the titration process. The reaction product TPE-TLE-O was confirmed using 1 H NMR (Fig. S1). The AIE material TPE-TLE was capable of detecting H 2 O 2 by an aggregation emission method.
Sensitivity was an important criterion for developing highly sensitive fluorescence imaging agents 31 . Mainly reason was that the pathophysiologic ally relevant concentrations of H 2 O 2 were in the low to medium micro molar concentrations 32 . We further investigate sensitive of probe in order to prove whether TPE-TLE can detect H 2 O 2 in intercellular environment. As shown in Fig. 6b, the detection limit of the probe was 6.0 μM, indicating that the probe TPE-TLE exhibited highly sensitive to H 2 O 2 by an aggregation emission method. We expect that the probe TPE-TLE was able to detect low micro molar concentrations of H 2 O 2 and possessed potentially applicate in medical and biological.
To demonstrate the selectivity of TPE-TLE to H 2 O 2 , TPE-TLE was treated with a wide variety of cations, anions, and oxidants, and then fluorescence spectra were further measured. As shown in Fig. 6c and d, the probe By contrast, other species induced a very low ratiometric response with I 550 /I 435 . This clearly indicated that the probe TPE-TLE had an excellent selectivity to H 2 O 2 over the other analytes. The AIE material TPE-TLE was capable of highly selective detecting H 2 O 2 by an aggregation emission method. In addition, the time-dependent fluorescence intensity changes from the probe TPE-TLE to H 2 O 2 were further studied. As shown in Fig. S2, the probe TPE-TLE to H 2 O 2 revealed that the reaction can be completed about 3 h.
We conducted another experiment condition to show that the probe can detect H 2 O 2 in a ratiometric fashion in vitro. To examine the ratiometric fluorescence response of probe TPE-TLE to H 2 O 2 , the probe was titrated with different equiv. of H 2 O 2 in 90% PBS buffer solution (containing 10% DMF). As shown in Fig. 4a and inset, the free probe displayed an emission peak with maximum at around 450 nm. By contrast, upon addition of H 2 O 2 , the intensity of the emission peak at 450 nm gradually decreased with the simultaneous appearance of a new blue-shifted emission band centered at 560 nm. This result was similar with response of probe TPE-TLE to H 2 O 2 in 50% PBS buffer solution (containing 50% DMF). Moreover, as shown in Fig. S4b, the probe TPE-TLE exhibited higher sensitivity (Detection limit: 1 μM) in PBS buffer solution (containing 10% DMF) than 50% PBS buffer solution (containing 50% DMF). Furthermore, selectivity experiment indicated that the probe has high selectivity for H 2 O 2 in 10% PBS buffer solution (containing 10% DMF) ( Fig. S4c and d). The above results demonstrated that probe TPE-TLE could ratiometric detect H 2 O 2 in 10% PBS buffer solution (containing 10% DMF).
We investigated influence of the probe TPE-TLE to H 2 O 2 under different pH conditions. The results revealed that the pH value of solution had a notable influence on the probe response to H 2 O 2 . As shown in Fig. S4, in the presence of H 2 O 2 , the fluorescence intensity at 550 nm of probe response to H 2 O 2 had no changes with the value increase of pH in the range of 5.0-8.0. In the absence of H 2 O 2 , the free probe TPE-TLE exhibited weak fluorescence intensity in the wide pH range. The above results demonstrated that the TPE-TLE can respond well to H 2 O 2 without dramatic influenced by pH. Living cell imaging studies in RAW264.7 macrophages. In order to be useful as ratiometric imaging agents, ratiometric fluorescent probe should have low cytotoxicity. Thus, we investigated the potential toxicities of TPE-TLE against a representative cell: RAW264.7 macrophages. The living cells were incubated with various concentrations of the new ratiometric probe for 24 h, and then the cell viability was determined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays; The results indicated that the ratiometric probe do not exhibit marked cytotoxicity (Fig. S5).
As we know, H 2 O 2 widely exists in RAW 264.7 macrophages and plays key role in apoptosis and oxidative stress 33 . Thus, to prove the capability of the probe TPE-TLE for H 2 O 2 ratiometric fluorescence imaging in living cells, TPE-TLE (8 µM) was incubated with living RAW 264.7 macrophages for 0.5 h at 37 °C, and then confocal imaging was carried out. As shown in Fig. 7, cells emitted a strong blue fluorescence signal and weak red fluorescence in the blue (Fig. 7b) and red channels (Fig. 7c), respectively. Because endogenous H 2 O 2 will be produced by stimulating RAW264.7 macrophage cells using 12-myristate-13-acetate (PMA). Then TPE-TLE (8 µM) was incubated with living RAW 264.7 macrophages treated with PMA for 0.5 h at 37 °C, and further achieved cells imaging. Imaging results demonstrated that cells displayed a slight blue emission (Fig. 7e) and a strong red emission (Fig. 7f), consistent with the H 2 O 2 -induced ratiometric fluorescent response in vitro. Moreover, we further obtained ratiometric fluorescence imaging picture of both channels in living RAW 264.7 macrophages (Fig. 7g). The above results indicated that the probe TPE-TLE was capable of ratiometric imaging endogenous H 2 O 2 in living RAW 264.7 macrophages by an aggregation emission method. The ratios of emission intensities profiles further prove that the TPE-TLE emitted stronger fluorescent intensity in living RAW 264.7 macrophages treated with PMA than RAW 264.7 macrophages untreated with PMA (Fig. 7h). This result was agreement with H 2 O 2 -induced ratiometric fluorescent response in vitro.
Living cell imaging studies in cancer cells HepG2. It has been known for many years that H 2 O 2 plays a major role in the progression of human illnesses, perhaps most importantly, cancer 34 . To demonstrate the probe TPE-TLE ratiometric imaging exogenous H 2 O 2 , the living cancer cells HepG2 and HepG2 treated with PMA were prepared. Imaging results indicated that cells untreated with PMA gave a strong blue fluorescence signal and a weak red signal in blue and red channels, respectively (Fig. 8a-c). However, images of cells treated with PMA results demonstrated that cells displayed a strong blue emission (Fig. 8e) and a strong red emission (Fig. 8f). Similarly with RAW 264.7 macrophages, ratiometric fluorescence imaging picture (Fig. 8g) and fluorescence emission profiles (Fig. 8h) were obtained. Thus, the above results establish that TPE-TLE is cell-membrane permeable ratiometric probe with AIE property, and suitable for the ratiometric imaging enogenous H 2 O 2 in the living HepG2 cells by an aggregation emission method. Compared to RAW 264.7 macrophages, both channels exhibited stronger fluorescence in living HepG2 cells than RAW 264.7 macrophages in the absence and presence of PMA. Moreover, the TPE-TLE probe emitted stronger red fluorescence in HepG2 cells treated with PMA than RAW 264.7 macrophages. Furthermore, the TPE-TLE exhibited higher ratio of emission intensities in HepG2 cells treated with PMA than RAW 264.7 macrophages. The above results demonstrated that cancer cells should contain more H 2 O 2 than macrophages.
To the best of our knowledge, no reports to date have been published on the construction of ratiometric H 2 O 2 fluorescent probes with AIE property for ratiometric fluorescent imaging endogenously H 2 O 2 in RAW 264.7 macrophage and HepG2 cells.

Conclusions
In summary, we introduced a thiazole group and a borate moiety on the TPE-core to afford a novel ratiometric H 2 O 2 probe TPE-TLE with AIE property for the first time. For sensing mechanism of TPE-TLE, in the presence of H 2 O 2 , the oxidation reaction of H 2 O 2 to the borate moiety will provide TPE-TLE-O, which will decrease aggregation fluorescence signal of TPE-TLE to induce aggregation fluorescence signal of TPE-TLE-O. Furthermore, the TPE-TLE exhibits excellent properties including the well-resolved emission peaks, high sensitivity, high selectivity, low cytotoxicity, and good cell-membrane permeability. The above mechanism and attributes enable us to demonstrate, for the first time, the ratiometric imaging of endogenously produced H 2 O 2 in macrophages and cancer cells. Moreover, by comparing to fluorescence and ratio of emission intensities of both cells, it is firstly found that cancer cells should contain much more endogenous H 2 O 2 than macrophages. We expect that TPE-TLE will be useful fluorescent platform for the development of a variety of ratiometric fluorescent probes with AIE property, and further achieve unique biological applications.

Methods
General procedure for the spectral measurement. The stock solution of the probe TPE-TLE was prepared at 1 mM in DMSO. The PBS (pH = 7.4) solutions with 50% DMSO was prepared. The solutions of various testing species were prepared from NaCl, KCl, CaCl 2 , glutathione (GSH), H 2 O 2 , homocysteine (Hcy), cysteine (Cys), NaNO 2 , NaOH, t-butylhydroperoxide, NaClO, in the twice-distilled water. The test solution of the probe TPE-TLE (5 μM) were 50% PBS buffer solution (containing 50% DMF) and 90% PBS buffer solution (containing 10% DMF). The resulting solution was shaken before measuring the spectra. For titration and selectivity experiments of probe, both experiments were carried out using excitation wavelength of 405 nm, and the excitation and emission slit widths of the spectral measurement were 5 and 5 nm, respectively. In this formula, s and r stand for the sample and the reference, respectively. Ф stands for quantum yield; F and A stands for the integrated emission intensity and the absorbance, respectively; n is refractive index 35 . Cytotoxicity assay. Living RAW264.7 cells were cultivated in per well of 96-well plate. After 24 h, the culture medium was changed using new culture medium containing different concentration (0 μM, 1 μM, 5 μM, 10 μM, 20 μM) TPE-TLE. After 8 h, the culture medium and the excess probes were removed, and then 10 μL MTT (5 mg/mL in PBS) was added to the above medium. Subsequently, the culture medium was removed, and 100 μL DMSO was added to the 96-well plate in order to dissolve the formazan crystal product. The plate containing Living RAW264.7 cells was shaken for 10 min, and then the plate containing RAW264.7 cells was measured at 490 nm using the microplate reader. Firstly, control experiment was further carried out. The culture medium of the cells was added to a fresh medium containing 50.0 μg/mL 12-myristate-13-acetate (PMA) and incubated for 3 h. Secondly, the medium was removed and washed three times with PBS in order to remove the excess PMA. After that, 1 mL of the medium containing 8 μM TPE-TLE was added and further incubated at 37 °C for 30 min. Finally, the confocal imaging was carried out in the blue (λ ex = 405 nm, λ em = 425-475 nm) and green channels (λ ex = 405 nm, λ em = 570-620 nm) using Nikon AMP1 confocal microcopy.
Synthesis of 1. The compound was synthesized by refer literature 38 .

Synthesis of TPE-TLE-O.
In a round-bottomed flask (25 mL) equipped with a magnetic stirrer, a solution of the compound 1 (0.37, 1.0 mmol) and 2-aminothiophenol (0.15 g, 1.2 mmol) in methanol (10 mL) was prepared. Then, 30% H 2 O 2 (6.0 mmol) and 37% HCl (3.0 mmol) were added and the mixture was stirred at room temperature for 2 h. The mixture was quenched by adding H 2 O (10 mL), extracted with EtOAc (3 5 mL), and the combined extracts were dried (Na 2 SO 4 ). The corresponding benzothiazoles were obtained after removal of solvents and purified by silica gel chromatography (eluent: n-hexane/EtOAc = 4:1). The yield of TPE-TLE-O was 65%. 1   Synthesis of TPE-TLE. Compound 2 (0.24 g, 0.5 mmol) was added into a flask containing a mixture of 4-bromomethylphenyl boronic acid (0.15 g, 0.5 mmol), K 2 CO 3 (0.07 g, 0.5 mmol), and 10 mL of DMF with nitrogen at room temperature for 6 h, then poured into H 2 O (500 mL) and extracted with EtOAc The organic phase was separated, dried with MgSO 4 , and removed by vacuum distillation. The product was obtained as a yellow solid with a yield of 60% after purified by column chromatography with ethyl acetate/petroleum ether (2:1, v/v) as eluent. Melting point: 142-146 °C. 1