An aie fluorescent probe for monitoring ferroptosis and a preparation method and application thereof

Abstract

The application provides an AIE fluorescent probe for monitoring ferroptosis and a preparation method and application thereof, and belongs to the technical field of functional fluorescent molecule synthesis for biomedicine. The application provides a fluorescent probe with a novel structure and an aggregation-induced emission effect and a preparation method thereof, and also provides application of the AIE fluorescent probe in in-situ and visualized lipid droplet monitoring of cell ferroptosis. The fluorescent probe provided by the application can in-situ image changes of lipid droplets in cells and can monitor cell ferroptosis in real time, and has a good application prospect in the fields of photoelectricity, sensing and biomedicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional fluorescent molecule synthesis for biomedical use, and particularly relates to an AIE fluorescent probe for monitoring ferroptosis and a preparation method and application thereof. BACKGROUND

[0002] Ferroptosis is a new type of cell death mode caused by excessive accumulation of lipid peroxides in an iron ion-dependent manner, which is different from apoptosis, necrosis and autophagy in morphology, biochemistry and genetics. In recent years, it has been found that ferroptosis is closely related to the pathophysiological processes of cardiovascular diseases, neurodegenerative diseases, tumors, organ damage and other diseases. Professor Wang Fuhe et al. first revealed in 2019 that ferroptosis is the main cause of myocardial injury caused by ischemia-reperfusion, and ferroptosis inhibitors can effectively alleviate myocardial ischemia-reperfusion injury. Drug-resistant cancer cells, especially mesenchymal stem cells and cancer cells prone to metastasis, are prone to ferroptosis. Therefore, inducing or inhibiting ferroptosis has great potential in the treatment of tumor drug resistance and cardiovascular diseases. However, real-time monitoring of ferroptosis to determine the sensitivity of cells to ferroptosis is a major challenge in the development of ferroptosis targeted treatment methods. The lack of ferroptosis sensitivity biomarkers further highlights this problem.

[0003] When ferroptosis occurs, ferrous ions and hydrogen peroxide produce a large number of hydroxyl radicals or lipid radicals through Fenton reaction. Then, the unsaturated lipids form lipid peroxides by reacting with the radicals, which directly or indirectly destroy the structure and function of the cell membrane and cause cell death. Unsaturated lipids are mainly stored in lipid droplets, and lipid droplets can regulate ferroptosis by regulating the transport of polyunsaturated lipids. Studies have confirmed that the level of lipid droplets in cells is negatively correlated with ferroptosis. Accumulation of lipid droplets can inhibit the level of reactive oxygen species and inhibit the oxidation of polyunsaturated fatty acids, ultimately inhibiting ferroptosis. Decomposition of lipid droplets can increase the content of free polyunsaturated fatty acids and promote ferroptosis. Based on the above analysis, we believe that visualizing the dynamic changes of lipid droplets during ferroptosis is not only important for monitoring the progress of ferroptosis, but also crucial for in-depth understanding of the biological effects of lipid droplets during ferroptosis. Therefore, it is urgent to develop a method for real-time, in situ visualization of monitoring the dynamic changes of lipid droplets during ferroptosis. SUMMARY

[0004] The purpose of the present application is to provide an AIE fluorescent probe for monitoring ferroptosis and a preparation method and application thereof, which can monitor cell ferroptosis in real time by in situ imaging changes of lipid droplets in cells.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The application provides a fluorescent probe with an aggregation-induced emission effect, and a structural formula of the fluorescent probe is shown in the following.

[0007]

[0008] R1 is one of

[0009] .

[0010] The application further provides a preparation method of the fluorescent probe, and the preparation method comprises the following steps.

[0011] (1) pyridine-4-yl boronic acid, 4,7-dibromo benzo [c] [1,2,5] thiazole and a catalyst are dissolved in an organic solvent, a substitution reaction is carried out under an alkaline environment and inert gas protection, after the reaction is completed, an organic phase is extracted, and after the organic phase is dried, concentrated and purified, 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole is obtained.

[0012] (2) 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole prepared in step (1) and compound a and a catalyst are dissolved in an organic solvent, a substitution reaction is carried out under an alkaline environment and inert gas protection, after the reaction is completed, the reaction product is concentrated and purified, and the fluorescent probe is obtained.

[0013] The structural formula of the compound a is

[0014]

[0015] R1 is one of .

[0016] Preferably, the molar ratio of the pyridine-4-yl boronic acid to the 4,7-dibromo benzo [c] [1,2,5] thiazole is 1.0:1.0-1.3.

[0017] Preferably, in step (1), the temperature of the substitution reaction is 75-85 DEG C, and the time is 20-28 h.

[0018] Preferably, the molar ratio of the 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole to the compound a is 1.0:1.0-1.3.

[0019] Preferably, in step (2), the temperature of the substitution reaction is 75-85 DEG C, and the time is 10-14 h.

[0020] The application further provides application of the fluorescent probe or the fluorescent probe obtained by the preparation method in monitoring ferroptosis.

[0021] Preferably, the fluorescent probe monitors ferroptosis by in situ imaging lipid droplets.

[0022] The AIE fluorescent probe for monitoring ferroptosis provided by the application has the following beneficial effects:

[0023] The application provides a fluorescent probe with an aggregation-induced emission effect, which can monitor cell ferroptosis by in situ imaging lipid droplets. During ferroptosis, the content of lipid droplets in cells gradually increases within 0-9 h, reaches a peak at 9 h, and then starts to decrease. The increase of lipid droplets in the early stage of ferroptosis may be due to the fact that cells store polyunsaturated fatty acids to resist ferroptosis by synthesizing a large amount of lipid droplets. In the late stage of ferroptosis, the decomposition of lipid droplets exceeds the synthesis, finally leading to a decrease in the content of lipid droplets. Therefore, the change of lipid droplets in cells can be monitored in real time by in situ imaging.

[0024] The AIE probe for monitoring ferroptosis prepared by the application has a large Stokes shift, which can avoid the cross interference of excitation light and emission light; the probe has red light emission characteristics, which can effectively reduce the interference of cell autofluorescence background signals; the probe has strong lipophilicity, and the ClogP value is located in 7.16-8.65, and has high cell membrane permeability; in addition, the probe has weak fluorescence in polar solvents and strong fluorescence emission in hydrophobic lipid droplet environment, so that the imaging has a high signal-to-background ratio.

[0025] The application also provides a preparation method of a fluorescent probe with a novel structure and an aggregation-induced emission effect (AIE), and provides an application of the probe in in situ and visualized lipid droplet monitoring of cell ferroptosis. The fluorescent probe with an excited state intramolecular proton transfer and an aggregation-induced emission effect provided by the application has a good application prospect in the fields of optoelectronics, sensing, and biomedicine. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The fluorescence emission spectrum of the probe TPABTBP (10 μM) in different organic solvents in Example 4;

[0027] Figure 2 The fluorescence emission spectrum (A) of the compound TPABTBP (10 μM) in a mixed system of tetrahydrofuran with different water contents and water in Example 5, and the correlation between the fluorescence intensity of the compound TPABTBP at 625 nm and different water contents (B);

[0028] Figure 3 The cell imaging diagram of Hela cells after being respectively incubated with different concentrations of the probe TPABTBP and the commercial lipid droplet probe BODIPY493 / 503 in Example 6, and the colocalization analysis of the imaging of the two; the nucleus is stained with DAPI, and the scale is 10 μm;

[0029] Figure 4The images show foam macrophages in Example 7 after co-incubation with different concentrations of the probe TPABTBP and the commercial lipid droplet probe BODIPY493 / 503, as well as the co-localization analysis of the two images; the cell nuclei were stained with DAPI, and the scale bar is 10 μm.

[0030] Figure 5 The images shown are confocal images of H9C2 cells in Example 8 after incubation with 10 μM probes TPIBTBP and BTDPP, respectively; the commercial lipid droplet probe BODIPY493 / 503 was used as a control; colocalization analysis was performed on the fluorescent signals of the probes and the commercial probes; cell nuclei were stained with DAPI. Scale bar is 10 μm.

[0031] Figure 6 The images are laser confocal images of HeLa, RAW264.7 and H9C2 cells induced by Erastin, the ferroptosis inducer in Example 9, after 24 hours of incubation with the probe TPABTBP for 30 minutes; cell nuclei were stained with DAPI, and the scale bar is 10 μm.

[0032] Figure 7 This is a laser confocal imaging image of H9C2 cells induced by Erastin, the ferroptosis inducer, for 24 hours and incubated with the probe TPIBTBP for 30 minutes. The cell nuclei were stained with DAPI. The scale bar is 10 μm.

[0033] Figure 8 This is a laser confocal imaging image of HeLa cells after incubation with probe BTDPP for 30 min 24 hours after induction with the ferroptosis inducer Erastin in Example 11. The cell nuclei are stained with DAPI. The scale bar is 10 μm.

[0034] Figure 9 The images show laser confocal images of H9C2 cells induced with Erastin at different time points and incubated with the probe TPABTBP for 30 min in Example 12; cell nuclei were stained with DAPI, and the scale bar is 10 μm.

[0035] Figure 10 The fluorescence intensity of lipid droplets in H9C2 cells induced with Erastin at different time points in Example 12 was quantified after incubation with the probe TPABTBP for 30 min. Detailed Implementation

[0036] This invention provides a fluorescent probe with aggregation-induced emission effect that can monitor cell ferroptosis through in situ imaging of lipid droplets, the structural formula of which is:

[0037]

[0038] Wherein, R1 is selected from

[0039]

[0040] one of the three structures.

[0041] The application further provides a preparation method of the fluorescent probe, comprising the following steps:

[0042] (1) pyridine-4-yl boronic acid, 4,7-dibromo benzo [c] [1,2,5] thiazole and a catalyst are dissolved in an organic solvent, and a substitution reaction is carried out under an alkaline environment and inert gas protection; after the reaction is completed, an organic phase is extracted; and after the organic phase is dried, concentrated and purified, 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole is obtained.

[0043] (2) 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole prepared in step (1) and compound a, a catalyst are dissolved in an organic solvent, and a substitution reaction is carried out under an alkaline environment and inert gas protection; after the reaction is completed, the reaction product is concentrated and purified to obtain the fluorescent probe.

[0044] The structural formula of the compound a is

[0045]

[0046] Among them, R1 is

[0047] one of the three structures.

[0048] In the application, pyridine-4-yl boronic acid, 4,7-dibromo benzo [c] [1,2,5] thiazole and a catalyst are dissolved in an organic solvent, and a substitution reaction is carried out under an alkaline environment and inert gas protection; after the reaction is completed, an organic phase is extracted; and after the organic phase is dried, concentrated and purified, 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole is obtained.

[0049] In the application, the molar ratio of the pyridine-4-yl boronic acid to the 4,7-dibromo benzo [c] [1,2,5] thiazole is 1.0:1.0-1.3, preferably 1.0:1.1.

[0050] In the application, the catalyst is preferably Pd(PPh3)4.

[0051] In the application, the mass ratio of the catalyst Pd(PPh3)4 to pyridine-4-yl boronic acid is preferably 1:8-12, and is further preferably 1:10.

[0052] In the application, the organic solvent is preferably N,N-dimethylformamide aqueous solution (DMF).

[0053] In the present application, the volume ratio of N,N-dimethylformamide to water in the aqueous N,N-dimethylformamide solution is preferably 10:1-3, and more preferably 10:2.

[0054] In the present application, the mass-to-volume ratio of pyridine-4-ylboronic acid to the aqueous N,N-dimethylformamide solution is preferably 200 mg: 10-14 mL, and more preferably 200 mg: 12 mL.

[0055] In the present application, the basic environment is preferably created by adding potassium carbonate.

[0056] In the present application, the mass ratio of potassium carbonate to the pyridine-4-ylboronic acid is preferably 220-270:200, and more preferably 250:200.

[0057] In the present application, the inert gas is preferably nitrogen or argon, and more preferably nitrogen.

[0058] In the present application, the temperature of the substitution reaction is 75-85℃, and preferably 80℃; and the time is 20-28 h, and preferably 24 h.

[0059] In the present application, the mixture after the above reaction is preferably cooled to room temperature, and then the organic phase is extracted.

[0060] In the present application, the extractant for the extracted organic phase is preferably ethyl acetate, and extraction is preferably performed 3 times.

[0061] In the present application, the organic phases after the three extractions are combined, dried with anhydrous sodium sulfate, and the filtrate is concentrated under reduced pressure to remove the organic solvent, to obtain a crude product.

[0062] In the present application, the purification is preferably performed by silica gel column chromatography using petroleum ether / ethyl acetate (25:1, v / v) as the eluent.

[0063] After purification, the intermediate product 4-bromo-7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazole (PBTBBr) is obtained.

[0064] In the present application, 4-bromo-7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazole prepared in step (1) is dissolved in an organic solvent with compound a and a catalyst, and a substitution reaction is performed under a basic environment and protection of an inert gas. After the reaction is completed, the reaction product is concentrated and purified to obtain the fluorescent probe.

[0065] The structural formula of the compound a is

[0066]

[0067] wherein R1 is

[0068] one of the following.

[0069] In the present application, the molar ratio of the 4-bromo-7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazole to the compound a is 1.0:1.0-1.3, preferably 1.0:1.1.

[0070] In the present application, the catalyst is preferably Pd(PPh3)4.

[0071] In the present application, the mass ratio of the catalyst Pd(PPh3)4 to the 4-bromo-7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazole is preferably 1:8-12, further preferably 1:10.

[0072] In the present application, the organic solvent is preferably N,N-dimethylformamide.

[0073] In the present application, the mass-volume ratio of the 4-bromo-7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazole to the N,N-dimethylformamide is preferably 100 mg:4-6 mL, further preferably 100 mg:5 mL.

[0074] In the present application, the basic environment is preferably created by adding potassium carbonate.

[0075] In the present application, the mass ratio of the potassium carbonate to the 4-bromo-7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazole is preferably 40-60:100, further preferably 50:100.

[0076] In the present application, the inert gas is preferably nitrogen or argon, further preferably nitrogen.

[0077] In the present application, the temperature of the substitution reaction is 75-85℃, preferably 80℃; the time is 10-14 h, preferably 12 h.

[0078] In the present application, the purification is preferably silica gel column chromatography purification with petroleum ether / ethyl acetate (25:1, v / v) as the eluent.

[0079] After purification, the fluorescent probe is obtained.

[0080] The present application also provides a use of the fluorescent probe or the fluorescent probe obtained by the preparation method in monitoring ferroptosis.

[0081] In the present application, the fluorescent probe monitors ferroptosis by in situ imaging of lipid droplets.

[0082] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present application.

[0083] Example 1

[0084] The probe with an aggregation-induced emission effect for in-situ imaging of lipid droplets in this example is 4-methoxy-N-(4-methoxyphenyl)-N-(4-(7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazol-4-yl)phenyl)aniline (TPABTBP).

[0085] In this example The synthetic route is as follows:

[0086]

[0087] 1. Synthesis of compound PBTBBr:

[0088] Into a 100 mL round-bottom flask equipped with a condenser was added pyridin-4-ylboronic acid (200 mg, 1.62 mmol), 4,7-dibromobenzo[c][1,2,5]thiadiazole (472.8 mg, 1.62 mmol), potassium carbonate 250 mg and Pd(PPh3)4(20 mg) to obtain a mixed system; 10 mL DMF and 2 mL water were added to the mixed system to obtain a mixture; the mixture was stirred (400 rpm) and heated to reflux at 80°C for 24 h; after cooling to room temperature (25°C), the reaction mixture was extracted with ethyl acetate three times; the organic phase was collected and dried with anhydrous sodium sulfate to remove the water therein; the filtrate was concentrated under reduced pressure (vacuum degree 0.1 Mpa) to remove the organic solvent therein to obtain a crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (25:1, v / v) as the eluent to obtain solid PBTBBr (226.2 mg, yield 48%). 1 H NMR (600 MHz, CDCl3), δ (ppm): 8.78-8.79 (m, 2H), 7.98-7.99 (d, 1H), 7.86-7.87 (d, 2H), 7.69-7.70 (d, 1H); 13 C NMR (150 MHz, CDCl3), δ (ppm): 153.96, 152.48, 150.22, 143.94, 132.12, 130.89, 128.92, 123.48, 115.42.

[0089] 2. Synthesis of compound TPABTBP:

[0090] Compound PBTBBr (55.1 mg, 0.19 mmol) and compound (4-(bis(4- methoxyphenyl)amino)phenyl)boronic acid (66.3 mg, 0.19 mmol) were dissolved in 5 mL DMF, and stirred at room temperature (25 °C) at 600 rpm; then 1 mL aqueous solution containing 50 mg K2CO3 was added to the reaction system, and 10 mg of catalyst Pd(PPh3)4 was added; the reaction system was refluxed at 80 °C under nitrogen protection for 12 hours, and TLC plate was used to monitor the completion of the reaction. After the reaction was completed, the solvent was removed by distillation under reduced pressure (vacuum degree was 0.2 rpm), and silica gel column chromatography (petroleum ether / ethyl acetate (25:1, v / v) as eluent) was used to separate the product TPABTBP. The yield was 32.8%. 1 H NMR (600 MHz, CDC13), δ (ppm): 8.92-8.93 (d, 2H), 8.58-8.57 (d, 2H), 8.07-8.09 (d, 1H), 7.88-7.90 (d, 2H), 7.83-7.85 (d, 1H), 7.16-7.17 (m, 4H), 7.05-7.06 (d, 2H), 6.88-6.90 (d, 4H), 3.83 (s, 6H); 13 C NMR (150 MHz, CDC13), δ (ppm): 156.63, 154.11, 153.19, 152.36, 150.13, 142.52, 139.92, 137.88, 130.87, 130.26, 127.47, 127.02, 125.83, 125.29, 124.72, 118.92, 114.91, 55.53. HRMS (ESI+): calcd for C 31 H 25 N4O2S + [M+H] + : 517.1693; found: 517.1689.

[0091] Example 2

[0092] The probe with aggregation-induced emission effect for in-situ imaging of lipid droplets in this example is 4-(4-(4,5-bis(4-methoxyphenyl)-1-phenyl-1H-imidazol-2-yl)phenyl)-7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazole (TPIBTBP).

[0093] In this example The synthetic route is as follows:

[0094]

[0095] 1. Synthesis of compound PBTBBr, same as example 1.

[0096] 2. Synthesis of compound TPIBTBP:

[0097] Compound PBTBBr (150 mg, 0.52 mmol) and compound (4-(4,5-bis(4- methoxyphenyl)-1-phenyl-1H-imidazol-2-yl)phenyl)boronic acid (245.5 mg, 0.52 mmol) were weighed and dissolved in 5 mL DMF, stirred well at room temperature (25 °C) (600 rpm); then 1 mL aqueous solution containing 80 mg K2CO3 was added to the reaction system, and 20 mg of catalyst Pd(PPh3)4 was added. The reaction system was refluxed at 80 °C under nitrogen protection for 12 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure (vacuum degree 0.2 Mpa), and the product TPIBTBP was separated by silica gel column chromatography. The yield was 32.8%. 1 HNMR (600 MHz, CDC13), δ (ppm): 8.94 (s, 2H), 8.50 (s, 2H), 7.94-8.09 (m, 4H), 7.73-7.74 (m, 2H), 7.39-7.54 (m, 5H), 7.20-7.21 (m, 2H), 7.04-7.08 (m, 2H), 6.88-6.89 (d, 2H), 6.81-6.82 (d, 2H), 3.80-3.82 (m, 6H); 13 C NMR (150 MHz, CDC13), δ (ppm): 159.24, 158.50, 153.91, 153.54, 152.08, 150.09, 145.76, 144.9, 142.69, 138.16, 137.29, 136.43, 135.88, 135.22, 134.42, 132.39, 130.34, 130.15, 129.26, 129.02, 128.93, 128.85, 128.79, 128.56, 128.54, 128.41, 127.78, 124.78, 124.02, 123.59, 123.48, 122.73, 115.92, 113.88, 55.22, 55.15. HRMS (ESI+): calcd for C 40 H 29 N5O2S: 643.2042; found: 643.2053.

[0098] Example 3

[0099] The probe with aggregation-induced emission effect for in-situ imaging of lipid droplets in this embodiment is 4-methoxy-N-(4-methoxyphenyl)-N-(4-(7-(pyridin-4-yl)benzo[c][1,2,5]thiadiazol-4-yl)phenyl)aniline (BTDPP).

[0100] In this example The synthesis of compound BTDPP is as follows:

[0101]

[0102] 1. Synthesis of compound PBTBBr, same as Example 1.

[0103] 2. Synthesis of compound BTDPP:

[0104] Compound PBTBBr (100 mg, 0.22 mmol) and compound (4-(diphenylamino)phenyl)boronic acid (63.6 mg, 0.22 mmol) were weighed and dissolved in 5 mL of DMF, and stirred well at room temperature (25°C) (700 rpm); then 1 mL of an aqueous solution containing 55 mg of K2CO3 was added to the reaction system, and 8 mg of catalyst Pd(PPh3)4 was added. The reaction system was refluxed at 80°C under nitrogen protection for 12 hours, and after the reaction was completed as monitored by TLC plate, the solvent was removed by distillation under reduced pressure (vacuum degree 0.3 Mpa), and the product BTDPP was separated by silica gel column chromatography. The yield was 32.8%. 1 H NMR (600 MHz, CDCl3), δ (ppm): 8.77-8.78 (d, 2H), 7.95-7.96 (d, 2H), 7.88-7.90 (m, 3H), 7.79-7.80 (d, 1H), 7.29-7.32 (m, 4H), 7.07-7.10 (m, 2H); 13 C NMR (150 MHz, CDCl3), δ (ppm): 154.07, 153.63, 150.12, 148.51, 147.33, 144.80, 134.74, 132.33, 130.91, 130.16, 130.06, 129.42, 129.26, 128.97, 128.85, 126.88, 125.10, 123.55, 122.55.

[0105] Example 4 Fluorescence emission spectrum of probe TPABTBP in different organic solvents

[0106] The probe TPABTBP was dissolved in toluene, tetrahydrofuran, ethyl acetate, chloroform, dichloromethane, DMF, DMSO, acetonitrile and methanol, respectively, to prepare a system with a concentration of 10 μM. The fluorescence emission spectrum was measured by a fluorescence spectrophotometer with an excitation wavelength of 480 nm.

[0107] As shown in Figure 1 , the probe has weak fluorescence emission in polar solvents such as tetrahydrofuran, ethyl acetate, chloroform, dichloromethane, DMF, DMSO, acetonitrile and methanol, and strong fluorescence emission in weakly polar solvents such as toluene (its polarity is reported to be similar to that of lipid droplets). The above results confirm that the probe has weak fluorescence in polar solvents and strong fluorescence emission in hydrophobic lipid droplet environments, and is suitable for lipid droplet imaging and is expected to have a high signal-to-background ratio. As shown in Figure 1 , the probe TPABTBP has an emission wavelength of 625 nm and an excitation wavelength of 480 nm, with a Stokes shift of 145 nm, indicating that the probe TPABTBP has a large Stokes shift, which can avoid the cross interference of excitation light and emission light, making the detection result more accurate.

[0108] Example 5 Study on the aggregation-induced emission effect of probe TPABTBP

[0109] TPABTBP was dissolved in THF solution to prepare a stock solution with a concentration of 1 mM. 10 μL of the stock solution was accurately taken and placed in a mixture of pure water and THF (with different water contents), with a total volume of 1 mL and a concentration of 10 μM. The water content percentages were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%, respectively.

[0110] As shown in Figure 2 , TPABTBP has very weak fluorescence in pure THF solution. When the water content is 10%-70%, the probe still has weak fluorescence. When the water content increases to 80%, the fluorescence intensity at 610 nm increases by 23 times compared with that in THF solution, indicating that the molecules have aggregated at this time. After aggregation, the intramolecular rotation pathway is restricted, so strong fluorescence emission can be produced under light excitation. When the water content further increases to 95%, the fluorescence decreases. This phenomenon indicates that TPABTBP forms irregular large-size aggregates and precipitates from the aqueous solution, resulting in a decrease in fluorescence intensity. The above results show that TPABTBP has a significant AIE effect.

[0111] Example 6 Targeted imaging of endogenous lipid droplets in Hela cells by probe TPABTBP

[0112] The probe TPABTBP was dissolved in DMSO solution to prepare a 1 mM stock solution, which was then diluted to obtain 5 μM, 10 μM and 30 μM probe TPABTBP solutions.

[0113] Live HeLa cells were incubated for 30 min for 30 min with 5 μM, 10 μM, and 30 μM probes TPABTBP in serum-free conditions (high-glucose Gibco DMEM medium containing 1% penicillin and antibiotics). After discarding the original medium, commercial probes BODIPY493 / 503 (Thermo Fisher Invitorgen, catalog number D3922) were added at concentrations of 5 μM, 10 μM, and 30 μM in serum-free working solution (high-glucose Gibco DMEM medium containing 1% penicillin and antibiotics) and incubated for 30 min. After washing three times with PBS, the cells were fixed with 4% neutral paraformaldehyde for 10 min, washed three times with PBS, stained with DAPI at room temperature for 10 min, washed three times with PBS, and then imaged using a confocal laser scanning microscope.

[0114] like Figure 3 The red light channel is shown to exhibit a significant dotted distribution of red fluorescence, and it shows excellent co-localization with the green fluorescence channel of the commercial lipid droplet probe BODIPY493 / 503, with Pearson constants of 0.94, 0.90, and 0.94, respectively. Furthermore, compared to commercial probes, TPABTBP exhibits stronger specificity for lipid droplet imaging, while BODIPY493 / 503 can non-specifically stain other components in the cytoplasm. The probe's red light emission properties effectively reduce interference from the cell's autofluorescence background signal.

[0115] Example 7: TPABTBP probe-targeted imaging of endogenous lipid droplets in foam macrophages

[0116] The probe TPABTBP was dissolved in DMSO solution to prepare a 1 mM stock solution, which was then diluted to obtain 5 μM, 10 μM and 30 μM probe TPABTBP solutions.

[0117] To further confirm the universality of probe TPABTBP for imaging lipid droplets in cells, RAW264.7 cells were induced by 2 mg / mL oxidized low-density lipoprotein for 24 h to form foam macrophages, and then the foam macrophages were incubated with 5 μM, 10 μM and 30 μM of probe TPABTBP for 30 min under serum-free conditions (high-glucose Gibco DMEM medium containing 1% double antibodies). After the original culture medium was discarded, BODIPY493 / 503 (5 μM, 10 μM and 30 μM) serum-free working solution (high-glucose Gibco DMEM medium containing 1% double antibodies) was added and incubated for 30 min, and then washed with PBS for 3 times, fixed with 4% neutral paraformaldehyde for 10 min, washed with PBS for 3 times, then stained with DAPI at room temperature for 10 min, washed with PBS for 3 times, and then imaged by a confocal laser scanning microscope.

[0118] As shown in FIG. 8, the red fluorescence of the probe was well co-localized with the green fluorescence of BODIPY, and the co-localization coefficients were 0.981, 0.906 and 0.9014, respectively. The above results showed that the probe TPABTBP could specifically image lipid droplets. Figure 4

[0119] Example 8 Targeted imaging of endogenous lipid droplets in cells by probes TPIBTBP and BTDPP

[0120] The probes TPIBTBP and BTDPP were dissolved in DMSO to prepare a stock solution with a concentration of 1 mM, and then the probes TPABTBP and BTDPP were diluted to 10 μM, respectively.

[0121] Live H9C2 cells were incubated with 10 μM of probes TPIBTBP and BTDPP for 30 min under serum-free conditions (high-glucose Gibco DMEM medium containing 1% double antibodies). After the original culture medium was discarded, commercial probe BODIPY493 / 503 (Thermo Fisher Invitorgen, product number D3922) was added at a concentration of 10 μM and incubated for 30 min, and then washed with PBS for 3 times, fixed with 4% neutral paraformaldehyde for 10 min, washed with PBS for 3 times, then stained with DAPI at room temperature for 10 min, washed with PBS for 3 times, and then imaged by a confocal laser scanning microscope.

[0122] As shown in FIG. 9, the red fluorescence of probes TPIBTBP and BTDPP was well co-localized with the green fluorescence of BODIPY, and the co-localization coefficients were 0.964 and 0.861, respectively. The above results showed that the probes TPIBTBP and BTDPP had good ability to specifically image lipid droplets. Figure 5

[0123] ​​Example 9: TPABTBP Targeted Imaging Lipid Droplet Monitoring of Ferrocytes

[0124] 0.5 × 10⁶ seeds were seeded in confocal microplates. 4 HeLa cells, Raw264.7 cells, and H9C2 cells were induced for 24 h in a medium containing the ferroptosis inducer Erastin (5 μM) (high-glucose Gibco DMEM complete medium containing 10% Gibco FBS (fetal bovine serum) and 1% penicillin antibiotics), after which the medium was discarded. A serum-free 10 μM probe working solution (high-glucose Gibco DMEM medium containing 1% penicillin antibiotics) was prepared and incubated with the probe for 30 min. Cells were then fixed with 4% neutral paraformaldehyde for 10 min, washed three times with PBS, stained with DAPI at room temperature for 10 min, washed three times with PBS, and observed using a confocal laser scanning microscope. The same procedure was performed, with a control group lacking the ferroptosis inducer.

[0125] like Figure 6 As shown, before Erastin induction, HeLa cells, Raw264.7 cells, and H9C2 cells exhibited significant lipid droplet distribution. After 24 hours of Erastin induction, the fluorescence intensity of lipid droplets in the cells was significantly enhanced, the number of lipid droplets increased significantly, and their size was significantly larger. These results indicate that the abnormal increase in the number of lipid droplets can indicate ferroptosis, and the probe TPABTBP can indicate ferroptosis by imaging lipid droplets.

[0126] Example 10: Probe-guided TPIBTBP targeted imaging of lipid droplets to monitor ferroptosis

[0127] Inoculate 0.5 × 10⁻⁶ seeds in confocal microplates. 4 H9C2 cells were induced for 24 h with a medium containing the ferroptosis inducer Erastin (5 μM) (high-glucose Gibco DMEM complete medium containing 10% Gibco FBS (fetal bovine serum) and 1% penicillin-free antibiotics), and then the medium was discarded. A serum-free 10 μM probe working solution (high-glucose Gibco DMEM medium containing 1% penicillin-free antibiotics) was prepared and incubated with the cells for 30 min. The cells were then fixed with 4% neutral paraformaldehyde for 10 min, washed three times with PBS, stained with DAPI at room temperature for 10 min, washed three times with PBS, and observed using a confocal laser scanning microscope. The same procedure was performed, with a control group lacking the ferroptosis inducer.

[0128] like Figure 7 As shown, a small number of lipid droplets were observed in H9C2 cells before Erastin induction. After 24 h of Erastin induction, the fluorescence intensity of lipid droplets in the cells was significantly enhanced, the number of lipid droplets increased significantly, and their size was significantly larger. The results indicate that the probe TPIBTBP can indicate ferroptosis by imaging lipid droplets.

[0129] Example 11 Probe BTDPP Targeting Lipid Droplet Imaging to Monitor Ferroptosis

[0130] In a confocal dish, 0.5 x 10 4 Hela cells were induced with ferroptosis inducer Erastin (5 mM) for 24 h in culture medium (high glucose Gibco DMEM complete medium containing 10% Gibco FBS (fetal bovine) serum, 1% double-antibiotic), and then the culture medium was discarded; 10 mM probe working solution (high glucose Gibco DMEM medium containing 1% double-antibiotic) without serum was prepared and incubated with the cells for 30 min, followed by 4% neutral paraformaldehyde fixation for 10 min, PBS washing three times, DAPI staining at room temperature for 10 min, PBS washing three times, and observation with a confocal laser scanning microscope. The same operation was performed as above, and a control group without ferroptosis inducer was used.

[0131] As shown in FIG. 11, a small amount of lipid droplets was observed in Hela cells before induction with Erastin. The fluorescence intensity of lipid droplets in the cells was significantly enhanced, and the number of lipid droplets was significantly increased after induction with Erastin for 24 h. The results showed that the abnormal increase in the number of lipid droplets can indicate ferroptosis, and probe BTDPP can indicate ferroptosis by imaging lipid droplets. Figure 8

[0132] Example 12 Probe TPABTBP to Monitor Dynamic Changes of Lipid Droplets in Ferroptosis Process

[0133] H9C2 was seeded at a density of 1 x 10 4 After the cells adhered, 5 mM Erastin medium (high glucose Gibco DMEM complete medium containing 10% Gibco FBS (fetal bovine) serum, 1% double-antibiotic) was added for incubation, and the induction was terminated at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h, respectively, followed by the addition of 10 mM serum-free probe working solution (high glucose Gibco DMEM medium containing 1% double-antibiotic) for incubation for 30 min, 4% neutral paraformaldehyde fixation for 10 min, PBS washing three times, and finally DAPI staining at room temperature for 10 min, PBS washing, and image acquisition with a confocal laser scanning microscope.

[0134] To indicate the degree of ferroptosis induced by Erastin at different time points, BODIPY TM 581 / 591 C11 probe (Thermo Fisher Invitrogen, product number D3861) was added to detect the degree of lipid peroxidation in the cells. ​

[0135] As shown in Figure 9 and Figure 10 At 0h, weak fluorescence was observed in the cells, indicating the presence of lipid droplets. After the cells were induced with 5μM Erastin for 3h, the fluorescence intensity of the cells increased, indicating an increase in the number of lipid droplets. The fluorescence intensity further increased when the induction time was extended to 6h and 9h. However, when the time was extended to 12h and 24h, the fluorescence intensity in the cells decreased to some extent compared to that at 9h.

[0136] The results of the lipid peroxidation probe imaging showed that the degree of lipid peroxidation in the cells significantly increased as the induction time was extended from 3h to 24h. The above results confirmed that the content of lipid droplets in the cells gradually increased within 0-9h during ferroptosis, and the content of lipid droplets reached a peak at 9h and then began to decrease. The increase in lipid droplets in the early stage of ferroptosis may be due to the fact that the cells store polyunsaturated fatty acids by synthesizing a large number of lipid droplets to resist ferroptosis. In the late stage of ferroptosis, the decomposition of lipid droplets exceeds the synthesis, eventually leading to a decrease in the content of lipid droplets. Therefore, the change in the lipid droplets in the cells in situ imaging can monitor the ferroptosis of the cells in real time.

[0137] From the above examples, the present application provides a preparation method of a fluorescent probe with a novel structure having an aggregation-induced emission effect (AIE), and simultaneously provides the application of the probe in monitoring the ferroptosis of cells in situ and visualizing lipid droplets.

[0138] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A fluorescent probe having an aggregation-induced emission effect, characterized in that, The structural formula of the fluorescent probe is as follows: R1 is 2. A method for preparing the fluorescent probe according to claim 1, characterized by, The method comprises the following steps: (1) pyridine-4-yl boronic acid, 4,7-dibromo benzo [c] [1,2,5] thiazole and a catalyst are dissolved in an organic solvent, and a substitution reaction is carried out under alkaline environment and inert gas protection, after the reaction is completed, the organic phase is extracted, and after drying, concentration and purification, 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole is obtained; (2) 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole prepared in step (1) and compound a, a catalyst are dissolved in an organic solvent, and a substitution reaction is carried out under alkaline environment and inert gas protection, after the reaction is completed, the reaction product is concentrated and purified to obtain the fluorescent probe; The structural formula of the compound a is R1 is 3. The preparation method according to claim 2, characterized in that, The molar ratio of the pyridine-4-yl boronic acid to the 4,7-dibromo benzo [c] [1,2,5] thiazole is 1.0:1.0-1.

3.

4. The production method according to claim 3, characterized by, In step (1), the temperature of the substitution reaction is 75-85 DEG C, and the time is 20-28 h.

5. The preparation method according to claim 4, characterized in that, The molar ratio of the 4-bromo-7-(pyridine-4-yl) benzo [c] [1,2,5] thiazole to the compound a is 1.0:1.0-1.

3.

6. The method of any one of claims 2 to 5, wherein the method further comprises, In step (2), the temperature of the substitution reaction is 75-85 DEG C, and the time is 10-14 h.

7. The use of the fluorescent probe of claim 1 or the fluorescent probe obtained by the preparation method of any one of claims 2-6 in the preparation of a drug for monitoring ferroptosis.

8. Use according to claim 7, characterized in that, The fluorescent probe monitors ferroptosis by in situ imaging lipid droplets. The fluorescent probe monitors ferroptosis by in situ imaging lipid droplets.

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