Non-reactive near-infrared mitochondrial ridge tracing super-resolution fluorescent probe and application thereof

Abstract

The invention belongs to the field of mitochondrial super-resolution fluorescent probes, and relates to a non-reactive near-infrared mitochondrial ridge tracing super-resolution fluorescent probe and application thereof. The probe comprises a nitrobenzofuran-containing compound or pharmaceutically acceptable salts, hydrates, solvates, optical isomers, racemes and the like of the nitrobenzofuran-containing compound, and the chemical structural formula of the nitrobenzofuran-containing compound is as shown in a formula (I) # imgabs0 #. Particularly, when the mitochondrial membrane potential is reduced or disappears, the probe still can be fixed on the mitochondria, which indicates that the probe can track the dynamic change of the mitochondria; the probe can also realize visualization of the mitochondrial ridge change in the physiological and pathological process. Compared with other probes in the prior art, the probe provided by the invention has the characteristics of high brightness, strong light stability, low cytotoxicity and the like, can be a useful tool for further research on the mitochondrial ridge morphology under physiological and pathological conditions, and can promote basic research in related fields.

Description

Technical Field

[0001] The invention belongs to the field of mitochondrial super-resolution fluorescent probes, and relates to a non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Mitochondrial cristae maintain a dynamic structure to adapt to various physiological and pathological processes (such as mitophagy and ferroptosis), so visualizing and tracking different changes in cristae is of great significance for physiological and pathological studies. Cristae are densely packed ultrastructures within mitochondria, and the distance between adjacent cristae is usually less than 100nm. Therefore, for many years, the methods and techniques for visualizing mitochondrial cristae have been limited to transmission electron microscopy (TEM). Although researchers have gained a deep understanding of mitochondrial cristae using transmission electron microscopy, the inability to observe living samples and obtain dynamic information limits its wide application. In the past few decades, fluorescent probe-assisted fluorescence imaging technology has been widely used in labeling and visualizing mitochondrial cristae due to its unique advantages of being applicable to living samples, near-nondestructive detection, real-time and in situ visualization.

[0004] In recent years, super-resolution imaging technology has attracted increasing attention due to its nanoscale ultra-high resolution, which is suitable for observing the ultrastructure of living cells. Currently, there are three main super-resolution imaging techniques: single-molecule localization microscopy (SMLM), structured illumination microscopy (SIM), and stimulated emission depletion (STED) microscopy. Among them, SMLM improves the resolution to a few nanometers, but it requires the collection of a large number of images for data analysis and reconstruction, which is computationally intensive and time-consuming, limiting its application in tracking cellular dynamic processes. Although SIM can achieve fast imaging, its spatial resolution is 90-120nm, which is not enough to observe a single mitochondrial cristae. In contrast, STED microscopy can provide a spatial resolution of ~50nm and a temporal resolution of 1s, which makes it a useful tool for capturing mitochondrial cristae. However, strong dissipative lasers can easily lead to photobleaching of the probe, which places higher requirements on fluorescent probes.

[0005] A variety of physiological and pathological processes related to mitochondria are accompanied by a decrease in mitochondrial membrane potential (MMP), so monitoring the changes in mitochondrial cristae during these processes requires MMP-independent probes. Several MMP-independent probes have been developed to observe mitochondrial cristae. Tian et al. reported a unique probe, Py-BODIPY, to reveal how the mitochondrial 3D ultrastructure changes under oxidative phosphorylation conditions. Yamaguchi et al. observed cristae fusion in single mitochondria using the ultra-photostable probe MitoPB Yellow. Xi et al. rationally designed MitoESq-635 to observe the morphology of cristae during mitochondrial fusion and fission. The above MMP-independent probes can be fixed in mitochondria by reacting with the thiol groups of mitochondrial proteins and exhibit high cytotoxicity. Compared with reactive-based probes, non-reactive probes that bind to phospholipids through hydrophobic interactions have theoretically better biocompatibility. According to the inventors' research, there are no reports on the non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe containing NBD and its application in marking and displaying the distribution of mitochondrial cristae in normal living cells and in mitochondria of living cells with reduced mitochondrial membrane potential, or in observing intracellular mitochondrial autophagy and cellular ferroptosis. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe and its application, which can mark or display the morphology and spacing of mitochondrial cristae in physiological and pathological processes under a STED microscope, and can observe mitochondrial autophagy and cell ferroptosis in cells.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, a nitrobenzofuran-containing compound, the chemical structure of which is shown in formula (I);

[0009]

[0010] The compound is named: 1-ethyl-2-((Z)-(3-(Z)-1-ethyl-3,3-dimethylindolin-2-ylidene)methyl)-2-((3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)propyl)thio)-4-oxocyclobut-2-ene-1-ylidene)methyl)-3,3-dimethyl-3H-indole-1-bromide, abbreviated as CSN.

[0011] In a second aspect, a method for preparing the above-mentioned nitrobenzofuran-containing compound comprises the steps of preparing a target compound using 2,3,3-trimethyl-3H-indole and ethyl bromide as reaction raw materials according to the following reaction route;

[0012]

[0013] Specifically, the process is as follows: 2,3,3-trimethyl-3H-indole and ethyl bromide undergo quaternization reaction to generate N-1; then N-1 reacts with squaric acid to generate N-2; then N-2 reacts with Lawesson's reagent to generate N-3; N-3 and N-4 react at room temperature to obtain CSN.

[0014] More specifically, 2,3,3-trimethyl-3H-indole and ethyl bromide react at 80 to 90° C. to generate N-1.

[0015] More specifically, the reaction of N-1 and squaric acid to produce N-2 is carried out at a temperature of 91 to 99°C.

[0016] More specifically, the solvent used for the reaction of N-1 with squaric acid to generate N-2 is an ethanol solution.

[0017] More specifically, N-2 and Lawesson's reagent react at room temperature to generate N-3. The room temperature in the present invention refers to the indoor environment temperature, which can be 15-30°C, generally 25°C.

[0018] More specifically, 4-chloro-7-nitro-1,2,3-benzoxadiazole and bromopropylamine hydrobromide are reacted to produce N-4.

[0019] In a third aspect, a non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe comprises the above-mentioned nitrobenzofuran-containing compound or its pharmaceutically acceptable salt, hydrate, solvate, optical isomer, racemate, etc.

[0020] The fluorescent probe CSN of the present invention is obtained through novel and ingenious design and construction: 1. The cationic dimethyl indole salt can provide positive charge for the molecule; 2. The large conjugated system composed of the cationic indole salt, vinyl and squaric acid ensures that CSN has a large ionic radius and an emission wavelength suitable for STED microscopy, thereby ensuring the good membrane permeability and mitochondrial targeting of the probe CSN; 3. The lipophilic group NBD is introduced to generate hydrophobic interaction with the phospholipid bilayer of the mitochondrial inner membrane, thereby ensuring the MMP independence of the probe CSN.

[0021] The pharmaceutically acceptable salts described in the present invention include, for example, non-toxic salts or quaternary ammonium salts formed from non-toxic inorganic acids or organic acids; wherein the inorganic acids include sulfuric acid, phosphoric acid, nitric acid, etc., and the organic acids include acetic acid, propionic acid, succinic acid, tartaric acid, citric acid, etc.

[0022] In some embodiments, pharmaceutically acceptable excipients are also included. Specifically, the excipients include but are not limited to pH regulators, stabilizers, antioxidants, buffers, preservatives, and the like.

[0023] In a fourth aspect, a detection kit comprises the above-mentioned nitrobenzofuran-containing compound or the non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe, and a solvent.

[0024] In a fifth aspect, a use of the above-mentioned nitrobenzofuran-containing compound or non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe or detection kit in monitoring cell mitochondria or preparing a detection product for monitoring cell mitochondria.

[0025] In some embodiments, monitoring of cell mitochondria is performed using a laser confocal fluorescence microscope or a stimulated emission depletion microscope; or, the detection product is used in conjunction with a laser confocal fluorescence microscope or a stimulated emission depletion microscope for detection.

[0026] In some embodiments, the detection product is used to mark or display mitochondria in normal living cells, living cells with reduced mitochondrial membrane potential, or cells with disappeared mitochondrial membrane potential.

[0027] Specifically, the normal cells refer to animal cells or cancer cells whose mitochondrial membrane potential is at a normal value.

[0028] Among them: animal cells: primary mouse fibroblasts, African green monkey kidney fibroblast-like cell line (COS-7), mouse embryonic fibroblasts (3t3) or human normal mammary epithelial cells (MCF-10A); cancer cells: cervical cancer cells (HeLa), human breast cancer cells (MCF-7) or human non-small cell lung cancer cells (A549).

[0029] Experimental findings show that the NBD-containing non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe of the present invention can highly selectively label mitochondria in normal living cells, living cells with reduced mitochondrial membrane potential, or cells with disappeared mitochondrial membrane potential.

[0030] In some embodiments, the detection product is used to mark or display the distribution of mitochondrial cristae in mitochondria in normal living cells.

[0031] The experimental results confirmed that CSN can image mitochondrial cristae in different cell lines with a resolution of 52nm.

[0032] In some embodiments, the detection product is used to observe changes in mitochondrial cristae during cell ferroptosis and mitochondrial autophagy.

[0033] The experimental results confirmed that mitochondria first experienced swelling and cristae breakage, then partial vacuolation, and finally complete vacuolation during autophagy, while mitochondria experienced a decrease in cristae number, then partial breakage, and finally vacuolation during ferroptosis.

[0034] The beneficial effects of the present invention are:

[0035] The present invention discloses a non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe containing NBD and its biological application. A large number of experiments have shown that the fluorescent probe CSN described in the present invention can stain mitochondria in normal living cells, especially when the mitochondrial membrane potential is reduced or disappeared, it can still be fixed on the mitochondria, indicating that CSN can track the dynamic changes of mitochondria. At the same time, under the STED microscope, the mitochondrial cristae can be clearly observed using CSN, and it is observed that the distance between mitochondrial cristae is different in different cell lines. The non-reactive mitochondrial tracking fluorescent probe described in the present invention has low toxicity and can track the changes of mitochondrial cristae during mitochondrial autophagy and cell ferroptosis in real time. Using CSN, it is successfully observed that during mitochondrial autophagy, mitochondria first experience swelling and cristae breakage, then partial vacuolation, and finally complete vacuolation, while during cell ferroptosis, mitochondria will experience a decrease in the number of cristae, then partial breakage, and finally vacuolation. These results show that CSN may be a useful tool for further studying mitochondrial cristae under physiological and pathological conditions, and can promote basic research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 : Confocal images obtained by staining with CSN (150nM) and Mito Tracker GreenFM (200nM) in A549 cells, HeLa cells, and MCF-7 cells. Scale bar: 10μm. Pearson colocalization coefficients are shown in the right column. Excitation wavelength: 640nm for CSN, 488nm for MTG; acquisition band: 650-700nm for CSN, 505-550nm for MTG. The above results confirm that the staining behavior of the probe CSN is not affected by the cell type.

[0038] Figure 2 : Confocal fluorescence images of normal active HeLa cells, CCCP-treated HeLa cells, and paraformaldehyde-fixed HeLa cells stained with CSN and Mitochondria Tracking Red (MTR). The colocalization coefficients of CSN and MTR are marked in the superimposed images (Merge). Excitation wavelength: 640nm for CSN and 561nm for MTR; acquisition band: 650-700nm for CSN and 575-625nm for MTR. This indicates that CSN can image mitochondria in cells with normal membrane potential, mitochondria in cells with reduced membrane potential, and mitochondria in cells with lost membrane potential.

[0039] Figure 3 : The survival rate of live HeLa cells incubated with different concentrations of CSN and MTDR, indicating that CSN has low cytotoxicity.

[0040] Figure 4 : a STED image of mitochondrial cristae in living HeLa cells labeled with CSN. Scale bar: 2 μm. Magnified image of the middle white area (i) in (a). Magnified image of the yellow area in (a) (right). Scale bar: 1 μm. b Fluorescence intensity analysis of the position marked in (a) and Gaussian fitting calculation of the half-peak width. Confocal (left) and STED (right) images of mitochondria of different cells labeled with CSN: MCF-7 (c); MCF-10A (d); HeLa (e); COS-7 (f); primary mouse fibroblasts (PMF) (g) and 3t3 (h). Scale bar: 2 μm. All STED data have been deconvoluted. The above results confirm that CSN can clearly depict mitochondrial cristae.

[0041] Figure 5 :a Changes in mitochondrial morphology and cristae after treatment with carbonyl cyanide m-chlorobenzene (CCCP) for different time periods. The first row is a confocal image; the second row is a STED image; b is an enlarged image of the region of interest. All STED data have been deconvolved. Scale bar: 2 μm. The results show that during mitophagy, mitochondria first undergo swelling and cristae breakage, then partial vacuolization, and finally complete vacuolization. This indicates that CSN can be used to track changes in mitochondrial cristae during physiological processes.

[0042] Figure 6 :a Changes in mitochondrial morphology and cristae after treatment with Erastin for different time periods. The first row is a confocal image; the second row is a STED image. The third row is a magnified image of the region of interest. b Mitochondrial length and cristae number of control and Erastin-treated cells. All STED data have been deconvolved. Scale bar: 2μm. The results show that during cell ferroptosis, mitochondria experience a decrease in the number of cristae, followed by partial breakage and finally vacuolization. This indicates that CSN can be used to track changes in mitochondrial cristae during pathological processes. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0044] In the following examples, the materials, reagents, etc. used were obtained from commercial sources unless otherwise specified.

[0045] Example 1: Synthesis of probe CSN.

[0046] The synthetic route of the probe CSN is as follows:

[0047]

[0048] The specific process is as follows:

[0049] (1) Synthesis of N-1:

[0050] 2,3,3-trimethyl-3H-indole (3.18 g, 20 mmol) and iodoethane (2.16 g, 20 mmol) were mixed in 50 mL of ethanol and then refluxed under an argon atmosphere for 12 hours, then the heating was stopped and cooled. The precipitate was then filtered through a Buchner funnel, the solid product was washed with ether and dried in vacuo to obtain a pink product.

[0051] (2) Synthesis of N-1:

[0052] Place square acid (1.14 g, 10 mmol) in a 250 mL round-bottom flask, then add anhydrous ethanol (100 mL) to the flask; heat the obtained reaction mixture to reflux under an argon atmosphere until the white square acid is completely dissolved and the solution becomes transparent; then add N-ethyl-2,3,3-trimethylindolium bromide (5.34 g, 20 mmol) to the reaction mixture until all the raw materials are consumed to obtain a dark blue solution. N-2 is purified by silica gel column chromatography with a DCM (dichloromethane) / MeOH (methanol) gradient eluent, and then a blue solid is obtained.

[0053] (3) Synthesis of N-3:

[0054] Lawesson's reagent (2.2 g, 5 mmol) was added to a mixed solvent of DCM (5 mL) and THF (10 mL) containing N-2 (1.3 g, 3 mmol). The solution was stirred at 40 °C for 5 hours. After the solution was cooled to room temperature, the solution was removed under reduced pressure and no further purification was required for the next reaction.

[0055] (4) Synthesis of N-4:

[0056] First, 4-chloro-7-nitro-1,2,3-benzoxadiazole (2 g, 10 mmol) was dissolved in methanol and stirred for 10 minutes, and then 3-bromopropionamide (2.19 g, 10 mmol) was added. Next, the reaction system was stirred at room temperature for 8 hours. The mixture was then extracted from water with dichloromethane. Subsequently, the organic compound was washed twice with water and dried over anhydrous sodium sulfate overnight. Finally, ethyl acetate / petroleum ether (1:10, v / v) was used as the eluent, and the purified product was obtained by column chromatography.

[0057] (5) Synthesis of CSN:

[0058] (Z)-4-((1-ethyl-3,3-dimethyl-3H-indol-1-ium-2-yl)methylene)-2-(((Z)-1-ethyl-3,3-dimethylindolin-2-ylidene)methyl)-3-oxocyclobutene-1-sulfate (N-3, 500 mg, 1 mmol) and MeCN (acetonitrile, 5 mL) were added to N-(3-bromopropyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (N-4, 300 mg, 1 mmol) at 85° C. overnight. CSN was purified by silica gel column chromatography using DCM (dichloromethane) / MeOH (methanol) gradient eluent, and a green solid was obtained.

[0059] 1 H NMR (400MHz, CDCl3) δ7.39-7.30(m,6H),7.25(d,J=7.4Hz,2H),7.14(d,J=7.9Hz,2H),5.70(s,2H),4.27(d,J=7.1Hz,4H),3.98( t,J=7.1Hz,2H),3.78(dd,J=7.2,2.0Hz,1H),2.35(d,J=6.4Hz,2H),1.62(s,12H),1.19(d,J=8.4Hz,6H),0.81(t,J=6.9Hz,2H). 13 C NMR (101MHz, CDCl3) δ174.65,173.02,141.40,139.78,127.61,125.26,121.37,110.27 ,87.44,49.36,39.44,28.84,28.67,24.91,24.83,11.40.HRMS(m / z):[M]+calculated for C 39 H 41 N6O4S + ,689.2905;found,689.2780.

[0060] Example 2: Preparation of living cell samples for testing.

[0061] The MCF-7, COS-7, 3t3 and HeLa cells used were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C, 5% CO2 atmosphere. The primary mouse fibroblasts used were cultured in Dulbecco's modified Eagle's medium, which contained 50% Ham's F12 nutrient medium, 10% FBS and 1% penicillin and streptomycin, and cultured at 37°C, 5% CO2 atmosphere. The A549 cells used were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin and streptomycin at 37°C, 5% CO2 atmosphere. The MCF-10A cells used were cultured in MCF-10A-specific medium. When the cells grow to the logarithmic phase, culture the coverslips: ① Soak the coverslips in anhydrous ethanol for 30 minutes, dry them with an alcohol lamp, and place them in a disposable 35mm culture dish for later use; ② Wash the cells in the 100mL cell bottle with PBS three times, digest them with 1mL 0.25% trypsin for 3-5 minutes, pour out the trypsin carefully, add fresh culture medium, blow and mix evenly, and count the cells. The cell density is controlled by the amount of culture medium added, so that the final cell concentration is 1×10 5 / mL, and then inoculated into the above-mentioned culture dish containing the cover glass, and placed in a 5% CO2 incubator at 37°C to culture the cells to allow them to grow on the cover glass. When the cells have grown all over the cover glass, living cell samples of HeLa cells, MCF-7 cells, COS-7 cells, 3t3 cells, primary mouse fibroblasts, A549 cells and MCF-10A cells growing on the cover glass are obtained and used for the experiments in the subsequent embodiments.

[0062] Example 3: Testing and verifying the staining effect of the probe CSN on MCF-7 cells, HeLa cells and A549 cells.

[0063] First, prepare a 1mM CSN DMSO solution as a master solution. After the cells have grown all over the coverslip, stain the cells with 150nM CSN and 200nM MTG (Mito Tracker Green FM) and incubate in a CO2 incubator for 30 minutes. After staining, wash away the excess probe, cover the cell growth surface down on the slide, and observe the staining results under a laser confocal fluorescence microscope, including cell staining location, brightness, etc.

[0064] The experimental results are shown in Figure 1 The experimental results show that the probe CSN can stain mitochondria in A549 cells, and the same results are observed in HeLa cells and MCF-7 cells, indicating that the staining behavior of CSN is not affected by cell types and can be used as a useful tool for imaging mitochondria.

[0065] Example 4 The mitochondrial membrane potential independence of the probe CSN was verified by co-localization experiments.

[0066] In live cells: active HeLa cells were first incubated with 0.2μM MTR (mitochondrial dye, Mito-Tracker RedCMXRos) for 30min, then incubated with 150nM CSN for 30min; in live cells with reduced mitochondrial membrane potential: active HeLa cells were first incubated with 200nM MTR for 90min, then incubated with 150nM CSN for 30min; then treated with 10μM CCCP (carbonyl cyanide m-chlorophenylhydrazone) for 90min; in cells with lost mitochondrial membrane potential: active HeLa cells were first incubated with 200nM MTR for 90min, then incubated with 150nM CSN for 30min; then fixed with 4% paraformaldehyde for 30min. Before staining another probe each time, cells were washed 3 times with PBS.

[0067] The experimental results are shown in Figure 2 The experimental results showed that the fluorescence signal of CSN overlapped well with that of MTR, and their colocalization coefficients were 0.95 in living cells, 0.86 in CCCP-treated cells, and 0.85 in fixed cells, indicating that CSN can be firmly fixed in mitochondria regardless of whether the mitochondrial membrane potential is reduced or eliminated. This indicates that CSN can image mitochondria in cells with normal mitochondrial membrane potential, mitochondria in cells with reduced mitochondrial membrane potential, and mitochondria in cells with eliminated mitochondrial membrane potential.

[0068] Example 5: Cytotoxicity test experiment.

[0069] The toxicity test of CSN was performed using MTT reagent.

[0070] HeLa cells were seeded in a 96-well plate with an average cell density of 10,000 cells / mL per well and grown for 24 hours. Then, 200 μL of CSN and MTDR (mitochondrial dye, MitoTracker DeepRed) at concentrations of 2, 1, and 0.5 μM were added to the experimental group, and DMEM high-glucose medium (200 μL / well) was added to the control group. Then the 96-well plate was placed in a 37°C constant temperature incubator containing 5% CO2 for 24 hours. Then 10 μL of MTT (thiazolyl blue, 5 mg / mL) was added to the experimental group and the control group, respectively. After 4 hours in the incubator, the liquid in each well was removed and 200 μL of DMSO was added to dissolve the generated purple crystals. After 20 minutes, the absorbance of each well at 490 nm was tested with an ELISA reader.

[0071] The experimental results are shown in Figure 3 The experimental results show that the cytotoxicity of CSN is lower than that of MTDR.

[0072] Example 6: Imaging mitochondrial cristae of different cell lines under STED microscopy using CSN.

[0073] HeLa cells, COS-7 cells, MCF-7 cells, MCF-10A cells, 3t3 cells and mouse primary fibroblasts were stained with 150 nM CSN and incubated in a CO2 incubator for 30 min. After staining, the excess probe was washed away and the staining results were observed under a STED microscope.

[0074] The experimental results are shown in Figure 4 The experimental results showed that the probe CSN clearly depicted mitochondrial cristae in HeLa cells with a resolution of 52nm. At the same time, mitochondrial cristae were also observed in different cell lines using the probe CSN.

[0075] Example 7: Using CSN to image the changes of mitochondrial cristae during mitophagy under STED microscope.

[0076] HeLa cells were stained with 150 nM CSN and incubated in a CO2 incubator for 30 min. After staining, the excess probe was washed away and the cells were treated with 10 μM CCCP for different time periods. The staining results were observed under a STED microscope. CCCP could not be removed during fluorescence imaging.

[0077] The results are as follows Figure 5 As shown in the figure. In the control group, mitochondrial cristae were clearly observed, while in cells treated with CCCP for 40 minutes, mitochondria were swollen and cristae were ruptured. As the treatment time increased, some mitochondria were vacuolated and cristae disappeared. After 150 minutes of treatment, mitochondria were completely vacuolated. The above experimental results show that mitochondrial autophagy is usually accompanied by mitochondrial vacuolation and loss of cristae.

[0078] Example 8: Using CSN to image the changes of mitochondrial cristae during cell ferroptosis under STED microscope.

[0079] HeLa cells were stained with 150 nM CSN and incubated in a CO2 incubator for 30 min. After staining, the excess probe was washed away and the cells were treated with 10 μM erastin for different time periods. The staining results were observed under a STED microscope. Erastin cannot be removed during fluorescence imaging.

[0080] The experimental results are as follows Figure 6As shown. The experimental results showed that after treating cells with erastin, the mitochondrial cristae gradually decreased as a whole during ferroptosis compared with the control group. The number of mitochondrial cristae and the length of mitochondria in the control group and cells treated with erastin were statistically analyzed, and it was found that with the extension of treatment time, the length of mitochondria gradually shortened and the number of mitochondrial cristae decreased. Subsequently, mitochondrial fragmentation was observed after 90 minutes of treatment, and the mitochondria were completely vacuolated after 100 minutes. The above experimental results show that cell ferroptosis will cause a significant decrease in the number of mitochondrial cristae, but mitochondrial cristae remain until mitochondrial vacuolization.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nitrobenzofuran-containing compound, characterized in that: Its chemical structural formula is shown in formula (I); 2. A method for preparing the nitrobenzofuran-containing compound according to claim 1, characterized in that: The method comprises the steps of using 2,3,3-trimethyl-3H-indole and ethyl bromide as reaction raw materials to prepare a target compound according to the following reaction route; 3. The preparation method according to claim 2, characterized in that: The process is as follows: 2,3,3-trimethyl-3H-indole and ethyl bromide undergo quaternization reaction to generate N-1; then N-1 reacts with squaric acid to generate N-2; then N-2 reacts with Lawesson's reagent to generate N-3; N-3 and N-4 react at room temperature to obtain CSN.

4. The preparation method according to claim 3, characterized in that: 2,3,3-Trimethyl-3H-indole and ethyl bromide react at 80-90°C to produce N-1; Alternatively, the reaction of N-1 and squaric acid to produce N-2 is carried out at a temperature of 91 to 99°C; Or, the solvent used in the reaction of N-1 and squaric acid to generate N-2 is an ethanol solution; Alternatively, N-2 and Lawesson's reagent react at room temperature to produce N-3; Alternatively, 4-chloro-7-nitro-1,2,3-benzoxadiazole and bromopropylamine hydrobromide react to produce N-4.

5. A non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe, characterized in that: The invention comprises the nitrobenzofuran-containing compound according to claim 1 or a pharmaceutically acceptable salt, hydrate, solvate, optical isomer or racemate thereof.

6. The non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe according to claim 5, characterized in that: Pharmaceutically acceptable excipients are also included.

7. A detection kit, characterized in that: The method comprises the nitrobenzofuran-containing compound according to claim 1 or the non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe according to claim 5 or 6, and a solvent.

8. Use of the nitrobenzofuran-containing compound of claim 1, the non-reactive near-infrared mitochondrial cristae tracking super-resolution fluorescent probe of claim 5 or 6, or the detection kit of claim 7 in monitoring cell mitochondria or preparing a detection product for monitoring cell mitochondria.

9. The use according to claim 8, characterized in that: Monitoring of cell mitochondria is performed using a laser confocal fluorescence microscope or a stimulated emission depletion microscope; or, the detection product is used in conjunction with a laser confocal fluorescence microscope or a stimulated emission depletion microscope for detection.

10. The use according to claim 8, characterized in that: The detection product is used to mark or display mitochondria in normal living cells, living cells with reduced mitochondrial membrane potential, or cells with disappeared mitochondrial membrane potential; Or, the detection product is used to mark or display the distribution of mitochondrial cristae in mitochondria in normal living cells; Alternatively, the detection product is used to observe changes in mitochondrial cristae during cell ferroptosis and mitochondrial autophagy.

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