A dual-color fluorescent probe for monitoring mitochondrial membrane potential by self-reference and its application

Abstract

The application relates to a mitochondrial membrane potential detection probe technology, in particular to a dual-color fluorescence probe for self-referenced monitoring of mitochondrial membrane potential and application thereof.The probe comprises a compound CBTH or pharmaceutically acceptable salts, hydrates, solvates, optical isomers, racemates and the like of the compound CBTH.The chemical structural formula of the compound CBTH is shown as formula (I).The probe CBTH can be imaged simultaneously in dual colors, the fluorescence intensity does not disappear with the change of the cell state, and the change of the mitochondrial membrane potential can be monitored semi-quantitatively by calculating the colocalization coefficient.The probe has low toxicity, good biocompatibility, reduces the cumbersome operation of co-staining of two probes, and is expected to be widely applied in the research on diseases and physiological processes related to mitochondria.

Description

Technical Field

[0001] This invention relates to mitochondrial membrane potential detection probe technology, specifically to a self-referenced dual-color fluorescent probe for monitoring mitochondrial membrane potential and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Mitochondria are vital sites for providing energy to cells. The mitochondrial membrane potential (MMP) participates in ordered physiological activities such as ATP synthesis, the formation of electrical potential and proton gradients, and is fundamental to cellular metabolism. Abnormalities in the MMP may indicate disruption of the mitochondrial electron transport chain, leading to conditions such as brain necrosis, Parkinson's disease, diabetes, and cancer. Therefore, efficient and convenient monitoring of MMP changes in living cells has practical value in biomedical research and the diagnosis of related diseases.

[0004] Biofluorescent probes offer advantages such as high sensitivity and non-invasiveness, enabling in-situ real-time detection of micronutrients (MMPs) in live cells. However, most fluorescent probes can only qualitatively detect changes in MMPs. Some migratory probes target mitochondria when MMPs are normal and the nucleus when MMPs decrease. While the fluorescence intensity ratio reflects MMP changes, it cannot exclude the influence of photobleaching, viscosity, and staining concentration on fluorescence intensity. Colocalization assays are widely used to validate organelle targeting of fluorescent molecules. Colocalization coefficients allow for quantitative analysis of MMP changes, with minimal impact from photostability and imaging conditions. Existing fluorescent probes that monitor MMPs using colocalization coefficients migrate to lysosomes when MMPs disappear, and colocalization coefficients are calculated using commercially available lysosomal probes. However, the combined use of multiple probes introduces several problems, such as cumbersome staining processes, asynchronous light responses, and targeting competition between probes, affecting imaging quality and efficiency. A single probe that achieves the effect of two probes, using self-reference to calculate the colocalization coefficient, would be more advantageous than using multiple probes.

[0005] Therefore, developing probes with stable self-reference parameters and the ability to quantify MMP fluctuations through physical parameter ratios is of great value. The inventors' research indicates that the alkaline environment of mitochondria (pH approximately 8.0) allows molecules to partially circularize within the mitochondria, resulting in dual emission. Furthermore, the open-circular form exhibits a certain affinity for nuclear DNA, enabling it to target the nucleus when MMP levels decrease. However, probes that quantify MMP detection using the co-localization coefficient between the dual emission channels have not yet been reported. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a self-referenced dual-color fluorescent probe for monitoring MMPs and its applications. The probe CBTH provided by this invention can perform simultaneous dual-color imaging, and its fluorescence intensity does not disappear with changes in cell state. Furthermore, it can semi-quantitatively monitor changes in MMPs by calculating co-localization coefficients. Moreover, this probe exhibits low toxicity and good biocompatibility, reducing the cumbersome operation of co-staining with two probes.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] Firstly, a compound CBTH has the chemical structural formula shown in formula (I):

[0009]

[0010] The compound is named (E)-2-(2-(7-(diethylamino)2-oxo-2H-benzopyran-3-yl)vinyl)3-benzo[d]thiazolyl-3-onium, abbreviated as CBTH.

[0011] On the other hand, a method for preparing the above-mentioned compound CBTH includes the step of obtaining CBTH according to the following reaction route;

[0012] .

[0013] The process is as follows: 4-(diethylamino)salicylaldehyde is reacted with diethyl malonate to obtain compound 1. Compound 1 is reacted in a mixture of phosphorus oxychloride and N,N-dimethylformamide to generate compound 2. 2-methylbenzothiazole is reacted with iodoethanol to obtain compound 3. Then, compound 2 and compound 3 are reacted with Knoevenagel to synthesize the product. Finally, the product is purified by column chromatography, and the resulting purple solid product is CBTH.

[0014] In some embodiments, 4-(diethylamino)salicylaldehyde and diethyl malonate are first reacted under alkaline conditions at 70-90 °C, and then under acidic conditions at 110-130 °C to obtain the product. Specifically, 4-(diethylamino)salicylaldehyde, diethyl malonate, and piperidine are added to ethanol, heated under reflux, then the ethanol is removed, glacial acetic acid and concentrated hydrochloric acid are added, and the mixture is heated to 110-130 °C to proceed with the reaction.

[0015] In some implementations, the molar ratio of 4-(diethylamino)salicylaldehyde to diethyl malonate is 1:2.0-3.0.

[0016] In some embodiments, phosphorus oxychloride and N,N-dimethylformamide are reacted in an inert atmosphere by heating to 45-55°C; then compound 1 is added, and the mixture is heated to 55-65°C to obtain compound 2.

[0017] In some embodiments, the reaction temperature of 2-methylbenzothiazole with iodoethanol is 75-85 °C.

[0018] In some implementations, the molar ratio of 2-methylbenzothiazole to iodoethanol is 1:2.5~3.5.

[0019] In some embodiments, the molar ratio of compound 2 to compound 3 is 1:0.9~1.1.

[0020] In some embodiments, the reaction temperature of compound 2 and compound 3 is 70-80 °C.

[0021] Thirdly, a dual-color fluorescent probe for self-reference monitoring of MMPs includes the above-mentioned compound CBTH or its pharmaceutically acceptable salts, hydrates, solvates, optical isomers, racemates, etc.

[0022] This invention employs the compound CBTH as a self-referenced dual-color fluorescent probe for monitoring MMPs, which consists of two parts: 7-diethylaminocoumarinaldehyde and hydroxyethylbenzothiazole salt. Hydroxyethylbenzothiazole salt is an important family of pH-responsive probes; its positive charge allows for selective staining of mitochondria through electrostatic interactions. Simultaneously, benzothiazole salt probes, such as anthocyanin dyes and thiazole orange, can insert into DNA and exhibit strong binding affinity to DNA production in the cell nucleus. Furthermore, DNA in the cell nucleus exhibits basophilic properties (pH = ~7.2), while mitochondria maintain an alkaline environment under normal conditions (pH = ~8.0). This pH difference between these two organelles allows the self-referenced dual-color fluorescent probe CBTH described in this invention to label mitochondria with two different emission colors and migrate monochromatic to the cell nucleus when MMP levels decrease.

[0023] Specifically, the compound CBTH, as a dual-color fluorescent probe, exists in an open-ring form (structural formula as shown in I) under acidic conditions, and undergoes cyclization to transform into a closed-ring form (structural formula as shown in II) under alkaline conditions, as shown in the following reaction formula:

[0024]

[0025] Taking HeLa cells as an example, the fluorescent probe CBTH enters live cells in an open-ring form and accumulates in the mitochondria through electrostatic interactions. Since the pH of mitochondria in live cells is around 8, some probes undergo cyclization in the weakly alkaline environment of the mitochondria, transforming into a closed-ring form. Under excitation at 405 nm, the closed-ring form II emits green fluorescence, while under excitation at 543 nm, the open-ring form I emits red fluorescence. Therefore, in cells with normal MMP levels, mitochondria exhibit red and green fluorescence, while the nucleus remains dark. In cells with reduced MMP levels, mitochondria exhibit red and green fluorescence, while the nucleus exhibits weak red fluorescence. In cells where MMP is absent, mitochondria exhibit green fluorescence, and the nucleus exhibits red fluorescence. Using the green fluorescence channel of mitochondria as a reference, changes in MMP can be monitored by calculating the colocalization coefficient between the red and green fluorescence channels.

[0026] Experimental results confirm that the self-referenced MMP monitoring dual-color fluorescent probe CBTH described in this invention exists in a circular form in weakly alkaline mitochondria, emitting green fluorescence. At the same time, the open-circular form of the probe can migrate between the mitochondria and the cell nucleus depending on changes in MMP, emitting red fluorescence.

[0027] The selectivity of the aforementioned dual-color fluorescent probe CBTH on cells has been rigorously demonstrated. Based on cell staining, the dual-color fluorescent probe CBTH described in this invention was first tested with a commercially available mitochondrial probe (Mito-Tracker Deep Red FM) in a counterstaining experiment, confirming its high co-localization coefficient. This confirmed that the fluorescence in both red and green channels in cells with normal MMP, as well as the green fluorescence in fixed cells or cells with reduced MMP (CCCP-treated cells), all originated from mitochondria. Furthermore, a counterstaining experiment with a commercially available nuclear probe (Hoechst 33342) confirmed its high co-localization coefficient, further confirming that the red fluorescence in fixed cells or cells with reduced MMP (CCCP-treated cells) originated from the cell nucleus.

[0028] The pharmaceutically acceptable salts described in this invention include, for example, non-toxic salts or quaternary ammonium salts formed from non-toxic inorganic acids or organic acids; wherein, inorganic acids include phosphoric acid, sulfuric acid, nitric acid, etc., and organic acids include acetic acid, oxalic acid, citric acid, fumaric acid, etc.

[0029] In some implementations, pharmaceutically acceptable excipients are also included. Specifically, these excipients include, but are not limited to, pH adjusters, stabilizers, antioxidants, buffers, and preservatives.

[0030] Fourthly, a detection kit comprising the aforementioned compound CBTH or a dual-color fluorescent probe for self-reference monitoring of MMPs, and a solvent.

[0031] In some embodiments, the solvent is dimethyl sulfoxide and / or Britton-Robinson (BR) buffer solution.

[0032] Fifthly, the application of the above-mentioned compound CBTH or a dual-color fluorescent probe or detection kit for self-reference monitoring of MMPs in the preparation of a formulation that simultaneously uses two fluorescent colors to label or image mitochondria in live cells, or a formulation that simultaneously uses two fluorescent colors to label or image mitochondria and nuclei in live cells.

[0033] Specifically, the live cells are HeLa cells; some probes cyclize in the weakly alkaline environment of mitochondria when incubating live cells, and the cyclized molecules emit green fluorescence when excited by light at 405 nm; the open-ring probes target mitochondria via cations and emit red fluorescence when excited by light at 543 nm. As MMP decreases, the red fluorescence gradually shifts from the mitochondria to the cell nucleus.

[0034] Sixthly, the application of the above-mentioned compound CBTH or a two-color fluorescent probe or detection kit for self-reference monitoring of MMPs in the preparation of formulations for monitoring changes in MMPs in living cells.

[0035] Specifically, the live cells are HeLa cells; the dual-color fluorescent probe CBTH targets mitochondria in live cells with normal MMP activity, with both fluorescent channels at excitation light of 405 nm and 543 nm targeting mitochondria; in live cells with MMP damage, the dual-color fluorescent probe CBTH targets mitochondria with the fluorescent channel at excitation light of 405 nm, and targets both mitochondria and the cell nucleus with the fluorescent channel at excitation light of 543 nm; in cells where MMP is almost completely absent, the dual-color fluorescent probe CBTH targets mitochondria with the fluorescent channel at excitation light of 405 nm, and targets the cell nucleus with the fluorescent channel at excitation light of 543 nm.

[0036] In some embodiments, the formulation displays changes in MMPs in living cells via a colocalization coefficient between two fluorescence channels.

[0037] Seventhly, the application of the above-mentioned compound CBTH or a two-color fluorescent probe or detection kit for self-reference monitoring of MMPs in the preparation of formulations for identifying healthy live cells and MMP-damaged cells.

[0038] Specifically, the active cells are HeLa cells; in healthy living cells, the dual-color fluorescent probe targets mitochondria simultaneously with the fluorescence of the red channel (excitation light of 543 nm) and the green channel (excitation light of 405 nm), resulting in a high co-localization coefficient between the two channels; while in cells with decreased MMP, the red channel fluorescence targets the cell nucleus, and the green channel fluorescence targets the mitochondria, resulting in a decreased co-localization coefficient between the two channels.

[0039] Experiments have confirmed that the dual-color fluorescent probe described in this invention can monitor intracellular MMP changes using dual-color fluorescence self-reference. In healthy living cells, this dual-color fluorescent probe can image mitochondria using red and green fluorescence. In cells where CCCP treatment induces a decrease in MMP, the green fluorescence of the dual-color fluorescent probe maintains mitochondrial targeting, while the red fluorescence gradually migrates from the mitochondria to the nucleus, and the Pearson colocalization coefficient between the two channels gradually decreases. In rotenone-induced apoptotic cells, the probe also maintains green fluorescence targeting mitochondria, while the red fluorescence gradually migrates from the mitochondria to the nucleus, and the Pearson colocalization coefficient between the two channels gradually decreases.

[0040] The beneficial effects of this invention are as follows:

[0041] Compared to existing MMP probes, the self-referenced dual-color fluorescent probe CBTH for MMP monitoring in this invention can simultaneously image in both colors, without any fluorescence intensity in any particular channel disappearing with changes in cell state. Therefore, it can calculate co-localization coefficients through self-reference, enabling semi-quantitative monitoring of MMP changes. Furthermore, this probe has low toxicity and good biocompatibility, reducing the cumbersome operation of co-staining the cell nucleus and mitochondria with two probes and minimizing the resulting cytotoxicity. In addition, the spatial distribution and co-localization coefficients of the dual-color fluorescence can distinguish healthy live cells from MMP-damaged cells, and it can also be used to study the distribution and morphological changes of mitochondria and the cell nucleus during apoptosis, making it of significant experimental application value. Attached Figure Description

[0042] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0043] Figure 1 (a) Absorption spectra of probe CBTH in Britton-Robinson (BR) buffer solutions containing 2% DMSO at different pH values. Probe concentration: 10 μmol / L. (bc) Fluorescence emission spectra of probe CBTH at different pH values ​​(b: λ) ex = 440 nm, c: λ ex = 530 nm, solvent is BR buffer containing 2% DMSO). (d) I 625 / I 498Relationship with different pH values. (e) Photostability of the probe under continuous laser irradiation in different pH buffers. (f) Cell viability of HeLa cells after incubation with different concentrations of the probe CBTH for 24 hours. The results showed that after incubation with 5 μM CBTH for 24 hours, the cell viability was still as high as 96%, indicating that the probe has very low toxicity.

[0044] Figure 2 (a) Two-channel confocal microscopy image of live HeLa cells stained with 1 μM CBTH probe. (bc) Confocal microscopy image of HeLa cells stained with CBTH (1 μM, 30 min) and a commercial probe (200 nM MTDR, 10 min). CBTH was excited at 405 nm in the green channel and collected at 500-550 nm; in the red channel, it was excited at 543 nm and collected at 600-700 nm. MTDR was excited at 633 nm and collected in the range of 650-700 nm. The image shows that CBTH can simultaneously image the mitochondria of live cells in both green and red light. Furthermore, the colocalization coefficient between the red and green channels was 0.91, the colocalization coefficient between the red channel and MTDR was 0.91, and the colocalization coefficient between the green channel and MTDR was 0.87.

[0045] Figure 3 (a) Dual-channel confocal microscopy image of HeLa cells fixed in 4% paraformaldehyde for 30 min after staining with 1 μM probe CBTH. (bc) Confocal microscopy image of HeLa cells fixed in 4% paraformaldehyde for 30 min after co-staining with CBTH (1 μM, 30 min) and commercial probes (5 μM Hoechst 33342, 10 min; 200 nM MTDR, 10 min). CBTH was excited at 405 nm in the green channel and collected at 500-550 nm; in the red channel, it was excited at 543 nm and collected at 600-700 nm. Hoechst 33342 was excited at 405 nm and collected at 450-480 nm; MTDR was excited at 635 nm and collected at 650-700 nm. As can be seen from the image, the probe CBTH can target mitochondria with green fluorescence and image the cell nucleus with red fluorescence in the fixed cells. Furthermore, the colocalization coefficients of CBTH in the nucleus and mitochondria were 0.90 and 0.91, respectively (Merged plot).

[0046] Figure 4Confocal fluorescence images of HeLa cells stained with 1 μM CBTH treated with CCCP (10 μM). CBTH was excited at 405 nm in the green channel and collected at 500-550 nm; and excited at 543 nm in the red channel and collected at 600-700 nm. The results showed that the red fluorescence in CCCP-treated cells gradually separated from the green fluorescence, with the red fluorescence migrating from the mitochondria to the nucleus, and the co-localization coefficient between the two fluorescence channels significantly decreased.

[0047] Figure 5 Confocal fluorescence images of untreated and rotenone-pretreated HeLa cells were obtained by staining with 1 μM CBTH probe. CBTH was used to excite the green channel at 405 nm and collect fluorescence at 500-550 nm; in the red channel, it was excited at 543 nm and collected at 600-700 nm. The results showed that the red and green channels overlapped significantly in untreated cells, with a high co-localization coefficient. In treated cells, the red channel fluorescence shifted from the mitochondria to the nucleus, and the co-localization coefficient between the two channels decreased significantly. Detailed Implementation

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] Example 1: Synthesis of probe CBTH

[0051] 1) Synthesis of 7-diethylaminocoumarin (compound 1)

[0052] 4-Diethylaminosalicylic acid aldehyde (0.97 g, 4 mmol), diethyl malonate (1.6 g, 10 mmol), and piperidine (1.0 mL) were mixed in anhydrous ethanol (30 mL) and stirred under reflux at 80 °C for 6 hours. Ethanol was removed under reduced pressure, followed by the addition of glacial acetic acid (20 mL) and concentrated hydrochloric acid (12 mol / L, 20 mL) and stirring for 8 hours (120 °C). The solution was cooled to room temperature and poured into 50 mL of ice water. A 1.0 mol / L sodium hydroxide solution was added dropwise to adjust the pH to 5, and the mixture was filtered and washed with water.

[0053] 2) Synthesis of 7-diethylaminocoumarinaldehyde (compound 2)

[0054] POCl3 (0.4 mL, 4.2 mmol, 1.4 equivalents) was carefully added to anhydrous DMF (N,N-dimethylformamide, 4 mL) under an argon atmosphere. The reaction mixture was stirred at 50 °C for 45 min. 7-Diethylaminocoumarin (650 mg, 3 mmol, 1 equivalent) dissolved in anhydrous DMF (3 mL) was added to the reaction mixture. The reaction mixture was stirred at 60 °C for 2 h. After the reaction was complete, 100 mL of ice water was added. The reaction mixture was stirred for 1 h until an orange precipitate appeared. The precipitate was filtered and washed twice with water. The crude product was co-evaporated twice under vacuum with ethanol to give 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde.

[0055] 1 H NMR (400 MHz, DMSO- d 6), δ (ppm): 9.90 (s, 1H), 8.41 (s, 1H), 7.67(s, 1H), 6.82 (s, 1H), 6.61 (s, 1H), 3.51 (q, J = 7.1 Hz, 4H), 1.15 (t, J = 7.0Hz, 6H).

[0056] 3) Synthesis of benzothiazole iodide (compound 3)

[0057] 2-Iodoethanol (7.74 g, 45 mmol) and 2-methylbenzothiazole (2.24 g, 15 mmol) were dissolved in acetonitrile solution (20 mL). The mixture was refluxed at 80 °C for 12 hours. After cooling and filtration, the solid was washed with petroleum ether to give compound 2 (1.95 g, 67% yield), a pale yellow solid.

[0058] 1 H NMR (400 MHz, DMSO- d 6), δ (ppm): 8.46 (dd, J = 8.1, 1.2 Hz, 1H), 8.33 (dd, J = 8.4, 1.0 Hz, 1H), 7.89 (ddd, J = 8.6, 7.3, 1.3 Hz, 1H), 7.81(ddd, J = 8.3, 7.3, 1.1 Hz, 1H), 5.24 (s, 1H), 4.87 (dd, J = 6.6, 3.5 Hz, 2H), 3.90 (d, J= 4.6 Hz, 2H), 3.23 (s, 3H).

[0059] 4) Synthesis of probe CBTH

[0060] Compound 2 (0.49 g, 2 mmol) and compound 3 (0.64 g, 2 mmol) were added to a flask containing 20 mL of ethanol. The mixture was stirred for 10 minutes, then piperidine (200 μL) was added, and the mixture was refluxed at 75 °C for 8 hours. After cooling to room temperature, the deep purple solid was filtered off, and the probe CBTH was washed with cooled diethyl ether. (Mass: 0.8 g, Yield: 73%)

[0061] 1 H NMR (400 MHz, DMSO- d 6), δ (ppm): 8.59 (s, 1H), 8.39 (dd, J = 8.1, 1.3 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 15.4 Hz, 1H), 8.00 (d, J =15.4 Hz, 1H), 7.83 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 7.75 (ddd, J = 8.2, 7.2,1.1 Hz, 1H), 7.58 (d, J = 9.0 Hz, 1H), 6.88 (dd, J = 9.1, 2.4 Hz, 1H), 6.69 (d, J = 2.4 Hz, 1H), 5.21 (t, J = 5.8 Hz, 1H), 4.86 (t, J = 4.6 Hz, 2H), 3.92 (q, J = 5.3 Hz, 2H), 3.54 (q, J = 7.0 Hz, 4H), 1.17 (t, J = 7.0 Hz, 6H).

[0062] The preparation reaction formula is as follows:

[0063]

[0064] Example 2: pH Titration Experiment

[0065] Prepare BR buffer solutions containing 2% DMSO (dimethyl sulfoxide) with different pH values ​​(pH = 2.0–12.0). Prepare test solutions containing 10 μM CBTH using the above buffer solutions. Measure the absorption spectra and fluorescence imaging of the above solutions using a UV-Vis spectrophotometer and a fluorescence spectrometer.

[0066] The results are as follows Figure 1 As shown in the figures, in (a), the absorption peak of probe CBTH at 440 nm increases with increasing pH, while the absorption peak at 530 nm decreases with increasing pH; in (b), when excited at 530 nm, the fluorescence emission of probe CBTH decreases with increasing pH; in (c), when excited at 440 nm, the fluorescence emission of probe CBTH increases with increasing pH; the ratio of the fluorescence intensity at 625 nm in (b) to the fluorescence intensity at 498 nm in (c) exhibits a Z-shaped distribution with pH, ​​as shown in (d); and in (e), the change in the ratio of the fluorescence intensity at 625 nm when probe CBTH is excited at 530 nm to the fluorescence intensity at 498 nm when excited at 440 nm under continuous laser irradiation. This indicates that probe CBTH is a pH-sensitive, cyclization-responsive dual-color fluorescent probe, and the switching ring of probe CBTH is almost unaffected by light irradiation.

[0067] Example 3: Toxicity test of probe CBTH

[0068] Cytotoxicity in live cells was determined using the standard MTT assay. HeLa cells in logarithmic growth phase were seeded into 96-well plates (approximately 1 × 10⁻⁶ cells / well). 4 Cells were inoculated into wells at 37°C and 5% CO2 for 24 hours. Then, 0.2, 0.5, 1, 2, and 5 μM CBTH were added to each well as experimental groups. Additionally, DMEM culture medium containing 0.2% DMSO was added as a control group. Cells were incubated at 37°C and 5% CO2 for 24 hours. Then, MTT (thiazolyl blue, 5 mg / mL) was added to each well. After incubation at 37°C for 4 hours, 100 μL of DMSO was added. After another 20 minutes of incubation, the absorbance of each well was measured at 490 nm using a microplate reader. The cytotoxicity assay was repeated four times.

[0069] Cell viability can be calculated using the following formula:

[0070]

[0071] Among them, A sample A represents the absorbance of the experimental group. c The absorbance of the control group, A bThe absorbance of the blank group is shown.

[0072] See results Figure 1 (f) in the figure: cell survival rate of HeLa cells incubated with different concentrations of the probe CBTH for 24 hours.

[0073] Experimental results showed that after incubating HeLa cells with 5 μM CBTH for 24 hours, the cell survival rate was still as high as 96%, indicating that the probe had very low toxicity.

[0074] Example 4: Culture of HeLa cells

[0075] HeLa cells were cultured in a high-glucose medium containing 10% fetal bovine serum in a 37°C, 5% CO2 saturated humidity incubator. The medium was changed every 2-3 days, and the cells were passaged. Once the cells reached the logarithmic growth phase, they were grafted onto slides for further culture.

[0076] ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and then place it in a disposable 35 mm petri dish for later use;

[0077] ② Wash the confluent cells in a 100 mL cell culture flask three times with PBS (phosphate-buffered saline), digest with 1 mL of 0.25% trypsin for 3-5 minutes, carefully pour off the trypsin, add fresh culture medium, mix well by pipetting, and count the cells. Control the cell density by adding culture medium to achieve a final cell concentration of 1 × 10⁶ cells per mL. 5 Each cell was seeded and then inoculated into a culture dish containing a coverslip, and placed in a 5% CO2 incubator to allow the cells to grow in close contact with the culture dish. Once the HeLa cells have grown and completely covered the coverslip, they can be used for cell experiments.

[0078] Example 5: Colocalization experiment of probe CBTH in viable HeLa cells

[0079] First, a 1 mM probe stock solution was prepared using DMSO. After HeLa cells had fully grown onto the coverslip, the viable HeLa cells were incubated in culture medium containing 1 μM CBTH for 30 minutes. After rinsing twice with PBS, 200 nM MTDR was added to the culture medium and incubated for 10 minutes. The cells were then imaged using a fluorescence confocal microscope.

[0080] See results Figure 2 .

[0081] Example 6: Using the probe CBTH to distinguish between normal cells and MMP-damaged cells

[0082] Using Example 4 as the control group, viable HeLa cells were first incubated in culture medium with 1 μM CBTH for 30 minutes, washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed twice with PBS, and then incubated with 5 μM Hoechst33342 or 200 nM MTDR for 10 minutes. The cells were then imaged using a fluorescence confocal microscope.

[0083] See results Figure 2 In the figure 'a': fluorescent targeting of CBTH in untreated cells. Figure 3 In the figure a: Fluorescent targeting of CBTH in cells immobilized with paraformaldehyde to eliminate MMPs.

[0084] The excitation wavelength of CBTH in the green channel was 405 nm, and the fluorescence collection wavelength was 500-550 nm; the excitation wavelength in the red channel was 543 nm, and the fluorescence collection wavelength was 600-700 nm. The results are as follows: Figure 2 and Figure 3 As shown, in untreated cells, CBTH targets mitochondria in two colors, while in cells immobilized with paraformaldehyde to eliminate MMPs, the green channel of CBTH targets mitochondria, and the red channel targets the nucleus. This demonstrates that the CBTH probe can be used to differentiate between healthy live cells and cells with damaged mitochondrial membrane potential.

[0085] Example 7: Displaying MMP changes in living cells and quantifying them using colocalization coefficients.

[0086] Viable HeLa cells were stained with 1 μM CBTH for 30 minutes, and then observed using a fluorescence confocal microscope. In the experimental group, CCCP was first dissolved in DMSO to obtain a 10 mM stock solution. HeLa cells were cultured in glass-bottomed culture dishes for 24 hours. After incubating HeLa cells for another 24 hours, cells without CCCP treatment were imaged, and then 1 μL of the CCCP stock solution was added and mixed thoroughly. Imagery was performed using a laser confocal microscope at regular intervals.

[0087] Another group, similar to the CCCP treatment, first dissolved rotenone in DMSO to obtain a 5 mM stock solution. HeLa cells were cultured in glass-bottomed culture dishes for 24 hours. Viable HeLa cells were stained with 1 μM CBTH for 30 minutes, and then observed using a fluorescence confocal microscope. In the experimental group, after adding 1 μL of rotenone, HeLa cells were incubated for different times, then stained with 1 μM CBTH for 30 minutes, and finally imaged using a laser confocal microscope.

[0088] See results Figure 4 , Figure 5Confocal fluorescence images of untreated, CCCP-pretreated, and rotenone-pretreated HeLa cells were obtained by staining with 1 μM probe CBTH.

[0089] The CBTH probe was excited at 405 nm in the green channel and collected at 500-550 nm; in the red channel, it was excited at 543 nm and collected at 600-700 nm. Results showed that untreated cells targeted mitochondria with both fluorescence. In the experimental group, cells treated with CCCP and paclitaxel showed no change in green fluorescence targeting, while red fluorescence gradually shifted from mitochondria to the nucleus. This may be due to the decrease in MMP levels after drug treatment. Furthermore, the co-localization coefficient of the two fluorescence channels gradually decreased during drug treatment. This clearly indicates that the probe of this invention can monitor MMP changes in living cells and quantify them through co-localization coefficients.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a compound comprising a compound CBTH of formula (I) or a self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe or a detection kit in the preparation of a preparation for simultaneously labeling or imaging mitochondria in living cells with two fluorescent colors or a preparation for simultaneously labeling or imaging mitochondria and nucleus in living cells with two fluorescent colors. The self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe comprises a compound CBTH of formula (I) or a pharmaceutically acceptable salt thereof. The detection kit comprises a compound CBTH of formula (I) or the self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe, and a solvent. The chemical structure of the compound of formula (I) is as follows: 。 2. Use according to claim 1, wherein the compound is ###0001### or a pharmaceutically acceptable salt thereof. The preparation method of the compound CBTH of formula (I) comprises the following steps of obtaining CBTH according to the following reaction route: 。 3. Use according to claim 2, wherein the compound is ###0002### 4-(diethylamino)salicylaldehyde and diethyl malonate are first reacted under alkaline conditions and heated to 70-90 ℃, and then reacted under acidic conditions and heated to 110-130 ℃ to obtain compound 1. Alternatively, the molar ratio of 4-(diethylamino)salicylaldehyde to diethyl malonate is 1:2.0-3.

0. Alternatively, phosphorus oxychloride and N,N-dimethylformamide are reacted under an inert atmosphere and heated to 45-55 ℃; then compound 1 is added and reacted at 55-65 ℃ to obtain compound 2. Alternatively, the reaction temperature of 2-methylbenzothiazole and iodoethanol is 75-85 ℃. Alternatively, the molar ratio of 2-methylbenzothiazole to iodoethanol is 1:2.5-3.

5. Alternatively, the molar ratio of compound 2 to compound 3 is 1:0.9-1.

1. Alternatively, the reaction temperature of compound 2 and compound 3 is 70-80 ℃.

4. The use according to claim 1, wherein the compound is ###00002### or a salt thereof. The self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe further comprises a pharmaceutically acceptable excipient.

5. Use of a compound comprising a compound CBTH of formula (I) or a self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe or a detection kit in the preparation of a preparation for monitoring changes in mitochondrial membrane potential in living cells. The self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe comprises a compound CBTH of formula (I) or a pharmaceutically acceptable salt thereof. The detection kit comprises a compound CBTH of formula (I) or the self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe, and a solvent. The chemical structure of the compound of formula (I) is as follows: 。 6. The use according to claim 5, characterized in that The preparation displays changes in mitochondrial membrane potential in living cells by a colocalization coefficient between two fluorescent channels.

7. Use of a compound comprising a compound CBTH of formula (I) or a self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe or a detection kit in the preparation of a preparation for distinguishing healthy living cells and cells with damaged mitochondrial membrane potential. The self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe comprises a compound CBTH of formula (I) or a pharmaceutically acceptable salt thereof. The detection kit comprises a compound CBTH of formula (I) or the self-reference monitoring mitochondrial membrane potential dual-color fluorescent probe, and a solvent. The chemical structure of the compound of formula (I) is as follows: 。

Images