Double-color fluorescent probe for self-reference monitoring of mitochondrial membrane potential and application of double-color fluorescent probe

Abstract

The invention relates to a mitochondrial membrane potential detection probe technology, in particular to a double-color fluorescent probe for self-reference monitoring of mitochondrial membrane potential and application of the double-color fluorescent probe. Comprising a compound CBTH or pharmaceutically acceptable salts, hydrates, solvates, optical isomers, racemes and the like. The chemical structural formula of the compound CBTH is as shown in formula (I): # imgabs0 #. The probe CBTH disclosed by the invention can be used for simultaneously imaging two colors, the fluorescence intensity does not disappear along with the change of the cell state, and the change of the mitochondrial membrane potential can be semi-quantitatively monitored by calculating a co-localization coefficient. The probe is low in toxicity and good in biocompatibility, the tedious operation of co-dyeing of the two probes is reduced, and the probe is expected to be widely applied to the research of mitochondrial related diseases and physiological processes.

Description

Technical Field

[0001] The invention relates to a mitochondrial membrane potential detection probe technology, and in particular to a dual-color fluorescent probe for self-reference monitoring of mitochondrial membrane potential 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] Mitochondria are important places for providing energy to cells. Mitochondrial membrane potential (MMP) participates in orderly physiological activities such as synthesizing ATP, forming electric potential and proton gradient, and is also the basis of cell metabolism. Abnormal MMP may mean the destruction of the mitochondrial electron transport chain, leading to brain necrosis, Parkinson's syndrome, diabetes and cancer. Therefore, efficient and convenient monitoring of MMP changes in living cells has practical value in biomedical research and diagnosis of related diseases.

[0004] Bioluminescent probes have the advantages of high sensitivity and non-invasiveness. Fluorescent probes can be used to detect MMP in situ and in real time in living cells. Most fluorescent probes can only qualitatively detect changes in MMP. Some migration probes target mitochondria when MMP is normal and target the nucleus when MMP is reduced. The change of MMP is reflected by the fluorescence intensity ratio, but the influence of photobleaching, viscosity and staining concentration on fluorescence intensity cannot be ruled out. Colocalization experiments are widely used to verify the organelle targeting of fluorescent molecules. The colocalization coefficient can be used to quantitatively analyze the changes of MMP, and the parameters are less affected by photostability and imaging conditions. Existing fluorescent probes that use the colocalization coefficient to monitor MMP migrate to lysosomes when MMP disappears, and the colocalization coefficient is calculated by introducing commercial lysosomal probes. However, the combination of multiple probes will bring many problems, such as cumbersome staining process, asynchronous light response, and target competition between probes, which will affect the quality and efficiency of imaging. If a single probe can achieve the effect of two probes, and the colocalization coefficient can be calculated by self-reference, it will be more advantageous than using multiple probes.

[0005] Therefore, it is of great value to develop a probe with stable self-reference parameters and capable of quantifying MMP fluctuations through the ratio of physical parameters. The inventors have studied and found that the alkaline environment of the mitochondrial pH value of about 8.0 is used to partially cyclize the molecule in the mitochondria to produce dual emission, and the open ring form has a certain affinity for nuclear DNA and can target the nucleus when MMP decreases. There is no report on a probe that quantifies the detection of MMP through the colocalization coefficient between dual emission channels. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a dual-color fluorescent probe for self-reference monitoring of MMP and its application. The probe CBTH provided by the present invention can image dual colors simultaneously, the fluorescence intensity will not disappear with the change of cell state, and the change of MMP can be semi-quantitatively monitored by calculating the co-localization coefficient. In addition, the probe has low toxicity and good biocompatibility, which reduces the cumbersome operation of co-staining of two probes.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] In the first aspect, a compound CBTH, whose chemical structure is 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]thiazol-3-ium, abbreviated as CBTH.

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

[0012]

[0013] The process is as follows: 4-(diethylamino) salicylaldehyde and diethyl malonate are mixed to react to obtain compound 1, and compound 1 is reacted in a mixture of phosphorus oxychloride and N,N-dimethylformamide to generate compound 2; 2-methylbenzothiazole and iodoethanol are mixed to react to obtain compound 3; then compound 2 and compound 3 are reacted through Knoevenagel reaction to synthesize a product; finally, the product is purified through column chromatography, and the obtained purple solid product is CBTH.

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

[0015] In some embodiments, 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 at 45-55° C. in an inert atmosphere; then compound 1 is added and heated to 55-65° C. to react to obtain compound 2.

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

[0018] In some embodiments, 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 to 1.1.

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

[0021] In a third aspect, a dual-color fluorescent probe for self-reference monitoring of MMP includes the above-mentioned compound CBTH or a pharmaceutically acceptable salt, hydrate, solvate, optical isomer, racemate, etc. thereof.

[0022] The present invention adopts compound CBTH as a dual-color fluorescent probe for self-reference monitoring of MMP, which is composed of two parts: 7-diethylaminocoumarin aldehyde and hydroxyethylbenzothiazole salt. Hydroxyethylbenzothiazole salt is an important family of pH-responsive probes, and the positive charge carried can selectively stain mitochondria through electrostatic interaction. At the same time, benzothiazole salt probes, such as anthocyanin dyes and thiazole orange, can be inserted into DNA and produce a strong binding force with DNA in the cell nucleus. In addition, DNA in the cell nucleus exhibits basophilic properties (pH=~7.2), while mitochondria maintain an alkaline environment (pH=~8.0) under normal circumstances. The difference in pH values ​​of these two organelles enables the dual-color fluorescent probe CBTH for self-reference monitoring of MMP described in the present invention to mark mitochondria with two different emission colors respectively, and migrate to the cell nucleus in a single color when MMP decreases.

[0023] Specifically, the compound CBTH is used as a dual-color fluorescent probe. In an acidic environment, it exists in an open-ring form (structural formula I). ​​In an alkaline environment, the probe undergoes cyclization and is converted into a closed form (structural formula II). The reaction formula is shown below:

[0024]

[0025] Taking HeLa cells as an example, the fluorescent probe CBTH enters living cells in an open-ring form and is enriched in mitochondria through electrostatic interactions. Since the pH value of mitochondria in living cells is about 8, some probes undergo cyclization and transformation into closed-ring forms in the weakly alkaline environment of mitochondria. Under 543nm excitation, closed-ring form II emits red fluorescence, and under 405nm excitation, open-ring form I emits green fluorescence. Therefore, in cells with normal MMP, mitochondria exhibit red and green fluorescence, and the nucleus exhibits no light. In cells with reduced MMP, mitochondria exhibit red and green fluorescence, and the nucleus exhibits weak red fluorescence. In cells where MMP disappears, 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 of the red fluorescence channel and the green fluorescence channel.

[0026] The experimental results confirm that the dual-color fluorescent probe CBTH for self-reference monitoring of MMP described in the present invention exists in a cyclized form in weakly alkaline mitochondria, emitting green fluorescence, while the open-ring form of the probe can migrate between mitochondria and cell nuclei depending on changes in MMP, emitting red fluorescence.

[0027] The selectivity of the above-mentioned dual-color fluorescent probe CBTH on cells has been strictly proven. Based on the cell staining, the dual-color fluorescent probe CBTH described in the present invention was firstly counterstained with a commercial mitochondrial probe (Mito-Tracker Deep Red FM) to confirm that it has a high colocalization coefficient, thereby confirming that the red and green fluorescence of the normal MMP channel, the green fluorescence of fixed cells or cells with reduced MMP (CCCP (carbonyl cyanide m-chlorophenylhydrazone) treatment) are all from mitochondria. Further, a counterstained experiment was performed with a commercial nuclear probe (Hoechst 33342), confirming that it has a high colocalization coefficient, thereby confirming that the red fluorescence of fixed cells or cells with reduced MMP (CCCP treatment) comes from the cell nucleus.

[0028] 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 phosphoric acid, sulfuric acid, nitric acid, etc., and the organic acids include acetic acid, oxalic acid, citric acid, fumaric acid, etc.

[0029] 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.

[0030] In a fourth aspect, a detection kit comprises the above-mentioned compound CBTH or a dual-color fluorescent probe for self-reference monitoring of MMP, and a solvent.

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

[0032] In a fifth aspect, a dual-color fluorescent probe or detection kit for self-reference monitoring of MMP or the above-mentioned compound CBTH is used in the preparation of a preparation for simultaneously marking or imaging mitochondria in living cells with two fluorescent colors, or a preparation for simultaneously marking or imaging mitochondria and nuclei in living cells with two fluorescent colors.

[0033] Specifically, the living cells are HeLa cells; some probes undergo cyclization in the weakly alkaline environment of mitochondria when incubating living cells, and the cyclized molecules emit green fluorescence when the excitation light is 405nm; the open-ring form of the probe targets mitochondria through cations and emits red fluorescence when the excitation light is 543nm. When MMP is reduced, the red fluorescence gradually transfers from the mitochondria to the cell nucleus.

[0034] In a sixth aspect, a use of the above-mentioned compound CBTH or a dual-color fluorescent probe or detection kit for self-reference monitoring of MMP in the preparation of a preparation for monitoring changes in MMP in living cells.

[0035] Specifically, the living cells are HeLa cells; the dual-color fluorescent probe CBTH dual-color targets mitochondria in living cells with normal MMP, and both fluorescence channels with excitation light of 405nm and 543nm target mitochondria; in living cells with MMP damage, the fluorescence channel with excitation light of 405nm targets mitochondria, and the fluorescence channel with excitation light of 543nm targets mitochondria and cell nuclei; in cells in which MMP has almost completely disappeared, the fluorescence channel with excitation light of 405nm targets mitochondria, and the fluorescence channel with excitation light of 543nm targets cell nuclei.

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

[0037] In a seventh aspect, a use of the above-mentioned compound CBTH or a dual-color fluorescent probe or detection kit for self-reference monitoring of MMP in the preparation of a preparation for distinguishing healthy living 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 due to the red channel (excitation light is 543nm) and green channel (excitation light is 405nm) fluorescence, and the colocalization coefficient between the two channels is calculated to be high; while in cells with decreased MMP, the red channel fluorescence targets the cell nucleus, and the green channel fluorescence targets the mitochondria, and the colocalization coefficient between the two channels is reduced.

[0039] Experiments have confirmed that the dual-color fluorescent probe of the present invention can monitor intracellular MMP changes through dual-color fluorescence self-reference. In healthy living cells, the dual-color fluorescent probe can image mitochondria with red and green fluorescence. In cells induced by CCCP treatment to decrease MMP, the green fluorescence of the dual-color fluorescent probe remains mitochondrial-targeted, the red fluorescence gradually shifts from the mitochondria to the nucleus, and the Pearson colocalization coefficient between the two channels gradually decreases. In apoptotic cells induced by rotenone, the green fluorescence can also be targeted to the mitochondria, 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 the present invention are:

[0041] Compared with the existing MMP probe, the dual-color fluorescent probe CBTH for monitoring MMP with self-reference of the present invention can image dual colors simultaneously, and the fluorescence intensity of a certain fluorescent channel will not disappear with the change of cell state, so the co-localization coefficient can be calculated by self-reference to semi-quantitatively monitor the change of MMP. And the toxicity of the probe is low, and the biocompatibility is good, which can reduce the cumbersome operation of two probes to dye the nucleus and mitochondria together, and reduce the cytotoxicity caused thereby. In addition, the spatial distribution and co-localization coefficient of dual-color fluorescence can be used to distinguish healthy living cells from MMP-damaged cells, and the distribution and morphological changes of mitochondria and nuclei during cell apoptosis can also be studied, which makes it have important experimental application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] 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.

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

[0044] Figure 2 :(a) Dual-channel confocal microscopy image of living HeLa cells stained with 1μM probe CBTH. (bc) Confocal microscopy image of HeLa cells co-stained with CBTH (1μM, 30 minutes) and commercial probe (200nM MTDR, 10 minutes). The excitation wavelength of CBTH in the green light channel is 405nm, and the fluorescence collection wavelength is 500-550nm; the excitation wavelength in the red light channel is 543nm, and the fluorescence collection wavelength is 600-700nm; the excitation wavelength of MTDR is 633nm, and the light collection range is 650-700nm; It can be seen from the figure that the probe CBTH can simultaneously image the mitochondria of living cells in green and red. In addition, the colocalization coefficient of the red and green channels is 0.91, the colocalization coefficient of the red channel and MTDR is 0.91, and the colocalization coefficient of the green channel and MTDR is 0.87.

[0045] Figure 3 :(a) Dual-channel confocal microscopy images of HeLa cells fixed with 4% paraformaldehyde for 30 min after staining with 1 μM probe CBTH. (bc) Confocal microscopy images of HeLa cells fixed with 4% paraformaldehyde for 30 min co-stained with CBTH (1 μM, 30 min) and commercial probes (5 μM Hoechst 33342, 10 min; 200 nM MTDR, 10 min). The excitation wavelength of CBTH in the green light channel is 405 nm, and the fluorescence collection wavelength is 500-550 nm; the excitation wavelength in the red light channel is 543 nm, and the fluorescence collection wavelength is 600-700 nm; the excitation wavelength of Hoechst 33342 is 405 nm, and the light collection range is 450-480 nm; the excitation wavelength of MTDR is 635 nm, and the light collection range is 650-700 nm; it can be seen from the figure that the probe CBTH can target mitochondria with green fluorescence and image the nucleus with red fluorescence in fixed cells. In addition, the colocalization coefficients of CBTH in the nucleus and mitochondria were 0.90 and 0.91, respectively (Merged figure).

[0046] Figure 4 :Confocal fluorescence images of HeLa cells stained with 1μM CBTH treated with CCCP (10μM). The excitation wavelength of CBTH in the green light channel is 405nm, and the fluorescence collection wavelength is 500-550nm; the excitation wavelength in the red light channel is 543nm, and the fluorescence collection wavelength is 600-700nm. The results show that the red fluorescence of cells treated with CCCP gradually separates from the green fluorescence position, the red fluorescence migrates from the mitochondria to the nucleus, and the colocalization coefficient between the two fluorescence channels is significantly reduced.

[0047] Figure 5: Confocal fluorescence images of untreated and rotenone-pretreated HeLa cells were stained with 1 μM probe CBTH. The excitation wavelength of CBTH in the green light channel is 405 nm, and the fluorescence collection wavelength is 500-550 nm; the excitation wavelength in the red light channel is 543 nm, and the fluorescence collection wavelength is 600-700 nm. The results showed that the red and green channels of untreated cells overlapped, and the colocalization coefficient was high. After treatment, the red channel fluorescence of the cells was transferred from the mitochondria to the nucleus, and the colocalization coefficient between the two channels decreased significantly. DETAILED DESCRIPTION

[0048] 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.

[0049] In the following examples, the materials, reagents, etc. used were obtained from commercial sources unless otherwise specified.

[0050] Example 1: Synthesis of probe CBTH

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

[0052] 4-Diethylamino salicylaldehyde (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 at 80 ° C for 6 hours. The ethanol was removed under reduced pressure and then glacial acetic acid (20 mL) and concentrated hydrochloric acid (12 mol / L, 20 mL) were added to react and stirred for 8 hours (120 ° C). The solution was cooled to room temperature and poured into 50 mL of ice water. 1.0 mol / L sodium hydroxide solution was added dropwise to adjust the pH to 5, filtered, and washed with water.

[0053] 2) Synthesis of 7-diethylaminocoumarin aldehyde (Compound 2)

[0054] POCl3 (0.4 mL, 4.2 mmol, 1.4 eq) 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 minutes. 7-diethylaminocoumarin (650 mg, 3 mmol, 1 eq) dissolved in anhydrous DMF (3 mL) was added to the reaction mixture. The reaction mixture was stirred at 60 ° C for 2 hours. After the reaction was completed, 100 mL of ice water was added. The reaction mixture was stirred for 1 hour until an orange precipitate appeared. The precipitate was filtered and washed with water twice. The crude product was co-evaporated twice with ethanol under vacuum to obtain 7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde.

[0055] 1H NMR (400MHz, DMSO-d6), 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 = 7.1Hz, 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 obtain a light yellow solid compound 2 (mass 1.95 g, yield 67%).

[0058] 1 H NMR (400MHz, DMSO-d6), δ (ppm): 8.46 (dd, J=8.1, 1.2Hz, 1H), 8.33 (dd, J=8.4, 1.0Hz, 1H), 7.89 (ddd, J=8.6, 7.3, 1.3Hz ,1H),7.81(ddd,J=8.3,7.3,1.1Hz,1H),5.24(s,1H),4.87(dd,J=6.6,3.5Hz,2H),3.90(d,J=4.6Hz,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. After the mixture was stirred for 10 minutes, piperidine (200 μL) was added and then refluxed at 75 ° C for 8 hours. After cooling to room temperature, the dark purple solid was filtered and the probe CBTH was washed with a cooled ether solution. (Mass: 0.8 g, yield: 73%).

[0061] 1H NMR (400MHz, DMSO-d6), δ (ppm): 8.59 (s, 1H), 8.39 (dd, J = 8.1, 1.3Hz, 1H), 8.24 (d, J = 8.4Hz, 1H), 8.1 4(d,J=15.4Hz,1H),8.00(d,J=15.4Hz,1H),7.83(ddd,J=8.5,7.2,1.3Hz,1H),7.75(ddd,J=8.2,7.2 ,1.1Hz,1H),7.58(d,J=9.0Hz,1H),6.88(dd,J=9.1,2.4Hz,1H),6.69(d,J=2.4Hz,1H),5.21(t,J=5. 8Hz, 1H), 4.86 (t, J = 4.6Hz, 2H), 3.92 (q, J = 5.3Hz, 2H), 3.54 (q, J = 7.0Hz, 4H), 1.17 (t, J = 7.0Hz, 6H).

[0062] The preparation reaction formula is as follows:

[0063]

[0064] Example 2: pH titration experiment

[0065] BR buffer containing 2% DMSO (dimethyl sulfoxide) at different pH values ​​(pH=2.0-12.0) was prepared, and a test solution containing 10 μM CBTH was prepared with the above buffer. The above solution was tested for its absorption spectrum and two-color fluorescence imaging using a UV-visible spectrophotometer and a fluorescence spectrometer.

[0066] The results are as follows Figure 1 As shown, Figure 1 In (a), the absorption peak of the probe CBTH at 440 nm increases with increasing pH, and the absorption peak at 530 nm decreases with increasing pH; Figure 1 In (b), when excited at 530 nm, the fluorescence emission of the probe CBTH decreases with increasing pH; Figure 1 In (c), when excited at 440 nm, the fluorescence emission of the probe CBTH increases with the increase of pH; Figure 1 (b) Fluorescence intensity at 625 nm and Figure 1 In (c), the fluorescence intensity ratio at 498 nm shows a sigmoidal distribution as the pH changes. Figure 1 (d) Figure 1In (e), the change in the ratio of the fluorescence intensity at 625 nm when the probe CBTH is excited at 530 nm to the fluorescence intensity at 498 nm when it is excited at 440 nm under continuous laser irradiation indicates that the probe CBTH is a pH-sensitive dual-color fluorescent probe that can undergo cyclization reaction, and the on-off ring of the probe CBTH is almost unaffected by light.

[0067] Example 3: Toxicity test of probe CBTH

[0068] The cytotoxicity of live cells was determined by the standard MTT assay. HeLa cells growing in logarithmic phase were seeded in 96-well plates (approximately 1×10 4 cells / well), and the small wells were filled with cell-free culture medium as a blank group. The inoculated cells were placed in a 37°C, 5% CO2 incubator for 24 hours, and then 0.2, 0.5, 1, 2, and 5 μM concentrations of CBTH were added to the wells as the experimental group. In addition, DMEM culture medium with a final concentration of 0.2% DMSO was added as a control group. The cells were incubated at 37°C, 5% CO2 for 24 hours. MTT (thiazolyl blue, 5 mg / mL) was then 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 at 490 nm was tested using an enzyme reader, and the cytotoxicity experiment was repeated 4 times.

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

[0070]

[0071] Among them, A sample is the absorbance of the experimental group, A c is the absorbance of the control group, A b is the absorbance of the blank group.

[0072] Results Figure 1 (f): Cell viability of HeLa cells incubated with different concentrations of probe CBTH for 24 h.

[0073] The 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 toxicity of the probe was very low.

[0074] Example 4: Cultivation of HeLa cells

[0075] HeLa cells were cultured in a high-glucose medium containing 10% fetal bovine serum at 37°C in a saturated humidity incubator with 5% CO2. The medium was changed every 2-3 days and subcultured. When the cells grew to the logarithmic phase, they were cultured in slices:

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

[0077] ② Wash the cells in the 100mL cell bottle three times with PBS (phosphate buffered saline), digest 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. Control the cell density by adding culture medium so that the final cell concentration is 1×10 per mL. 5 Then, inoculate the culture dish containing the cover glass and place it in a 5% CO2 incubator to allow the cells to grow close to the culture dish. After the HeLa cells grow on the plate and cover the cover glass, they are used for cell experiments.

[0078] Example 5: Co-localization experiment of probe CBTH in active HeLa cells

[0079] First, DMSO was used to prepare a probe stock solution with a concentration of 1 mM. After the HeLa cells had grown all over the coverslip, the active HeLa cells were incubated in a culture medium containing 1 μM CBTH for 30 minutes, rinsed twice with PBS, and then 200 nM MTDR was added to the culture medium and incubated for 10 minutes. The cells were imaged using a fluorescence confocal microscope.

[0080] Results Figure 2 .

[0081] Example 6: Using the probe CBTH to distinguish normal cells from MMP-impaired cells

[0082] Using Example 4 as a control group, active HeLa cells were first incubated with 1 μM CBTH in culture medium for 30 minutes, rinsed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes, rinsed twice with PBS, 5 μM Hoechst 33342 or 200 nM MTDR was added to the culture medium and incubated for 10 minutes, and the cells were imaged using a fluorescence confocal microscope.

[0083] Results Figure 2 a: Fluorescence targeting of CBTH in untreated cells. and Figure 3 a: Fluorescence targeting of CBTH in cells fixed with paraformaldehyde to eliminate MMP.

[0084] The excitation wavelength of CBTH in the green light channel is 405nm, and the fluorescence collection wavelength is 500-550nm; the excitation wavelength in the red light channel is 543nm, and the fluorescence collection wavelength is 600-700nm. Figure 2 and Figure 3As shown, in untreated cells, CBTH dual-color targets mitochondria, and in cells fixed with paraformaldehyde to eliminate MMP, CBTH green channel targets mitochondria and red channel targets nuclei. Thus, it can be confirmed that the probe CBTH can be used to distinguish healthy living cells from cells with damaged mitochondrial membrane potential.

[0085] Example 7: MMP changes in living cells were shown and quantified by colocalization coefficient.

[0086] Active HeLa cells were stained with 1μM 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 10mM stock solution. HeLa cells were cultured in a glass-bottomed dish for 24 hours. HeLa cells were then incubated for 24 hours, and after imaging the cells that were not treated with CCCP, 1μL of CCCP stock solution was added and mixed evenly. Images were taken at intervals using a laser confocal microscope.

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

[0088] Results Figure 4 , Figure 5 , Confocal fluorescence images of HeLa cells untreated, pretreated with CCCP, and pretreated with rotenone stained with 1 μM probe CBTH.

[0089] The excitation wavelength of CBTH in the green light channel is 405nm, and the fluorescence collection wavelength is 500-550nm; the excitation wavelength in the red light channel is 543nm, and the fluorescence collection wavelength is 600-700nm. The results showed that the untreated cells dual-color targeted mitochondria. In the experimental group, the cells treated with CCCP and paclitaxel had unchanged green fluorescence targeting, and the red fluorescence gradually shifted from the mitochondria to the nucleus, which may be due to the reduction of MMP after drug treatment. In addition, the colocalization coefficient of the two fluorescence channels gradually decreased during drug treatment. It is clearly suggested that the probe of the present invention can monitor the changes of MMP in living cells and quantify them by the colocalization coefficient.

[0090] 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 compound CBTH, characterized in that Its chemical structure is shown in formula (I):

2. A method for preparing the compound CBTH according to claim 1, characterized in that: The method comprises the steps of obtaining CBTH according to the following reaction scheme; 3. The preparation method according to claim 2, characterized in that: 4-(Diethylamino) salicylaldehyde and diethyl malonate are first heated to 70-90°C under alkaline conditions for reaction, and then heated to 110-130°C under acidic conditions for reaction to obtain; Or, the molar ratio of 4-(diethylamino) salicylaldehyde to diethyl malonate is 1:2.0-3.0; Alternatively, phosphorus oxychloride and N,N-dimethylformamide are heated to 45-55° C. in an inert atmosphere for reaction; then compound 1 is added and heated to 55-65° C. for reaction to obtain compound 2; Or, the temperature for the reaction of 2-methylbenzothiazole and iodoethanol is 75-85°C; Or, the molar ratio of 2-methylbenzothiazole to iodoethanol is 1:2.5-3.5; Or, 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°C.

4. A dual-color fluorescent probe for self-reference monitoring of mitochondrial membrane potential, characterized in that: It includes the compound CBTH according to claim 1 or its pharmaceutically acceptable salt, hydrate, solvate, optical isomer, racemate.

5. The dual-color fluorescent probe for self-reference monitoring of mitochondrial membrane potential as claimed in claim 4, characterized in that: Pharmaceutically acceptable excipients are also included.

6. A detection kit, characterized in that: The invention comprises the compound CBTH as claimed in claim 1 or the dual-color fluorescent probe for self-reference monitoring of mitochondrial membrane potential as claimed in claim 4 or 5, and a solvent.

7. Use of the compound CBTH according to claim 1, the dual-color fluorescent probe for self-reference monitoring of mitochondrial membrane potential according to claim 4 or 5, or the detection kit according to claim 6 in the preparation of a preparation for simultaneously marking or imaging mitochondria in living cells with two fluorescent colors, or a preparation for simultaneously marking or imaging mitochondria and nuclei in living cells with two fluorescent colors.

8. Use of the compound CBTH according to claim 1, the dual-color fluorescent probe for monitoring mitochondrial membrane potential with self-reference according to claim 4 or 5, or the detection kit according to claim 6 in the preparation of a preparation for monitoring changes in mitochondrial membrane potential in living cells.

9. The use according to claim 8, characterized in that: The preparation shows the changes of mitochondrial membrane potential in living cells through the colocalization coefficient between two fluorescence channels.

10. Use of the compound CBTH according to claim 1, the dual-color fluorescent probe for monitoring mitochondrial membrane potential with self-reference according to claim 4 or 5, or the detection kit according to claim 6 in the preparation of a preparation for distinguishing healthy living cells from cells with damaged mitochondrial membrane potential.

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