Application of a benzothiazole fluorescent probe in simultaneous observation of multiple organelles
By designing benzothiazole fluorescent probes, the problem of simultaneous imaging of lysosomes, mitochondria and nucleoli in existing technologies has been solved, achieving low-toxicity and high-efficiency multi-organ imaging, which is suitable for the preparation of tumor diagnosis and imaging reagents.
Patent Information
- Application Number
- CN202311470943.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-07
- Publication Date
- 2025-09-16
- Estimated Expiration
- 2043-11-07
AI Technical Summary
Existing technologies make it difficult to simultaneously image lysosomes, mitochondria, and nucleoli, and co-staining with multiple probes can increase cytotoxicity.
A benzothiazole fluorescent probe has been developed, which introduces cationic structures such as benzothiazole into its structure. It can simultaneously observe and image lysosomes, mitochondria and nucleoli, and can achieve simultaneous imaging of the three using a single excitation light.
It achieves simultaneous imaging of multiple organs with low cytotoxicity, improves observation efficiency, and reduces damage to cells by probes.
Smart Images
Figure CN117510432B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of chemical medicine technology and relates to the application of a benzothiazole fluorescent probe in the simultaneous observation of multiple organelles, particularly the application of a benzothiazole fluorescent probe in the simultaneous observation and imaging of lysosomes, mitochondria, and nucleoli. Background Art
[0002] Lysosomes are crucial organelles within cells, specialized vesicles that serve as a crucial site for the digestion of waste products. Lysosomes contain hydrolases that break down and degrade waste and harmful substances within the cell. Lysosomal disorders primarily affect the proper functioning of lysosomes. Deficiency or dysfunction of specific lysosomal enzymes can impair the proper degradation and processing of substances, leading to the accumulation of intracellular waste. Lysosomal disorders can lead to a variety of diseases, such as Garni's disease and Cohen's disease. Mitochondria are the energy production sites of the cell, providing energy for cellular metabolism. Mutations or defects in mitochondrial DNA or mitochondrial proteins lead to mitochondrial abnormalities, which in turn affect many organs and tissues in the body, including the heart, muscles, and liver, causing a variety of diseases, such as movement disorders and muscle weakness. The nucleolus is a site within the cell where RNA is concentrated. It plays an important role in transcriptional regulation, ribosome assembly, RNA processing and modification, and cell cycle regulation. Therefore, observing and imaging these three organelles is of great significance in cell biology and medicine.
[0003] Currently, these organelles can be observed using electron microscopy, but this can cause cell damage. Fluorescence imaging, due to its high sensitivity and excellent staining, allows for in situ, real-time observation of intracellular organelles, and is gradually becoming an important observation method. Currently, researchers have developed many probes that can image lysosomes, mitochondria, and nucleoli individually. However, simultaneous imaging can only be achieved through double staining, which can produce fluorescence bleed-through, and co-staining with multiple probes can increase cytotoxicity.
[0004] Therefore, researchers have been working to develop a single probe that can simultaneously image different organelles. Currently, there is no single fluorescent probe that can simultaneously image lysosomes, mitochondria, and nucleoli. Summary of the Invention
[0005] Purpose of the invention: The purpose of the present invention is to provide a benzothiazole fluorescent probe for use in the simultaneous observation of multiple organelles. The benzothiazole fluorescent probe provided by the present invention can simultaneously observe and image lysosomes, mitochondria, and nucleoli.
[0006] The fluorescent probe of the present invention can simultaneously distinguish and image lysosomes, mitochondria, and nucleoli. Since these three organelles are associated with various diseases (such as cancer), the fluorescent probe of the present invention can be used to prepare tumor diagnostic reagents, fluorescent imaging reagents, or radioactive imaging reagents for tumors.
[0007] Technical solution: The purpose of the present invention is achieved through the following technical solution:
[0008] The present invention provides an application of a benzothiazole fluorescent probe in the simultaneous observation of multiple organelles. The chemical name of the fluorescent probe is (E)-2-(3-chloro-4-hydroxy-5-methoxyphenylvinyl)-3-methylbenzothiazole-3-iodine salt; its structural formula is:
[0009]
[0010] The fluorescent probe can be used to observe cellular oxidative damage.
[0011] The present invention also provides the use of the benzothiazole fluorescent probe in simultaneously imaging multiple organelles.
[0012] The organelles are lysosomes, mitochondria and nucleoli.
[0013] The concentration of the fluorescent probe for staining cells is 1-15 μM. This concentration has low cytotoxicity and good imaging effect.
[0014] The fluorescent probe described herein is excited by a single excitation light source. When imaging multiple organelles, conventional staining requires multiple fluorescent probes to be imaged separately, thus requiring multiple excitation light sources. The fluorescent probe described herein requires only a single excitation light source to simultaneously image lysosomes, mitochondria, and nucleoli. This makes the fluorescent probe described herein more convenient and quicker to use, with minimal damage to the probe.
[0015] The benzothiazole fluorescent probe of the present invention can be used to prepare tumor diagnostic reagents, and its structural formula is:
[0016]
[0017] The benzothiazole fluorescent probe of the present invention can be used to prepare fluorescent imaging agents or radioactive imaging agents for tumors.
[0018] Beneficial effects:
[0019] The fluorescent probe of the present invention introduces cationic structures such as benzothiazole, which give the molecule good optical properties. In addition, the introduction of the cationic structure makes the probe easy to target mitochondria. Because the probe structure is of suitable size, it can be inserted into the RNA chain, so the probe can target the nucleolus. In addition, the hydroxyl structure of the probe makes it easy to target the lysosome. Because these three probes are related to various diseases (cancer, etc.), the fluorescent probe of the present invention can be used to prepare tumor diagnostic reagents, fluorescent imaging reagents or radioactive imaging reagents for preparing tumors. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 The fluorescence spectra and fluorescence fitting curves of TBCL (10 μM) in solutions with different pH concentrations are shown in Figure 2. Figure 1 (A) Fluorescence spectra of TBCL (10 μM) in solutions with different pH concentrations; Figure 1 (B) is the corresponding fluorescence fitting curve.
[0021] Figure 2 This confocal image shows TBCL in HeLa cells. Green channel, 490-560nm; red channel, 560-660nm. Merged is a superposition of the previous images. DIC is differential interference contrast imaging.
[0022] Figure 3 This image shows the colocalization of TBCL in HeLa cells. The green channel has an absorption range of 490-560 nm; the red channel has an absorption range of 560-660 nm. Merged is a superposition of the previous images. LTDR is a lysosome-tracking red fluorescent probe; MTDR is a mitochondrial deep red fluorescent probe.
[0023] Figure 4 This is a picture of TBCL in detecting cell oxidative damage. Figure 4 (A) is the unoxidized picture; Figure 4 (B) is the oxidation picture of 3 mM H2O2; Figure 4 (C) is the oxidation picture of 6 mM H2O2. DETAILED DESCRIPTION
[0024] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.
[0025] The substances in each example are all commercially available products.
[0026] The absorption spectrum was measured using a Hitachi U-2910 spectrophotometer, the fluorescence spectrum was measured using a Hitachi F-2700 spectrophotometer, and the cell imaging instrument was a Lecia confocal microscope.
[0027] Example 1 Synthesis of benzothiazole fluorescent probe molecules (abbreviated as TBCL)
[0028]
[0029] Wherein, the substituent R is an alkyl group. In this embodiment, R is selected from methyl group.
[0030] Benzothiazole iodide (0.291 g, 1 mmol) and 3-chloro-4-hydroxy-5-methoxybenzaldehyde (0.186 g, 1 mmol) were dissolved in 20 mL of methanol and stirred in a flask for 1 hour. After stirring, a few drops of piperidine were added, and the mixture was refluxed at 85°C for 8 hours. After cooling to room temperature, the mixture was washed with petroleum ether. Column chromatography using a mixture of CH2Cl2 and CH3OH (CH2Cl2:CH3OH volume ratio of 10:1 to 6:1) as the eluent yielded 0.16 g of a red solid, chemically designated (E)-2-(3-chloro-4-hydroxy-5-methoxyphenyl)-3-methylbenzothiazole-3-iodide, a benzothiazole fluorescent probe molecule, abbreviated as TBCL.
[0031] The structure of the compound was confirmed to be correct by analyzing the compound's hydrogen and carbon spectra. The characterization results are as follows:
[0032] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.33 (d, J = 8.0Hz, 1H), 8.11 (d, J = 8.0Hz, 1H), 8.0 (d, J = 16 .0Hz,1H),7.78-7.82(m,2H),7.68-7.72(m,2H),7.55(s,1H),4.26(s,3H),3.92(s,3H).
[0033] 13 C NMR (400MHz, DMSO-d6), δ (ppm): 170.82, 150.03, 148.21, 142.51, 129.45, 127.99, 127 .50,126.83,126.78,124.39,124.27,121.91,116.45,111.33,109.22,56.90,36.26.C 17 H 15 ClINO2S
[0034] Example 2 Photophysical properties test of fluorescent probe TBCL in different solvents
[0035] A 1 mM concentration of the fluorescent probe TBCL stock solution was prepared using DMSO. The pH of the PBS solution was adjusted with hydrochloric acid and NaOH to prepare solvents with different pH values (pH = 3, 4, 5, 6, 7, and 8). TBCL was then added to prepare test solutions containing 10 μM TBCL. The fluorescence emission spectrum was measured using a fluorescence spectrometer, and the fluorescence spectrum was fitted to obtain the corresponding curve. The results are shown in Figure 1 . Figure 1 (A) Fluorescence spectra of TBCL (10 μM) in solutions with different pH concentrations; Figure 1 (B) is the corresponding fluorescence fitting curve.
[0036] As can be seen from the figure, when excited with a single excitation light, two fluorescence peaks appear, namely 530nm and 590nm. As the pH value increases, the fluorescence intensity at 530nm decreases, while the fluorescence intensity at 590nm remains unchanged. The fitting plot shows that the pKa of the probe is 5.0.
[0037] According to the above image results, it can be found that the probe has two fluorescence peaks at different pH values, indicating that the probe can emit two types of fluorescence. Therefore, the probe can be used for dual-color imaging.
[0038] Example 3 Cell Imaging Experiment of Fluorescent Probe TBCL
[0039] The probe stock solution was prepared with DMSO at a concentration of 1 mM. After HeLa cells (ATCC) were grown on the coverslip, the active HeLa cells were incubated in 5% calf serum-containing high glucose medium (DMEM) at 37°C and 5% CO2 for 30 minutes. After being washed twice with PBS, the cells were imaged using a fluorescence confocal microscope. The results are shown in Figure 2. Figure 2 .
[0040] The green channel's light absorption range is 490-560nm, while the red channel's range is 560-660nm. As can be seen in the image, the probe in the green channel displays dot-like structures resembling lysosomes, while the red channel displays structures resembling mitochondria and nucleoli. This demonstrates that the probe can stain lysosomes, mitochondria, and nucleoli simultaneously.
[0041] Example 4 Co-localization Cell Experiment of Fluorescent Probe TBCL
[0042] Use DMSO to prepare a probe stock solution with a concentration of 1mM. After the HeLa cells have grown all over the coverslip, the active HeLa cells are incubated in a high-glucose culture medium containing 5μM TBCL and 5% calf serum at 37°C and 5% CO2 for 30 minutes. After rinsing twice with PBS, the cells are imaged using a fluorescence confocal microscope. Another group of active HeLa cells were incubated in a culture medium containing 5μM TBCL for 30 minutes, and then 2nM LTDR was added to the culture medium and incubated for 10 minutes. The cells were imaged using a fluorescence confocal microscope. For the mitochondrial counterstaining experiment, active HeLa cells were incubated in a culture medium containing 5μM TBCL for 30 minutes, and then 2nM MTDR was added to the culture medium and incubated for 10 minutes. The cells were imaged using a fluorescence confocal microscope. The results are shown in. Figure 3 .
[0043] in, Figure 3 (A) Confocal microscopy images of viable HeLa cells co-stained with the probes TBCL (5 μM, 15 min) and LTDR (2 nM, 15 min). Figure 3 (B) is a confocal microscopy image of active HeLa cells co-stained with the probes TBCL (5μM, 15 min) and MTDR (2nM, 15 min). The excitation wavelength of TBCl in the green light channel is 488nm, and the fluorescence collection wavelength is 490-560nm; the red light channel collection range is 560-660nm; the excitation wavelength of LTDR in the red light channel is 633nm, and the fluorescence collection wavelength is 640-740nm. As can be seen from the figure: the probes TBCl and LTDR have a large overlap, with colocalization coefficients of 0.88 and 0.89, respectively (Merged figure), which fully demonstrates that the probe TBCL of the present invention can simultaneously image active HeLa lysosomes, mitochondria, and nucleoli.
[0044] Example 5 Experiment on detecting cell oxidative damage using fluorescent probe TBCL
[0045] A 1mM probe stock solution was prepared with DMSO. After HeLa cells had grown all over the coverslip, the active HeLa cells were pretreated with 3mM and 6mM H2O2 for 1 hour. After staining with TBCL (5μM) for 30 minutes, the cells were imaged using a fluorescence confocal microscope. Figure 4 The excitation wavelength is 488nm, the green channel fluorescence collection wavelength is 490-560nm, and the red channel collection range is 560-620nm.
[0046] in, Figure 4(A) Confocal microscopy images of viable HeLa cells stained with the probe TBCL (5 μM, 15 min). As can be seen from the image, the probe can simultaneously image lysosomes, mitochondria, and nucleoli. Figure 4 (B) Cells pretreated with 3 mM H2O2 were stained with the probe TBCL (5 μM, 15 min). As can be seen from the figure, the fluorescence intensity of the red channel decreased and the filamentous mitochondrial morphology disappeared. Figure 4 (C) Cells pretreated with 6 mM H2O2 were stained with the probe TBCL (5 μM, 15 min). As can be seen from the image, the red channel only reveals the morphology of the nucleolus. Therefore, the fluorescent probe TBCL can be used to observe the process of cellular oxidative damage.
[0047] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims
1. A benzothiazole fluorescent probe for the simultaneous observation of multiple organelles for non-disease diagnosis and treatment purposes, characterized in that: The chemical name of the fluorescent probe is (E)-2-(3-chloro-4-hydroxy-5-methoxyphenyl)-3-methylbenzothiazole-3-iodine salt; its structural formula is: ； The organelles are lysosomes, mitochondria and nucleoli.
2. The use according to claim 1, characterized in that The fluorescent probe is used to observe cellular oxidative damage.
3. Use of a benzothiazole fluorescent probe in the preparation of a reagent for simultaneous imaging of multiple organelles, characterized in that: The structural formula of the fluorescent probe is: ； The organelles are lysosomes, mitochondria and nucleoli.
4. The use according to claim 1 or 3, characterized in that The concentration of the fluorescent probe for staining cells is 1-15 μM.
5. The use according to claim 1 or 3, characterized in that The fluorescent probe is excited with a single excitation light.
6. Use of a benzothiazole fluorescent probe in the preparation of a tumor diagnostic reagent, characterized in that: The structural formula of the fluorescent probe is: 。 7. Use of a benzothiazole fluorescent probe in the preparation of a fluorescent imaging agent for tumors, characterized in that: The structural formula of the fluorescent probe is: 。