Pyridine fluorescent probe and application thereof

Abstract

The application discloses a pyridine fluorescent probe and application thereof. The chemical name of the fluorescent probe is (Z)-3-(6-methoxynaphthalene acetic amide)-2-(pyridine)-3-acrylonitrile. The probe has obvious fluorescence change in an aggregated state, and can realize double-channel imaging of lysosomes. The fluorescent probe can be used for observing lysosome damage through fluorescence change in cell oxidative damage. Therefore, the fluorescent probe can be used for preparing a tumor diagnosis reagent, and can be used for preparing a fluorescent imaging reagent or a radioactive imaging reagent for tumors.

Description

Technical Field

[0001] This invention belongs to the field of chemical and pharmaceutical technology, specifically relating to a pyridine-based fluorescent probe and its applications. Background Technology

[0002] As an important organelle, lysosomes have the function of dissolving and breaking down substances inside and outside the cell. When lysosomes are damaged or malfunction, their normal decomposition and dissolution functions are affected. Lysosomal damage can cause intracellular metabolic disorders, release of cytotoxic substances, exacerbation of inflammatory responses, and abnormal immune regulation, thereby leading to various diseases such as infections, inflammation, and tumors. Therefore, studying lysosomal damage can provide important evidence for cell biology and disease development, and promote the advancement of biology and medicine.

[0003] Currently, lysosomal autophagy can be observed using imaging methods such as electron microscopy. However, this can damage the sample. Fluorescence methods, on the other hand, have attracted widespread attention because they are easy to operate and can observe the dynamic processes of organelles in situ, non-destructively, and in real time.

[0004] Current lysosomal fluorescent probes are often pH-sensitive, allowing them to target lysosomes due to their strong acidity. However, lysosomal damage is observed by monitoring changes in fluorescence intensity during oxidative damage; this observation is significantly affected by excitation light and instrument errors. Dual-color fluorescent probes can reduce measurement errors by varying the ratio of fluorescence intensity between the two channels, enabling dual-channel observation of lysosomal damage. However, currently, probes capable of dual-color observation of lysosomal damage are relatively few. Summary of the Invention

[0005] Objective of this invention: The objective of this invention is to provide a pyridine-based fluorescent probe and its applications. This fluorescent probe can detect oxidative damage to lysosomes through fluorescence changes in two channels, avoiding interference from other fluorescence errors. Since lysosomal damage is associated with various diseases (such as cancer), this fluorescent probe can be used to prepare tumor diagnostic reagents for lysosomal oxidative damage, and to prepare fluorescent imaging reagents or radioactive imaging reagents for tumors.

[0006] Technical solution: The objective of this invention is achieved through the following technical solution:

[0007] This invention provides a pyridine-based fluorescent probe with the chemical name (Z)-3-(6-methoxynaphthylacetamide)-2-(pyridine)-3-acrylonitrile and the structural formula:

[0008]

[0009] This invention also provides the application of the above-mentioned pyridine fluorescent probes in imaging lysosomes of live cells cultured in vitro.

[0010] The fluorescent probe of the present invention is a probe with good membrane permeability. When in use, the fluorescent probe comes into contact with the cell and passes through the cell membrane. After the cell is stained, it can be used to image lysosomes.

[0011] This invention also provides the application of the above-mentioned pyridine fluorescent probes in the specific labeling of organelles.

[0012] The organelles mentioned are lysosomes.

[0013] This invention also provides the application of the above-mentioned pyridine fluorescent probes in the preparation of tumor diagnostic reagents.

[0014] This invention also provides the application of the above-mentioned pyridine fluorescent probes in the preparation of fluorescent imaging reagents or radioactive imaging reagents for tumors.

[0015] The concentration of the fluorescent probe used to stain cells is 1-15 μM. This concentration of staining exhibits low cytotoxicity and provides good imaging results.

[0016] The fluorescent probe observes lysosomal damage by detecting changes in fluorescence during cellular oxidative damage.

[0017] Beneficial effects:

[0018] The fluorescent probe of this invention possesses excellent optical properties by introducing a nitrile pyridine derivative. Furthermore, the introduction of a naphthalene ring causes a redshift in fluorescence. This redshift in aqueous solution enables two-color imaging of lysosomes. Since lysosomes are associated with various diseases (such as cancer), the fluorescent probe of this invention can be used to prepare tumor diagnostic reagents, fluorescent imaging reagents for tumors, or radioactive imaging reagents. Attached Figure Description

[0019] Figure 1 Absorption spectra of 3-PN (10 μM) in different solvents ( Figure 1 A) and fluorescence spectrum ( Figure 1 B).

[0020] Figure 2 Confocal fluorescence images of normally cultured and fixed HeLa cells stained with 3-PN (5 μM).

[0021] Figure 3 Fluorescence images of HeLa cells co-stained with 3-PN (5 μM) and LTDR (0.2 μM) and fluorescence intensity curves at the dashed positions in the fluorescence channels.

[0022] Figure 4 Confocal fluorescence images of normally cultured and oxidatively damaged HeLa cells stained with 3-PN (5 μM) and the corresponding fluorescence intensity ratios of the blue and green channels. Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0024] The substances used in each embodiment are all derived from commercially available products.

[0025] Among them, 3-pyridine acetonitrile and other substances were purchased from Bailingwei Technology Co., Ltd.

[0026] The absorption spectroscopy instrument was a Hitachi U-2910 spectrophotometer; the fluorescence spectroscopy instrument was a Hitachi F-2700 spectrophotometer; and the cell imaging instrument was a Lecia SP5.

[0027] Example 1: Synthesis of a fluorescent probe (abbreviated as 3-PN)

[0028]

[0029] 3-Pyridineacetonitrile (1 mmol) and 6-methoxy-2-naphthaldehyde (1 mmol) were dissolved in 20 mL of methanol and stirred at room temperature for 1 h in a flask. After stirring, 3 drops of piperidine were added, and the mixture was refluxed at 85 °C for 8 h. After cooling to room temperature, the solution was washed with petroleum ether. The solution was purified by column chromatography using a mixture of CH2Cl2 and CH3OH (volume ratio of CH2Cl2 to CH3OH 10:1 to 5:1) as the eluent to obtain 0.23 g of a yellow solid, chemically named (Z)-3-(6-methoxynaphthylacetamide)-2-(pyridine)-3-acrylonitrile, which is an organic compound molecule, abbreviated as 3-PN.

[0030] Analysis of the proton NMR spectrum of the compound confirmed its correct structure. The results are as follows:

[0031] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.97 (s, 1H), 8.65 (d, J = 4.0Hz, 1H), 8.25 (s, 1H), 8.12 (q, J = 3.0Hz, 1H), 8.01 (d, J = 4.0Hz, 1H), 7.83 (q, J = 2.6Hz, 2H), 7.69 (s, 1H), 7.42 (q, J = 4.0Hz, 1H), 7.17-7.23 (m, 2H), 3.97 (s, 3H).

[0032] Example 2: Photophysical property testing experiment of fluorescent probe 3-PN in different solvents

[0033] Different types of organic solvents were used (see [link to organic solvent list]). Figure 1A test solution containing 10 μM 3-PN was prepared. The absorption spectrum of the solution was measured using a UV spectrophotometer, and its fluorescence spectrum was measured using a fluorescence spectrophotometer. The results are shown below. Figure 1 .

[0034] from Figure 1 It can be seen that the fluorescent probe 3-PN has a large absorbance value in the range of 300-420 nm. Figure 1 A) exhibits a large fluorescence peak in the 410-610 nm range. Figure 1 B) This indicates that the fluorescent compound can be excited by light in the range of 300-420 nm, and its emission spectrum ranges from 410-610 nm.

[0035] Example 3: Imaging experiments of fluorescent probe 3-PN in live and fixed cells.

[0036] 1. Culture of HeLa cells

[0037] Human cervical cancer cells (HeLa) originated from Thermo Fisher Scientific.

[0038] HeLa cells were cultured in a high-glucose culture medium (ThermoFisher) containing 10% fetal bovine serum and in a saturated humidity incubator at 37°C and 5% CO2. The culture medium was changed every 2-3 days and the cells were passaged.

[0039] Once the cells have grown to the logarithmic growth phase, mount them onto a slide for culture.

[0040] ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol lamp, and then place it in a disposable 35mm petri dish for later use;

[0041] ② Wash the confluent cells in a 100mL cell culture flask three times with PBS, digest with 1mL of 0.25% trypsin (gibco) 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 / mL. 5 Each cell was seeded and then inoculated into a culture dish containing a coverslip. The dish was then incubated in a 5% CO2 incubator to allow the cells to grow in close contact with the culture dish. Once the HeLa cells had grown and completely covered the coverslip, they were used for cell experiments.

[0042] 2. Live-cell imaging

[0043] 3-PN was prepared into a 1 mM solution using DMSO. After HeLa cells had fully colonized the coverslip, the viable HeLa cells were incubated for 20 min in a culture medium containing 5 μM 3-PN (high glucose culture medium containing 10% fetal bovine serum, Thermo Fisher) (incubation conditions: 37℃, 5% CO2). The cells were then washed twice with PBS (8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 dissolved in 800 mL of water). The cells were then imaged using a fluorescence confocal microscope. This experiment served as a control group. The excitation wavelength was 405 nm, and the absorption range was 410-470 nm (blue channel) and 500-600 nm (green channel).

[0044] 3. Imaging of fixed cells

[0045] After HeLa cells had fully grown onto the coverslip, viable HeLa cells were treated with 4% paraformaldehyde at room temperature for 2.5 h, washed twice with PBS, and then stained and fixed with PBS solution containing 5 μM 3-PN for 20 min (condition: room temperature). After rinsing twice with PBS, the cells were imaged using a fluorescence confocal microscope. This experiment was the experimental group, with an excitation wavelength of 405 nm and a receiving range of 410-470 nm (blue channel) and 500-600 nm (green channel).

[0046] Experimental results:

[0047] Figure 2 Confocal fluorescence images of normally cultured and fixed HeLa cells stained with 3-PN (5 μM).

[0048] from Figure 2 As can be seen, the control group cells showed stronger fluorescence intensity in the green channel and weaker fluorescence intensity in the blue channel, with a dotted distribution; in the fixed cells of the experimental group, the blue channel fluorescence intensity was enhanced, and the cells also showed a dotted distribution. Therefore, this probe can be used to distinguish between normal cells and dead cells.

[0049] Example 4: Colocalization assay of fluorescent probe 3-PN in viable HeLa cells

[0050] The culture method for HeLa cells is the same as in Example 3.

[0051] After HeLa cells had fully colonized the coverslip, the viable HeLa cells were incubated in culture medium containing 5 μM 3-PN (high glucose culture medium containing 10% fetal bovine serum, Thermo Fisher) for 20 min (incubation conditions: 37℃, 5% CO2). After washing twice with PBS, 200 nM LTDR (lysosomal deep red fluorescent probe, Beyotime) was added to the culture medium and incubated for another 20 min. The cells were then imaged using a fluorescence confocal microscope. Excitation wavelengths: 405 nm for the green channel and 638 nm for the red channel; absorption range: 500-600 nm for the green channel and 640-740 nm for the red channel.

[0052] See results Figure 3 .in, Figure 3 Image A shows the fluorescence of HeLa cells co-stained with 3-PN (5 μM) and LTDR (0.2 μM); Figure 3 B represents the fluorescence intensity curve at the doodled position in the fluorescence channel.

[0053] exist Figure 3 In the study, cells emitted 3-PN fluorescence in the green channel and LTDR fluorescence in the red channel, with significant overlap between the green and red light. Figure 3 As shown in B, the fluorescence intensity of the red and green channels is consistent. Their colocalization coefficient is 86%, indicating that the probe stains lysosomes.

[0054] This indicates that the fluorescent probe 3-PN can image lysosomes, has excellent lysosomal tracking effect, and can specifically label intracellular lysosomes.

[0055] Example 5: Observation of Lysosomal Oxidative Damage by Fluorescent Probe 3-PN

[0056] The culture method for HeLa cells is the same as in Example 3.

[0057] Control group: After HeLa cells had fully colonized the coverslip, viable HeLa cells were incubated in culture medium containing 5 μM 3-PN (high glucose culture medium containing 10% fetal bovine serum, Thermo Fisher) for 20 min (incubation conditions: 37℃, 5% CO2). After washing twice with PBS, the cells were imaged using a fluorescence confocal microscope. Excitation wavelength: 405 nm; absorption range: 410-470 nm (blue channel) and 500-600 nm (green channel).

[0058] Experimental group: After HeLa cells had fully grown onto the coverslip, they were treated with 5 mmol hydrogen peroxide (to induce oxidative damage) for 30 min, then stained with PBS containing 5 μM 3-PN at room temperature for 20 min. Finally, imaging was performed using a fluorescence confocal microscope. Excitation wavelength: 405 nm; absorption range: 410-470 nm (blue channel) and 500-600 nm (green channel).

[0059] See results Figure 4 .in, Figure 4 A shows fluorescence images of normal cells and oxidatively damaged cells. Figure 4 B is Figure 4 The fluorescence intensity ratio of the blue channel to the green channel corresponding to A. Figure 4 In Figure A, the control group cells showed stronger fluorescence intensity in the green channel and weaker fluorescence intensity in the blue channel. After treatment with 5 mmol of hydrogen peroxide, the fluorescence intensity in the blue channel significantly increased. Figure 4 The fluorescence intensity ratio in B increases (by directly reading the fluorescence intensity values ​​of the two channels and calculating the fluorescence intensity ratio of the blue channel to the green channel). This indicates that the probe can observe the lysosomal oxidative damage process.

[0060] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A pyridine-based fluorescent probe, characterized in that, Its chemical name is (Z)-3-(6-methoxynaphthylacetamide)-2-(pyridine)-3-acrylonitrile; its structural formula is: 。 2. The application of the pyridine fluorescent probe according to claim 1 in imaging lysosomes of live cells cultured in vitro.

3. The use of the pyridine fluorescent probe of claim 1 in the preparation of fluorescent probes for labeling specific organelles, wherein the organelle is a lysosome.

4. The use of the pyridine fluorescent probe according to claim 1 in the preparation of fluorescent imaging reagents or radioactive imaging reagents for tumors.

5. The application according to any one of claims 2 to 4, characterized in that, The concentration of the fluorescent probe stained cells was 1-15 μM.

6. The application according to claim 4, characterized in that, The fluorescent probe observes lysosomal damage by detecting changes in fluorescence during cellular oxidative damage.

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