Lysosome fluorescent probe and application thereof

Abstract

The invention discloses a lysosome fluorescent probe and application thereof. The chemical name of the fluorescent probe disclosed by the invention is (Z)-4-(6-methoxynaphthalene acetamide)-2-(pyridine)-4-acrylonitrile. The probe has obvious fluorescence change in an aggregation state, and can realize two-channel imaging of lysosome. The fluorescent probe can be used for observing lysosome damage. Therefore, the compound can be used for preparing a tumor diagnosis reagent, a tumor fluorescence imaging reagent or a radioactive imaging reagent.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical medicine, and in particular relates to a lysosome fluorescent probe and application thereof. Background Art

[0002] As an important organelle, lysosomes have the function of dissolving and decomposing substances inside and outside the cell. When lysosomes are damaged or dysfunctional, their normal decomposition and dissolution functions are affected. Lysosomal damage can cause metabolic disorders of intracellular substances, release of cytotoxic substances, aggravated inflammatory responses, abnormal immune regulation, etc., which in turn cause a variety of diseases, such as infection, inflammation and tumors. Therefore, studying lysosomal damage can provide important basis for cell biology and disease development, and promote the development of biology and medicine.

[0003] Currently, the lysosomal autophagy process can be observed through imaging methods such as electron microscopy. However, this will damage the sample. The fluorescence method has attracted widespread attention because of its ease of operation and the ability to observe the dynamic process of organelles in situ, non-destructively, and in real time.

[0004] The current lysosomal fluorescent probes are often pH-sensitive probes, which can target lysosomes through the strong acidity of lysosomes. Lysosomal damage is observed by changes in lysosomal fluorescence intensity during oxidative damage, and the observation results are greatly affected by the excitation light and instrument errors. Dual-color fluorescent probes can reduce measurement errors by changing the ratio of fluorescence intensities of two channels, and realize dual-channel observation of lysosomal damage. However, at present, there are few probes that can observe lysosomal damage in two colors. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a lysosomal fluorescent probe and its application. The fluorescent probe of the present invention can detect lysosomal oxidative damage through fluorescence changes in two channels, avoiding interference from other fluorescence errors. Since lysosomal damage is related to a variety of diseases (cancer, etc.), the fluorescent probe of the present invention 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 purpose of the present invention is achieved through the following technical solution:

[0007] The present invention provides a lysosomal fluorescent probe, whose chemical name is: (Z)-4-(6-methoxynaphthylacetamide)-2-(pyridine)-4-acrylonitrile; its structural formula is:

[0008]

[0009] The present invention also provides the use of the fluorescent probe in imaging lysosomes of living cells cultured in vitro.

[0010] The fluorescent probe of the present invention is a probe with good membrane permeability. When used, the fluorescent probe contacts with cells, passes through the cell membrane, and can image lysosomes after the cells are stained.

[0011] The present invention also provides the use of the fluorescent probe in specifically marking cell organelles.

[0012] The cell organelle is a lysosome.

[0013] The present invention also provides the use of the fluorescent probe in preparing tumor diagnostic reagents.

[0014] The present invention also provides the use of the fluorescent probe in preparing fluorescent imaging agents or radioactive imaging agents for tumors.

[0015] The concentration of the fluorescent probe for staining cells is 1-15 μM. This concentration has low cytotoxicity and good imaging effect.

[0016] The fluorescent probe is used to observe lysosomal damage through fluorescence changes when cells are damaged by oxidation.

[0017] Beneficial effects:

[0018] The fluorescent probe of the present invention introduces nitrile pyridine derivatives, so that the molecule has good optical properties. In addition, the introduction of the naphthalene ring causes its fluorescence to red-shift. Since the probe red-shifts in aqueous solution, dual-color imaging of lysosomes is achieved. Since lysosomes are related to various diseases (cancer, etc.), the fluorescent probe of the present invention can be used to prepare tumor diagnostic reagents, and to prepare fluorescent imaging reagents or radioactive imaging reagents for tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The absorption spectra of 4-PN (10 μM) in different solvents ( Figure 1 A) and fluorescence spectra ( Figure 1 B).

[0020] Figure 2 Confocal fluorescence images of normal cultured and fixed HeLa cells stained with 4-PN (5 μM).

[0021] Figure 3 Fluorescence images of HeLa cells co-stained with 4-PN (5 μM) and LTDR (0.2 μM) and the fluorescence intensity curve at the underlined position in the fluorescence channel.

[0022] Figure 4 Confocal fluorescence images of normal cultured and oxidatively damaged HeLa cells stained with 4-PN (5 μM) and the corresponding fluorescence intensity ratio of the blue light channel to the green light channel. DETAILED DESCRIPTION

[0023] 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.

[0024] The substances in each example are all commercially available products.

[0025] Among them, 4-pyridineacetonitrile and others were purchased from J.B. & K. Technology Co., Ltd.

[0026] The absorption spectrum was tested by a Hitachi U-2910 spectrophotometer, the fluorescence spectrum was tested by a Hitachi F-2700 spectrophotometer, and the cell imaging instrument was a Lecia SP5.

[0027] Example 1 Synthesis of fluorescent probe (abbreviated as 4-PN)

[0028]

[0029] Dissolve 4-pyridineacetonitrile (1mmol) and 6-methoxy-2-naphthaldehyde (1mmol) in 20mL methanol and stir at room temperature in a flask for 1h. After stirring, add 3 drops of piperidine, reflux at 85℃ for 8h, cool to room temperature, and wash with petroleum ether. Use a mixture of CH2Cl2 and CH3OH (the volume ratio of CH2Cl2 and CH3OH is 9:1 to 6:1) as the eluent, perform column chromatography separation and purification, and obtain 0.22g of yellow solid, chemically named: (Z)-4-(6-methoxynaphthylacetamide)-2-(pyridine)-4-acrylonitrile, that is, an organic compound molecule, referred to as 4-PN.

[0030] By analyzing the hydrogen spectrum of the compound, it is confirmed that the structure of the above compound is correct. The results are as follows:

[0031] 1 H NMR (400MHz, CDCl3) δ (ppm): 8.71 (d, J = 4.0Hz, 2H), 8.26 (s, 1H), 8.12-8.15 (m, 1H),7.81-7.84(m,3H),7.60(d,J=1.0Hz,2H),7.16-7.32(m,2H),2.95(s,3H).

[0032] Example 2 Photophysical properties test experiment of fluorescent probe 4-PN in different solvents

[0033] Use different types of organic solvents (see Figure 1 ) Prepare a test solution containing 10 μM 4-PN, test the absorption spectrum of the solution with a UV spectrophotometer, and measure the fluorescence spectrum with a fluorescence spectrophotometer. Figure 1 .

[0034] from Figure 1 It can be seen that the fluorescent probe 4-PN has a large absorbance value at 300-420nm ( Figure 1 A), with a larger fluorescence peak at 410-610nm ( Figure 1 B), which indicates that the fluorescent compound can be excited by light of 300-420nm, and its emission spectrum ranges from 410-610nm.

[0035] Example 3 Imaging experiments of fluorescent probe 4-PN in living cells and fixed cells

[0036] 1. Culture of HeLa cells

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

[0038] HeLa cells were adherently cultured in a high-glucose culture medium (ThermoFisher) containing 10% fetal bovine serum in a 37° C., 5% CO 2 saturated humidity incubator. The culture medium was replaced every 2-3 days and subculture was performed.

[0039] When the cells grow to the logarithmic phase, culture them in sections:

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

[0041] ② Wash the cells in the 100mL cell bottle three times with PBS, digest with 1mL 0.25% trypsin (gibco) for 3-5 minutes, pour out the trypsin carefully, add fresh culture medium, blow evenly and count the cells, and control the cell density by adding culture medium to make the final cell concentration of 1×10 per mL. 5 Then, the cells were inoculated into the culture dish containing the cover glass and placed in a 5% CO2 incubator to allow the cells to grow close to the culture dish. After the HeLa cells grew on the plate and covered the cover glass, they were used for cell experiments.

[0042] 2. Live cell imaging:

[0043] 4-PN was prepared into a 1mM solution with DMSO. After the HeLa cells crawled and covered the coverslip, the active HeLa cells were incubated in a culture medium containing 5μM 4-PN (high-glucose culture medium containing 10% fetal bovine serum, ThermoFisher) (incubation conditions: 37°C, 5% CO2) for 20 minutes, and then rinsed twice with PBS (8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4 dissolved in 800mL water), and the cells were imaged using a fluorescent confocal microscope. This experiment was the control group, with an excitation wavelength of 405nm and a light collection range of 410-470nm (blue light channel) and 500-600nm (green light channel).

[0044] 3. Fixed cell imaging:

[0045] After the HeLa cells have grown all over the coverslip, the active HeLa cells were treated with 4% paraformaldehyde at room temperature for 2.5 hours, washed twice with PBS, and then stained with PBS solution containing 5μM 4-PN for 20 minutes (condition: room temperature). After rinsing twice with PBS, the cells were imaged with a fluorescence confocal microscope. This experiment is the experimental group, with an excitation wavelength of 405nm and a light collection range of 410-470nm (blue light channel) and 500-600nm (green light channel).

[0046] Figure 2 Confocal fluorescence images of normal cultured and fixed HeLa cells stained with 4-PN (5 μM).

[0047] exist Figure 2 In the experiment, the fluorescence intensity of the cells in the control group was stronger in the green channel, weaker in the blue channel, and distributed in dots; in the fixed cells of the experimental group, the fluorescence intensity of the blue channel was enhanced and distributed in dots. Therefore, this probe can be used to distinguish normal cells from dead cells.

[0048] Example 4 Co-localization test of fluorescent probe 4-PN

[0049] The culture method of HeLa cells is the same as that in Example 3.

[0050] After HeLa cells have grown all over the coverslip, the active HeLa cells were incubated in a culture medium containing 5μM 4-PN (high-glucose culture medium containing 10% fetal bovine serum, ThermoFisher) (incubation conditions: 37°C, 5% CO2) for 20 minutes. After rinsing twice with PBS, 200nM LTDR (lysosomal deep red fluorescent probe, Bio-Sky) was added to the culture medium and incubated for 20 minutes. The cells were imaged using a fluorescence confocal microscope. Excitation wavelength: 405nm for green channel and 638nm for red channel; light collection range: 500-600nm for green channel and 640-740nm for red channel.

[0051] Results Figure 3 .in, Figure 3 A is the fluorescence image of HeLa cells co-stained with 4-PN (5 μM) and LTDR (0.2 μM); Figure 3 B is the fluorescence intensity curve at the underlined position in the fluorescence channel.

[0052] exist Figure 3 In the image, the cells emit 4-PN fluorescence in the green channel and LTDR fluorescence in the red channel, and the green and red lights overlap to a large extent. Figure 3 As can be seen in B, the fluorescence intensity of the red and green channels is consistent. The colocalization coefficient is 88%, indicating that the probe stains lysosomes.

[0053] This indicates that the fluorescent probe 4-PN can image lysosomes, has excellent lysosome tracing effects, and can specifically label lysosomes in cells.

[0054] Example 5 Experiment of observing lysosomal oxidative damage using fluorescent probe 4-PN

[0055] The culture method of HeLa cells is the same as that in Example 3.

[0056] Control group: After HeLa cells have grown all over the coverslip, the active HeLa cells were incubated in a culture medium containing 5μM 4-PN (high-glucose culture medium containing 10% fetal bovine serum, ThermoFisher) (incubation conditions: 37°C, 5% CO2) for 20 minutes, and after rinsing twice with PBS, the cells were imaged using a fluorescence confocal microscope. Excitation wavelength: 405nm, light collection range: 410-470nm (blue light channel) and 500-600nm (green light channel).

[0057] Experimental group: After HeLa cells have grown all over the coverslip, they were treated with 5mmol hydrogen peroxide (to induce cellular oxidative damage) for 30min, then stained with PBS solution containing 5μM 4-PN at room temperature for 20min, and finally imaged with a fluorescence confocal microscope. Excitation wavelength: 405nm, light collection range: 410-470nm (blue light channel) and 500-600nm (green light channel).

[0058] The experimental results are shown in Figure 4 .in, Figure 4 A is the fluorescence image of normal cells and oxidatively damaged cells. Figure 4 B is Figure 4 The fluorescence intensity ratio of the blue light channel to the green light channel corresponding to A. Figure 4 In A, the fluorescence intensity of the cells in the control group was stronger in the green light channel and weaker in the blue light channel. After treatment with 5mmol hydrogen peroxide, the fluorescence intensity of the blue light channel was significantly enhanced. Figure 4 The fluorescence intensity ratio in B increases (the fluorescence intensity values ​​of the two channels are directly read, and the fluorescence intensity ratio of the blue light channel to the green light channel is calculated). This shows that the probe can observe the lysosomal oxidative damage process.

[0059] 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 in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A lysosomal fluorescent probe, characterized in that: Its chemical name is: (Z)-4-(6-methoxynaphthoacetamide)-2-(pyridine)-4-acrylonitrile; its structural formula is:

2. Use of the lysosomal fluorescent probe according to claim 1 in imaging lysosomes of living cells cultured in vitro.

3. Use of the lysosomal fluorescent probe according to claim 1 in specifically labeling organelles.

4. The use according to claim 3, characterized in that: The cell organelle is a lysosome.

5. Use of the lysosomal fluorescent probe according to claim 1 in the preparation of tumor diagnostic reagents.

6. Use of the lysosomal fluorescent probe according to claim 1 in preparing fluorescent imaging agents or radioactive imaging agents for tumors.

7. The use according to any one of claims 2 to 6, characterized in that: The concentration of the fluorescent probe for staining cells is 1-15 μM.

8. The use according to claim 5 or 6, characterized in that: The fluorescent probe is used to observe lysosomal damage through fluorescence changes when cells are damaged by oxidation.

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