A wash-free lipid droplet fluorescent probe, its preparation method and application

Abstract

This invention belongs to the field of chemical and pharmaceutical technology, specifically relating to a wash-free lipid droplet fluorescent probe, its preparation method, and its application. The wash-free lipid droplet fluorescent probe of this invention is structurally non-fluorescent in cell culture medium. After rapid endocytosis, it quickly enters the cell, rapidly enters the lipid droplet, and releases bright red fluorescence. It possesses advantages such as large Stokes shift, good biocompatibility, high photostability, and strong fluorescence signal-to-noise ratio, making it suitable for lipid droplet imaging. The preparation method of this invention for a large Stokes shift, long duration, wash-free lipid droplet fluorescent probe has advantages such as fewer synthesis steps and simple post-processing.

Description

Technical Field

[0001] This invention belongs to the field of chemical and pharmaceutical technology, specifically relating to a wash-free lipid droplet fluorescent probe, its preparation method, and its application. Background Technology

[0002] Lipid droplets are the main intracellular lipid storage organs and are associated with many physiological processes, various metabolic diseases, and cancer. Therefore, tracking the state and behavior of lipid droplets is of great significance. Studying the dynamic interactions between lipid droplets and other organelles can help us better understand the function of lipid droplets, and appropriate lipid droplet tools can specifically trace lipid droplets, which is the molecular basis.

[0003] Fluorescent probes play a crucial role in scientific research and applications, offering advantages such as real-time imaging and depth visualization, making them widely used in fields like bioimaging, drug delivery, and biomolecular sensing. Currently, several commercially available lipid droplet-targeting probes, such as Nile Red and BODIPY 493 / 503, are widely used for specific lipid droplet imaging. However, these probes still have several shortcomings: 1) The Stokes shift of these probes is generally small, easily causing fluorescence crosstalk, which hinders the differentiation of fluorescence signals and makes multicolor imaging difficult; 2) The probes are easily expelled by lipid droplets, making it difficult to achieve long-term lipid droplet fluorescence imaging, thus hindering real-time monitoring of intracellular lipid droplet metabolism; 3) In cell culture media, high background noise easily generates non-specific fluorescence, requiring cumbersome washing steps to remove background interference, affecting observation results. Therefore, there is an urgent need to develop lipid droplet fluorescent probes with large Stokes shifts, long durations, and wash-free characteristics to enable specific imaging of lipid droplets and real-time monitoring of intracellular metabolism. Summary of the Invention

[0004] One of the objectives of this invention is to provide a wash-free lipid droplet fluorescent probe with advantages such as large Stokes shift and good biocompatibility.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A wash-free lipid droplet fluorescent probe, the fluorescent probe having the following structural formula:

[0007]

[0008] The second objective of this invention is to provide a method for preparing a wash-free lipid droplet fluorescent probe.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The above-described method for preparing the wash-free lipid droplet fluorescent probe includes the following steps:

[0011]

[0012] (1) Compound 1 was dissolved in organic solvent 1, ethyl acetoacetate was added, and the reaction was stirred to obtain compound 2;

[0013] (2) Dissolve the above compound 2 in organic solvent 2, add Lawson's reagent, and stir the reaction to obtain compound 3;

[0014] (3) Dissolve the above compound 3 in organic solvent 3, add triethylamine, malononitrile and silver nitrate, and stir to react to obtain a fluorescent probe.

[0015] Further, in step (1), the stirring reaction temperature is 100-120℃ and the stirring reaction time is 5-6h; in step (2), the stirring reaction temperature is 100-120℃ and the stirring reaction time is 3-4h; in step (3), the stirring reaction temperature is room temperature and the stirring reaction time is 3-4h.

[0016] Further, the molar ratio of compound 1 and ethyl acetoacetate in step (1) is 1:(1-2).

[0017] Furthermore, the molar ratio of compound 2 and Lawson's reagent in step (2) is 1:(1-3).

[0018] Further, the molar ratio of compound 3, triethylamine, malononitrile and silver nitrate in step (3) is 1:(0.5-0.6):(1-3):(1-3).

[0019] Further, the organic solvent 1 is p-toluenesulfonic acid; the organic solvent 2 is toluene; and the organic solvent 3 is acetonitrile.

[0020] The third objective of this invention is to provide an application of a wash-free lipid droplet fluorescent probe in the specific labeling of lipid droplets.

[0021] To achieve the above objectives, the present invention adopts the following technical solution:

[0022] The above-described application of the wash-free lipid droplet fluorescent probe in the specific labeling of lipid droplets.

[0023] Compared with the prior art, the beneficial effects of this invention are as follows:

[0024] (1) The fluorescent probe of the present invention is a wash-free type. It is in a fluorescent quenched state in the cell culture medium. After rapid entry into the cell through endocytosis, the probe molecules quickly enter the lipid droplet and release bright red fluorescence, which can intuitively and dynamically achieve the purpose of lipid droplet-specific fluorescence imaging.

[0025] (2) The fluorescent probe of the present invention has the advantages of good biocompatibility, large Stokes shift, high photostability, strong targeting performance and high fluorescence signal-to-noise ratio.

[0026] (3) The preparation method of the fluorescent probe of the present invention has the advantages of fewer synthesis steps and simple post-processing. Attached Figure Description

[0027] Figure 1 This is a high-resolution mass spectrometry of the fluorescent probe prepared in Example 1;

[0028] Figure 2 These are the fluorescence spectra of the fluorescent probe prepared in Example 1 in solvents of different polarities;

[0029] Figure 3 These are the fluorescence spectra of the fluorescent probe prepared in Example 1 in solvents of different viscosities;

[0030] Figure 4 This is a cytotoxicity diagram of the fluorescent probe prepared in Example 1;

[0031] Figure 5 These are lipid droplet imaging cell images of the fluorescent probe prepared in Example 1 and the comparative fluorescent probe;

[0032] Figure 6 This is a cell image of a long-term lipid droplet imaging using a fluorescent probe prepared in Example 1. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0034] Example 1

[0035] A wash-free lipid droplet fluorescent probe, wherein the structural formula of the wash-free lipid droplet fluorescent probe is as follows:

[0036]

[0037] A method for preparing a wash-free lipid droplet fluorescent probe, the specific steps of which are as follows:

[0038] (1) Compound 1 (206 mg, 1 mmol) was dissolved in p-toluenesulfonic acid solution (10 mL), and ethyl acetoacetate (190 μL, 1.5 mmol) was added. The mixture was stirred at 100 °C for 5 h. The precipitate was rapidly filtered, washed with ice water, and the solvent was removed under reduced pressure. The mixture was then separated by column chromatography and dried under reduced pressure to obtain compound 2 (yield 73.5%).

[0039]

[0040] (2) Compound 2 (272 mg, 1 mmol) was dissolved in toluene solution (5 mL), Lawson's reagent (808 mg, 2 mmol) was added, and the mixture was stirred at 100 °C for 3 h. The mixture was extracted with water and ethyl acetate, the organic layer was collected, evaporated under reduced pressure, separated by column chromatography, and dried under reduced pressure to obtain compound 3 (yield 65.4%).

[0041]

[0042] (3) Compound 3 (288 mg, 1 mmol) was dissolved in acetonitrile solution (5 mL), and triethylamine (70 μL, 0.5 mmol), silver nitrate (255 mg, 1.5 mmol), and malononitrile (99 mg, 1.5 mmol) were added. The mixture was stirred at room temperature for 3 h, extracted with water and ethyl acetate, and the organic layer was collected. The organic layer was evaporated to dryness under reduced pressure, separated by column chromatography, and the solvent was removed under reduced pressure to obtain the fluorescent probe (yield 68.7%). 1 H NMR (300MHz, CDCl3) δ10.11(s,1H),6.97(s,1H),6.00(s,1H),3.82(s,3H),3.40(s,8H),1.16(dt,J=7.0Hz,6H). 13 C NMR(75MHz,DMSO-d6)δ170.07,151.83,149.29,142.05,133.98,116.58,115.90,111.14,107.60,102.67,95.05,47.70,4 6.90,45.72,45.09,44.31,40.85,40.57,40.30,40.14,40.02,39.74,39.46,39.19,18.97,10.69,9.82.ESI-MS(positive ion mode):m / z[M+H] + :calcd:386.1829; obsd:386.1841. For high-resolution mass spectrometry details, please refer to [link to high-resolution mass spectrometry]. Figure 1 ).

[0043]

[0044] Example 2

[0045] A wash-free lipid droplet fluorescent probe, wherein the structural formula of the wash-free lipid droplet fluorescent probe is as follows:

[0046]

[0047] A method for preparing a wash-free lipid droplet fluorescent probe, the specific steps of which are as follows:

[0048] (1) Compound 1 (206 mg, 1 mmol) was dissolved in p-toluenesulfonic acid solution (10 mL), ethyl acetoacetate (127 μL, 1 mmol) was added, and the mixture was stirred at 100 °C for 5 h. The precipitate was rapidly filtered, washed with ice water, and the solvent was removed under reduced pressure. The mixture was then separated by column chromatography and dried under reduced pressure to obtain compound 2 (yield 72.4%).

[0049]

[0050] (2) Compound 2 (272 mg, 1 mmol) was dissolved in toluene solution (5 mL), Lawson's reagent (404 mg, 1 mmol) was added, and the mixture was stirred at 100 °C for 3 h. The mixture was extracted with water and ethyl acetate, the organic layer was taken, evaporated under reduced pressure, separated by column chromatography, and dried under reduced pressure to obtain compound 3 (yield 64.7%).

[0051]

[0052] (3) Compound 3 (288 mg, 1 mmol) was dissolved in acetonitrile solution (5 mL), and triethylamine (70 μL, 0.5 mmol), silver nitrate (170 mg, 1 mmol) and malononitrile (66 mg, 1 mmol) were added. The mixture was stirred at room temperature for 3 h, extracted with water and ethyl acetate, and the organic layer was collected. The organic layer was evaporated under reduced pressure, separated by column chromatography, and the fluorescent probe was obtained after removing the solvent under reduced pressure (yield 64.3%).

[0053]

[0054] Example 3

[0055] A wash-free lipid droplet fluorescent probe, wherein the structural formula of the wash-free lipid droplet fluorescent probe is as follows:

[0056]

[0057] A method for preparing a wash-free lipid droplet fluorescent probe, the specific steps of which are as follows:

[0058] (1) Compound 1 (206 mg, 1 mmol) was dissolved in p-toluenesulfonic acid solution (10 mL), and ethyl acetoacetate (254 μL, 2 mmol) was added. The mixture was stirred at 100 °C for 5 h. The precipitate was rapidly filtered, washed with ice water, and the solvent was removed under reduced pressure. The mixture was then separated by column chromatography and dried under reduced pressure to obtain compound 2 (yield 71.9%).

[0059]

[0060] (2) Compound 2 (272 mg, 1 mmol) was dissolved in toluene solution (5 mL), Lawson's reagent (1212 mg, 3 mmol) was added, and the mixture was stirred at 100 °C for 3 h. The mixture was extracted with water and ethyl acetate, the organic layer was collected, evaporated under reduced pressure, separated by column chromatography, and dried under reduced pressure to obtain compound 3 (yield 62.9%).

[0061]

[0062] (3) Compound 3 (288 mg, 1 mmol) was dissolved in acetonitrile solution (5 mL), and triethylamine (58 μL, 0.6 mmol), silver nitrate (510 mg, 3 mmol) and malononitrile (198 mg, 3 mmol) were added. The mixture was stirred at room temperature for 3 h, extracted with water and ethyl acetate, and the organic layer was collected. The organic layer was evaporated under reduced pressure, separated by column chromatography, and the fluorescent probe was obtained after removing the solvent under reduced pressure (yield 60.9%).

[0063]

[0064] Comparative Example 1

[0065] Comparative Example 1 provides a green fluorescent lipid droplet probe, BODIPY 493 / 503.

[0066] Experimental Example 1

[0067] Fluorescence spectroscopy characterization:

[0068] (1) Preparation of test solution

[0069] The fluorescent probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM test stock solution for later use.

[0070] (2) Experimental process

[0071] The fluorescence spectral properties of the fluorescent probe in PBS buffer, solvents of different polarities, and environments of different viscosities were tested using a fluorescence spectrometer. The results are as follows: Figure 2 , Figure 3 As shown.

[0072] Figure 2 These are fluorescence spectra of the fluorescent probes prepared in the examples in different polar solvents. Figure 3 These are fluorescence spectra of the fluorescent probe prepared in Example 1 under different viscosity environments. The results show that the fluorescent probe prepared in Example 1 exhibits almost no fluorescence in PBS solution, but strong fluorescence in viscous or polar environments. This oil-specific fluorescence enhancement property of the fluorescent probe prepared in Example 1 eliminates interference from the fluorescent probe itself, which is beneficial for improving the signal-to-noise ratio. This also indirectly demonstrates the significant characteristic of the fluorescent probe being wash-free for lipid droplet imaging.

[0073] Experimental Example 2

[0074] Cytotoxicity assay:

[0075] (1) HepG2 cells were cultured in high-glucose DMEM medium containing 10% newborn calf serum and placed in an incubator at 37°C, 5% CO2, and 80% humidity. When the HepG2 cells reached 85% confluence, the old culture medium was discarded, and the HepG2 cells were washed twice with phosphate-buffered saline and digested with 1 mL of 0.25% trypsin. During this process, the cell status was carefully observed. When the cells detached in sheets, 2 mL of fresh culture medium was added immediately to stop the digestion. The cell suspension was transferred to a 4 mL centrifuge tube and centrifuged at 1000 rpm / min for 5 min at room temperature. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 mL of the appropriate culture medium to disperse the cells into a single state. 10 μL of the well-mixed cell resuspension was taken from a cell counting chamber and added to the well of the cell counting chamber for cell counting under a microscope. The cell concentration (number of cells per milliliter of cell suspension) and cell viability (percentage of live cells to total cells) were recorded based on the observed cell number.

[0076] (2) HepG2 cells were seeded at a density of 4000 cells / well in 96-well plates and cultured for 24 h in an incubator at 37°C, 5% CO2, and 80% humidity until confluence reached 70%. Then, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, and 50 μM of the fluorescent probe prepared in Example 1 were added. An equal volume of high-glucose DMEM medium was added to the blank control group. Each treatment was repeated in triplicate. After 24 h of treatment, MTT reagent was added to each well to a final concentration of 0.5 mg / mL. The plates were incubated in the dark for another 6 h. Then, 100 μL of DMSO was added to each well to dissolve the formazan precipitate. The optical density (OD) value was measured at 490 nm using a microplate reader. The results are as follows: Figure 4 As shown.

[0077] Figure 4 The figure shows the survival rate of HepG2 cells after adding different concentrations of the fluorescent probe prepared in Example 1. As can be seen from the figure, after incubating the fluorescent probe with HepG2 cells for 24 hours, the cell survival rate was still above 85% (0-50 μM), indicating that the fluorescent probe prepared in Example 1 has good biocompatibility.

[0078] Experimental Example 3

[0079] Lipid droplet imaging capability detection:

[0080] After counting the HepG2 cells obtained in step (1) of Experimental Example 2, they were added to a confocal microplate and cultured for 12 hours in an incubator at 37°C, 5% CO2, and 80% humidity. The comparative green fluorescent lipid droplet probe BODIPY 493 / 503 was then added and incubated for 30 minutes. After incubation, the fluorescent probe molecules were removed, and the cells were washed three times with PBS. The HepG2 cells obtained in step (1) of Experimental Example 2 were added to 1 mL of cell culture medium containing the fluorescent probe from Example 1 (10 μM) and incubated for 30 minutes in an incubator at 37°C and 5% CO2. No washing was required. The cells were then imaged under a laser confocal microscope + STED ONE laser confocal microscope. Fluorescence images of the lipid droplet fluorescent probes from the examples and the comparative lipid droplet fluorescent probes were collected. The collected fluorescence images were processed using ImageJ software, and superimposed or compared with changes in fluorescence intensity in the same region to determine the subcellular organelle location of the fluorescent probes. The results are shown in the figure.

[0081] Figure 5 The images show the imaging results using the lipid droplet fluorescent probe from Example 1 and the comparative lipid droplet fluorescent probe. Figure 6 This is a cell image obtained from long-term lipid droplet imaging using a fluorescent probe in Example 1. Figure 5 It can be seen that the colocalization coefficient between the fluorescent probe of Example 1 and the comparative fluorescent probe is 0.92. This indicates that they have similar accuracy in recognizing lipid droplets, and preliminarily verifies the effectiveness of the fluorescent probe of the example. Figure 6 It is evident that the fluorescent probe of this embodiment can specifically indicate lipid droplets 12 hours after staining. This indicates that the fluorescent probe of this embodiment can accurately image to cellular lipid droplets and also has good stability, maintaining its fluorescent properties for a long time, and can be used for long-term imaging of cellular lipid droplets. In addition, the experimental results also show that the background fluorescence of this fluorescent probe is very weak when applied to cells, and the probe with low background fluorescence can perform wash-free imaging, thereby reducing the damage to cells caused by repeated washing.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A wash-free lipid droplet fluorescent probe, characterized in that, The structural formula of the fluorescent probe is: 。 2. The method for preparing the wash-free lipid droplet fluorescent probe according to claim 1, characterized in that, The preparation method includes the following steps: (1) Compound 1 was dissolved in organic solvent 1, ethyl acetoacetate was added, and the reaction was stirred to obtain compound 2; (2) Dissolve the above compound 2 in organic solvent 2, add Lawson's reagent, and stir to react to obtain compound 3; (3) Dissolve the above compound 3 in organic solvent 3, add triethylamine, malononitrile and silver nitrate, and stir to obtain a fluorescent probe.

3. The method for preparing the wash-free lipid droplet fluorescent probe according to claim 2, characterized in that, In step (1), the stirring reaction temperature is 100-120℃ and the stirring reaction time is 5-6h; in step (2), the stirring reaction temperature is 100-120℃ and the stirring reaction time is 3-4h; in step (3), the stirring reaction temperature is room temperature and the stirring reaction time is 3-4h.

4. The method for preparing the wash-free lipid droplet fluorescent probe according to claim 2, characterized in that, The molar ratio of compound 1 and ethyl acetoacetate in step (1) is 1:(1-2).

5. The method for preparing the wash-free lipid droplet fluorescent probe according to claim 2, characterized in that, The molar ratio of compound 2 and Lawson's reagent in step (2) is 1:(1-3).

6. The method for preparing the wash-free lipid droplet fluorescent probe according to claim 2, characterized in that, The molar ratio of compound 3, triethylamine, malononitrile and silver nitrate in step (3) is 1:(0.5-0.6):(1-3):(1-3).

7. The method for preparing the wash-free lipid droplet fluorescent probe according to claim 2, characterized in that, Organic solvent 1 is p-toluenesulfonic acid; organic solvent 2 is toluene; and organic solvent 3 is acetonitrile.

8. The application of the wash-free lipid droplet fluorescent probe according to claim 1 in the specific labeling of lipid droplets for non-disease diagnosis or treatment purposes.

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