Washing-free lipid droplet fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of chemical medicines, and particularly relates to a washing-free lipid droplet fluorescent probe as well as a preparation method and application thereof. The washing-free lipid droplet fluorescent probe is in a non-fluorescence state in a cell culture solution, can quickly enter lipid droplets after quickly entering cells through endocytosis, can release bright red fluorescence, and has the advantages of large Stokes shift, good biocompatibility, high light stability, strong fluorescence signal-to-noise ratio, high sensitivity, high sensitivity and the like. And the method is suitable for lipid droplet imaging. The preparation method of the large-Stokes-shift, long-time-history and no-wash type lipid droplet fluorescent probe has the advantages of few synthesis steps, simple post-treatment process and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine, and particularly relates to a wash-free lipid droplet fluorescent probe and a preparation method and application thereof. Background Art

[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. Their advantages, such as real-time imaging and deep visualization, have led to their widespread application in fields such as bioimaging, drug delivery, and biomolecular sensing. Currently, a variety of commercial lipid droplet-targeted probes, such as Nile Red and BODIPY 493 / 503, are widely used for specific lipid droplet imaging. However, these probes still suffer from several drawbacks: 1) The probes' generally small Stokes shifts can easily cause fluorescence crosstalk, hindering the differentiation of fluorescent signals and complicating multicolor imaging; 2) the probes are easily expelled from lipid droplets, making long-term lipid droplet fluorescence imaging difficult, hindering real-time monitoring of intracellular lipid droplet metabolism; 3) the high background noise in cell culture media can easily generate nonspecific fluorescence, requiring tedious washing steps to remove background interference, which can compromise observational quality. Therefore, there is an urgent need to develop lipid droplet fluorescent probes with large Stokes shifts, long-term fluorescence duration, and wash-free properties to enable specific lipid droplet imaging 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 object, the present invention adopts the following technical solutions:

[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 object, the present invention adopts the following technical solutions:

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

[0011]

[0012] (1) dissolving compound 1 in organic solvent 1, adding ethyl acetoacetate, and stirring to react to obtain compound 2;

[0013] (2) dissolving the above compound 2 in an organic solvent 2, adding Lawesson's reagent, and stirring to react to obtain compound 3;

[0014] (3) The compound 3 is dissolved in an organic solvent 3, triethylamine, malononitrile and silver nitrate are added, and the mixture is stirred to react to obtain a fluorescent probe.

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

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

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

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

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

[0020] A third object of the present invention is to provide a wash-free lipid droplet fluorescent probe for use in specifically labeling lipid droplets.

[0021] To achieve the above object, the present invention adopts the following technical solutions:

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

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The fluorescent probe of the present invention is a wash-free type and is in a fluorescence quenching state in cell culture medium. After rapidly entering the cell through endocytosis, the probe molecule quickly enters the lipid droplets and releases 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 is a high-resolution mass spectrum of the fluorescent probe prepared in Example 1;

[0028] Figure 2 is the fluorescence spectrum of the fluorescent probe prepared in Example 1 in solvents of different polarities;

[0029] Figure 3 is the fluorescence spectrum of the fluorescent probe prepared in Example 1 in solvents of different viscosities;

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

[0031] Figure 5 This is a lipid droplet imaging cell image of the fluorescent probe prepared in Example 1 and the comparative example 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 DESCRIPTION

[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 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 quickly filtered and washed with ice water. The solvent was removed under reduced pressure, and the mixture was 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, see 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), Lawesson'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, and the organic layer was evaporated to dryness under reduced pressure. The mixture was 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 evaporated to dryness under reduced pressure. The mixture was separated by column chromatography, and the solvent was removed under reduced pressure to obtain a fluorescent probe (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), Lawesson'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, and the organic layer was evaporated to dryness under reduced pressure. The mixture was 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 evaporated to dryness under reduced pressure. The mixture was separated by column chromatography, and the solvent was removed under reduced pressure to obtain a fluorescent probe (yield: 60.9%).

[0063]

[0064] Comparative Example 1

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

[0066] Test 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) Test process

[0071] The fluorescence spectrometer was used to test the fluorescence spectrum properties of the fluorescent probe in PBS buffer, solvents of different polarities, and environments of different viscosities. The results are as follows: Figure 2 、 Figure 3 As shown.

[0072] Figure 2 1 is the fluorescence spectrum of the fluorescent probe prepared in the example in solvents of different polarities. Figure 3 The following are fluorescence spectra of the fluorescent probe prepared in Example 1 in different viscosity environments. These results demonstrate that the fluorescent probe prepared in Example 1 exhibits almost no fluorescence in PBS solution, but exhibits strong fluorescence in more viscous or polar environments. This lipid-specific fluorescence enhancement of the fluorescent probe prepared in Example 1 eliminates interference from the fluorescent probe itself, improving the signal-to-noise ratio. This also indirectly demonstrates the fluorescent probe's remarkable wash-free nature for lipid droplet imaging.

[0073] Test 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 confluence of HepG2 cells reached 85%, the old culture medium was discarded, the HepG2 cells were rinsed twice with phosphate buffer, and 1 mL of 0.25% trypsin was added for digestion. During the process, the state of the cells was carefully observed. When the cells fell off in pieces, 2 mL of fresh culture medium was immediately added to stop the digestion. The cell suspension was transferred to a 4 mL centrifuge tube and centrifuged at 1000 rpm / min at room temperature for 5 minutes. After centrifugation, the supernatant was discarded and the cells were resuspended in 1 mL of the corresponding culture medium to disperse the cells into a single state. Take a cell counting plate, aspirate 10 μL of the mixed cell suspension, add it dropwise to the well of the cell counting plate, and count the cells under a microscope. According to the number of cells observed, the cell concentration (the number of cells in each mL of cell suspension) and cell viability (the proportion of live cells to total cells) were recorded.

[0076] (2) HepG2 cells were seeded into 96-well plates at a density of 4000 cells / well and cultured in an incubator at 37°C, 5% CO2, and 80% humidity for 24 h. When the confluence reached 70%, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, and 50 μM of the fluorescent probe prepared in Example 1 were added, respectively. The blank control group was added with an equal amount of high-glucose DMEM medium. Three replicate wells were set for each treatment. After 24 h of treatment, methyl thiazolium tetrazolium (MTT) reagent was added to each well to a final concentration of 0.5 mg / mL. The cells were incubated in the dark for another 6 h. 100 μL of DMSO was added to each well to dissolve the generated formazan precipitate. The optical density (OD) value was measured at 490 nm using a microplate reader. The results are shown in FIG. Figure 4 shown.

[0077] Figure 4 This figure shows the survival rate of HepG2 cells incubated with different concentrations of the fluorescent probe prepared in Example 1. As shown in the figure, after 24 hours of incubation with the fluorescent probe, the cell survival rate was still above 85% (0-50 μM), indicating that the fluorescent probe prepared in Example 1 has good biocompatibility.

[0078] Test Example 3

[0079] Lipid droplet imaging capability detection:

[0080] After the HepG2 cells obtained in step (1) of Experimental Example 2 were counted, they were added to a confocal dish and cultured in an incubator at 37°C, 5% CO2, and 80% humidity for 12 hours. The comparative green fluorescent lipid droplet probe BODIPY 493 / 503 was added and incubated for 30 minutes. After the incubation, the fluorescent probe molecules were removed and rinsed 3 times with PBS. The HepG2 cells obtained in step (1) of Experimental Example 2 were added with 1 mL of cell culture solution containing the fluorescent probe (10 μM) of Example 1 and incubated in an incubator at 37°C and 5% CO2 for 30 minutes without washing. The cells were placed under a laser confocal microscope + STED ONE laser confocal microscope for imaging. Fluorescent images of the example lipid droplet fluorescent probe and the comparative example lipid droplet fluorescent probe were collected respectively. The collected fluorescent images were processed using Image J software, and the fluorescence intensity changes in the same area were compared to determine the subcellular organelle position of the fluorescent probe. 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 of the fluorescent probe in Example 1 and the comparative fluorescent probe is 0.92. This shows that they have similar accuracy in identifying lipid droplets, which preliminarily verifies the effectiveness of the fluorescent probe in the example. Figure 6 As can be seen, the fluorescent probe of this example can specifically identify lipid droplets 12 hours after staining. This indicates that the fluorescent probe of this example can accurately image cellular lipid droplets and has good stability, maintaining its fluorescence properties for a long time, making it suitable 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. The probe with low background fluorescence can be used for wash-free imaging, thereby reducing the damage caused by multiple cell washes.

[0082] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions 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 comprises the following steps: (1) dissolving compound 1 in organic solvent 1, adding ethyl acetoacetate, and stirring to react to obtain compound 2; (2) dissolving the above compound 2 in an organic solvent 2, adding Lawesson's reagent, and stirring to react to obtain compound 3; (3) The compound 3 is dissolved in an organic solvent 3, triethylamine, malononitrile and silver nitrate are added, and the mixture is stirred to react 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° C., and the stirring reaction time is 5-6 h; in step (2), the stirring reaction temperature is 100-120° C., and the stirring reaction time is 3-4 h; in step (3), the stirring reaction temperature is room temperature, and the stirring reaction time is 3-4 h.

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 to 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 to Lawesson'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: The organic solvent 1 is p-toluenesulfonic acid; the organic solvent 2 is toluene; and the organic solvent 3 is acetonitrile.

8. Use of the wash-free lipid droplet fluorescent probe according to claim 1 in specific labeling of lipid droplets.

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