A phenothiazine-based near-infrared lipid droplet fluorescent dye, a preparation method and application thereof

Abstract

This invention discloses a phenothiazine-based near-infrared lipid droplet fluorescent dye, its preparation method, and its applications, belonging to the field of fluorescence sensing and detection technology. The phenothiazine-based near-infrared lipid droplet fluorescent dye is compound II with the following structural formula. In a low-polarity environment, the emission wavelength of this phenothiazine-based near-infrared lipid droplet fluorescent dye is located in the near-infrared region, exhibiting a large Stokes shift and strong fluorescence intensity. It can be used for specific imaging of lipid droplets within living cells, demonstrating strong specificity for lipid droplets and enabling imaging analysis of intracellular lipid droplet division and lipid migration. Leveraging the advantages of near-infrared emission, this dye can be used for in situ imaging of mouse breast cancer. Furthermore, the near-infrared lipid droplet fluorescent dye of this invention exhibits strong stability and significantly superior photobleaching resistance compared to Nile Red.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescent sensing detection technology, and particularly relates to a near-infrared lipid droplet fluorescent dye based on phenothiazine as well as a preparation method and application thereof. BACKGROUND

[0002] Lipid droplets are a class of organelles wrapped by phospholipid membranes, with a large number of proteins distributed on the surface and a large number of neutral lipids, including triglycerides and cholesterol esters, stored inside. Therefore, lipid droplets are considered as energy storage organelles. Compared with the hydrophilic environment in other organelles, the lipid droplet microenvironment has the characteristics of strong hydrophobicity, small polarity and high viscosity. Studies have shown that abnormal behavior of lipid droplet generation in cells is related to a variety of lipid metabolism diseases, such as obesity, fatty liver, cardiovascular disease and diabetes. Therefore, high-sensitivity imaging analysis of lipid droplets is helpful for the pathological research of related diseases.

[0003] At present, the fluorescence emission spectrum of commercially available lipid droplet fluorescent dyes covers the red, yellow and green regions. These dyes can be used for imaging research at the cell and tissue levels, but due to the short emission wavelength and poor light stability, they are not suitable for in vivo imaging analysis and dynamic analysis of lipid droplets. Moreover, the common commercial lipid droplet dyes Nile Red and BODIPY493 / 503 also have staining effects on other hydrophobic regions in cells (such as endoplasmic reticulum), and have poor specificity. In recent years, researchers have reported some lipid droplet-specific fluorescent dyes by taking advantage of the unique microenvironment of lipid droplets, but most of the dyes have a short emission wavelength (<600nm), which is not conducive to in vivo imaging (Chemical Communications, 2022, 58, 1495-1509); in addition, the stability and specificity of the dyes are also insufficient, and they are prone to react with active substances (Sensors and Actuators: B. Chemical, 2022. 365, 131937), which greatly limits the research of tissues and in vivo lipid droplets. Therefore, it is particularly urgent to develop a high-stability fluorescent lipid droplet dye with a wavelength in the near-infrared (650-900nm) region to improve the imaging depth of the dye. SUMMARY

[0004] In view of the deficiencies of the prior art, one of the purposes of the present application is to provide a near-infrared lipid droplet fluorescent dye based on phenothiazine, which has strong stability, near-infrared fluorescence emission in a small-polarity solvent, a large Stokes shift, and can be used for imaging analysis of the splitting and lipid migration behavior of intracellular lipid droplets; and solves the problems of short emission wavelength of commercial red lipid droplet dyes, poor in vivo imaging, poor stability and insufficient specificity in the prior art.

[0005] The purpose of the present application is achieved by the following technical solutions.

[0006] A near-infrared lipid droplet fluorescent dye based on phenothiazine, the chemical structural formula of which is shown below:

[0007]

[0008] The phenothiazine-based near-infrared lipid droplet fluorescent dye of this invention emits in the near-infrared region under low polarity conditions, exhibits a large Stokes shift, and possesses strong fluorescence intensity. This allows for specific imaging of lipid droplets within living cells, demonstrating high specificity for lipid droplets and enabling imaging analysis of intracellular lipid droplet division and lipid migration. Furthermore, the near-infrared lipid droplet fluorescent dye of this invention exhibits strong stability and significantly superior photobleaching resistance compared to Nile Red.

[0009] Another object of the present invention is to provide a method for preparing the phenothiazine-based near-infrared lipid droplet fluorescent dye, comprising the following steps:

[0010] 2-Methoxy-10-ethyl-10H-phenthiazine-3-carboxaldehyde (compound I), 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile and an organic base were dissolved in an anhydrous polar organic solvent and reacted at 50–90 °C for 4–12 h to obtain the near-infrared lipid droplet fluorescent dye (compound II).

[0011] The preparation method of the near-infrared lipid droplet fluorescent dye based on phenothiazine of the present invention is as follows:

[0012]

[0013] The present invention provides a simple and high-yield method for preparing near-infrared lipid droplet fluorescent dyes based on phenothiazine.

[0014] Preferably, the molar ratio of 2-methoxy-10-ethyl-10H-phenthiazine-3-carboxaldehyde, 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile to the organic base is (0.8-1.0):1:(1.1-1.5); and the molar volume ratio of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile to the anhydrous polar organic solvent is 1 mmol:(10-20) mL.

[0015] Preferably, the anhydrous polar organic solvent includes at least one of anhydrous acetonitrile, anhydrous methanol, anhydrous ethanol, acetone, tetrahydrofuran, dichloromethane, dimethylformamide, and dimethyl sulfoxide.

[0016] Preferably, the organic base includes at least one of aziridine and 1,4-oxaziridine.

[0017] Preferably, the near-infrared lipid droplet fluorescent dye is obtained after vacuum distillation and column chromatography purification after the reaction is completed.

[0018] Another object of the present invention is to provide the application of the phenothiazine-based near-infrared lipid droplet fluorescent dye in the field of fluorescence imaging of lipid droplets in living cells and in vivo. The near-infrared lipid droplet fluorescent dye can specifically label lipid droplets and achieve fluorescence imaging in living cells and in vivo.

[0019] Another object of the present invention is to provide the application of the phenothiazine-based near-infrared lipid droplet fluorescent dye in in situ imaging of mouse breast cancer.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The phenothiazine-based near-infrared lipid droplet fluorescent dye of this invention exhibits near-infrared fluorescence emission in a low-polarity environment, with an emission wavelength in the near-infrared region, a large Stokes shift (185 nm in 1,4-dioxane), and strong fluorescence intensity (fluorescence quantum yield of 0.55 in 1,4-dioxane). This allows for specific imaging of lipid droplets, demonstrating high specificity and enabling imaging analysis of intracellular lipid droplet division and lipid migration. Leveraging the advantages of near-infrared emission, this dye can be used for in situ imaging of mouse breast cancer. Furthermore, the near-infrared lipid droplet fluorescent dye of this invention exhibits strong stability and significantly superior photobleaching resistance compared to Nile Red. This addresses the problems of existing commercially available red lipid droplet dyes, such as short emission wavelengths, unsuitability for in vivo in situ imaging, poor stability, and insufficient specificity. Attached Figure Description

[0022] Figure 1 The 1H NMR spectrum of compound II prepared in Example 1;

[0023] Figure 2 The carbon NMR spectrum of compound II prepared in Example 1;

[0024] Figure 3 The absorption spectra of compound II prepared in Example 1 in different solvents are shown, where the horizontal axis represents wavelength and the vertical axis represents absorbance.

[0025] Figure 4 The fluorescence spectra of compound II prepared in Example 1 in different solvents are shown, where the horizontal axis represents wavelength and the vertical axis represents relative fluorescence intensity.

[0026] Figure 5 The fluorescence spectra of compound II prepared in Example 1 in mixed systems of 1,4-dioxane-tetrahydrofuran at different volume ratios are shown, where the horizontal axis represents wavelength and the vertical axis represents fluorescence intensity.

[0027] Figure 6 The image shows the fluorescence imaging of compound II prepared in Example 1 and BODIPY 493 / 503 after co-culturing in oleic acid-stimulated 4T1 cells. BODIPY 493 / 503 is a commercially available lipid droplet dye (green channel), and compound II is a near-infrared lipid droplet fluorescent dye (red channel). The overlay image is the superposition of the two channels, and the correlation is the calculation of the co-localization coefficient.

[0028] Figure 7 Fluorescence images of compound II prepared in Example 1 and Nile Red, each scanned 50 times in 4T1 cells;

[0029] Figure 8 The fluorescence intensity change curves of compound II prepared in Example 1 and Nile Red were each scanned 50 times in 4T1 cells;

[0030] Figure 9 Fluorescence imaging of lipid droplets in 4T1 cells of compound II prepared in Example 1;

[0031] Figure 10 This is an in situ imaging image of compound II prepared in Example 1 in a mouse breast cancer model. Detailed Implementation

[0032] The applicant will now provide a detailed description of the method of the present invention with reference to specific embodiments, in order to enable those skilled in the art to clearly understand the present invention. However, the following embodiments should not be construed in any way as limiting the scope of protection claimed in the present invention.

[0033] The method for synthesizing the phenothiazine-based near-infrared lipid droplet fluorescent dye of the present invention is as follows:

[0034]

[0035] Compound I was prepared according to the method described in the literature (Chem.Commun.,2020,56,2759-2762), and 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile was a commercially available product.

[0036] In some embodiments, the molar ratio of 2-methoxy-10-ethyl-10H-phenthiazine-3-carboxaldehyde, 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile to the organic base is (0.8-1.0):1:(1.1-1.5); and the molar volume ratio of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile to the anhydrous polar organic solvent is 1 mmol:(10-20) mL.

[0037] In some embodiments, the anhydrous polar organic solvent includes at least one of anhydrous acetonitrile, anhydrous methanol, anhydrous ethanol, acetone, tetrahydrofuran, dichloromethane, dimethylformamide, and dimethyl sulfoxide; the organic base includes azircyclohexane and / or 1,4-oxazircyclohexane.

[0038] Example 1

[0039] This embodiment provides a method for preparing a near-infrared lipid droplet fluorescent dye based on phenothiazine, the steps of which are as follows:

[0040] 2-Methoxy-10-ethyl-10H-phenthiazine-3-carboxaldehyde (compound I), 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile, and azircyclohexane in a molar ratio of 0.9:1:1.3 were dissolved in anhydrous acetonitrile (the molar volume of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile to anhydrous acetonitrile was 1 mmol: 15 mL). The mixture was then reacted at 70 °C for 8 h. After removing the solvent under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether: dichloromethane = 1:4, v / v) to obtain the near-infrared lipid droplet fluorescent dye (compound II) in yield of 50%–80%.

[0041] Example 2

[0042] This embodiment provides a method for preparing a near-infrared lipid droplet fluorescent dye based on phenothiazine, the steps of which are as follows:

[0043] 2-Methoxy-10-ethyl-10H-phenthiazine-3-carboxaldehyde (compound I), 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile, and azircyclohexane in a molar ratio of 0.8:1:1.1 were dissolved in anhydrous methanol (the molar volume of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile to anhydrous acetonitrile was 1 mmol: 10 mL). The mixture was then reacted at 50 °C for 12 h. After removing the solvent under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether: dichloromethane = 1:4, v / v) to obtain the near-infrared lipid droplet fluorescent dye (compound II).

[0044] Example 3

[0045] This embodiment provides a method for preparing a near-infrared lipid droplet fluorescent dye based on phenothiazine, the steps of which are as follows:

[0046] 2-Methoxy-10-ethyl-10H-phenthiazine-3-carboxaldehyde (compound I), 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile and 1,4-oxazacyclohexane in a molar ratio of 1:1:1.5 were dissolved in anhydrous ethanol (the molar volume of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile to anhydrous acetonitrile was 1 mmol: 20 mL). The mixture was then reacted at 90 °C for 4 h. After removing the solvent under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether: dichloromethane = 1:4, v / v) to obtain the near-infrared lipid droplet fluorescent dye (compound II).

[0047] Figure 1 and Figure 2 The images show the 1H and 1C NMR spectra of the near-infrared lipid droplet fluorescent dye (compound II) prepared in Example 1, respectively.

[0048] The near-infrared lipid droplet fluorescent dye (compound II) from Example 1 was dissolved in dimethyl sulfoxide to prepare a 5 mmol / L stock solution. Then, different test solutions were prepared as needed to detect changes in its fluorescence spectrum and fluorescence imaging within cells.

[0049] (1) Absorption spectra of compound II prepared in Example 1 in different solvents

[0050] Each time, 6 μL of the stock solution was taken and added to 3 mL of water, dimethyl sulfoxide, acetonitrile (MeCN), N,N-dimethylformamide (DMF), methanol (MeOH), 1,4-dioxane, tetrahydrofuran (THF), dichloromethane (DCM), and glycerol (Glycerol) to prepare a 10 μmol / L fluorescent probe test solution, and the ultraviolet absorption spectrum was measured.

[0051] Figure 3 The graphs show the absorption spectra of compound II prepared in Example 1 in different solvents, where the horizontal axis represents wavelength and the vertical axis represents absorbance. As can be seen from the graphs, the fluorescent dye of this invention exhibits similar ultraviolet absorption spectra in organic solvents other than water and glycerol, with the maximum absorption peak located between 496 nm and 532 nm.

[0052] (2) Fluorescence spectra of compound II prepared in Example 1 in different solvents

[0053] Each time, 6 μL of the stock solution was taken and added to 3 mL of water, dimethyl sulfoxide, acetonitrile (MeCN), N,N-dimethylformamide (DMF), methanol (MeOH), 1,4-dioxane, tetrahydrofuran (THF), dichloromethane (DCM), and glycerol (Glycerol) respectively to prepare a 10 μmol / L fluorescent probe test solution. The fluorescence spectrum was then measured, with the excitation wavelength λ... ex =470nm, and the excitation / emission slit width is 2nm.

[0054] Figure 4 The figures show the fluorescence spectra of compound II prepared in Example 1 in different solvents, where the horizontal axis represents wavelength and the vertical axis represents relative fluorescence intensity. As can be seen from the figures, the fluorescence intensity of compound II is greatly affected by solvent polarity. In low-polarity environments, such as in 1,4-dioxane, the fluorescence intensity is extremely strong, with a fluorescence emission wavelength of 685 nm and a Stokes shift of 185 nm. However, in solvents with slightly higher polarity, such as tetrahydrofuran, the fluorescence intensity is very weak. This indicates that compound II has a very sensitive response to low-polarity environments. Considering the low-polarity environment inside lipid droplets, the inventors hypothesize that compound II can be used for specific imaging of lipid droplets.

[0055] (3) Fluorescence spectra of compound II prepared in Example 1 in mixed systems of 1,4-dioxane-tetrahydrofuran at different volume ratios

[0056] Each time, 6 μL of the stock solution was taken and added to 11 separate mixtures of 1,4-dioxane-tetrahydrofuran (the total volume of 1,4-dioxane-tetrahydrofuran in each mixture was 3 mL, and the volume percentages of tetrahydrofuran in the mixtures were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%) to prepare a 10 μmol / L fluorescent probe test solution. The fluorescence spectrum was then measured, with the excitation wavelength λ... ex =470nm, and the excitation / emission slit width is 2nm.

[0057] Figure 5 The figures show the fluorescence spectra of compound II prepared in Example 1 in mixed systems of 1,4-dioxane-tetrahydrofuran at different volume ratios. The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity. As can be seen from the figures, the polarity of the system gradually increases with the increase of the volume percentage of tetrahydrofuran, leading to a gradual decrease in the fluorescence intensity of compound II.

[0058] (4) Fluorescence imaging of compound II prepared in Example 1 and BODIPY 493 / 503 co-cultured in oleic acid-stimulated 4T1 cells.

[0059] 1 μL of the stock solution was diluted to 1 mL with culture medium to obtain a 5 μmol / L solution of compound II. This solution was then added to 4T1 cells and incubated for 30 min. Next, commercially available lipid droplet dye BODIPY 493 / 503 (200 nmol / L) was added, and incubation continued for another 30 min. The cells were then washed three times with PBS and observed under a confocal microscope. The excitation wavelength λ for compound II was specified. ex =488nm, fluorescence emission wavelength λ em 663-738nm; BODIPY 493 / 503: excitation wavelength λ ex =488nm, fluorescence emission wavelength λ em 500-550nm;

[0060] Figure 6 The image shows a fluorescence imaging result of compound II prepared in Example 1 of this invention co-cultured with BODIPY 493 / 503 in oleic acid-stimulated 4T1 cells. BODIPY 493 / 503 is a commercially available lipid droplet dye (green channel), and compound II is a near-infrared lipid droplet fluorescent dye (red channel). The overlay image shows the superposition of the two channels, and the correlation is calculated using the co-localization coefficient. The image shows good overlap between compound II and BODIPY 493 / 503, with a Pearson correlation coefficient of 0.93, demonstrating the excellent lipid droplet imaging specificity of compound II.

[0061] (5) Anti-photobleaching properties of compound II prepared in Example 1 and Nile Red

[0062] 4T1 cells were incubated in two confocal culture dishes for 24 h. One dish was stained with compound II (5 μmol / L) for 30 min, and the other was stained with Nile Red (1 μmol / L) for 30 min. After washing three times with PBS, fluorescence images were obtained by continuous laser scanning 50 times under a confocal microscope. Nile Red: excitation wavelength λ ex =488nm, fluorescence emission wavelength λ em The wavelength is 570-620 nm; Compound II: excitation wavelength λ ex =488nm, fluorescence emission wavelength λ em The wavelength is 663-738nm;

[0063] Figure 7 Fluorescence images of compound II prepared in Example 1 and Nile Red, each scanned 50 times in 4T1 cells; Figure 8The graph shows the fluorescence intensity changes after 50 scans. As can be seen from the graph, after 50 consecutive laser scans, the relative fluorescence intensity of compound II became 93% of its original value, while the fluorescence intensity of Nile Red became 53% of its original value; this indicates that compound II has significantly better anti-photobleaching properties than Nile Red.

[0064] (6) Fluorescence imaging of lipid droplets in 4T1 cells using compound II prepared in Example 1.

[0065] 4T1 cells were incubated in confocal culture dishes for 24 h, then incubated with compound II (5 μmol / L) in an incubator for 30 min, washed three times with PBS, and observed under a confocal microscope. The excitation wavelength λ was [not specified]. ex =488nm, fluorescence emission wavelength λ em The wavelength is 663-738nm;

[0066] Figure 9 The first row of images shows the fluorescence imaging of lipid droplets in 4T1 cells using compound II prepared in Example 1. The first row shows lipid migration between adjacent droplets a and b: after 2 minutes, droplet a shows decreased lipid levels and weaker fluorescence, while droplet b shows increased lipid levels and stronger fluorescence. Growth of droplet c can also be observed. After 4 minutes, droplet fusion occurs. The second row shows that droplet d gradually splits into two droplets within 4 minutes, while droplet e gradually weakens, possibly due to lipolysis caused by lipases in the cytoplasm. This indicates that compound II, due to its excellent photostability, can be used to observe the dynamic changes in lipid droplet structure.

[0067] (7) In situ imaging of compound II prepared in Example 1 in a mouse breast cancer model

[0068] 4T1 tumor cells (1×10) 6 One cell was subcutaneously injected into the left axilla using 100 μL of PBS. Compound II prepared in Example 1 was dissolved in DMSO to prepare a 1 mmol / L solution. After 10 days, 10 μL of this solution was subcutaneously injected into the tumor-bearing left axilla and normal tissue in the right axilla of 4T1 tumor-bearing mice. One hour later, the mice were anesthetized with isoflurane, and observation was performed using a real-time optical imaging system. The excitation wavelength λ... ex =500nm, fluorescence emission wavelength λ em It is 690nm;

[0069] Figure 10 This is an in situ imaging image of compound II prepared in Example 1 in a mouse breast cancer model. The image shows that the fluorescence intensity in the tumor is significantly higher than that in normal tissue, indicating that compound II can be used for in situ imaging of mouse tumors.

[0070] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A near-infrared lipid droplet fluorescent dye based on phenothiazine, characterized in that, The chemical structural formula of the near-infrared lipid droplet fluorescent dye is shown below: 。 2. The method for preparing a near-infrared lipid droplet fluorescent dye based on phenothiazine as described in claim 1, characterized in that, Includes the following steps: 2-Methoxy-10-ethyl-10H-phenthiazine-3-carboxaldehyde, 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile and an organic base are dissolved in an anhydrous polar organic solvent and reacted at 50-90°C for 4-12 hours to obtain the near-infrared lipid droplet fluorescent dye.

3. The method for preparing a near-infrared lipid droplet fluorescent dye based on phenothiazine according to claim 2, characterized in that, The 2-methoxy-10-ethyl-10 H The molar ratio of phenthiazine-3-carboxaldehyde, 2-(3,5,5-trimethylcyclohexyl-2-ene-1-yl)malonitrile to organic base is (0.8~1.0):1:(1.1~1.5); the molar volume ratio of 2-(3,5,5-trimethylcyclohexyl-2-ene-1-yl)malonitrile to anhydrous polar organic solvent is 1 mmol:(10~20) mL.

4. The method for preparing a near-infrared lipid droplet fluorescent dye based on phenothiazine according to claim 2, characterized in that, The anhydrous polar organic solvent includes at least one of anhydrous acetonitrile, anhydrous methanol, anhydrous ethanol, acetone, tetrahydrofuran, dichloromethane, dimethylformamide, and dimethyl sulfoxide.

5. The method for preparing a near-infrared lipid droplet fluorescent dye based on phenothiazine according to claim 2, characterized in that, The organic base includes at least one of aziridine and 1,4-oxaziridine.

6. The method for preparing a near-infrared lipid droplet fluorescent dye based on phenothiazine according to claim 2, characterized in that, After the reaction was completed, the near-infrared lipid droplet fluorescent dye was obtained by vacuum distillation and column chromatography purification.

7. The application of the phenothiazine-based near-infrared lipid droplet fluorescent dye as described in claim 1 in intracellular lipid droplet fluorescence imaging for non-diagnostic purposes.

8. The application of the phenothiazine-based near-infrared lipid droplet fluorescent dye as described in claim 1 in in situ imaging of mouse breast cancer for non-diagnostic purposes.

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