Fluorescent probe with dynamically changed polarity of lipid droplet, preparation method and application of fluorescent probe

Abstract

The invention belongs to the technical field of organic molecules and fluorescence detection, and discloses a fluorescent probe for dynamically changing the polarity of a lipid droplet, a preparation method and application of the fluorescent probe. The fluorescent probe has intramolecular charge transfer (ICT) capacity based on a fluorophore of a D-pi-A molecular configuration, is formed by taking fluorodipyrrole (BODIPY) as an electron acceptor and 6-methoxy-2-naphthaldehyde as a donor and connecting the donor and the acceptor through a pi bridge, and is used for targeted LDs polarity detection in a low-polarity environment. Under the condition of low polarity, weak interaction with solvent molecules causes small charge separation, so that the solution is pink and emits strong fluorescence, the sensitivity to polarity change is high, the chromogenic reaction is only carried out under the condition of low polarity, and the color development effect is good. Other common cations, anions, active oxygen, amino acids and enzyme species do not generate interference.

Description

Technical Field

[0001] The present invention relates to the technical field of organic molecules and fluorescence detection, and in particular to a fluorescent probe for dynamic changes in lipid droplet polarity, a preparation method and an application thereof. Background Art

[0002] Heart failure (HF) is the ultimate pathological outcome of most cardiovascular diseases, and poor prognosis can lead to serious consequences. The role of lipid droplets (LDs) in cardiovascular disease has also garnered increasing attention. Abnormal changes in LD polarity can disrupt lipid metabolism and are closely associated with the development and progression of HF. However, no fluorescent probe visualization technology has been developed to detect changes in LD polarity in HF cell and animal models. Summary of the Invention

[0003] To overcome the problems existing in the related art, the embodiments disclosed in the present invention provide a fluorescent probe for the dynamic change of LDs polarity, a preparation method and its application. Specifically, it relates to the application of organic molecules and fluorescence diagnostic technology in the early diagnosis of HF disease. A fluorescent probe for detecting polarity to monitor the dynamic changes of cardiomyocyte LDs is of great significance for understanding the biological characteristics of LDs and the physiological status of cells. In the present invention, a probe WT-X based on the BODIPY skeleton was designed and synthesized, and the emission wavelength showed a good linear relationship with the polarity range of 0.229-0.301. Therefore, the probe provides a potential tool for accurately detecting the polarity changes of LDs in chemical solvents or cardiomyocytes.

[0004] The technical solution is as follows: a fluorescent probe for detecting dynamic changes in LDs polarity in HF cells and animal models, the fluorescent probe is WT-X, and its structural formula is:

[0005]

[0006] Furthermore, the fluorescent probe is based on a fluorophore with a D-π-A molecular configuration that has intramolecular charge transfer (ICT) capability. Fluorinated dipyrrole (BODIPY) is used as an electron acceptor, 6-methoxy-2-naphthaldehyde is used as a donor, and the donor and acceptor are connected by a π bridge. It is used for targeted LDs polarity detection in a low-polarity environment of Δf = 0.3011 to 0.2291.

[0007] Another object of the present invention is to provide an application of the fluorescent probe in preparing reagents for identifying LDs and testing solution polarity.

[0008] Furthermore, the fluorescent probe for detecting the dynamic changes of LDs polarity in HF cells and animal models is used in the early detection of targeted LDs in heart failure cells, inflammatory cell models, and zebrafish inflammation models.

[0009] Another object of the present invention is to provide a method for preparing a fluorescent probe for detecting dynamic changes in LD polarity in HF cells and animal models. The method is used to prepare the fluorescent probe for detecting dynamic changes in LD polarity in HF cells and animal models, and the method comprises the following steps:

[0010] S1. Mixing 2,4-dimethylpyrrole represented by Formula I with benzaldehyde in the presence of a catalyst at room temperature for 12-24 hours, then adding 2,3-dichloro-5,6-dicyanobenzoquinone and stirring at room temperature for 4 hours, and finally adding triethylamine to provide an alkaline environment. Adding boron trifluoride etherate and stirring at room temperature for 12 hours in an ice bath to obtain a compound represented by Formula II.

[0011] The molecular formulas of Formula I and Formula II are:

[0012]

[0013]

[0014] S2. Dissolve 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroborylguanidine described in Formula II and 6-methoxy-2-naphthaldehyde in toluene at 100°C-120°C, add piperidine and mix well to react for 4-6 hours to obtain WT-X.

[0015] In step S1, the catalyst is at least one of an organic base and an inorganic base;

[0016] The organic base is at least one of triethylamine and pyridine;

[0017] The inorganic base is at least one of potassium carbonate, sodium acetate, sodium carbonate and sodium bicarbonate.

[0018] In step S1, the molar ratio of 2,4-dimethylpyrrole of formula I to benzaldehyde and catalyst is 2:1:0.5.

[0019] In step S2, the molar ratio of 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroborylguanidine described in formula II to 6-methoxy-2-naphthaldehyde is 1:4:0.5.

[0020] In step S1, the reaction is carried out in an organic solvent, wherein the organic solvent is tetrahydrofuran, and the catalyst is triethylamine and trifluoroacetic acid.

[0021] In step S2, the reaction is carried out in an organic solvent, and the organic solvent is at least one of acetic acid and toluene.

[0022] In combination with all the above technical solutions, the beneficial effects possessed by the present invention are as follows: the fluorescent probe of the present invention is WT-X, and the present invention is based on fluorodipyrrole (BODIPY) as an electron acceptor (A), 6-methoxy-2-naphthaldehyde as a donor (D), and the donor and the acceptor are connected by a π-bridge. The fluorophore with the D-π-A molecular configuration has intramolecular charge transfer (ICT) capability, and is very sensitive to changes in polarity. Under low polarity conditions, weak interactions with solvent molecules cause small charge separations that can make the solution appear pink and emit strong fluorescence, with high sensitivity to polarity changes. The color development reaction only occurs under low polarity conditions, and other common cations, anions, reactive oxygen species, amino acids, and enzyme species do not interfere.

[0023] This paper develops fluorescent probe visualization technology for the first time to detect changes in LDs polarity in HF cells and animal models, and studies lipid consumption and accumulation during disease progression by monitoring changes in LDs during the disease process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure;

[0025] Figure 1 This is a flow chart of a method for preparing a fluorescent probe for detecting the polarity of LDs provided in an embodiment of the present invention;

[0026] Figure 2 is a hydrogen spectrum of WT-X provided in an embodiment of the present invention (400 MHz, CDCl3, 298 K);

[0027] Figure 3 This is the carbon spectrum of WT-X provided in an embodiment of the present invention (400 MHz, CDCl3, 298 K);

[0028] Figure 4 is a mass spectrum of WT-X provided in an embodiment of the present invention (100 MHz, CDCl3, 298 K);

[0029] Figure 5 1 is a fluorescence emission spectrum of the fluorescent probe WT-X provided in an embodiment of the present invention in different solvents;

[0030] Figure 6 : This is the UV normalized spectra of the fluorescent probe WT-X provided in the embodiment of the present invention in different solvents;

[0031] Figure 7This is a fluorescence emission spectrum of the prepared WT-X provided in an embodiment of the present invention and a mixed system of 1,4-dioxane-H2O at different ratios;

[0032] Figure 8 is a linear relationship diagram between the emission intensity of WT-X prepared according to an embodiment of the present invention and solvent parameters;

[0033] Figure 9 This is a fluorescence response diagram of WT-X prepared in an embodiment of the present invention to an interfering substance in a PBS environment;

[0034] Figure 10 This is a fluorescence response diagram of WT-X prepared in an embodiment of the present invention to an interfering substance in a 1,4-dioxane environment;

[0035] Figure 11 This is a graph showing changes in fluorescence intensity of WT-X prepared in PBS environments with different pH values ​​and in a 50% 1,4-dioxane environment, as provided in an embodiment of the present invention;

[0036] Figure 12 This is a graph showing the changes in the photostable fluorescence intensity of WT-X prepared by the present invention in two different systems: 1,4-dioxane and H2O;

[0037] Figure 13 This is a bio-imaging image of LDs specificity of WT-X prepared by the present invention in H9c2 cells;

[0038] Figure 14 This is a fluorescence imaging diagram of the WT-X prepared by the present invention for detecting the polarity of LDs in H9c2 cells induced by isoproterenol;

[0039] Figure 15 This is a fluorescence imaging diagram of the detection of LDs polarity in H9c2 cells induced by lipopolysaccharide by WT-X prepared by the present invention;

[0040] Figure 16 This is a fluorescence imaging diagram of LDs polarity detection in an inflammatory zebrafish model using WT-X prepared by the present invention;

[0041] Figure 17 This is a fluorescence imaging diagram of the WT-X prepared by the present invention for detecting the polarity of LDs in a heart failure mouse model. DETAILED DESCRIPTION

[0042] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0043] The novelty of this invention lies in the fact that the probe designed and synthesized exhibits excellent photostability, biocompatibility, and polarity selectivity. Under confocal imaging microscopy, changes in cell polarity in a heart failure model were detected, as well as in the yolk sac region of inflamed zebrafish. The probe also demonstrated remarkable sensitivity in identifying polarity abnormalities in LDs in the myocardium of heart failure mice. This discovery provides new insights and tools for the research and diagnosis of heart failure.

[0044] Example 1, a fluorescent probe for detecting the polarity of LDs, the fluorescent probe is WT-X, and its structural formula is as follows:

[0045]

[0046] Example 2, as Figure 1 As shown, the preparation method of the fluorescent probe for detecting the polarity of LDs includes the following steps:

[0047] S1. In the presence of a catalyst, 2,4-dimethylpyrrole represented by Formula I and benzaldehyde are mixed and reacted at room temperature for 12 to 24 hours. Subsequently, 2,3-dichloro-5,6-dicyanobenzoquinone is added and stirred at room temperature for 4 hours. Finally, triethylamine is added to provide an alkaline environment. Boron trifluoride etherate is added in an ice bath and stirred at room temperature for 12 hours to obtain the compound represented by Formula II.

[0048]

[0049] S2. Dissolve 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroborylguanidine described in Formula II and 6-methoxy-2-naphthaldehyde in toluene at 100°C-120°C, add piperidine and mix well to react for 4-6 hours to obtain WT-X.

[0050] Illustratively, in step S1, the catalyst is at least one of an organic base and an inorganic base; the organic base is at least one of triethylamine and pyridine; and the inorganic base is at least one of potassium carbonate, sodium acetate, sodium carbonate and sodium bicarbonate.

[0051] Illustratively, in step S1, the molar ratio of 2,4-dimethylpyrrole of Formula I to benzaldehyde and catalyst is 2:1:0.5.

[0052] Illustratively, in step S2, the molar ratio of 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroborylguanidine described in Formula II to 6-methoxy-2-naphthaldehyde is 1:4:0.5.

[0053] Illustratively, in step S1, the reaction is carried out in an organic solvent, the organic solvent is tetrahydrofuran, and the catalyst is triethylamine or trifluoroacetic acid.

[0054] Illustratively, in step S2, the reaction is carried out in an organic solvent, and the organic solvent is at least one of acetic acid and toluene.

[0055] Example 3. The present invention proposes an application of a fluorescent probe WT-X that targets LDs and is polarity-sensitive for detecting the polarity of LDs. The probe molecule can be used to prepare reagents for identifying lipid droplets and testing solution polarity.

[0056] For example, the maximum emission wavelength of the fluorescent probe WT-X is at 597 nm, and the maximum Stokes shift can reach 22 nm. There is a large wavelength difference between the maximum emission wavelength and the maximum excitation wavelength, low background interference, and high detection sensitivity.

[0057] Specifically, the maximum emission wavelength of the fluorescent probe WT-X is at 597 nm, and the main absorption peak of WT-X in a mixed solution of water and 1,4-dioxane is concentrated around 574 nm. In addition, in a 1,4-dioxane-H2O mixed solvent, the polarity Δf is 0.3011 (70% H2O), 0.2796 (60% H2O), 0.2695 (50% H2O), 0.2594 (40% H2O), 0.2493 (30% H2O), 0.2392 (20% H2O), and 0.2291 (10% H2O). There is an excellent linear relationship between the maximum fluorescence intensity of WT-X and the polarity Δf (R 2 =0.985).

[0058] Preferably, the emission spectra of the fluorescent probe WT-X in solvents of different polarities, such as dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, 1,4-dioxane and toluene, are significantly different, and the emission peak of WT-X is closely related to the empirical parameter of solvent polarity (E T (30)) has a good linear relationship (R 2 =0.984).

[0059] Exemplarily, the color development reaction occurs only in a low-polarity environment, and other common cations, anions, reactive oxygen species, amino acids, and enzyme species do not interfere.

[0060] For example, in the low-polarity environment of LDs, the fluorescence of the probe WT-X is significantly enhanced, enabling efficient and accurate targeting of LDs and detecting changes in LD number and polarity within cells. The fluorescent probe obtained in this invention has demonstrated excellent biosafety and photostability when applied to heart failure cells, inflammatory cell models, and zebrafish inflammation models, and can efficiently and accurately target LDs. The probe WT-X provides a reliable tool for further studying the dynamic changes in LD polarity and the development and progression of heart failure (HF), showing great potential for early detection in HF disease models.

[0061] From the above examples, it can be seen that the fluorophore based on the D-π-A molecular configuration of the present invention has the ability of intramolecular charge transfer (ICT), and uses fluorodipyrrole (BODIPY) as the electron acceptor (A) and 6-methoxy-2-naphthaldehyde as the donor (D). The donor and the acceptor are connected by a π bridge and have good fluorescence properties: high stability, extremely high sensitivity to polarity and strong fluorescence emission. A fluorescent probe targeting LDs is designed. Experiments have found that the fluorescence intensity of the probe itself is very weak. In a low-polarity environment, based on the intramolecular charge transfer mechanism, the probe is turned on, the solution fluorescence is significantly enhanced and the color changes from almost colorless to pink, indicating that this method can be used for polarity detection. In order to verify the practicality of this method, it was applied to the test of solution polarity and the detection of LDs polarity in heart failure cell models and inflammatory cell models. The probe WT-X designed in this invention appears pink in low-polarity solutions and emits strong fluorescence, with high sensitivity. The color reaction only occurs under low-polarity conditions, and other common reactive oxygen species, amino acids, and enzyme species do not interfere.

[0062] The present invention has the advantages of simple operation, low cost, rapidity, high efficiency and sensitivity, and will become a powerful research tool in related fields such as modern biology, physiology and medicine.

[0063] Example 4, the steps for preparing WT-X are as follows:

[0064] Under the catalysis of 120 μL of trifluoroacetic acid, 2,4-dimethylpyrrole (249 μL, 2.4 mmol) and benzaldehyde (127 μL, 1.2 mmol) were dissolved in tetrahydrofuran (15 mL) and stirred at room temperature for 12 hours under inert gas for condensation reaction. DDQ (270 mg, 1.2 mmol) was then added to the reaction mixture and allowed to react at room temperature for 4 hours. Triethylamine (3 mL) and boron trifluoride etherate (3 mL) were then added and stirring continued at room temperature for another 12 hours. After completion of the reaction, the solvent was concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography using petroleum ether / ethyl acetate (70:1, v / v) as the eluent to yield 29.6 mg of Formula II.

[0065] The product of Formula II (32.4 mg, 0.1 mmol) and 6-methoxy-2-naphthaldehyde (74.4 mg, 0.4 mmol) were dissolved in toluene (5 mL), and piperidine (0.5 mL) was added. The mixture was reacted at 110°C for 5 hours. After completion of the reaction, the solvent was cooled and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using petroleum ether / ethyl acetate (20:1, v / v) as the eluent to obtain 25 mg of the product WT-X.

[0066] The structural characterization data of the product are as follows:

[0067] 1 H NMR (400MHz, CDCl3, 298K), δ (ppm): 7.83 (s, 1H), 7.78-7.69 (m, 4H), 7.47 (d, J = 5.2Hz, 3H), 7.37-7.28 (m, 3H),7.11(d,J=8.4Hz,2H),6.63(s,1H),5.99(s,1H),3.91(s,3H),2.59(s,3H),1.42(s,3H),1.37(s,3H).

[0068] 13 C NMR (100MHz, CDCl3, 298K), δ (ppm): 158.37, 136.50, 135.02, 130.92, 129.91, 129.12, 128.96, 128.85, 128.23, 127.32, 124.64, 119.21, 106.04,65.58,55.37,53.43,31.94,30.58,29.71,29.67,29.53,29.37,29.33,29.25,26.72,25.53,22.70,19.19,14.13,13.74,1.03.

[0069] ESI HRMS (m / z):calcd for C 31 H 27 BF2N2NO[M+H] + 493.2257, found 493.2277. See the results Figure 2 , Figure 3 , Figure 4 .

[0070] Example 5, spectral properties of the compound WT-X in reaction with solutions of different polarities.

[0071] 4.54 mg of the pure probe prepared in Example 4 was accurately weighed on an analytical balance and dissolved in 8 mL of dimethyl sulfoxide solvent to prepare a probe stock solution with a final concentration of 1 mM. In this experiment, five solvents with different dielectric constants were selected to measure the fluorescence emission spectra and UV normalized spectra of the WT-X probe in 1 mL of dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, 1,4-dioxane, and toluene solution systems. The results are shown in Figure 2. Figure 5 、 Figure 6 As the polarity of the solvent decreases, the position of the fluorescence emission peak changes significantly. Under 540nm excitation, the fluorescence intensity of WT-X increases with the decrease of solvent polarity. The proportion of water in the 1,4-dioxane-H2O solution is gradually reduced from 70% to 10% to simulate different polar solvent environments. The fluorescence emission spectra of the probe in different polar solvents are measured. The test results are shown in Figure 7 、 Figure 8 As the volumetric content of H2O decreased, the polarity of the solution increased and the fluorescence intensity decreased. A good linear relationship between Δf and fluorescence intensity was observed, indicating that the probe is polarity-responsive. The optimal excitation wavelength for the fluorescent probe WT-X was 540 nm, and the optimal emission wavelength was 597 nm. The excitation and emission slit widths were 5 nm, and the voltage was 700 V.

[0072] Example 6, fluorescence emission spectra obtained when the fluorescent probe WT-X reacts with other interfering substances.

[0073] Various substances were added to PBS and 1,4-dioxane solution respectively: (1) blank; (2) Hg + (100μM); (3)K + (100 μM); (4) Cu 2+ (100μM); (5)Ca 2+ (100μM); (6)Cr 3+ (100 μM); (7) Mg 2+ (10μM); (8)Bi 3+ (10μM); (9)Ag + (10μM); (10)Fe 2+ (10μM); (11)Fe 3+ (10μM); (12)Ni 3+ (10μM); (13)Pb 2+ (10μM); (14)S 2-(10μM); (15)GSH (100μM); (16)Gal (4U / mL); (17) Cys (100μM); (18) Hcy (100μM); (19)NTR (500U / m L); (20) CES (0.5U / mL); (21) ALDH (10μM); (22) Ser (100μM); (23) Trp (10μM); (24) Leu (50μM); (25) 1 O2(10μM);(26)ONOO - (10μM); (27) H2O2 (100μM); (28) HClO (10μM); (29) BSA (0.1g / L); (30) HAS (0.1g / L); (31) 1,4-Dioxane. After reacting at 37°C for 30 minutes, the fluorescence emission spectrum was measured. When measuring the fluorescence emission spectrum, the excitation wavelength was 540nm; the slit width of the excitation and emission was 5nm; the voltage was 700V, and 10μL of WT-X mother solution (1mM) was added to 1mL of the solution mixed with the above substances. Figure 9 、 Figure 10 , the experimental results showed that only WT-X was highly selective for polarity, while the presence of other species did not interfere with the determination of polarity.

[0074] Example 7: Fluorescent probe WT-X is affected by pH environment.

[0075] The fluorescent probe WT-X prepared in Example 4 was prepared into a DMSO stock solution with a final concentration of 1 mM. A series of solutions with different pH values ​​(pH = 3.46, 4.27, 5.35, 6.26, 7.35, 8.40, 10.33, 10.33, 11.57 and 12.68) were prepared using 0.2 mol / L PBS buffer, and their fluorescence spectra were measured. The effect of different pH environments on their fluorescence intensity was evaluated based on the change in fluorescence intensity at 597 nm. The results are shown in Figure 2. Figure 11 As shown in the figure, within the pH range of 3.46-12.68, the change in the fluorescence intensity of the probe is almost unaffected by the pH environment, indicating that the probe has good stability.

[0076] Example 8, photostability of fluorescent probe WT-X.

[0077] WT-X was irradiated continuously for 2000s in two solvent systems, H2O and 1,4-dioxane, and it can be seen that the fluorescence intensity has always remained within a stable range. The experimental results show that the probe WT-X can detect changes in polarity in the microenvironment under continuous illumination conditions and has good photostability. Figure 12 .

[0078] Example 9, LDs-specific bioimaging of fluorescent probe WT-X in H9c2 cells.

[0079] 1) Cell culture:

[0080] H9c2 cells were grown in glass-bottom culture dishes using DMEM liquid culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured in a constant temperature incubator set at 37°C and 5% CO2 concentration.

[0081] 2) Colocalization experiments:

[0082] Co-localization experiments were conducted in H9c2 cells with commercial dyes targeting mitochondria (labeled with Rhodamine 123), LDs (stained with BODIPY 493 / 503), and lysosomes (labeled with LysoTracker Green DND-26). x =496 nm, collection wavelength 500-550 nm) to verify the ability of probe WT-X to locate LDs. At the same time, the commercial lysosomal probe LysoTracker Green DND-26 and the commercial mitochondrial probe Rhodamine 123 were used as controls. Rhodamine 123 (2 μM, E x =496 nm, collection wavelength 500-570 nm) to locate mitochondria, LysoTracker Green DND-26 (1 μM, E x =496nm, collection wavelength is 500-550nm) to locate lysosomes. Figure 13 As shown, the red fluorescence of the probe molecule can overlap well with the green fluorescence of BODIPY to form yellow fluorescence, and the colocalization coefficient reaches 0.88, showing good targeting ability to LDs.

[0083] Example 10, Fluorescence imaging of fluorescent probe WT-X for detecting LDs polarity in isoproterenol-induced H9c2 cells.

[0084] H9c2 cells were stimulated with different concentrations of isoproterenol (20 μM and 30 μM) for 24 h and then treated with 5 μM probe WT-X for 40 min. Figure 14 As shown, in isoproterenol-stimulated H9c2 cells, the fluorescence intensity increased significantly after incubation with WT-X, and the fluorescence intensity gradually increased with increasing isoproterenol concentrations.

[0085] Example 11, fluorescence imaging of the fluorescent probe WT-X for detecting LDs polarity in H9c2 cells induced by lipopolysaccharide.

[0086] H9c2 cells were incubated with different concentrations of lipopolysaccharide (3, 8, and 13 μM) for 6 hours to establish a simplified inflammation model, and then incubated with WT-X (5 μM) for 40 minutes. Figure 15 As shown, compared with the weak fluorescence of cells treated with WT-X alone, the fluorescence gradually increased with increasing LPS concentration. Compared with H9c2 cells treated with WT-X alone, the fluorescence intensity signal increased by 0.5 times after 6 hours of stimulation with 13 μM drug.

[0087] Example 12, fluorescence imaging of LDs polarity detection in an inflammatory zebrafish model using the fluorescent probe WT-X.

[0088] A zebrafish inflammation model was established and corresponding imaging experiments were performed. Seven-day-old zebrafish with the same growth status were selected and randomly divided into two groups for fluorescence imaging analysis. The first group served as the control group and only received WT-X treatment for 60 minutes. The second group served as the experimental group and was first pretreated with 50 μM lipopolysaccharide for 2 hours and then treated with WT-X for 60 minutes. Figure 16 Results showed that control zebrafish exhibited very weak fluorescence signals in the yolk sac region. However, after LPS treatment, significant fluorescence was observed in the yolk sac. These results demonstrate that WT-X can be used for zebrafish imaging and can detect changes in LD polarity within the yolk sac.

[0089] Example 13, Fluorescence imaging of LDs polarity detection in a heart failure mouse model using the fluorescent probe WT-X.

[0090] Eight-week-old C57BL / 6J female mice were divided into three groups using different injection solutions and injection times. (1) The control group mice were intraperitoneally injected with normal saline for 4 consecutive days; (2) the model group mice were intraperitoneally injected with isoproterenol for 4 consecutive days; (3) the model group mice were intraperitoneally injected with isoproterenol for 8 consecutive days. The heart failure mice in the control group and experimental group were sacrificed and the hearts were obtained. After rinsing with PBS, the hearts of the control group and model group mice were immersed in normal saline solution containing the probe WT-X (30μM) for 1 hour, and then fluorescence imaging of the isolated organs was performed on a small animal in vivo imaging system. Figure 17 As shown, compared to the untreated control group, the WT-X probe-treated mouse hearts showed weak fluorescence. In contrast, strong fluorescence was observed in the heart failure model group. These results demonstrate that WT-X can be used for in vitro imaging of the heart in heart failure mice and can detect changes in LDs in these mice.

[0091] The above description is only a preferred specific implementation method of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A fluorescent probe for detecting dynamic changes in lipid droplet polarity in heart failure cells and animal models, characterized in that: The fluorescent probe is WT-X, and its structural formula is:

2. The fluorescent probe according to claim 1, wherein This fluorescent probe is based on a fluorophore with a D-π-A molecular configuration and has intramolecular charge transfer (ICT) capability. It uses fluorodipyrrole (BODIPY) as an electron acceptor and 6-methoxy-2-naphthaldehyde as a donor. The donor and acceptor are connected by a π bridge and are used for targeted lipid droplet polarity detection in a low-polarity environment of Δf = 0.3011 to 0.2291.

3. Use of the fluorescent probe according to any one of claims 1 to 2 in the preparation of reagents for identifying lipid droplets and testing solution polarity.

4. The use according to claim 3, characterized in that The fluorescent probe for detecting dynamic changes in lipid droplet polarity in heart failure cells and animal models is used in the early detection of targeted lipid droplets in heart failure cells, inflammatory cell models, and zebrafish inflammation models.

5. A method for preparing a fluorescent probe for detecting dynamic changes in lipid droplet polarity in heart failure cells and animal models, characterized in that: The preparation method is used to prepare the fluorescent probe for detecting dynamic changes in lipid droplet polarity in heart failure cells and animal models according to any one of claims 1-2. The following steps are involved: S1. Mixing 2,4-dimethylpyrrole represented by Formula I with benzaldehyde in the presence of a catalyst at room temperature for 12-24 hours; then adding 2,3-dichloro-5,6-dicyanobenzoquinone and stirring at room temperature for 4 hours; finally, adding triethylamine to provide an alkaline environment; and adding boron trifluoride etherate in an ice bath and stirring at room temperature for 12 hours to obtain a compound represented by Formula II; The molecular formulas of Formula I and Formula II are: S2. Dissolve 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroborylguanidine described in Formula II and 6-methoxy-2-naphthaldehyde in toluene at 100°C-120°C, add piperidine and mix well to react for 4-6 hours to obtain WT-X.

6. The preparation method according to claim 5, characterized in that In step S1, the catalyst is at least one of an organic base and an inorganic base; The organic base is at least one of triethylamine and pyridine; The inorganic base is at least one of potassium carbonate, sodium acetate, sodium carbonate and sodium bicarbonate.

7. The preparation method according to claim 5, characterized in that In step S1, the molar ratio of 2,4-dimethylpyrrole of formula I to benzaldehyde and catalyst is 2:1:0.

5.

8. The preparation method according to claim 5, characterized in that In step S2, the molar ratio of 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroborylguanidine described in formula II to 6-methoxy-2-naphthaldehyde is 1:4:0.

5.

9. The preparation method according to claim 5, characterized in that In step S1, the reaction is carried out in an organic solvent, wherein the organic solvent is tetrahydrofuran, and the catalyst is triethylamine and trifluoroacetic acid.

10. The preparation method according to claim 5, characterized in that In step S2, the reaction is carried out in an organic solvent, and the organic solvent is at least one of acetic acid and toluene.

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