Polarity response type lipid droplet targeting fluorescent probe and application thereof

Abstract

The invention belongs to the technical field of fluorescent probes and biosensing, and discloses a polarity response type lipid droplet targeting fluorescent probe and application thereof. The structural formula of the fluorescent probe is # imgabs0 #. The fluorescent probe has the advantages of excellent biocompatibility, high sensitivity and the like. The probe has ultra-strong fluorescent response to polarity change, and can be further enriched in lipid droplets of living cells through passive targeting, so that real-time washing-free imaging with specific response to the lipid droplets is realized, and the internal polarity change of the lipid droplets of the living cells and the form, size and number change of the lipid droplets are dynamically detected; by using the probe, specific distinguishing of liver cancer cells from other cells can be realized according to different fluorescence emission intensities of the probe in the cells. In view of the excellent properties of the probe, the probe is applied to real-time dynamic detection of the polarity change process of the lipid droplets in liver cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probes and biosensor technology, in particular to a lipid droplet-targeting fluorescent probe. Background Art

[0002] Lipid droplets are spherical organelles composed of a single layer of phospholipid molecules encapsulating neutral lipids, ranging in size from nanometers to micrometers. As abundant organelles in adipocytes, lipid droplets are directly involved in the body's lipid metabolism and energy supply. They not only serve as a transit point for lipid storage and migration but also respond to changes in external metabolic levels. On the one hand, they can store excess free fatty acids in the form of glycerides within their hydrophobic core, thereby alleviating lipotoxicity. On the other hand, when needed, they can release fatty acids to provide energy and nutrients, maintaining metabolic homeostasis. Furthermore, lipid droplets not only play a key role in lipid storage but also participate in important life activities such as lipid metabolism, immune regulation, and signal transduction.

[0003] Polarity is a key parameter of the cellular microenvironment and is crucial for the normal function and regulation of cells. Changes in intracellular polarity can reflect physiological or pathological states, and studies have shown that abnormal polarity is closely associated with diseases such as diabetes, depression, and cancer. For example, due to significant changes in lipid metabolism in cancer cells, the polarity of their lipid droplets is often lower than that of normal cells. In addition, many physiological processes, such as protein denaturation, enzyme catalysis, peptide aggregation, membrane fusion, and signal transduction, rely on precise regulation of polarity. Lipid droplets, as highly dynamic energy storage organelles, participate in a variety of critical cellular activities. Abnormal polarity can lead to lipid metabolism disorders, further causing functional impairment and related diseases. The liver is a core organ for lipid metabolism, and the lipid droplets of liver cancer cells differ significantly from those of normal liver cells in terms of polarity, size, and morphology. Therefore, real-time monitoring of dynamic changes in lipid droplet polarity is of great significance for studying the mechanisms of occurrence and development of polarity-related diseases and potential diagnostic strategies.

[0004] Fluorescent probes have the advantages of simple operation, high selectivity, and high sensitivity. They are the most widely used fluorescence analysis method and are widely used in pharmacology, medicine, physiology, and biology. Based on classic fluorescent dyes such as Nile red, coumarin, hemicyanine, naphthalimide, tetraphenylethylene, flavonoids, and intramolecular charge transfer mechanisms, a variety of fluorescent probes for identifying and sensing lipid droplets have been reported. However, the fluorescence emission wavelengths of the fluorescent probes reported so far mostly red-shift with increasing polarity, which can easily cause errors in the results of monitoring the polarity of lipid droplets using a single wavelength. In addition, the fluorescence emission of lipid droplet-targeted probes whose emission wavelengths red-shift with polarity covers a wide range of the visible region. When co-stained with other organelle dyes, spectral overlap will cause cross-coloring, making them unsuitable for multi-color imaging in cells. Prior art CN113940021A discloses a class of high-brightness, multi-color wash-free lipid droplet fluorescent probes. This type of probe shows very strong fluorescence in dichloromethane (Δf = 0.2171), cannot be used for ultra-low polarity detection, and cannot distinguish liver cancer cells. Therefore, the development of fluorescent probes with highly sensitive polarity response, good lipid droplet targeting, and absorption and fluorescence emission wavelengths that do not change with polarity is of great significance for the study of lipid droplet physiological mechanisms and related diseases, but also poses great challenges. Summary of the Invention

[0005] In response to the above technical problems, the present invention proposes a polarity-responsive lipid droplet-targeted fluorescent probe and its application.

[0006] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0007] A polarity-responsive lipid droplet-targeting fluorescent probe named PAN-BODIPY (or fluorescent probe PAN-BODIPY) has the following structural formula:

[0008]

[0009] Furthermore, the polarity-responsive lipid droplet-targeting fluorescent probe has lipid droplet targeting capability, and has polarity-responsive capability when the polarity is .

[0010] Furthermore, the preparation method of the polarity-responsive lipid droplet-targeted fluorescent probe comprises the following steps:

[0011] (1) 2-Methoxy-5-nitrobenzaldehyde (Compound 1) and 2,4-dimethylpyrrole were dissolved in dichloromethane (CH2Cl2). The system was stirred at room temperature under a nitrogen atmosphere for 20 minutes. Trifluoroacetic acid (TFA) was then slowly injected into the system, and the reaction mixture was stirred overnight.

[0012] (2) After confirming complete conversion of the starting materials by thin-layer chromatography, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was added and stirred at room temperature for 2 h. The reaction solution was washed with water, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to obtain a solid.

[0013] (3) The solid obtained in step (2) and triethylamine (TEA) were dissolved in anhydrous CH2Cl2 solution and stirred at room temperature under nitrogen for 30 minutes. BF3·OEt2 was added and stirring was continued for 10 minutes. Deionized water and NaOH solution were added to the system in sequence to quench the reaction. The reaction solution was extracted with CH2Cl2, and the organic phases were combined and dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (EA / PE = 1:5) to obtain an orange solid intermediate 2.

[0014] (4) The intermediate 2 obtained in step (3) and Pd-C were dissolved in anhydrous CH2Cl2 solution and stirred at room temperature under a hydrogen atmosphere for 3 h. After the reaction was completed, the mixture was filtered and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (EA / PE = 1:5) to obtain an orange solid product, namely, the polarity-responsive lipid droplet-targeted fluorescent probe PAN-BODIPY.

[0015] In the above step (1), the molar ratio of 2-methoxy-5-nitrobenzaldehyde to 2,4-dimethylpyrrole is 1:2. The molar-volume ratio of 2-methoxy-5-nitrobenzaldehyde to trifluoroacetic acid is 1 mmol:1.5-2L.

[0016] In the above step (2), the molar ratio of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to 2-methoxy-5-nitrobenzaldehyde is 1:1.

[0017] In the above step (3), the volume-molar ratio of triethylamine to 2-methoxy-5-nitrobenzaldehyde is 1 mmol:2 L, and the volume ratio of BF3·OEt2 to triethylamine is 1:1.

[0018] The concentration of Pd-C in the above step (4) is 10%, and the mass ratio of 10% Pd-C to intermediate 2 is 2:1.

[0019] The polarity-responsive lipid droplet-targeted fluorescent probe is used to detect the polarity of a test solution for non-disease diagnosis purposes. The steps are: adding a stock solution of the polarity-responsive lipid droplet-targeted fluorescent probe to the test solution, and detecting the fluorescence intensity of the test solution at an excitation wavelength of 490 nm.

[0020] More specifically, the steps are: (a) dissolving the fluorescent probe PAN-BODIPY in acetonitrile solution (ACN) to prepare a probe stock solution;

[0021] (b) adding the probe stock solution to solvents of different polarities to form a series of test solutions, and then performing fluorescence emission spectroscopy at an excitation wavelength of 490 nm;

[0022] (c) A mixed gradient solution of ethanol / n-hexane with different volume ratios was prepared. The probe stock solution was added to the mixed solution to form a series of gradient standard solutions. The fluorescence emission spectrum was then measured at an excitation wavelength of 490 nm, and a working curve was plotted between the fluorescence intensity of the solution and the polarity parameter (Δf) of the solution.

[0023] Wherein, the concentration of the probe stock solution in the above step (a) is 2 mM;

[0024] The final concentration of the polarity-responsive lipid droplet-targeted fluorescent probe in the above steps (b) and (c) is 5 μM.

[0025] The polarity-responsive lipid droplet-targeted fluorescent probe is used to prepare a rapid, wash-free imaging reagent for intracellular lipid droplet imaging. The steps are: incubate the probe stock solution with cells for 15 minutes, and observe the wash-free imaging effect of the probe using laser confocal imaging. The final concentration of the fluorescent probe is 4 μM, and the cells are human hepatoma cell lines SMMC-7721.

[0026] The polarity-responsive lipid droplet-targeted fluorescent probe is used to prepare a reagent for visually monitoring changes in lipid droplet polarity in living cells.

[0027] The polarity-responsive lipid droplet-targeted fluorescent probe is used to test lipid droplet targeting effects and prepare detection reagents for distinguishing liver cancer cells from other cells. The specific steps are as follows:

[0028] S1. Combine the probe stock solution and the commercial lipid droplet dye HCS Lipid TOX TM Deep Red was incubated with cells for 15 minutes, and the lipid droplet targeting effect of the probe was observed by laser confocal imaging.

[0029] S2. Incubate the probe stock solution with normal cells and cancer cells for 15 minutes, and obtain the imaging effect of the probe in different normal cells and cancer cells by laser confocal imaging.

[0030] The final concentration of the fluorescent probe in the above steps S1 and S2 is 4 μM.

[0031] The cells in the above step S1 are human hepatoma cell lines SMMC-7721.

[0032] The normal human cells used in the above step S2 are HUVCE, HL-7702 and HEK293, and the human cancer cells are MCF-7, HeLa and SMMC-7721.

[0033] The beneficial effects produced by the present invention are:

[0034] (1) The present invention uses BODIPY fluorescent dye as the matrix and introduces a p-methoxyaniline group at the meso-position of the dye based on the principle of photoinduced electron transfer (PET) to design and synthesize a polarity-sensitive lipid droplet-targeting fluorescent probe PAN-BODIPY. The introduction of this group gives the probe good polarity responsiveness. In a strongly polar solvent, the probe quenches the fluorescence of the BODIPY dye due to the PET process of the probe molecule, resulting in a solution without fluorescence. However, in a non-polar or less polar solvent, the PET is inhibited, causing the fluorescence of the BODIPY molecule to recover and emit strong fluorescence, thereby realizing the recognition and sensing of polarity in solution and cells.

[0035] (2) Since the probe has almost no fluorescence in medium-to-strong polar solutions such as water, the probe can achieve rapid, wash-free imaging of lipid droplets, thus avoiding damage to cells caused by repeated washing during the imaging process.

[0036] (3) The introduction of the p-methoxyaniline functional group also makes the probe have good lipid solubility, which can specifically target lipid droplets, thereby effectively avoiding nonspecific staining and greatly improving the effect of lipid droplet imaging.

[0037] (4) Since the fluorescent probe has excellent targeting to lipid droplets and has a good response to the ultra-small polarity range Δf = 0-0.013, the probe can perform real-time in situ detection of changes in the polarity, quantity and morphology of lipid droplets in living cells, and can effectively distinguish liver cancer cells from other cells based on the changes in fluorescence intensity caused by polarity differences in the cells.

[0038] (5) The probe has good biocompatibility and good stability under physiological conditions, which facilitates real-time in situ long-term monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 This is the synthetic route of the fluorescent probe PAN-BODIPY.

[0041] Figure 2 This is the 1H NMR spectrum of the fluorescent probe PAN-BODIPY.

[0042] Figure 3(a) The UV-visible absorption spectra and (b) fluorescence emission spectra of 5 μM probe in solutions of different polarities, as well as the linear relationship between the fluorescence intensity of the solution and the solution polarity parameter (Δf) (c) are shown. The excitation wavelength is 490 nm.

[0043] Figure 4 The UV-visible absorption spectra (a) and fluorescence emission spectra (b) of a 5 μM probe in mixed solutions of ethanol and n-hexane at different volume ratios (0-100%), as well as the linear relationship between the solution polarity parameter (Δf) and fluorescence intensity (c) are shown. The excitation wavelength is 490 nm.

[0044] Figure 5 UV-visible absorption spectra (a) and fluorescence emission spectra (b) of 5 μM probe in BR solutions with different pH values (2-12), with an excitation wavelength of 490 nm.

[0045] Figure 6 Cell imaging images (ae) of SMMC-7721 cells co-incubated with 4 μM fluorescent probe for different time periods and fluorescence intensity change graph (f) of SMMC-7721 cells co-incubated with 4 μM fluorescent probe for different time periods.

[0046] Figure 7 Confocal laser scanning images of SMMC-7721 cells incubated with different concentrations of the probe (1, 2, 4, 6, and 10 μM) (af) and a linear relationship graph (g) between fluorescence intensity and probe incubation concentration. The excitation wavelength was 500 nm, and the collection wavelength range was 520-560 nm.

[0047] Figure 8 Colocalization images of 4 μM PAN-BODIPY probe and the commercial lipid droplet dye HCS LipidTOX™ Deep Red in SMMC-7721 cells. (a) shows the bright field of the cell, (b) shows the distribution of the probe in the cell, (c) shows the distribution of HCS LipidTOX™ Deep Red in the cell, (d) shows the overlay of (b) and (c), (e) shows the correlation scatter plot of the colocalization intensity between (b) and (c), and (f) shows the fluorescence intensity distribution of the green line in (d). The probe was excited at 490 nm, and the collection wavelength range was 520-560 nm. The HCS LipidTOX™ Deep Red was excited at 637 nm, and the collection wavelength range was 650-670 nm.

[0048] Figure 9Figure 4 shows fluorescence imaging of 4 μM in normal cells (HUVCE, HL-7702, and HEK293) and cancer cells (MCF-7, HeLa, SMMC-7721) (a) and a comparison of cell fluorescence intensity (b). The excitation wavelength of the probe is 490 nm, and the collection wavelength range is 520-560 nm. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0050] Example 1

[0051] The preparation method of the polarity-responsive lipid droplet-targeted fluorescent probe of this embodiment is as follows: Figure 1 , the steps are as follows:

[0052]

[0053] (1) Synthesis of Intermediate 2: 2-Methoxy-5-nitrobenzaldehyde (3.00 g, 16.6 mmol) and 2,4-dimethylpyrrole (3.15 g, 33.5 mmol) were added to a 500 mL round-bottom flask containing CH2Cl2. The mixture was stirred at room temperature for 20 min under nitrogen protection, and trifluoroacetic acid (30 μL) was added and stirred overnight. When thin-layer chromatography showed that the starting material was completely consumed, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 3.9 g, 16.6 mmol) was dissolved in CH2Cl2 (40 mL) and added to the reaction mixture, and stirring was continued for 2 h. The reaction mixture was washed with water, dried over Na2SO4, filtered, and evaporated.

[0054] (2) The solid obtained in step (1) and triethylamine (33 mL) were dissolved in anhydrous CH2Cl2 and stirred at room temperature for 30 min. BF3·OEt2 (33 mL) was added and the mixture was stirred for 10 min. Water and 2M NaOH were added to the reaction mixture and extracted with CH2Cl2. The organic phase was dried over anhydrous Na2SO4, filtered, and evaporated to obtain a crude intermediate. The crude product was purified by column chromatography (EA / PE = 1:5) to obtain intermediate 2 as an orange solid (2.30 g, yield 34.8%).

[0055] (3) Synthesis of the fluorescent probe PAN-BODIPY: Intermediate 2 (0.5 g, 1.25 mmol) and 10% Pd-C (1 g) were dissolved in 50 mL of CH2Cl2. H2 was introduced at room temperature with continuous stirring for 3 h. The reaction mixture was filtered, and the filtrate was rotary evaporated to obtain a crude solid product. The crude product was purified by silica gel column chromatography to obtain PAN-BODIPY (310 mg, 67% yield) as an orange solid. 1 HNMR(600MHz,Methanol-d4)δ6.94(d,J=8.8Hz,1H),6.89(dd,J=8.8,2.8Hz,1H ),6.58(d,J=2.8Hz,1H),6.02(s,2H),3.71(s,3H),2.47(s,6H),1.56(s,6H), such as Figure 2 shown.

[0056] Implementation effect examples

[0057] (1) Optical properties of fluorescent probe PAN-BODIPY in organic solvents

[0058] The fluorescent probe PAN-BODIPY prepared in Example 1 was dissolved in acetonitrile to prepare a 2 mM stock solution. The solvation effect of the probe molecule PAN-BODIPY in common solvents, including PBS, methanol, acetonitrile, ethanol, ethylene glycol, N,N-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, toluene, cyclohexane, and n-hexane, was further investigated. Figure 3 As shown. Figure 3 As can be seen in a, the absorption peak of PAN-BODIPY in the solvent is located at around 490nm, and the position of the maximum absorption peak of the probe remains basically unchanged. Figure 3 b It can be clearly seen that the emission wavelength of the probe PAN-BODIPY is around 514 nm, and the fluorescence intensity of PAN-BODIPY is significantly higher in low-polarity organic reagents such as n-hexane and cyclohexane. Figure 3 As can be seen in Figure c, the probe has a good response to the ultra-small polarity range Δf = 0-0.013. Therefore, 490 nm was selected as the optimal excitation wavelength for the probe, and 514 nm was selected as the optimal emission wavelength for PAN-BODIPY for subsequent studies.

[0059] (2) Polarity response ability test of fluorescent probe PAN-BODIPY

[0060] Add 2 mL of n-hexane solution containing different proportions (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%) of ethanol to the cuvette, add 5 μL of 2 mM probe stock solution to make a mixed solution with a probe concentration of 5 μM, and perform fluorescence spectrum test. UV-visible absorption spectrum ( Figure 4 a) and fluorescence spectra ( Figure 4 b), ( Figure 4 c) is the relationship between fluorescence intensity and solvent polarity. The experimental results show that as the polarity (Δf) increases from 0.0 to 0.3, the fluorescence of the probe at 514 nm decreases sharply, indicating that the probe recognizes sensing polarity.

[0061] (III) pH stability of fluorescent probe PAN-BODIPY

[0062] Add 2 mL of BR buffer solution (0.2 M) with different pH values (2-12) to the cuvette, add 5 μL of 2 mM probe stock solution to prepare a mixture with a probe concentration of 5 μM, and test the UV-visible absorption spectrum of the mixture at different pH values ( Figure 5 a) and fluorescence spectra ( Figure 5 b). It can be seen that the probe has high stability to pH.

[0063] (IV) Screening of the cell entry time of the fluorescent probe PAN-BODIPY

[0064] SMMC-7721 cells were seeded in a confocal microplate, grown in a high-glucose medium containing 10% serum, and cultured in a constant-temperature incubator at 37°C, 5% carbon dioxide, and 95% air for 48 hours. The cells were first washed three times with PBS to remove metabolic waste during cell growth. The probe (4 μM) was co-incubated with SMMC-7721 cells, and the time when the probe entered the cells was determined by laser confocal imaging. The results are shown in Figure 2. Figure 6 As shown in the figure, the fluorescence intensity of the probe gradually increased with increasing incubation time with SMMC-7721 cells, reaching a plateau after 15 minutes. Therefore, a 15-minute incubation time was selected as the optimal time for probe entry. In subsequent cell imaging experiments, 15 minutes was used as the optimal incubation time for the probe to enter the cell. During this process, a gradual increase in green fluorescent spots was observed in the cytoplasm, with a morphology and appearance similar to lipid droplets, preliminarily confirming the rationality of the probe design. After 15 minutes of incubation, the fluorescence intensity remained essentially unchanged, indicating that the probe has good cell membrane permeability.

[0065] (V) Screening of the intracellular concentration of the fluorescent probe PAN-BODIPY

[0066] The specific operating conditions are as follows: SMMC-7721 cells were grown in a culture medium containing 10% serum and cultured in a 37°C constant temperature incubator containing 5% carbon dioxide and 95% air for 24 hours. Before confocal imaging, the attached cells were washed 2-3 times with PBS to remove metabolic waste during cell growth. Then, fresh culture medium containing different concentrations (1, 2, 4, 6, 10 μM) of fluorescent probes was added and incubated for 15 minutes before confocal imaging experiments.

[0067] The results are as follows Figure 7 As shown in the figure, with the increase of the concentration of fluorescent probe incubated with SMMC-7721, the fluorescence intensity of intracellular lipid droplets first gradually increased, and then after the probe concentration reached 2 μM, the fluorescence intensity of lipid droplets gradually stabilized. After the fluorescent probe concentration reached 6 μM, the fluorescence intensity in the cell began to decrease significantly. Therefore, the cell incubation concentration of the probe was selected as 4 μM, and the subsequent cell imaging experiments all selected 4 μM as the optimal concentration for the probe to enter the cell.

[0068] (VI) Co-localization test of fluorescent probe PAN-BODIPY in cells

[0069] After the adherent SMMC-7721 cells were washed with PBS 2-3 times, fresh culture medium containing the fluorescent probe (4 μM) and the commercial dye HCS Lipid TOXTM Deep Red (2 μM) was added and incubated in a constant temperature incubator at 37°C, 5% carbon dioxide and 95% air for 15 minutes. The culture medium was removed, the cells were washed with PBS 2-3 times, and fresh culture medium was added for laser confocal imaging.

[0070] like Figure 8 As shown in the figure, a is the cell bright field, b is the distribution of the probe in the cell, c is the distribution of HCS Lipid TOX™ Deep Red in the cell, d is the superimposed field of b and c, and f is the fluorescence intensity distribution of the green line in figure d. The distribution of the fluorescent probe and HCS Lipid TOX™ Deep Red in the cell has good overlap, with a colocalization coefficient exceeding 0.93, indicating that the fluorescent probe prepared by the present invention can be enriched in lipid droplets and achieve lipid droplet targeting.

[0071] (VII) Fluorescent probe PAN-BODIPY imaging in normal cells and cancer cells

[0072] The culture medium containing the probe (4 μM) was incubated in normal cells (HUVCE, HL-7702, and HEK293) and cancer cells (MCF-7, HeLa, and SMMC-7721) for 15 minutes, and then imaged using a laser confocal microscope.

[0073] pass Figure 9 Normal cells have relatively few lipid droplets and exhibit weaker fluorescence. Cancer cells, however, exhibit weaker fluorescence, except for SMMC-7721 cells, despite having more lipid droplets. This indicates that SMMC-7721 cells can be specifically identified by changes in fluorescence intensity. This suggests that the inventive probe can distinguish SMMC-7721 cells from other cells based on changes in fluorescence intensity caused by differences in intracellular polarity.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polarity-responsive lipid droplet-targeting fluorescent probe, characterized in that: The structural formula is as follows:

2. The polarity-responsive lipid droplet-targeting fluorescent probe according to claim 1, characterized in that The fluorescent probe has lipid droplet targeting capability and highly sensitive polarity response capability when the polarity Δf is 0-0.

013.

3. Use of the polarity-responsive lipid droplet-targeted fluorescent probe according to claim 1 for detecting the polarity of a test solution for non-disease diagnosis purposes.

4. The use according to claim 3, characterized in that The steps are: adding a stock solution of a polarity-responsive lipid droplet-targeted fluorescent probe to a test solution, and detecting the fluorescence intensity of the test solution at an excitation wavelength of 490 nm.

5. The use according to claim 4, characterized in that The final concentration of the polarity-responsive lipid droplet-targeted fluorescent probe is 5 μM.

6. Use of the polarity-responsive lipid droplet-targeting fluorescent probe according to claim 1 in the preparation of a rapid, wash-free imaging reagent for intracellular lipid droplets.

7. The use according to claim 6, characterized in that The steps are: incubating the stock solution of the polarity-responsive lipid droplet-targeted fluorescent probe with cells, and observing the wash-free imaging effect of the probe through laser confocal imaging.

8. The use according to claim 7, characterized in that The co-incubation time is 15 minutes, and the final concentration of the polarity-responsive lipid droplet-targeted fluorescent probe is 4 μM.

9. Use of the polarity-responsive lipid droplet-targeted fluorescent probe according to claim 1 in preparing a reagent for visually monitoring changes in lipid droplet polarity in living cells.

10. Use of the polarity-responsive lipid droplet-targeted fluorescent probe according to claim 1 in preparing a detection reagent for distinguishing liver cancer cells from other cells, characterized in that: The other cells are normal cells, MCF-7 cells or HeLa cells.

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