Application of a polarity-resistant blue-shifting lipid droplet-targeting fluorescent probe in polarity detection

Abstract

This invention discloses the application of a polarity-resistant, blue-shift-resistant, lipid droplet-targeting fluorescent probe in the preparation of lipid droplet labeling, monitoring of lipid droplet dynamic changes, and intradroplet polarity changes. The fluorescent probe exhibits a strong fluorescence response to polarity, with the fluorescence emission intensity gradually decreasing as polarity increases, while the maximum fluorescence emission wavelength remains unaffected by red-shift or blue-shift. The fluorescent probe can accumulate in lipid droplets within living cells, enabling real-time, wash-free imaging of lipid droplets. It can specifically label the morphology of lipid droplets and monitor intradroplet polarity. Based on its excellent polarity response and lipid droplet targeting, it is applied to monitor the dynamic changes of lipid droplets in oleic acid-stimulated and bisphenol A-induced non-alcoholic fatty liver disease. The probe of this invention possesses good biocompatibility, high photostability, and deep tissue penetration, making it suitable for research at the cellular and in vivo levels.

Description

Technical Field

[0001] This invention relates to the fields of fluorescent probes and biosensing technology, specifically to the application of a polarity-resistant blue-shifting lipid droplet-targeting fluorescent probe in polarity detection. Technical Background

[0002] Lipid droplets are highly dynamic subcellular organelles for storing and supplying lipids within cells, composed of a hydrophobic core of neutral lipids such as cholesterol esters and triglycerides. Previously, lipid droplets were simply considered inert fat particles. However, recent research indicates that lipid droplets not only store excess lipids in cells but also participate in various intracellular life activities, including energy balance, lipid metabolism, membrane protein expression, and membrane transport. Abnormalities in lipid droplets are closely related to various diseases such as fatty liver, type II diabetes, obesity, atherosclerosis, and cancer. The cellular microenvironment, including polarity, viscosity, pH, and temperature, is an important parameter for assessing the metabolic state of an organism. Abnormal lipid droplet polarity disrupts the homeostasis of intracellular lipid metabolism, undermines the normal functioning of various intracellular life activities, and leads to lipid droplet dysfunction and various diseases. Therefore, there is an urgent need to develop tools for real-time monitoring of changes in intracellular lipid droplet polarity.

[0003] Fluorescent probes are widely used in biomedical sensing due to their advantages such as simple synthesis, high sensitivity, good biocompatibility, and high spatiotemporal resolution. They provide effective tools for long-term, real-time observation of living cells and the interactions of various life activities within organisms. Fluorescent probes play a crucial role in tracking changes in lipid droplet behavior. However, most reported lipid droplet-targeting fluorescent probes are designed based on intramolecular charge transfer effects. Their inherently broad emission bands and full width at half maximum (FWHM), along with the blue or red shift of the maximum emission wavelength due to changes in microenvironment polarity, lead to inaccurate collection of fluorescence signals and erroneous analysis of chemical and biological information. In particular, in live samples where molecular oxygen concentration levels cannot be controlled and in single-molecule imaging, the spectral stability of fluorescent probes is crucial for the identification and tracking of observed targets, as well as the integrity and reproducibility of collected research data. Therefore, developing spectrally stable lipid droplet-targeting fluorescent probes with fluorescence illumination modes is necessary for real-time monitoring of lipid droplet behavior and elucidating the physiological and pathological functions of lipid droplets. Summary of the Invention

[0004] This invention proposes an application of a polarity-resistant blue-shift lipid droplet-targeting fluorescent probe in polarity detection. Using BODIPY fluorescent dye as the core, a polarity-resistant blue-shift fluorescent probe was designed and synthesized by introducing a strongly electron-withdrawing nitrile group at its meso- position. The fluorescence emission intensity of this probe gradually decreases with increasing polarity, while the maximum fluorescence emission wavelength remains unchanged. After entering the cell, this fluorescent probe can rapidly penetrate the cell membrane and specifically accumulate in lipid droplets, enabling specific recognition and sensing of lipid droplets and real-time dynamic monitoring of changes in the number, diameter, and polarity of intracellular lipid droplets under physiological and pathological conditions.

[0005] The technical solution for realizing the present invention is as follows:

[0006] Application of a polarity-resistant blue-shift lipid droplet-targeting fluorescent probe in polarity detection, wherein the fluorescent probe has the following structural formula: The fluorescent probe is targeted to lipid droplets, enabling real-time wash-free imaging of lipid droplets, and exhibits a sensitive fluorescence response to polarity.

[0007] The above application specifically includes the following steps:

[0008] (1) Dissolve the fluorescent probe in acetonitrile to prepare a probe stock solution;

[0009] (2) Take water and 1,4-dioxane in different volume ratios to prepare a series of solutions with different polarities;

[0010] (3) Place the solutions of different polarities prepared in step (2) into multiple fluorescent cuvettes, add the probe stock solution prepared in step (1) to each fluorescent cuvette, measure the fluorescence spectrum of each solution with 575nm as the excitation wavelength, and observe the change of fluorescence spectrum with polarity.

[0011] The concentration of the probe stock solution in step (1) is 2 mM, and the final concentration of the fluorescent probe in step (3) is 4 μM.

[0012] Changes in fluorescence emission spectrum: As the polarity of the solution increases, the fluorescence intensity of the fluorescent probe at 615 nm gradually decreases, but the emission wavelength remains basically unchanged. The maximum emission wavelength of the fluorescent probe does not undergo a blue shift or red shift with the change in polarity.

[0013] The fluorescent probe is used in reagents for labeling lipid droplets, monitoring the dynamic changes of lipid droplets, and monitoring the polarity changes within lipid droplets. The fluorescent probe can be enriched in lipid droplets in living cells, enabling real-time wash-free imaging of lipid droplets. It can specifically label the morphology of lipid droplets and monitor the dynamic changes in the number, size, and polarity of lipid droplets in physiological and pathological conditions.

[0014] The specific steps of the above application are as follows: the culture medium containing 4 μM fluorescent probe is incubated with SMMC-7721 cells in a constant temperature incubator at 37℃, 5% carbon dioxide and 95% air for 40 min, and the changes in the number, size and fluorescence intensity of lipid droplets are observed by laser confocal imaging.

[0015] The fluorescent probe can monitor changes in the number, size, and polarity of intracellular lipid droplets induced by drugs.

[0016] The drug includes oleic acid and bisphenol A.

[0017] The steps for monitoring the dynamic changes of intracellular lipid droplets under oleic acid stimulation using the fluorescent probe are as follows: first, stimulate the test cells with oleic acid, then incubate the oleic acid-stimulated test cells with a culture medium containing the fluorescent probe for 40 min, and observe the changes in the number, size and fluorescence intensity of intracellular lipid droplets by laser confocal imaging.

[0018] The steps for monitoring the dynamic changes of lipid droplets in bisphenol A-induced non-alcoholic fatty liver disease using the fluorescent probe are as follows: stimulate the test cells with the same concentration of bisphenol A for different durations, or stimulate the test cells with different concentrations of bisphenol A for the same duration, and then incubate the bisphenol A-stimulated test cells with a culture medium containing the fluorescent probe for 40 min. Observe the changes in the number, size and fluorescence intensity of intracellular lipid droplets by laser confocal imaging.

[0019] The beneficial effects of this invention are:

[0020] (1) The fluorescent probe provided by the present invention has excellent spectral stability. Under high intensity excitation, the maximum emission wavelength of the fluorescent probe will not change with the change of environmental parameters, so that quantitative analysis of the target and tracking of various life activities in cells can be realized based on the change of fluorescence signal at the maximum emission wavelength, and accurate research data can be collected to provide reliable chemical biology information.

[0021] (2) The present invention uses the BODIPY basic structure with high quantum yield and narrow half-peak width as the fluorescent group. After introducing a nitrile group in the middle position, the maximum absorption and emission wavelengths of the fluorescent probe are red-shifted by nearly 100 nm compared with traditional BODIPY dyes. This effectively avoids interference from biological background fluorescence, reduces damage to cells, and improves its applicability in vivo.

[0022] (3) The fluorescent probe provided by this invention has good biocompatibility and strong tissue penetration. MTT experiments showed that SMMC-7721 cells still had a survival rate of over 90% when the probe concentration reached 10 μM, indicating that the probe has good biocompatibility; red light emission endows the probe with strong tissue penetration ability.

[0023] (4) The fluorescent probe provided by the present invention can realize the lipid droplet-free imaging, which simplifies the cell imaging process and avoids damage to cell morphology during PBS washing. It has broad application prospects in the field of wash-free imaging. Attached Figure Description

[0024] 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.

[0025] Figure 1 A shows the fluorescence emission spectra of the probe (final concentration of 4 μM) in mixed solutions of water and 1,4-dioxane at different volume fractions. Figure 1 B is a graph showing the linear relationship between the fluorescence intensity of the fluorescent probe at 615 nm and the polarity of the solution when the excitation wavelength is 575 nm.

[0026] Figure 2 A is a bar chart showing the relative fluorescence intensity of the fluorescent probe at 615 nm under the presence of different interfering substances when the excitation wavelength is 575 nm. Figure 2 B is a bar chart showing the relative fluorescence intensity of the fluorescent probe at 615 nm in BR buffer solutions with different pH values, with an excitation wavelength of 575 nm;

[0027] Figure 3 The results show the cytotoxicity of different concentrations of fluorescent probes (final concentrations of 0, 2, 4, 6, 8, and 10 μM) in SMMC-7721 cells.

[0028] Figure 4 A shows cell imaging images of SMMC-7721 cells co-incubated with a 4μM fluorescent probe for different time periods; Figure 4 B shows the changes in fluorescence intensity when a 4 μM fluorescent probe is co-incubated with SMMC-7721 cells for different time periods.

[0029] Figure 5 The images show the photostability test results of a 4 μM fluorescent probe and a 2 μM commercial dye BODIPY493 / 503 in SMMC-7721 cells.

[0030] Figure 6 Colocalization imaging of a 4 μM fluorescent probe and a 2 μM commercial dye BODIPY 493 / 503 in SMMC-7721 cells;

[0031] Figure 7Laser confocal imaging (A), bar graph of lipid droplet number in a single cell (B), bar graph of lipid droplet diameter (C), and bar graph of fluorescence intensity of a single lipid droplet were obtained after stimulating SMMC-7721 cells with 0.5 mM oleic acid for 24 h.

[0032] Figure 8 Laser confocal imaging (A), bar graph of intracellular lipid droplet number and cell fluorescence intensity (B), and bar graph of lipid droplet diameter and single lipid droplet fluorescence intensity (C) are shown for SMMC-7721 cells stimulated with 20 μM bisphenol A for different time periods and then co-incubated with 4 μM fluorescent probe for 40 min.

[0033] Figure 9 Laser confocal imaging (A), bar graph of intracellular lipid droplet number and cell fluorescence intensity (B), and bar graph of lipid droplet diameter and single lipid droplet fluorescence intensity (C) were obtained after stimulating SMMC-7721 cells with different concentrations of bisphenol A for 24 h and then incubating the cells with a 4 μM fluorescent probe for 40 min. Detailed Implementation

[0034] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Example 1: Fluorescence spectroscopy test of fluorescent probe in solutions of different polarities

[0036] The experimental steps are as follows:

[0037] (1) Weigh a certain mass of fluorescent probe, dissolve it in acetonitrile, and prepare a 2mM probe stock solution. The CAS number of the fluorescent probe used in this invention is 157410-23-6, which was purchased from Tokyo Chemical Industry Co., Ltd.

[0038] (2) A series of solutions with different polarities (Δf) were prepared by water and 1,4-dioxane, with volume ratios of water to 1,4-dioxane of 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0, respectively.

[0039] (3) Take 2 mL of the solutions with different polarities prepared in step (2) and place them in multiple fluorescent cuvettes. Add 4 μL of the probe stock solution prepared in step (1) to each fluorescent cuvette, so that the final concentration of the fluorescent probe in each solution is 4 μM. Under excitation at 575 nm, test the fluorescence intensity of each solution at 615 nm. All solutions are tested in quartz cuvettes at room temperature.

[0040] Figure 1 A shows the fluorescence emission spectra of the fluorescent probe in water / 1,4-dioxane polar test solutions at different volume ratios. The curves from top to bottom correspond to the water percentages in the solutions as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

[0041] like Figure 1 As shown in Figure A, with increasing water content, the polarity of the solution gradually increases, and the fluorescence intensity of the fluorescent probe gradually decreases, but the fluorescence emission wavelength remains essentially unchanged. Figure 1 As shown in Figure B, when the solution polarity is in the range of Δf = 0.27-0.31, the fluorescence intensity of the fluorescent probe at 615 nm in the solution exhibits a good linear relationship with the polarity (Rf). 2 =0.9863).

[0042] The above experiments demonstrate that the fluorescent probe is sensitive to polarity, and the maximum emission wavelength of the probe does not undergo a blue shift or red shift with changes in environmental polarity.

[0043] Example 2: Investigation of the selectivity of fluorescent probes and fluorescence response at different pH levels

[0044] (1) Selective experimental procedure:

[0045] Prepare aqueous solutions of the relevant analytes, including metal ions (K+, K ... + Ca 2+ Na + Mg 2+ Al 3+ Zn 2+ Fe 2 + Fe 3+ Cu 2+ Mn 2+ ), amino acids (Ser, Asn, Val, Ile, His, Lys, Gln, Asp, Thr, Glu, GSH, Cys), ascorbic acid (AA), hydrogen peroxide (H2O2), and hypochlorite (ClO2) - );

[0046] Liposomes and probe stock solution were added to one cuvette to make a total volume of 2 mL, with final concentrations of 4 μM for the fluorescent probe and 4 mM for the liposomes. Another cuvette was taken, and probe stock solution and PBS solution were added to make a total volume of 2 mL, with a final concentration of 4 μM for the fluorescent probe, serving as the control group. Then, multiple cuvettes were taken, and different analyte solutions and probe stock solutions were added to each cuvette to make a total volume of 2 mL, with final concentrations of 4 μM for the fluorescent probe and 1 mM for the analyte, serving as the experimental group. The fluorescence intensity of each solution was measured at 615 nm, with an excitation wavelength of 575 nm.

[0047] Figure 2 A is a bar chart showing the relative fluorescence intensity of fluorescent probes in the presence of different analytes. Figure 2 From left to right in image A: Liposome, PBS (control group), K + Ca 2+ Na + Mg 2+ Al 3+ Zn 2+ Fe 2+ Fe 3+ Cu 2+ Mn 2+ ,Ser,Asn,Val,Ile,His,Lys,Gln,Asp,Thr,Glu,GSH,Cys,AA,H2O2,ClO - .

[0048] The results are as follows Figure 2 As shown in Figure A, compared to the fluorescence intensity of the control group, the fluorescence intensity of the photoprobe remained essentially unchanged after the addition of various analytes. Only in liposomes did the probe release a significant fluorescence signal.

[0049] (2) Fluorescence response in different pH environments

[0050] Prepare BR buffer solutions with different pH values ​​(4-10);

[0051] 4 μL of probe stock solution was diluted in 2 mL of buffer solutions with different pH values ​​to make the final concentration of fluorescent probe 4 μM. The excitation wavelength was 575 nm, and the fluorescence intensity of each solution was measured at 615 nm.

[0052] The results are as follows Figure 2 As shown in Figure B, in BR buffer solutions with pH 4-10, the fluorescence intensity of the probe did not change significantly with changes in solution pH.

[0053] The above experimental results show that the fluorescence intensity of the probe remains essentially unchanged in the presence of relevant analytes, and also remains essentially unchanged under different pH conditions. This verifies that the fluorescent probe described in this invention can be used to sense polarity in complex organisms without being interfered with.

[0054] Example 3: Cytotoxicity test of fluorescent probe

[0055] SMMC-7721 cells were cultured in 96-well plates with 100 μL of DMEM (1% penicillin and streptomycin and 10% FBS) at 37°C (5% CO2 and 95% air) for 24 h. Then, the culture medium in the 96-well plates was removed, and fresh culture medium containing different concentrations of fluorescent probes (final concentrations of 0, 2, 4, 6, 8, and 10 μM) was added. The cells were cultured for another 24 h, and 10 μL of MTT solution (5 mg / mL) was added to each well. -1 Continue culturing for 4 hours, dissolve the generated formazan in 100 μL of DMSO, place the 96-well plate on a mixer and shake for 10 minutes, and use an ELISA reader to detect the absorbance of each well at 570 nm.

[0056] Cell viability (%) = (OD) sample -OD blank ) / (OD control -OD blank )×100%

[0057] OD sample SMMC-7721 cells incubated in culture medium containing different concentrations of fluorescent probes.

[0058] OD control SMMC-7721 cells incubated in a culture medium without fluorescent probes.

[0059] OD blank Culture medium without SMMC-7721 cells.

[0060] The results are as follows Figure 3 As shown, when the probe concentration reaches 10 μM, SMMC-7721 cells still have a cell survival rate of over 90%, indicating that the fluorescent probe described in this invention has good biocompatibility.

[0061] Example 4: Screening of Cell Entry Time for Fluorescent Probes

[0062] The experimental steps are as follows:

[0063] (1) SMMC-7721 cells were seeded in a special confocal dish and 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 24 hours.

[0064] (2) The 4μM probe was co-incubated with SMMC-7721 cells, and the time of probe entry into the cells was determined by laser confocal imaging.

[0065] The specific operating conditions were as follows: SMMC-7721 cells were grown in a 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 adherent cells were washed with PBS 2-3 times to remove metabolic waste generated during cell growth. Then, fresh medium containing 4 μM of fluorescent probe was added, and confocal imaging experiments were performed after incubation for different times.

[0066] The results are as follows Figure 4 As shown, the fluorescence intensity of the fluorescent probe gradually increased with the increase of co-incubation time with SMMC-7721 cells, and tended to stabilize after 40 min. Therefore, 40 min was selected as the optimal incubation time for probe entry into cells in subsequent cell imaging experiments. During this process, the number of red fluorescent spots in the cytoplasm gradually increased, and their morphology and appearance were similar to lipid droplets, which preliminarily proved the rationality of the probe design.

[0067] Example 5: Photostability Test of Fluorescent Probes in Cells

[0068] Two groups of adherent SMMC-7721 cells were washed 2-3 times with PBS, and then added to fresh culture medium containing a fluorescent probe and the commercial dye BODIPY493 / 503, respectively, to achieve final concentrations of 4 μM for the fluorescent probe and 2 μM for the commercial dye. The cells were incubated at 37°C in a constant temperature incubator with 5% CO2 and 95% air. The co-incubation time between the fluorescent probe and SMMC-7721 cells was 40 min, and the co-incubation time between BODIPY493 / 503 and SMMC-7721 cells was 15 min. Laser confocal imaging was then performed. The excitation wavelength of the probe was 550 nm, and the collection wavelength was 580-640 nm. The excitation wavelength of BODIPY493 / 503 was 488 nm, and the collection wavelength was 500-540 nm. The continuous laser scanning time was 15 min.

[0069] The results are as follows Figure 5As shown, a bright fluorescence signal was observed in the cells at 0 min of laser scanning. With increasing laser scanning time, after 15 min of continuous scanning, the fluorescence intensity of the probe within the cells still retained more than 70% of its initial value. In contrast, cells stained with the commercial lipid droplet dye BODIPY493 / 503 showed only 20% of their initial intensity after 15 min of continuous scanning. These experimental results indicate that the probe also exhibits good stability within cells and is unaffected by the internal environment.

[0070] Example 6: Colocalization test of fluorescent probes in cells

[0071] After adhering SMMC-7721 cells were washed 2-3 times with PBS, they were incubated with a 4 μM probe in a constant temperature incubator at 37°C, 5% CO2 and 95% air for 40 min. Then, fresh medium containing 2 μM BODIPY 493 / 503 was added and incubated for 15 min. After removing the medium, the cells were washed 2-3 times with PBS and fresh medium was added for laser confocal imaging.

[0072] Imaging results as follows Figure 6 As shown, a is the bright field of the cell, b is the distribution of the fluorescent probe in the cell, c is the distribution of BODIPY493 / 503 in the cell, d is the superposition field of b and c, and f is the fluorescence intensity distribution of the white line in figure d. Figure 6 As shown in Figure e, the distribution of the fluorescent probe and BODIPY 493 / 503 in the cells shows good overlap, with a co-localization coefficient exceeding 0.96, indicating that the probe can be enriched in lipid droplets, achieving lipid droplet targeting.

[0073] The above experimental results demonstrate that the probe has good lipid droplet targeting ability.

[0074] Example 7: Monitoring the dynamic changes of lipid droplets under oleic acid treatment with fluorescent probes

[0075] Two groups of adherent SMMC-7721 cells were incubated for 24 hours in a constant temperature incubator at 37°C, 5% CO2, and 95% air using fresh culture medium containing 0.5 mM oleic acid. Before imaging, the cells were washed three times with PBS to remove excess oleic acid. Subsequently, oleic acid-stimulated SMMC-7721 cells were co-incubated with culture medium containing 4 μM fluorescent probe and 2 μM BODIPY493 / 503 in a constant temperature incubator at 37°C, 5% CO2, and 95% air. The co-incubation time between the fluorescent probe and SMMC-7721 cells was 40 min, and the co-incubation time between BODIPY493 / 503 and SMMC-7721 cells was 15 min. Laser confocal imaging was then performed. The excitation wavelength of the probe is 550 nm, and the collection wavelength is 580-640 nm. The excitation wavelength of BODIPY 493 / 503 is 488 nm, and the collection wavelength is 500-540 nm.

[0076] The results are as follows Figure 7 As shown in Figures A, B, C, and D, quantitative analysis revealed that the number and size of lipid droplets in the two groups of cells treated with oleic acid increased by approximately 2.7-fold and 1.8-fold, respectively. Furthermore, co-incubation of SMMC-7721 cells stimulated with oleic acid using a fluorescent probe resulted in a 2.4-fold decrease in the fluorescence intensity of individual lipid droplets. However, co-incubation of BODIPY493 / 503 cells with SMMC-7721 cells stimulated with oleic acid showed no significant change in the fluorescence intensity of individual lipid droplets. These experimental results indicate that oleic acid can stimulate an increase in the polarity of intracellular lipid droplets.

[0077] Example 8: Monitoring the dynamic changes of lipid droplets in bisphenol A-induced non-alcoholic fatty liver disease using fluorescent probes.

[0078] (1) SMMC-7721 cells were first incubated with culture medium containing 20 μM bisphenol A in a constant temperature incubator at 37°C, 5% carbon dioxide, and 95% air for different times (0 h, 12 h, 24 h). Then, SMMC-7721 cells were incubated with culture medium containing 4 μM fluorescent probe in a constant temperature incubator at 37°C, 5% carbon dioxide, and 95% air for 40 min after bisphenol A stimulation. Laser confocal imaging was then performed. The excitation wavelength was 550 nm, and the collection wavelength was 580-640 nm.

[0079] The results are as follows Figure 8 As shown, with the increase of bisphenol A stimulation time, the number of intracellular lipid droplets and the fluorescence intensity of the cells gradually increased, while the size of the lipid droplets and the fluorescence intensity of a single lipid droplet remained basically unchanged.

[0080] (2) First, SMMC-7721 cells were incubated for 24 h in a constant temperature incubator at 37°C, 5% carbon dioxide and 95% air with culture medium containing different concentrations of bisphenol A (0, 10 μM, 20 μM). Then, SMMC-7721 cells stimulated with different concentrations of bisphenol A for 24 h were incubated for 40 min in a constant temperature incubator at 37°C, 5% carbon dioxide and 95% air with culture medium containing 4 μM fluorescent probe. Then, laser confocal imaging was performed.

[0081] The results are as follows Figure 9 As shown, with the increase of bisphenol A stimulation concentration, the number of lipid droplets in the cell and the cell fluorescence intensity gradually increase, while the size of the lipid droplets and the fluorescence intensity of a single lipid droplet remain basically unchanged.

[0082] The results of this experiment show that the number of lipid droplets increases in bisphenol A-induced non-alcoholic fatty liver disease, but the polarity and diameter of the lipid droplets remain unchanged.

[0083] 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 within the protection scope of the present invention.

Claims

1. The application of a polarity-resistant blue-shift lipid droplet-targeting fluorescent probe in the preparation of lipid droplet labeling, monitoring of lipid droplet dynamic changes and intradroplet polarity changes, characterized in that, The structural formula of the fluorescent probe is as follows: The fluorescent probe is targeted to lipid droplets.

2. The application according to claim 1, characterized in that, Includes the following steps: The fluorescent probe was dissolved in acetonitrile to prepare a probe stock solution; A series of solutions with different polarities were prepared by taking water and 1,4-dioxane in different volume ratios. The solutions with different polarities prepared in step (2) were placed in multiple fluorescent cuvettes. The probe stock solution prepared in step (1) was added to each fluorescent cuvette. The fluorescence spectrum of each solution was measured with 575 nm as the excitation wavelength, and the change of fluorescence spectrum with polarity was observed.

3. The application according to claim 2, characterized in that, The concentration of the probe stock solution in step (1) is 2 mM, and the final concentration of the fluorescent probe in step (3) is 4 mM. .

4. The application according to claim 2 or 3, characterized in that, As the polarity of the solution increases, the fluorescence intensity of the fluorescent probe gradually decreases, while the emission wavelength remains essentially unchanged.

5. The application according to claim 1, characterized in that, The steps are as follows: The culture medium containing the fluorescent probe is incubated with the cells in a constant temperature incubator at 37°C, 5% carbon dioxide and 95% air. The changes in the number, size and fluorescence intensity of lipid droplets are observed by laser confocal imaging.

6. The application according to claim 5, characterized in that, The cells were SMMC-7721 cells, and the final concentration of the fluorescent probe in the culture medium was 4. The co-incubation time is 40 minutes.

7. The application according to any one of claims 5-6, characterized in that, The fluorescent probe can monitor changes in the number, size, and polarity of intracellular lipid droplets induced by oleic acid and bisphenol A.

Images