A viscosity-responsive lipid droplet-targeted fluorescent probe and its application

Abstract

The present invention discloses a viscosity-responsive lipid droplet-targeted fluorescent probe and its application. The structural formula of the probe is such that the fluorescent probe has a strong fluorescent response to viscosity and can be enriched in the lipid droplets of living cells, realizing real-time washing-free imaging of lipid droplets, capable of specifically labeling the morphology of lipid droplets and monitoring the viscosity inside the lipid droplets. Based on its good viscosity responsiveness and lipid droplet targeting property, the probe is applied to the real-time dynamic monitoring of the viscosity fluctuation inside lipid droplets in non-alcoholic fatty liver disease; using this probe, the effective differentiation between normal hepatocytes and hepatoma cells can also be achieved; the fluorescent probe prepared by the present invention has good biocompatibility, high photostability and strong tissue penetration ability, and can be used for research at the cellular and in vivo levels.

Description

Technical Field

[0001] The present invention relates to the technical fields of fluorescent probes and biosensing, and particularly relates to a viscosity-responsive lipid droplet-targeted fluorescent probe and its application. Technical Background

[0002] Lipid droplets are highly dynamic organelles that play important roles in important life processes such as lipid metabolism, immunity, and signal transduction, and bear the heavy responsibility of storing biological lipids in cells. Abnormal lipid droplets are closely related to various metabolic diseases, such as diabetes, fatty liver, atherosclerosis, and cancer. Viscosity, as an important microenvironmental parameter in cells, plays important roles in membrane fusion, material transport, signal transduction, autophagy, apoptosis, ferroptosis, and oxidative stress. Abnormal viscosity within lipid droplets will disrupt the balance of lipid metabolism, thereby causing disorders of intracellular physiological processes and life activities, and further leading to the occurrence of a series of lipid droplet-related diseases such as lipid storage diseases. Therefore, dynamic monitoring of the viscosity of intracellular lipid droplets helps to explore the physiological and pathological processes of lipid droplet-related diseases, clarify the physiological and pathological functions of lipid droplets, and promote the pathological research of related metabolic diseases.

[0003] Fluorescent probes have advantages such as high spatiotemporal resolution and high sensitivity, and are suitable for long-term real-time tracing in cells and organisms, becoming one of the essential tools in the field of biomedical research. BODIPY dyes have received extensive attention due to their advantages such as large molar extinction coefficient, high fluorescence quantum yield, good chemical stability, and strong biocompatibility. At present, the application of BODIPY dye-based lipid droplet-targeted probes in organisms is greatly limited, mainly because of their short emission wavelength and small Stokes shift. For example, the Stokes shift of the commercial lipid droplet dye BODIPY 493 / 503 is only 20 nm, and there is significant background light interference from biological substances and easy self-absorption during in vivo imaging, which will affect the accurate judgment of researchers' analysis and research of chemical biology information. Therefore, rationally constructing a fluorescent probe with high sensitivity to viscosity, rapidly crossing the cell membrane and specifically enriching in lipid droplets, to achieve specific recognition and sensing of the viscosity within lipid droplets and real-time dynamic monitoring of the changes in the number and viscosity of intracellular lipid droplets under pathological conditions is of great significance for the study of the occurrence and development of lipid droplet-related diseases in clinical practice. Summary of the Invention

[0004] The present invention provides a viscosity-responsive lipid droplet-targeted fluorescent probe and its application. Using a BODIPY fluorescent dye as the core, a small-molecule fluorescent probe with lipid droplet-targeting ability and viscosity sensitivity is designed and synthesized by introducing a trifluoromethyl rotor group at its meso-position. This probe can rapidly penetrate the cell membrane and specifically accumulate in lipid droplets, achieving specific recognition and sensing of the viscosity within lipid droplets, as well as real-time dynamic monitoring of the changes in the number and viscosity of intracellular lipid droplets under pathological conditions. Based on the differences in lipid droplet viscosity between normal hepatocytes and hepatoma cells, this probe can also effectively distinguish between normal hepatocytes and hepatoma cells.

[0005] The technical solution to achieve the present invention is as follows:

[0006] A viscosity-responsive lipid droplet-targeted fluorescent probe, the structural formula of the fluorescent probe is:

[0007]

[0008] The preparation method of the fluorescent probe includes the following steps:

[0009] (1) Add 2,4-dimethylpyrrole and CF3COOH to a CH2Cl2 solution respectively, stir and mix evenly, then add PhSiCl3, stir at room temperature for 10 min, then continue to add Et3N to the above solution and stir for another 15 min, and then add BF3·Et2O to the stirred solution and continue to stir for 3 h;

[0010] (2) After the reaction mixture obtained in step (1) is washed with H2O, the aqueous phase is extracted with CH2Cl2, the combined organic phases are dried with magnesium sulfate, and the crude product is obtained after filtration and evaporation;

[0011] (3) The crude product is purified by silica gel column chromatography, dried and evaporated to obtain a dark red solid.

[0012] The synthetic route of the fluorescent probe is as follows:

[0013]

[0014] The application of the fluorescent probe in viscosity detection.

[0015] The above application includes the following steps:

[0016] (1) Dissolve the fluorescent probe in DMSO to prepare a 2 mM probe stock solution;

[0017] (2) Take different volumes of methanol and glycerol respectively to prepare a series of solutions with different viscosities (0.65 cP - 953 cP);

[0018] (3) Take the solutions with different viscosities prepared in step (2) and place them in multiple fluorescence cuvettes. Add the probe stock solution prepared in step (1) to each fluorescence cuvette so that the final concentration of the fluorescent probe in each cuvette is 2 μΜ. Under the excitation at 550 nm, measure the fluorescence intensity of each solution at 614 nm.

[0019] Fluorescence intensity change: As the viscosity of the solution increases, the fluorescence intensity of the probe gradually increases.

[0020] Application of the fluorescent probe in lipid droplet labeling and monitoring lipid droplet viscosity change reagents. The fluorescent probe can be enriched in the lipid droplets of living cells, realizing real-time washing-free imaging of lipid droplets, being able to specifically label the morphology of lipid droplets, and monitor the viscosity inside the lipid droplets.

[0021] The fluorescent probe can monitor the changes in the number and viscosity of intracellular lipid droplets under drug induction.

[0022] The drugs include nystatin and bisphenol A.

[0023] The steps for monitoring the change in intracellular lipid droplet viscosity under nystatin stimulation using the fluorescent probe are as follows: First, stimulate the cells to be tested with nystatin, and then incubate the cells to be tested after nystatin stimulation with a medium containing the fluorescent probe for 15 min. Observe the change in the fluorescence intensity of the probe in the cells through laser confocal imaging.

[0024] The steps for monitoring the change in lipid droplet viscosity in non-alcoholic fatty liver disease induced by bisphenol A using the fluorescent probe are as follows: Stimulate the cells to be tested with the same concentration of bisphenol A for different times, or stimulate the cells to be tested with different concentrations of bisphenol A for the same time. Then incubate the cells to be tested after bisphenol A stimulation with a medium containing the fluorescent probe for 15 min. Observe the changes in the number of intracellular lipid droplets and the fluorescence intensity of the probe through laser confocal imaging.

[0025] The fluorescent probe can image the lipid droplets in cancer cells and detect the viscosity inside the lipid droplets. According to the viscosity difference of lipid droplets in normal cells and cancer cells, effective differentiation between normal cells and cancer cells can be achieved. The specific content is as follows: Under the same conditions, incubate human normal liver cells HL-7702 and human liver cancer cells SMMC-7721 with a medium containing the fluorescent probe for 15 min respectively, and then observe the fluorescence intensity of the probe in the above cells through laser confocal imaging.

[0026] The beneficial effects of the present invention are:

[0027] (1) The present invention uses a lipid-soluble BODIPY fluorescent dye as the parent nucleus, and designs and synthesizes a small molecule fluorescent probe with lipid droplet targeting ability and viscosity sensitivity by introducing a trifluoromethyl rotor group at its meso-position. In a non-viscous or low-viscosity environment, the trifluoromethyl rotor group can rotate freely and release energy through non-radiative transition, resulting in fluorescence quenching; under high-viscosity conditions, the rotation of the trifluoromethyl rotor is blocked, non-radiative transition is inhibited, and the fluorescence signal of the fluorophore is released; based on the sensitivity of the probe to viscosity and its targeting to lipid droplets, the fluctuation of viscosity inside lipid droplets can be monitored in real time and dynamically by the change of fluorescence intensity, and it is successfully used to monitor the dynamic changes of lipid droplets in bisphenol A-induced non-alcoholic fatty liver disease. In addition, by combining the difference in viscosity inside lipid droplets between normal hepatocytes and hepatoma cells, the probe releases a significantly stronger fluorescence signal in hepatoma cells than in normal hepatocytes, thus realizing the effective distinction between the two.

[0028] (2) The probe provided by the present invention has good stability. Under the condition that the excitation wavelength is 550 nm, the fluorescence intensity of the probe at 610 nm in glycerol and PBS solutions basically remains unchanged with the extension of the illumination time. After continuous illumination for 30 min, more than 92% of the fluorescence remains, indicating that the probe has good photostability in both low-viscosity and high-viscosity environments; secondly, the presence of common metal ions and amino acids in cells will not interfere with the fluorescence signal of the probe, indicating that the probe has high chemical stability and can be used for long-term imaging in vivo.

[0029] (3) The probe provided by the present invention has good biocompatibility and strong tissue permeability. The MTT experiment proves that when the probe concentration reaches 5 μΜ, more than 90% of the SMMC-7721 cells still have viability, indicating that the probe has good biocompatibility; the red light emission endows the probe with strong tissue penetration ability.

[0030] (4) The probe provided by the present invention has better biological applicability. Most lipid droplet-targeted fluorescent probes based on BODIPY dyes have relatively short emission wavelengths and small Stokes shifts, which are greatly affected by the background fluorescence of biomaterials and the self-absorption of the molecule itself during bioimaging, and have relatively high limitations in in vivo applications. For example, for commercial lipid droplet dyes such as BODIPY 493 / 503, its Stokes shift is only 20 nm. The introduction of meso-trifluoromethyl shifts its emission wavelength from the visible region to the red light region, and the Stokes shift can reach 60 nm, increasing the signal-to-noise ratio during bioimaging, reducing the interference of biomaterial background light, and improving the applicability in vivo. Description of the Drawings

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0032] Figure 1 For the fluorescent probe prepared in Example 1 1 1H NMR spectrum;

[0033] Figure 2 In, A is the fluorescence emission spectrum of the fluorescent probe prepared in Example 1 (final concentration of 2 μM) in methanol / glycerol mixed solutions with different volume fractions; Figure 2 In, B is the linear relationship diagram between the logarithm of the fluorescence intensity of the fluorescent probe at 614 nm and the logarithm of the solution viscosity in each solution when the excitation wavelength is 550 nm;

[0034] Figure 3 In, A is the change diagram of the fluorescence intensity of the fluorescent probe prepared in Example 1 at 610 nm with the illumination time in glycerol and PBS solutions (the final concentration of the probe is 2 μM) when the excitation wavelength is 550 nm; Figure 3 In, B is the bar chart of the relative fluorescence intensity of the fluorescent probe at 610 nm in the presence of different interferents;

[0035] Figure 4 Are the cytotoxicity test results of fluorescent probes with different concentrations (final concentrations are 0.5, 1, 2, 3, 5 μM) in SMMC-7721 cells;

[0036] Figure 5 In, A is the cell imaging diagram of 2 μM fluorescent probe co-incubated with SMMC-7721 cells for different times; Figure 5 In, B is the change diagram of the fluorescence intensity of 2 μM fluorescent probe co-incubated with SMMC-7721 cells for different times;

[0037] Figure 6 Are the photosensitivity test result diagrams of 2 μM fluorescent probe and 2 μM commercial dye BODIPY 493 / 503 in SMMC-7721 cells respectively;

[0038] Figure 7 Are the co-localization imaging diagrams of 2 μM fluorescent probe and 1 μM commercial dye HCS Lipid TOX TM Deep Red in SMMC-7721 cells;

[0039] Figure 8Laser confocal imaging (A) and fluorescence intensity histogram (B) of cells after 20 μΜ nystatin-stimulated SMMC-7721 cells for 1.5 h and then co-incubated with 2 μΜ fluorescent probe for 15 min;

[0040] Figure 9 Laser confocal imaging (A) and fluorescence intensity histogram (B) of single lipid droplets after 2 μΜ fluorescent probe co-cultured with human normal liver cells HL-7702 and human hepatoma cells SMMC-7721 for 15 min;

[0041] Figure 10 Laser confocal imaging (A), histogram of the number of intracellular lipid droplets and cell fluorescence intensity (B), and histogram of lipid droplet diameter and single lipid droplet fluorescence intensity (C) after 20 μΜ bisphenol A-stimulated SMMC-7721 cells for different times and then co-incubated with 2 μΜ fluorescent probe for 15 min;

[0042] Figure 11 Laser confocal imaging (A), histogram of the number of intracellular lipid droplets and cell fluorescence intensity (B), and histogram of lipid droplet diameter and single lipid droplet fluorescence intensity (C) after SMMC-7721 cells were stimulated with different concentrations of bisphenol A for 24 h and then co-incubated with 2 μΜ fluorescent probe for 15 min; Detailed implementation mode

[0043] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0044] Example 1 Synthesis of viscosity-responsive lipid droplet-targeted fluorescent probe

[0045] Synthesis steps of the probe: Add 2,4-dimethylpyrrole (0.5 mL, 4.86 mM) and CF3COOH (0.18 mL, 2.36 mM) to 6 mL of CH2Cl2, stir and mix evenly, then add PhSiCl3 (0.38 mL, 2.34 mM), stir at room temperature for 10 min, and then add Et3N (0.75 mL, 5 mM) and stir again for 15 min; Subsequently, add BF3·Et2O (1 mL, 8 mM) to the stirred solution and continue to stir for 3 h. The reaction mixture is washed twice with 60 mL of water, the aqueous phase is extracted with CH2Cl2, the combined organic phases are dried with magnesium sulfate, filtered, and evaporated to obtain the crude product. The crude product is purified by silica gel column chromatography (dichloromethane / n-hexane, 1:1) to obtain 122.7 mg of dark red solid, with a yield of 8%.

[0046] Figure 1 1H NMR spectrum of the fluorescent probe synthesized in Example 1, showing the following data in the spectrum: 1 1H NMR (600 MHz, CDCl3) δ 6.15 (s, 2H), 2.54 (s, 6H), 2.30 (q, J = 3.4 Hz, 6H).

[0047] It can be seen from the above spectrum that the viscosity-responsive lipid droplet-targeted fluorescent probe of the present invention has been successfully synthesized.

[0048] Fluorescence spectrum test of the fluorescent probe in Example 2 in solutions with different viscosity coefficients

[0049] The experimental steps are as follows:

[0050] (1) Weigh a certain amount of the fluorescent probe prepared in Example 1 and dissolve it in DMSO to prepare a 2 mM probe stock solution;

[0051] (2) Prepare viscosity test solutions with different volume ratios of methanol and glycerol. The volume ratios of methanol to glycerol are 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0, and the corresponding viscosity values are 953 cP, 395 cP, 155 cP, 72 cP, 37 cP, 7.8 cP, 4.5 cP, 2.3 cP, 1.56 cP, 0.97 cP, and 0.65 cP respectively;

[0052] (3) Take 2 mL of the viscosity test solutions with different viscosities prepared in step (2) and place them in multiple fluorescence cuvettes. Add 2 μL of the probe stock solution prepared in step (1) to each cuvette so that the final concentration of the fluorescent probe in each solution is 2 μM. Under the excitation at 550 nm, measure the fluorescence intensity of each solution at 614 nm. All solutions are placed in quartz cuvettes for testing, and the test temperature is room temperature.

[0053] Figure 2 In, A is the fluorescence emission spectrum of the fluorescent probe in methanol / glycerol viscosity test solutions with different volume ratios. The curves correspond to the glycerol ratios in the solutions from bottom to top as 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% in sequence.

[0054] As Figure 2 shown in A in, as the glycerol content increases, the fluorescence intensity of the fluorescent probe in the solution gradually increases. Especially when the glycerol ratio is in the range of 70% - 100%, the fluorescence intensity increases significantly; as Figure 2As shown in B of , when the solution viscosity is in the range of 0.65 cP - 953 cP, the logarithm of the fluorescence intensity of the fluorescent probe in the solution at 614 nm shows a good linear relationship with the logarithm of the viscosity (R 2 = 0.9702).

[0055] The above experiments show that in a low-viscosity environment, the trifluoromethyl rotor group can rotate freely and release energy through non-radiative transitions, resulting in fluorescence quenching; under high-viscosity conditions, the rotation of the trifluoromethyl rotor group is hindered, non-radiative transitions are inhibited, and the fluorescence signal of the fluorophore is released. Sensing of different viscosity environments is achieved based on the change in the fluorescence intensity of the probe.

[0056] Example 3 Photostability Test and Selectivity Experiment of Fluorescent Probe in Solutions with Different Viscosities

[0057] (1) The steps of the photostability experiment are as follows: Add 2 μL of the probe stock solution prepared in Example 2 to 2 mL of glycerol and 2 mL of PBS solution respectively, so that the final concentration of the probe is 2 μM in both cases. Under the condition of an excitation wavelength of 550 nm, measure the change in the fluorescence intensity at 610 nm of the above two solutions within 30 min respectively.

[0058] The results are as Figure 3 shown in A of . When the excitation wavelength is 550 nm, the fluorescence intensity of the fluorescent probe at 610 nm in glycerol remains basically unchanged with the extension of the illumination time, and the fluorescence intensity of the fluorescent probe at 610 nm in the PBS solution also remains basically unchanged with the extension of the illumination time. After continuous illumination for 30 min, more than 92% of the fluorescence of the fluorescent probe remains in both glycerol and PBS solutions.

[0059] The above experimental results show that the probe has good photostability in both high-viscosity and low-viscosity environments and can be used for long-term imaging in vivo.

[0060] (2) Steps of the selectivity experiment:

[0061] Prepare aqueous solutions of relevant analytes. The analytes include metal ions (K + , Ca 2+ , Na + , Mg 2+ , Al 3+ , Zn 2+ , Fe 2 + , Cu 2+ , Mn 2+ ), amino acids (Ser, Asn, Val, Ile, His, Lys, Gln, Asp, Thr, Glu, GSH, Cys, Hcy), ascorbic acid (AA), and ATP;

[0062] Add the probe stock solution and PBS solution to one of the cuvettes to make the total volume 2 mL and the final concentration of the fluorescent probe 2 μM as the control group; then take another cuvette, add glycerol and the probe stock solution to make the total volume 2 mL and the final concentration of the fluorescent probe 2 μM, and then take multiple cuvettes, add different analyte solutions and the probe stock solution respectively to make the total volume of the solution in each cuvette 2 mL, and the final concentrations of the fluorescent probe and the analyte are 2 μM and 1 mM respectively as the experimental group; measure the fluorescence intensity of each solution at 610 nm, and the excitation wavelength is 550 nm.

[0063] Figure 3 B in [Figure] is the bar chart of the relative fluorescence intensity of the fluorescent probe in the presence of different analytes, Figure 3 In B in [Figure], from left to right are: PBS (control group), K + , Ca 2+ , Na + , Mg 2+ , Zn 2+ , Fe 2+ , Al 3+ , Mn 2+ , Ser, Asn, Val, Ile, His, Lys, Gln, Asp, Thr, Glu, GSH, Cys, Hcy, AA, ATP and Glycerol.

[0064] The results are as shown in Figure 3 B in [Figure]. Compared with the fluorescence intensity of the control group, after adding various analytes, the fluorescence intensity of the probe remained basically unchanged, and only in glycerol, the probe released a significant fluorescence signal.

[0065] The above experimental results show that in the presence of relevant analytes, the fluorescence intensity of the probe basically does not change, verifying that the fluorescent probe prepared by the present invention can be used for sensing the viscosity in complex organisms without being interfered.

[0066] Example 4 Cytotoxicity Test of Fluorescent Probe

[0067] Culture SMMC-7721 cells with 100 μL of DMEM (1% penicillin-streptomycin and 10% FBS) in a 96-well plate in a constant temperature incubator at 37 °C (95% air and 5% carbon dioxide) for 24 h. Then, remove the medium in the 96-well plate, add fresh medium containing different concentrations of the fluorescent probe (the final concentrations of the fluorescent probe are 0, 0.5, 1, 2, 3, and 5 μM respectively) and continue to culture for 24 h. Add 10 μL of MTT solution (5 mg / mL -1) Incubate for another 4 h. Dissolve the generated formazan with 100 μL of DMSO. Place the 96-well plate on a shaker and shake for 10 min. Measure the absorbance of each well at 570 nm using a microplate reader.

[0068] Cell survival rate (%) = (OD sample - OD blank ) / (OD control - OD blank ) × 100%

[0069] OD sample : SMMC-7721 cells incubated with media containing different concentrations of fluorescent probe.

[0070] OD control : SMMC-7721 cells incubated with media without fluorescent probe.

[0071] OD blank : Media without SMMC-7721 cells.

[0072] The results are as Figure 4 shown. When the probe concentration reaches 5 μM, more than 90% of the SMMC-7721 cells still have a survival rate, indicating that the fluorescent probe prepared by the present invention has good biocompatibility.

[0073] Example 5 Screening of the time for the fluorescent probe to enter cells

[0074] The experimental steps are as follows:

[0075] (1) Seed SMMC-7721 cells in a dedicated confocal dish and grow them in high-glucose medium containing 10% serum in a constant temperature incubator at 37 °C, 5% carbon dioxide, and 95% air for 24 h;

[0076] (2) Incubate 2 μM of the probe with SMMC-7721 cells and determine the time for the probe to enter the cells through laser confocal imaging.

[0077] The specific operating conditions are as follows: SMMC-7721 cells are grown in medium containing 10% serum, placed in a constant temperature incubator at 37 °C with 5% carbon dioxide and 95% air for 24 h. Before confocal imaging, the adherent cells are washed 2 - 3 times with PBS to remove the metabolic wastes during cell growth, and then fresh medium containing 2 μM of the fluorescent probe is added. After incubation for different times, confocal imaging experiments are carried out.

[0078] The results are as Figure 5As shown, with the increase in the co-incubation time of the fluorescent probe with SMMC-7721 cells, the fluorescence intensity of the probe gradually increases and stabilizes after 15 min. Therefore, the cell entry time was selected as 15 min, and 15 min was chosen as the optimal incubation time for the probe to enter the cells in the subsequent cell imaging experiments. During this process, it can be observed that the red fluorescent dots in the cytoplasm gradually increase, and their morphology and appearance are similar to lipid droplets, preliminarily proving the rationality of the probe design.

[0079] Example 6 Photostability Test of Fluorescent Probe in Cells

[0080] After washing the two groups of adherent SMMC-7721 cells with PBS 2-3 times, fresh medium containing the fluorescent probe and the commercial dye BODIPY 493 / 503 was added respectively, so that the final concentrations of the fluorescent probe and the commercial dye in the medium were both 2 μM. Then, they were incubated in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 15 min for laser confocal imaging. The excitation wavelength of the probe was 550 nm, the collection wavelength was 590 - 630 nm, the excitation wavelength of BODIPY 493 / 503 was 488 nm, the collection wavelength was 500 - 540 nm, and the number of laser scans was 60 times.

[0081] The results are as Figure 6 shown. When the number of laser scans was 0, bright fluorescence signals could be observed in the cells. As the number of laser scans continued to increase, after 60 consecutive scans, more than 70% of the fluorescence of the probe in the cells was still retained; in contrast, the intensity of the cells stained with the commercial lipid droplet dye BODIPY 493 / 503 was only 40% after 60 consecutive scans. The experimental results show that the probe prepared by the present invention also has good stability in cells and is not affected by the internal environment.

[0082] Example 7 Colocalization Test of Fluorescent Probe in Cells

[0083] After washing the adherent SMMC-7721 cells with PBS 2-3 times, fresh medium containing the fluorescent probe and the commercial dye HCSLipid TOX TM Deep Red was added, so that the concentrations of the fluorescent probe and the commercial dye HCS Lipid TOX TM Deep Red in the medium were 2 μM and 1 μM respectively. They were co-incubated in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 15 min, the medium was removed, washed with PBS 2-3 times, and fresh medium was added for laser confocal imaging. The excitation wavelength of the probe was 550 nm, the collection wavelength was 590 - 630 nm, HCS Lipid TOX TMThe excitation wavelength of Deep Red is 637 nm, and the collection wavelength is 650 - 670 nm.

[0084] The imaging results are as Figure 7 shown, where a is the bright field of the cells, b is the distribution of the probe in the cells, c is HCS LipidTOX TM The distribution of Deep Red in the cells, d is the superimposed field of b and c, and f is the fluorescence intensity distribution diagram of the white line in Figure d. As Figure 7 shown, the fluorescence probe and HCS Lipid TOX TM The distribution of Deep Red in the cells has good overlap, and the co-localization coefficient reaches above 0.98, indicating that the fluorescent probe prepared by the present invention can be enriched in lipid droplets, realizing the targeting of lipid droplets.

[0085] The above experimental results show that the probe has good lipid droplet targeting.

[0086] Example 8 Monitoring the dynamic changes of lipid droplets in cells stimulated by nystatin using a fluorescent probe

[0087] First, incubate SMMC-7721 cells in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air with a medium containing 20 μM nystatin for 1.5 h. After being stimulated by nystatin, the SMMC-7721 cells are then incubated with a medium containing 2 μM fluorescent probe in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for another 15 min, and then laser confocal imaging is performed. The excitation wavelength is 550 nm, and the collection wavelength is 590 - 630 nm.

[0088] The results are as Figure 8 shown. After being stimulated by nystatin, the fluorescence of SMMC-7721 cells increased significantly, while the diameter of the lipid droplets hardly changed. This indicates that the intracellular viscosity will increase under the stimulation of nystatin. The experimental results show that the probe can be used to monitor the dynamic changes of intracellular lipid droplet viscosity.

[0089] Example 9 Confocal imaging of a fluorescent probe in normal hepatocytes and hepatoma cells

[0090] The adherent HL-7702 and SMMC-7721 cells are washed 2 - 3 times with PBS respectively, and then added with fresh medium containing a probe concentration of 2 μM and incubated together in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 15 min, and then laser confocal imaging is performed. The excitation wavelength is 550 nm, and the collection wavelength is 590 - 630 nm.

[0091] As Figure 9As shown in A above, after the probe enters the above two types of cells, it is enriched in the lipid droplets inside the cells, and the fluorescence intensity in SMMC-7721 cells is significantly higher than that in HL-7702 cells; similarly, as Figure 9 shown in B above, the fluorescence intensity of a single lipid droplet of the probe in liver cancer cells is significantly higher than that in normal liver cells.

[0092] Example 10 Monitoring the change of lipid droplet viscosity in bisphenol A-induced non-alcoholic fatty liver disease by fluorescence probe

[0093] (1) First, incubate SMMC-7721 cells with a 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), and then incubate the SMMC-7721 cells after being stimulated with bisphenol A for different times with a medium containing 2 μM fluorescence probe in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 15 min, and then perform laser confocal imaging. The excitation wavelength is 550 nm, and the collection wavelength is 590 - 630 nm.

[0094] The results are as Figure 10 shown. As the stimulation time of bisphenol A increases, the number of lipid droplets inside the cells and the cell fluorescence intensity gradually increase, while the size of the lipid droplets and the fluorescence intensity of a single lipid droplet remain unchanged.

[0095] (2) First, incubate SMMC-7721 cells with a medium containing different concentrations of bisphenol A (0, 10 μM, 20 μM) in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 24 h, and then incubate the SMMC-7721 cells after being stimulated with different concentrations of bisphenol A for 24 h with a medium containing 2 μM fluorescence probe in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 15 min, and then perform laser confocal imaging.

[0096] The results are as Figure 11 shown. As the stimulation concentration of bisphenol A increases, the number of lipid droplets inside the cells and the cell fluorescence intensity gradually increase, while the size of the lipid droplets and the fluorescence intensity of a single lipid droplet remain unchanged.

[0097] The experimental results show that in bisphenol A-induced non-alcoholic fatty liver, the number of lipid droplets increases, but the viscosity and diameter of the lipid droplets remain unchanged.

[0098] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a viscosity-responsive lipid droplet-targeted fluorescent probe in the preparation of a reagent for lipid droplet labeling and monitoring changes in lipid droplet viscosity, characterized in that, The structural formula of the fluorescent probe is as follows: .

2. The application according to claim 1, characterized in that The fluorescent probe can monitor the changes in the number and viscosity of intracellular lipid droplets induced by drugs.

3. The application according to claim 2, wherein, The drugs include nystatin and bisphenol A.

4. The application according to claim 1, wherein: The fluorescent probe can image the lipid droplets in cancer cells and detect the viscosity inside the lipid droplets, and distinguish normal cells from cancer cells according to the viscosity difference inside the lipid droplets in normal cells and cancer cells.

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