A lipid droplet-targeted fluorescent probe for specifically detecting liver cancer cells and its application

Abstract

The present invention discloses a lipid droplet-targeted fluorescent probe for specifically detecting liver cancer cells and its application. The structural formula of the fluorescent probe is as follows. The probe is sensitive to polarity, has near-infrared emission, a large Stokes shift, excellent photostability, wash-free imaging, and a high signal-to-noise ratio. It can specifically enrich in lipid droplets in cells and monitor the dynamic behavior changes of lipid droplets for a long time. Compared with normal cells, the number of lipid droplets in cancer cells is higher and the polarity is lower, which has gradually become the diagnostic basis for normal cells and cancer cells. Combining with confocal imaging technology, the probe has been successfully applied to specifically distinguish liver cancer cells in different types of cells. There is a good linear relationship between the emission wavelength of the probe and the polarity parameter, and it has been successfully applied to quantify the polarity of lipid droplets in different cells, revealing the polarity heterogeneity of lipid droplets in different types of cells. It has also been further applied to the imaging research of liver cancer at the organ and in vivo levels.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes and bioimaging, and particularly relates to a lipid droplet-targeted fluorescent probe for specifically detecting liver cancer cells and its application. Technical Background

[0002] Hepatocellular carcinoma (HCC), as an aggressive liver malignancy, is the fourth leading cause of cancer-related death, which has gradually attracted a research boom. Liver cancer has gradually become synonymous with death due to its high incidence and mortality. However, more and more evidence shows that this is indeed the case. Liver cancer causes nearly 1 million deaths globally every year because most liver cancer patients can only be diagnosed at an advanced stage, missing the best treatment opportunity. If cancer can be diagnosed at an early stage, many cancers can be cured and the survival rate can be improved. Therefore, accurately distinguishing liver cancer cells or tissues is crucial for early clinical diagnosis but is also a difficult challenge.

[0003] As cancer progresses, the number and microenvironment of disease-related organelles in cancer cells will undergo some specific changes compared with normal cells, which has gradually become the criterion for differentiating cancer cells. Lipid droplets are the main organelles for intracellular lipid storage and metabolism, and play an important role in physiological processes such as lipid storage and metabolism signal transduction, protein degradation, and membrane trafficking. More and more evidence shows that in cancer cells, due to their need for more energy supply, the number of lipid droplets is higher than that in normal cells; the change in lipid metabolism mode leads to lower polarity of their lipid droplets. Importantly, in hepatocytes, lipid droplets are highly dynamic organelles and are in continuous proliferation and differentiation. Therefore, the lipid droplets in liver cancer cells may show a higher number and lower polarity. Lipid droplets, as an emerging diagnostic and therapeutic target for normal cells and cancer cells, are receiving extensive attention.

[0004] Fluorescence imaging technology has the advantages of high sensitivity, high spatio-temporal resolution, fast response, good biocompatibility, and good photostability, and has gradually become one of the essential tools in the field of biomedical imaging. Therefore, in view of the differences in lipid droplets between liver cancer cells and other cells, it is meaningful to develop a lipid droplet-targeted fluorescent probe with sensitive response to polarity to accurately distinguish liver cancer cells from other tumor cells and normal cells. In addition, quantifying the polarity of lipid droplets in different cells is crucial for revealing polarity heterogeneity and pathological information of lipid droplet-related diseases such as liver cancer. Designing and synthesizing probes that can reveal the polarity heterogeneity of lipid droplets in different cells needs to meet the following two conditions: 1. The probe has a very sensitive response to polarity; 2. The emission wavelength of the probe has a good linear relationship with polarity. Therefore, developing new polarity-responsive fluorescent probes for revealing the polarity heterogeneity of lipid droplets in different types of cells and specifically distinguishing liver cancer cells is of great significance and is also a difficult challenge. Summary of the Invention

[0005] The present invention provides a lipid droplet-targeted fluorescent probe for specifically detecting liver cancer cells and its application. The fluorescent probe can target lipid droplets and be used for long-term imaging of lipid droplets, can specifically label or display the morphology and distribution of lipid droplets in living cells, is sensitive to polarity, and based on the differences in the number of lipid droplets and the polarity of lipid droplets in liver cancer cells and normal cells, the fluorescent probe can specifically distinguish liver cancer cells.

[0006] The technical solution to achieve the present invention is as follows:

[0007] A lipid droplet-targeted fluorescent probe for specifically detecting liver cancer cells, characterized in that the structural formula of the fluorescent probe is

[0008] The application of the fluorescent probe in polarity detection.

[0009] The above application specifically includes the following steps:

[0010] (1) Dissolve the fluorescent probe in dimethyl sulfoxide to prepare a 2 mM probe stock solution;

[0011] (2) Prepare mixed solutions of ethanol and n-hexane with different volume ratios (ethanol content 0 - 10%), take different ratios of the mixed solutions of ethanol and n-hexane in a cuvette, add the probe stock solution prepared in step (1), the final concentration of the probe is 8 μM, and measure the fluorescence emission spectra of each solution at an excitation wavelength of 440 nm.

[0012] Fluorescence spectrum changes: As the ethanol content increases from 0 to 10%, the polarity parameter Δf of the solution ranges from 0 to 0.16, the emission wavelength of the probe gradually redshifts from 602 nm to 661 nm, the fluorescence intensity at 602 nm decreases by 47.3 times, and there is a good linear relationship between the emission wavelength of the probe and the polarity parameter Δf.

[0013] The application of the fluorescent probe in lipid droplet imaging, and the fluorescent probe can target lipid droplets to achieve real-time washing-free imaging of lipid droplets.

[0014] The fluorescent probe can specifically label the morphology of lipid droplets and detect the polarity inside the lipid droplets.

[0015] The fluorescent probe can image lipid droplets in living cells, ex vivo organs and in vivo, specifically distinguish liver cancer cells according to the differences in the number of lipid droplets and fluorescence intensity in cells, and distinguish tumor tissues and normal tissues according to the differences in the fluorescence intensity of the probe in ex vivo organs and in vivo.

[0016] The method of the above application is specifically as follows:

[0017] (1) After co - incubating the culture medium containing the fluorescent probe with live cells in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 50 min, confocal microscopy imaging was performed to observe the number of lipid droplets in the cells and the fluorescence intensity of individual lipid droplets.

[0018] (2) After washing the excised organs such as the heart, liver, spleen, lung, kidney and tumor with PBS, they were immersed in a PBS solution containing 12 μM fluorescent probe for 0, 10, 20 and 30 minutes. After washing with PBS, imaging was performed on a mouse imager to observe the fluorescence intensity of the fluorescent probe in each excised organ.

[0019] (3) Equal amounts of PBS solution containing 50 μM fluorescent probe were injected into the mouse tumor region and the symmetric normal region respectively, and imaging was performed on them with a mouse imager to observe the fluorescence intensity of the probe at the tumor region and the symmetric normal region.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The probe of the present invention is designed based on the ketocyanine resonance mechanism, with fluorene as the fluorophore. By modifying the electron donor and acceptor respectively, a high - performance lipid droplet - targeting fluorescent probe with very sensitive polarity response is successfully designed. The probe is very sensitive to polarity. In a mixed system of n - hexane and ethanol with different ratios, the presence of only 1% ethanol can cause the emission wavelength of the probe to redshift by 18 nm and the fluorescence intensity to decrease by 4.1 times, thus realizing the recognition and sensing of polarity in solution and cells. Due to the lipophilicity and electro - neutral structure of the probe, as well as its excellent photostability, the probe can specifically target lipid droplets and be used for long - time imaging of lipid droplets.

[0022] 2. The probe provided by the present invention has good selectivity for polarity. pH, metal cations, reactive oxygen species, reactive sulfur species and amino acids have little effect on the fluorescence of the probe, and it can be used for the selective recognition and sensing of lipid droplet polarity in cells and organisms. In addition, the probe has good biocompatibility. The cytotoxicity experiment proves that when the probe concentration is as high as 10 μM, the survival rate of SMMC - 7721 cells is still more than 86%, indicating that the probe has excellent biocompatibility. The probe has near - infrared emission, which can overcome the spontaneous background fluorescence in organisms and be applied to in - vivo research.

[0023] 3. The probe provided by the present invention has a sensitive response to polarity, and there is a good linear relationship between its emission wavelength and polarity. The probe quantifies the polarity heterogeneity of different intracellular lipid droplets. In addition, the probe has also successfully achieved the specific differentiation of liver cancer cells and realized the specific imaging of liver cancer at the organ and tumor - bearing mouse levels. Description of the Drawings

[0024] 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.

[0025] Figure 1 It is the synthetic route diagram of the probe prepared in Example 1.

[0026] Figure 2 It is the 1 1H NMR spectrum of the probe prepared in Example 1.

[0027] Figure 3 It is the 1 13C NMR spectrum of the probe prepared in Example 1.

[0028] Figure 4 It is the high-resolution mass spectrum of the probe prepared in Example 1.

[0029] Figure 5 It is the fluorescence spectrum (A) of the probe (8 μM) in different polar solvents, the normalized fluorescence spectrum (B), the linear relationship (C) between the emission wavelength of the probe and the solvent polarity parameter ET(30), the fluorescence emission spectrum (D) of the probe in a mixed system of different ratios of n-hexane and ethanol, the change diagram (E) of fluorescence intensity with the polarity parameter Δf, the linear relationship diagram (F) between the emission wavelength of the probe and the solution polarity parameter Δf, and the excitation wavelength is 440 nm.

[0030] Figure 6 It is the change diagram (A) of the fluorescence intensity of the probe (8 μM) at 700 nm in different pH buffer solutions, the bar chart (B) of the fluorescence intensity of the probe at 700 nm in the presence of different bioactive substances (such as reactive oxygen species, reactive sulfur species, and amino acids, etc.), and the experimental result diagram (C) of the cytotoxicity of the probe at different concentrations (0, 2, 4, 6, 8, and 10 μM) in SMMC-7721 cells.

[0031] Figure 7 It is the cell imaging diagram (A) and the change diagram (B) of fluorescence intensity when the 6 μM probe is co-incubated with SMMC-7721 cells for different times, the cell imaging diagram (C) and the change diagram (D) of fluorescence intensity when different concentrations of the probe are co-incubated with SMMC-7721 cells for 50 min. The excitation wavelength is 580 nm, and the collection wavelength range is 630 - 680 nm.

[0032] Figure 8It is the co-localization imaging diagram (A), signal-to-noise ratio (B), cell imaging diagram (C) after irradiation for different times in the photostability test, and its relative retained fluorescence intensity (D) of the probe (6 μM) and the commercial lipid droplet dye BODIPY493 / 503 (2 μM) in SMMC-7721. The excitation wavelength of the probe is 580 nm, and the collection wavelength range is 630 - 680 nm. The excitation wavelength of BODIPY493 / 503 is 488 nm, and the collection wavelength range is 500 - 540 nm.

[0033] Figure 9 It is the fluorescence imaging diagram (A), whole-cell fluorescence intensity comparison diagram (B), single lipid droplet fluorescence intensity comparison diagram (C) in the cell, and statistical chart of the number of lipid droplets in the cell (D) of the probe (6 μM) in liver cancer cells (SMMC-7721, HepG2, and Huh-7) and normal cells (HL-7702 and HEK293). The excitation wavelength of the probe is 580 nm, and the collection wavelength range is 630 - 680 nm.

[0034] Figure 10 It is the fluorescence imaging diagram (A), whole-cell fluorescence intensity comparison diagram (B), single lipid droplet fluorescence intensity comparison diagram (C) in the cell, and statistical chart of the number of lipid droplets in the cell (D) of the probe (6 μM) in liver cancer cells (SMMC-7721, HepG2, and Huh-7) and other cancer cells (A549, HeLa, and MCF-7). The excitation wavelength of the probe is 580 nm, and the collection wavelength range is 630 - 680 nm.

[0035] Figure 11 It is the fluorescence imaging diagram (A) of the probe in ex vivo organs such as the heart, liver, spleen, lung, kidney, and tumor, and the in-situ imaging diagram (B) of the tumor in SMMC-7721 tumor-bearing mice. The excitation wavelength of the probe in the mouse imager is 605 nm, and the collection wavelength is 700 nm.

[0036] Figure 12 It is the in-situ fluorescence spectra diagrams (A - H) of the probe in liver cancer cells (SMMC-7721, HepG2, and Huh-7), other cancer cells (A549, HeLa, and MCF-7), and normal cells (HL-7702 and HEK293), the bar chart of the emission wavelengths of the probe in different cells (I), and the polarity parameter data (J). The excitation wavelength of the probe is 580 nm, and the collection wavelength range is 600 - 720 nm. Detailed implementation

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1 Synthesis of probe

[0039] The technical synthesis route of lipid droplet-targeted fluorescent probes for specific detection of liver cancer cells is as follows: Figure 1 As shown, the specific steps are as follows:

[0040] In a 250 ml three-necked flask, 3,6-dibromo-9H-fluorene-9-one (338 mg, 1 mmol), bisbenzylideneacetone palladium (29 mg, 0.05 mmol), tri-tert-butylphosphine (20 mg, 0.10 mmol), sodium tert-butoxide (288 mg, 3 mmol), and diethylamine (216 mg, 3 mmol) were added to 6 mL of toluene and heated under reflux at 110 ° C for 12 h under nitrogen protection. After cooling to room temperature, the reaction system was filtered on diatomaceous earth, and the filtrate was dried by spin drying. The crude product was purified by silica gel column chromatography (eluent, petroleum ether: ethyl acetate = 30:1 to 4:1, all containing 1% triethylamine, and the 6:1 eluent was collected) to obtain an orange intermediate probe 1a (yield: 63%).

[0041] Compound 1a (0.161 g, 0.5 mmol) and malononitrile (0.099 g, 1.5 mmol) were dissolved in 10 mL of anhydrous ethanol, and piperidine (catalytic amount: 50 μL) was added, and the reaction system was refluxed at 85° C. for 12 h. The solvent was removed under vacuum, and the crude mixture was purified by column chromatography (eluent, petroleum ether: dichloromethane = 1:1 to pure dichloromethane, collecting the eluent of 1:10) to obtain a black final product probe (yield: 60%).

[0042] The black solid obtained in this example was characterized by nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high-resolution mass spectrum, indicating that the fluorescent probe has been successfully synthesized. Figures 2 - 4 As shown:

[0043] 1 H NMR (600MHz, CDCl3) δ 8.06 (d, J = 8.9 Hz, 2H), 6.72 (d, J = 2.2 Hz, 2H), 6.37 (dd, J1 = 8.9, J2 = 2.2 Hz, 2H), 3.49 (q, J = 7.1 Hz, 8H), 1.26 (t, J = 7.1 Hz, 12H).

[0044] 1313C NMR (151 MHz, CDCl3) δ 160.02, 151.76, 144.53, 128.29, 123.04, 116.57, 109.90, 102.88, 61.92, 44.90, 12.89.

[0045] HRMS (ESI) [M + H] + calcd for C 24 H 27 N4371.2235, found 371.2223.

[0046] Spectral Tests of the Probe in Example 2 in Different Polar Organic Solvents and Ethanol / n-Hexane Mixed Solutions with Different Volume Ratios

[0047] (1) Spectral Tests of the Probe in Different Polar Organic Solvents

[0048] Dissolve the fluorescent probe prepared in Example 1 in chromatographically pure dimethyl sulfoxide to prepare a 2 mM probe stock solution. Add 2 mL of different polar organic solvents to a cuvette, and then add 8 μL of the probe stock solution respectively so that the final concentration of the probe in each solution is 8 μM. Subsequently, perform fluorescence spectral tests with an excitation wavelength of 440 nm.

[0049] The organic solvents added are n-hexane, toluene, 1,4-dioxane, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, and dimethyl sulfoxide, and the corresponding polarity parameters ET(30) are 31.0, 33.9, 36.0, 37.4, 40.7, 43.2, and 45.1 respectively.

[0050] The experimental results are as shown in Figure 5 A - C below. As the polarity of the solvent increases, that is, the polarity parameter ET(30) of the solvent increases from 31.0 to 45.1, the emission wavelength of the probe gradually redshifts from 602 nm to 704 nm, and the fluorescence intensity at 602 nm decreases by 82,000 times; and the emission wavelength of the probe has a good linear relationship with the polarity parameter ET(30) (R 2 = 0.9981), indicating that the probe has a very sensitive response to polarity.

[0051] (2) Spectral Tests of the Probe in Ethanol / n-Hexane Mixed Solutions with Different Volume Ratios

[0052] The fluorescent probe prepared in Example 1 was dissolved in chromatographically pure dimethyl sulfoxide to prepare a 2 mM probe stock solution. Hexane solutions containing different proportions of ethanol (0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%) with a final volume of 2 mL were added to cuvettes respectively, and then 8 μL of the probe stock solution was added to each solution so that the final concentration of the probe in each solution was 8 μM. Subsequently, fluorescence spectroscopy tests were carried out with an excitation wavelength of 440 nm.

[0053] The experimental results are as Figure 5 shown in D-F of 2 . Only 1% ethanol can cause a 18 nm red shift in the emission wavelength of the probe and a 4.1-fold decrease in fluorescence intensity. As the ethanol content ranges from 0 to 10%, that is, the polarity parameter Δf of the solvent ranges from 0 to 0.16, the emission wavelength of the probe gradually redshifts from 602 nm to 661 nm, and the fluorescence intensity at 602 nm decreases by 47.3 times. In addition, there is a good linear relationship (R

[0054] Example 3: Stability, anti-interference experiment and cytotoxicity experiment of the probe in solutions with different pH values

[0055] (1) Stability of the probe in solutions with different pH values

[0056] B-R buffer solutions with pH values ranging from 6 to 12 were added to cuvettes respectively, and then 8 μL of the probe stock solution was added to each solution so that the final concentration of the probe in each solution was 8 μM. Subsequently, fluorescence spectroscopy tests were carried out with an excitation wavelength of 455 nm, and the changes in the fluorescence intensity of the probe at 700 nm in each solution were observed.

[0057] The results are as Figure 6 shown in A of

[0058] (2) Anti-interference experiment

[0059] Aqueous solutions of relevant analytes (such as metal ions, reactive oxygen species, reactive sulfur species and amino acids, etc.) were prepared, where the concentration of amino acids was 20 mM, and the solution concentrations of metal ions, reactive oxygen species and reactive sulfur species were 10 mM;

[0060] In the control group, only the probe and PBS buffer solution (pH 7.4, 10 mM) were added to the cuvette No. 1, and the final concentration of the probe was 8 μM. In the cuvettes of the experimental group, a certain amount of analyte solution and probe solution were added respectively, so that the final concentrations of the probe and the analyte were 8 μM and 1 mM. The fluorescence spectra of each solution were measured with an excitation wavelength of 455 nm, and the change in the fluorescence intensity of the probe at 700 nm in each solution was observed.

[0061] Figure 6 In the bar graph of B in, from left to right are as follows: 1: probe, 2 - 6: metal ions (K + 、Ca 2+ 、Na + 、Mg 2+ 、Cu 2+ ), 7 - 16: amino acids (Glu, Thr, Ala, Tyr, His, Ser, Val, Lys, Phe, and Met), 17: ascorbic acid AA, 18 - 19: reactive sulfides (Cys and GSH), 20: reactive oxygen species (H2O2).

[0062] The results are as shown in Figure 6 B in. In the presence of high concentrations of metal ions, reactive oxygen species, reactive sulfides, and amino acids, the fluorescence intensity of the probe did not change significantly, indicating that the probe has good anti - interference ability and can specifically sense polarity in complex organisms without being interfered by other bioactive substances.

[0063] (3) Cytotoxicity experiment

[0064] The classic 3 - (4,5 - dimethyl - 2 - thiazolyl) - 2,5 - diphenyl - 2 - H - tetrazolium bromide (MTT) experiment was used to evaluate the cytotoxicity of the probe to cells. First, 100 μL of DMEM (1% penicillin - streptomycin and 10% FBS) was added to a 96 - well plate, and then SMMC - 7721 cells were added and the 96 - well plate was placed in an incubator at 37 °C (95% air and 5% CO2) for 24 h. Next, the culture medium in the wells was aspirated, and fresh culture medium solutions containing different concentration gradients of the probe (0, 2, 4, 6, 8, and 10 μM) were added to the 96 - well plate respectively and cultured for 24 h. Next, 10 μL of MTT solution (concentration 5 mg·mL-1) was added to each well in the 96 - well plate and cultured for 4 h to produce sufficient formazan, and then 100 μL of DMSO was used to dissolve the formazan. Finally, the 96 - well plate was placed on a shaker and shaken left and right for about 10 minutes, and the absorbance value of each well in the 96 - well plate at 490 nm was measured on an enzyme - linked immunosorbent assay (ELISA) reader.

[0065] Cell survival rate (%) = (ODsample -OD blank ) / (OD control -OD blank )×100%

[0066] OD sample : SMMC-7721 cells incubated with media containing different concentration gradients of probes.

[0067] OD control : SMMC-7721 cells incubated with media without probes.

[0068] OD blank : Pure media (without antibiotics and serum) without SMMC-7721 cells.

[0069] The results are as shown in C of Figure 6 . When the probe concentration reaches 10 μM, the SMMC-7721 cells still maintain a survival rate of more than 86%, indicating that the fluorescent probe prepared by the present invention has good biocompatibility.

[0070] Example 4: Screening experiment on the optimal time and concentration for the probe to enter cells

[0071] (1) Screening of the time for entering cells

[0072] Inoculate SMMC-7721 cells into a confocal dish, grow them in DMEM medium containing 10% serum, and culture them in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 48 h. First, wash the cells 3 times with PBS, incubate the SMMC-7721 cells with the medium containing the probe (final concentration of 6 μM), and determine the time for the probe to enter the cells through laser confocal imaging.

[0073] The results are as shown in A of Figure 7 and B of Figure 7 . As the incubation time prolongs, the red fluorescence in the cells gradually increases. After incubating for 50 minutes, the fluorescence intensity basically remains unchanged, and the optimal time for entering cells is determined to be 50 min.

[0074] (2) Screening of the concentration for entering cells

[0075] Inoculate SMMC-7721 cells into a confocal dish, grow them in DMEM medium containing 10% serum, and culture them in a constant temperature incubator at 37 °C, 5% carbon dioxide and 95% air for 48 h. First, wash the cells 3 times with PBS, incubate the SMMC-7721 cells with the medium containing different concentrations of the probe (final concentrations are 0, 2, 4, 6, and 8 μM) for 50 min, and determine the concentration of the probe entering the cells through laser confocal imaging.

[0076] The results are as Figure 7 shown in C of Figure 7 and D of

[0077] As shown, with the increase of the incubation concentration, the fluorescence intensity in the cells gradually increases with the increase of the probe concentration, and the staining positions are all dot-like substances similar to lipid droplets, indicating that the probe will not cause non-specific staining due to the increase in concentration. Combining the results of the cytotoxicity experiment and the fluorescence intensity, the optimal incubation concentration of the probe was finally determined to be 6 μM.

[0078] Example 5: Co-localization experiment of the probe and intracellular photostability test

[0079] After incubating SMMC-7721 cells with DMEM medium containing the probe (6 μM) for 35 minutes, the commercial lipid droplet dye BODIPY493 / 503 (final concentration of 2 μM) was added and co-incubated for 15 min in the dark. After washing twice with PBS solution, laser confocal microscopy imaging was immediately performed.

[0080] The results are as Figure 8 shown in A of Figure 8 where the staining area of the red channel of the probe and the staining area of the green channel of the commercial lipid droplet dye have good overlap, and the Pearson coefficient is 0.90, indicating that the probe can selectively target cell lipid droplets;

[0081] (2) Photostability test

[0082] After incubating SMMC-7721 cells with DMEM medium containing the probe (6 μM) for 50 minutes, the cells were continuously scanned every 10 s within 0 - 10 min in the xyt mode on the confocal microscope.

[0083] The results are as Figure 8 shown in C of

[0084] Example 6: Imaging diagrams of the probe in liver cancer cells and normal cells

[0085] The experimental procedure was as follows: Liver cancer cells (SMMC-7721, HepG2, and Huh-7) and normal cells (HL-7702 and HEK293) were incubated with the medium containing the probe (6 μM) for 50 minutes, and then imaged with a confocal microscope.

[0086] The results are as Figure 9As shown, the probe emits obvious fluorescence only in liver cancer cells, while showing weak fluorescence in normal cells ( Figure 9 A in Figure 9 ); statistical analysis was performed on the cell imaging map of A in Figure 9 As shown in B in Figure 9 , the fluorescence intensity of the probe in liver cancer cells is 2.5 to 5.9 times that in normal cells, higher than the medical diagnostic threshold of 2 times; in addition, the fluorescence intensity of individual lipid droplets in liver cancer cells ( Figure 9 C in

[0087] Example 7: Imaging maps of the probe in liver cancer cells and other cancer cells

[0088] The experimental steps were as follows: Liver cancer cells (SMMC-7721, HepG2, and Huh-7) and other cancer cells (A549, HeLa, and MCF-7) were incubated with a medium containing the probe (6 μM) for 50 minutes, and then imaged with a confocal microscope.

[0089] The results are as Figure 10 shown. The probe emits obvious fluorescence only in liver cancer cells, while showing weak fluorescence in other cancer cells ( Figure 10 A in Figure 10 ); statistical analysis was performed on the cell imaging map of A in Figure 10 As shown in B in Figure 10 , the fluorescence intensity of the probe in liver cancer cells is 2.9 to 4.9 times that in other cancer cells; in addition, the fluorescence intensity of individual lipid droplets in liver cancer cells ( Figure 10 C in

[0090] Example 8: Imaging maps of the probe in ex vivo organs and tumor-bearing mice

[0091] (1) Imaging of the probe in ex vivo organs

[0092] The experimental procedure was as follows: ex vivo organs such as the heart, liver, spleen, lung, kidney, and tumor were washed twice with PBS, and then immersed in a PBS solution containing the probe (12 μM) for 0, 10, 20, and 30 minutes. After that, each organ was washed with PBS two to three times, and then imaged on a mouse imager. The tumor was prepared by subcutaneous injection of cancer cells into the axilla of nude mice. The diameter of the tumor was 0.5 cm, and the inoculated cancer cells were human liver cancer cells SMMC-7721.

[0093] The results were as Figure 11 shown in A of [reference]. As the immersion time increased, the probe emitted more obvious fluorescence in the tumor than in other ex vivo organs, indicating that the probe could specifically accumulate in the tumor region.

[0094] (2) Imaging of the probe in tumor-bearing mice

[0095] The experimental procedure was as follows: 100 μL of a PBS solution containing 50 μM of the probe was injected into the tumor region of the mouse (inoculated with SMMC-7721 cells, the following set of pictures) and the symmetric normal region (the above set of pictures) respectively, and then imaged on a mouse imager.

[0096] The results were as Figure 11 shown in B of [reference]. As the incubation time of the probe increased, the fluorescence intensity of the probe at the tumor site was higher than that at the symmetric normal region, indicating that the probe had good staining specificity for the tumor region and could achieve tumor imaging and differentiation based on the fluorescence intensity difference in vivo.

[0097] Example 9: In-situ fluorescence spectra of the probe in different cells

[0098] The experimental procedure was as follows: a culture medium solution containing the probe (6 μM) was incubated in different cells for 50 minutes, and then imaged with a confocal microscope.

[0099] The results were as Figure 12 shown. The emission wavelength of the probe in cancer cells was generally lower than that in normal cells, and the emission wavelength of the probe in liver cancer cells was significantly shorter than that in other cancer cells. Specifically, from Figure 5 the linear relationship between the emission wavelength of the probe and the solvent polarity, it could be inferred that the polarity parameters Δf of different cells were about 0.064 to 0.103, and the ET(30) values were 32.1 to 34.3 ( Figure 12 J in [reference]). Therefore, based on this powerful probe, it could be inferred that the polarity of intracellular lipid droplets showed the following order: liver cancer cells < other cancer cells < normal cells.

[0100] Combined Figures 9 - 12Results. Due to the excellent polar response ability of this probe, there is a good linear relationship between its emission wavelength and the solution polarity parameter. This probe can not only specifically distinguish liver cancer cells in different cells, but also quantify the polarity of lipid droplets in different cells through in-situ fluorescence spectroscopy, revealing the polarity heterogeneity of lipid droplets in different cells.

[0101] 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 lipid droplet-targeted fluorescent probe for specifically detecting liver cancer cells in lipid droplet imaging for non-diagnostic and non-therapeutic purposes, characterized in that, The structural formula of the fluorescent probe is .

2. The application according to claim 1, wherein The fluorescent probe can specifically label the morphology of lipid droplets and detect the polarity within the lipid droplets.

3. The application according to claim 1 or 2, characterized in that, The fluorescent probe can image lipid droplets in living cells, ex vivo organs, and in vivo. Based on the differences in the number of lipid droplets and fluorescence intensity within the cells, liver cancer cells can be specifically distinguished. Based on the differences in the fluorescence intensity of the probe in ex vivo organs and in vivo, tumor tissues and normal tissues can be distinguished.

4. The application according to claim 3, wherein The application method is as follows: (1) After co-incubating the culture medium containing the fluorescent probe with living cells in an incubator at 37°C, 5% carbon dioxide, and 95% air, image using a confocal microscope to observe the number of lipid droplets within the cells and the fluorescence intensity of individual lipid droplets; (2) Wash the ex vivo organs of the heart, liver, spleen, lungs, kidneys, and tumors with PBS, then soak them in a PBS solution containing the fluorescent probe. After washing with PBS, image using a mouse imager to observe the fluorescence intensity of the fluorescent probe in each ex vivo organ; (3) Inject equal amounts of the PBS solution containing the fluorescent probe into the tumor region and the symmetrical normal region of the mouse respectively, and image it using a mouse imager to observe the fluorescence intensity of the probe in the tumor region and the symmetrical normal region.

Images