A super-bright fluorescent probe for cellular lipid droplets and its application in the preparation of bioimaging reagents

Abstract

A super-bright fluorescent probe for cellular lipid droplets and its application in the preparation of bioimaging reagents, belonging to the field of bioimaging technology. Aiming at the deficiencies of the existing fluorescent imaging probes for cellular lipid droplets in terms of insufficient photostability and insufficient selectivity for lipid droplet staining, the present invention selects a fluorescent molecule based on stilbene derivatives in the development of a new type of fluorescent probe for lipid droplet imaging. While exhibiting high photostability, this fluorescent molecule has excellent selectivity for lipid droplet staining. This fluorescent probe can be used to prepare reagents for specifically labeling lipid droplets in cells, visually tracking the dynamic trajectories of cellular lipid droplets, and quantitatively measuring the movement speed of cellular lipid droplets. The fluorescent probe Lipi-Bright described in the present invention has excellent photostability and staining selectivity. Under the same staining and imaging conditions, Lipi-Bright exhibits more excellent photostability and staining selectivity than commercial fluorescent probes for lipid droplets, and has great application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioimaging, and particularly relates to a super-bright cell lipid droplet fluorescent probe based on stilbene derivatives and its applications in the preparation of reagents for specifically labeling lipid droplets in cells, visually tracking the dynamic trajectories of cell lipid droplets, and quantifying the movement speed of cell lipid droplets. Background Art

[0002] Lipid droplets were only regarded as fat accumulation granules in cells some years ago. It was not until recent years that the research on lipid droplets became more in-depth, and it was found that lipid droplets are spherical organelles composed of a neutral lipid core (triglyceride and cholesterol ester) and a phospholipid monolayer membrane, on which various proteins such as perilipin and adipose differentiation-related protein are fixed, and are widely distributed in almost all organisms from prokaryotes to humans. Lipid droplets are involved in many important cellular activities, such as membrane synthesis and transport, protein storage and degradation, and inflammation. Dysfunction of lipid droplets can lead to the occurrence of various diseases, such as obesity, diabetes, fatty liver, atherosclerosis, and cancer. Therefore, the research on lipid droplets in the past decade has become the most popular field in cell biology.

[0003] As is well known, fluorescence imaging is a powerful method for monitoring the behavior of subcellular organelles, with the ability of in-situ and real-time detection. So far, many lipid droplet fluorescent probes have been developed for lipid droplet imaging and dynamic tracking. However, due to the limitations of insufficient photostability and / or relatively low lipid droplet staining specificity, these lipid droplet fluorescent probes are usually not suitable for long-term fluorescence imaging. Therefore, tracking the dynamics of lipid droplets in a long-term, in-situ, and real-time manner is a very important but challenging task. Summary of the Invention

[0004] Aiming at the deficiencies in the existing cell lipid droplet imaging fluorescent probe technology, the problem to be solved by the present invention is to provide a super-bright (Example 6) cell lipid droplet fluorescent probe based on stilbene derivatives and its applications in the preparation of reagents for specifically labeling lipid droplets in cells (Example 5), visually tracking the dynamic trajectories of cell lipid droplets, and quantifying the movement speed of cell lipid droplets (Examples 7 and 8).

[0005] A super-bright cell lipid droplet fluorescent probe (Lipi-Bright) based on stilbene derivatives of the present invention has the following structural formula:

[0006]

[0007] Its preparation reaction formula is as follows:

[0008]

[0009] In view of the insufficient photo-stability of existing fluorescence imaging probes for cellular lipid droplets and the lack of selectivity in lipid droplet staining, the present invention selects a fluorescent molecule based on stilbene derivatives in the development of a new type of lipid droplet imaging fluorescent probe. While exhibiting high photo-stability, this fluorescent molecule has excellent selectivity for lipid droplet staining. The present invention has successfully used this probe to visually track the dynamic trajectory of cellular lipid droplets and quantify the movement speed of cellular lipid droplets.

[0010] The cell described in the present invention is a HeLa cell.

[0011] Experimental results confirm that the fluorescent probe Lipi-Bright described in the present invention has excellent photo-stability and staining selectivity. Under the same staining and imaging conditions, Lipi-Bright exhibits better photo-stability and staining selectivity than commercial lipid droplet fluorescent probes BODIPY493 / 503 and Nile Red. Therefore, the fluorescent probe Lipi-Bright can be further used to prepare reagents for specifically labeling lipid droplets in cells, visually tracking the dynamic trajectory of cellular lipid droplets, and quantifying the movement speed of cellular lipid droplets. Description of the Drawings

[0012] Figure 1 : Absorption-emission spectrum of the fluorescent probe Lipi-Bright prepared in Example 1 of the present invention in toluene solvent; the left dotted part is the absorption spectrum, and the right solid line part is the emission spectrum.

[0013] Figure 2 : Bar chart of the cell survival rate of HeLa cells stained with different concentrations of the fluorescent probe Lipi-Bright for 24 hours; the cell survival rate is above 90%, demonstrating the good biocompatibility of the fluorescent probe.

[0014] Figure 3 : Co-localization photo of the fluorescent probe Lipi-Bright and the lipid droplet fluorescent probe LDs-Red (Anal. Chem. 2022, 94, 35, 12095~12102) in HeLa cells;

[0015] Among them, Figure a is the fluorescence photo of the Lipi-Bright and the nuclear dye Hoechst 33342 channels; Figure b is the fluorescence photo of the LDs-Red and the nuclear dye Hoechst 33342 channels; Figure c is the superimposed photo of Figures a and b; Figure d is the picture of the Pearson correlation coefficient (0.97) calculated by ImageJ software.

[0016] Figure 4: Quantitative fluorescence graphs of the fluorescent probe Lipi - Bright and the lipid droplet fluorescent probes BODIPY 493 / 503 and Nile Red in HeLa cells (BODIPY 493 / 503 and Nile Red are the most commonly used commercial lipid droplet fluorescent probes);

[0017] Among them, Figure a is the fluorescence photograph of HeLa cells stained with the fluorescent probe Lipi - Bright and BODIPY 493 / 503, Nile Red under different laser powers (0.05%, 1% or 2%); Figure b is the fluorescence intensity of HeLa cells measured through a flow cytometer in the FITC channel and the PE channel; indicating that the probe Lipi - Bright has excellent staining fluorescence brightness.

[0018] Figure 5 : Images of the fluorescent probe Lipi - Bright labeling lipid droplets, tracking the dynamic trajectory of lipid droplets, and quantifying the movement speed of lipid droplets; among them, Figure a is a dual - color image in a specified field of view; Figure b is a schematic diagram of continuous imaging of 500 images in this field of view; Figure c is the information on the dynamic trajectory of lipid droplets in the enlarged area of Figure b processed by software; Figure d is the movement speed of nuclear lipid droplets and cytoplasmic lipid droplets after normal or oleic acid stimulation (10 parallel experiments were conducted and the average value was finally statistically analyzed).

[0019] Figure 6 : Images of the fluorescent probe Lipi - Bright labeling lipid droplets, tracking the dynamic trajectory of lipid droplets under starvation conditions, and quantifying the movement speed of lipid droplets; among them, Figure a is a dual - color image and an experimental protocol schematic diagram at different time points in a specified field of view; Figure b is a schematic diagram of continuous imaging of 500 images in this field of view at different time points; Figures c and d are the information on the dynamic trajectory of lipid droplets in the enlarged area of Figure a processed by software at 0 hour and 1 hour time points; Figure e is the movement speed of nuclear lipid droplets and cytoplasmic lipid droplets under starvation conditions at different time points (the average value was calculated and statistically analyzed for all lipid droplets in Figure a). Detailed implementation mode

[0020] Example 1:

[0021] 1. Synthesis of Compound 2

[0022] A mixture of compound 1 (benzo[1,2-b:4,5-b']dithiophene-4,8-dione (4.40 g, 20.0 mmol)), zinc powder (2.73 g, 42 mmol), sodium hydroxide (12.0 g, 30.0 mmol) and water (100 mL) was heated under reflux for 2 h. Then, 1-bromo-3-methoxypropane (9.09 g, 59.4 mmol) and a catalytic amount of tetrabutylammonium bromide were added. After the reaction mixture was refluxed for 12 h, it was poured into water and extracted several times with CH2Cl2. The organic layer was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to obtain 4.45 g (19.80 mmol, 61%) of a white solid as compound 2.

[0023] 1 H NMR (500 MHz, CDCl3): δ 7.51 (d, J = 5.5 Hz, 2H), 7.40 (d, J = 5.5 Hz, 2H), 4.40 (t, J = 6.2 Hz, 4H), 3.72 (t, J = 6.2 Hz, 4H), 3.44 (s, 6H), 2.21–2.13 (m, 4H).

[0024] 2. Synthesis of compound 3

[0025] Cooled in an ice-water bath, a mixture of liquid bromine (3.05 g, 19.1 mmol) and CH2Cl2 (10 mL) was slowly added to a solution of compound 2 (3.50 g, 9.55 mmol) in CH2Cl2 (20 mL) within 30 minutes, and then stirred at room temperature for 12 h. After removing the solvent under reduced pressure, it was purified by silica gel column chromatography to obtain 2.85 g (5.44 mmol, 57%) of a white solid as compound 3

[0026] 1 H NMR (500 MHz, CDCl3): δ 7.46 (d, J = 5.0 Hz, 2H), 4.29 (s, 2H), 3.66 (t, J = 6.1 Hz, 4H), 3.42 (s, 6H), 2.21-2.13 (m, 4H).

[0027] 3. Synthesis of compound 4

[0028] To a mixture of compound 3 (2.00 g, 3.81 mmol), 2-methoxycarbonylphenylboronic acid (4.0 g, 15.3 mmol), Pd(PPh3)4 (0.174 g, 0.150 mmol) and K2CO3 (1.66 g, 12.0 mmol) was added a degassed solvent mixture of toluene, acetonitrile and water (50 mL, volume ratio = 8:1:1). The reaction mixture was then refluxed for 12 h. After cooling to room temperature, the mixture was filtered to remove inorganic salts. The filtrate was poured into water and extracted three times with CH2Cl2. The combined organic layers were washed with brine, then dried over anhydrous MgSO4 and filtered. The mixture obtained after concentrating the filtrate under reduced pressure was purified by silica gel column chromatography to give 1.34 g (2.11 mmol, 55%) of compound 4 as a yellow solid.

[0029] 1 H NMR (500 MHz, CD2Cl2): δ 7.79 (s, 2H), 7.64 (s, 2H), 7.59 (s, 2H), 7.49 (s, 2H), 4.39 (s, 4H), 3.74 (s, 6H), 3.65 (s, 4H), 3.34 (s, 6H), 2.12 (s, 4H).

[0030] 4. Synthesis of compound 5

[0031] Under ice-bath conditions, methylmagnesium bromide (2.0 M in tetrahydrofuran, 0.63 mL, 1.26 mmol) was added dropwise to a solution of compound 4 (400 mg, 0.63 mmol) in dry tetrahydrofuran (20 mL). After heating to room temperature, the reaction mixture was stirred for 12 h. The mixture was poured into water and extracted three times with CH2Cl2. The organic layer was washed with brine, then dried over anhydrous MgSO4, filtered, and the white solid obtained after filtration was concentrated under reduced pressure for use in the next step without further purification.

[0032] A solution of the above white solid in CH2Cl2 (10 mL) was slowly added to a solution of BF3·Et2O (0.5 mL) in CH2Cl2 (10 mL). Then, the reaction mixture was stirred at room temperature for 2 h. After quenching the reaction with water, the mixture was extracted three times with CH2Cl2. The organic layer was washed with brine, then dried over anhydrous MgSO4 and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to give 94.0 mg (0.14 mmol, 23%) of compound 5 as a yellow solid.

[0033] 11H NMR (500 MHz, CDCl3): δ 7.45 (s, 4H), 7.39 - 7.29 (m, 4H), 4.55 (s, 4H), 3.80 (t, J = 6.3 Hz, 4H), 3.50 (s, 6H), 2.36 - 2.30 (m, 4H), 1.73 (s, 12H).

[0034] 5. Synthesis of Lipi - Bright

[0035] m - CPBA (164 mg, 0.95 mmol) was slowly added to a solution of compound 5 (90 mg, 0.136 mmol) in CH2Cl2 (10 mL) within 30 minutes. Then the mixture was refluxed for 48 h, quenched with saturated aqueous NaHCO3 solution (10 ml), and the organic layer was washed with aqueous NaHCO3 solution, then dried over anhydrous MgSO4, filtered. After concentrating the filtrate under reduced pressure, the resulting mixture was purified by silica gel column chromatography to obtain 48.6 mg (0.077 mmol, 56%) of yellow solid as Lipi - Bright.

[0036] 1 1H NMR (500 MHz, CD2Cl2): δ 7.58 (d, J = 7.3 Hz, 2H), 7.48 (d, J = 7.3 Hz, 2H), 7.41 - 7.37 (m, 4H), 4.68 (t, J = 6.7 Hz, 4H), 3.65 (t, J = 6.3 Hz, 4H), 3.39 (s, 6H), 2.34 - 2.27 (m, 4H), 1.67 (s, 12H). ESI - MS: 661.7 [M - H] + (calcd: 661.2).

[0037] Example 2: Determination of the Absorption - Emission Spectrum of the Fluorescent Probe Lipi - Bright Prepared in Example 1

[0038] The fluorescent probe Lipi - Bright synthesized in Example 1 was formulated into a 10 μM concentration solution with 10 mL of solvent. The absorption spectrum was scanned with a UV - Vis spectrophotometer in the wavelength range of 300 - 700 nm, and the fluorescence emission spectrum was collected with a fiber - optic fluorescence spectrometer under 470 nm excitation. The data was processed with Origin software to obtain the absorption - emission spectrum of the fluorescent probe Lipi - Bright in solution as shown in Figure 1 (the left dotted line part is the absorption spectrum, and the right solid line part is the emission spectrum). This figure shows the absorption - emission peak positions and its large Stokes shift of the fluorescent probe Lipi - Bright prepared in the present invention. Among them, the absorption peak is located at 463 nm, the emission peak is located at 559 nm, and the Stokes shift is 96 nm.

[0039] Example 3: Cultivation of HeLa Cells

[0040] All percentages in this example are volume fractions.

[0041] The HeLa cell line was cultured in an incubator at 37 °C with a CO2 concentration of 5%. The culture medium was high-glucose DMEM containing 10% fetal bovine serum and 1% double antibody (a mixture of penicillin and streptomycin). Among them, fetal bovine serum, double antibody, and high-glucose DMEM were directly purchased from a biological reagent company.

[0042] When the cells grew to the logarithmic phase, the cells were passaged: After sucking away the original 5 mL of culture medium in the cell culture flask, the cell surface was washed with 2 mL of DMEM culture medium without fetal bovine serum. After sucking away this culture medium, the cells were digested with 0.5 mL of trypsin for 2 minutes. After most of the cells detached from the wall, 2 mL of high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% double antibody was added and pipetted evenly. An appropriate amount of this cell dispersion was transferred to new cell culture flasks and culture dishes respectively, and then placed in a CO2 cell incubator for culture. The cells in the culture dish were used for confocal or super-resolution imaging experiments after the concentration was appropriate.

[0043] Example 4: Testing the Cytotoxicity of the Fluorescent Probe Lipi-Bright Prepared in Example 1

[0044] We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to test the cytotoxicity of the fluorescent probe Lipi-Bright. HeLa cells were seeded in a 96-well plate (1×10 4 cells per well) and cultured in a CO2 cell incubator for 24 hours. Then, the culture medium in the middle 60 wells was replaced with a culture medium containing different concentrations (0, 0.5, 1.0, 2.0, 5.0, and 10.0 μM) of the fluorescent probe Lipi-Bright and 1% (volume fraction) DMSO (10 parallel experiments were set for each concentration). After culturing for another 24 hours, MTT reagent (10 μL per well) was added to these wells and then returned to the cell incubator for continued culture for 4 hours. After removing the original culture medium in these wells, DMSO (100 μL per well) was added to dissolve the formed formazan crystals. After standing at room temperature for 30 minutes, the absorbance values of each well were measured at 490 nm with an enzyme-linked immunosorbent assay reader. Since only living cells can react with MTT reagent to form formazan crystals, we can calculate the cell survival rate by comparing the average absorbance value of the wells with different concentrations in each group with the average absorbance value of the control group (10 wells with a probe concentration of 0). The results are as Figure 2As shown, it shows that the fluorescent probe Lipi-Bright has little cytotoxicity, and the fluorescent probe Lipi-Bright at a concentration of 10.0 μM will not affect the normal growth of HeLa cells within 24 hours. Example 5: Co-staining experiment of the fluorescent probe Lipi-Bright prepared in Example 1 and the lipid droplet fluorescent probe LDs-Red in HeLa cells

[0045] We cultured HeLa cells in a 20 mm diameter glass-bottomed culture dish and propagated them in a CO2 incubator for 2 days. After taking it out of the incubator, remove the original DMEM culture medium in the culture dish, add 1 mL of DMEM culture medium containing 20 μM Hoechst 33342, and place it in the incubator for 20 minutes. After washing again with fresh culture medium, add 1 mL of DMEM culture medium containing Lipi-Bright (500 nM), LDs-Red (2 μM) and 1% (volume fraction) DMSO, and continue to culture in a cell culture incubator for 2 hours. After taking it out, wash it 3 times with HBSS solution, and then perform fluorescence imaging in HBSS solution. Figure 3 As shown, we can clearly observe that the fluorescent probe Lipi-Bright prepared in Example 1 and the lipid droplet fluorescent probe LDs-Red can achieve good co-localization in HeLa cells, indicating that the fluorescent probe Lipi-Bright prepared in Example 1 has excellent specificity for marking cell lipid droplets.

[0046] Example 6: Staining brightness test of the fluorescent probe Lipi-Bright prepared in Example 1

[0047] After we took out the three culture dishes filled with HeLa cells in Example 5 from the cell culture incubator, we removed the original DMEM culture medium, added DMEM culture medium (containing 1% DMSO) containing 2μM Lipi-Bright, 2μM BODIPY 493 / 503 and 2μM Nile Red respectively, put them back into the incubator and continued to culture for 2 hours, then took out the three culture dishes, washed them three times with HBSS solution, and then performed fluorescence imaging. It was found that the laser power required for Lipi-Bright to excite the fluorescence signal saturation was much lower than that of BODIPY493 / 503 and Nile Red. The stained cultured cells were tested by flow cytometry, and the fluorescence signal intensity of Lipi-Bright in both detection channels was much higher than that of BODIPY 493 / 503 and Nile Red. This illustrates the excellent staining brightness of Lipi-Bright, such as Figure 4 shown.

[0048] Example 7: Observation of lipid droplet dynamics using the fluorescent probe Lipi-Bright prepared in Example 1

[0049] After taking out the culture dish covered with HeLa cells in Example 5 from the incubator, the original DMEM culture medium was removed, and DMEM culture medium containing 2 μM Lipi-Bright and 1% DMSO was added. After putting it back into the incubator and culturing for 2 hours, it was taken out. After continuously imaging 500 images in the selected area, the movement trajectory and movement speed of lipid droplets were recorded. The movement speed of cytoplasmic lipid droplets was about 50% faster than that of nuclear lipid droplets, and the movement speed of lipid droplets was about 20% faster after oleic acid stimulation, as Figure 5 shown.

[0050] Example 8: Observation of lipid droplet dynamics with the fluorescent probe Lipi-Bright prepared in Example 1

[0051] After taking out the culture dish covered with HeLa cells in Example 5 from the incubator, the original DMEM culture medium was removed, and DMEM culture medium containing 2 μM Lipi-Bright and 1% DMSO was added. After putting it back into the incubator and culturing for 2 hours, it was taken out. In-situ imaging was performed in the selected area, and after continuously imaging 500 images at 1-hour intervals, the movement trajectory and movement speed of lipid droplets were recorded. The movement speed of lipid droplets showed a downward trend under starvation conditions, as Figure 6 shown.

Claims

1. A cell lipid droplet fluorescence imaging probe based on stilbene derivatives, and its structural formula is shown as follows:

2. Use of the cell lipid droplet fluorescence imaging probe based on stilbene derivatives according to claim 1 in the preparation of a reagent for specifically labeling lipid droplets in cells, visually tracking the dynamic trajectory of cell lipid droplets, and quantitatively measuring the movement speed of cell lipid droplets, wherein the cell is a HeLa cell.

Images