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

Abstract

The present invention belongs to the field of analytical chemistry and relates to a viscosity-responsive lipid droplet-targeting fluorescent probe and its application. The structural formula of the fluorescent probe is such that its phospholipid group not only has strong electron-withdrawing ability but also has a large steric hindrance. By introducing a phospholipid group at the meso -position of BODIPY, the absorption and emission wavelengths of the probe are red-shifted to 580 nm and 621 nm, endowing the probe with good viscosity responsiveness. In a methanol and glycerol mixture, as the glycerol ratio increases from 0% to 100%, the fluorescence intensity of the probe increases by 33 times. The probe can also selectively target lipid droplets to achieve rapid wash-free imaging of lipid droplets. The probe is suitable for real-time visualization monitoring of lipid droplet viscosity. Based on the differences in lipid droplet viscosity and quantity between cancer cells and normal cells, combined with confocal imaging technology, the probe is used to visually distinguish normal cells and cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and relates to a viscosity-responsive lipid droplet-targeting fluorescent probe and its application. Background Art

[0002] Lipid droplets are a very widely distributed organelle, the main storage site of neutral lipids in cells, and exist in most prokaryotes and almost all eukaryotes. A lipid droplet is an organelle containing a neutral lipid core surrounded by a phospholipid monolayer and some proteins. In the past, lipid droplets were only considered to be inert fat particles. Recent studies have shown that lipid droplets not only store excess lipids in cells, but also play a key role in a variety of cellular functions, including energy balance, lipid metabolism, membrane protein expression, membrane trafficking, etc. The cellular microenvironment is an important parameter reflecting the metabolic status of cells and the body. As an important physical and chemical parameter of the cellular microenvironment, cellular viscosity plays an important role in many key physiological processes such as material transport, signal transduction, and membrane fusion in cells and organisms. The viscosity of intracellular lipid droplets directly affects the physiological functions of lipid droplets. Abnormalities in lipid droplet viscosity are closely related to diseases such as diabetes, fatty liver, atherosclerosis, and cancer. Real-time and accurate measurement of the viscosity inside lipid droplets not only helps to clarify the physiological and pathological functions of lipid droplets and their viscosity microenvironment, but also holds promise for the development of rapid detection methods for these major diseases. Therefore, it is of great significance to develop a method for visualizing and detecting the viscosity inside lipid droplets.

[0003] Fluorescent probes have the advantages of high sensitivity, good selectivity, etc., and can be used for real-time imaging of living cells and in vivo with high spatio-temporal resolution, becoming one of the essential tools in the current biomedical field. Coumarin, rhodamine, cyanine, and boron dipyrromethene (BODIPY) are currently the most actively studied classical fluorescent dyes. By modifying these classical fluorescent dyes and introducing recognition and sensing sites, a series of dyes and probes with high photophysical and recognition and sensing properties can be obtained. In recent years, fluorescent dyes based on meso -substituted BODIPY have received increasing attention. The introduction of meso -functional groups in BODIPY can not only improve the optical properties of the dye, but also introduce recognition and sensing sites to construct fluorescent probes. These properties make meso-substituted BODIPY dyes an excellent platform for constructing fluorescent probes. Patent CN109535190A discloses a meso-pyrrolidone BODIPY dye viscosity probe and its preparation method and application. The molecular structural formula of this type of meso-pyrrolidone BODIPY probe is , and by setting substituents at both the 1,7 positions of the molecular skeleton, the response to viscosity is achieved. Although meso-Position substituted BODIPY dyes have developed rapidly, but there are few reported dye probes that can obtain emission wavelengths exceeding 600 nm and are located in the deep infrared region. The existence of these problems greatly limits the application of fluorescent probes in the field of biomedical imaging. Therefore, it is very necessary to design and develop fluorescent probes with emission wavelengths in the deep infrared region, which is beneficial to broadening and improving the performance of fluorescent probes. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes a viscosity-responsive lipid droplet-targeted fluorescent probe and its application.

[0005] The technical solution of the present invention is realized as follows:

[0006] A viscosity-responsive lipid droplet-targeted fluorescent probe, whose structural formula is as follows:

[0007] 。

[0008] The preparation method of the above viscosity-responsive lipid droplet-targeted fluorescent probe comprises the steps of:

[0009] (1) 2,4-Dimethylpyrrole and N,N-diisopropylethylamine are successively added to a tetrahydrofuran solution, and after purging with nitrogen and cooling the temperature below 0 °C, a tetrahydrofuran solution containing triphosgene is added dropwise, and the reaction is carried out at 25 °C for 6 hours. After rotary evaporation and column chromatography separation, a white solid, namely compound 3, is obtained.

[0010] (2) Under nitrogen protection, POCl is added to the 1,2-dichloroethane of compound 3 3 , and the mixed solution is refluxed for 3 hours. Then the temperature is cooled below 0 °C, and triethylamine is added dropwise. After about 30 minutes, boron trifluoride diethyl etherate is added dropwise. Finally, the mixture is stirred at 25 °C for 4 hours, and after extraction, drying and column chromatography separation, compound 2 is obtained.

[0011] (3) Under nitrogen protection, compound 2 and triisopropyl phosphite are mixed and refluxed for 30 minutes, and then the heating is stopped. The solvent is removed, and the probe 1 is obtained by column chromatography separation.

[0012] Preferably, in the step (1), the molar ratio of 2,4-dimethylpyrrole, triphosgene and N,N-diisopropylethylamine is 1:0.17:1.

[0013] Preferably, in the step (2), the molar ratio of compound 3, POCl 3 , triethylamine and boron trifluoride diethyl etherate is 1:2:10:11.

[0014] Preferably, in the step (3), the molar ratio of compound 2 and triisopropyl phosphite is 1:3.76.

[0015] Use of the above-mentioned fluorescent probe in detecting the viscosity of a detection solution for non-diagnostic purposes.

[0016] Use of the above-mentioned fluorescent probe in rapid non-washing imaging of intracellular lipid droplets for non-diagnostic purposes.

[0017] Use of the above-mentioned fluorescent probe in visual monitoring of changes in lipid droplet viscosity in living cells for non-diagnostic purposes.

[0018] Use of the above-mentioned fluorescent probe in differentiating cancer cells from normal cells for non-diagnostic purposes.

[0019] The above applications specifically include:

[0020] ① Weigh a certain mass of the probe and dissolve it in acetonitrile to prepare a 2 mM probe stock solution.

[0021] ② Prepare mixed solutions of methanol and glycerol with different volume ratios (glycerol content 0 - 100%). Take 2 mL of the mixed solutions of methanol and glycerol with different ratios in a cuvette, add 10 μL of the probe stock solution in step (1) to make the probe concentration 10 μM, and measure the fluorescence intensity of the probe in solutions with different viscosities at an excitation wavelength of 570 nm.

[0022] ③ Incubate the probe (6 μM) with cells for 15 minutes and observe the non-washing imaging effect of the probe through laser confocal imaging.

[0023] ④ Incubate the probe (6 μM) and the commercial lipid droplet dye BODIPY 493 / 503 with cells for 15 minutes and observe the lipid droplet targeting effect of the probe through laser confocal imaging.

[0024] ⑤ Incubate the probe (6 μM) with normal cells and cancer cells for 15 minutes, and obtain the imaging effects of the probe in different normal cells and cancer cells through laser confocal imaging, so as to achieve the purpose of differentiating normal cells from cancer cells.

[0025] The present invention has the following beneficial effects:

[0026] 1. The present invention uses a BODIPY fluorescent dye as the parent nucleus, by meso-A viscosity-sensitive lipid droplet-targeting fluorescent probe was designed and synthesized by introducing a phosphoester group with strong electron-withdrawing and large steric hindrance effects. The introduction of the phospholipid group played a four-in-one function. First, the introduction of the phospholipid group endows the probe with good viscosity responsiveness. The fluorescence intensity in high-viscosity glycerol is 33 times that in low-viscosity methanol, thus realizing the recognition and sensing of viscosity in solution and cells. And since the probe has almost no fluorescence in weak-viscosity solutions such as water, the probe can achieve rapid wash-free imaging of lipid droplets, thus avoiding the damage to cells and the interruption of the imaging process caused by repeated washing during imaging. Second, the introduction of the phospholipid group also makes the probe have good liposolubility, so it can specifically target lipid droplets, effectively avoiding non-specific staining and strongly enhancing the effect of lipid droplet imaging. Third, due to the strong electron-withdrawing effect of the phosphoester group, the emission wavelength of the probe is red-shifted to 621 nm, which is in the deep red region, effectively reducing biological background fluorescence and light scattering, thus enhancing the imaging effect. Finally, the strong electron-withdrawing ability of the phospholipid group also makes the probe have good photostability, which is helpful for long-term in-situ real-time monitoring.

[0027] 2. The probe provided by the present invention has good selectivity for viscosity and has no response to pH, polarity, reactive oxygen species, reactive sulfur species, and anions, and can be used for selective recognition and sensing of lipid droplet viscosity in solution, cells, and organisms.

[0028] 3. The probe provided by the present invention has good biocompatibility and cell membrane penetration ability, can achieve the best imaging effect within 15 minutes, and can also distinguish normal cells and cancer cells through the response to viscosity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order 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.

[0030] Figure 1 is the synthesis route of the probe in Example 1.

[0031] Figure 2 is the 1 1H NMR spectrum of the probe in Example 1.

[0032] Figure 3 is the 1 13C NMR spectrum of the probe in Example 1.

[0033] Figure 4UV-Vis absorption spectra (A) and fluorescence emission spectra (B) of the 10 μM probe in different polar solvents. The excitation wavelength was 570 nm.

[0034] Figure 5 Fluorescence emission spectra (A) of the probe (10 μM) in mixed solutions of glycerol and methanol with different volume ratios (0 - 100%) and the linear relationship graph (B) between solution viscosity and fluorescence intensity.

[0035] Figure 6 Fluorescence emission spectra (A) of the probe (10 μM) in glycerol and in the presence of metal ions, amino acids, reactive oxygen species, reactive sulfur species, and anions (1 mM) in PBS buffer solution (pH 7.4, 10 mM), and the bar graph (B) of the fluorescence intensity of the probe at 621 nm as a function of the interferent.

[0036] Figure 7 Fluorescence emission spectra of the 10 μM probe in B - R solutions with different pH values (2 - 12). The excitation wavelength was 570 nm.

[0037] Figure 8 Time screening of the probe (6 μM) entering SMMC - 7721 cells. The excitation wavelength was 580 nm, and the collection wavelength range was 610 - 660 nm.

[0038] Figure 9 Imaging diagrams of the probe (6 μM) in SMMC - 7721 cells at different scanning times. The excitation wavelength was 580 nm, and the collection wavelength range was 610 - 660 nm.

[0039] Figure 10 Co - localization imaging diagrams of the probe (6 μM) and the commercial lipid droplet dye BODIPY493 / 503 in SMMC - 7721 cells. The excitation wavelength of the probe was 580 nm, and the collection wavelength range was 610 - 660 nm. The excitation wavelength of BODIPY493 / 503 was 488 nm, and the collection wavelength range was 500 - 540 nm.

[0040] Figure 11 Fluorescence imaging diagrams (A) of the probe (6 μM) in normal cells (3T3, HL - 7702, and HEK293) and cancer cells (4T1, MCF - 7, HeLa, SMMC - 7721), (B) the comparison graph of cell fluorescence intensity, and (C) the comparison graph of intracellular lipid droplet fluorescence intensity. The excitation wavelength of the probe was 580 nm, and the collection wavelength range was 610 - 660 nm. Detailed implementation methods

[0041] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Embodiment

[0042] The synthesis route of the viscosity-responsive lipid droplet-targeted fluorescent probe in this embodiment is as Figure 1 , and the specific steps are as follows:

[0043] In a 1 L three-necked flask, add 300 mL of the solvent tetrahydrofuran, and successively add 2,4-dimethylpyrrole (15 g, 0.158 mol) and N,N N-diisopropylethylamine (20.4 g, 0.158 mol). 2 Replace three times, lower the temperature below 0 °C, and then dropwise add a tetrahydrofuran solution of 75 mL of triphosgene. Then react at 25 °C for 6 hours. Rotavapor to remove the tetrahydrofuran solvent, and obtain 5.4 g of white solid compound 3 by column chromatography separation, with a yield of 31.5%.

[0044] Add 1,2-dichloroethane (150 mL) and compound 3 (5 g, 23.12 mmol) into a 500 mL three-necked flask. Under N 2 protection, add POCl 3 (7.1 g, 46.24 mmol) and reflux for 3 hours until the raw materials basically disappear. Then cool down below 0 °C, dropwise add triethylamine (23.4 g, 231.2 mmol) and react for 30 minutes, and then dropwise add boron trifluoride diethyl etherate (36.1 g, 254.32 mmol). Then raise the temperature to 25 °C and stir and react for 4 hours to end the reaction. Pour the reaction solution into ice water, separate the layers, extract the aqueous phase twice with dichloromethane (50 mL), combine the obtained organic phases, add salt for drying, and rotary evaporate to dryness. The crude product is separated by column chromatography to obtain 3.1 g of compound 2, with a yield of 47.4%.

[0045] Add compound 2 (2 g, 7 mmol) and triisopropyl phosphite (6 mL, 24 mmol) into a 10 mL single-necked flask. Under N 2 protection, reflux for 30 minutes and stop heating. After cooling to room temperature, rotary evaporate to remove the solvent, then dissolve the sample with dichloromethane, and obtain 1.8 g of probe 1 by column chromatography separation, with a yield of 62.4%.

[0046] 1 H NMR (600 MHz, CDCl 3): δ 6.09 (s, 2H), 4.85 (dd, J = 12.4, 6.2 Hz, 2H), 2.50 (s, 6H), 2.46 (s, 6H), 1.40 (d, J = 6.2 Hz, 6H), 1.27 (d, J = 6.2 Hz, 6H).

[0047] 13 C NMR (151 MHz, CDCl 3 ): δ 157.52, 144.35, 137.04, 130.13, 128.92, 123.42, 72.69, 72.65, 23.92, 23.89, 23.52, 23.48, 16.45, 15.02.

[0048] Example of implementation effect

[0049] 1. UV-Vis absorption spectra and fluorescence emission spectra of the probe in solutions with different polarities and viscosities

[0050] The fluorescent probe prepared in the example was dissolved in acetonitrile to prepare a 2 mM stock solution. 2 mL of 1,4-dioxane, ethyl acetate, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, N,N N,N-dimethylformamide, ethylene glycol, ethanol, acetonitrile, methanol and glycerol were respectively added to a cuvette, and then 10 μL of the 2 mM probe stock solution was added to prepare a mixed solution with a probe concentration of 10 μM. After mixing, its UV-Vis absorption spectrum ( Figure 4 A in) and fluorescence spectrum ( Figure 4 B in) were measured. The experimental results showed that the probe showed bright fluorescence only in glycerol, indicating that the probe had a good response to viscosity.

[0051] 2. Fluorescence emission spectra of the probe in solutions with different viscosities and linear relationship between probe fluorescence intensity and viscosity change

[0052] 2 mL of methanol solutions containing different proportions (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%) of glycerol were respectively added to a cuvette, and 10 μL of the 2 mM probe stock solution was added to prepare a mixed solution with a probe concentration of 10 μM, and fluorescence spectrum measurement was carried out ( Figure 5 ). The experimental results showed that as the viscosity increased from 0.65 cP to 953 cP, the fluorescence of the probe at 621 nm gradually increased, and the fluorescence change before and after was as high as 33 times, indicating that the probe recognized and sensed viscosity.

[0053] 3. Stability of the probe

[0054] Prepare stock solutions of cations (K + , Ca 2+ , Na + , Mg 2+ , Fe 3+ , Fe 2+ ), anions (F - , HSO 3 - , NO 3 - , CO 3 2- , SO 4 2- ), reactive oxygen species (H 2 O 2 , ClO - ), reactive sulfur (Cys, Hcy, GSH), and amino acids (Lys, Asn, Ala, Asp, Glu, Pro, Ser, His, Arg, and Thr). The concentration of amino acids is 20 mM, and the concentrations of cations, anions, reactive oxygen species, and reactive sulfur are 10 mM. In the cuvette of the control group, only the probe and PBS buffer solution (pH 7.4, 10 mM) are added. In the cuvettes of the experimental groups, a certain amount of the analyte and the probe solution are added respectively, and the final concentrations of the probe and the analyte are 10 μM and 1 mM. (1) PBS; (2) K + ; (3)Ca 2+ ; (4) Na + ; (5) Mg 2+ ; (6) Fe 3+ ; (7) Fe 2+ ; (8) F - ; (9) HSO 3 - ; (10) NO 3 - ; (11) CO 3 2- ; (12)SO 4 2- ; (13) H 2 O 2 ; (14) ClO - ; (15) Cys; (16) Hcy; (17)GSH; (18) L; ys (19) Asn; (20)Ala; (21)Asp; (22) Glu; (23) Pro; (24) Ser; (25) His; (26) Arg; (27)Thr; (28) Gly. As Figure 6As shown, the probe did not react with the metal ions, anions, reactive oxygen species, reactive sulfur species, and amino acid substances being tested, and its fluorescence only increased significantly in glycerol, indicating that the probe has good stability.

[0055] 4. pH Stability of the Probe

[0056] Add 2 mL of B-R buffer solution (0.2 M) with different pH values (2 - 12) to a cuvette, add 10 μL of the probe stock solution with a concentration of 2 mM, and prepare a mixed solution with a probe concentration of 10 μM. Test the fluorescence emission spectrum varying with pH. As Figure 7 shown, the fluorescence of the probe did not change significantly at pH values from 2 to 9, indicating that the probe has good pH stability under physiological conditions.

[0057] 5. Screening of the Probe's Uptake Time

[0058] Inoculate SMMC-7721 cells into a confocal dish, grow them in high-glucose 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 to remove the metabolic waste during cell growth. Incubate the probe (6 μM) with SMMC-7721 cells, and determine the time when the probe enters the cells through laser confocal imaging. As Figure 8 shown, as the incubation time extends, the red fluorescence in the cells gradually increases. After 15 minutes of incubation, the fluorescence intensity basically remains unchanged, indicating that the probe has good cell membrane permeability and can rapidly perform wash-free imaging of lipid droplets.

[0059] 6. Test of the Probe's Photostability in Cells

[0060] After incubating the probe (6 μM) with SMMC-7721 cells in DMEM medium for 15 minutes, test the graph of cell fluorescence varying with the number of imaging times. As Figure 9 shown, after 100 scans, the fluorescence intensity of the cells can still reach more than 95%, indicating that the probe has good photostability in cells.

[0061] 7. Co-localization Test of the Probe in Cells

[0062] Incubate SMMC-7721 cells with DMEM medium containing the probe (6 μM) and the commercial lipid droplet dye BODIPY493 / 503 (2 μM) for 15 minutes, and then perform laser confocal microscopy imaging. As Figure 10 shown, the red channel and the green channel of the fluorescent probe can overlap very well, and the Pearson coefficient is 0.93. The experimental results show that the probe of the present invention can selectively target cell lipid droplets.

[0063] 8. Imaging of normal cells and cancer cells

[0064] The culture medium containing the probe (6 μM) was incubated in normal cells (3T3, HL-7702, and HEK293) and cancer cells (4T1, MCF-7, HeLa, and SMMC-7721) for 15 minutes and then imaged using a confocal microscope. Figure 11 It can be seen that normal cells have relatively few lipid droplets and show very weak fluorescence; cancer cells show strong fluorescence and have more lipid droplets. Statistics on the fluorescence intensity of cells and lipid droplets also verify that the viscosity of normal cells is lower than that of cancer cells. This shows that the invented probe can distinguish normal cells from cancer cells by changes in fluorescence intensity based on the difference in viscosity between normal cells and cancer cells. ( Figure 11 A in the figure is a cell imaging picture. Figure 11 B in the figure is a statistical chart of cell intensity of normal cells and cancer cells. Figure 11 (C) is a statistical diagram of lipid droplet intensity in normal cells and cancer cells).

[0065] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a viscosity-responsive lipid droplet-targeted fluorescent probe in the preparation of a detection reagent for differentiating cancer cells from normal cells, characterized in that, the structure of the viscosity-responsive lipid droplet-targeted fluorescent probe is as follows: 。 2. Use of the viscosity-responsive lipid droplet-targeted fluorescent probe according to claim 1 in the preparation of a detection reagent for differentiating cancer cells from normal cells, characterized in that: the final concentration of the viscosity-responsive lipid droplet-targeted fluorescent probe in the detection reagent is 6 μM.

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