Fluorescent probe based on BODIPY targeting lipid droplet and application of fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent probes, and discloses a fluorescent probe based on BODIPY targeted lipid droplets and application of the fluorescent probe. The structural formula of the fluorescent probe for targeting the lipid droplets is as follows: # imgabs0 # is as follows: due to strong electron withdrawing capability and relatively large steric hindrance of ester groups, the ester groups of the probe can rotate in an excited state, so that energy is lost through non-radiative transition, and fluorescence quenching is caused. The ester group of the probe can rotate in an excited state due to the strong electron withdrawing capability and large steric hindrance of the ester group, so that energy is lost through non-radiative transition, and fluorescence quenching is caused. And due to the introduction of the ester group, the fat solubility of the BODIPY dye is further enhanced, so that the lipid droplet targeting property of the probe is effectively improved, and no-wash imaging of lipid droplets is realized.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probes, and in particular to a BODIPY-based lipid droplet-targeting fluorescent probe and applications thereof. Background Art

[0002] Lipid droplets are the primary storage site for intracellular lipids. They are composed of a phospholipid monolayer and neutral lipids and are home to a variety of functional proteins. Lipid droplets are highly dynamic organelles that play important roles in physiological processes such as lipid metabolism, signal transduction, and immune responses. Abnormalities in lipid droplets are closely associated with a variety of diseases, including obesity, fatty liver disease, cirrhosis, diabetes, cardiovascular disease, and cancer. Abnormalities in lipid droplets can also reflect abnormalities in intracellular lipid metabolism. Real-time visualization of lipid droplets facilitates the study of the mechanisms of intracellular lipid metabolism disorders and aids in the diagnosis and treatment of major lipid droplet-related diseases. Intracellular viscosity is a crucial microenvironmental parameter in biological systems. Abnormal changes in viscosity significantly influence intracellular transport and signal transduction, interactions between biomolecules, and the diffusion of active metabolites. Apoptosis, autophagy, ferroptosis, oxidative stress, and other cellular functions are inextricably linked to viscosity. Lipid droplet viscosity influences lipid transport and is closely related to the physiological and pathological functions of lipid droplets. Therefore, the development of probes that can detect and sense lipid droplet viscosity is of great significance.

[0003] Currently, the most commonly used commercial lipid droplet staining reagent is BODIPY 493 / 503. Its excitation and emission wavelengths are compatible with the 488nm laser used in commercial laser confocal microscopes. It also boasts advantages such as high quantum yield and narrow half-width (FWHM), making it suitable for multicolor imaging studies. However, BODIPY 493 / 503 still has some drawbacks, such as poor photostability, high nonspecific staining, and the need for multiple cell washes during imaging to achieve optimal lipid droplet imaging. Although viscosity-responsive lipid droplet-targeting probes have been reported, the optimal excitation wavelengths of the probes reported so far do not match the laser, resulting in poor imaging results. Patent CN117069768A discloses a viscosity-responsive lipid droplet-targeting probe based on mesophospholipid-substituted BODIPY. However, this probe has an excitation wavelength of 580nm, which is poorly compatible with commercial lasers, and exhibits a large FWHM. Summary of the Invention

[0004] In response to the above technical problems, the present invention proposes a BODIPY-based fluorescent probe targeting lipid droplets and its application.

[0005] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0006] A fluorescent probe based on BODIPY targeting lipid droplets, with the following structural formula:

[0007] By modifying the BODIPY structure and adding a lipid group at the mesoposition, the team increased BODIPY's lipid solubility and broadened its absorption and emission wavelengths. In high-viscosity microenvironments, the rotation of the excited ester group is inhibited, releasing strong fluorescence from the probe and enabling fluorescence-enhanced viscosity sensing. Furthermore, the introduction of the ester group further enhances the lipid solubility of the BODIPY dye, effectively improving the probe's targeting of lipid droplets and enabling wash-free imaging of lipid droplets.

[0008] The above-mentioned BODIPY-based fluorescent probe targeting lipid droplets has viscosity-responsiveness, and the fluorescence intensity in glycerol is 38 times higher than that in methanol.

[0009] The application of the BODIPY-based lipid droplet-targeted fluorescent probe in detecting the viscosity of a test solution for non-disease diagnosis purposes is as follows: adding the BODIPY-based lipid droplet-targeted fluorescent probe to the test solution, and detecting the fluorescence intensity of the test solution at an excitation wavelength of 500 nm.

[0010] Furthermore, the final concentration of the BODIPY-based lipid droplet-targeting fluorescent probe is 0.2-10 μM. For example, the final concentration of the fluorescent probe is 0.2 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, or 10 μM, as long as it is any concentration between 0.2-10 μM.

[0011] Application of the above-mentioned BODIPY-targeted lipid droplet fluorescent probe in the field of fluorescence imaging or fluorescence sensing.

[0012] The application of the above-mentioned BODIPY-based lipid droplet-targeted fluorescent probe in a reagent for visually monitoring changes in lipid droplet viscosity in living cells.

[0013] The application of the above-mentioned BODIPY-based lipid droplet-targeting fluorescent probe in the preparation of reagents for rapid and wash-free imaging of intracellular lipid droplets.

[0014] The cells are SMMC-7721 cells; the final concentration of the fluorescent probe in the reagent is 6 μM.

[0015] The specific steps for targeting intracellular lipid droplets using a BODIPY-based fluorescent probe are as follows: After incubating cells with the fluorescent probe molecule, the cells themselves are non-fluorescent. Laser confocal imaging reveals that the fluorescence in the green channel gradually increases over time and stabilizes around 10 minutes. By observing the green fluorescence, changes in intracellular lipid droplets can be observed. The fluorescent probe molecule concentration is 6 μM, and the incubation time is 10 minutes.

[0016] The beneficial effects produced by the present invention are:

[0017] (1) The present invention proposes a fluorescent probe based on BODIPY targeting lipid droplets, which achieves efficient detection of intracellular lipid droplets. The fluorescent probe introduces an ester group into the BODIPY mesoposition, causing the ester group of the probe to rotate in the excited state, thereby dissipating energy through non-radiative transitions and causing fluorescence quenching, thereby achieving good viscosity responsiveness.

[0018] (2) The present invention proposes a fluorescent probe based on BODIPY targeting lipid droplets. Due to the introduction of ester groups, the lipid solubility of BODIPY dye is further enhanced, thereby effectively improving the lipid droplet targeting of the probe and realizing wash-free imaging of lipid droplets.

[0019] (3) This paper proposes a BODIPY-based lipid droplet-targeting fluorescent probe that exhibits good photostability, lipid solubility, and efficient lipid droplet targeting. This provides valuable insights and new strategies for the subsequent development of lipid droplet-targeting fluorescent probes.

[0020] (4) The present invention proposes a fluorescent probe based on BODIPY targeting lipid droplets, whose excitation wavelength is close to the excitation wavelength of the commercial lipid droplet staining reagent BODIPY 493 / 503. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Schematic diagram of the viscosity response of the BODIPY-based fluorescent probe targeting lipid droplets.

[0023] Figure 2 (a) UV-visible absorption spectra and (b) fluorescence spectra of the fluorescent probe in methanol-glycerol systems with different ratios.

[0024] Figure 3 UV-visible absorption spectra (a) and fluorescence spectra (b) of the fluorescent probe in different pH solutions.

[0025] Figure 4 Laser confocal imaging of SMMC-7721 cells after incubation with the probe (4 μM) over time (0-20 min) (af); graph of the fluorescence intensity of the cells changing over time (g). λex = 500 nm, λem = 520-560 nm. Scale bar: 20 μm.

[0026] Figure 5Laser confocal imaging of SMMC-7721 cells incubated with different concentrations (0μM-10μM) of probes (af); graph showing the change of cell fluorescence intensity with probe concentration (g).

[0027] Figure 6 Confocal laser scanning imaging of SMMC-7721 cells incubated with the probe (4 μM) and the commercial lipid droplet-targeting probe HCS Lipid TOX™ Deep Red. (a) Brightfield image of the cells; (b) Green channel image of the probe (λex = 500 nm, λem = 520-560 nm); (c) Red channel image of HCS Lipid TOX™ Deep Red (λex = 637 nm, λem = 650-670 nm); (d) Overlay of the green and red channels; (e) Pearson coefficient correlation plot of the green and red channels; (f) Fluorescence intensity of a selected region of the cell as a function of distance. Scale bar: 20 μm.

[0028] Figure 7 Confocal imaging images of SMMC-7721 cells incubated with the probe (4 μM) and irradiated with 500 nm laser for different times (ag); and the change of cell fluorescence intensity with laser irradiation time (h). DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0030] The fluorescent probe based on BODIPY targeting lipid droplets used in the present invention has the structural formula: It was prepared according to the preparation method in reference (DOI: 10.1002 / chem.201503040).

[0031] The imaging study of the fluorescent probe targeting lipid droplets in living cells includes the following steps:

[0032] S1: The optimal incubation time of the fluorescent probe in SMMC-7721 cells is 10 min;

[0033] S2: The optimal incubation concentration of the fluorescent probe in SMMC-7721 cells is 6 μM;

[0034] S3: The fluorescent probe has good photostability in SMMC-7721 cells;

[0035] S4: Use laser confocal imaging to collect fluorescence in the green channel to image the lipid droplets of living cells using fluorescent probes.

[0036] By modifying the BODIPY structure, adding a lipid group at the middle position can improve the lipid solubility of BODIPY and increase the absorption and emission wavelengths of BODIPY. In a high viscosity microenvironment, the rotation of the excited ester group is inhibited, thereby releasing strong fluorescence of the probe and realizing fluorescence-enhanced viscosity sensing, such as Figure 1 The introduction of the ester group further enhances the lipid solubility of the BODIPY dye, thereby effectively improving the lipid droplet targeting of the probe and achieving wash-free imaging of lipid droplets.

[0037] Example 1: Detection of probe response to solutions of different viscosities

[0038] Different volumes of methanol and glycerol were mixed to prepare glycerol / methanol mixed solutions with glycerol volume fractions of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. The probe was added to a cuvette containing 2 mL of methanol / glycerol system (the final concentration of the probe was 10 uM). Figure 2 Figure a is the UV-vis absorption spectrum of the probe in methanol / glycerol mixtures with different ratios. It can be seen from the figure that with the increase of glycerol concentration, the maximum absorption peak of the probe at 510nm gradually red-shifts to 514nm. Figure 2 As shown in Figure b, the fluorescence intensity of the probe at 542 nm increases with increasing glycerol concentration, and the fluorescence intensity in glycerol is 38 times higher than that in methanol. These results indicate that the probe has a good fluorescence response to changes in viscosity and can be used to detect changes in viscosity.

[0039] Example 2: Detection of probe response to solutions of different pH values

[0040] The pH of different organelles within the cell is significantly different, and changes in intracellular pH affect the physiological functions of the cell. In order to effectively identify the viscosity within the sensing cell, the probe should show good stability to pH under physiological conditions. The pH stability of the probe was investigated by fluorescence emission spectroscopy. The probe (final concentration of the probe is 10uM) was added to 2mL of Britton-Robinson solution (pH 3-10), mixed well, and the fluorescence emission spectrum of the solution was tested.

[0041] like Figure 3As shown in Figures 3a and 3b, the probe exhibits a maximum emission wavelength of 520 nm in buffer solution, with a small shoulder peak at 582 nm. The probe exhibits very weak fluorescence in buffer solutions with a pH range of 3-10, and its fluorescence does not change significantly with pH changes. This demonstrates the probe's excellent stability and potential for detection in living cells.

[0042] Example 3: Detection of the optimal time for probe incubation of cells

[0043] The optimal time for incubating cells with the probe was optimized by laser confocal imaging. After washing SMMC-7721 cells three times with PBS, phenol red-free DMEM medium containing 4 μM probe was added for real-time imaging. Since the probe has very weak fluorescence in aqueous solution, it only shows strong fluorescence in a high-viscosity microenvironment. Therefore, after the cells were incubated with the probe, there was no obvious fluorescence signal outside the cells, and there was a dot-like bright green fluorescence inside the cells, indicating that the probe is suitable for wash-free imaging of lipid droplets in living cells. Figure 4 As shown in ag, cells themselves are nonfluorescent. After incubation with the probe, fluorescence in the green channel gradually increases over time, reaching a plateau around 10 minutes. These results demonstrate that the probe has good cell membrane permeability and can be used for rapid fluorescence imaging of lipid droplets in living cells.

[0044] Example 4: Detection of the optimal concentration of probe for incubating cells

[0045] Next, the incubation concentration of the probe was screened. After washing the SMMC-7721 cells in the laser confocal microscopy dish with PBS three times, phenol red-free DMEM medium containing 0μM, 2μM, 4μM, 6μM, 8μM, and 10μM probes were added and laser confocal microscopy imaging was performed. Figure 5 As shown in af, when cells were incubated with 0μM, 2μM, 4μM, 6μM, 8μM, and 10μM probes for 10 minutes, the probes all showed bright green fluorescence in the cells. When the probe concentration was 6μM, the fluorescence intensity reached the maximum ( Figure 5 g), so a probe concentration of 6 μM was selected for subsequent experiments. These results indicate that the probe can be used for lipid droplet imaging in living cells over a wide concentration range.

[0046] Example 5: Detection of Probes Targeting Lipid Droplets

[0047] Lipid droplet targeting is an important parameter for characterizing lipid droplet probes. The targeting of the probe was investigated through co-localization experiments with commercial lipid droplet targeting dyes. Figure 6 As shown, after co-staining with the probe and the commercial lipid droplet-targeting probe HCS Lipid TOXTM Deep Red, the cells showed bright punctate fluorescence in both the green and red channels. Figure 6 d shows that the red and green channels have very good overlap, and the yellow dot fluorescence is the effective superposition of the green and red channels. The Pearson coefficient of the probe and the commercial HCS Lipid TOXTM Deep Red is 0.9236 ( Figure 6 e). The fluorescence variation diagram of the selected area in the cell shows that the green channel and the red channel have good overlap with the distance change ( Figure 6 f). The above results indicate that the probe has good lipid droplet targeting ability.

[0048] Example 6: Detection of photostability of cells incubated with probes

[0049] The most commonly used commercial lipid droplet probe BODIPY 493 / 503 has poor photostability and faces many problems in practical applications. In the probe molecule of the present invention, because an electron-withdrawing ester group is introduced, the electronic energy level of the BODIPY fluorescent dye can be reduced, thereby enhancing the photostability of the probe. The photostability of the probe in lipid droplet imaging of living cells was further investigated. After washing SMMC-7721 cells in a laser confocal dish with PBS three times, phenol red-free DMEM culture medium containing 4 μM probe was added and laser confocal imaging was performed under continuous laser irradiation. Figure 7 As shown in the figure, after 30 minutes of irradiation, the cells still showed strong fluorescence, indicating that the probe has good photostability and is an excellent lipid droplet targeting probe.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fluorescent probe based on BODIPY targeting lipid droplets, characterized in that: The structural formula of the fluorescent probe is as follows:

2. The BODIPY-targeted lipid droplet fluorescent probe according to claim 1, characterized in that: The fluorescent probe has viscosity-responsiveness, and the fluorescence intensity in glycerol is 38 times higher than that in methanol.

3. Use of the BODIPY-targeted lipid droplet fluorescent probe according to claim 1 in detecting the viscosity of a test solution for non-disease diagnosis purposes.

4. The use according to claim 3, characterized in that The steps are: adding a fluorescent probe based on BODIPY targeting lipid droplets to a solution to be tested, and detecting the fluorescence intensity of the solution to be tested at an excitation wavelength of 500 nm.

5. The use according to claim 4, characterized in that The final concentration of the BODIPY-based lipid droplet-targeting fluorescent probe is 0.2-10 μM.

6. Use of the BODIPY-targeted lipid droplet fluorescent probe according to claim 1 in the field of fluorescence imaging or fluorescence sensing.

7. Use of the BODIPY-based lipid droplet-targeting fluorescent probe according to claim 1 in a reagent for visually monitoring changes in lipid droplet viscosity in living cells.

8. Use of the BODIPY-based lipid droplet-targeting fluorescent probe according to claim 1 in preparing a reagent for rapid, wash-free imaging of intracellular lipid droplets.

9. The use according to claim 7 or 8, characterized in that The cells are SMMC-7721 cells.

10. The use according to claim 9, characterized in that The final concentration of the fluorescent probe in the reagent is 6 μM.

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