Lipid droplet fluorescent probe as well as synthesis method and application thereof

Abstract

The invention discloses a lipid droplet fluorescent probe and a synthesis method and application thereof.The synthesis method comprises the steps that 4-diethylaminosalicylaldehyde and 3-dicyanomethylene indanone are dissolved in an organic solvent, then N, N-diisopropylethylamine is added, stirring and heating reflux are conducted at the room temperature, precipitates are filtered and purified, and then a red solid, namely the lipid droplet fluorescent probe, is obtained; the fluorescent probe is named as a fluorescent probe B41. The probe prepared by the invention can be accurately positioned on a lipid droplet, has relatively high sensitivity, good optical stability and specific response, and is rapid in response and relatively short in dyeing time consumption; background interference in a living body can be avoided, and the applicability in the living body is improved; the compound shows extremely low cytotoxicity, has relatively good biocompatibility and is suitable for cell imaging experiments; the preparation method is simple, purification is simple, raw materials are easy to obtain, and industrial production is easy to realize.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a lipid droplet fluorescent probe and a synthesis method and application thereof. Background Art

[0002] Lipid droplets are oily droplets formed by neutral lipids wrapped in a monolayer of phospholipids. They can not only provide a material basis for the normal life activities of cells, but also regulate lipid homeostasis in cells. At the same time, studies have shown that lipid droplets can move along the cytoskeleton and interact with other organelles, playing an important role in the storage and transportation of lipids. Abnormal lipid droplets usually cause a variety of diseases, such as neurodegenerative diseases, Alzheimer's disease and cardiovascular diseases.

[0003] Microglia are immune effector cells of the central nervous system and play an important role in neurodegenerative diseases. Studies have shown that neutral lipid droplets are abnormally formed in microglia during neurodegenerative diseases, so lipid droplets are increasingly considered to be inflammatory organelles in peripheral myeloid cells.

[0004] Fluorescence imaging technology has become a powerful tool for the precise positioning of lipid droplets in living cells due to its advantages such as high sensitivity, high selectivity, real-time imaging, and convenient sample preparation. Lipid droplet-targeted fluorescent probes have become an important medium for studying lipid droplets. Currently, commercial lipid droplet fluorescent probes include Nile Red and BODIPY 493 / 503, but there are still many limitations. For example, Nile Red will stain other hydrophobic structures in cells at the same time; BODIPY 493 / 503 staining takes a long time and may cause serious crosstalk between the excitation spectrum and the emission spectrum. Therefore, it is crucial to develop lipid droplet-targeted fluorescent probes with excellent performance. Summary of the invention

[0005] In view of the problems existing in the current prior art, the present invention provides a lipid droplet fluorescent probe and a synthesis method and application thereof. The probe can accurately locate lipid droplets, has high sensitivity, good optical stability and specific response, rapid response, and short staining time.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] The present invention provides a lipid droplet fluorescent probe, which is named fluorescent probe B41 and has the following structural formula:

[0008]

[0009] In an optional embodiment, the method for preparing the lipid droplet fluorescent probe comprises the following steps: dissolving 4-diethylamino salicylaldehyde and 3-dicyanomethylene indone in an organic solvent, then adding N,N-diisopropylethylamine and stirring at room temperature, heating to reflux, and filtering and purifying the precipitate to obtain a red solid, which is a lipid droplet fluorescent probe, named fluorescent probe B41.

[0010] In an optional embodiment, the molar ratio of 4-diethylamino salicylaldehyde to 3-dicyano methylene indone is 1:1.2.

[0011] In an optional embodiment, the precipitate is filtered, rinsed with ethanol, dried, and then purified by column chromatography to obtain a purified product.

[0012] In an optional embodiment, the mobile phase for column chromatography separation is dichloromethane-methanol in a volume ratio of 30:1.

[0013] The present invention also provides an application of a fluorescent probe in preparing a reagent for monitoring abnormal lipid droplets in microglia.

[0014] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0015] 1. The probe prepared by the present invention can accurately locate the lipid droplets, has high sensitivity, good optical stability and specific response, rapid response, and short staining time.

[0016] 2. The probe B41 (715 nm) prepared by the present invention has the advantages of a near-infrared fluorescent probe, can avoid background interference in the body, and improves its applicability in the body.

[0017] 3. When the concentration of the probe prepared by the present invention reaches 80 μM, the cell survival rate of BV2 cells after incubation for 24 hours is still as high as 60%, showing extremely low cytotoxicity, good biocompatibility, and suitable for cell imaging experiments.

[0018] 4. The preparation method of the present invention is simple, the purification is simple, the raw materials are easily available, and industrial production is easy to achieve. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The fluorescence emission spectra of the fluorescent probe B41 in glycerol-PBS solution systems with different viscosities;

[0020] Figure 2 The toxicity test results of fluorescent probe B41;

[0021] Figure 3 This is the fluorescence test result of the fluorescent probe B41;

[0022] Figure 4 This is the test result of the lipid droplet staining performance of the fluorescent probe B41;

[0023] Figure 5 This is the flow cytometry detection result of fluorescent probe B41. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0025] Example 1: Synthesis of fluorescent probe B41

[0026] This embodiment provides a method for synthesizing a lipid droplet fluorescent probe, and the specific steps are as follows:

[0027]

[0028] 4-Diethylaminosalicylaldehyde (463 mg, 2.4 mmol) and 3-dicyanomethylideneindone (388 mg, 2 mmol) were dissolved in anhydrous ethanol, stirred and mixed, and then 0.1 ml N, N-diisopropylethylamine was added. The mixture was heated under reflux overnight. After the reaction, obvious precipitation was observed. The mixture was filtered while hot, rinsed three times with ethanol, dried, and purified by silica gel chromatography using dichloromethane / methanol (V:V=30:1) as the eluent to obtain a red solid product (442 mg, yield 52%), which was named fluorescent probe B41 (hereinafter referred to as B41).

[0029] B41(C 23 H 19 N3O2) H NMR spectrum determination: 1 H NMR(400MHz,DMSO)δ8.37(d,J=7.6Hz,1H),8.03–7.88(m,2H),7.76(dt,J=15.3,7.3Hz, 2H),7.43(d,J=8.8Hz,1H),6.62–6.39(m,2H),3.51–3.47(m,4H),1.15(t,J=7.0Hz,6H).

[0030] Experimental Example 1: Fluorescence spectrum test of B41 in solutions with different viscosity coefficients

[0031] The experimental steps are as follows: (1) Weigh 1 mg of B41 (369 g / mol) powder prepared in Example 1 and dissolve it in 2.71 ml of DMSO. Vortex mix to prepare a fluorescent probe stock solution with a concentration of 1 mmol / L.

[0032] (2) Use a pipette to draw 50 μL of B41 stock solution into a centrifuge tube, and use PBS buffer and glycerol to prepare 5 mL solutions with water contents of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, respectively. After the preparation, let it stand for 30 minutes, and then measure the fluorescence intensity of each solution.

[0033] The results are as follows Figure 1 As shown, in glycerol-PBS solutions with different water contents, the B41 molecule exhibited obvious fluorescence emission at 690 nm. Moreover, with the increase of the viscosity of the glycerol-PBS solution, the emission fluorescence intensity of B41 was significantly enhanced. A 47-fold enhancement of the fluorescence intensity was observed, indicating that B41 was dependent on viscosity.

[0034] Experimental Example 2: Cytotoxicity test of B41

[0035] Cytotoxicity is an important indicator in practical applications. The standard CCK-8 method was used to verify the cytotoxicity of B41.

[0036] The specific steps are as follows: mouse microglial cells BV2 were inoculated into 4 96-well plates (about 1.5×105 cells / well), blank wells were filled with cell-free culture medium as blank group, incubated the inoculated cells in a 37°C, 5% CO2 incubator for 24h, and then B41 at concentrations of 2.5, 5, 10, 20, 40, 80, and 160μM prepared with serum-free culture medium (DMEM) was added to the wells as the experimental group, and serum-free DMEM was added as the control group. The 4 plates of cells were incubated at 37°C, 5% CO2 for 1h, 2h, 4h, and 24h, respectively, and then CCK8 detection reagent was added and incubated at 37°C, 5% CO2 for 1h, and then the absorbance of each well at 450nm was detected using an enzyme reader. The cell survival rate can be calculated by the following formula:

[0037] Survival rate (%)=[(As-Ab) / (Ac-Ab)]×100%

[0038] Where As is the absorbance of the experimental group, Ab is the absorbance of the blank group, and Ac is the absorbance of the control group.

[0039] The results are as follows Figure 2As shown, in the figure, A is the cell survival rate of BV2 cells incubated with different concentrations of B41 for 1 hour; B is the cell survival rate of BV2 cells incubated with different concentrations of B41 for 2 hours; C is the cell survival rate of BV2 cells incubated with different concentrations of B41 for 4 hours; D is the cell survival rate of BV2 cells incubated with different concentrations of B41 for 24 hours.

[0040] Figure 2 In the figure, A, B, and C respectively show that the survival rates of BV2 cells are high after being incubated with different concentrations of B41 for 1h, 2h, and 4h. As can be seen from Figure D, the cell survival rate is still as high as 60% after BV2 cells are incubated with 80μM B41 for 24h, indicating that the probe prepared by the present invention has very low toxicity and good biocompatibility.

[0041] Experimental Example 3: Fluorescence Test of B41

[0042] BV2 cells were inoculated in two 96-well plates (about 1.5×105 cells / well), and the inoculated cells were placed in a 37°C, 5% CO2 incubator for 24 hours. The cells were divided into two groups, the CON group and the LPS (lipopolysaccharide) group. Then LPS (200 ng / mL) prepared in serum-free DMEM was added to the wells of the LPS (lipopolysaccharide) group. In addition, serum-free DMEM was added to the wells of the CON group as a control group. After incubation at 37°C, 5% CO2 for 24 hours, B41 at a concentration of 2.5 μM and B41 at a concentration of 10 μM were added, respectively. After the two plates were incubated at 37°C, 5% CO2 for 0.5 hours and 2 hours, respectively, images were acquired using a fluorescence microscope.

[0043] The results are as follows Figure 3 As shown, Figure A is the fluorescence image of B41 (2.5 μM) staining at 0.5h and 2h; Figure B is the fluorescence image of B41 (10 μM) staining at 0.5h and 2h.

[0044] from Figure 3 It can be seen that B41 can be used to stain cells at lower concentrations (2.5μM, 10μM) to obtain fluorescence images, and the staining effect becomes stronger and stronger with increasing concentration, indicating that B41 has high sensitivity. Moreover, fluorescence can be maintained after 0.5h and 2h of staining, and becomes stronger and stronger with time, indicating that B41 has good optical stability and specific response, and the fluorescence effect can be further enhanced by treating cells with LPS.

[0045] Experimental Example 4: Cell Imaging Test of B41

[0046] BV2 cells were inoculated in laser confocal special dishes (about 1.5×105 cells / well), and the inoculated cells were placed in a 37℃, 5% CO2 incubator for 24h. The cells were divided into two groups: CON group and LPS (5μg / mL) group, and then LPS (5μg / mL) prepared in serum-free DMEM was added to the wells of the LPS (5μg / mL) group. In addition, serum-free DMEM was added to the wells of the CON group as a control group. After incubation at 37℃, 5% CO2 for 18h, B41 (10μM) was added at the same time, and incubated at 37℃, 5% CO2 for 0.5h, washed twice with PBS, and the adherent cells were placed on a slide; then fluorescence imaging was performed using a fluorescence microscope. At the same time, a commercial lipid droplet probe (BODIPY493 / 503) was used for co-localization experiments. Images were then acquired using a laser confocal microscope.

[0047] The results are as follows Figure 4 As shown, Figure 4 A is an image of lipid droplets in BV2 cells stained with B41. From left to right in the figure are DAPI imaging, B41 imaging, and DAPI and B41 superimposed images; as can be seen from A, B41 can clearly stain lipid droplets in BV2 cells.

[0048] Among them, the DAPI image is a cell nucleus stained with DAPI dye, showing blue fluorescence, the purpose is to locate the position of the target molecule in the nucleus, avoid signal interference from other organelles, and improve the accuracy of detection. The B41 image is stained with B41, in which the bright red represents lipid droplets, and the Merge image shows that the lipid droplets in the stained cells avoid interference from other organelles.

[0049] Figure 4 B is a co-staining image of commercial fluorescent probes BODIPY and B41. From left to right in the figure, DAPI imaging, BODIPY493 / 503 probe imaging, B41 imaging, and superimposed imaging of DAPI, BODIPY493 / 503 probe, and B41 are shown respectively; from the co-staining image, it can be seen that B41 has the same ability to stain lipid droplets as the commercial fluorescent probe BODIPY, and the staining overlap rate of B41 and the commercial fluorescent probe BODIPY reaches 100%, indicating that B41 of the present invention can be used for biological imaging of lipid droplets, and because the wavelength of B41 (715nm) is close to infrared, it can also effectively avoid background interference in the body.

[0050] Among them, the DAPI image is a cell nucleus stained with DAPI dye, showing blue fluorescence, the purpose is to locate the position of the target molecule in the nucleus, avoid signal interference from other organelles, and improve the accuracy of detection. The BODIPY image is stained with a commercial BODIPY fluorescent probe, and the green light represents lipid droplets. The B41 image is stained with B41, and the red light represents lipid droplets. The Merge image shows that the lipid droplets in the stained cells avoid interference from other organelles.

[0051] Experimental Example 5: Flow cytometry detection of B41

[0052] BV2 cells were inoculated in a 12-well plate (approximately 1.5×105 cells / well), and the inoculated cells were placed in a 37°C, 5% CO2 incubator for incubation for 24 hours. The cells were divided into two groups: a CON group and an LPS (5 μg / mL) group. LPS (5 μg / mL) prepared in serum-free DMEM was then added to the wells of the LPS (5 μg / mL) group. In addition, serum-free DMEM was added to the wells of the CON group as a control group. After incubation at 37°C, 5% CO2 for 18 hours, the plates were washed twice with PBS, and B41 (10 μM) was added at the same time. The plates were incubated at 37°C, 5% CO2 for 0.5 hours, and BV2 cells were detected using a flow cytometer.

[0053] The results are as follows Figure 5 As shown in the figure, A is a comparison of the fluorescence intensity of B41. From A, it can be seen that the fluorescence intensity of the LPS group is significantly stronger than that of the CON group; Figure B is a statistical graph of fluorescence intensity. From B, it can be seen that there is a significant difference in fluorescence intensity between the LPS group and the CON group. This shows that B41 has a high sensitivity and has a good staining effect on lipid droplets.

[0054] Although the present invention has been described using the above preferred embodiments, it is not intended to limit the scope of protection of the present invention. Any person skilled in the art who makes various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention still fall within the scope of protection of the present invention.

Claims

1. A lipid droplet fluorescent probe, characterized in that The lipid droplet fluorescent probe is named fluorescent probe B41, and the structural formula is as follows:

2. A method for preparing a lipid droplet fluorescent probe as claimed in claim 1, characterized in that: The following steps are involved: 4-Diethylaminosalicylaldehyde and 3-dicyanomethylindone were dissolved in an organic solvent, and then N,N-diisopropylethylamine was added and stirred at room temperature. The mixture was heated to reflux, and the precipitate was filtered and purified to obtain a red solid, which is a lipid droplet fluorescent probe named fluorescent probe B41.

3. The method for preparing a lipid droplet fluorescent probe according to claim 2, characterized in that: The molar ratio of the 4-diethylamino salicylaldehyde to the 3-dicyano methylene indone is 1:1.

2.

4. The method for preparing a lipid droplet fluorescent probe according to claim 2, characterized in that: The precipitate is filtered, washed with ethanol, dried, and purified by column chromatography to obtain a purified product.

5. The method for preparing a lipid droplet fluorescent probe according to claim 4, characterized in that: The mobile phase for column chromatography separation is dichloromethane-methanol, with a volume ratio of 30:

1.

6. Use of the lipid droplet fluorescent probe according to claim 1 in preparing a reagent for monitoring lipid droplet abnormalities in microglia.

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