A fatty acid-sensitive fluorescent lifetime probe, preparation method and biological application

Abstract

The application discloses a kind of fatty acid sensitive fluorescent lifetime probe, preparation method and biological application, the fluorescent probe is used to mark intracellular lipid droplet (LD), the content of fatty acid in lipid droplet is detected and its dynamic process in real time by the mode of fluorescent lifetime imaging.9-chloro acridine and diphenylamine are reacted, and the obtained fluorescent probe (DPA-AD) can well mark lipid droplet in living cell.The probe can specifically produce with fatty acid (FAs) in lipid droplet due to the existence of two lone pair electrons of N atom on acridine group, and does not interact with other lipids in lipid droplet, so as to provide a means that can real-time quantification fatty acid level in lipid droplet.DPA-AD is specific, biocompatible, and has low cytotoxicity.Therefore, the fluorescent probe of the application is used for real-time imaging of lipid droplet in living cell, and the level of fatty acid content in lipid droplet can be quantified in real time by fluorescent lifetime imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of material synthesis and biological imaging analysis, in particular to a fatty acid-sensitive fluorescent lifetime probe, a preparation method and biological application. BACKGROUND

[0002] Lipid droplets (LDs) are one of the most active organelles in mammalian cells, which are composed of a lipid core and a phospholipid layer with embedded proteins / enzymes. In the core, there are various neutral lipids, including triglycerides (TGs), diglycerides (DGs), free fatty acids (FFAs), cholesterol (CHOL) and cholesteryl esters (CEs), etc. On the surface of LDs, proteins and enzymes are involved in the hydrolysis and transformation of neutral lipids, providing various lipids for other organelles.

[0003] Fluorescent probes have been widely used for cell imaging, but cannot dynamically monitor the evolution of FAs levels in living cells to study cellular lipid metabolism. The main reasons are: (1) the previously reported probes lack the ability to bind long alkyl chain FAs; (2) they cannot detect subtle FAs fluctuations in small organelles such as LDs due to photobleaching and diffusion; (3) the fluorescence intensity signal mainly depends on the interaction or reaction between the probe and the analyte, which will change the metabolic kinetics of FAs (FAs are transformed into triglycerides, triglycerides release FAs, and FAs are transferred to mitochondria). Therefore, the problem of fluorescence signal after the interaction of the fluorescent probe with the target molecule will seriously affect the real-time observation of the slight changes of the signal molecule. SUMMARY

[0004] (I) Technical problems solved

[0005] In view of the deficiencies of the prior art, the present application provides a fatty acid-sensitive fluorescent lifetime probe, a preparation method and biological application, which solves the problem that the prior art cannot detect the slight changes of lipid droplet fatty acids in real time, and designs and develops a fluorescent lifetime probe sensitive to fatty acids. Because the fluorescence lifetime is an absolute value, it is not affected by the concentration of the probe and photobleaching. In addition, the interaction of the probe with its target (FAs) should be reversible and should not affect lipid metabolism and the transport of FAs into mitochondria. In this case, the probe meets these design criteria at the same time. First, under normal physiological conditions, almost no intermolecular interaction with FAs is detected. Only in the excited state, the probe can accept protons from FAs, thus producing a lifetime reaction. It releases protons when returning to the ground state. Second, the probe quantitatively responds to the FAs level in LDs during lipid metabolism and transport. Third, the probe does not affect the degradation of triglycerides to FAs and the transport of FAs from LDs to mitochondria. In summary, the combination of fluorescence lifetime and ESPT mechanism in probe design can expand the application of FLIM in molecular cell biology.

[0006] (ii) Technical Solution

[0007] To achieve the above object, the present application is implemented by the following technical solution: a fatty acid sensitive fluorescent lifetime probe, taking 9-chloroacridine and diphenylamine as raw materials, under the action of potassium tert-butoxide and tris-dibenzalacetone palladium, the target product is obtained, and its structural formula is as follows:

[0008]

[0009] The oil-water distribution coefficient (LogP value) of the fluorescent probe of the present application is 3.68, and the lipophilicity is good, so it can be well positioned in lipid droplets. The content and kinetic process of fatty acids in lipid droplets are detected in real time through fluorescence lifetime imaging, and after the action of two commonly used polyunsaturated fatty acids (DHA / EPA), it is found that DHA promotes the decomposition of lipid droplet lipids, and the presence of EPA can offset the decomposition effect of DHA on lipid droplet lipids.

[0010] Preferably, the probe can well mark the lipid droplets in the cells, and only the fatty acids in the lipid droplets emit excited state proton transfer (ESPT), so as to cause the change of the fluorescence lifetime of the probe, through in vitro simulation of the lipid droplet environment, the probe is placed in triglyceride, and different concentrations of several fatty acids are added, and it is found that the relationship between the fluorescence lifetime of the probe and the concentration of different fatty acids is the same as the fitting equation. The probe can well quantify the fatty acids, and the content of the fatty acids can be calculated according to the size of the fluorescence lifetime by using the simulated equation.

[0011] The present application also provides a preparation method of a fatty acid sensitive fluorescent lifetime probe, specifically: diphenylamine, 9-chloroacridine and potassium tert-butoxide are added to an organic solvent in a molar ratio of 1:1.5:2-1:2:5, tris-dibenzalacetone palladium is used as a catalyst, the reaction liquid is removed after reflux stirring for 6-8 hours, and the target product is obtained after purification.

[0012] Preferably, the molar ratio of the diphenylamine, 9-chloroacridine and potassium tert-butoxide is 1:1.5:2.

[0013] Preferably, the molar ratio of the diphenylamine, 9-chloroacridine and potassium tert-butoxide is 1:2:5.

[0014] Preferably, the organic solvent is a solvent composed of toluene, benzene, dioxane and N-N dimethylformamide.

[0015] Preferably, the purification refers to purification by column chromatography, and the eluent is petroleum ether, ethyl acetate or a mixture thereof.

[0016] The present application designs a fluorescent lifetime probe sensitive to fatty acids, which is used for observing real-time changes of fatty acid content in lipid droplet dynamics.

[0017] The fluorescent probe of the present application is added into cells to make the final concentration 2 μM, and then incubated at 37°C under 5% CO2 for 20 min, and then laser confocal and fluorescence lifetime imaging are performed.

[0018] (III) Beneficial effects

[0019] The present application provides a fatty acid sensitive fluorescent lifetime probe, a preparation method and biological applications. Compared with the prior art, the following beneficial effects are achieved:

[0020] (1) The fatty acid sensitive fluorescent lifetime probe, the preparation method and the biological applications, the fluorescent probe synthesized by the present application is obtained by one-step reaction of diphenylamine and 9-chloroacridine, which has less reaction steps, uses inexpensive materials, is simple to synthesize, and has mild reaction conditions. Because the fluorescence lifetime is an absolute value, it is not affected by the probe concentration and light bleaching. It is only related to the environment of the probe. The designed probe can well locate the lipid droplets and observe the dynamic process.

[0021] (2) The fatty acid sensitive fluorescent lifetime probe, the preparation method and the biological applications, the interaction between the probe and its target (FAs) is reversible, which should not affect the lipid metabolism and the transport of FAs into mitochondria. Under normal physiological conditions, almost no intermolecular interaction with FAs is detected. Only in the excited state, the probe can accept protons from FAs, thereby producing a lifelong reaction. It releases protons when returning to the ground state.

[0022] (3) The fatty acid sensitive fluorescent lifetime probe, the preparation method and the biological applications, the probe can quantitatively respond to the FAs level in LDs during lipid metabolism and transport, and the probe does not affect the degradation of triglycerides to FAs and the transport of FAs from LDs to mitochondria. The fluorescent probe synthesized by the present application has extremely low phototoxicity and almost no effect on the physiological state of cells. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A-B is the change of fluorescence and fluorescence lifetime of the fluorescent probe (DPA-AD) of the present application in the triglyceride environment after adding oleic acid (OA). Figure 1 A is a photograph taken under irradiation of a 365 nm ultraviolet lamp.

[0024] Figure 2 A mechanism diagram of the probe of the present application.

[0025] Figure 3 A fluorescence lifetime and concentration relationship diagram of the present application; wherein, Figure 3A is the plot of fluorescence lifetime and concentration relationship of the fluorescent probe of the present application in tricaprylin with different fatty acids added, Figure 3 B is the plot of fluorescence lifetime and concentration relationship of different triglycerides with oleic acid added. The inserted equation is fitted according to the relationship between fluorescence lifetime and fatty acid. Error bars represent standard error of the mean.

[0026] Figure 4 is the response of fluorescence lifetime of several common lipids in lipid droplets in the environment of triglycerides.

[0027] Figure 5 is the plot of viscosity non-response of the probe.

[0028] Figure 6 is the schematic diagram of selective analysis of the probe to various analysis substrates contained in cells.

[0029] Figure 7 is the schematic diagram of cytotoxicity analysis of the probe.

[0030] Figure 8 is the plot of fluorescence lifetime relationship of the fluorescent probe.

[0031] Figure 9 is the co-localization analysis of the probe. Fluorescence imaging (pseudocolor) of DPA-AD and commercial LDs-tracker stained Hep G2 cells. Cells were incubated with DPA-AD (2 μM) for 20 minutes, and then treated with LDs-tracker (2 μM) for 20 minutes.

[0032] Figure 10 is the time imaging of lipid droplets in single cells.

[0033] Figure 11 is the confocal and FLIM imaging of triglyceride lipolysis in lipid droplet-mitochondria contact. Hep G2 cells were first incubated with DPA-AD (2 μM) and Mito-tracker (1 μM) for 20 minutes, and then cultured in serum-free medium.

[0034] Figure 12 is the schematic diagram of the probe of the present application.

[0035] Figure 13 is the high-resolution mass spectrum of DPA-AD of the present application.

[0036] Figure 14 is the first nuclear magnetic hydrogen spectrum of DPA-AD of the present application.

[0037] Figure 15 is the second nuclear magnetic carbon spectrum of DPA-AD of the present application. DETAILED DESCRIPTION

[0038] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0039] Please refer to Figures 1-15 The embodiments of the present application provide three technical solutions: a fatty acid sensitive fluorescent lifetime probe, a preparation method and biological applications.

[0040] Embodiment 1:

[0041] Diphenylamine (1.2 g, 7.09 mmol), 9-chloroacridine (1.01 g, 4.73 mmol), tris-dibenzylideneacetone palladium (0.20 g, 0.99 mmol), and potassium tert-butoxide (0.80 g, 7.09 mmol) were mixed, and then 50 ml of benzene was added. After the temperature was raised to 60°C, 450 μl of tri-tert-butyl phosphine was added. After refluxing for 6-8 hours, the obtained product was spin-dried, and then column chromatography was used for purification with ethyl acetate as the eluent. 1.23 g of product was obtained, with a yield of 75.1%.

[0042] 1 H NMR (400 MHz, CDCl3, δ) 8.31-8.29 (d, J = 8 Hz, 2H), 8.07-8.05 (d, J = 8 Hz, 2H), 7.75-7.71 (t, 2H), 7.41-7.39 (t, 2H), 7.22-7.18 (t, 4H), 7.05-7.03 (d, J = 8 Hz, 4H), 6.97-6.94 (t, 2H). 13 C NMR (100 MHz, CDCl3, δ) 149.42, 147.24, 146.64, 129.34, 128.79, 128.49, 125.48, 124.29, 123.67, 121.36, 120.22. HR-MS (m / z, ESI): Calcd for C 25 H 18 N2] m / z = 347.1543 Found m / z = 347.1535.

[0043] 2. Application of the fluorescent probe in triglycerides.

[0044] The probe was dissolved in a mixed system of triglycerides and different fatty acids, with a probe concentration of 10 μM and a test temperature of 37°C.

[0045] 3. Fluorescence and fluorescence lifetime imaging of the fluorescent probe in cells.

[0046] All cell imaging probes were used at a concentration of 2 μM for 20 min. After probe treatment, cells were washed three times with culture medium or PBS, and then subjected to confocal imaging and FLIM imaging (excitation at 470 nm, collection range 520-580 nm. HyD SMD 3 detector was used with a pulse frequency of 10 MHz).

[0047] 4. Probe observation of the dynamics of lipid droplet and mitochondria interaction.

[0048] The medium containing 2 μM probe was prepared, and after the cells were cultured in the medium at 37℃ and 5% CO2 for 20 min, 1 μM Mito-tracker was used for 20 min, and then washed three times with PBS. Subsequently, the medium was replaced with serum-free medium, and the dynamics of lipid droplet and mitochondria interaction were observed by FLIM imaging. The imaging and statistical results are shown in Figure 5 .

[0049] Example 2:

[0050] A fatty acid-sensitive fluorescence lifetime probe is prepared from 9-chloroacridine and diphenylamine as raw materials under the action of potassium tert-butoxide and tris(dibenzylideneacetone)dipalladium to obtain the target product, and the structural formula is as follows:

[0051]

[0052] The fluorescence probe in the embodiment of the application has a good lipophilicity with a LogP value of 3.68, and can be well positioned in lipid droplets. The content of fatty acids in the lipid droplets and the dynamics thereof can be detected in real time by means of fluorescence lifetime imaging, and it is found that DHA promotes the decomposition of lipid droplet lipids, and the presence of EPA can offset the decomposition effect of DHA on the lipid droplet lipids.

[0053] In the embodiment of the application, the probe can well mark the lipid droplets in the cells, and only the fatty acids in the lipid droplets emit excited state proton transfer (ESPT), so that the fluorescence lifetime of the probe changes. By simulating the lipid droplet environment in vitro, the probe is placed in triglycerides, and different concentrations of several fatty acids are added, and it is found that the relationship between the fluorescence lifetime of the probe and the concentration of different fatty acids is fitted with the same equation. The probe can well quantify the fatty acids, and the simulated equation can be used to calculate the content of the fatty acids according to the size of the fluorescence lifetime.

[0054] The embodiment of the present application also provides a preparation method of the fatty acid sensitive fluorescent lifetime probe, specifically comprising: adding diphenylamine, 9-chloroacridine and potassium tert-butoxide in a molar ratio of 1:1.5:2 into an organic solvent, using tris(dibenzylideneacetone)dipalladium as a catalyst, stirring under reflux for 7 hours, removing the solvent in the reaction solution, and obtaining the target product after purification.

[0055] In the embodiment of the present application, the organic solvent is a solvent composed of toluene, benzene, dioxane and N-N dimethylformamide.

[0056] In the embodiment of the present application, the purification refers to purification by column chromatography, and the eluent is petroleum ether, ethyl acetate or a mixture thereof.

[0057] The present application designs a fatty acid sensitive fluorescent lifetime probe for observing real-time changes of the fatty acid content in lipid droplet dynamics.

[0058] The fluorescent probe of the present application is added into cells to make the final concentration 2 μM, and then laser confocal and fluorescent lifetime imaging are performed after incubation at 37 DEG C for 20 min in a 5% CO2 environment.

[0059] Embodiment 3:

[0060] A fatty acid sensitive fluorescent lifetime probe, which is obtained from 9-chloroacridine and diphenylamine under the action of potassium tert-butoxide and tris(dibenzylideneacetone)dipalladium, has the following structural formula:

[0061]

[0062] The fluorescent probe of the present application has a good lipophilicity with an oil-water partition coefficient (LogP value) of 3.68, and can be well positioned in lipid droplets. The content and kinetic process of fatty acids in lipid droplets can be detected in real time by means of fluorescent lifetime imaging, and it is found that DHA promotes the decomposition of lipid droplet lipids, and the presence of EPA can offset the decomposition effect of DHA on the lipid droplet lipids.

[0063] In the embodiment of the present application, the probe can well mark the lipid droplets in cells, and only emits excited state proton transfer (ESPT) for the fatty acids in the lipid droplets, so that the fluorescent lifetime of the probe changes. By simulating the lipid droplet environment in vitro, the probe is placed in triglycerides, and different concentrations of several fatty acids are added, and it is found that the relationship between the fluorescent lifetime of the probe and the concentration of different fatty acids is the same as the fitting equation. The probe can well quantify the fatty acids, and the content of the fatty acids can be calculated according to the size of the fluorescent lifetime by using the simulated equation.

[0064] The embodiment of the present application also provides a preparation method of the fatty acid sensitive fluorescent lifetime probe, specifically comprising: adding diphenylamine, 9-chloroacridine and potassium tert-butoxide in a molar ratio of 1:2:5 into an organic solvent, using tris-dibenzylideneacetone palladium as a catalyst, stirring under reflux for 6 hours, removing the solvent in the reaction solution, and obtaining the target product after purification.

[0065] In the embodiment of the present application, the organic solvent is a solvent composed of toluene, benzene, dioxane and N-N dimethylformamide.

[0066] In the embodiment of the present application, the purification refers to purification by column chromatography, and the eluent is petroleum ether, ethyl acetate or a mixture thereof.

[0067] The present application designs a fluorescent lifetime probe sensitive to fatty acids, which is used for observing real-time changes of the fatty acid content in lipid droplet dynamics.

[0068] The fluorescent probe of the present application is added into cells to make the final concentration 2 μM, and then laser confocal and fluorescent lifetime imaging are performed after incubation at 37 DEG C and 5% CO2 for 20 min.

[0069] In summary, the fluorescent probe synthesized in the present application is obtained by one-step reaction of diphenylamine and 9-chloroacridine, and has the advantages of less reaction steps, inexpensive materials, simple synthesis, mild reaction conditions, etc. Since the fluorescent lifetime is an absolute value, it is not affected by the probe concentration and light bleaching. It is only related to the environment of the probe. The designed probe can well locate the lipid droplets and observe the dynamic process. The interaction between the probe and its target (FAs) is reversible, and should not affect the lipid metabolism and the transport of FAs into mitochondria. Under normal physiological conditions, almost no intermolecular interaction with FAs is detected. Only in the excited state, the probe can accept protons from FAs, thereby producing a lifetime reaction. It releases protons when returning to the ground state. The probe can quantitatively respond to the FAs level in LDs during the lipid metabolism and transport process. The probe does not affect the degradation of triglycerides to FAs and the transport of FAs from LDs to mitochondria. The fluorescent probe synthesized in the present application has extremely low phototoxicity and almost no effect on the physiological state of cells.

[0070] Meanwhile, the contents not described in detail in the specification all belong to the prior art known by those skilled in the art.

[0071] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting; it is not intended to exclude myriad other embodiments of the present application that other inventors can develop based on the same general inventive concepts embodied by the described embodiments. That is, although the present application is described in terms of particular embodiments and illustrative figures, it should be apparent that the scope of the present application is not limited to these specific embodiments.

[0072] While the embodiments of the application have been shown and described herein, it will be understood by those skilled in the art that many changes, modifications, substitutions and alterations to these embodiments can be made without departing from the principles and spirits of the application, and it is intended that the scope of the application be limited solely by the scope of the appended claims and the equivalents thereof.

Claims

1. A fatty acid-sensitive fluorescence lifetime probe for the visualization and real-time detection of fatty acids in an in vitro oily environment for non-disease diagnosis and treatment purposes, characterized by: The probe structure is: .

2. A fatty acid-sensitive fluorescent lifetime probe for use in visualizing and monitoring changes in lipid droplet fatty acids in cells for non-disease diagnosis and treatment purposes, characterized in that: The probe structure is: .

3. Use of a fatty acid-sensitive fluorescence lifetime probe for real-time observation of fatty acid transport between lipid droplets and mitochondria for non-disease diagnosis and treatment purposes, characterized in that: The probe structure is: .

Images