A mitochondria nadh detection and lipid droplet imaging bifunctional fluorescent probe and a preparation method and use thereof

Abstract

The application discloses a kind of mitochondrial NADH detection and lipid droplet imaging bifunctional fluorescent probe and its preparation method and purposes, wherein the structure of mitochondrial NADH detection and lipid droplet imaging bifunctional fluorescent probe is as shown in the following: The bifunctional fluorescent probe of the application can produce effective optical response under the activation of NADH.The photophysical property experiment in vitro reflects that the probe can sensitively detect the level fluctuation of NADH (detection limit is as low as 12.83 nM), and confocal fluorescence microscopic imaging experiment shows that the probe has good light stability for G2 cell, and can effectively locate mitochondrion in cell (location coefficient is 0.92). With the lapse of time, the probe gradually transfers from mitochondrion to lipid droplet, and good lipid droplet localization (co-localization coefficient is 0.92) is achieved after one hour, so that fluorescence tracing of lipid droplet is realized.

Description

Technical Field

[0001] The present invention relates to a dual-function fluorescent probe for mitochondrial NADH detection and lipid droplet imaging, as well as a preparation method and application thereof, so as to realize fluorescence imaging of NADH detection in mitochondria in vitro and in cells, and lipid droplet tracing imaging in cells, and has the advantages of high sensitivity, high selectivity, efficient detection and good biocompatibility. Background Art

[0002] Reduced nicotinamide adenine dinucleotide (NADH) is an essential coenzyme in living cells, involved in numerous cellular metabolic processes, including energy metabolism, mitochondrial function, immunity, biosynthesis, gene expression, cell death, and aging. NADH / NAD+ is a key electron carrier in living cells, transferring hydrogen atoms and electrons from one metabolite to another in numerous cellular redox reactions. NADH participates in the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and plays a crucial role in the metabolism of reactive sulfur species (RSS) and other important biological species. Alterations in NADH levels are closely associated with various metabolic pathologies, including diabetes and cancer. The spatiotemporal tracking of NADH metabolism in living cells is crucial for understanding the role of this cofactor in cell biology. For example, in cancer cells, aberrant oncogene-driven increases in glycolysis lead to excessive NADH production. This highlights the importance of real-time, dynamic monitoring of NADH in living cells for studying and understanding its contributions to physiological and pathological processes. Therefore, methods for real-time monitoring of NADH levels in living cells and animals are highly important. However, NADH levels are affected by multiple pathways, including mitochondrial respiration, glycolysis, and the tricarboxylic acid cycle, making accurate monitoring in vivo challenging. To date, many in vitro methods have been developed to measure NADH, including enzymatic cycling assays, electrochemical analysis, high-performance liquid chromatography, and capillary electrophoresis. However, these methods have limitations in detecting changes in NADH levels in living cells and tissues in real time.

[0003] Lipid droplets are unique organelles in eukaryotic cells, composed of a neutral lipid core surrounded by a phospholipid monolayer and decorated with integrins and peripheral proteins. They have the ability to mitigate nutrient stress fluctuations in cells. A notable characteristic of cancer cells compared to normal cells is the high concentration of lipid droplets within them. These droplets provide the energy and lipids necessary for the rapid proliferation of cancer cells. They also provide the phospholipid membranes necessary for the synthesis of organelles within cancer cells, enabling them to adapt to their rapid growth. Summary of the Invention

[0004] In response to the deficiencies of the above-mentioned prior art, the present invention provides a dual-functional fluorescent probe for mitochondrial NADH detection and lipid droplet imaging, as well as its preparation method and use. The technical problem to be solved is to obtain a mitochondrial targeted response NADH through molecular design that can be transferred to the lipid droplets to achieve fluorescent tracing imaging of intracellular lipid droplets.

[0005] The present invention takes NADH's strong reducing property, which can reduce many heterocyclic compounds, as its starting point. Quinoline is selected as the recognition unit for the construction of the probe. The 2-[1-(6-methoxy-2-naphthyl)ethylidene]malononitrile group and quinolyl salt form a donor-π-acceptor-π-acceptor configuration. At the same time, the positively charged quinolyl salt structure gives the probe good water solubility, thereby constructing the LZO fluorescent probe. When the probe recognizes NADH, quinoline will be reduced to an electron-rich amine, and the donor-π-acceptor-π-acceptor configuration will be transformed into a donor-π-acceptor-π-donor configuration, showing strong fluorescence emission. In addition, due to the positive charge of the quinolyl salt part, the probe molecule has the ability to target mitochondria. After the response is completed, the probe molecule will show neutrality. At this time, the probe has the ability to target lipid droplets, transfer from mitochondria to lipid droplets, and play a role in lipid droplet tracer imaging. Through optical property testing, it is shown that the probe can specifically detect NADH and can well reflect the level of NADH. This study evaluated the feasibility of the probe LZO for imaging NADH levels and lipid droplets in living cells through analysis of cell confocal fluorescence imaging. The aim is to provide a means for visual detection of intracellular NADH levels and fluorescent tracing imaging of lipid droplets, and to provide a convenient chemical tool for the study of related diseases.

[0006] The dual-function fluorescent probe for mitochondrial NADH detection and lipid droplet imaging of the present invention is abbreviated as LZO, with 2-[1-(6-methoxy-2-naphthyl)ethylidene]malononitrile as the parent and quinolate as the detection group. Its structural formula is shown below:

[0007] .

[0008] The preparation method of the dual-function fluorescent probe for mitochondrial NADH detection and lipid droplet imaging of the present invention comprises the following steps:

[0009] Step 1: 2 g of 6-methoxy-2-acetonaphthalene and 0.793 g of malononitrile were dissolved in 15 mL of acetic acid. After stirring at room temperature for 10 minutes, 1.94 g of hexamethyldisilazane (HMDS) was added dropwise. The mixture was then heated to 80°C and reacted for 12 hours. After the reaction was completed, the mixture was cooled and poured into 100 mL of deionized water. Sodium carbonate was added while stirring. After the reaction stopped bubbling, the reaction mixture was extracted with dichloromethane to obtain a brown-yellow liquid. The product was separated by column chromatography (eluent: petroleum ether:ethyl acetate = 20:1, v / v) to obtain a yellow solid intermediate 1.

[0010] Step 2: Dissolve 1.24 g of intermediate 1 and 0.185 g of quinoline-3-carboxaldehyde in 20 mL of acetonitrile. Add 1 mL of pyridine while stirring. Heat to 80°C and react for 4 h. After the reaction, separate the product by column chromatography (eluent: petroleum ether:ethyl acetate = 10:1, v / v) to obtain yellow solid intermediate 2.

[0011] Step 3: Dissolve 1.16 g of intermediate 2 in 10 mL of dichloromethane. Add 0.492 g of methyl trifluoromethanesulfonate dropwise while stirring. React under a nitrogen atmosphere for 24 h. After the reaction, analyze the product by column chromatography (eluent: dichloromethane:ethyl acetate = 1:1, v / v) to obtain the target product LZO as a dark yellow solid.

[0012] The synthesis route of the dual-functional fluorescent probe LZO for mitochondrial NADH detection and lipid droplet imaging of the present invention is as follows:

[0013] .

[0014] The invention discloses an application of a dual-function fluorescent probe in the preparation of an NADH detection reagent.

[0015] The detection reagent can target mitochondria before responding to NADH, and can target lipid droplets after the response is completed, thereby achieving transfer from mitochondria to lipid droplets, playing a role in lipid droplet tracing and imaging.

[0016] The dual-function fluorescent probe of the present invention has the advantages of high selectivity and high sensitivity. Cell confocal fluorescence imaging tests show that the NADH detection probe of the present invention has high cell compatibility and has good mitochondrial and lipid droplet targeting capabilities before and after responding to NADH.

[0017] There is a linear relationship between the fluorescence intensity of the detection reagent at 590 nm and the NADH concentration.

[0018] The detection limit of the detection reagent for NADH is 12.83 nM.

[0019] The detection method is as follows:

[0020] The LZO of the present invention was dissolved in DMSO to prepare a 2 mM mother solution, and 15 μL of the mother solution was taken in 3 mL of PBS buffer solvent with different NADH concentrations to obtain the UV and fluorescence spectra of 10 μM LZO in different test solutions. The UV results showed that as the NADH concentration increased, the absorption peaks of LZO gradually decreased at 320 nm and 360 nm, and gradually increased at 550 nm until the reaction endpoint was reached. The fluorescence results showed that the fluorescence intensity gradually increased at 590 nm until the reaction endpoint was reached. And in response to low concentrations of NADH, the fluorescence intensity increased linearly, R 2 The detection limit reached 0.9928, with a detection limit as low as 12.83 nM. These results indicate that LZO can effectively detect NADH levels in vitro. A commercial mitochondrial dye (MitoTracker Deep Red) was used to colocalize with LZO in G2 cells. The results showed that the fluorescence images of LZO and MitoTracker Deep Red overlapped well, and the colocalization coefficient was calculated to be 0.92, reflecting that LZO can effectively enter mitochondria to detect NADH levels. One hour after LZO was added to the cells, a commercial lipid droplet dye (BODIPY) was used to colocalize with LZO in G2 cells. The results showed that the fluorescence images of LZO and BODIPY overlapped well, and the Pearson colocalization coefficient of LZO and BODIPY was calculated to be 0.92. These results indicate that LZO can effectively transfer to lipid droplets in living cells for fluorescence imaging after responding to NADH. To explore the ability of LZO to produce and detect NADH in cells, LZO was added to G2 cells and G2 cells incubated with exogenous NADH, respectively. Then, through confocal fluorescence imaging, it can be seen that the fluorescence intensity of cells incubated with exogenous NADH was significantly higher than that of cells without NADH, and the fluorescence intensity gradually increased with the increase of NADH concentration, which reflects that LZO can effectively detect changes in the level of intracellular NADH.

[0021] This dual-function fluorescent probe for mitochondrial NADH detection and lipid droplet imaging can effectively detect NADH levels in both solution and cells, with high sensitivity and selectivity. The probe exhibits excellent cell compatibility, and confocal fluorescence microscopy experiments demonstrate that LZO can effectively localize to lipid droplets after response, with a Pearson colocalization coefficient of 0.92, making it suitable for lipid droplet fluorescence imaging in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This figure investigates the feasibility of LZO (10 µM) for detecting NADH. (a) UV absorption spectra of LZO (10 µM) and a PBS solution containing NADH (100 µM); (b) Fluorescence emission spectra of LZO (10 µM) and a PBS solution containing NADH (100 µM).

[0023] Figure 2 Figures 1 and 2 show HPLC-MS data of LZO before and after the NADH reaction, analyzing the changes in the substances before and after the reaction. (a) UV absorption peak positions in the HPLC chromatograms of the substances before and after the reaction; (b) Mass spectrum corresponding to the peak position at 1.08 min on the HPLC; (c) Mass spectrum corresponding to the peak position at 1.78 min on the HPLC; (d) Mass spectrum corresponding to the peak position at 3.16 min on the HPLC.

[0024] Figure 3 This is a schematic diagram of the mechanism of LZO responding to NADH, indicating the structural changes and targeting ability changes of LZO before and after responding to NADH.

[0025] Figure 4 Figure 1 shows the ability of LZO (10 µM) to detect NADH levels in vitro. (a) Changes in the UV absorption spectrum of LZO (10 µM) after addition to PBS solutions containing NADH (0-100 µM); (b) Changes in fluorescence intensity of LZO (10 µM) after addition to PBS solutions containing NADH (0-100 µM); (c) The linear relationship between the peak fluorescence intensity at 590 nm and the NADH concentration when LZO is added to PBS solutions containing different NADH concentrations; (d) The time dependence of the response of LZO to NADH (100 µM) in PBS solution.

[0026] Figure 5 This is an investigation of the selectivity of LZO (10 µM) for NADH. The changes in fluorescence intensity after co-incubation of common intracellular ions (100 µM), amino acids (100 µM) and LZO (10 µM) were analyzed.

[0027] Figure 6 This image shows confocal fluorescence imaging of mitochondria in G2 cells co-stained with LZO (10 µM) and a commercial mitochondrial probe (MitoTracker Deep Red) at 1 µM. This image demonstrates the mitochondrial targeting ability of LZO.

[0028] Figure 7This is a confocal fluorescence image of lipid droplets in G2 cells co-stained with LZO (10 µM) and 1 μM of a commercial lipid droplet probe (BODIPY) for 1 hour. This study investigates the lipid droplet targeting ability of LZO.

[0029] Figure 8 Confocal fluorescence images of cells incubated with LZO (10 µM) in response to varying concentrations of NADH (0–40 µM) were used to investigate the ability of LZO to detect intracellular NADH levels. (a) Confocal fluorescence images of G2 cells incubated with LZO (10 µM) and varying concentrations of NADH; (b) Quantified fluorescence intensity of the fluorescence images.

[0030] Figure 9 Confocal fluorescence images of normal and cancer cells after LZO (10 µM) was added to investigate the ability of LZO to distinguish between normal and cancer cells. (a) Confocal fluorescence images of normal (AML-12) and cancer cells (G2) after LZO (10 µM) was added; (b) Quantified fluorescence intensity of the fluorescence images. DETAILED DESCRIPTION

[0031] The present invention will be further described below by way of examples.

[0032] Example 1: Synthesis of LZO

[0033] 1.161 g of intermediate 2 was dissolved in 10 mL of dichloromethane. 0.492 g of methyl trifluoromethanesulfonate was added dropwise with stirring. The mixture was allowed to react under a nitrogen atmosphere for 24 hours. After the reaction, the product was analyzed by column chromatography to obtain 1.185 g of the desired product, LZO, as a dark yellow solid (yield: 73%).

[0034] 1 H NMR (600 MHz, DMSO- d ) δ 9.95 (s, 1H), 9.64 (s, 1H), 8.50 (d, J =8.8 Hz, 1H), 8.42 (d, J = 8.1 Hz, 1H), 8.30 (t, J = 7.7 Hz, 1H), 8.15 (s,1H), 8.05 (d, J = 15.4 Hz, 3H), 8.00 (d, J = 8.9 Hz, 1H), 7.61 (d, J = 8.3Hz, 1H), 7.51 (s, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.22 (d, J = 15.7 Hz, 1H), 4.61 (s, 3H), 3.95 (s, 3H).13 C NMR (151 MHz, DMSO- d ) δ 170.46, 159.66, 151.17,145.80, 142.47, 138.49, 136.96, 136.30(s), 131.62, 131.10, 130.95, 130.19,129.44, 129.30, 128.84, 128.23, 128.08, 126.93, 122.29, 120.47, 119.82, 114.44, 113.40, 106.69, 84.71, 56.07, 46.01.

[0035] Example 2: Feasibility of LZO in detecting NADH in vitro

[0036] In order to explore the feasibility of LZO in detecting NADH levels, the optical properties of the probe for detecting NADH were tested in PBS solution in vitro. Figure 1 As shown in the figure, when LZO is added to a PBS buffer solution containing NADH, the UV absorption peaks at 320 nm and 360 nm disappear, and a new absorption peak appears at 550 nm, indicating that LZO reacts to form new molecules. The fluorescence graph shows that after the reaction, the fluorescence turns on at 590 nm, demonstrating the feasibility of LZO for NADH fluorescence detection.

[0037] Example 3: Verification of LZO response mechanism

[0038] In order to explore the mechanism of LZO responding to NADH, high performance liquid chromatography was used to analyze the changes in substances before and after the reaction. Figure 2 As shown in the figure, the peak positions of LZO and NADH before the reaction were 1.78 min and 1.02 min respectively. After LZO responded to NADH, a new peak appeared in the liquid chromatography at 3.16 min, which means that a small polar compound was produced after LZO responded. Therefore, we believe that the mechanism of LZO responding to NADH is as follows: Figure 3 shown.

[0039] Example 4: Ability of LZO to detect NADH levels in vitro

[0040] To explore the ability of LZO to detect NADH levels in vitro, LZO was added to PBS buffer solutions containing different concentrations of NADH, and then UV and fluorescence were tested. Figure 4As shown in the figure, as the NADH concentration continues to increase, the absorption peaks at 320 nm and 360 nm in the UV spectrum gradually disappear, while the absorption peak at 550 nm gradually increases until it reaches its peak intensity. Simultaneously, the fluorescence intensity at 590 nm in the fluorescence spectrum gradually increases until it reaches its peak. The detection limit of LZO for NADH can be determined in low-concentration fluorescence intensity titrations, as shown in the figure. Its detection limit for NADH is as low as 12.83 nM, demonstrating its high sensitivity to NADH and its ability to accurately reflect NADH levels. Furthermore, the time response results show a good response speed, reaching the reaction endpoint in approximately 20 minutes.

[0041] Example 5: Selectivity of LZO for NADH

[0042] LZO was added to a PBS buffer solution containing common intracellular ions and amino acids, and the fluorescence intensity was detected to see if there was any change. Figure 5 As shown in the figure, after adding LZO to these common ion and amino acid solutions, the fluorescence intensity of the solution did not change significantly, which reflects that LZO has good specificity for NADH and can specifically detect NADH in cells without interference from other ions and molecules.

[0043] Example 6: Cell localization test

[0044] To investigate the ability of LZO to detect mitochondrial entry, a commercial mitochondrial probe (MitoTracker Deep Red) was used to colocalize with LZO in G2 cells. em = 570-610 nm, λ ex = 510 nm) and the red channel of MitoTracker Deep Red (λ em = 655-695 nm, λ ex = 644 nm) fluorescence images overlapped well, and the colocalization coefficient was calculated to be 0.92 ( Figure 6 To investigate the targeting ability of LZO to lipid droplets after detecting NADH, a commercial lipid droplet probe (BODIPY) was used to co-localize with LZO in G2 cells 1 h after LZO detected NADH. The results showed that the red channel (λ em = 570-610 nm, λ ex = 510 nm) and the green channel of BODIPY (λ em =490-530 nm, λ ex= 488 nm) overlapped well, and the Pearson colocalization coefficient of LZO and BODIPY was calculated to be 0.92 ( Figure 7 These results indicate that when LZO responds to NADH, it can be well transferred to lipid droplets and perform fluorescence imaging of lipid droplets.

[0045] Example 7: Ability of LZO to Recognize NADH Levels in Cells

[0046] In order to explore the ability of LZO to detect intracellular NADH levels, 10 μM LZO was added to G2 cells incubated with different concentrations of NADH (0-40 μM), and then the changes in fluorescence intensity were observed by confocal fluorescence imaging. Figure 8 As can be seen in the figure, the fluorescence intensity of the cells increases with the increase of the concentration of added NADH, which reflects that LZO can well detect the NADH level in the cells.

[0047] Example 8: Ability of LZO to distinguish between normal cells and cancer cells

[0048] In order to explore the ability of LZO to distinguish normal cells from cancer cells, 10 μM LZO was added to normal mouse cells (AML-12) and human liver cancer cells (G2), and then the difference in fluorescence between the two was observed by confocal fluorescence imaging. Figure 9 It can be seen that there is an obvious difference in fluorescence intensity between AML-12 cells and G2 cells. The fluorescence brightness of G2 cells is visibly higher than that of AML-12 cells, which reflects that LZO has the ability to distinguish normal cells from cancer cells.

Claims

1. A dual-functional fluorescent probe for mitochondrial NADH detection and lipid droplet imaging, characterized by: The bifunctional fluorescent probe is abbreviated as LZO, with 2-[1-(6-methoxy-2-naphthyl)ethylidene]malononitrile as the parent and quinolate as the detection group. Its structural formula is shown below: 。 2. The method for preparing the dual-function fluorescent probe according to claim 1, characterized in that The steps include: Step 1: 6-methoxy-2-acetonaphthalene and malononitrile were dissolved in acetic acid, stirred at room temperature for 10 minutes, and then hexamethyldisilazane was added dropwise thereto, and the temperature was raised to react. After the reaction, the reaction solution was cooled, poured into deionized water, and sodium carbonate was added with stirring. After the generation of bubbles stopped, the reaction solution was extracted with dichloromethane to obtain a brown-yellow liquid. The product was separated by column chromatography to obtain a yellow solid intermediate 1. Step 2: Dissolve intermediate 1 and quinoline-3-carboxaldehyde in acetonitrile, add pyridine while stirring, and heat to react; after the reaction, separate the product by column chromatography to obtain yellow solid intermediate 2; Step 3: Dissolve the intermediate 2 in dichloromethane, add methyl trifluoromethanesulfonate dropwise thereto while stirring, and react under a nitrogen atmosphere; after the reaction, separate the product by column chromatography to obtain the target product LZO as a dark yellow solid; The synthetic route is as follows: 。 3. Use of the dual-function fluorescent probe according to claim 1 in the preparation of an NADH detection reagent.

4. The use according to claim 3, characterized in that: The detection reagent can target mitochondria before responding to NADH, and can target lipid droplets after the response is completed, thereby achieving transfer from mitochondria to lipid droplets, playing a role in lipid droplet tracing and imaging.

5. The use according to claim 3, characterized in that: There is a linear relationship between the fluorescence intensity of the detection reagent at 590 nm and the NADH concentration.

6. The use according to claim 3, characterized in that: The detection limit of the detection reagent for NADH is 12.83 nM.

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