Mitochondrial NADH detection and lipid droplet imaging bifunctional fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a bifunctional fluorescent probe for mitochondrial NADH detection and lipid droplet imaging as well as a preparation method and application of the bifunctional fluorescent probe. The structure of the bifunctional fluorescent probe for mitochondrial NADH detection and lipid droplet imaging is as follows: # imgabs0 #. The bifunctional fluorescent probe disclosed by the invention can generate effective optical response under the activation of NADH (Nicotinamide Adenine Dinucleotide). An in-vitro photophysical property experiment shows that the probe can sensitively detect the horizontal fluctuation of NADH (the detection limit is as low as 12.83 nM), and a confocal fluorescence microscopic imaging experiment shows that the probe has good light stability on G2 cells and can effectively position mitochondria in the cells (the positioning coefficient is 0.92). As time goes on, the probe is gradually transferred into the lipid droplet from the mitochondria, good lipid droplet positioning is achieved after one hour (the co-positioning coefficient is 0.92), and fluorescent tracing of the lipid droplet is achieved.

Description

Technical Field

[0001] The present invention relates to a dual-function fluorescent probe for mitochondrial NADH detection and lipid droplet imaging, and a preparation method and use thereof, so as to realize fluorescent 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 important coenzyme in living cells and is involved in many cellular metabolic processes, including energy metabolism, mitochondrial function, immune function, 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 many cellular redox reactions. NADH is involved in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and plays an important role in the metabolism of reactive sulfur species (RSS) and other important biological species. Changes in NADH levels are closely related to a variety of metabolic pathological states such as diabetes and cancer. Spatiotemporal tracking of NADH metabolism in living cells plays a crucial role in understanding the role of this cofactor in cell biology. For example, in cancer cells, the abnormal increase in glycolysis driven by oncogenes leads to excessive production of NADH. This highlights that real-time, dynamic monitoring of NADH in living cells is essential for studying and understanding the contribution of NADH to physiological and pathological processes. Therefore, methods for real-time monitoring of NADH levels in living cells and animals are very 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 methods have been developed to detect NADH in vitro, 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. They are surrounded by a phospholipid monolayer and consist of a neutral lipid core decorated with integrins and peripheral proteins. They have the ability to alleviate nutritional stress fluctuations in cells. Compared with normal cells, a notable feature of cancer cells is that they contain a large number of lipid droplets. Lipid droplets can provide energy and necessary lipid prerequisites for the rapid proliferation of cancer cells. They can also provide phospholipid membranes for the synthesis of organelles in cancer cells to adapt to the rapid growth of cancer cells. Summary of the invention

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

[0005] The present invention takes the strong reducing property of NADH as a starting point to reduce many heterocyclic compounds, selects quinoline as a recognition unit to construct a probe, and uses 2-[1-(6-methoxy-2-naphthyl)ethylidene]malononitrile group and quinoline salt to form a donor-π-acceptor-π-acceptor configuration, and the positively charged quinoline salt structure gives the probe good water solubility, thereby constructing a 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 converted into a donor-π-acceptor-π-donor configuration, showing a strong fluorescence emission. In addition, due to the positive charge of the quinoline salt part, the probe molecule has the ability to target mitochondria. After the response is completed, the probe molecule will present electrical neutrality, and the probe has the ability to target lipid droplets, which is transferred from mitochondria to lipid droplets, and plays a role in lipid droplet tracing imaging. Through the test of optical properties, it is shown that the probe can specifically detect NADH and can better reflect the level of NADH. This study evaluated the feasibility of using the probe LZO for imaging NADH levels and lipid droplets in living cells through analysis of cell confocal fluorescence imaging, aiming 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 quinoline salt as the detection group, and its structural formula is as follows: .

[0007] The method for preparing the dual-functional fluorescent probe for mitochondrial NADH detection and lipid droplet imaging of the present invention comprises the following steps: Step 1: 2 g of 6-methoxy-2-acetonaphthalene and 0.793 g of malononitrile were dissolved in 15 mL of acetic acid, stirred at room temperature for 10 minutes, and then 1.94 g of hexamethyldisilazane (HMDS) was dropped thereinto, and then the temperature was raised to 80°C and the reaction was carried out for 12 h; after the reaction was completed, the reaction solution was cooled, and then poured into 100 mL of deionized water, and sodium carbonate was added thereto while stirring. After no more bubbles were generated, the reaction solution was extracted with dichloromethane to obtain a brown-yellow liquid; the product was separated by column chromatography (the eluent was petroleum ether:ethyl acetate = 20:1, v / v) to obtain a yellow solid intermediate 1.

[0008] Step 2: Take 1.24 g of intermediate 1 and 0.185 g of quinoline-3-carboxaldehyde and dissolve them in 20 mL of acetonitrile. Add 1 mL of pyridine while stirring, raise the temperature 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.

[0009] Step 3: Take 1.16 g of intermediate 2 and dissolve it in 10 mL of dichloromethane. Add 0.492 g of methyl trifluoromethanesulfonate dropwise therein while stirring, and react for 24 h under a nitrogen atmosphere. 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.

[0010] 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: .

[0011] The invention discloses an application of a dual-function fluorescent probe in preparing a NADH detection reagent.

[0012] 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 imaging.

[0013] The bifunctional fluorescent probe of the present invention has the advantages of high selectivity and high sensitivity. Cell confocal fluorescence imaging test shows 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.

[0014] There is a linear relationship between the fluorescence intensity of the detection reagent at 590 nm and the NADH concentration.

[0015] The detection limit of the detection reagent for NADH is 12.83 nM.

[0016] The detection method is as follows: 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 with the increase of NADH concentration, the absorption peak 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 Reached 0.9928, the detection limit was as low as 12.83 nM. The above results show that LZO can effectively detect the level of NADH in vitro. A commercial mitochondrial dye (MitoTrackerDeep Red) was used to co-localize with LZO in G2 cells. The results showed that the fluorescence images of LZO and MitoTracker Deep Red overlapped well, and the co-localization coefficient was calculated to be 0.92, reflecting that LZO can enter the mitochondria well to detect the level of NADH. One hour after LZO was added to the cells, a commercial dye for lipid droplets (BODIPY) was used to co-localize with LZO in G2 cells. The results showed that the fluorescence images of LZO and BODIPY overlapped well, and the Pearson co-localization coefficient of LZO and BODIPY was calculated to be 0.92. The above results show that LZO can be well transferred to the lipid droplets of living cells for fluorescence imaging after responding to NADH. To investigate 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. Confocal fluorescence imaging showed that the fluorescence intensity of cells incubated with exogenous NADH was significantly higher than that of cells without NADH, and that the fluorescence intensity gradually increased with the increase in NADH concentration, which reflects that LZO can effectively detect changes in the level of intracellular NADH.

[0017] The dual-function fluorescent probe for mitochondrial NADH detection and lipid droplet imaging of the present invention can effectively detect NADH levels in solutions and cells, and has high sensitivity and selectivity. The probe has good compatibility with cells, and confocal fluorescence microscopy experiments show that LZO can be effectively localized in lipid droplets after response, with a Pearson colocalization coefficient of 0.92, and is suitable for lipid droplet fluorescence imaging of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The feasibility of LZO (10 µM) in detecting NADH is explored. (a) UV absorption spectra of LZO (10 µM) and PBS solution containing NADH (100 µM); (b) Fluorescence emission spectra of LZO (10 µM) and PBS solution containing NADH (100 µM).

[0019] Figure 2 The following are the HPLC-MS data of LZO before and after the response to NADH, analyzing the changes in the substances before and after the reaction. (a) The position of the ultraviolet absorption peak of the HPLC chromatogram of the substances before and after the reaction; (b) The mass spectrum corresponding to the peak position of the HPLC 1.08 min; (c) The mass spectrum corresponding to the peak position of the HPLC 1.78 min; (d) The mass spectrum corresponding to the peak position of the HPLC 3.16 min.

[0020] Figure 3 It 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.

[0021] Figure 4 is the ability of LZO (10 µM) to detect NADH levels in vitro. (a) Changes in UV absorption spectrum of LZO (10 µM) after adding PBS solution containing NADH (0-100 µM); (b) Changes in fluorescence intensity of LZO (10 µM) after adding PBS solution containing NADH (0-100 µM); (c) The linear relationship between the emission peak fluorescence intensity at 590 nm and the change in NADH concentration when LZO is added to PBS solution containing different concentrations of NADH; (d) The time relationship of LZO in response to NADH (100 µM) in PBS solution.

[0022] 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.

[0023] Figure 6 This is a confocal fluorescence image of mitochondria in G2 cells co-stained with LZO (10 µM) and 1 μM commercial mitochondrial probe (MitoTracker Deep Red). This study explored the mitochondrial targeting ability of LZO.

[0024] Figure 7This is a confocal fluorescence image of lipid droplets in G2 cells after co-staining LZO (10 µM) and 1 μM commercial lipid droplet probe (BODIPY) for 1 h. This study explored the lipid droplet targeting ability of LZO.

[0025] Figure 8 The confocal fluorescence images of cells incubated with LZO (10 µM) in response to different 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 different concentrations of NADH; (b) Quantified fluorescence intensity of the fluorescence images.

[0026] Fig. 9 These are confocal images of LZO (10 µM) added to normal cells and cancer cells to explore the ability of LZO to distinguish normal cells from cancer cells. (a) Confocal fluorescence images of LZO (10 µM) added to normal cells (AML-12) and cancer cells (G2); (b) Quantified fluorescence intensity of the fluorescence image. DETAILED DESCRIPTION

[0027] The present invention will be further described below by way of examples. Example 1: Synthesis of LZO

[0028] 1.161 g of intermediate 2 was dissolved in 10 mL of dichloromethane, and 0.492 g of methyl trifluoromethanesulfonate was added dropwise thereto during stirring, and the mixture was reacted for 24 h under a nitrogen atmosphere. After the reaction, the product was analyzed by column chromatography to obtain 1.185 g of the target product LZO as a dark yellow solid (yield: 73%).

[0029] 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). 13C 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. Example 2: Feasibility of LZO in detecting NADH in vitro

[0030] In order to explore the feasibility of LZO for 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 the 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, which means that LZO reacts to form new molecules. The fluorescence graph shows that after the response, the fluorescence turns on at 590 nm, which reflects the feasibility of LZO for NADH fluorescence detection. Example 3: Verification of LZO response mechanism

[0031] 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. Example 4: Ability of LZO to detect NADH levels in vitro

[0032] 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, with the continuous increase of NADH concentration, the absorption peaks at 320 nm and 360 nm in the UV spectrum gradually disappear, and the absorption peak at 550 nm gradually increases until it reaches the highest intensity. At the same time, the fluorescence intensity at 590 nm in the fluorescence spectrum gradually increases until it reaches the highest. In the low-concentration fluorescence intensity titration, the detection limit of LZO for NADH can be obtained as shown in the figure. Its detection limit for NADH is as low as 12.83 nM, which reflects that LZO has a high sensitivity to NADH and can accurately reflect the level of NADH. And from the time response results, its response speed is good, and the reaction endpoint is reached in about twenty minutes. Example 5: Selectivity of LZO for NADH

[0033] LZO was added to a PBS buffer solution containing common ions and amino acids in cells, and the fluorescence intensity was detected to see if there was any change. Figure 5 As shown, 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. Example 6: Cell localization test

[0034] To explore the ability of LZO to detect mitochondrial entry, a commercial mitochondrial probe (MitoTrackerDeep Red) was used to co-localize 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) overlapped well, and the colocalization coefficient was calculated to be 0.92 ( Figure 6 ). In order to explore 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. Example 7: Ability of LZO to recognize NADH levels in cells

[0035] 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 It can be seen that 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. Example 8: Ability of LZO to distinguish between normal cells and cancer cells

[0036] 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. Fig. 9 It can be seen that there is an obvious difference in fluorescence intensity between AML-12 cells and G2 cells. The fluorescence brightness in G2 cells is visibly higher than that in AML-12 cells, which reflects that LZO has the ability to distinguish normal cells from cancer cells.

Claims

1. A dual-function fluorescent probe for mitochondrial NADH detection and lipid droplet imaging, characterized in that: The bifunctional fluorescent probe is abbreviated as LZO, with 2-[1-(6-methoxy-2-naphthyl)ethylidene]malononitrile as the parent and quinoline salt as the detection group, and its structural formula is as follows: 。 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 are dissolved in acetic acid, stirred at room temperature for 10 minutes, and then hexamethyldisilazane is added dropwise thereto, and the temperature is raised for reaction; after the reaction is completed, the reaction solution is cooled, poured into deionized water, and sodium carbonate is added under stirring, and the reaction solution is extracted with dichloromethane after no more bubbles are generated to obtain a brown-yellow liquid; the product is separated by column chromatography to obtain a yellow solid intermediate 1; Step 2: Dissolve the intermediate 1 and quinoline-3-carboxaldehyde in acetonitrile, add pyridine during stirring, and heat to react; after the reaction, separate the product by column chromatography to obtain a yellow solid intermediate 2; Step 3: Dissolve the intermediate 2 in dichloromethane, drop methyl trifluoromethanesulfonate into the mixture while stirring, and react under a nitrogen atmosphere; after the reaction, analyze 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 bifunctional fluorescent probe according to claim 1 in preparing a 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 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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