A mitochondrial-targeted two-photon fluorescent probe with a double salt structure and its preparation method and use

Abstract

The present invention discloses a mitochondrial-targeted two-photon fluorescent probe with a double salt structure, a preparation method and a use thereof, wherein the structure of the mitochondrial-targeted two-photon fluorescent probe with a double salt structure is as follows: The mitochondrial-targeted two-photon fluorescent probe with a double salt structure of the present invention exhibits good responsiveness to NADH in an in vitro experiment and has good two-photon absorption performance. Cytotoxicity tests show that the fluorescent probe has low biological toxicity, and confocal fluorescence microscopy experiments show that the fluorescent probe has good imaging performance for HeLa cells, can effectively locate mitochondria in cells (the localization coefficient is 0.91), and is suitable for confocal fluorescence imaging of NADH in cells.

Description

Technical Field

[0001] The present invention relates to a mitochondrial-targeted two-photon fluorescent probe with a double salt structure, a preparation method and application thereof, so as to realize fluorescent imaging detection of changes in NADH levels in cell mitochondria, and has the advantages of high selectivity, high sensitivity, low biological toxicity and the like. Background Art

[0002] Mitochondria are maternally inherited cytoplasmic organelles of eukaryotic cells. They are the site of oxidative metabolism in eukaryotic organisms and the final site of energy release from oxidation of carbohydrates, fats, and amino acids. Therefore, mitochondria play a vital role in cellular metabolism, organs, tissues, and the entire body. 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. Abnormal levels of NADH are associated with tumors, cancer, ischemia, and diabetes. In particular, NADH located in mitochondria is essential for the production of ATP by the mitochondrial respiratory chain, as well as for maintaining the mitochondrial antioxidant defense system and regulating mitochondrial membrane potential. Abnormal levels of NADH in mitochondria induce mitochondrial dysfunction, generate reactive oxygen species (ROS) and DNA damage, and are associated with stroke, Alzheimer's disease, and Parkinson's disease. In addition, it has recently been reported that mitochondrial NADH is overexpressed in various tumor cancers. Therefore, this highlights that real-time and dynamic monitoring of NADH in living cells is essential for investigating and understanding the contribution of NADH to physiological and pathological processes.

[0003] To date, many methods have been developed for the detection of NADH in vitro, including enzyme cycling assays, electrochemical analysis, high performance liquid chromatography, and capillary electrophoresis. Capillary electrophoresis. However, these methods have limitations in real-time detection of changes in NADH levels in living cells and tissues. Fluorescent probes have gained widespread attention in the scientific community due to their many advantages, such as high sensitivity, high selectivity, rapid response rate, low detection limit, and ease of operation. In the past few decades, attention has been focused on the development of fluorescent probes for the detection of NADH in living cells, even in vivo. These fluorescent probes have been successfully used to distinguish normal cells from cancer cells and detect cancer cell death through reductive stress induced by natural antioxidants. Two-photon fluorescent probes have the advantages of low phototoxicity and photobleaching, and large tissue penetration depth. Therefore, the development of two-photon fluorescent probes for the detection of NADH in mitochondria of living cells will be of great significance in disease diagnosis and prevention. Summary of the invention

[0004] The present invention aims to provide a mitochondrial-targeted two-photon fluorescent probe with a double salt structure, a preparation method and use thereof. The technical problem to be solved is to obtain a probe molecular structure that can specifically target mitochondria and specifically respond to NADH fluorescence through molecular design, so as to realize fluorescent detection of NADH in mitochondria of living cells. The probe has the advantages of high selectivity, good sensitivity, excellent two-photon absorption performance and good photostability. Cytotoxicity tests show that the fluorescent probe has good cell compatibility.

[0005] The mitochondrial-targeted two-photon fluorescent probe of the present invention with a double salt structure is abbreviated as Mito-FCC, which uses 1,1,2-trimethyl-1H-benzo[e]indole salt and quinoline salt as mitochondrial targeting units, and quinoline salt is also a response unit of NADH, and its structural formula is as follows:

[0006]

[0007] The preparation method of the mitochondrial-targeted two-photon fluorescent probe with a double salt structure of the present invention comprises the following steps:

[0008] Step 1: Dissolve 1 g of 1,1,2-trimethyl-1H-benzo[e]indole and 1.36 g of CH3I in 10 mL of CH3CN, stir at 25°C for 24 h, and after the reaction is completed, filter and wash with petroleum ether to obtain intermediate 1, 1.47 g, with a yield of 87.5%.

[0009] Step 2: 0.67 g of intermediate 1 and 0.3 g of 3-quinolinecarboxaldehyde were used as reactants, 8 mL of anhydrous ethanol was used as solvent, and 3 drops of piperidine were added as catalyst. The reaction was carried out under argon atmosphere and temperature of 60°C for 6 hours. After the reaction was completed, the crude product was filtered off and then washed with petroleum ether, ether and ethyl acetate to obtain intermediate 2, 0.60 g, with a yield of 64%.

[0010] Step 3: 0.25 g of intermediate 2 and 0.17 g of methyl trifluoromethanesulfonate were used as reactants, 10 mL of dichloromethane was used as solvent, and stirred for 24 hours under an argon atmosphere at a temperature of 25°C; after the reaction was completed, the crude product was filtered off, and then the crude product was washed with petroleum ether, ether and dichloromethane to obtain the target product Mito-FCC, 0.253 g, with a yield of 75.83%.

[0011] The synthesis route of the mitochondrial-targeted two-photon fluorescent probe with a double salt structure of the present invention is as follows:

[0012]

[0013] The purpose of the mitochondria-targeted two-photon fluorescent probe with a double salt structure of the present invention is to prepare a detection reagent for detecting changes in NADH levels in mitochondria of living cells.

[0014] There is a linear relationship between the fluorescence intensity of the detection reagent at 580 nm and the NADH concentration.

[0015] Furthermore, the detection limit of the detection reagent is 0.26 μM.

[0016] The specific detection methods are as follows:

[0017] The Mito-FCC of the present invention was dissolved in DMSO to prepare a 2mM mother solution, and 15 μL of the mother solution was taken in 3 mL of PBS (pH=7.4) solvent with different NADH concentrations to obtain the fluorescence and UV spectra of Mito-FCC (10 μM) in test solutions with different NADH concentrations. As the NADH concentration increased, the absorbance of Mito-FCC increased at 557nm, decreased at 360nm, and the fluorescence increased continuously at 580nm. The NADH concentration in the solution and the fluorescence intensity showed a good linear relationship (R 2 =0.977). Then, the selectivity and response time of Mito-FCC were also tested in PBS (pH=7.4) buffer, and it was found that Mito-FCC was less affected by other interferents, indicating its good selectivity, and the response time reached the maximum fluorescence intensity in about 10 minutes. Therefore, the above results show that Mito-FCC effectively responds to NADH in solution and can be used as a fluorescent probe for detecting NADH. Then, a commercial mitochondrial dye (Mito-Tracker Green) was used to conduct a co-localization study with Mito-FCC in HeLa cells. The results showed that the fluorescence images of Mito-FCC and Mito-Tracker Green overlapped well, and the Pearson co-localization coefficient of Mito-FCC and Mito-TrackerGreen was calculated to be 0.91. The above results show that Mito-FCC can be well localized in the mitochondria of living cells.

[0018] The mitochondria-targeted two-photon fluorescent probe with a double salt structure of the present invention has good response to NADH in both solution and cells. Cytotoxicity tests show that Mito-FCC has good cell compatibility, and confocal fluorescence microscopy experiments show that Mito-FCC can effectively locate mitochondria (localization coefficient is 0.91), and is suitable for mitochondrial confocal fluorescence imaging and in situ detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 (a) UV absorption spectrum and (b) fluorescence emission spectrum of Mito-FCC (10 μM) at different NADH concentrations (0-90 μM) and the color and fluorescence changes of the solution before and after adding NADH.

[0020] Figure 2 The graphs are time response curves of Mito-FCC (10 μM) before and after the addition of (a) NADH (100 μM) and (b) selectivity test bar graphs of various analytes at 100 μM.

[0021] Figure 3 is the fluorescence intensity of Mito-FCC (10 μM) (I 580nm (a) Linear relationship between the concentration of NADH (0-10 μM) and (b) broken line relationship between the pH value (4.5-9) before and after the addition of NADH (100 μM).

[0022] Figure 4 This is a line graph showing the relationship between the effective two-photon absorption cross section and wavelength of Mito-FCC (1 mM) in the presence of NADH (1 mM).

[0023] Figure 5 It is a graph showing the survival rate of HeLa cells under the action of Mito-FCC at different concentrations (0 μM, 10 μM, 20 μM and 30 μM).

[0024] Figure 6 This is a confocal fluorescence image of mitochondria in HeLa cells co-stained with Mito-FCC (10 μM) and 0.5 μM commercial mitochondrial fluorescent probe (Mito-Tracker Green). This is to explore the mitochondrial targeting ability of Mito-FCC.

[0025] Figure 7 (a) Confocal fluorescence imaging and (b) fluorescence intensity histogram of Mito-FCC (10 μM) and different concentrations of NADH (0.2 mM, 0.5 mM, 1 mM) incubated in HeLa cells to explore the ability of Mito-FCC to image NADH exogenously.

[0026] Figure 8 The (a) confocal fluorescence imaging and (b) fluorescence intensity bar graph of Mito-FCC (10 μM) and its incubation with 20 mM glucose and 5 mM pyruvate in HeLa cells were used to investigate the changes in NADH levels during glycolysis.

[0027] Fig. 9 The following are (a) confocal fluorescence imaging and (b) fluorescence intensity histogram of Mito-FCC (10 μM) in tumor cells (HeLa, HepG2, A549) and normal cells (3T3, 293T, MH-S). The ability of Mito-FCC to distinguish between normal cells and tumor cells was explored.

[0028] Fig.10Fluorescence imaging of Mito-FCC (10 μM) in (a) tumor-bearing mice and (b) their organ tissues. To explore the responsiveness of Mito-FCC to NADH in tumor tissues. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further illustrated by the following embodiments.

[0030] Example 1: Synthesis of Mito-FCC

[0031] Compound 2 (0.25 g, 0.51 mmol) and methyl trifluoromethanesulfonate (0.17 g, 1.03 mmol) were used as reactants, 10 mL of dichloromethane was used as solvent, and stirred for 24 hours under an argon atmosphere at 25°C. After the reaction was completed, the crude product was filtered off and then washed with petroleum ether, ether and dichloromethane to obtain the target product Mito-FCC, 0.253 g, with a yield of 74.93%. 1 H NMR(400MHz, DMSO-d6)δ10.21(d,J=1.9Hz,1H),9.99-9.93(m,1H),8.71-8.59(m,2H),8.57-8.49(m,2H),8.46-8.34(m,2H),8.31-8 .24(m,1H),8.23-8.14(m,2H),8.10(d,J=16.7Hz,1H),7.91-7.82(m,1H),7.82-7.75(m,1H),4.73(s,3H),4.40(s,3H),2.08(s,6H). 13 C NMR(101MHz,DMSO-d6)δ182.33,151.45,146.77,144.31,140.06,139.66,138.94,137.63,134.14,131.88,131.71,131.53,130.6 3,129.25,129.17,128.92,128.31,127.11,124.13,122.82,120.21,119.62,117.50,114.18,54.85,46.56,36.38,25.02.ESI-MS m / z:1 / 2{[Mito-FCC-I-OTf] 2+}1 / 2{[C 27 H 26 N2] 2+}calcd,189.1043; found,189.1052.

[0032] Example 2: Spectral Testing of Mito-FCC

[0033] The Mito-FCC of the present invention was dissolved in DMSO to prepare a 2 mM mother solution, and 15 μL of the mother solution was added to 3 mL of PBS (pH=7.4) solvent with different NADH concentrations to obtain the UV absorption spectra of 10 μM Mito-FCC in test solutions with different NADH concentrations ( Figure 1 (a)) and fluorescence emission spectra ( Figure 1 (b)). As the concentration of NADH increases, the absorbance of Mito-FCC increases at 557nm, decreases at 360nm, and the fluorescence increases at 580nm. Secondly, the response time of Mito-FCC was also tested in PBS (pH = 7.4) buffer ( Figure 2 (a)) and analytes (PBS, Ca 2+ , ClO - , Cu 2+ , Fe 3+ 、H2O2、HCO3 - , K + Mg 2+ , S 2- , Cys, GSH, HCy, glycine, tyrosine, serine, NADH) selectivity ( Figure 2 (b)), it was found that Mito-FCC was less affected by other interferents, indicating its good selectivity, and the response time reached the maximum fluorescence intensity in about 10 min. In addition, the fluorescence intensity (I 580nm ) has a good linear relationship with NADH concentration in the range of 0 to 10 μM (R 2 =0.977), and the detection limit of Mito-FCC for NADH was calculated to be 0.26 μM ( Figure 3 (a)). Finally, the pH stability of the fluorescent probe Mito-FCC was tested ( Figure 3 (b)) It was found that in the presence of NADH, the fluorescent probe showed stable and significant fluorescence enhancement, indicating that Mito-FCC can be used to detect NADH in weakly alkaline mitochondria. Therefore, the above results show that Mito-FCC can effectively respond to NADH in solution and can be used as a fluorescent probe for detecting NADH.

[0034] Example 3: Two-photon absorption performance test of Mito-FCC

[0035] In order to study the two-photon absorption performance of the probe Mito-FCC, when the test solution contained NADH (1 mM), the broken line relationship between the effective two-photon absorption cross section (Φδ) and the wavelength was measured. It was found that the maximum value appeared at the excitation wavelength of 775 nm, which was 28 GM ( Figure 4). This indicates that Mito-FCC has good two-photon absorption properties and can be used for two-photon confocal fluorescence imaging of NADH in mitochondria of living cells.

[0036] Example 4: Cytotoxicity test

[0037] Before using the fluorescent probe Mito-FCC for cell imaging, the toxicity of the fluorescent probe needs to be tested. The MTT method was used for the experiment. 0μM, 10μM, 20μM and 30μM fluorescent probe Mito-FCC were added to culture HeLa cells for 24h. The results are as follows Figure 5 As shown, it was found to show low cytotoxicity and thus can be used in biological applications.

[0038] Example 5: Cell localization test

[0039] In order to study the mitochondrial localization performance of Mito-FCC, a commercial mitochondrial dye (Mito-Tracker Green) was used to co-localize with Mito-FCC in HeLa cells. The results showed that the channel (λ em =560-600nm, λ ex =488nm) and Mito-Tracker Green channel (λ em =500-540nm, λ ex =488nm) were almost completely overlapped, and the Pearson colocalization coefficient was calculated to be 0.91 ( Figure 6 ). These results indicate that Mito-FCC can be well localized in the mitochondria of living cells.

[0040] Example 6: Mito-FCC ability to image NADH exogenously in cells

[0041] In order to explore the ability of Mito-FCC to image exogenous NADH, Mito-FCC (10 μM) and different concentrations of NADH (0.2 mM, 0.5 mM, 1 mM) were cofocally imaged in HeLa cells. Figure 7 ) The results showed that as the concentration of exogenous NADH increased, its intracellular fluorescence was enhanced, indicating that Mito-FCC has a good ability to image exogenous NADH in cells.

[0042] Example 7: Monitoring NADH changes in glycolysis using the fluorescent probe Mito-FCC

[0043] In order to explore the effect of Mito-FCC on monitoring the level of NADH changes in glycolysis, HeLa cells were cultured with Mito-FCC (10 μM), Mito-FCC (10 μM) and 20 mM glucose, and Mito-FCC (10 μM) and 5 mM pyruvate. According to literature reports, the presence of glucose will increase NADH levels due to glycolysis, while pyruvate will consume NADH through intracellular lactate dehydrogenase. As expected ( Figure 8 ), cells treated with glucose showed significantly stronger fluorescence intensity, while cells treated with pyruvate showed weaker red fluorescence signals compared with the control group, indicating that Mito-FCC is able to monitor the changes in NADH in glycolysis.

[0044] Example 8: Differentiation of normal cells and tumor cells using the fluorescent probe Mito-FCC

[0045] In order to compare the difference in NADH levels between normal cells and tumor cells, Mito-FCC (10 μM) was used to perform fluorescence imaging on three types of tumor cells (HeLa, HepG2, A549) and three types of normal cells (3T3, 293T, MH-S). It was found that the fluorescence emission signals in tumor cells were stronger than those in normal cells, indicating that the concentration level of NADH in tumor cells was higher ( Fig. 9 ). Therefore, the fluorescent probe Mito-FCC can distinguish tumor cells from normal cells.

[0046] Example 9: Mito-FCC imaging of tumor mice and their organs

[0047] To investigate the imaging ability of Mito-FCC at mouse tumors, Mito-FCC solution (10 μM) was injected into the left leg (control site) and right hind leg (tumor site) of ICR mice bearing mouse hepatocarcinoma H22 tumors, as shown in Figure 2A. Fig.10 (a) The results show that the tumor site has obvious fluorescence, indicating that the tumor site has a higher NADH level than the normal site. Then the tumor mice were dissected, and the tumor tissues and heart, liver, spleen, lungs, and kidneys were separated. Then Mito-FCC was used to image the various organ tissues. Fig.10 (b) shows that there is obvious fluorescence in the tumor site compared with other organs, indicating that NADH is overexpressed in tumors. Therefore, Mito-FCC can be used to detect NADH in tumor cells and perform tumor imaging in vivo.

Claims

1. A dual-salt structure mitochondrial-targeted two-photon fluorescent probe, referred to as Mito-FCC, characterized in that Its structural formula is as follows:

2. A method for preparing the mitochondrial-targeted two-photon fluorescent probe with a double salt structure according to claim 1, characterized in that The steps include: Step 1: Dissolve 1,1,2-trimethyl-1H-benzo[e]indole and CH3I in CH3CN, stir at 25°C for 24 hours, and after the reaction is completed, filter and wash with petroleum ether to obtain intermediate 1; Step 2: intermediate 1 and 3-quinolinecarboxaldehyde are used as reactants, anhydrous ethanol is used as a solvent, and piperidine is added dropwise as a catalyst, and the mixture is reacted at 60° C. for 6 hours under an argon atmosphere; after the reaction is completed, the crude product is filtered off and then washed with petroleum ether, ether and ethyl acetate to obtain intermediate 2; Step 3: intermediate 2 and methyl trifluoromethanesulfonate were used as reactants, dichloromethane was used as solvent, and the mixture was stirred at 25°C for 24 hours under an argon atmosphere; after the reaction was completed, the crude product was filtered out, and then washed with petroleum ether, ether and dichloromethane to obtain the target product Mito-FCC; The synthetic route is as follows:

3. A use of the mitochondrial-targeted two-photon fluorescent probe with a double salt structure according to claim 1, characterized in that: The two-photon fluorescent probe is used to prepare a detection reagent, and the detection reagent is used to detect changes in NADH levels in mitochondria of living cells.

4. The use according to claim 3, characterized in that: There is a linear relationship between the fluorescence intensity of the detection reagent at 580 nm and the NADH concentration.

5. The use according to claim 3 or 4, characterized in that: The detection limit of the detection reagent is 0.26 μM.

6. Use of the double salt structure mitochondrial targeted two-photon fluorescent probe according to claim 1, characterized in that: The two-photon fluorescent probe is used to prepare a detection reagent, and the detection reagent realizes in vivo tumor imaging through mitochondrial confocal fluorescence imaging.

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