Double-response near-infrared fluorescent probe and preparation method thereof

Abstract

The invention belongs to the technical field of probes. The invention provides a dual-response near-infrared fluorescent probe and a preparation method thereof. The chemical structural formula of the probe is shown as a formula (1) or a formula (2). The double-response near-infrared fluorescent probe provided by the invention can be used for simultaneously monitoring the viscosity of living cells and NAFLD, ONOO <-> and ONOO-induced LDs; the mitochondria can be spontaneously migrated to the lipid droplet, so that the detection accuracy and sensitivity are remarkably improved. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of probes, and particularly relates to a dual-responsive near-infrared fluorescent probe and a preparation method thereof. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a serious liver disease that is prone to develop into chronic liver disease. Research shows that changes in the viscosity microenvironment and peroxynitrite (ONOO - ) levels of different subcellular organelles, as well as the formation of lipid droplets (LDs), are closely related to the progression of NAFLD. Therefore, simultaneously using highly sensitive real-time detection of viscosity, ONOO - and lipid droplets will be beneficial for monitoring and evaluating the treatment of NAFLD.

[0003] Fluorescence imaging is an emerging non-invasive imaging technology due to its characteristics of high spatio-temporal resolution, high specificity, and high sensitivity. In recent years, a large number of high-performance fluorescent probes have been developed for monitoring the fluctuations of viscosity, ONOO - and targeting LDs respectively.

[0004] Although significant progress has been made in the prior art, most probes are designed to simultaneously detect viscosity and ONOO in a single organelle - , and rarely explore their migration from one organelle to another after response. In addition, there are few reports that fluorescent probes respond to both viscosity and ONOO in the near-infrared (NIR) region (>650 nm). To better evaluate NAFLD and explore the potential connections between biomarkers, the probe should meet the following requirements: (1) It should be able to localize to mitochondria to detect ONOO- and viscosity changes, so that the resulting reaction products can subsequently target lipid droplets, enabling the simultaneous detection of the aforementioned three biomarkers; (2) It should respond to both viscosity and ONOO - in the NIR region, thus greatly improving the in vivo imaging effect for the diagnosis and treatment evaluation of NAFLD. Summary of the Invention

[0005] Aiming at the defects of the prior art, the purpose of the present invention is to provide a dual-responsive near-infrared fluorescent probe that can simultaneously monitor viscosity, ONOO - and lipid droplets and a preparation method thereof.

[0006] The present invention provides the following technical solutions:

[0007] A dual-responsive near-infrared fluorescent probe, the chemical structural formula of the probe is shown in Formula (1) or Formula (2):

[0008]

[0009] The probe is used to simultaneously monitor viscosity, ONOO - and lipid droplets.

[0010] In particular, the structural formula of the probe is shown in Formula (2) and is used to detect the degree of hepatic steatosis.

[0011] A preparation method of a dual-responsive near-infrared fluorescent probe, the chemical structural formula of the probe is as described in Formula (1) in Claim 1, and the preparation method includes the following steps:

[0012] S1. React the compound of Formula (3) and the compound of Formula (4) to obtain the product of Formula (5);

[0013] S2. React the product of Formula (5) and the compound of Formula (6) to obtain the probe shown in Formula (1);

[0014]

[0015] Preferably, in step S1, 3 mmol of the compound of Formula (3) and 3.2 mmol of the solution of the compound of Formula (4) are refluxed in dry toluene for 16 hours, then the mixture is cooled to room temperature, and then the solid is filtered and washed with toluene, and dried in vacuum to obtain the product of Formula (5).

[0016] Preferably, in step S2, 0.5 mmol of the product of Formula (5) and 0.5 mmol of the compound of Formula (6) are mixed with ethanol, then piperidine is added to the mixture, and the reaction mixture is refluxed under nitrogen for 3 hours; after cooling to room temperature, the solvent is removed, and the probe shown in Formula (1) is obtained by column chromatography purification.

[0017] A preparation method of a dual-responsive near-infrared fluorescent probe, the chemical structural formula of the probe is as described in Formula (2) in Claim 1, and the preparation method includes the following steps:

[0018] S1. React the compound of Formula (3) and the compound of Formula (7) to obtain the product of Formula (8);

[0019] S2. React the product of Formula (8) and the compound of Formula (9) to obtain the probe shown in Formula (2);

[0020]

[0021] Preferably, 3 mmol of the compound of Formula (3) and 3.2 mmol of the solution of the compound of Formula (7) are refluxed in dry toluene for 16 hours, then the mixture is cooled to room temperature, and then the solid is filtered and washed with toluene, and dried in vacuum to obtain the product of Formula (8).

[0022] Preferably, in step S2, 0.5 mmol of the product of formula (8) and 0.5 mmol of the compound of formula (9) are mixed with ethanol, then piperidine is added to the mixture, and the reaction mixture is refluxed for 24 hours under nitrogen; after cooling to room temperature, the solvent is removed, and the probe shown in formula (2) is obtained by column chromatography purification.

[0023] Preferably, in step S2, the addition amounts of each substance are as follows: 220 mg of the product of formula (8), 178 g of the compound of formula (9), 10 mL of ethanol, and 10 μL of piperidine.

[0024] Advantages of the present invention:

[0025] The dual-responsive near-infrared fluorescence probe provided by the present invention can simultaneously monitor the viscosity of living cells and NAFLD, ONOO⁻, and ONOO-induced LDs; it can spontaneously migrate from mitochondria to lipid droplets, significantly improving the accuracy and sensitivity of detection. Description of the drawings

[0026] Figure 1 1H NMR spectrum of the probe HX-VP-1 of the present invention;

[0027] Figure 2 13C NMR spectrum of the probe HX-VP-1 of the present invention;

[0028] Figure 3 HRMS value of the probe HX-VP-1 of the present invention;

[0029] Figure 4 1H NMR spectrum of the probe HX-VP-2 of the present invention;

[0030] Figure 5 13C NMR spectrum of the probe HX-VP-2 of the present invention;

[0031] Figure 6 HRMS value of the probe HX-VP-2 of the present invention;

[0032] Figure 7 1H NMR spectrum of the compound product;

[0033] Figure 8 13C NMR spectrum of the compound product;

[0034] Figure 9 HRMS value of the compound product;

[0035] Figure 10Experimental results of the probe performance of the present invention. Among them, part (A) shows the fluorescence change of HX-VP-2 (5 μM) with different viscosities (0.59 - 945 cP); part (B) shows the linear relationship between the fluorescence intensity (5 μM) of HX-VP-2 and viscosity (4.83 - 107 cP); part (C) shows the selectivity of HX-VP-2 (5 μM) to viscosity; part (D) shows the fluorescence change of HX-VP-2 (5 μM) with different concentrations of ONOO - (0 - 9.58 μM); part (E) shows the linear relationship between HX-VP-2 (5 μM) and the fluorescence intensity of ONOO - (0.58 - 9.58 μM); part (F) shows the selectivity of HX-VP-2 (5 μM) to ONOO -

[0036] Figure 11 This is the experimental result diagram of the staining experiment of the present invention. Among them, part (A) shows that HepG2 cells are treated with Mito-tracker Green (200 nM) and HX-VP-2 (10 μM) for 30 min; part (B) shows that HepG2 cells are treated with Bodipy 493 / 503 (1 μM) and HX-VP-2 (10 - μM) for 30 min; part (C) shows that HepG2 cells are treated with HX-VP-2 (10 μM) and SIN-1 (500 μM) for 30 min, and then incubated with Bodipy 493 / 503 (1 μM); part (D) shows the magnified image; part (E) shows the treatment of the product (10 μM) and Bodipy 493 / 503 (1 μM) for 30 min; part (F) shows the measurement of ONOO-induced LDs migration by HX-VP-2; Red channel: HX-VP-2 and product, λex = 450 nm, λem = 570 - 670 nm; Green channel: Mito-tracker Green, λex = 488 nm, λem = 500 - 550 nm, Bodipy - ; λex = 493 nm, λem = 500 - 550 nm; Scale bar = 10 μm; 493 / 503

[0037] Figure 12 This is the fluorescence imaging result diagram of the present invention. Among them, part (A) shows the fluorescence imaging of viscosity and ONOO-in NAFLD cell models treated with different concentrations of OA and PA. ONOO channel: λex = 450 nm, λem = 570 - 670 nm, viscosity channel: λex = 520 nm, λem = 690 - 790 nm, Scale bar = 10 μm; part (B) shows the detection of the fluorescence intensity of cells in the ONOO- and viscosity channels; part (C) shows the PCR detection of the expression of related genes in different groups;​​

[0038] Figure 13 For the viscosity and ONOO of the NAFLD mouse model of the present invention - Fluctuation experiment results, wherein part (A) is the fluorescence imaging, H&E and Oil Red O staining of viscosity and ONOO at different stages of NAFLD - ; ONOO- channel: λex = 470 nm, λem = 650 nm, viscosity channel: λex = 530 nm, λem = 750 nm, scale bar = 100 μm; part (B) is the fluorescence intensity of cells in the ONOO - channel; part (C) is the fluorescence intensity of cells in the viscosity channel; part (D) is the body weight, liver weight, and liver index of different groups; part (E) is the ALT, AST, TG analysis between different groups;

[0039] Figure 14 For the imaging experiment results of human liver tissue of the present invention, wherein part (A) is the fluorescence imaging, H&E and Oil Red O staining of human liver tissue viscosity and ONOO - , scale bar = 100 μm; part (B) is the fluorescence intensity of cells in the ONOO - channel and viscosity channel;

[0040] Figure 15 For the absorption spectrum and fluorescence spectrum results of HX-VP-1 of the present invention, wherein part (A) is the absorption spectrum of HX-VP-1 (5 μM) in methanol and glycerol; part (B) is the fluorescence spectrum of HX-VP-1 (5 μM) in methanol and glycerol; λex = 500 nm;

[0041] Figure 16 For the absorption spectrum and fluorescence spectrum results of HX-VP-2 of the present invention, wherein part (A) is the absorption spectrum of HX-VP-2 (5 μM) in methanol and glycerol; part (B) is the fluorescence spectrum of HX-VP-2 (5 μM) in methanol and glycerol; λex = 500 nm;

[0042] Figure 17 For the fluorescence stability experiment results of HX-VP-1 of the present invention, the figure shows the fluorescence stability of HX-VP-1 (5 μM) in methanol and 75% glycerol-methanol solution; λex = 500 nm;

[0043] Figure 18 For the fluorescence stability experiment results of HX-VP-2 of the present invention, the figure shows the fluorescence stability of HX-VP-2 (5 μM) in methanol and 75% glycerol-methanol solution; λex = 500 nm;

[0044] Figure 19Effect of pH on the reaction between HX-VP-1 (5 μM) and ONOO - (10 μM); 50 mM PBS, 30% DMF-PBS, λex = 450 nm;

[0045] Figure 20 Effect of pH on the reaction between HX-VP-2 (5 μM) and ONOO - (10 μM); 50 mM PBS, 30% DMF-PBS, λex = 450 nm;

[0046] Figure 21 Fluorescence spectra of HX-VP-1 (5 μM) against different concentrations of ONOO - (0 - 10 μM), 50 mM PBS, pH 8.0, 30% DMF-PBS, λex = 450 nm;

[0047] Figure 22 In part (A) are the absorption spectra of HX-VP-1 (5 μM) before and after reaction with ONOO-; in part (B) are the fluorescence spectra of HX-VP-1 (5 μM) before and after reaction with ONOO-; 50 mM PBS, pH 8.0, 30% DMF-PBS, λex = 450 nm;

[0048] Figure 23 In part (A) are the absorption spectra of HX-VP-2 (5 μM) before and after reaction with ONOO-; in part (B) are the fluorescence spectra of HX-VP-2 (5 μM) before and after reaction with ONOO-; 50 mM PBS, pH 8.0, 30% DMF-PBS, λex = 450 nm;

[0049] Figure 24 Effect of pH on the reaction between HX-VP-1 (5 μM) and ONOO- (10 μM); 50 mM PBS, 30% DMF-PBS, λex = 450 nm;

[0050] Figure 25 Effect of pH on the reaction between HX-VP-2 (5 μM) and ONOO- (10 μM); 50 mM PBS, 30% DMF-PBS, λex = 450 nm;

[0051] Figure 26 Fluorescence spectra of HX-VP-1 (5 μM) against different concentrations of ONOO- (0 - 10 μM), 50 mM PBS, pH 8.0, 30% DMF-PBS, λex = 450 nm;

[0052] Figure 27The linear relationship of the fluorescence intensity of HX-VP-1 (5 μM) with ONOO- (0.1 - 10 μM), 50 mM PBS, pH 8.0, 30% DMF-PBS, and λex = 450 nm;

[0053] Figure 28 The reaction results of HX-VP-1 (5 μM) with ONOO- and different analytes; 10 μM ONOO - 、100 μM (Na + 、K + 、Ca 2+ 、Cu 2+ 、Zn 2+ 、Mg 2+ 、Fe 3+ 、S 2- 、GSH, Cys), 25 μM (HOCl, . OH, NO), 50 μM H2O2, glycerol, 10 mM PBS, pH 7.4, λ ex =500 nm, λ em =730 nm;

[0054] Figure 29 The results of the cell viability experiment of HepG2 cells treated with different concentrations of HX-VP-2 in the present invention;

[0055] Figure 30 The experimental results of the effect of fluorescence imaging on the viscosity change of HepG2 cells. Among them, part (a) is the control group only using HX-VP-2 (10 μM); part (b) is the monensin group first using nystatin (20 μM), and then using HX-VP-2 (10 μM); part (c) is the nystatin group first using nystatin (20 μM), and then using HX-VP-2 (10 μM); part (d) is the relative fluorescence intensity of parts (a), (b), and (c); λex = 520 nm; λem = 690 - 790 nm; scale bar = 10 μm;

[0056] Figure 31 The experimental results of the fluorescence imaging of HepG2 cells with different concentrations of ONOO-. Among them, after HepG2 cells were treated with HX-VP-2 (10 μM) for 30 min, they were then treated with different concentrations of SIN-1 ((a) 0 μM; (b) 250 μM; (c) 500 μM; (d) 1000 μM) for another 30 min; part (e) is the average fluorescence intensity of (a) - (d); the fluorescence channel of HX-VP-2: λex = 450 nm, λem = 570 - 670 nm; scale bar = 10 μm;

[0057] Figure 32Fluorescence imaging experimental results showing viscosity and ONOO⁻ changes in different channels; among them, part (a) shows cells treated with HX-VP-2 (10 μM) for 30 min; part (b) shows HepG2 cells treated with HX-VP-2 (10 μM) for 30 min and then treated with SIN-1 (500 μM) for another 30 min; part (c) shows the average fluorescence intensity in different channels for (a) and (b); Viscosity channel: λex = 520 nm; λem = 690 - 790 nm; ONOO⁻ channel: λex = 450 nm, λem = 570 - 670 nm; Scale bar = 10 μm;

[0058] Figure 33 Co-localization results of HX-VP-2 and compound products; HepG2 cells were treated with (A) Mito-tracker Green (200 nM) and HX-VP-2 (10 μM) for 30 min; (B) compound products (10 μM) and Bodipy 493 / 503 (1 μM) for 30 min; Red channel: HX-VP-2 and product, λex = 450 nm, λem = 570 - 670 nm; Green channel: Mito-tracker Green, λex = 488 nm, λem = 500 - 550 nm, Bodipy493 / 503, λex = 493 nm, λem = 500 - 550 nm; Scale bar = 10 μm;

[0059] Figure 34 H&E staining results of organs after treatment with HX-VP-2 (500 μM, 5 μL / g) for 24 h; Scale bar = 100 μm;

[0060] Figure 35 Liver photos of each group. Detailed implementation manners

[0061] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the protection scope of the present invention.

[0062] Example 1

[0063] Synthesis of HX-VP-1:

[0064]

[0065] As shown in the above reaction, a solution of Compound 1 (888 mg, 3 mmol) and Compound 2 (298 mg, 3.2 mmol) was refluxed in dry toluene (5 mL) for 16 h. The mixture was then cooled to room temperature, and the solid was filtered and washed with toluene and dried in vacuo to give Compound 3. The crude product was further used in the next step without further purification. Compound 3 (194 mg, 0.5 mmol) and Compound 4 (178 mg, 0.5 mmol) were mixed with ethanol (10 mL). Then, piperidine (10 μL) was added to the mixture. The reaction mixture was refluxed under nitrogen for 3 h. After cooling to room temperature, the solvent was removed. The crude product was purified by column chromatography to give HX-VP-1 (97.3 mg, yield: 26.8%). 1H NMR (400 MHz, CDCl3) δ (ppm): 9.05 (2H, d, J = 6.4 Hz), 7.81 (5H, m), 7.51 (2H, d, J = 8.0 Hz), 7.44 (2H, d, J = 8.8 Hz), 7.36 (1H, d, J = 4.0 Hz), 7.28 (4H, m), 7.18 (1H, d, J = 3.6 Hz), 7.07 (8H, m), 6.70 (1H, d, J = 15.6 Hz), 6.04 (2H, s), 1.31 (12H, s). 13C nuclear magnetic resonance (100 MHz, CDCl3) δ (ppm): 153.82, 150.17, 149.06, 147.48, 144.30, 138.74, 136.38, 86, 135.37, 129.93, 129.1, 1.01, 127.34, 126.95, 125.53, 124.23, 123.94, 123.64, 123.00, 120.04, 84.55, 63.61, 25.31. HRMS m / z: calculated value 647.2898, measured value 647.2898 [M] + .

[0066] Synthesis of HX-VP-2:

[0067]

[0068] As shown in the above reaction, a solution of Compound 1 (888 mg, 3 mmol) and Compound 5 (458 mg, 3.2 mmol) was refluxed in dry toluene (5 mL) for 16 h. Then, the mixture was cooled to room temperature, and the solid was filtered and washed with toluene and dried in vacuo to obtain Compound 6. The crude product was further used in the next step without further purification. Compound 6 (220 mg, 0.5 mmol) and Compound 4 (178 mg, 0.5 mmol) were mixed in ethanol (10 mL). Then, piperidine (10 μL) was added to the mixture. The reaction mixture was refluxed under nitrogen at 50 °C for 24 h. After cooling to room temperature, the solvent was removed. The crude product was purified by column chromatography to give HX-VP-2 (79.2 mg, yield: 10.2%). 1H nuclear magnetic resonance (400 MHz, CDCl3) δ (ppm): 10.20 (1H, d, J = 6.8 Hz), 8.47 (2H, m), 8.23 (1H, d, J = 15.6 Hz), 8.10, 10 (1 = 8.8 Hz, J = 8.0 Hz), 7.80, 1 (1H, t, J = 8.0 Hz), 7, 51, 1, 1), 7.30 (4H, m), 7.31 (1), 7.23 (2H, m), 7.12, 1.29 (12H, 1). 13C nuclear magnetic resonance (100 MHz, CDCl3) δ (ppm): 153.60, 150.94, 149.30, 149.16, 147.44, 139.43, 138.71, 138.13, 136.70, 136.60, 136.30, 129.36, 129.36, 127.45, 127.15, 126.85, 126, 126.29, 125.62, 124.34, 122.92, 119.82, 116.65, 116.36, 84.49, 61.11, 25.30. HRMS m / z: calculated value 697.3055, measured value 697.3060 [M] + .

[0069] Synthesis of compound product:

[0070]

[0071] As shown in the above reaction, benzoyl chloride (116 μL, 1 mmol) was added to dry DMF (15 mL) of compound 5 (286 mg, 2 mmol), and the resulting mixture was stirred at room temperature for 20 min. Then, compound 4 (355 mg, 1 mmol) was added to the mixture, and the mixture was refluxed at 160 °C for another 5 h. After cooling to room temperature, the mixture was diluted with EA (80 mL), and then washed with water (50 mL × 3). The solution was added to anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the product (188 mg, yield: 39%). 1H nuclear magnetic resonance (400 MHz, DMSO-d6) δ (ppm): 8.86 (1H, d, J = 4.8 Hz), 8.41 (1H, d, J = 8.4 Hz), 8.03 (1H, d, J = 8.0 Hz), 7.73 (7H, m), 7.45 (2H, s), 7.34 (4H, t, J = 8.0 Hz), 7.09 (6H, m), 7.00 (2H, d, J = 8.8 Hz). 13C nuclear magnetic resonance (100 MHz, DMSO-d6) δ (ppm): 150.08, 148.35, 147.18, 146.71, 141.11, 141.1, 130.37, 120.01, 129.36, 129.09, 127.12, 12, 126.56, 126.48, 125.40, 124.43, 123.67, 123.57, 122.74, 120.49, 116.17. HRMS m / z: calculated value 481.1733, measured value 481.1730 [M + H] + .

[0072] HX-VP-1 and HX-VP-2 are typical D-π-A conjugated molecules, including a triphenylamine derivative as an electron donor, a conjugated carbon chain as a π-bridge, a pyridinium or quinolinium cation salt as an electron acceptor, and a phenylborate ester group. Compared with the pyridinium salt structure of HX-VP-1, HX-VP-2 adopts a quinolinium salt structure, aiming to further redshift the absorption / emission band. The rotation of single bonds in the triphenylamine derivative and the pyridinium or quinolinium cation salt makes HX-VP-1 and HX-VP-2 sensitive to viscosity in the NIR region. In addition, the phenylborate unit is considered to be an ideal ONOO - reaction moiety, which can trigger an elimination reaction to release a product with a shorter wavelength. In addition, the cationic salt structure tends to target mitochondria, and the lipophilicity of HX-VP-1 and HX-VP-2 with ONOO− may enter LDs. Therefore, HX-VP-1 and HX-VP-2 are expected to simultaneously detect ONOO− and viscosity in the NIR region and may be used to explore the ONOO−-induced LDs migration generated by mitochondria. As Figures 1-9As shown, the inventors synthesized HX-VP-1 and HX-VP-2, and determined their structures by HR-MS and NMR.

[0073] Example 2

[0074] (I) Experimental methods

[0075] 1. Materials and instruments

[0076] Unless otherwise specified, materials were obtained from commercial suppliers and used without further purification. 1H NMR and 13C NMR spectra were measured using a Bruker AM400 NMR spectrometer. The proton chemical shifts of the nuclear magnetic resonance spectra were in ppm relative to the internal reference TMS (1H, 0.00 ppm). HRMS spectral data were obtained using a Bruker Daltonics Bio TOF mass spectrometer; absorption spectra and photoluminescence spectra were obtained using an Agilent Cary 3500 UV-Vis and fluorescence original + fluorescence spectrophotometer; cell imaging was obtained using a white light laser under a Leica TCS SP8 confocal microscope. Tissue imaging was obtained using a Nikon Ni-E multiphoton laser scanning confocal microscope. Animal experiments were conducted on an IVIS IVIS spectrum.

[0077] 2. Preparation of cationic and anionic solutions

[0078] 10 mmol of inorganic salts (sodium chloride, potassium chloride, CaCl2·2H2O, copper chloride, zinc chloride, MgCl2·6H2O, FeCl3·6H2O, glutathione (GSH), cysteine (Cys)) were dissolved in distilled water (50 mL) to obtain the corresponding aqueous solutions. When needed, they were diluted to the desired concentration with water.

[0079] 3. Preparation of reactive oxygen species (ROS) and reactive nitrogen species (RNS)

[0080] Hypochlorous acid was prepared by diluting commercial sodium hypochlorite solution with deionized water, and was measured using a ε292nm = 350 M -1 cm -1 spectrophotometer. Hydrogen peroxide was prepared by directly diluting commercial hydrogen peroxide solution. Nitric oxide (NO) was prepared using the NOC-9 method. Peroxynitrite stock solution (ONOO - ), and the peroxynitrite concentration was measured using a ε302nm = 1670 M -1 cm -1 spectrophotometer. Hydroxyl radicals were prepared by the Fenton chemical method by adding a solution containing excess hydrogen peroxide to an Fe 2+ solution. Superoxide was generated from potassium superoxide in a potassium superoxide saturated solution.

[0081] 4. Fluorescence analysis

[0082] Dissolve HX-VP-1 and HX-VP-2 in dimethyl sulfoxide (DMSO) to prepare a stock solution (10 mM). Prepare the final test solutions (5 μM) of HX-VP-1 and HX-VP-2 by diluting the stock solution. Analytes used in the viscosity selection experiment: 10 μM ONOO - 、100 μM (Na + 、K + 、Ca 2+ 、Cu 2+ 、Zn 2+ 、Mg 2+ 、Fe 3+ 、S 2- 、GSH, Cys), 25 μM (hypochlorous acid,. Oh, no), 50 μM hydrogen peroxide, glycerol. Except for the selectivity experiment, viscosity detection was carried out in MeOH and glycerol systems, HX-VP-1: λ ex = 500 nm, λ em = 722 nm, HX-VP-2: λ ex = 500 nm, λ em = 777 nm. For detecting ONOO - : 50 mM PBS, pH 8.0, 30% DMF-PBS, HX-VP-1: λ ex = 450 nm, λ em = 620 nm, HX-VP-2: λ ex = 450 nm, λ em = 657 nm. Before recording the spectrum, shake the resulting solution at room temperature.

[0083] Statistical analysis

[0084] Perform statistical analysis on the data using GraphPad Prism software (version 9; GraphPad Prism, San Diego, CA, USA). The data are expressed as mean ± standard deviation (SD). The differences between groups were analyzed by two-tailed Welch's t-test, *P < 0.05, which was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0085] 5. Cell culture and cytotoxicity assay

[0086] HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics (including 100 U / ml penicillin and 100 μg / ml streptomycin) in a 37 °C, 5% CO2 / 95% air incubator. The cytotoxicity was evaluated by the CCK-8 method. HepG2 cells were seeded into 96-well plates (1 × 104 (Cells / well). After incubation at 37 °C for 24 h, the cells were treated with 100 μL of fresh medium containing different concentrations of HX-VP-2 (0, 5, 10, 20, 40 μM) for 24 h. After removing the medium, the cells were treated with fresh medium supplemented with CCK-8 (at a ratio of 10:1) at 37 °C for 2 h. Finally, the absorbance was measured at 450 nm.

[0087] 6. Cell Viscosity Response

[0088] HepG2 cells were seeded in confocal dishes and incubated at 37 °C for 24 h. Then the medium was replaced with fresh medium containing 10 μM nystatin (NYS) or monensin (Mon) for 30 min. After washing twice with PBS, the cells were incubated with HX-VP-2 (10 μM) for 30 min. Confocal imaging was performed.

[0089] 7. Cellular ONOO - Reaction

[0090] HepG2 cells were seeded in confocal dishes and incubated at 37 °C for 24 h. Then the medium was replaced with fresh medium containing different concentrations of 3-morpholinosydnonimine (SIN-1) (0, 0.25, 0.5, 1 mM) and cultured for 30 min. After washing twice with PBS, the cells were incubated with HX-VP-2 (10 μM) for 30 min. Confocal imaging was performed.

[0091] 8. Subcellular Localization of Cells

[0092] HepG2 cells were seeded in confocal dishes and incubated at 37 °C for 24 h. After that, the cells were co-incubated with 10 μM HX-VP-2 and different commercial organelle dyes (mito-tracker green, Bodipy 493 / 503 ) for 15 min. Confocal imaging was performed.

[0093] 9. Subcellular Localization after Reaction with ONOO -

[0094] HepG2 cells were seeded in confocal dishes and incubated at 37 °C for 24 h. Then the medium was replaced with fresh medium containing 0.5 mM SIN-1 and the cells were treated for 30 min. Then the cells were co-incubated with HX-VP-2 (10 μM) and Bodipy 493 / 503 (500 nM) for 30 min. After that, confocal imaging was completed.

[0095] 10. Real-Time Fluorescent Quantitative PCR Analysis

[0096] ​Total RNA was extracted using an animal total RNA isolation kit (pre-protein isolation kit). According to the manufacturer's instructions, reverse transcription reactions were performed on 2 μg of RNA using qPCR Master Mix. The products were amplified using Taq pro Universal SYBR qPCR Master Mix (Vazyme). The expression levels of target gene mRNAs were detected using a real-time fluorescence quantitative PCR system. The target mRNA levels were normalized to the Gapdh gene as an endogenous control. The primer sequence list is shown in the following table:

[0097] Table 1

[0098]

[0099] 11. NAFLD mouse model

[0100] All animal experiments were conducted strictly in accordance with the relevant laws and guidelines issued and approved by the Animal Ethics Committee for Laboratory Animal Care and Use of West China Hospital (20240118002). Briefly, acute toxicity was first evaluated. Male C57BL / 6J mice (8 weeks old) were injected once via the tail vein with 100 μM HX-VP-2 (50 μL per 10 g body weight). After 24 observations, the mice were euthanized, and tissues of the heart, liver, spleen, lungs, and kidneys were collected for hematoxylin and eosin (H&E) staining. Male C57BL / 6J mice (6 - 8 weeks old) were purchased from Jinfa Technology Co., Ltd. (Jiangsu, China). After one week of adaptation, the mice were randomly divided into 5 groups (n = 5 per group). Control group: normal diet for 16 weeks; HFD 4w group: changed to a high-fat diet (HFD, D12492, Research Diet) after 12 weeks of normal diet and fed for 4 weeks; HFD 8w group: changed to HFD after 8 weeks of normal diet and fed for 8 weeks; HFD 16w group: fed HFD for 16 weeks; HFD16w + exercise 8w group: fed HFD for 16 weeks and started treadmill training from the 8th week. The mice started running on the treadmill at a speed of 8 m / min, increasing by 1 m / min every two days until reaching 12 m / min, for 1 hour per day or until exhaustion (defined as the mouse staying in an electric shock current of 1 mA for more than 10 seconds), five days a week for a total of 8 weeks. All mice were weighed once a week. All mice were injected via the tail vein with 100 μM HX-VP-2 (50 μL per 10 g body weight). One hour later, in vivo imaging was performed using an in vivo imaging system (PerkinElmer). After fluorescence imaging, they were euthanized, and liver tissues were collected. After weighing the liver tissues, H&E staining and Oil Red O staining were performed respectively.

[0101] 12. NAFLD cell model.

[0102] HepG2 cells were seeded in confocal dishes and incubated at 37 °C for 24 h. Then, the medium was replaced with fresh medium containing different concentrations of oleic acid (OA) and palmitic acid (PA) (in a 2:1 ratio, 0, 1.5, 3 mM), and the cells were incubated for another 24 h. Then, the cells were incubated with HX-VP-2 (10 μM) for 30 min and imaged.

[0103] 13. Fluorescence imaging of human liver tissues.

[0104] All human liver tissue samples involved in this study were from "discarded" donor livers for liver transplantation, including livers discarded due to severe hepatic steatosis and excessive donor liver volume requiring reduction surgery. This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of West China Hospital, Sichuan University (2024-694). Written informed consent was obtained from the family of each donor. Donor liver tissues of approximately 1×1×1 cm size were immersed in 50 μM probe solution for 1 h and then washed twice with PBS. Then, the liver tissues were cut into 1-mm thick slices and subjected to fluorescence imaging using a confocal microscope. Additionally, some liver tissue samples were reserved for H&E and Oil Red O staining.

[0105] (II) Experimental results

[0106] 1. Response to viscosity

[0107] To evaluate whether HX-VP-1 and HX-VP-2 are sensitive to viscosity, the inventors first detected their optical properties in PBS and glycerol. As Figure 15 and 16 shown, the absorption peaks of HX-VP-1 and HX-VP-2 in methanol were at 496 nm and 554 nm, respectively, and those in glycerol were at 505 nm and 566 nm, respectively. For methanol to glycerol, the emission wavelengths at 722 nm and 777 nm increased significantly. HX-VP-1 and HX-VP-2 showed stable fluorescence in both high-viscosity and low-viscosity environments ( Figure 17 and 18 ). Next, the inventors evaluated the sensitivity of HX-VP-1 and HX-VP-2 to viscosity. As Figure 10 Part A and Figure 19 shown, the fluorescence intensities of HX-VP-1 and HX-VP-2 at 722 nm and 777 nm increased with viscosity from 0.59 cP to 945 cP, respectively. In addition, there was a strong linear correlation between the fluorescence intensities of HX-VP-1 (R 2 = 0.990) and HX-VP-2 (R 2 = 0.986) and the viscosity change ( Figure 10 Part B in and Figure 20)。Finally, the inventors conducted selective experiments to eliminate potential interference from biomolecules. As Figure 10 part C in Figure 21 shows, after adding biomolecules, the fluorescence changes of HX-VP-1 and HX-VP-2 are very small, but the fluorescence is significantly enhanced at 722 nm and 777 nm, and changes only with the increase in viscosity. These results indicate that both HX-VP-1 and HX-VP-2 can be used as effective probes for detecting viscosity changes.

[0108] 2. Reaction with ONOO -

[0109] After determining the responsiveness to viscosity, we further investigated the recognition performance of HX-VP-1 and HX-VP-2 towards ONOO-. As Figure 22 and 23 show, the absorption spectra of HX-VP-1 and HX-VP-2 shift from 482 nm and 548 nm to 418 nm and 422 nm respectively, showing blue shifts of 64 nm and 126 nm. And the fluorescence of HX-VP-1 and HX-VP-2 at 620 nm and 657 nm is significantly enhanced. Fortunately, the fluorescence spectrum of HX-VP-2 during the reaction with ONOO - is still within the NIR region. The inventors then evaluated the influence of pH on the reaction of ONOO - . As Figure 24 and 25 show, after adding ONOO - and the pH value reaches 8, the fluorescence intensities of HX-VP-1 and HX-VP-2 increase significantly, which is beneficial for detecting the expression of ONOO - in mitochondria. Subsequently, fluorescence detection of HX-VP-1 and HX-VP-2 was carried out to detect different concentrations of ONOO - . As Figure 10 part D in Figure 26 shows, both HX-VP-1 and HX-VP-2 show weak fluorescence, and with the addition of different concentrations of ONOO - , the fluorescence intensities of both probes gradually increase. In addition, the fluorescence intensities of HX-VP-1 (R 2 = 0.981) and HX-VP-2 (R 2 = 0.983) have good linear correlations with the ONOO concentrations of 0.1 - 10 μM and 0.58 - 9.58 μM respectively, and the detection limits are 32 nM and 12.8 nM ( Figure 10 part E in Figure 27 ). In addition, the inventors also studied the selectivity of the two probes towards ONOO-. None of all substances, including reactive substances and metal ions, caused significant fluorescence changes, only ONOO​- can cause strong fluorescence enhancement ( Figure 10 in the F part and Figure 28 ). Both HX-VP-1 and HX-VP-2 can be used as effective probes for detecting viscosity and ONOO- changes. However, HX-VP-2 not only has a longer emission wavelength and a larger Stokes shift during viscosity monitoring, but also exhibits NIR fluorescence and a lower detection limit after reacting with ONOO - .

[0110] 3. Possible mechanism and theoretical calculation of the reaction of HX-VP-2 with viscosity and ONOO -

[0111] To verify that the fluorescence enhancement result is due to the release of the pyridine structure after the reaction of the phenylborate group with ONOO - , the inventors carried out HR-MS analysis. The mass spectrum of HX-VP-2 showed a dominant peak at 697.3046 m / z. After reacting with ONOO - , the main mass spectrum peak shifted to 481.1735 m / z, indicating the formation of the compound product. In addition, density functional theory (DFT) calculations were also carried out to further study the sensing mechanism of HX-VP-2 towards ONOO- and viscosity. The calculated oscillator strength of HX-VP-2 in the excited-state planar conformation (about 0°) was 1.2346. When the rotation of the single bond formed an orthogonal conformation (about 90°), the oscillator strength decreased significantly (0.0009), making electron transition challenging and sensitive to viscosity. The HOMO and LUMO of HX-VP-2 were calculated to be 2.06 eV, indicating a relatively strong intramolecular charge transfer (ICT) effect through its π-conjugated pathway compared to the product formed by the reaction of HX-VP-2 and ONOO-, with an energy gap of 2.69 eV. These theoretical calculation results were consistent with the dual-response behavior of HX-VP-2 towards viscosity and ONOO-

[0112] 4. Fluorescence imaging of viscosity and ONOO-

[0113] In view of the good test performance of HX-VP-2, the inventors further explored the imaging properties of living cells. First, the inventors used CCK-8 assay to evaluate the cytotoxicity of HX-VP-2 ( Figure 29 ). After HepG2 cells were cultured with different concentrations of HX-VP-2 (0 μM, 5 μM, 10 μM, 10 μM, 20 μM) for 24 hours, the cell viability exceeded 85%, indicating that the probe had low toxicity. Next, the inventors evaluated the ability of HX-VP-2 to detect changes in cell viscosity. HepG2 cells were incubated with 20 μM monensin (MON) or nystatin (NYS), both of which are known to increase intracellular viscosity. Compared with the control group, the inventors observed enhanced fluorescence after treatment​Figure 30 ), indicating that HX-VP-2 can effectively sense the changes in intracellular viscosity. Subsequently, the inventors evaluated the ability of the probe HX-VP-2 to respond to intracellular ONOO. HepG2 cells were incubated with different concentrations of SIN-1, a radical donor that is commonly used in many in vivo and in vitro studies to continuously generate peroxynitrite. As Figure 31 shown, cells exposed to SIN-1 showed a significant increase in fluorescence. It has been reported that a large amount of ROS can induce changes in intracellular viscosity. Therefore, the inventors explored the dual response of HX-VP-2 to viscosity and ONOO - treatment. As Figure 32 shown, HepG2 cells showed an increase in fluorescence in both the ONOO - and viscosity channels. In addition, new multi-punctate structures could also be observed in the ONOO - channel. This result prompted the inventors to further study the subcellular distribution of HX-VP-2 and the product. As Figure 11 shown in part A and Figure 33 shown, the red fluorescence emitted by HX-VP-2 almost completely overlapped with the fluorescence of Mito-Tracker in the green channel, with a Pearson correlation coefficient of 0.82. However, the green fluorescence of Bodipy493 / 503 ( Figure 11 in part B) hardly overlapped with HX-VP-2, indicating that HX-VP-2 has good mitochondrial targeting ability in living cells. After treatment with SIN-1, the red fluorescence of HX-VP-2 partially merged with the green fluorescence emitted by Bodipy493 / 503 ( Figure 11 in parts C and D). As Figure 11 shown in part E, the fluorescence of the product completely overlapped with Bodipy493 / 503, with a Pearson correlation coefficient as high as 0.88, indicating that the product has the targeting ability to LDs. These results indicate that HX-VP-2 exhibits good mitochondrial targeting, and the product between HX-VP-2 and ONOO− can migrate from mitochondria to LDs, thus improving the sensitivity ( Figure 11 in part F).

[0114] 5. Viscosity and ONOO - imaging of OA- and PA-induced NAFLD cell models.

[0115] It has been reported that oleic acid (OA) and palmitic acid (PA) can induce the metabolism of NAFLD in HepG2 cells after a large intake of lipids. Therefore, in order to verify the diagnostic effect of HX-VP-2 on the NAFLD cell model in vitro, the inventors co-incubated HepG2 cells with OA and PA at a ratio of 2:1 for 24 h at concentrations of 1.5 mM and 3 mM, respectively, to establish the model. AsFigure 12 As shown in parts A and B, ONOO - The fluorescence intensity in the channel increased with the increase in the concentrations of OA and PA, and simultaneously the number of high-intensity LDs increased significantly. In the viscosity channel, the fluorescence intensity of the model cells was significantly higher than that of the control group. The fluorescence intensity of the 3 mM group was lower than that of the 1.5 mM group. The inventors attributed this to the increase in intracellular ONOO - levels due to the increase in the concentrations of OA and PA, resulting in an increase in the consumption of HX-VP-2. To explore its possible mechanism of action, the inventors performed PCR analysis. As Figure 12 shown in part C, after adding OA and PA, the expressions of CD36 and Fabp4 related to fatty acid transport both increased. Similarly, the expressions of genes involved in de novo lipid synthesis, such as Fasn, were inhibited due to the increase in lipid uptake. These results confirmed the successful establishment of the NAFLD cell model and were consistent with the imaging results.

[0116] 6. Observe the viscosity and ONOO of the NAFLD mouse model - fluctuations

[0117] After successfully detecting the viscosity and ONOO of the OA- and PA-induced NAFLD cell model - , the inventors further explored its potential role in different stages of NAFLD in mice. The cytotoxicity evaluation of injecting HX-VP-2 via the tail vein into C57BL6 mice was the least toxic ( Figure 34 ). Subsequently, NAFLD mouse models at different stages were established by feeding a high-fat diet (HFD) for 4, 8, and 16 weeks. Another group started exercising from the 8th week while continuing the HFD for 16 weeks. As the feeding time increased, the body weight, liver weight, liver index, and biochemical markers (ALT, AST, TG) all gradually increased ( Figure 13 in parts D and E). Observed from the gross liver specimens, compared with the control group, as the HFD feeding time prolonged, the liver volume gradually increased and the color turned red ( Figure 35 ). At the same time, H&E and Oil Red O staining showed an increase in the number and size of lipid droplets, hepatocyte swelling, and inflammatory response ( Figure 13 in part A). Interestingly, these abnormalities were significantly alleviated by exercise. These results indicated that the model had been successfully established.

[0118] To study the diagnostic potential of the probe HX-VP-2 in NAFLD and its ability to detect early NAFLD, the inventors performed fluorescence imaging by injecting HX-VP-2 via the tail vein. As Figure 13As shown in Part A, the fluorescence signal was mainly concentrated in the liver, mainly due to the metabolic function of the liver. Compared with the control group, the fluorescence signals in the ONOO - and viscosity channels of the mice fed with HFD were significantly enhanced ( Figure 13 Parts B and C). Even in the mice fed with HFD for only 4 weeks, more obvious fluorescence signals were observed compared with the control group, and the fluorescence signals gradually increased with the prolongation of the feeding time. After endurance exercise, the liver fluorescence signal was significantly weakened. These fluorescence results were consistent with the pathological results, and the fluorescence change of HX-VP-2 could effectively reflect the severity and early stage of NAFLD.

[0119] 7. Human liver tissue imaging experiment

[0120] Considering that donor liver steatosis is an independent risk factor affecting the prognosis of liver transplant recipients, the inventors also explored the potential of using HX-VP-2 to evaluate the degree of donor liver steatosis. As shown in Table 2, the basic clinical characteristics of the liver donors were obtained. After the fresh liver was incubated with HX-VP-2 for 1 hour, confocal imaging was performed. As Figure 14 shown in Parts A and B, the tissue of Case 1 had red fluorescence in the viscosity channel, indicating that the viscosity of Case 1 was higher, and the increase in the ONOO - level caused the product to form target LDs. In addition, corresponding H&E and Oil Red O staining were also performed. As Figure 14 shown in Part A, Case 1 had obvious steatosis with a large amount of lipid accumulation, and the lipid accumulation in Case 2 was mild compared with Case 3. These results were consistent with the imaging results, indicating that HX-VP-2 had the potential to be used as a tool for evaluating steatosis of donor livers for transplantation.

[0121] Table 2

[0122]

[0123] Note: BMI: body mass index, TBIL: total bilirubin; ALT: alanine aminotransferase; AST: aspartate aminotransferase.

Claims

1. A dual-responsive near-infrared fluorescence probe, characterized in that, The chemical structural formula of the probe is shown in Formula (1) or Formula (2):

2. The dual-responsive near-infrared fluorescence probe according to claim 1, wherein The probe is used to simultaneously monitor viscosity, ONOO - and lipid droplets.

3. A dual-responsive near-infrared fluorescence probe according to claim 1, wherein, The structural formula of the probe is shown in Formula (2) and is used to detect the degree of hepatic steatosis.

4. A preparation method of a dual-responsive near-infrared fluorescent probe, characterized in that, The chemical structural formula of the probe is as described in Formula (1) of Claim 1, and the preparation method comprises the following steps: S1. React the compound of Formula (3) with the compound of Formula (4) to obtain the product of Formula (5); S2. React the product of Formula (5) with the compound of Formula (6) to obtain the probe shown in Formula (1); 5. The preparation method according to claim 4, characterized in that, In step S1, 3 mmol of the compound of Formula (3) and 3.2 mmol of the solution of the compound of Formula (4) are refluxed in dry toluene for 16 hours, then the mixture is cooled to room temperature, and the solid is filtered and washed with toluene and dried in vacuo to obtain the product of Formula (5).

6. The preparation method according to claim 4, wherein In step S2, 0.5 mmol of the product of Formula (5) and 0.5 mmol of the compound of Formula (6) are mixed with ethanol, then piperidine is added to the mixture, and the reaction mixture is refluxed under nitrogen for 3 hours; after cooling to room temperature, the solvent is removed, and the probe shown in Formula (1) is obtained by column chromatography purification.

7. A preparation method of a dual-responsive near-infrared fluorescent probe, characterized in that, The chemical structural formula of the probe is as described in Formula (2) of Claim 1, and the preparation method comprises the following steps: S1. React the compound of Formula (3) with the compound of Formula (7) to obtain the product of Formula (8); S2. React the product of Formula (8) with the compound of Formula (9) to obtain the probe shown in Formula (2); 8. The preparation method according to claim 7, characterized in that, In step S1, 3 mmol of the compound of Formula (3) and 3.2 mmol of the solution of the compound of Formula (7) are refluxed in dry toluene for 16 hours, then the mixture is cooled to room temperature, and the solid is filtered and washed with toluene and dried in vacuo to obtain the product of Formula (8).

9. The preparation method according to claim 7, characterized in that, In step S2, 0.5 mmol of the product of Formula (8) and 0.5 mmol of the compound of Formula (9) are mixed with ethanol, then piperidine is added to the mixture, and the reaction mixture is refluxed under nitrogen for 24 hours; after cooling to room temperature, the solvent is removed, and the probe shown in Formula (2) is obtained by column chromatography purification.

10. The preparation method according to claim 7, characterized in that In step S2, the addition amounts of each substance are as follows: 220 mg of the product of Formula (8), 178 g of the compound of Formula (9), 10 mL of ethanol, and 10 μL of piperidine.

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