Near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a near-infrared fluorescent probe as well as a preparation method and application thereof. The near-infrared fluorescent probe is a novel near-infrared fluorescent probe with large Stokes shift (262 nm), is used for detecting hydrogen peroxide in mitochondria, and combines benzonitro-heteroanthracene as a fluorophore and a borate group as a recognition unit. The borate group inhibits an intramolecular charge transfer (ICT) effect, and the near-infrared fluorescent probe does not emit fluorescence at first; however, borate groups are removed by hydrogen peroxide to expose hydroxyl groups and produce intense ICT-induced fluorescence. The near-infrared fluorescent probe disclosed by the invention can be used for specifically detecting the change of hydrogen peroxide in mitochondria.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a near-infrared fluorescent probe and a preparation method and application thereof. Background Art

[0002] Drug-induced liver injury (DILI) refers to the adverse reactions of hepatotoxic chemicals, biological products, Chinese patent medicines or other drugs used in the clinical treatment of diseases. One of the main causes of DILI is the irrational use of acetaminophen (APAP). APAP is metabolized by hepatocytes to N-acetyl-p-benzoquinoneimine (NAPQI, a highly active toxic compound), which can induce oxidative stress, leading to the production of high levels of reactive oxygen species. This cascade reaction leads to mitochondrial dysfunction and sterile inflammation, and the liver may eventually progress to irreversible liver fibrosis (HF) if it is exposed to damaging agents for a long time. Recent studies have shown that hydrogen peroxide is an important marker in the process of liver injury. Therefore, detecting the content of hydrogen peroxide in hepatocyte mitochondria can help to make early and accurate diagnosis of drug-induced liver injury, which has important guiding significance for clinical treatment.

[0003] In recent years, the rapid development of fluorescence analysis technology has helped realize the application of small molecule fluorescent probes in the biomedical field. Compared with traditional methods, fluorescence analysis offers several advantages, including easy operation, high sensitivity, high resolution, and the ability to detect in situ in real time. Although some fluorescent probes that can detect DILI by detecting intracellular hydrogen peroxide have been reported in the prior art, these probes cannot specifically target mitochondria and cannot reflect changes in hydrogen peroxide levels in mitochondria. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a near-infrared fluorescent probe and a preparation method and application thereof. The near-infrared fluorescent probe of the present invention can specifically detect changes in hydrogen peroxide in mitochondria.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a near-infrared fluorescent probe having a structure shown in Formula 1:

[0007]

[0008] The present invention also provides a method for preparing the near-infrared fluorescent probe described in the above technical solution, comprising the following steps:

[0009] The near-infrared fluorescent probe is obtained by mixing a compound having a structure shown in Formula 2, 4-bromomethylphenylboronic acid pinacol ester, a catalyst and a solvent, and then performing a substitution reaction;

[0010]

[0011] Preferably, the solvent includes one or more of acetone, acetonitrile and N,N-dimethylformamide.

[0012] Preferably, the catalyst includes one or more of potassium carbonate, triethylamine, p-dimethylaminopyridine and sodium carbonate.

[0013] Preferably, the molar ratio of the compound having the structure of formula 2 to 4-bromomethylphenylboronic acid pinacol ester is 1:(1.5-2.5).

[0014] Preferably, the molar ratio of the compound having the structure shown in Formula 2 to the catalyst is 1:(2-4).

[0015] Preferably, the mixing comprises mixing the compound having the structure shown in Formula 2, 4-bromomethylphenylboronic acid pinacol ester and a solvent, and then adding a catalyst.

[0016] Preferably, the temperature of the substitution reaction is 50-80°C and the time is 1.5-3h;

[0017] The substitution reaction is carried out under stirring and reflux conditions.

[0018] Preferably, after the substitution reaction is completed, the process further comprises concentration under reduced pressure and purification by silica gel column chromatography.

[0019] The present invention also provides the use of the near-infrared fluorescent probe described in the above technical solution or the near-infrared fluorescent probe prepared by the preparation method described in the above technical solution in the preparation of a preparation specifically reflecting the detection of hydrogen peroxide in mitochondria.

[0020] The present invention provides a near-infrared fluorescent probe having a structure shown in Formula 1:

[0021]

[0022] The near-infrared fluorescent probe of the present invention is a novel near-infrared fluorescent probe with a large Stokes shift (262 nm) for detecting hydrogen peroxide in mitochondria. The near-infrared fluorescent probe combines benzonitro-anthracene as a fluorescent group and a boronate group as a recognition unit. The boronate group inhibits the intramolecular charge transfer (ICT) effect, and the near-infrared fluorescent probe does not fluoresce initially; however, the boronate group is removed by hydrogen peroxide to expose the hydroxyl group and generate strong ICT-induced fluorescence (such as Figure 1As shown). Therefore, the hydrogen peroxide level can be quantitatively monitored by a unique "up" fluorescent signal. Using super-resolution imaging, the near-infrared fluorescent probe has good lipid solubility, and can effectively visualize the complex structure of mitochondria at the sub-organelle level by passively transporting across the cell membrane and directly aggregating on active mitochondria. Therefore, the near-infrared fluorescent probe is able to detect the fluctuation of hydrogen peroxide levels in L02 cell mitochondria induced by DILI, which is of great significance for the visual diagnosis of DILI and the screening of effective treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the working principle of the near-infrared fluorescent probe of the present invention;

[0024] Figure 2 The absorption spectra of the SML described in Example 1 in a PBS buffer solution with a pH of 7.4 in the presence and absence of hydrogen peroxide;

[0025] Figure 3 Spectral response of the SML described in Example 1 (the concentration in the test solution is 10 μM) to different concentrations of hydrogen peroxide;

[0026] Figure 4 is a curve showing the relationship between the fluorescence intensity of the SML described in Example 1 and the concentration of hydrogen peroxide;

[0027] Figure 5 is the fluorescence intensity of the SML described in Example 1 in PBS buffers of different pH values ​​in the presence or absence of hydrogen peroxide;

[0028] Figure 6 HPLC spectra of a 10 μM SML solution of Example 1, a mixed solution of a 10 μM SML of Example 1 and a 100 μM hydrogen peroxide solution, and a 10 μM dye SML-OH solution;

[0029] Figure 7 SML / H 2 O 2 High-resolution mass spectra (HRMS) of the mixture;

[0030] Figure 8 The reaction mechanism of SML and hydrogen peroxide described in Example 1;

[0031] Fig. 9 The toxicity test results of SML and SML-OH at different concentrations on L02 cells (n=3);

[0032] Fig.10Co-localization imaging of the SML (at a concentration of 10 μM) with mitochondria (wherein, green channel: λem=500-550 nm (λex=488 nm), red channel: λem=651-720 nm (λex=640 nm). n=3);

[0033] Fig.11 SIM images of the SML detecting endogenous hydrogen peroxide in L02 cells after different pretreatments (A is the SIM image, and B is the corresponding fluorescence intensity in image A);

[0034] Fig.12 SIM images and fluorescence intensities of APAP-induced L02 cells after incubation with the SML (A is the SIM image, and B is the corresponding fluorescence intensity in image A);

[0035] Fig.13 SIM images of APAP-induced L02 cells after incubation with the SML-OH (red channel: λem=651-720nm (λex=640nm));

[0036] Fig.14 Fluorescence imaging of drug-induced liver injury in mice (A), fluorescence intensity (B), in vitro fluorescence images of major organs (heart, liver, spleen, lung, and kidney) of mouse dissection (C), actual images of liver organs, and H&E staining of liver tissue sections (D) (scale bars: 200 μm (middle); 20 μm (bottom), n=3);

[0037] Fig.15 Figure 2 is the fluorescence tracking imaging of SML injection after 24 hours of treatment with various therapeutic drugs in the above 24-hour DILI mouse model (red channel: λ em =695-773nm(λ ex =649nm), n=3);

[0038] Fig.16 Actual images of liver organs and H&E staining of liver tissue sections in the NAC group, GSH group, SILY group, and Metadoxine group (D) (scale bars: 200 μm (middle); 20 μm (bottom), n=3);

[0039] Fig.17 Fluorescence imaging of liver fibrosis in mice of the control group and HF model (A), fluorescence intensity (B), ex vivo fluorescence images of major organs (heart, liver, spleen, lung, and kidney) of mouse dissection (C), and actual images of liver organs and H&E staining of liver tissue sections (D) (scale bars: 200 μm (middle); 20 μm (bottom), n=3);

[0040] Fig.18 The SML-OH described in Example 11 HNMR spectrum;

[0041] Fig.19 The SML described in Example 1 1 HNMR spectrum;

[0042] Fig. 20 The SML described in Example 1 13 C spectrum;

[0043] Fig.21 This is the HRMS spectrum of the SML described in Example 1. DETAILED DESCRIPTION

[0044] The present invention provides a near-infrared fluorescent probe (SML for short) having a structure shown in Formula 1:

[0045]

[0046] The present invention also provides a method for preparing the near-infrared fluorescent probe described in the above technical solution, comprising the following steps:

[0047] A compound having a structure shown in Formula 2 (abbreviated as SML-OH), 4-bromomethylphenylboronic acid pinacol ester, a catalyst and a solvent are mixed and then subjected to a substitution reaction to obtain the near-infrared fluorescent probe;

[0048]

[0049] In the present invention, unless otherwise specified, all preparations are commercially available products well known to those skilled in the art.

[0050] In the present invention, the compound having the structure shown in Formula 2 is preferably prepared by referring to the prior art (Analytical Chemistry, 2022, 94, 14, 5514-5520 or Chemical Communication, 2018, 54, 11590-11593).

[0051] In the present invention, the catalyst preferably includes one or more of potassium carbonate, triethylamine, p-dimethylaminopyridine and sodium carbonate, and more preferably potassium carbonate; when the catalyst is two or more of the above specific selections, the present invention has no special restrictions on the ratio of the above specific substances, and they can be mixed in any ratio. In an embodiment of the present invention, the catalyst can be potassium carbonate.

[0052] In the present invention, the solvent preferably includes one or more of acetone, acetonitrile and N,N-dimethylformamide, more preferably acetone; when the solvent is N,N-dimethylformamide, the N,N-dimethylformamide is preferably dry N,N-dimethylformamide; when the solvent is two or more of the above specific selections, the present invention has no special restrictions on the ratio of the above specific substances, and they can be mixed in any ratio. In an embodiment of the present invention, the solvent can be dry N,N-dimethylformamide.

[0053] In the present invention, the molar ratio of the compound having the structure of formula 2 and 4-bromomethylphenylboronic acid pinacol ester is preferably 1: (1.5-2.5), more preferably 1: 2. The molar ratio of the compound having the structure of formula 2 and the catalyst is preferably 1: (2-4), more preferably 1: 3. In the present invention, the amount ratio of the compound having the structure of formula 2 and the solvent is preferably 0.1mmol: (5-15)mL, more preferably 0.1mmol: 10mL. In an embodiment of the present invention, the molar ratio of the compound having the structure of formula 2 and 4-bromomethylphenylboronic acid pinacol ester can be 1: 2, the molar ratio of the compound having the structure of formula 2 and the catalyst can be 1: 3, and the amount ratio of the compound having the structure of formula 2 and the solvent can be 0.1mmol: 10mL.

[0054] In the present invention, the mixing preferably includes mixing the compound having the structure shown in Formula 2, 4-bromomethylphenylboronic acid pinacol ester and a solvent, and then adding a catalyst.

[0055] In the present invention, the temperature of the substitution reaction is preferably 50-80°C, more preferably 60°C; the time is preferably 1.5-3h, more preferably 2h; the substitution reaction is preferably carried out under stirring and reflux conditions; the temperature of the substitution reaction is preferably obtained by heating in an oil bath. In an embodiment of the present invention, the temperature of the substitution reaction can be 60°C and the time can be 2h.

[0056] After the substitution reaction is completed, the present invention also preferably includes vacuum concentration and silica gel column chromatography purification in sequence; the present invention has no special limitation on the vacuum concentration process, and it can be carried out using a process well known to those skilled in the art. In the present invention, the mobile phase used for the silica gel column chromatography purification is preferably dichloromethane.

[0057] The present invention also provides the use of the near-infrared fluorescent probe described in the above technical solution or the near-infrared fluorescent probe prepared by the preparation method described in the above technical solution in the preparation of a preparation for detecting hydrogen peroxide specifically in mitochondria. The present invention does not have any special limitation on the method of the application, and the method well known to those skilled in the art can be used.

[0058] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] Example 1

[0060] SML-OH was prepared by referring to the prior art (Analytical Chemistry, 2022, 94, 14, 5514-5520 or Chemical Communication, 2018, 54, 11590-11593), Fig.18 For the SML-OH 1 HNMR spectrum, 1 HNMR (400 MHz, DMSO-d 6 )δ7.75-7.66(m,1H),7.62(d,J=15.6Hz,H),7.07(d,J=8.3Hz,1H),6.70(s,1H),6.65(d,J=1.7Hz, 2H),6.60-6.49(m,2H),2.52-2.48(m,6H),2.44(t,J=5.8Hz,2H),1.75-1.66(m,2H),1.02(s,6H);

[0061] 39.6 mg (0.1 mmol) of the SML-OH and 59.2 mg (0.2 mmol) of 4-bromomethylphenylboronic acid pinacol ester were dissolved in 10 mL of acetone, and then 41.4 mg (0.3 mmol) of potassium carbonate was added. After heating and stirring in an oil bath at 60° C. for 2 hours, the mixture was concentrated under reduced pressure and purified by silica gel column chromatography using dichloromethane as the mobile phase to obtain 41.6 mg of a blue-purple solid product (SML. The yield was 68%). The characterization results of the SML ( Fig.19 For the SML 1 HNMR spectrum, Fig. 20 For the SML 13 C spectrum, Fig.21 is the HRMS spectrum of the SML): 1 H NMR (500 MHz, CDCl 3)δ7.84(d,J=8.0Hz,2H),7.58(d,J=15.6Hz,1H),7.43(d,J=8.0Hz,2H),6.98(d,J=8.4Hz,1H),6.71(s,1H),6.70(d,J=2.3Hz,1H),6.64(dd,J=8 .4,2.4Hz,1H),6.40(s,1H),6.35(d,J=15.7Hz,1H),5.12(s,2H),2.57( s,2H),2.53-2.40(m,6H),1.84-1.76(m,2H),1.35(s,12H),1.09(s,6H). 13 CNMR (101MHz, CDCl 3 )δ167.75,158.89,154.60,152.96,150.56,138.45,134.20,134.08,131.34, 127.28,126.94,125.83,125.48,124.38,122.41,120.54,114.82,113.57,11 2.77,110.69,109.26,100.85,82.87,73.65,69.18,52.41,42.00,38.18,31. 00,28.68,27.05,23.85,23.62,21.67,19.82,13.11.MS(ESI):calcd.m / zofC 39 H 42 BN 2 O 4 ([M+H] + )613.3238; found,613.3271.

[0062] Test Case

[0063] Spectral response to hydrogen peroxide:

[0064] Figure 2 is the absorption spectrum of the SML described in Example 1 in PBS buffer with a pH of 7.4 in the presence and absence of hydrogen peroxide, Figure 2 It can be seen that when hydrogen peroxide is not added, SML shows a relatively weak absorption peak at 510nm. After adding hydrogen peroxide, the absorption peak at 510nm increases significantly.

[0065] Figure 3The spectral response of the SML (with a concentration of 10 μM in the test solution) of Example 1 to different concentrations of hydrogen peroxide, wherein a is the fluorescence spectrum of the SML (with a concentration of 10 μM in the test solution) in a PBS buffer solution with a pH of 7.4 and different concentrations of hydrogen peroxide (0 to 100 μM); b is the change in fluorescence intensity (772 nm) of the SML (with a concentration of 10 μM in the test solution) in a PBS buffer solution with a pH of 7.4 and different concentrations of hydrogen peroxide (0 μM, 50 μM and 100 μM) over time; c is the fluorescence intensity of the SML in the presence of hydrogen peroxide and other analytes (1.Na2O3) with a concentration of 100 μM. + ; 2.K + ; 3.Ca 2+ ; 4.Mg 2+ ; 5.Zn 2+ ; 6.Fe 2+ ; 7.OH - ; 8.Cl - ; 9.SO 4 2- ; 10.CO 3 2- ; 11.NO; 12.NO 2 - ; 13.GSH; 14.ClO - ; 15. 1 O 2 ;16.·OH;17.Cys;18.Hcy;19.ONOO - ; 20.H 2 O 2 .λ ex =510 nm.) in the presence of fluorescence spectrum; Figure 3 As shown in A, with the increase of hydrogen peroxide concentration, the fluorescence at 772nm increased significantly, with the maximum enhancement reaching 41 times; Figure 3 As shown in B, the fluorescence intensity at 772 nm gradually increases with time and reaches a stable level within 60 s. Figure 3 From the C in FIG, it can be seen that only hydrogen peroxide can cause a significant change in fluorescence intensity, and the effects of other analytes on the probe are negligible; thus, the SML exhibits high selectivity and rapid responsiveness to hydrogen peroxide;

[0066] Figure 4 is the relationship curve between the fluorescence intensity of the SML and the hydrogen peroxide concentration described in Example 1, Figure 4It can be seen that the detection limit (3σ / k) of the SML for hydrogen peroxide is 17 nM; it can be seen that the SML can quantitatively detect hydrogen peroxide with high sensitivity. It is worth noting that, according to the calculation of the data (maximum emission wavelength minus maximum absorption wavelength), there is a significant Stokes shift at 262 nm, which is conducive to reducing self-quenching;

[0067] Figure 5 is the fluorescence intensity of the SML described in Example 1 in PBS buffers of different pH values ​​in the presence or absence of hydrogen peroxide; Figure 5 It can be seen that the SML exhibits excellent fluorescence response to hydrogen peroxide in the range of pH=6.0 to pH=10.0, which shows that the SML is suitable for biological imaging under physiological conditions;

[0068] HPLC test was performed on the SML solution of Example 1 with a concentration of 10 μM, a mixture of the SML of Example 1 with a concentration of 10 μM and hydrogen peroxide with a concentration of 100 μM, and a dye SML-OH solution with a concentration of 10 μM (test conditions: eluent: CH 3 CN / H 2 O (v / v, 7 / 3); flow rate: 1 mL / min; temperature: 25°C; detection wavelength: 510 nm), Figure 6 HPLC spectra of the SML solution of Example 1 with a concentration of 10 μM, a mixed solution including the SML of Example 1 with a concentration of 10 μM and hydrogen peroxide with a concentration of 100 μM, and a dye SML-OH solution with a concentration of 10 μM, wherein (a) is the HPLC spectra of the SML solution of Example 1 with a concentration of 10 μM, (b) is the HPLC spectra of the mixed solution including the SML of Example 1 with a concentration of 10 μM and hydrogen peroxide with a concentration of 100 μM, (c) is the HPLC spectra of the dye SML-OH solution with a concentration of 10 μM, Figure 6 It can be seen that the retention peaks of the SML solution and the dye SML-OH are at 12.08min and 5.73min, respectively. After adding hydrogen peroxide, the SML / H 2 O 2 The mixture obtained was consistent with the expected product SML-OH (such as Figure 7 As shown), it can be seen that the reaction mechanism of the SML and hydrogen peroxide is as follows Figure 8 As shown in Figure 2, the SML initially did not fluoresce due to the inhibition of the intramolecular charge transfer (ICT) effect by the boronate group. 2 O 2 The borate group leaves, resulting in a strong ICT effect and emitting fluorescence. 2 O 2Levels can be quantitatively monitored by a unique "on" fluorescent signal. ;

[0069] Live cell fluorescence imaging:

[0070] Cytotoxicity experiment: The cell technology kit-8 (CCK-8) method was used to evaluate the cytotoxicity of SML to L02 cells: the L02 cells were seeded in a 96-well plate, and different concentrations of SML solutions (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM) were added to the wells and incubated for 24 h. 10 μL of CCK-8 kit was added to each well and incubated at 37° C. for 4 hours. The absorbance was measured at 510 nm. Each group of experiments was repeated 3 times. Fig. 9 are the toxicity test results of different concentrations of the SML and SML-OH on L02 cells (n=3), wherein A is the toxicity test result of different concentrations of the SML on L02 cells, B is the toxicity test result of different concentrations of the SML-OH on L02 cells, Fig. 9 It can be seen that both SML and SML-OH showed low toxicity;

[0071] The targeting ability of the SML was examined using super-resolution structured illumination microscopy (SIM) and confocal imaging:

[0072] L02 cells were transferred to the confocal culture dish and cultured for 24 hours. Then 100 μM H 2 O 2 , and incubate for another 20 minutes. After washing three times with PBS, SML (10 μM) and Mito-Tracker Green (1 μM) were added and incubated for 30 minutes. Confocal and SIM images were taken using a Nikon A1-SIM super-resolution microscope; Fig.10 : is the co-localization imaging of the SML (at a concentration of 10 μM) on mitochondria (wherein, green channel: λem=500-550nm (λex=488nm), red channel: λem=651-720nm (λex=640nm). n=3), wherein A1 is a SIM image of L02 cells pretreated with hydrogen peroxide stained by SML, A2 is a SIM image of L02 cells pretreated with hydrogen peroxide stained by MTG, -A3 is an overlay of images A1 and A2, and A4 is a co-localization coefficient diagram of images A1 and A2; B1 is a confocal microscope image of L02 cells pretreated with hydrogen peroxide stained by SML, B2 is a confocal microscope image of L02 cells pretreated with hydrogen peroxide stained by MTG, B3 is an overlay of images B1 and B2, and B4 is a co-localization coefficient diagram of images B1 and B2, Fig.10It can be seen that there is a significant overlap between the SML and MTG channels, indicating that there is a high degree of colocalization between SML and the cell's selective fluorescent marker; at the same time, compared with the blurred image obtained by confocal imaging, the SIM image provides a clearer mitochondrial structure; in confocal mode, the Pearson colocalization coefficient of the SML and MTG is 0.91, while in SIM mode, it increases to 0.98. Therefore, SIM imaging can improve the accuracy of probe positioning within organelles;

[0073] Super-resolution fluorescence imaging was used to evaluate the ability of the SML to detect endogenous hydrogen peroxide in L02 cells: L02 cells were transferred to a confocal culture dish and cultured for 24 hours. Subsequently, the cells were divided into four groups. The first group was not pretreated, the second group was pretreated with lipopolysaccharide (LPS) (50μg / mL) for 12 hours, and then incubated with phorbol ester (PMA) (5μg / mL) for 1 hour. The third group was pretreated with LPS (100μg / mL) for 12 hours and then incubated with PMA (5μg / mL) for 1 hour. The fourth group was pretreated with N-acetylcysteine ​​(NAC) (1mM) for 2 hours, incubated with LPS (100μg / mL) for 12 hours, and incubated with PMA (5μg / mL) for 1 hour. After washing three times with PBS buffer, the four groups of cells were incubated with SML (10μM) for 30 minutes. SIM images were recorded using a Nikon A1-SIM super-resolution microscope; Fig.11 SIM images of the SML detecting endogenous hydrogen peroxide in L02 cells after different pretreatments (A is the SIM image, B is the corresponding fluorescence intensity in image A); Fig.11 It can be seen that when untreated L02 cells were incubated with the SML (10 μM) (corresponding to a1), no fluorescence signal was obtained in the red channel. When L02 cells were pretreated with different concentrations of LPS (5 μg / mL (corresponding to a2) and 100 μg / mL (corresponding to a3)) for 12 hours, then incubated with PMA (5 μg / mL) for 1 hour, and finally incubated with the SML (10 μM) for 30 minutes, a moderate red fluorescence signal was observed in the mitochondria. And with the increase of LPS concentration, the red fluorescence in mitochondria gradually increased; when L02 cells were pretreated with NAC (1 mM) for 2 hours, and then pretreated with LPS (100 μg / mL) for 12 hours and PMA (5 μg / mL) for 1 hour (corresponding to a4), the red fluorescence in mitochondria weakened. The main reason for the weakening of the red fluorescence is that NAC can remove most of the hydrogen peroxide from the cells and reduce the production of endogenous hydrogen peroxide; In summary, the SML can achieve effective visualization of endogenous hydrogen peroxide in L02 cells;

[0074] L02 cells were transferred to a confocal culture dish and divided into four groups after culturing for 24 hours. The first group was not pretreated, and the second group was pretreated with APAP (0.5mM) for 12 hours. The third group was pretreated with APAP (1mM) for 12 hours. The fourth group was pretreated with NAC (1mM) for 2 hours and then incubated with APAP (1mM) for 12 hours. After washing three times with PBS buffer, the four groups of cells were incubated with SML (10μM) for 30 minutes. The above experiment was repeated in HeLa cells to observe the changes in mitochondrial morphology, and SIM images were recorded using a Nikon A1-SIM super-resolution microscope; Fig.12 SIM images and fluorescence intensities of L02 cells induced by APAP after incubation with the SML (A is a SIM image, and B is the corresponding fluorescence intensity in image A), wherein a1 is a SIM image of L02 cells not induced by APAP after incubation with the SML for 30 minutes, a2 is a SIM image of L02 cells induced with 0.5 mM APAP for 12 hours after incubation with the SML for 30 minutes, a3 is a SIM image of L02 cells induced with 1 mM APAP for 12 hours after incubation with the SML for 30 minutes, and a4 is a SIM image of L02 cells first incubated with 1 mM NAC for 2 hours and then treated with 1 mM APAP for 12 hours after incubation with the SML for 30 minutes; Fig.12 It can be seen that a1 did not show significant fluorescence phenomenon, and L02 cells induced by APAP showed obvious fluorescence signals after incubation with the SML, and the fluorescence intensity of a3 was stronger. This observation confirmed that APAP can induce the production of excessive hydrogen peroxide in cells. Since NAC is a liver protective agent, it can reduce APAP-induced liver damage and oxidative stress. The fluorescence signal of the experimental group pretreated with NAC was significantly weakened;

[0075] Fig.13 SIM images of L02 cells induced by APAP after incubation with the SML-OH (red channel: λem=651-720nm(λex=640nm)), wherein a1 is the SIM imaging of L02 cells not induced by APAP after incubation with the SML-OH with a concentration of 10μM for 30min, a2 is the SIM imaging of L02 cells induced by APAP with a concentration of 0.5mM and incubated with the SML-OH with a concentration of 10μM for 30min, a3 is the SIM imaging of L02 cells induced by APAP with a concentration of 1mM and incubated with the SML-OH with a concentration of 10μM for 30min, a4 is the SIM imaging of L02 cells pretreated with NAC with a concentration of 1mM for 2 hours and induced with APAP with a concentration of 1mM for 12 hours and incubated with the SML-OH with a concentration of 10μM for 30min, Fig.13It can be seen that with the increase of APAP concentration, the mitochondrial morphology changes from slender rods to shorter rods, and the final product is a spherical structure. It can be seen that the SML can be used for real-time imaging of mitochondria in drug-induced hepatotoxicity; and the SML shows excellent sensitivity and selectivity in detecting hydrogen peroxide activity in cell models, which has important potential in drug toxicity assessment and further diagnosis of DILI;

[0076] In vivo imaging in mice:

[0077] The ability of SML to monitor fluctuations in endogenous hydrogen peroxide levels in the body in the DILI mouse model: C57BL / 6 mice were randomly divided into three groups. The first group was the control group and was intraperitoneally injected with 200 μL of normal saline. The second and third groups established DILI models: mice were intraperitoneally injected with acetaminophen (APAP) (200 μL, 500 mg / kg), dissolved in normal saline, and administered for 12 (denoted as the 12-hour model group) or 24 hours (denoted as the 24-hour model group). Subsequently, all mice were intravenously injected with 100 μL of normal saline (pH = 7.4, containing xx% DMSO and 10 μM probe SML), and imaging was performed using the IVIS Lumina LT in vivo imaging system at 10, 20, 30 and 60 minutes; among them Fig.14 Fluorescence imaging of drug-induced liver injury in mice (A), fluorescence intensity (B), in vitro fluorescence images of major organs (heart, liver, spleen, lung, and kidney) dissected from mice (C), actual pictures of liver organs, and H&E staining of liver tissue sections (D) (Scale bars: 200 μm (middle); 20 μm (bottom), n=3), wherein Saline24h in A is the fluorescence imaging at 10, 20, 30, and 60 min after the injection of probe SML 24 hours after intraperitoneal injection of normal saline in mice, APAP12h in A is the fluorescence imaging at 10, 20, 30, and 60 min after the injection of probe SML 12 hours after intraperitoneal injection of 500 mg / kg APAP in mice, and APAP24h in A is the fluorescence imaging at 10, 20, 30, and 60 min after the injection of probe SML 24 hours after intraperitoneal injection of 500 mg / kg APAP in mice; Fig.14 As shown in A and B, the control group of mice did not show fluorescence 24 hours after the injection of saline. However, the fluorescence signal of mice treated with APAP was significantly enhanced at 12 hours, and the fluorescence signal intensity at 24 hours was further enhanced, which was significantly different from that at 12 hours. Fig.14As shown in C, the organs of the mice in the control group did not show fluorescence, while the liver tissue of the DILI mouse model (DILI mouse model treated with APAP) showed significant fluorescence; the fluorescence intensity of the 24-hour model group was significantly stronger than that of the 12-hour model group. It can be seen that the SML can effectively detect the DILI mouse model produced by in situ hydrogen peroxide; Fig.14 As shown in D, the fresh liver of the control group mice was dark in color, with clear structure, dense and smooth membrane, and clear outline of liver lobes. In contrast, the fresh liver of the DILI mouse model showed signs of collapse and lighter color after 12 hours, with unclear lobes, loose capsules, and thin and fragile tissue edges; the liver lesions in the 24-hour model group were more obvious than those in the 12-hour model group, with white spots on the surface, loose texture, flat collapsed areas, pale color, and thin edges; H&E staining was then used to stain the liver pathological sections, and the liver cells in the control group were neatly arranged, with no pathological changes. In contrast, liver tissue treated with APAP showed signs of hepatocyte necrosis, nucleolar shrinkage, cellular inflammatory infiltration, and slight bleeding (arrows ae). It can be seen that the results of the above liver tissue observations confirmed the successful establishment of the DILI model;

[0078] APAP-induced DILI mice were randomly divided into 4 groups. The first group was intraperitoneally injected with 300 mg / kg NAC (100 μL), the second group was intraperitoneally injected with 300 mg / kg GSH (100 μL), the third group was orally administered with 300 mg / kg SILY (100 μL), and the fourth group was orally administered with 300 mg / kg Metadoxine (100 μL) for 24 hours to alleviate APAP-induced liver injury (respectively recorded as NAC group, GSH group, SILY group and Metadoxine group), and then 100 μL of 10 μM SML was intravenously injected, and imaging was performed 60 minutes later; Fig.15 Figure 2 is the fluorescence tracking imaging of SML injection after 24 hours of treatment with various therapeutic drugs in the above 24-hour DILI mouse model (red channel: λ em =695-773nm(λ ex =649nm), n=3), wherein A is the fluorescence imaging diagram of NAC group, GSH group, SILY group and Metadoxine group, B is the fluorescence intensity diagram of NAC group, GSH group, SILY group and Metadoxine group, C is the in vitro fluorescence image of the main organs (heart, liver, spleen, lung and kidney) of mice dissected in NAC group, GSH group, SILY group and Metadoxine group; Fig.15 It can be seen that the fluorescence intensity decreased in mice treated with therapeutic drugs. Notably, the NAC, GSH and SILY treatment groups showed a significant decrease in fluorescence intensity. Fig.16 Figure 2 shows the actual pictures of liver organs and H&E staining of liver tissue sections in the NAC group, GSH group, SILY group and Metadoxine group (D) (scale bar: 200 μm (middle); 20 μm (bottom), n=3), where the arrow corresponding to a indicates hepatocyte swelling and disordered arrangement, the arrow corresponding to b indicates red blood cell extravasation, the arrow corresponding to c indicates inflammatory cell infiltration, the arrow corresponding to d indicates hepatocyte nuclear condensation and dissolution, and the arrow corresponding to e indicates the concentrated area of ​​lesions; Fig.16 It can be seen that the macroscopic appearance of the liver and the histological examination of the sections confirmed the efficacy of the drug in alleviating liver damage.

[0079] To further evaluate the diagnostic ability of SML in various liver injury models, carbon tetrachloride (CCl 4 )-induced HF model, and the mice were divided into two groups: the HF model was injected with 20% carbon tetrachloride olive oil solution (5mL / kg); the control group was injected with the same volume of olive oil; the light-dark cycle was maintained for 12 hours, and food and water were available ad libitum. All mice were injected twice a week for a total of 8 weeks, and 100μL of SML at a concentration of 10μM was injected intravenously. After 60 minutes, images were collected using the IVIS Lumina LT in vivo imaging system; pathological examination of liver tissue sections showed that compared with the control group, the mice in the HF model group had pathological changes such as fatty degeneration of hepatocytes, degeneration and necrosis of hepatocytes, infiltration of inflammatory cells, and congestion and dilation of hepatic sinusoids. The results showed that the HF model was successfully established. Subsequently, the two groups of mice were intravenously injected with the SML (100μL, 10μM) and imaged 60 minutes later. Fig.17 Fluorescence imaging of liver fibrosis in control group and HF model mice (A), fluorescence intensity (B), in vitro fluorescence images of major organs (heart, liver, spleen, lung, kidney) of mouse dissection (C), actual images of liver organs and H&E staining of liver tissue sections (D) (scale bars: 200 μm (middle); 20 μm (bottom), n=3), by Fig.17 It can be seen that the peritoneal cavity of mice treated with carbon tetrachloride showed high-intensity fluorescence signals, while the control mice showed no fluorescence signals. After euthanasia, the two groups were dissected to obtain their various organs. The liver of mice in the HF model showed obvious fluorescence, while no fluorescence was detected in other organs. No organ in the control group showed any fluorescence. Therefore, the SML can be used as a powerful tool for in vivo visualization diagnosis and treatment evaluation.

[0080] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe, characterized in that: It has the structure shown in formula 1:

2. The method for preparing the near-infrared fluorescent probe according to claim 1, characterized in that: The following steps are involved: The near-infrared fluorescent probe is obtained by mixing a compound having a structure shown in Formula 2, 4-bromomethylphenylboronic acid pinacol ester, a catalyst and a solvent, and then performing a substitution reaction; 3. The preparation method according to claim 2, characterized in that: The solvent includes one or more of acetone, acetonitrile and N,N-dimethylformamide.

4. The preparation method according to claim 2, characterized in that: The catalyst comprises one or more of potassium carbonate, triethylamine, p-dimethylaminopyridine and sodium carbonate.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the compound having the structure of formula 2 to 4-bromomethylphenylboronic acid pinacol ester is 1:(1.5-2.5).

6. The preparation method according to any one of claims 2 to 5, characterized in that: The molar ratio of the compound having the structure shown in Formula 2 to the catalyst is 1:(2-4).

7. The preparation method according to claim 6, characterized in that: The mixing comprises mixing the compound having the structure shown in Formula 2, 4-bromomethylphenylboronic acid pinacol ester and a solvent, and then adding a catalyst.

8. The preparation method according to claim 7, characterized in that: The temperature of the substitution reaction is 50-80°C and the time is 1.5-3h; The substitution reaction is carried out under stirring and reflux conditions.

9. The preparation method according to claim 8, characterized in that: After the substitution reaction is completed, the steps include sequentially performing reduced pressure concentration and silica gel column chromatography purification.

10. Use of the near-infrared fluorescent probe according to claim 1 or the near-infrared fluorescent probe prepared by the preparation method according to any one of claims 2 to 9 in preparing a preparation for detecting hydrogen peroxide specifically in mitochondria.

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