Hydrazino near-infrared fluorescent probe and application thereof in detection of peroxynitrite

Abstract

The invention provides a hydrazino near-infrared fluorescent probe and application of the hydrazino near-infrared fluorescent probe in detection of peroxynitrite, and belongs to the field of biological detection. The emission wavelength of the hydrazino near-infrared fluorescent probe developed by the invention is 730nm, and the hydrazino near-infrared fluorescent probe has high selectivity, high sensitivity (the detection limit is as low as 91nM) and quick response ability to ONOO-, and has low cytotoxicity and excellent in-vivo imaging ability, so that visual observation of ONOO-in a living body CIRI model mouse body is successfully realized; the method has great potential in the aspect of real-time tracking of ONOO-related pathological states.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a hydrazine-based near-infrared fluorescent probe and its application in detecting ONOO - Application in. Background Art

[0002] Peroxynitrite (ONOO - ) is a highly reactive nitrogen species and a potent oxidant that can cause extensive damage to biomacromolecules including DNA, lipids, and proteins, further inducing apoptosis and cell death in biological systems. ONOO is produced in many physiological metabolic processes. - , especially cerebral ischemia-reperfusion injury (CIRI). In CIRI, neurons are subjected to severe oxidative stress, which leads to ONOO - Overproduction of ONOO disrupts the redox balance in the brain and damages neural signal transduction. These disturbances often manifest as severe neurological damage and disorders unique to stroke patients, which not only affects patients but also brings huge socioeconomic burdens to families and society. - To prevent serious consequences of cell damage, it is imperative to develop a method for real-time detection of ONOO during the development of CIRI. - methods to improve the understanding of the pathophysiological mechanisms underlying CIRI.

[0003] Fluorescent probes have the ability of non-invasive imaging, and a variety of fluorescent probes have been developed to monitor ONOO in biological tissues. – However, the wavelengths of most existing probes are in the visible light range, and short-wavelength light has limited penetration into tissues, hindering effective in vivo fluorescence imaging. To address this challenge, there is an urgent need for fluorescent probes that can detect ONOO - Near-infrared (NIR) fluorescent probes. Near-infrared emission (650nm-900nm) has multiple advantages, including deeper tissue penetration, reduced background fluorescence, improved signal-to-noise ratio, and enhanced image clarity. Among the molecular structures of near-infrared fluorescent probes, rhodamine derivatives are particularly noteworthy due to their excellent optical properties, including high quantum yield, excellent photostability, and a wide spectrum. The fluorescence emission of rhodamine derivatives can be precisely tuned to the near-infrared region by introducing various substituents. In addition, functionalization with different recognition groups has been used to synthesize near-infrared probes that respond to different target molecules.

[0004] Hydrazine hydrate has been shown to selectively recognize ONOO - , and several rhodamine-based fluorescent probes using hydrazine hydrate have been reported (see Figure 1For example, Wu et al. developed a cell-permeable fluorescent probe, Probe 2, to detect ONOO in bone marrow neutrophils of mice infected with the PAO1 strain of Pseudomonas aeruginosa. - Mao et al. designed a two-photon excitation probe, Probe 4, which is suitable for observing ONOO in various biological systems. - Xia et al. synthesized Probe 5, which can selectively detect ONOO in lysosomes. - , and monitor changes in lysosomal pH. Although there are many - A near-infrared probe for imaging, but specifically for real-time tracking of ONOO in cerebral ischemia-reperfusion injury (CIRI) - However, there are few probes for ischemic stroke. Existing studies have shown that after reperfusion of ischemic tissue, ROS levels increase significantly, leading to increased oxidative stress and ONOOˉ production. Therefore, it is necessary to perform real-time imaging of ONOOˉ in order to diagnose ischemia-reperfusion injury in a timely manner. However, the shortcomings of existing technologies have hindered the diagnosis of stroke and the progress of related drug research. Summary of the invention

[0005] In view of this, the present invention provides a novel near-infrared fluorescent probe with a longer emission wavelength and stronger tissue penetration, which can effectively detect ONOO in the CIRI mouse model. - The level changes of ONOO in CIRI-related pathologies are monitored in real time. - Level provides a powerful tool.

[0006] In the first aspect, the present invention provides a hydrazine-based near-infrared fluorescent probe (denoted as TJO), the structure of which is shown below:

[0007]

[0008] The probe TJO uses hydrazine hydrate as the recognition group. By introducing an asymmetric electronic structure and an extended conjugated system into the rhodamine structure, the probe fluorescence emission wavelength is significantly red-shifted to 730nm, and the Stokes shift is increased by 167nm, making it suitable for ONOO in biological systems. - Specific imaging.

[0009] In a second aspect, the present invention provides an application of a probe TJO, comprising at least the following:

[0010] 1) In vitro qualitative or quantitative detection of ONOO - ;

[0011] 2) ONOO in the body - Fluorescence imaging, and the fluorescence intensity reflects ONOO- Level fluctuations;

[0012] 3) Preparation and detection of ONOO - products.

[0013] In the above applications, organisms include cells and animals. For example, in one embodiment of the present invention, the cells are normal SH-SY5Y cells; in another embodiment, the cells are specifically a cerebral ischemia-reperfusion injury cell model established with SH-SY5Y cells. The above animals include animal tissues and living animals. For example, in a specific embodiment of the present invention, the probe TJO was used to perform fluorescence imaging of exogenous or endogenous ONOOˉ in normal mice and cerebral ischemia-reperfusion injury model mice. The experimental results show that the probe TJO can accurately reflect the regulated ONOOˉ levels in cells and animals, highlighting its potential as an important tool for studying oxidative stress-related pathologies.

[0014] In a third aspect, the present invention provides a method for preparing a probe TJO, comprising the following steps: reacting compound 1, hydrazine and BOP reagent (Carter condensation agent) in dichloromethane to obtain a crude product of the probe TJO; wherein the structural formula of compound 1 is as follows:

[0015]

[0016] In the above preparation method, the reaction temperature can be room temperature, i.e. 10 to 40°C.

[0017] Preferably, in the above preparation method, the molar ratio of compound 1, hydrazine and BOP reagent is 1:(180-220):(1.5-3).

[0018] Preferably, in the above preparation method, the following steps are also included:

[0019] The product was purified by silica gel column chromatography, wherein the eluent was CH2Cl2:MeOH=100:1-10:1.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The fluorescent probe TJO developed in the present invention has a large Stokes shift and an emission wavelength of 730 nm, which is significantly higher than the near-infrared fluorescent probe based on rhodamine structure disclosed in the prior art, so it is suitable for biological imaging.

[0022] (2) The fluorescent probe TJO is stable under physiological conditions and has excellent optical stability. - It has high sensitivity (detection limit of 91nM) and high selectivity, and can identify ONOO in complex environments (such as biological environments).- ; At the same time, it has a fast response capability to ONOOˉ, so it can monitor ONOO in real time - Changes in levels.

[0023] (3) The present invention successfully monitored ONOO in cells and CIRI models (including cells and living mice) using the probe TJO. - The level of change makes up for the shortcomings of existing technologies and provides an effective tool for the diagnosis of stroke and the research of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 The fluorescent probe TJO of the present invention and other detection methods of ONOO - Structural comparison of rhodamine fluorescent probes;

[0026] Figure 2 The fluorescent probe TJO of the present invention detects ONOO - Schematic diagram of the mechanism;

[0027] Figure 3 The synthetic route of the fluorescent probe TJO in the embodiment of the present invention;

[0028] Figure 4 is the fluorescent probe TJO in the embodiment of the present invention. 1 HNMR spectrum;

[0029] Figure 5 is the fluorescent probe TJO in the embodiment of the present invention. 13 CNMR spectrum;

[0030] Figure 6 HR-MS (ESI) spectrum of probe TJO in the embodiment of the present invention;

[0031] Figure 7 The probes TJO and ONOO in the embodiment of the present invention are - Related optical experimental results, where a is TJO and ONOO - Absorption spectra of TJO and ONOO - The emission spectra of TJO and other substances are shown in Figure 5, c is the selectivity experimental results of TJO and other substances, d is the selectivity experimental results of TJO and ONOO - The anti-interference experimental results in the presence of other substances, e is TJO and ONOO -The titration experiment results are shown in Figure 2, where f is the ratio of TJO and ONOO - Linear relationship at 730nm;

[0032] Figure 8 Graph showing the stability test results of the probe TJO in an embodiment of the present invention, wherein a is the fluorescence intensity of 10 μM probe TJO in PBS buffers of different pH values ​​containing 10% DMSO, and b is the optical stability test result graph of the probe TJO under constant excitation at 620 nm;

[0033] Fig. 9 The DFT calculation results (a) and the molecular orbital electrostatic potential map (b) of the probe TJO (left) and its product (right) in the embodiment of the present invention;

[0034] Fig.10 The results of HPLC-MS analysis (50% ACN and 50% H2O (containing 0.1% formic acid)) of the probe TJO and its product in the embodiment of the present invention, wherein a is the HPLC-MS analysis of 1 mM TJO, and b is the HPLC-MS analysis of 1 mM TJO and 2 mM ONOO - HPLC-MS analysis of the reaction products;

[0035] Fig.11 The probe TJO in the present invention is used to image ONOOˉ in SH-SY5Y cells pretreated with SIN-1 (a) or 2-ME (b);

[0036] Fig.12 The probe TJO in the present invention is used to image ONOOˉ in SH-SY5Y cells pretreated with UA (a) or Tiron (b);

[0037] Fig.13 This is a graph showing the CCK-8 test results of SH-SY5Y cells after OGD / R treatment in an embodiment of the present invention (the cell viability of the control group is defined as 1);

[0038] Fig.14 The imaging of ONOOˉ in SY5Y cells during OGD / R by the probe TJO in the embodiment of the present invention;

[0039] Fig.15 The imaging of ONOOˉ in OGD / R cells treated with EDA by the probe TJO in an embodiment of the present invention;

[0040] Fig.16 The imaging of ONOOˉ in OGD / R cells treated with SOD by the probe TJO in the embodiment of the present invention;

[0041] Fig.17The imaging of exogenous ONOOˉ in living mice by the probe TJO in the embodiment of the present invention;

[0042] Fig.18 This is the imaging of ONOOˉ in the cerebral ischemia-reperfusion mouse model by the probe TJO in the embodiment of the present invention. DETAILED DESCRIPTION

[0043] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs; the terms "including" and "having" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0045] Although the prior art has reported a number of near-infrared fluorescent probes developed based on rhodamine structure and hydrazine hydrate, the emission wavelength is still relatively short (e.g. Figure 1 As shown), ONOO can generally only be achieved in cells - imaging, and lacks the ability to track ONOO in real time in CIRI. - The pathological process of CIRI is very complicated. We used a highly specific and selective method to detect endogenous ONOO produced in the cerebral ischemia-reperfusion injury model. - It is crucial to understand its complex pathological processes.

[0046] In order to solve the above technical problems, the present invention develops a novel near-infrared fluorescent probe based on the rhodamine structure, which is sensitive to ONOO at 730nm. - The detection is highly selective and therefore very suitable for in vivo imaging.

[0047] Since the HOMO and LUMO of traditional rhodamine dyes have unique symmetry, their vibration orbits will decrease, resulting in minimal Stokes shift (<30nm) and reduced signal-to-noise ratio. The present invention introduces an asymmetric electronic structure into the rhodamine dye, and the excited electrons will transition to a lower energy vibration state, thereby increasing the Stokes shift of the dye; specifically, the present invention replaces nitrogen atoms with sulfur atoms as electron energy supply groups in rhodamine derivatives, while expanding the conjugated system, significantly red-shifting the fluorescence emission wavelength to 730nm, and increasing the Stokes shift by 167nm. In the probe TJO provided by the present invention, hydrazine hydrate serves as a recognition group, and the oxidation of hydrazine will rapidly oxidize and hydrolyze the spirolactam ring of rhodamine into acid, thereby opening the rhodamine molecule and releasing fluorescence (such as Figure 2 shown).

[0048] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0049] The chemicals and solvents used in the following examples were of analytical grade. HPLC-MS analysis was performed on a Waters LCMS system. NMR spectra (1H and 13C) were recorded on a Bruker AV-400 spectrometer with tetramethylsilane (TMS) as an internal reference. UV-visible absorption spectra were obtained by a Hitachi UV-3010 spectrophotometer, and fluorescence emission spectra were collected by a Hitachi F-2500 spectrofluorimeter. The absorption and fluorescence test results were recorded at 37°C in 10 mM phosphate buffered saline (PBS) containing 10% DMSO. Cell fluorescence imaging was performed using a Leica confocal microscope, blue channel: λex = 405 nm, λem = 430-500 nm; red channel: λex = 590 nm, λem = 700-780 nm, scale bar: 10 μm. Data were analyzed using one-way analysis of variance, and the mean values ​​of the experimental groups were compared with those of the control group (n = 3; ***, p < 0.001).

[0050] The 2-month-old male C57BL / 6J mice used in the following examples were purchased from Wuhan Mouse Bailey Biotechnology Co., Ltd., and all animal experiments were performed in accordance with the institutional animal use and care regulations approved by the Ethics Committee of Jianghan University.

[0051] If no specific techniques or conditions are specified in the following examples, the procedures were carried out according to the techniques or conditions described in the literature in the art or according to the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0052] Example 1

[0053] This example provides a method for synthesizing a probe TJO, such as Figure 3 As shown, the following steps are included:

[0054] A mixture of compound 1 (NovaFluor Yellow 730Dye, 0.19 mmol), hydrazine (38.4 mmol) and BOP reagent (0.38 mmol) in 100 ml of dichloromethane was stirred at room temperature overnight. Then, the reaction mixture was washed with water (3×100 mL) and the organic layer was concentrated under reduced pressure. The preliminary product was purified by silica gel column chromatography (eluent: CH2Cl2:MeOH=100:1-10:1) to obtain probe TJO (white solid, 360 mg, yield 79%).

[0055] The obtained probe TJO was tested as follows:

[0056] Figure 4 The probe TJO in CDCl3 1 HNMR spectrum. Specifically, 1 HNMR (400MHz, Chloroform-d) δ7.95-7.92(1H), 7.51-7.42(m,2H), 7.13-7.10(1H), 7.11-7.00(m,2H), 6.88-6.70(m,3H), 6.43(t,J=6 .6Hz,2H), 6.36(s,1H), 6.31(d,J=8.6Hz,1H), 3.92(q,J=6.9Hz,2H), 3.34(q,J=7.2Hz,4H), 1.51-1.39(m,3H), 1.16(t,J=7.2Hz,6H).

[0057] Figure 5 The probe TJO in CDCl3 13 CNMR spectrum. Specifically, 13 CNMR(101MHz,Chloroform-d)δ166.32,153.46,152.49,150.91,146.35,143.81,132.83,129.78,128.57,128.0 6,127.38,125.09,123.85,123.22,122.69,118.89,115.23,112.44,103.33,65.44,42.10,31.66,29.71,12.58.

[0058] Figure 6 is the HR-MS (ESI) spectrum of probe TJO, and the calculation is: C 32 H 31 N4O2S[M+H] +,535.2168;Found value:535.2160.

[0059] Example 2

[0060] This example demonstrates the effect of probe TJO on ONOO by optical experiments. - The following experiments were all carried out in PBS (pH 7.4) buffer containing 10% DMSO, with an excitation wavelength of 620 nm and a slit of 10 nm / 10 nm.

[0061] (1) Absorption spectrum detection.

[0062] Probe TJO (10 μM) and ONOO - The absorption spectrum of (100μM) is as follows Figure 7 As shown in a, due to the closed lactam ring structure of rhodamine, the probe TJO has no absorption peak; however, after adding 100 μM ONOO - Afterwards, the initially colorless TJO solution turned blue, and a new absorption peak appeared at 580 nm, which indicated that the rhodamine lactam ring was opened.

[0063] (2) Fluorescence spectrum detection.

[0064] Probe TJO (10 μM) and ONOO - The emission spectrum of (100 μM) is as follows Figure 7 As shown in b, ONOO was added to the TJO solution. - After that, the fluorescence was significantly enhanced, which indicated that the probe TJO - The detection has excellent fluorescence response.

[0065] In order to evaluate the response time, a fluorescence time-dependency experiment was also performed in this example. The results showed that when 100 μM ONOO was added to 10 μM TJO solution, - After that, the fluorescence of the probe TJO reached saturation within 2 minutes, indicating that the probe has the ability to monitor ONOO in real time. - ability.

[0066] (3) Specific detection

[0067] In this case, common metal ions, anions, ROS, RNS and other substances were selected for selective experiments to determine the effect of probe TJO on ONOO in complex environments. - The selectivity of the probe TJO (10 μM) and 100 μM of other substances is shown in the following table. Figure 7 c, where 1-20 are Fe 2+ ,Fe 3+ ,Ca 2+ ,Na+ ,Zn 2+ ,Cu 2+ ,CO3 2- ,NO3 2- ,SO4 2- ,HCO3 - ,GSH,Cys,Hcy,Na2S2O5,NaSH,H2O2,O2 - ,HNO and ClO - and ONOO - The results showed that only ONOO - It can react with TJO to produce obvious fluorescence changes.

[0068] Furthermore, the probe TJO (10 μM) and ONOO - The results of the anti-interference experiment in the presence of other substances (100 μM) are as follows Figure 7 d, where 1-19 are Fe 2+ ,Fe 3+ ,Ca 2+ ,Na + ,Zn 2+ ,Cu 2+ ,CO3 2- ,NO3 2- ,SO4 2- ,HCO3 - ,GSH,Cys,Hcy,Na2S2O5,NaSH,H2O2,O2 - ,HNO and ClO - The results show that the probe has a strong affinity for ONOO in the presence of other interfering substances. - The specificity is not affected.

[0069] The results of selectivity experiments and anti-interference experiments show that the probe TJO can identify ONOO in complex environments - , which is expected to accurately identify ONOO in biological environments - .

[0070] (4) Quantitative feasibility analysis.

[0071] In this case, the probe TJO was detected by reacting with different concentrations of ONOO - The fluorescence intensity after the reaction was evaluated by the probe TJO to quantify ONOO - The results are as follows Figure 7 e and 7f, with ONOO - As the concentration increases, the emission intensity at 730 nm also increases, and the fluorescence intensity is similar to that of ONOO -The concentrations of ONOO and TJO were strongly linearly correlated. The standard formula LOD = 3σ / S (σ refers to the standard deviation of the blank sample signal, S refers to the slope of the calibration curve) was used to calculate the probe TJO to ONOO - The detection limit was 91 nM.

[0072] (5) Optical stability testing.

[0073] In view of the pH variation in the human body and the significant pH sensitivity conferred by the unique rhodamine lactam ring structure, it is necessary to confirm the stability of the probe TJO in different pH ranges and its interaction with ONOO - reaction stability.

[0074] The results are as follows Figure 8 As shown in a, in the absence of ONOO - In the case of TJO, the emission increases significantly below pH 7.0, indicating that TJO is sensitive to acidic conditions as the helical structure opens; however, when the pH exceeds 7, the fluorescence of the probe changes little, and the pKa value is 3.82, indicating that it is stable under physiological conditions around pH 7.4. In addition, after 620nm excitation for 1 hour, the emission fluorescence intensity of the probe can still maintain more than 95%, showing its excellent optical stability ( Figure 8 b). The above results indicate that the stability of the probe TJO makes it suitable for in vivo application.

[0075] Example 3

[0076] Given that the probe TJO is effective for ONOO - This case provides relevant experimental evidence to support the specific detection ability of Figure 2 The probe TJO for ONOO is shown - The mechanism of reaction.

[0077] The HOMO and LUMO energies of the probe TJO and its reaction products were examined using DFT calculations at the B3LYP / 6-31G level. Fig. 9 As shown in a, the relatively high LUMO (-0.07 eV) of the probe TJO hinders the effective energy transfer of the photoexcited state, resulting in the suppression of ICT and the quenching of fluorescence. - After the reaction, the LUMO was significantly reduced to -2.33 eV, and the electron density distribution of the HOMO and LUMO was more uniform, which promoted ICT and restored fluorescence. In addition, the electron density of the probe was mainly concentrated on the lactam ring, while the electron density of the reaction product was dispersed throughout the molecule, which was beneficial to ICT and subsequent fluorescence emission ( Fig. 9 b). Meanwhile, the energy gap between HOMO and LUMO decreases from 6.19 eV to 4.18 eV, further facilitating energy transfer and fluorescence recovery.

[0078] The above mechanism was also confirmed by HPLC-MS analysis of the reaction mixture. - In the case of , the probe TJO has a peak at 7.24 minutes ( Fig.10 a). Add ONOO - After that, the probe TJO peak disappeared and a new peak appeared at 5.68 min, corresponding to the expected reaction product with a molecular weight of 521 ( Fig.10 b).

[0079] Example 4

[0080] This example uses human neuroblastoma cells (SH-SY5Y) as an example to demonstrate the effect of probe TJO on ONOO in cells. - imaging capabilities.

[0081] The SH-SY5Y cells used in this example were cultured in complete medium containing 1% penicillin-streptomycin, 10% fetal bovine serum, 89% MEM and Ham's F12 (MEM:F12=1:1), and all cell plates and culture dishes were placed in a humidified incubator at 37°C, 5% CO2 and 95% O2.

[0082] Before the imaging experiment, the MTT test was used to evaluate the cytotoxicity of TJO in SY5Y cells. The specific operation was as follows: the cultured SH-SY5Y cells were placed in a 96-well cell culture plate. After 24 hours, the cells were treated with different concentrations of TJO (0-20μM) for 24 hours; then the old culture medium was removed, DMSO (150μL per well) was added, and after oscillation for 5-10 minutes, the absorbance was measured at 570nm using a microplate reader. The results showed that the cytotoxicity of TJO was low. Even after 24 hours of treatment with 20μM TJO, more than 80% of the SH-SY5Y cells were still alive, indicating that the probe has good biocompatibility.

[0083] Monitoring exogenous ONOO in SH-SY5Y cells using TJO - Specifically, 0-1 mM exogenous ONOO was added to the cultured SH-SY5Y cells. - The donor 3-morpholinosydnonimine (SIN-1) was incubated for one hour, and then TJO (10 μM) was added and incubated for another 30 minutes. Fig.11 As shown in a, with the increase of SIN-1 concentration, the fluorescence intensity in SH-SY5Y cells increased, and the red fluorescence in the highest concentration group increased three times compared with the control group, indicating that TJO can effectively detect exogenous ONOO in cells. - .

[0084] 2-Methoxyestradiol (2-ME) was used as an endogenous ONOO - Inducers, in this case, TJO was used to monitor endogenous ONOO in SH-SY5Y cells. - Specifically, 0-5 μg / mL 2-ME was added to the cultured SH-SY5Y cells and incubated for 30 minutes, and then TJO (10 μM) was added and incubated for another 30 minutes. Fig.11 As shown in b, TJO can effectively detect endogenous ONOO in cells - , the fluorescence intensity increased up to fourfold when treated with 2-ME.

[0085] The probe TJO can not only effectively detect ONOO in cells - The increase of intracellular ONOO - In this example, uric acid (UA) and titanium iron reagent (Tiron) were used as ONOO - Scavenger, showing that the probe TJO detects intracellular ONOO - The specific operation is: after adding 2-ME to the cultured SH-SY5Y cells for one hour, UA (0-200μM) was added for one hour or Tiron (0-20μM) was added for 30 minutes, and then TJO (10μM) was added for another 30 minutes. Fig.12 As shown: With the increase of UA concentration, the red fluorescence of SH-SY5Y cells treated with 200 μM UA decreased by about two times ( Fig.12 a), which indicates that TJO can effectively detect ONOO in cells - reduction; Fig.12 b shows that the fluorescence intensity of SH-SY5Y cells treated with 20 μM Titan decreased by 3.5 times.

[0086] The above results confirm that the probe TJO is suitable for monitoring intracellular ONOO - level fluctuations.

[0087] Example 5

[0088] In this case, an ischemia-reperfusion cell model was established to verify the ability of the probe TJO to dynamically and real-time monitor the changes in ONOOˉ levels during ischemia-reperfusion injury.

[0089] First, SH-SY5Y cells were treated with glucose-oxygen deprivation / reperfusion (OGD / R) to establish a cell model of cerebral ischemia-reperfusion injury. SH-SY5Y cells were divided into five groups: control group, 4-hour hypoxia group (I4h), 4-hour hypoxia followed by 2-hour reperfusion group (I4h+R2h), 18-hour hypoxia group (I18h), and 18-hour hypoxia followed by 6-hour reperfusion group (I18h+R6h). The cell preparation method of each group was as follows: SH-SY5Y cells were cultured in a 96-well cell culture plate. After 24 hours, I18h, I4h, I4h+R2h, and I18h+R6h were placed in DMEM without glucose and cultured in a 37°C hypoxic incubator (oxygen concentration maintained at 1%-5%); I4h+R2h and I18h+R6h were placed in a normal complete medium and cultured in a normal incubator for another 2 hours and 6 hours, respectively.

[0090] The CCK-8 method was used to determine the viability of SH-SY5Y cells after OGD / R treatment. CCK-8 detection confirmed that the cells treated with OGD / R had different degrees of damage, proving that the cell model was successfully established ( Fig.13 ).

[0091] Each group of cells was added with the probe TJO (10 μM) and cultured for 30 minutes. Fig.14 As shown: the red fluorescence of the hypoxia and reperfusion groups was significantly stronger than that of the control group, and the red fluorescence of the reperfusion group was stronger than that of the hypoxia-only group, indicating that the probe TJO effectively detected the increase in ONOOˉ levels in the ischemia-reperfusion cell model.

[0092] In addition, the potent ONOOˉ scavenger edaravone (EDA) was used to detect the reduction of ONOOˉ in the ischemia-reperfusion injury model. Fig.15 As shown: The fluorescence intensity of the ischemia-reperfusion injury model cells treated with EDA was weaker than that of the OGD / R group. This indicates that TJO can detect the decrease in ONOOˉ levels in ischemia-reperfusion injury cells pretreated with EDA, further confirming the ability of TJO to dynamically monitor ONOOˉ in ischemia-reperfusion injury cell models.

[0093] Given the important role of superoxide dismutase (SOD) in regulating superoxide anion levels, this example also used SOD (1U / μL) to incubate SH-SY5Y cells pretreated with OGD / R. Cell imaging results showed that the red fluorescence of the SOD-treated group was significantly weaker than that of the OGD / R group ( Fig.16 ), that is, the ONOOˉ level decreased after SOD treatment.

[0094] The above results verified the ability of TJO to monitor ONOOˉ dynamics under different conditions and demonstrated its strong application potential in the study of ischemia-reperfusion injury.

[0095] Example 6

[0096] This example validates the ability of the probe TJO to monitor ONOOˉ fluctuations in an animal model, including the following experiments:

[0097] (1) The ability of the probe to cross the blood-brain barrier (BBB).

[0098] An important consideration for the application of probes in vivo is their ability to cross the BBB, which provides a stable environment for neural function. Lipophilicity quantified by the Log P value is a key parameter for predicting BBB permeability. Therefore, this example first uses the shake flask method to measure the lipophilicity of the probe TJO, specifically: TJO is placed in a mixture of n-octanol and ultrapure water (1:1, v / v) and shaken evenly for 72 hours, then centrifuged at 2500 rpm for 5 minutes, and the probe content in n-octanol and ultrapure water is evaluated by measuring the UV absorbance of the probe. Finally, the Log P value is calculated according to the following formula:

[0099]

[0100] In the formula, C 1-octanol and C water Represent the contents of the probe in the organic phase and the aqueous phase, respectively.

[0101] The log P value of TJO was found to be 2.04, which is in the optimal range of effective BBB permeability (2-4).

[0102] In this case, an in vitro BBB model was established using a Transwell system that simulates the BBB to further evaluate the blood-brain barrier permeability of the probe; calculations showed that 57% of TJO successfully passed through the model, confirming its potential to penetrate the BBB.

[0103] The BBB model used in this example was established by: inoculating hCMEC cells at 1.5×10 4 The density of the cells was inoculated in a 6-well Transwell plate, the average pore size of the membrane of the plate was 0.4 μm, the diameter of the insert culture plate was 24 mm, and the growth area of ​​the insert membrane was 4.67 cm 2 ; 5 days later, SW 1783 cells were expressed at 1.0×10 4 The cells were cultured at a density of 100 cells / well and studied again after 2 days of co-culture. The medium on the cell chamber side was replaced with fresh medium containing the probe TJO and cultured at 37°C for 24 hours; then, the medium on the cell chamber side and the basolateral side was collected and the probe permeability was evaluated by measuring its absorbance.

[0104] (2) Imaging of exogenous ONOOˉ in living mice.

[0105] Before the in vivo experiment, H&E staining was performed on the liver and kidney of mice injected with 100 μL 10 mg / kg TJO (experimental group) and 100 μL PBS (control group), respectively. No obvious pathological changes were found, indicating that the probe has good biosafety.

[0106] Another mouse was injected with SIN-1 (50 mg / kg, 100 μL) as ONOOˉ donor, and TJO (10 mg / kg, 100 μL) was injected intravenously 1 hour later. The control group was injected with an equal amount of PBS, and imaging was performed using the AniView imaging system (λex=675nm, λem=720-740nm). Fig.17 As shown, the fluorescence of the SIN-1 pretreatment group increased significantly, indicating that TJO can effectively detect exogenous ONOOˉ in living mice.

[0107] (3) Imaging of ONOOˉ in a mouse model of cerebral ischemia-reperfusion.

[0108] A mouse model of cerebral ischemia-reperfusion was established by middle cerebral artery occlusion (MCAO). The specific operation was as follows: after the mouse was anesthetized, the limbs and head were fixed on the operating table, the neck skin was incised, the subcutaneous muscle tissue was separated, the common carotid artery (CCA) was exposed and separated, and the proximal end of the CCA was ligated; after 1.5 hours of cerebral ischemia caused by suture embolism, the sutures were removed to restore blood perfusion.

[0109] The mice were randomly divided into four groups: control group, 1.5-hour ischemia group (I1.5H), 1.5-hour ischemia followed by reperfusion group (I1.5H+R), and EDA-treated ischemia-reperfusion group (I1.5H+R+EDA). After modeling, the MoorVMS laser Doppler imaging system was used to measure the cerebral blood flow of mice and indirectly evaluate the oxygen tension; the results showed that the blood flow on the ischemic side of MCAO was significantly reduced compared with the control group, confirming the decrease in oxygen tension after ischemia. At the same time, TTC staining was performed on the brain tissues of mice in the MCAO and control groups; the results confirmed that the brain tissues of mice in the MCAO group had obvious infarction, with an infarct area of ​​approximately 50%.

[0110] The live mice in the above groups were injected intravenously with TJO (10 mg / kg, 100 μL), and then the live imaging was performed using the AniView imaging system (λex=675nm, λem=720-740nm). Fig.18As shown: Compared with the ischemia group and the control group, the reperfusion group showed a stronger fluorescence signal, reflecting the increase in ONOOˉ levels after reperfusion; in the ischemia-reperfusion group, after EDA (100μL, 5mg / kg) treatment, the fluorescence signal was significantly reduced, indicating that ONOOˉ was effectively cleared.

[0111] The above results confirm that TJO can track the fluctuations of ONOOˉ levels in vivo.

[0112] In summary, the probe TJO provided by the present invention can selectively and sensitively detect ONOOˉ, has a rapid response capability to ONOOˉ, and has been successfully applied to ONOOˉ imaging in cell and animal models. It can accurately reflect the regulated ONOOˉ levels in cell and animal models, proving its application value in real-time monitoring of ONOOˉ in pathological conditions such as cerebral ischemia-reperfusion injury.

[0113] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A hydrazine-based near-infrared fluorescent probe, characterized in that: Its structure is as follows:

2. The hydrazine-based near-infrared fluorescent probe as claimed in claim 1 is used for in vitro ONOO - Application in detection.

3. The hydrazine-based near-infrared fluorescent probe as claimed in claim 1 is used in biological systems ONOO - Applications in imaging.

4. The use according to claim 3, characterized in that: The biological systems include cells and animals.

5. The use according to claim 4, characterized in that: The animals include cerebral ischemia-reperfusion injury model mice.

6. The use according to claim 4, characterized in that: The cells include SH-SY5Y cells.

7. The hydrazine-based near-infrared fluorescent probe as claimed in claim 1 is used to prepare ONOO - Detection of product applications.

8. A real-time monitoring method for ONOO in mice with cerebral ischemia-reperfusion injury - The method is characterized in that After the hydrazine-based near-infrared fluorescent probe according to claim 1 is intravenously injected into model mice, in vivo imaging is performed.

9. A method for preparing the hydrazine-based near-infrared fluorescent probe according to claim 1, characterized in that: Compound 1, hydrazine and BOP reagent are reacted in dichloromethane to obtain the compound 1, wherein the structural formula of compound 1 is:

10. The method according to claim 9, characterized in that The following steps are also included: The product was purified by silica gel column chromatography, wherein the eluent was CH2Cl2:MeOH=100:1-10:1.

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