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

Abstract

The invention provides a near-infrared fluorescent viscosity probe as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The near-infrared fluorescence viscosity probe has a structure as shown in a formula I; the invention also provides a preparation method and application of the near-infrared fluorescence viscosity probe. The near-infrared fluorescence viscosity probe disclosed by the invention has the characteristics of aggregation-induced emission, high viscosity sensitivity, pH stability and good biocompatibility, can realize rapid and accurate imaging of rheumatoid arthritis and osteoarthritis, and provides a novel tool for arthritis diagnosis and pathological research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a near-infrared fluorescent viscosity probe and a preparation method and application thereof. BACKGROUND

[0002] Arthritis, including rheumatoid arthritis (RA) and osteoarthritis (OA), is a globally high-incidence chronic and disabling disease, and its core pathological features include joint swelling, cartilage damage, synovitis, etc. If not diagnosed and intervened in time, it will lead to irreversible joint dysfunction. At present, the clinical diagnosis mainly relies on traditional imaging technologies such as ultrasound, X-ray, CT, MRI, etc. However, these methods have insufficient sensitivity in the early stage of the disease, and cannot reveal the pathological microenvironment changes (such as viscosity abnormalities) at the cellular and subcellular levels.

[0003] Fluorescence imaging technology has become a research hotspot for disease diagnosis due to its high sensitivity, rapid response and excellent spatiotemporal resolution. However, the traditional organic fluorescent probes used in fluorescence imaging technology have the problem of aggregation-caused quenching (ACQ), which limits their application in biological systems. Aggregation-induced emission (AIE) can emit strong fluorescence in an aggregated state, effectively overcoming the ACQ defect. In addition, intracellular viscosity, as a key microenvironment parameter, is closely related to the pathological process of arthritis and other diseases. However, there is currently a lack of AIE-type near-infrared probes that can specifically respond to viscosity changes and are suitable for arthritis imaging.

[0004] Currently, most arthritis-related fluorescent probes focus on the detection of inflammatory factors (such as NO and HOCl), and there is still no specific imaging tool for the correlation between viscosity and arthritis, which cannot meet the needs of early diagnosis and pathological mechanism research of the disease.

[0005] Therefore, developing a probe with high viscosity sensitivity, strong solid fluorescence, near-infrared emission and good biocompatibility is of great clinical significance for the precise diagnosis and basic research of arthritis. SUMMARY

[0006] The purpose of the present application is to provide a near-infrared fluorescent viscosity probe and a preparation method and application thereof. The near-infrared fluorescent viscosity probe has the characteristics of aggregation-induced emission, high viscosity sensitivity, pH stability and good biocompatibility, and can realize rapid and accurate imaging of rheumatoid arthritis and osteoarthritis, providing a new tool for arthritis diagnosis and pathological research. It can solve the problems of low sensitivity of current arthritis diagnosis technology, inability to monitor changes in cell microenvironment viscosity, and aggregation-caused quenching and lack of arthritis specificity of fluorescent probes.

[0007] To achieve the above purpose, the technical scheme adopted is to provide a near-infrared fluorescent viscosity probe, which has a structure as shown in formula I: .

[0008] The near-infrared fluorescent viscosity probe has a donor-π-acceptor (D-π-A) structure, the electron donor is a triphenylamine (TPA) group, has good electron donor ability and rotational freedom, and provides a molecular rotor basis for viscosity response; the electron acceptor is a pyrene oxadiazole structure, has strong electron accepting ability, and can enhance the near-infrared emission characteristics of the probe; the conjugated bridge (π) is an extended π-conjugated system, improves the optical performance of the probe, realizes a large Stokes shift (259 nm), and reduces the interference of excitation light. The structure gives the probe two core characteristics: one is the aggregation-induced emission effect, which avoids the quenching problem caused by aggregation, and emits strong solid fluorescence in an aggregated state (such as in cells and diseased tissues); the other is viscosity sensitivity, the rotational motion of the TPA group is limited as the viscosity increases, resulting in a significant increase in fluorescence intensity, realizing quantitative response to viscosity changes. The chemical structure of the near-infrared fluorescent viscosity probe corresponds to the characteristics that the molecule formula is C 56 H 58 N4O8S, and the molecular weight is 946.15.

[0009] The application further provides a preparation method of the near-infrared fluorescent viscosity probe, comprising the following steps: (1) dissolving 4,7-bis(2-bromo-4,5-bis((2-methoxyethoxy)methoxy)phenyl)-2,1,3-benzothiazole, 4-(diphenylamino)benzene boronic acid and cesium fluoride in a solvent, and then degassing to obtain a reaction solution; (2) under an inert atmosphere, adding a palladium catalyst into the reaction solution to react, and then purifying to obtain the near-infrared fluorescent viscosity probe shown in formula I; and the synthesis route is as follows: .

[0010] The beneficial effects of the above technical scheme are as follows: 4,7-bis(2-bromo-4,5-bis((2-methoxyethoxy)methoxy)phenyl)-2,1,3-benzothiazole, 4-(diphenylamino)benzene boronic acid and cesium fluoride are mixed and degassed, and then reacted under the action of a palladium catalyst; during the reaction process, the cesium fluoride acts as a base and an activator, and provides fluoride ions to promote the Suzuki coupling reaction.

[0011] Preferably, the solvent in step (1) is a tetrahydrofuran aqueous solution; the degassing mode is freeze, air extraction and freeze-thaw cycle degassing, and the degassing frequency is 4-6 times.

[0012] More preferably, the volume ratio of tetrahydrofuran to water in the tetrahydrofuran aqueous solution is 9:1.

[0013] More preferably, the degassing frequency is 5 times.

[0014] More preferably, the freezing, vacuumizing and thawing cycle degassing is specifically: using liquid nitrogen to freeze the reaction liquid into solid, then using a vacuum pump to vacuumize for 3-5 min, closing the vacuum system and introducing inert gas, and thawing the solid at room temperature.

[0015] More preferably, the inert gas is nitrogen or argon.

[0016] Preferably, the palladium catalyst in step (2) is bis(tri-tert-butylphosphine)palladium(0); the reaction is carried out under oil bath condition, the temperature is 70-80℃, and the time is 22-26 h.

[0017] More preferably, the reaction in step (2) is carried out under oil bath condition, the temperature is 75℃, and the time is 24 h.

[0018] Preferably, the molar ratio of 4,7-bis(2-bromo-4,5-bis((2-methoxyethoxy)methoxy)phenyl)-2,1,3-benzothiadiazole, 4-(diphenylamino)phenylboronic acid, cesium fluoride and palladium catalyst is (0.65-0.7):(0.6-0.65):(2-4):0.1.

[0019] More preferably, the molar ratio of 4,7-bis(2-bromo-4,5-bis((2-methoxyethoxy)methoxy)phenyl)-2,1,3-benzothiadiazole, 4-(diphenylamino)phenylboronic acid, cesium fluoride and palladium catalyst is 0.68:0.65:3:0.1.

[0020] Preferably, the purification comprises the following steps: after the reaction is cooled to room temperature, sequentially performing vacuum concentration, redissolution and filtration to obtain a filtrate; after the filtrate is concentrated, the purification is performed by rapid column chromatography, the target fraction is collected, and after vacuum drying, the product is obtained.

[0021] More preferably, the eluent used in the rapid column chromatography is a mixture of dichloromethane and ethyl acetate mixed in a volume ratio of 1:1.

[0022] The application also provides the use of the above-mentioned near-infrared fluorescent viscosity probe in the preparation of an arthritis diagnostic kit.

[0023] Preferably, the arthritis includes rheumatoid arthritis and osteoarthritis.

[0024] The application also provides an arthritis diagnostic kit comprising a near-infrared fluorescent viscosity probe having the structure shown in formula I.

[0025] Preferably, the molar concentration of the near-infrared fluorescent viscosity probe used for cell incubation is 4-6 μM, and the dosage used for intravenous injection is 0.4-0.6 mg / kg; the emission wavelength of the near-infrared fluorescent viscosity probe is 550-650 nm, and the imaging excitation wavelength is 480 nm.

[0026] More preferably, the molar concentration of the near-infrared fluorescent viscosity probe used for cell incubation is 5 μM, and the dosage used for intravenous injection is 0.5 mg / kg.

[0027] The application also provides the use of the near-infrared fluorescent viscosity probe in monitoring viscosity changes at the cellular and / or subcellular level.

[0028] The application has the following beneficial effects: (1) The near-infrared fluorescent viscosity probe of the application has a maximum absorption wavelength of 322 nm in toluene, a fluorescence emission wavelength of 581 nm, and a Stokes shift of 259 nm, which can effectively avoid excitation light interference; in a glycerol-ethanol mixed system, the fluorescence intensity is enhanced by 138 times when the viscosity increases from 0.893 cP to 945 cP, and the response sensitivity to viscosity is high; (2) The near-infrared fluorescent viscosity probe of the application has high fluorescence intensity in poor solvents, and emits bright yellow fluorescence under 365 nm ultraviolet light irradiation in a solid state, overcoming the aggregation-induced quenching defect of traditional organic fluorescent probes, and having significant aggregation-induced emission characteristics; (3) The near-infrared fluorescent viscosity probe of the application has good pH stability, and the fluorescence spectrum peak shape and intensity do not change significantly within the pH range (pH 5-9) covering the pathological microenvironment pH of arthritis, avoiding non-specific signal interference; and the near-infrared fluorescent viscosity probe of the application has excellent biocompatibility and no obvious cytotoxicity; (4) The near-infrared fluorescent viscosity probe of the application has outstanding imaging effect, can distinguish the viscosity difference between tumor cells and normal cells, and can track the viscosity changes in the processes of low temperature, dexamethasone stimulation, apoptosis, autophagy and inflammation; can realize the rapid visualization of rheumatoid arthritis and osteoarthritis lesion sites in a short time, and the fluorescence signal of the lesion tissue is significantly higher than that of normal joint tissue, and the signal is stable within 24 h. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1Figure for optical performance test results of the near-infrared fluorescent viscosity probe of Example 1; wherein A is a normalized ultraviolet-visible absorption and fluorescence emission spectrum diagram of the near-infrared fluorescent viscosity probe in water; B is a normalized ultraviolet-visible absorption spectrum diagram of the near-infrared fluorescent viscosity probe in different polar solvents; C is a fluorescence spectrum diagram of the near-infrared fluorescent viscosity probe in different polar solvents; D is a solid fluorescence diagram of the near-infrared fluorescent viscosity probe under natural light and 365 nm wavelength; E is a viscosity response diagram of the near-infrared fluorescent viscosity probe in a glycerol-ethanol system; F is a stability result diagram of the near-infrared fluorescent viscosity probe under different pH values; Figure 2 Figure for cytotoxicity test results of the near-infrared fluorescent viscosity probe of Example 1; Figure 3 Figure for imaging and fluorescence intensity quantitative analysis of the near-infrared fluorescent viscosity probe of Example 1 in different cell lines; wherein A is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe in HeLa cells; B is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe in RAW 264.7 cells; C is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe in ATDC5 cells; D is a statistical diagram of average fluorescence intensity of the near-infrared fluorescent viscosity probe in three groups of cells; Figure 4 Figure for tracking viscosity changes of the near-infrared fluorescent viscosity probe of Example 1 under low temperature and dexamethasone induction; wherein A is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe tracking 37℃ control group cells; B is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe tracking 4℃ low temperature group cells; C is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe tracking dexamethasone treatment group cells; D is a statistical diagram of average fluorescence intensity of the near-infrared fluorescent viscosity probe in three groups of cells; Figure 5 Figure for tracking viscosity changes of the near-infrared fluorescent viscosity probe of Example 1 in the process of cell apoptosis and autophagy; wherein A is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe tracking control group cells; B is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe tracking apoptosis group cells; C is fluorescence imaging, bright field imaging and fusion diagram of the near-infrared fluorescent viscosity probe tracking autophagy group cells; D is a statistical diagram of average fluorescence intensity of the near-infrared fluorescent viscosity probe in three groups of cells; Figure 6Fig. 1 is a graph of viscosity changes of inflammatory cells tracked by the near-infrared fluorescent viscosity probe of Example 1; wherein A is the fluorescent imaging, bright field imaging and fusion of the near-infrared fluorescent viscosity probe in the control group and the inflammatory group of HeLa cells; B is the fluorescent imaging, bright field imaging and fusion of the near-infrared fluorescent viscosity probe in the control group and the inflammatory group of RAW 264.7 cells; C is the fluorescent imaging, bright field imaging and fusion of the near-infrared fluorescent viscosity probe in the control group and the inflammatory group of ATDC5 cells; D is the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the control group and the inflammatory group of HeLa cells; E is the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the control group and the inflammatory group of RAW 264.7 cells; F is the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the control group and the inflammatory group of ATDC5 cells; Figure 7 Fig. 2 is the imaging and characterization of the near-infrared fluorescent viscosity probe of the RA model mice; wherein A is the transmission electron microscope of the near-infrared fluorescent viscosity probe; B is the particle size distribution graph of the near-infrared fluorescent viscosity probe; C is the imaging comparison graph of the near-infrared fluorescent viscosity probe in the RA joints and normal joints of the RA mouse model at different time points in vivo; D is the real object graph of the joint tissue after dissection; E is the fluorescent imaging graph of the joint tissue after dissection; F is the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe; Figure 8 Fig. 3 is the imaging graph of the OA model mice; wherein A is the micro-CT graph of the joints of the Ctrl mice and the OA mice; B is the HE staining graph of the joint tissue; C is the imaging comparison graph of the near-infrared fluorescent viscosity probe in the RA joints and normal joints of the OA mouse model at different time points in vivo; D is the fluorescent imaging graph of the biodistribution of the near-infrared fluorescent viscosity probe in the main organs; Figure 9 Fig. 4 is the HE staining result graph of the main organs. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present application clearer, the following further describes the present application in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application, i.e., the described embodiments are only a part of the embodiments of the present application, but not all the embodiments.

[0031] Therefore, the following detailed description of the embodiments of the present application provided herein is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.

[0032] The features and performances of the present application are further described in detail in combination with embodiments.

[0033] Example 1 A near-infrared fluorescent viscosity probe having a structure as shown in Formula I: .

[0034] The present embodiment also provides a preparation method of the above-mentioned near-infrared fluorescent viscosity probe, comprising the following steps: (1) Dissolve 4,7-bis(2-bromo-4,5-bis((2-methoxyethoxy)methoxy)phenyl)-2,1,3-benzothiadiazole (200 mg, 0.68 mmol), 4-(diphenylamino)benzeneboronic acid (189.23 mg, 0.65 mmol), cesium fluoride (458 mg, 3.0 mmol) in a tetrahydrofuran aqueous solution, and then degasify by 5 cycles of freeze-pumping-thaw to remove oxygen in the system, to obtain a reaction solution; the volume ratio of tetrahydrofuran to water in the tetrahydrofuran aqueous solution is 9:1; wherein the freeze-pumping-thaw cycle degassing is as follows: first, treat the reaction solution with liquid nitrogen to freeze it into a solid, then use a vacuum pump to pump for 3-5 min, close the vacuum system and introduce N2, and melt the solid at room temperature; (2) Under a nitrogen atmosphere, add bis(tri-tert-butylphosphine)palladium(0) (52.87 mg, 0.1 mmol) to the reaction solution, and stir to react in an oil bath at 75°C for 24 h; after the reaction is cooled to room temperature, remove the solvent by concentration under reduced pressure, redissolve the residue in dichloromethane, and filter to remove insoluble impurities; after the filtrate is concentrated, purify it by flash column chromatography, the eluent for flash column chromatography is dichloromethane / ethyl acetate (1:1, v / v), collect the target fraction, and dry under reduced pressure to obtain the near-infrared fluorescent viscosity probe TPA2PyO4 (orange solid, yield 64.63%) shown in Formula I; the synthesis route is as follows: .

[0035] Structural characterization: (1) 1 H NMR (400 MHz, DMSO-d6): δ 7.64-7.08 (m, 28H, aromatic protons), 4.10-4.06 (m, 4H, -OCH2-), 3.57-3.35 (m, 20H, methoxyethoxy chain protons), 3.18 (s, 6H, -OCH3); (2) 13C NMR (100 MHz, DMSO-d6): δ 152.66, 152.03, 147.50, 147.29, 132.31, 130.12, 130.03, 127.61, 124.85, 124.79, 123.87, 123.81, 123.54, 122.34 (aromatic ring and conjugated system carbon), 73.33, 71.70, 70.23, 70.18, 70.08 (ether bond carbon), 58.49 (methoxy carbon); (3) HRMS (ESI): m / z calcd for C 56 H 58 N4O8S Na + 969.38676, found 969.38684, the structure is correct.

[0036] Example 2 Optical performance test The optical performance of the near-infrared fluorescent viscosity probe was tested, and the fluorescence intensity, normalized UV-Vis absorption and fluorescence emission spectrum of the near-infrared fluorescent viscosity probe under different solvent conditions were tested. The specific test conditions are as follows: 5℃, quartz cuvette, probe concentration 5 μM, excitation wavelength 480 nm, and the results are shown in Figure 1 .

[0037] To characterize the spectral behavior of TPA2PyO4, the basic optical properties in aqueous solution, fluorescence characteristics in different polar solvents, fluorescence activity in solid state, potential viscosity response fluorescence characteristics and pH stability were tested in turn. The above tests were carried out at 5℃, and the concentration of TPA2PyO4 in the solution was 5 μM, and the excitation wavelength was 480 nm. The results are shown in Figure 1 .

[0038] As can be seen from Figure 1 A, the maximum absorption wavelength of TPA2PyO4 in water is 442 nm, the maximum emission wavelength is 581 nm, and the Stokes shift is 139 nm. Such a large Stokes shift is extremely advantageous in biological imaging applications, which can minimize the interference caused by excitation light and improve the signal-to-noise ratio of the detection system. As can be seen from Figure 1 B, in all test solvents, the maximum absorption wavelength is stable near 435 nm; but as the solvent polarity increases, the fluorescence emission peak shows a significant red shift Figure 1 C), and the emission intensity decreases significantly, which reveals that TPA2PyO4 has a twisted intramolecular charge transfer (TICT) effect.

[0039] At the same time, the probe also has fluorescence activity in solid state Figure 1(D) At room temperature, TPA2PyO4 is an orange solid. Under 365 nm ultraviolet light, it emits bright yellow fluorescence. This phenomenon may be due to its aggregation-induced emission properties and the difficulty in forming effective π-π stacking interactions in the solid state. This property allows TPA2PyO4 to circumvent the aggregation-induced quenching (ACQ) problem commonly found in traditional fluorescent dyes, achieving stable fluorescence emission even in the aggregated state. In other words, TPA2PyO4 is a near-infrared fluorescent probe suitable for viscosity detection, and its near-infrared emission characteristics are particularly beneficial for imaging studies in biological systems.

[0040] Secondly, from Figure 1 As can be seen from E, the fluorescence intensity of TPA2PyO4 gradually increases with the increase of glycerol ratio. When the system viscosity (η) increases from 0.893 cP under 0% glycerol conditions to 945 cP under 100% glycerol conditions, the fluorescence intensity of the probe at 581 nm increases by 138 times, which proves that TPA2PyO4 is highly sensitive to viscosity.

[0041] In addition, the effect of pH on the fluorescence properties of TPA2PyO4 was evaluated. Figure 1 As can be seen from F), within the pH range of 5-9, the fluorescence spectrum peak shape and fluorescence intensity fluctuation at 581 nm of the probe are less than 5%, indicating that its fluorescence response is not affected by physiological pH. The pH stability of the probe is crucial for biological applications. The physiological microenvironment of osteoarthritis (OA) and rheumatoid arthritis (RA) is usually weakly acidic to neutral. The probe of this invention remains stable under these conditions, avoiding non-specific signal interference, which is key to ensuring the accuracy of viscosity detection.

[0042] Example 3 Cell Experiment 1. Cell Culture HeLa (tumor cells), RAW 264.7 (macrophages), and ATDC5 (chondrocytes) were cultured in their respective culture media and grown adherently in a 37°C, 5% CO2 incubator.

[0043] 2. Cytotoxicity test (MTT assay) Cells were seeded in 96-well plates (1×10⁶ cells per well). 4 Cells / well were cultured for 24 h, and then different concentrations (0, 5, 10, 20, 40 μM) of the near-infrared fluorescent viscosity probe TPA2PyO4 were added, and the cells were cultured for another 24 h. 10 μL of MTT reagent was added to each well, and the cells were incubated at 37°C in the dark for 1 h. The absorbance at 450 nm was measured using a microplate reader, and the relative cell viability was calculated. The results are shown below. Figure 2 As shown.

[0044] from Figure 2As can be seen from Table 1, the cell survival rates of each concentration group are all higher than 85%, indicating that the near-infrared fluorescent viscosity probe TPA2PyO4 of the present application has no cytotoxicity.

[0045] 3 Cell Viscosity Imaging A control group (normal cells, ctrl), an inflammation group (HeLa / RAW 264.7 treated with LPS 200 ng / mL for 2 h, and ATDC5 treated with IL-1β 10 ng / mL for 12 h), an apoptosis group (treated with Cisplatin 1 mM for 6 h, Cisplatin), and an autophagy group (Starvation) were set up. After each group was fully grown, 5 μM TPA2PyO4 was added, and incubation was performed at 37°C for 30 min, followed by PBS washing for 3 times. Then, the inflammation Hela cells were treated at 4°C, 37°C, and with dexamethasone (DEX) for 1 h, followed by the addition of 5 μM TPA2PyO4, and incubation was performed at 37°C for 30 min, followed by PBS washing for 3 times. Laser confocal microscopy was used for each group, excitation was performed at 480 nm, and emission was performed at 550~650 nm, and the results are shown in Figures 3-6 ; Figures 3-6 In the imaging figures, the scale is 25 μm; Figure 5 In the imaging figures, Ctrl represents the control group, and LPS represents the inflammation group.

[0046] As can be seen from Table 1, the cell survival rates of each concentration group are all higher than 85%, indicating that the near-infrared fluorescent viscosity probe TPA2PyO4 of the present application has no cytotoxicity. Figure 3 As can be seen from Table 1, the cell survival rates of each concentration group are all higher than 85%, indicating that the near-infrared fluorescent viscosity probe TPA2PyO4 of the present application has no cytotoxicity. Figure 4 As can be seen from Table 1, the cell survival rates of each concentration group are all higher than 85%, indicating that the near-infrared fluorescent viscosity probe TPA2PyO4 of the present application has no cytotoxicity. Figures 5-6 As can be seen from Table 1, the cell survival rates of each concentration group are all higher than 85%, indicating that the near-infrared fluorescent viscosity probe TPA2PyO4 of the present application has no cytotoxicity.

[0047] Example 4 Imaging Experiment of Animal Model 1 Model Construction (1) RA model: The right knee joint of a mouse was injected with λ-carrageenan (20 μL, 5 mg / mL PBS solution), and joint swelling occurred after 24 h, indicating that the model was successfully constructed. (2) OA model: The mouse was subjected to medial meniscus instability (DMM) surgery, and after the surgery, joint space narrowing and osteophyte formation were observed by micro-CT, and synovial degeneration was shown by HE staining, indicating that the model was successfully constructed.

[0048] 2 In vivo Imaging The mice of the obtained RA model and OA model were injected with TPA2PyO4 (0.5 mg / kg) through the tail vein for administration; the mice of the control group (C57 mice from Huafukang Company, Ctrl) were injected with PBS buffer through the tail vein; imaging was performed at 1 h, 12 h and 24 h, respectively, using a small animal near-infrared imaging system (Caliper Life Sciences, IVIS Spectrum), excitation 480 nm, emission 550-650 nm; after imaging, the mice were sacrificed, and heart, liver, spleen, lung, kidney and joint tissues were taken for fluorescence imaging, and the results are shown in Figures 7-8 .

[0049] The results are shown in Figures 7-8 , the diseased joints of the RA / OA model mice showed obvious fluorescence signals within 1 h, and the signal was the strongest at 24 h, which was significantly higher than that of the normal joints; quantitative analysis of the fluorescence intensity of the diseased joint tissues after dissection showed that the difference was statistically significant compared with the normal joints (***p=0.001); at the same time, the near-infrared fluorescence viscosity probe mainly accumulated in the diseased joints, and the signal in the liver and kidney was weak (metabolic excretion pathway).

[0050] 3 Biocompatibility and biodistribution The heart, liver, spleen, lung and kidney of the control group (Ctrl) and OA model group (OA) mice sacrificed after 24 h of imaging were subjected to HE staining analysis, and the results are shown in Figure 9 . Figure 9 The scale bar is 50 μm.

[0051] From the HE staining of Figure 9 , it can be seen that there is no pathological damage in each main organ, and there is no significant difference from the Ctrl control group, indicating that the near-infrared fluorescence viscosity probe of the present application has good biocompatibility.

[0052] In summary, the near-infrared fluorescence viscosity probe TPA2PyO4 is successfully synthesized by reasonable molecular design, and its unique D-π-A structure endows it with excellent viscosity sensitivity, AIE characteristics, pH stability and biocompatibility; the probe can quickly and accurately realize the visualization imaging of the RA and OA diseased sites, solve the problem of low sensitivity of traditional diagnostic techniques and the inability to monitor changes in the cell microenvironment, provide a new tool for early diagnosis, pathological research and drug development of arthritis, and has important clinical application value and scientific research significance.

[0053] The present application is described in accordance with the above examples, it should be understood that the above examples do not limit the present application in any form, and any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of the present application.

Claims

1. A near-infrared fluorescent viscosity probe, characterized in that, The near-infrared fluorescent viscosity probe has the structure shown in Formula I: 。 2. The method for preparing the near-infrared fluorescent viscosity probe according to claim 1, characterized in that, Includes the following steps: (1) Dissolve 4,7-bis(2-bromo-4,5-bis((2-methoxyethoxy)methoxy)phenyl)-2,1,3-benzothiadiazole, 4-(diphenylamino)phenylboronic acid and cesium fluoride in a solvent, and then degas to obtain a reaction solution; (2) Under an inert atmosphere, palladium catalyst was added to the reaction solution to carry out the reaction, and after purification, the near-infrared fluorescent viscosity probe shown in Formula I was obtained; its synthetic route is as follows: 。 3. The method for preparing the near-infrared fluorescent viscosity probe as described in claim 2, characterized in that, The solvent in step (1) is an aqueous solution of tetrahydrofuran; the degassing method is a cycle of freezing, evacuation and thawing, with the number of degassing cycles being 4 to 6.

4. The method for preparing the near-infrared fluorescent viscosity probe as described in claim 2, characterized in that, In step (2), the palladium catalyst is bis(tri-tert-butylphosphine)palladium(0); the reaction is carried out in an oil bath at a temperature of 70-80°C for 22-26 h.

5. The method for preparing the near-infrared fluorescent viscosity probe as described in claim 2, characterized in that, The molar ratio of the 4,7-bis(2-bromo-4,5-bis((2-methoxyethoxy)methoxy)phenyl)-2,1,3-benzothiadiazole, 4-(diphenylamino)phenylboronic acid, cesium fluoride and palladium catalyst is (0.65~0.7):(0.6~0.65):(2~4):0.

1.

6. The application of the near-infrared fluorescent viscosity probe according to claim 1 in the preparation of an arthritis diagnostic kit.

7. An arthritis diagnostic kit, characterized in that, Including the near-infrared fluorescent viscosity probe as described in claim 1.

8. The arthritis diagnostic kit as described in claim 7, characterized in that, The near-infrared fluorescent viscosity probe is used at a molar concentration of 4-6 μM for cell incubation and at a dose of 0.4-0.6 mg / kg for intravenous injection; the emission wavelength of the near-infrared fluorescent viscosity probe is 550-650 nm and the imaging excitation wavelength is 480 nm.

9. The application of the near-infrared fluorescent viscosity probe of claim 1 in monitoring viscosity changes at the cellular and / or subcellular levels.

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