A near-infrared fluorescent viscosity probe and a preparation method and application thereof

Abstract

The application provides a near-infrared fluorescent viscosity probe, a preparation method and application thereof, and belongs to the technical field of biological medicines.The near-infrared fluorescent viscosity probe has a structure as shown in formula I; the application further provides a preparation method and application of the near-infrared fluorescent viscosity probe.The near-infrared fluorescent viscosity probe 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 new tool for arthritis diagnosis and pathological research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a near-infrared fluorescent viscosity probe, its preparation method, and its application. Background Technology

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

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

[0004] Existing fluorescent probes for arthritis mostly focus on the detection of inflammatory factors (such as NO and HOCl), and there are no specific imaging tools for the association between viscosity and arthritis, which cannot meet the needs of early diagnosis and pathological mechanism research.

[0005] Therefore, developing a probe that combines high viscosity sensitivity, strong solid-state fluorescence, near-infrared emission, and good biocompatibility is of great clinical significance for the accurate diagnosis and basic research of arthritis. Summary of the Invention

[0006] The purpose of this invention is to provide a near-infrared fluorescent viscosity probe, its preparation method, and its application. This near-infrared fluorescent viscosity probe has aggregation-induced emission properties, high viscosity sensitivity, pH stability, and good biocompatibility. It can achieve 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, inability to monitor changes in cell microenvironment viscosity, aggregation-induced quenching of fluorescent probes, and lack of arthritis specificity in existing arthritis diagnostic techniques.

[0007] The technical solution adopted to achieve the above objectives is to provide a near-infrared fluorescent viscosity probe, which has a structure as shown in Formula I:

[0008] .

[0009] The beneficial effects of the above-mentioned technical solution of this invention are as follows: The near-infrared fluorescent viscosity probe of this invention has a donor-π-acceptor (D-π-A) structure. The electron donor is a triphenylamine (TPA) group, which has good electron-donating ability and rotational freedom, providing a molecular rotor basis for viscosity response. The electron acceptor is a pyrene-xadiazole structure, which 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, which improves the optical performance of the probe, achieves a large Stokes shift (259 nm), and reduces excitation light interference. This structure endows the probe with two core characteristics: first, aggregation-induced emission effect, avoiding quenching problems caused by aggregation, and emitting strong solid-state fluorescence in the aggregated state (such as in cells and diseased tissues); second, viscosity sensitivity, the rotational motion of the TPA group is restricted as viscosity increases, resulting in a significant increase in fluorescence intensity, realizing a quantitative response to viscosity changes. The chemical structure of the near-infrared fluorescent viscosity probe of this invention is characterized by: containing a bis(triphenylamine) rotor and a pyridothiadiazole acceptor unit, with the molecular formula C 56 H 58 N4O8S has a molecular weight of 946.15.

[0010] The present invention also provides a method for preparing the above-mentioned near-infrared fluorescent viscosity probe, comprising the following steps:

[0011] (1) Dissolve 4,7-dibromo-5,6-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo[c][1,2,5]thiadiazole, 4-(diphenylamino)phenylboronic acid, and cesium fluoride in a solvent, and then degas to obtain a reaction solution;

[0012] (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:

[0013] .

[0014] The beneficial effects of the above technical solution are as follows: 4,7-dibromo-5,6-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo[c][1,2,5]thiadiazole, 4-(diphenylamino)phenylboronic acid and cesium fluoride are mixed, degassed and then reacted under the action of palladium catalyst; during the reaction, cesium fluoride acts as both a base and an activator, and promotes the Suzuki coupling reaction by providing fluoride ions.

[0015] Preferably, the solvent in step (1) is an aqueous solution of tetrahydrofuran; the degassing method is a cycle of freezing, evacuation and thawing, and the number of degassing cycles is 4 to 6.

[0016] More preferably, the volume ratio of tetrahydrofuran to water in the tetrahydrofuran aqueous solution is 9:1.

[0017] More preferably, the degassing is performed 5 times.

[0018] More preferably, the freezing, evacuation and thawing cycle degassing is specifically performed as follows: the reaction liquid is frozen into a solid by liquid nitrogen treatment, then a vacuum pump is used to evacuate for 3-5 minutes, the vacuum system is turned off and an inert gas is introduced, and the solid is melted at room temperature.

[0019] More preferably, the inert gas is nitrogen or argon.

[0020] Preferably, the palladium catalyst in step (2) is bis(tri-tert-butylphosphine)palladium(0); the reaction is carried out under oil bath conditions at a temperature of 70~80℃ for 22~26 h.

[0021] More preferably, the reaction in step (2) is carried out in an oil bath at a temperature of 75°C for 24 hours.

[0022] Preferably, the molar ratio of 4,7-dibromo-5,6-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo[c][1,2,5]thiadiazole, 4-(diphenylamino)phenylboronic acid, cesium fluoride and palladium catalyst is (0.65~0.7):(0.6~0.65):(2~4):0.1.

[0023] More preferably, the molar ratio of 4,7-dibromo-5,6-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo[c][1,2,5]thiadiazole, 4-(diphenylamino)phenylboronic acid, cesium fluoride and palladium catalyst is 0.68:0.65:3:0.1.

[0024] Preferably, the purification includes the following steps: after the reaction is cooled to room temperature, it is concentrated under reduced pressure, redissolved and filtered in sequence to obtain a filtrate; after the filtrate is concentrated, it is purified by rapid column chromatography, the target fraction is collected and dried under reduced pressure to obtain the final product.

[0025] More preferably, the eluent used in rapid column chromatography is a mixture of dichloromethane and ethyl acetate in a 1:1 volume ratio.

[0026] The present invention also provides the application of the above-mentioned near-infrared fluorescent viscosity probe in the preparation of arthritis diagnostic kits.

[0027] Preferably, arthritis includes rheumatoid arthritis and osteoarthritis.

[0028] The present invention also provides an arthritis diagnostic kit, comprising a near-infrared fluorescent viscosity probe with the structure shown in Formula I.

[0029] Preferably, the molar concentration of the near-infrared fluorescent viscosity probe for cell incubation is 4-6 μM, and the dose 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.

[0030] More preferably, the molar concentration of the near-infrared fluorescent viscosity probe used for cell incubation is 5 μM, and the dose used for intravenous injection is 0.5 mg / kg.

[0031] This invention also provides the application of near-infrared fluorescent viscosity probes in monitoring viscosity changes at the cellular and / or subcellular levels.

[0032] The present invention has the following beneficial effects:

[0033] (1) The near-infrared fluorescent viscosity probe of the present invention has a maximum absorption wavelength of 322 nm, a fluorescence emission wavelength of 581 nm, and a Stokes shift of 259 nm in toluene, which can effectively avoid excitation light interference; in the glycerol-ethanol mixed system, when the viscosity increases from 0.893 cP to 945 cP, the fluorescence intensity increases by 138 times, and the sensitivity to viscosity response is high.

[0034] (2) The near-infrared fluorescent viscosity probe of the present invention has high fluorescence intensity in poor solvents and emits bright yellow fluorescence under 365nm ultraviolet light in solid state, which overcomes the quenching defect caused by aggregation in traditional organic fluorescent probes and has significant aggregation-induced luminescence characteristics.

[0035] (3) The near-infrared fluorescent viscosity probe of the present invention has good pH stability. Within the pH range covering the pathological microenvironment of arthritis (pH 5~9), the peak shape and intensity of the fluorescence spectrum do not change significantly, thus avoiding non-specific signal interference. Furthermore, the near-infrared fluorescent viscosity probe of the present invention has excellent biocompatibility and no obvious cytotoxicity.

[0036] (4) The near-infrared fluorescent viscosity probe of the present invention has outstanding imaging effect, which can distinguish the viscosity difference between tumor cells and normal cells, and track viscosity changes during low temperature, dexamethasone stimulation, apoptosis, autophagy and inflammation. It can achieve rapid visualization of lesions of rheumatoid arthritis and osteoarthritis in a short time. The fluorescence signal of lesion tissue is significantly higher than that of normal joint tissue, and the signal remains stable within 24 hours. Attached Figure Description

[0037] Figure 1Figure 1 shows the optical performance test results of the near-infrared fluorescent viscosity probe in Example 1. A represents the normalized UV-Vis absorption and fluorescence emission spectra of the near-infrared fluorescent viscosity probe in water; B represents the normalized UV-Vis absorption spectra of the near-infrared fluorescent viscosity probe in different polar solvents; C represents the fluorescence spectra of the near-infrared fluorescent viscosity probe in different polar solvents; D represents the solid-state fluorescence spectra of the near-infrared fluorescent viscosity probe under natural light and at a wavelength of 365 nm; E represents the viscosity response of the near-infrared fluorescent viscosity probe in a glycerol-ethanol system; and F represents the stability results of the near-infrared fluorescent viscosity probe at different pH values.

[0038] Figure 2 This is a graph showing the cytotoxicity test results of the near-infrared fluorescent viscosity probe in Example 1;

[0039] Figure 3 The images shown are from Example 1, illustrating the imaging and quantitative fluorescence intensity analysis of the near-infrared fluorescent viscosity probe in different cell lines. Specifically, A represents the fluorescence imaging, bright-field imaging, and fusion diagram of the near-infrared fluorescent viscosity probe in HeLa cells; B represents the fluorescence imaging, bright-field imaging, and fusion diagram of the near-infrared fluorescent viscosity probe in RAW 264.7 cells; C represents the fluorescence imaging, bright-field imaging, and fusion diagram of the near-infrared fluorescent viscosity probe in ATDC5 cells; and D represents the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the three cell lines.

[0040] Figure 4 This is a graph from Example 1 showing the viscosity changes induced by low temperature and dexamethasone induced by a near-infrared fluorescent viscosity probe. A represents the fluorescence, bright-field, and fusion images of the near-infrared fluorescent viscosity probe tracking cells in the 37℃ control group; B represents the fluorescence, bright-field, and fusion images of the near-infrared fluorescent viscosity probe tracking cells in the 4℃ low-temperature group; C represents the fluorescence, bright-field, and fusion images of the near-infrared fluorescent viscosity probe tracking cells in the dexamethasone-treated group; and D represents the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the three groups of cells.

[0041] Figure 5 This is a graph showing viscosity changes during cell apoptosis and autophagy tracked by a near-infrared fluorescent viscosity probe in Example 1. A shows the fluorescence, bright-field, and fusion images of the control group cells tracked by the near-infrared fluorescent viscosity probe; B shows the fluorescence, bright-field, and fusion images of the apoptotic group cells tracked by the near-infrared fluorescent viscosity probe; C shows the fluorescence, bright-field, and fusion images of the autophagy group cells tracked by the near-infrared fluorescent viscosity probe; and D shows the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the three groups of cells.

[0042] Figure 6Example 1 shows the viscosity changes of near-infrared fluorescent viscosity probes tracking inflammatory cells. A represents the fluorescence, bright-field, and fusion images of the near-infrared fluorescent viscosity probe in the control and inflammation groups of HeLa cells; B represents the fluorescence, bright-field, and fusion images of the near-infrared fluorescent viscosity probe in the control and inflammation groups of RAW 264.7 cells; C represents the fluorescence, bright-field, and fusion images of the near-infrared fluorescent viscosity probe in the control and inflammation groups of ATDC5 cells; D represents the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the control and inflammation groups of HeLa cells; E represents the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the control and inflammation groups of RAW 264.7 cells; and F represents the statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe in the control and inflammation groups of ATDC5 cells.

[0043] Figure 7 Images of a rat model of respiratory retardation (RA) and characterization of near-infrared fluorescent viscosity probes are shown below. A is a transmission electron microscope image of the near-infrared fluorescent viscosity probe; B is a particle size distribution map of the near-infrared fluorescent viscosity probe; C is a comparison of RA joint and normal joint images at different time points in the RA mouse model using the near-infrared fluorescent viscosity probe; D is an image of the dissected joint tissue; E is a fluorescence image of the dissected joint tissue; and F is a statistical graph of the average fluorescence intensity of the near-infrared fluorescent viscosity probe.

[0044] Figure 8 Images of OA model mice: A is a micro-CT image of the joints of Ctrl mice and OA mice; B is an HE staining image of the joint tissue; C is a comparison of near-infrared fluorescent viscosity probe imaging of RA joints and normal joints at different time points in OA mouse models; D is a fluorescence imaging image of the biodistribution of near-infrared fluorescent viscosity probe in major organs.

[0045] Figure 9 Image showing the HE staining results of major organs. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are only a part of the embodiments of this invention, and not all of them.

[0047] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0048] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0049] Example 1

[0050] A near-infrared fluorescent viscosity probe has the structure shown in Formula I:

[0051] .

[0052] This embodiment also provides a method for preparing the above-mentioned near-infrared fluorescent viscosity probe, including the following steps:

[0053] (1) 4,7-Dibromo-5,6-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo[c][1,2,5]thiadiazole (200 mg, 0.68 mmol), 4-(diphenylamino)phenylboronic acid (189.23 mg, 0.65 mmol), and cesium fluoride (458 mg, 3.0 mmol) were dissolved in tetrahydrofuran aqueous solution, and then subjected to 5 freeze-evacuation-thawing cycles to remove oxygen from the system, to obtain the reaction solution; the volume ratio of tetrahydrofuran to water in the tetrahydrofuran aqueous solution was 9:1; the freeze-evacuation-thawing cycle degassing was specifically as follows: first, the reaction solution was frozen into a solid by liquid nitrogen treatment, then a vacuum pump was used to evacuate for 3~5 min, the vacuum system was turned off and N2 was introduced, and the solid was melted at room temperature;

[0054] (2) Under a nitrogen atmosphere, bis(tri-tert-butylphosphine)palladium(0) (52.87 mg, 0.1 mmol) was added to the reaction solution and stirred in an oil bath at 75 °C for 24 h. After the reaction was cooled to room temperature, the solvent was removed by vacuum concentration, the residue was redissolved with dichloromethane, and insoluble impurities were removed by filtration. The filtrate was concentrated and purified by rapid column chromatography with dichloromethane / ethyl acetate (1:1, v / v) as the eluent. The target fraction was collected, dried under reduced pressure, and the near-infrared fluorescent viscosity probe TPA2PyO4 (orange solid, yield 64.63%) shown in Formula I was obtained. The synthetic route is as follows:

[0055] .

[0056] Structural characterization:

[0057] (1) 1 ¹H NMR (400 MHz, DMSO-d6): δ 7.64–7.08 (m, 28H, aromatic ring proton), 4.10–4.06 (m, 4H, -OCH2-), 3.57–3.35 (m, 20H, methoxyethoxy chain proton), 3.18 (s, 6H, -OCH3);

[0058] (2) 13 C 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 carbons), 73.33, 71.70, 70.23, 70.18, 70.08 (ether bond carbons), 58.49 (methoxy carbons);

[0059] (3) HRMS (ESI): m / z calculated value C 56 H 58 N4O8S Na + 969.38676, measured value 969.38684, structural verification is correct.

[0060] Example 2 Optical Performance Testing

[0061] The optical properties of the near-infrared fluorescent viscosity probe were tested. The fluorescence intensity, normalized UV-Vis absorption, and fluorescence emission spectra of the probe were measured under different solvent conditions. Specific test conditions were: 5℃, quartz cuvette, probe concentration 5 μM, and excitation wavelength 480 nm. The results are as follows: Figure 1 As shown.

[0062] To characterize the spectral behavior of TPA2PyO4, its basic optical properties in aqueous solution, fluorescence characteristics in solvents of different polarities, fluorescence activity in the solid state, potential viscosity-responsive fluorescence characteristics, and pH stability were tested sequentially. All tests were conducted at 5℃, with a TPA2PyO4 concentration of 5 μM in the solution and an excitation wavelength of 480 nm. The results are as follows: Figure 1 As shown.

[0063] from Figure 1 As shown in Figure A, TPA₂PyO₄ exhibits a maximum absorption wavelength of 442 nm and a maximum emission wavelength of 581 nm in water, with a Stokes shift of 139 nm. Such a large Stokes shift is highly advantageous in bioimaging applications, minimizing interference from excitation light and improving the signal-to-noise ratio of the detection system. From... Figure 1 As can be seen from B, the maximum absorption wavelength remained stable around 435 nm in all tested solvents; however, with increasing solvent polarity, the fluorescence emission peak showed a significant red shift. Figure 1 C), while the emission intensity decreased significantly, a phenomenon that reveals the existence of the Twisted Intramolecular Charge Transfer (TICT) effect in TPA2PyO4.

[0064] Meanwhile, the probe also exhibits fluorescence activity in the 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.

[0065] 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.

[0066] 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.

[0067] Example 3 Cell Experiment

[0068] 1. Cell Culture

[0069] 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.

[0070] 2. Cytotoxicity test (MTT assay)

[0071] Cells were seeded in 96-well plates (1×10⁶ cells per well). 4Cells / 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.

[0072] from Figure 2 As can be seen, the cell survival rate of each concentration group is higher than 85%, indicating that the near-infrared fluorescent viscosity probe TPA2PyO4 of the present invention is non-cytotoxic.

[0073] 3. Cell viscosity imaging

[0074] The following groups were set up: a control group (normal cells, ctrl), an inflammation group (HeLa / RAW 264.7 cells treated with 200 ng / mL LPS for 2 h, ATDC5 cells treated with 10 ng / mL IL-1β for 12 h), an apoptosis group (cisplatin 1 mM treatment for 6 h), and an autophagy group (glucose-free DMEM culture for 12 h, Starvation). After each group reached confluence, 5 μM TPA2PyO4 was added, and the cells were incubated at 37°C for 30 min, followed by washing three times with PBS. Inflamed HeLa cells were then treated at 4°C, 37°C, and dexamethasone (DEX) for 1 h, followed by incubation at 37°C for 30 min with 5 μM TPA2PyO4, and then washed three times with PBS. All groups were analyzed using laser confocal microscopy, with excitation at 480 nm and emission at 550–650 nm. The results are shown below. Figures 3-6 As shown; Figures 3-6 The scale bar in the mid-image is 25 μm. Figure 5 In the middle, Ctrl represents the control group, and LPS represents the inflammation group.

[0075] from Figure 3 As can be seen, HeLa tumor cells have better viscosity than normal cells, resulting in higher fluorescence intensity after co-incubation; from Figure 4 It can be seen that low temperature and dexamethasone treatment increased cell viscosity, and probe incubation increased strength; while the... Figures 5-6 It can be seen that the fluorescence intensity of the inflammation group, apoptosis group, and autophagy group is significantly higher than that of the control group, and the fluorescence intensity of HeLa cells is higher than that of RAW264.7 and ATDC5 cells.

[0076] Example 4: Imaging Experiment of Animal Model

[0077] 1. Model Building

[0078] (1) RA model: λ-carrageenan (20 μL, 5 mg / mL PBS solution) was injected into the right knee joint cavity of mice. Joint swelling appeared 24 h later, and the model was successfully established.

[0079] (2) OA model: Mice underwent medial meniscus instability (DMM) surgery. Postoperatively, micro-CT showed joint space narrowing and osteophyte formation, and HE staining showed synovial degeneration. The model was successfully constructed.

[0080] 2. In vivo imaging

[0081] TPA2PyO4 (0.5 mg / kg) was administered via tail vein injection to mice with RA and OA models. Control mice (C57 mice, Ctrl, derived from Huafukang) were injected via tail vein injection with PBS buffer. Imaging was performed at 1 h, 12 h, and 24 h using a small animal near-infrared imaging system (Caliper Life Sciences, IVISSpectrum), with excitation at 480 nm and emission at 550-650 nm. After imaging, mice were sacrificed, and heart, liver, spleen, lung, kidney, and joint tissues were harvested for fluorescence imaging. The results are as follows: Figures 7-8 As shown.

[0082] The results are as follows Figures 7-8 As shown, the diseased joints of RA / OA model mice showed obvious fluorescence signals within 1 hour, with the strongest signal at 24 hours, significantly higher than that of normal joints; quantitative analysis of fluorescence intensity of diseased joint tissues after dissection showed that the difference was statistically significant compared with normal joints (***p=0.001); at the same time, the near-infrared fluorescent viscosity probe mainly accumulated in the diseased joints, while the signal was weaker in the liver and kidneys (metabolic excretion pathway).

[0083] 3. Biocompatibility and biological distribution

[0084] Heart, liver, spleen, lung, and kidney of control (ctrl) and OA model (OA) mice sacrificed 24 h after the above imaging were analyzed by HE staining. The results are as follows. Figure 9 As shown, Figure 9 The scale bar is 50 μm.

[0085] from Figure 9 HE staining showed no pathological damage to the major organs and no significant difference from the Ctrl control group, indicating that the near-infrared fluorescent viscosity probe of the present invention has good biocompatibility.

[0086] In summary, this invention successfully synthesized a near-infrared fluorescent viscosity probe, TPA2PyO4, through rational molecular design. Its unique D-π-A structure endows it with excellent viscosity sensitivity, AIE properties, pH stability, and biocompatibility. This probe can rapidly and accurately visualize lesions in RA and OA, solving the problems of low sensitivity and inability to monitor changes in the cellular microenvironment in traditional diagnostic techniques. It provides a novel tool for the early diagnosis, pathological research, and drug development of arthritis, and has significant clinical application value and scientific research significance.

[0087] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.

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-dibromo-5,6-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo[c][1,2,5]thiadiazole, 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-dibromo-5,6-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo[c][1,2,5]thiadiazole, 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.

Images