PH / viscosity double-lock activated afterglow luminescent small-molecule probe as well as preparation method and application of pH / viscosity double-lock activated afterglow luminescent small-molecule probe

Abstract

The invention relates to the technical field of biosensing, and discloses a pH / viscosity double-lock activated afterglow luminescent small molecule probe as well as a preparation method and application thereof. The structural formula of the afterglow luminescent small-molecule probe is shown in the specification. A rhodamine light-emitting unit and an afterglow light-emitting matrix unit SO are covalently connected through a chemical bond. The luminescent mechanism is as follows: under illumination, molecules generate singlet oxygen to oxidize and activate adjacent SO units, so that active oxygen mediated afterglow luminescence is generated. The afterglow luminescent small-molecule probe has a double-lock activation characteristic, that is, a strong afterglow signal can be generated only when a subacid environment and a high-viscosity condition are met at the same time. And the signal is weak under a single condition or a normal physiological environment. The probe provided by the invention has excellent environmental specificity and high signal-to-noise ratio imaging capability, and provides a powerful tool for realizing accurate imaging in a complex biological system.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosensing, and particularly relates to a persistent luminescence small-molecule probe. BACKGROUND

[0002] Molecular imaging is a real-time, non-invasive method for detecting the occurrence and development of diseases in vivo, which is expected to in-situ longitudinally monitor the molecular events in the body, which is difficult for in-vitro diagnostic methods based on static analysis. Among the many molecular imaging methods, low-background imaging in biological systems can achieve accurate measurement of specific analytes, which is crucial for basic biomedical research and clinical applications. Persistent luminescence (long-lasting luminescence) is a non-excitation imaging technique that stores the irradiated light energy and then slowly emits photons. Therefore, it can eliminate the disadvantages of fluorescence imaging and provide non-background molecular imaging in vivo. So far, persistent materials include inorganic persistent materials and organic persistent materials. Inorganic metal-based persistent luminescence materials have excellent persistent performance, but their inherent cytotoxicity limits their further application in biology. In contrast, organic persistent materials are considered to be more biocompatible, flexible in design, biodegradable, and conducive to biological imaging applications. However, organic persistent materials are relatively rare as a new type of imaging substrate, mainly in the form of multi-component nanoparticles.

[0003] In order to overcome the metabolic problems of nanoscale probes, researchers have begun to develop small organic molecule persistent probes. Such probes usually integrate target-responsive units, photon-sensitizing units, reactive oxygen species capturing units, and persistent luminescence units into a small molecule, with more optimal pharmacokinetic properties. Currently, the design strategy of organic persistent molecular probes mainly focuses on the response to a single physical or chemical parameter. However, the physiological and pathological processes in the body often involve the synergistic changes of multiple factors. For example, in the lesion area of inflammation and tumor, there are often characteristics of micro-acidity and local viscosity increase. However, the existing single-parameter responsive probes are prone to produce false positive signals due to the satisfaction of a single condition in this complex environment, and their specificity and accuracy face serious challenges.

[0004] Therefore, there is an urgent need in the art for a new molecular design strategy that can logically integrate the response mechanisms of two key parameters to construct an intelligent sensing system with "AND" gate characteristics. Under this strategy, the probe will only produce a significant output signal when two specific pathological signals (such as micro-acidity and high viscosity) exist simultaneously. This design is expected to achieve high selective recognition of the target pathological microenvironment and achieve a high signal-to-noise ratio in complex physiological background. SUMMARY

[0005] To address the aforementioned technical problems, this invention provides a persistent glow molecular probe with strict AND logic gating. Its afterglow luminescence intensity is significantly enhanced only when both pH and viscosity reach a set threshold, thereby fundamentally improving the specificity and accuracy of imaging. This invention proposes a pH / viscosity dual-lock activated persistent glow luminescence small molecule probe, its preparation method, and its applications.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows: A pH / viscosity dual-lock activated afterglow luminescent small molecule probe (RHSO) has the following structural formula: .

[0007] The core of the pH / viscosity dual-lock activated afterglow luminescence small molecule probe described above is a rhodamine-SO hybrid molecule. In this design, light excitation of the molecule generates and transfers reactive oxygen species to the adjacent SO luminescent matrix in situ, forming a high-energy intermediate. Then, energy is transferred to the rhodamine luminescent unit through chemical bonds, thereby efficiently initiating reactive oxygen species-mediated afterglow luminescence.

[0008] The above-mentioned method for preparing pH / viscosity dual-lock activated afterglow luminescence small molecule probe involves chemically integrating a rhodamine derivative with the afterglow luminescence matrix SO. The specific steps are as follows: S1. 4-Bromoacetophenone and SeO2 are added to a mixed solvent I of dioxane and water, and intermediate 1 is obtained by reaction I; S2. Intermediate 1 and N,N-dimethylaniline were added to a mixed solvent II of hexafluoroisopropanol and dichloromethane to react and obtain intermediate 2; S3. Intermediate 2 and mercaptoethanol were reacted with trimethylsilylchlorone to prepare intermediate 3; intermediate 3 and pinacol diboronate were reacted with a palladium complex catalyst under alkaline conditions to prepare intermediate 4. S4. 4-Bromo-2-carboxybenzoic acid, 3-diethylaminophenol and p-toluenesulfonamide are added to propionic acid, and intermediate 5 is obtained by reaction II; S5. Under the conditions of a palladium complex catalyst and an alkaline environment, intermediates 4 and 5 were added to a mixed solvent III of dioxane, water and anhydrous ethanol, and after reaction III, the afterglow luminescent small molecule probe RHSO was obtained. The structural formulas of intermediate 1, intermediate 2, intermediate 3, intermediate 4, and intermediate 5 are as follows: , , , , .

[0009] In step S1 above, the molar ratio of 4-bromoacetophenone to SeO2 is 1:1.2~1.5, and the volume ratio of dioxane to water in mixed solvent I is 30:0.5~1; in step S2, the molar ratio of intermediate 1 to N,N-dimethylaniline is 1.2~1.5:1; in step S3, 2~3 mol of intermediate 2 is added per L of mercaptoethanol; the molar ratio of intermediate 3 to pinacol diboronate is 1:2~2.5.

[0010] In step S4 above, the molar ratio of 4-bromo-2-carboxybenzoic acid, 3-diethylaminophenol and p-toluenesulfonamide is 1:2:0.2~0.5; in step S5, the molar ratio of intermediate 4 and intermediate 5 is 2:1~1.2; and the volume ratio of dioxane, water and anhydrous ethanol in mixed solvent III is 4:1:1~0.5.

[0011] The above-mentioned pH / viscosity dual-lock activated afterglow luminescence small molecule probes are used as afterglow luminescence imaging agents for purposes other than disease diagnosis and treatment.

[0012] Furthermore, the activation conditions for the above-mentioned afterglow luminescence are pH or viscosity.

[0013] The aforementioned pH / viscosity dual-lock activated afterglow luminescence small molecule probe is used as a dual-lock activated afterglow optical imaging agent for purposes other than disease diagnosis and treatment; wherein, dual-lock activation refers to activating a strong afterglow luminescence signal when both pH is below 7 and viscosity is greater than 1.76 mPa·s.

[0014] The above-mentioned pH / viscosity dual-lock activated afterglow luminescent small molecule probe is used in the preparation of imaging agents for inflammatory diseases for purposes other than disease diagnosis and treatment.

[0015] Furthermore, the aforementioned inflammatory diseases include joint inflammation.

[0016] The beneficial effects of this invention are: (1) A revolutionary "double-lock" activation mechanism has been achieved: This invention, through ingenious molecular design, couples the singlet oxygen generation capability (pH-dependent) and intramolecular motion restriction (viscosity response) of molecules, creating a strict "AND" logic gate. The probe can only efficiently generate reactive oxygen in a slightly acidic environment to act on the afterglow luminescent matrix SO. The released energy is then transferred to the rhodamine luminescent unit through chemical bonds. The excited-state rhodamine luminescent unit needs the synergistic effect of restricting its intramolecular rotation in a high-viscosity environment to effectively release photons and generate strong afterglow luminescence. This mechanism greatly eliminates false positive signals generated under single abnormal conditions, enabling accurate identification of complex biological microenvironments.

[0017] (2) Achieved ultra-high specificity and signal-to-noise ratio: Thanks to the above-mentioned "dual-lock" design, RHSO exhibits extremely high specificity in in vivo imaging. Experiments have shown that it can accurately distinguish lesion areas (such as inflamed joints) with abnormal pH / viscosity dual parameters from normal or other abnormal tissues, thereby obtaining specific imaging images with extremely low background and extremely high signal-to-noise ratio.

[0018] (3) It provides a powerful in vivo diagnostic tool: When RHSO is applied to a mouse model of arthritis, it can be specifically activated by the diseased joints and produce a strong afterglow signal, while the surrounding normal tissues show almost no afterglow signal. This indicates that RHSO is not only a novel molecular probe, but also an in vivo diagnostic tool with great potential for the early diagnosis and efficacy assessment of inflammatory diseases such as joint inflammation. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a synthetic route diagram for the afterglow molecular probe RHSO of this invention.

[0021] Figure 2 The above is the 1H NMR spectrum of RHSO in this embodiment of the invention.

[0022] Figure 3 This is the carbon NMR spectrum of RHSO in an embodiment of the present invention.

[0023] Figure 4 This is a high-resolution mass spectrum of RHSO in an embodiment of the present invention.

[0024] Figure 5 This is the UV-Vis absorption spectrum of RHSO in an embodiment of the present invention.

[0025] Figure 6 This is the fluorescence emission spectrum of RHSO in an embodiment of the present invention.

[0026] Figure 7 This is the afterglow emission spectrum of RHSO in an embodiment of the present invention.

[0027] Figure 8 This is a diagram showing the afterglow emission intensity of RHSO at different pH values ​​in an embodiment of the present invention.

[0028] Figure 9 This is a diagram showing the afterglow intensity of RHSO at different viscosities in a pH=5 buffer in an embodiment of the present invention.

[0029] Figure 10 This is a verification diagram of RHSO pH / viscosity dual-lock activation in an embodiment of the present invention.

[0030] Figure 11 This is a graph showing the RHSO cytotoxicity test in an embodiment of the present invention.

[0031] Figure 12 This is an image showing the imaging effect of RHSO on a mouse joint inflammation model in an embodiment of the present invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] This invention designs a pH / viscosity dual-lock activated afterglow luminescent small molecule probe RHSO, the structure of which is shown in formula (1). The aforementioned pH / viscosity-activated dual-lock activation afterglow luminescence small molecule probe, RHSO, has a rhodamine-SO hybrid molecule at its core. In this design, the photoexcited rhodamine unit can generate and transfer reactive oxygen species to the adjacent SO luminescent matrix in situ, thereby efficiently initiating afterglow luminescence. This probe employs a unique dual-lock activation mechanism, and the generation of its strong afterglow signal is strictly dependent on the coexistence of slightly acidic pH and high viscosity. This characteristic enables high-fidelity visualization of complex pathological microenvironments such as joint inflammation, providing a powerful tool for precise diagnosis at the in vivo level.

[0034] Example 1 The preparation method of the pH / viscosity dual-lock activated afterglow luminescent small molecule probe RHSO in this embodiment is shown in the synthetic route diagram below. Figure 1 As shown, the steps are as follows: S1: 4-Bromoacetophenone (10 mmol, 1.99 g) and SeO2 (15 mmol, 1.664 g) were added to a mixed solvent (31 mL) of dioxane and water, where V 二恶烷 V 水 =15:0.5, under nitrogen atmosphere, stirred at 100℃ for 5 h, after the reaction was completed, the reaction solution was filtered, the solvent was removed under vacuum, water was added and stirred until a solid appeared, filtered, and the mixture was pulped and filtered with petroleum ether to obtain intermediate 1.

[0035] S2: Intermediate 1 (1.2 mmol, 288 mg), N,N-dimethylaniline (1 mmol, 0.132 g), and hexafluoroisopropanol (2 mmol, 211 μL) were dissolved in 4 mL of dichloromethane, stirred at 0 °C for 12 h, and purified by column chromatography to obtain intermediate 2.

[0036] S3: Intermediate 2 (1 mmol, 333 mg), mercaptoethanol (0.5 mL), and trimethylsilylchlorone (0.5 mL) were dissolved in 15 mL of toluene, refluxed at 95 °C overnight under nitrogen protection, and purified by column chromatography to obtain intermediate 3.

[0037] S4: Intermediate 3 (0.42 mmol, 150 mg), pinacol diboronate (0.84 mmol, 211.65 mg), potassium acetate (1.26 mmol, 122.67 mg), and tetrakis(triphenylphosphine)palladium (0.006 mmol, 6 mg) were dissolved in 15 mL of dioxane. The mixture was stirred at 95 °C for 12 h under a nitrogen atmosphere, cooled to room temperature, extracted with ethyl acetate, dried and concentrated, and purified by column chromatography to obtain intermediate 4.

[0038] S5: 4-Bromo-2-carboxybenzoic acid (5 mmol, 1.15 g), 3-hydroxy-N,N-diethylaniline (10.05 mmol, 2.48 g), and p-toluenesulfonamide (1.0 mmol, 0.17 g) were dissolved in 15 mL of propionic acid, stirred at 90 °C for 18 h, and purified by column chromatography to obtain intermediate 5.

[0039] S6: Intermediate 4 (0.26 mmol, 105 mg), intermediate 5 (0.13 mmol, 62 mg), sodium carbonate (90 mg), and tetrakis(triphenylphosphine)palladium (0.012 mmol, 12 mg) were added to a mixed solvent (12 mL) of dioxane, anhydrous ethanol, and water, wherein V （二恶烷） V （无水乙醇） V （水） The mixture was stirred at 100°C for 24 h under a nitrogen atmosphere with a ratio of 8 mL: 2 mL: 2 mL, and purified by column chromatography to obtain RHSO.

[0040] The hydrogen NMR spectrum, carbon NMR spectrum, and high-resolution mass spectrum of the afterglow luminescent small molecule probe RHSO are shown below. Figures 2-4 As shown. 1 H NMR (600 MHz, MeOD) δ 8.22 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.21 (t, J= 12.9 Hz, 4H), 7.09 (dd, J = 14.6, 8.9 Hz, 4H), 6.99 (d, J = 2.2 Hz,2H), 6.96 – 6.90 (m, 4H), 6.47 (d, J = 8.7 Hz, 2H), 4.48 – 4.39 (m, 2H), 3.67(q, J = 7.1 Hz, 8H), 3.22 – 3.19 (m, 2H), 2.75 (s, 6H), 1.30 (dd, J = 13.6, 6.4Hz, 12H). 13 C NMR (151 MHz, MeOD) δ = 160.41,157.95, 155.62, 149.80, 143.75,143.40, 137.24, 136.02, 134.15, 131.42, 131.21, 130.63, 130.59, 129.15,129.13, 127.34, 127.32, 126.09, 125.77, 125.74, 113.90,113.86, 113.54,111.94, 111.92, 111.90, 109.74, 102.82, 95.81, 65.49, 48.18, 45.42, 39.09,31.66, 29.33, 29.05, 27.95, 22.32, 13.02, 11.52. HR-ESI-MSm / z:[M+H]+calcd.for694.3462; found,694.3483. Example 2 The preparation method of the pH / viscosity dual-lock activated afterglow luminescent small molecule probe RHSO in this embodiment is as follows: S1: 4-Bromoacetophenone (10 mmol, 1.99 g) and SeO2 (12 mmol) were added to a mixed solvent of dioxane and water (30.5 mL), where V 二恶烷 V 水 =30:0.5, under nitrogen atmosphere, stirred at 100℃ for 5 h, after the reaction was completed, the reaction solution was filtered, the solvent was removed under vacuum, water was added and stirred until a solid appeared, filtered, and the mixture was pulped and filtered with petroleum ether to obtain intermediate 1.

[0041] S2: Intermediate 1 (1.5 mmol), N,N-dimethylaniline (1 mmol, 0.132 g), and hexafluoroisopropanol (2 mmol, 211 μL) were dissolved in 4 mL of dichloromethane, stirred at 0 °C for 12 h, and purified by column chromatography to obtain intermediate 2.

[0042] S3: Intermediate 2 (1.5 mmol, 333 mg), mercaptoethanol (0.5 mL), and trimethylsilylchlorone (0.5 mL) were dissolved in 15 mL of toluene, refluxed at 95 °C overnight under nitrogen protection, and purified by column chromatography to obtain intermediate 3.

[0043] S4: Intermediate 3 (0.42 mmol, 150 mg), pinacol diboronate (1.05 mmol), potassium acetate (1.26 mmol, 122.67 mg), and tetrakis(triphenylphosphine)palladium (0.006 mmol, 6 mg) were dissolved in 15 mL of dioxane. The mixture was stirred at 95 °C for 12 h under a nitrogen atmosphere, cooled to room temperature, extracted with ethyl acetate, dried and concentrated, and purified by column chromatography to obtain intermediate 4.

[0044] S5: 4-Bromo-2-carboxybenzoic acid (5 mmol, 1.15 g), 3-hydroxy-N,N-diethylaniline (10.05 mmol, 2.48 g), and p-toluenesulfonamide (2.5 mmol) were dissolved in 15 mL of propionic acid, stirred at 90 °C for 18 h, and purified by column chromatography to obtain intermediate 5.

[0045] S6: Intermediate 4 (0.26 mmol, 105 mg), intermediate 5 (0.156 mmol), sodium carbonate (90 mg), and tetrakis(triphenylphosphine)palladium (0.012 mmol, 12 mg) were added to a mixed solvent (12 mL) of dioxane, anhydrous ethanol, and water, wherein V （二恶烷） V （无水乙醇） V （水） The mixture was prepared by mixing 8 mL, 2 mL, and 1 mL at 100 °C for 24 h under a nitrogen atmosphere and then purifying by column chromatography to obtain RHSO.

[0046] Example 3 The preparation method of the pH / viscosity dual-lock activated afterglow luminescent small molecule probe RHSO in this embodiment is as follows: S1: 4-Bromoacetophenone (10 mmol, 1.99 g) and SeO2 (14 mmol) were added to a mixed solvent of dioxane and water (30.7 mL), where V 二恶烷 V 水=30:0.7, under nitrogen atmosphere, stirred at 100℃ for 5 h, after the reaction was completed, the reaction solution was filtered, the solvent was removed under vacuum, water was added and stirred until a solid appeared, filtered, and the mixture was pulped and filtered with petroleum ether to obtain intermediate 1.

[0047] S2: Intermediate 1 (1.4 mmol), N,N-dimethylaniline (1 mmol, 0.132 g), and hexafluoroisopropanol (2 mmol, 211 μL) were dissolved in 4 mL of dichloromethane, stirred at 0 °C for 12 h, and purified by column chromatography to obtain intermediate 2.

[0048] S3: Intermediate 2 (1.25 mmol, 333 mg), mercaptoethanol (0.5 mL), and trimethylsilylchlorone (0.5 mL) were dissolved in 15 mL of toluene, refluxed at 95 °C overnight under nitrogen protection, and purified by column chromatography to obtain intermediate 3.

[0049] S4: Intermediate 3 (0.42 mmol, 150 mg), pinacol diboronate (0.96 mmol), potassium acetate (1.26 mmol, 122.67 mg), and tetrakis(triphenylphosphine)palladium (0.006 mmol, 6 mg) were dissolved in 15 mL of dioxane. The mixture was stirred at 95 °C for 12 h under a nitrogen atmosphere, cooled to room temperature, extracted with ethyl acetate, dried and concentrated, and purified by column chromatography to obtain intermediate 4.

[0050] S5: 4-Bromo-2-carboxybenzoic acid (5 mmol, 1.15 g), 3-hydroxy-N,N-diethylaniline (10.05 mmol, 2.48 g), and p-toluenesulfonamide (1.0 mmol, 0.17 g) were dissolved in 15 mL of propionic acid, stirred at 90 °C for 18 h, and purified by column chromatography to obtain intermediate 5.

[0051] S6: Intermediate 4 (0.26 mmol, 105 mg), intermediate 5 (0.143 mmol), sodium carbonate (90 mg), and tetrakis(triphenylphosphine)palladium (0.012 mmol, 12 mg) were added to a mixed solvent (12 mL) of dioxane, anhydrous ethanol, and water, wherein V （二恶烷） V （无水乙醇） V （水） The mixture was stirred at 100°C for 24 h under a nitrogen atmosphere with a ratio of 8 mL: 2 mL: 2 mL, and purified by column chromatography to obtain RHSO.

[0052] Implementation Results Example (1) Ultraviolet-visible absorption spectrum A 1 mM ethanol stock solution of RHSO was prepared, and a small amount was diluted to 100 μM. The absorption spectra of the probe in different solvents were measured using a UV-Vis spectrophotometer in the wavelength range of 300-800 nm. The measurements revealed a molecular absorption peak at 560 nm. Figure 5 ).

[0053] (2) Fluorescence emission spectrum RHSO was prepared as a 1 mM ethanol stock solution, and a small amount was diluted to 100 μM. Using a fluorescence spectrometer, the excitation wavelength was set to the maximum absorption wavelength of RHSO, and the fluorescence emission spectrum was recorded within the corresponding emission wavelength range. The molecular fluorescence emission peak was found to be at 600 nm (…). Figure 6 ).

[0054] (3) Afterglow emission spectrum Pipette 10 μL of the prepared 1 mM probe stock solution (ethanol) into a 500 μL centrifuge tube. Add 90 μL of pH 5 PBS buffer to the tube. Using the IVIS in vivo imaging system (bioluminescence mode), single-wavelength collection mode, collect the afterglow intensity at different emission wavelengths. The results are as follows: Figure 7 As shown in the figure, the afterglow emission wavelength of the probe is at 600 nm. By testing the afterglow decay time, the half-life of the probe's afterglow emission was found to be 32 s.

[0055] (4) Afterglow emission intensity at different pH values 10 μL of the prepared 1 mM probe stock solution (ethanol) was transferred to a 500 μL centrifuge tube. 90 μL of PBS buffer at different pH values ​​was added to the tube. The changes in afterglow intensity at different pH buffer values ​​were collected using an IVIS in vivo imaging system (bioluminescence mode). The results are as follows: Figure 8 As shown. Within the physiological pH range (5-8), the probe exhibits a weak afterglow emission signal when only the pH condition is changed.

[0056] (5) Afterglow intensity at different viscosities in a pH=5 buffer Solutions were prepared in pH 5 PBS buffer and glycerol at different volume ratios (V(glycerol)) of 0% (1.00 mPa·s), 10% (1.31 mPa·s), 20% (1.76 mPa·s), 30% (2.50 mPa·s), 40% (3.72 mPa·s), 50% (6.00 mPa·s), 60% (10.80 mPa·s), and 70% (22.50 mPa·s). 10 μL of the prepared 1 mM probe stock solution was added to 90 μL of the prepared solutions to obtain a series of uniformly concentrated (100 μM) sample solutions. The afterglow intensity signal of each sample was acquired and recorded using an IVIS in vivo imaging system (bioluminescence mode). Each condition was measured at least three times. Figure 9 As can be seen, at pH=5, the afterglow emission signal of the probe increases with increasing viscosity.

[0057] (6) RHSO pH / viscosity dual-lock activation verification Verification method for the AND gate response of the RHSO probe: Using PBS buffers at different pH values, the afterglow emission intensity of the probe was tested at viscosities without glycerol and with glycerol at a volume ratio of 50%. 10 μL of the prepared 1 mM probe stock solution was added to 90 μL of solutions with different pH values ​​and viscosities to prepare a series of sample solutions with uniform concentration (100 μM). The afterglow intensity signal of each sample was acquired and recorded using an IVIS in vivo imaging system (bioluminescence mode). Each condition was measured at least three times. Figure 10 As can be seen, viscosity can significantly enhance the afterglow intensity of the probe only under weakly acidic conditions, while under neutral or alkaline conditions, even with increased viscosity, the afterglow intensity of the probe remains very low. Therefore, within the physiological pH range (5-8), the probe must simultaneously satisfy both weak acidity and relatively high viscosity to obtain a strong afterglow signal.

[0058] (7) RHSO cytotoxicity test Cytotoxicity assay (MTT assay): HeLa cells in logarithmic growth phase were digested, resuspended, and the cell density was adjusted to 1 × 10⁶ cells / mL. 4 –1×10 5Cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well. The plates were incubated at 37°C with 5% CO2 for 24 hours to allow for full cell adhesion. After 24 hours, the original culture medium was discarded, and 100 μL of fresh culture medium containing a series of different concentrations (e.g., 0, 10, 20, 30, 40, 50, 60, 70 μM) of RHSO was added to each experimental group, with 3-6 replicates for each concentration. A negative control group (containing cells and drug-free culture medium) was also included. The plates were returned to the incubator for another 12 hours. After treatment, the supernatant was carefully aspirated from each well, and 90 μL of fresh culture medium and 10 μL of MTT solution (5 mg / mL, prepared with PBS) were added to bring the final MTT concentration to 0.5 mg / mL. The 96-well plates were then returned to the incubator and incubated in the dark for 4 hours. Four hours later, carefully aspirate the supernatant from each well. Add 100 μL of dimethyl sulfoxide (DMSO) to each well and place the culture plate on a shaker with low speed for 10 minutes to fully dissolve the purple formazan crystals. Measure the absorbance (OD) of each well at 490 nm using a microplate reader. 490 Cell viability is calculated using the following formula: Cell viability (%) = OD experimental group / OD negative control group × 100% The final results are expressed as mean ± standard deviation, and the cytotoxicity of RHSO was assessed by plotting cell viability-drug concentration curves. The results are as follows: Figure 11 As shown in the figure, the cell viability rate can still reach over 90% when the added probe concentration reaches 70 μM, indicating that the probe has good biocompatibility.

[0059] (8) The effect of RHSO on imaging of mouse joint inflammation model An acute arthritis model was established in C57BL / 6 mice by intra-articular injection of LPS. Control mice received an equal volume of sterile PBS. An RHSO probe (dissolved in physiological saline containing 10% DMSO) was injected orally into both the model and control mice. The mice were pre-illuminated with an incandescent lamp and placed in an IVIS in vivo imaging system (bioluminescence mode) to acquire afterglow signals at the joint sites. Quantitative analysis of the joint regions (ROI) was performed. Figure 12 As shown, compared with the control group, only the joints of the arthritis model group showed a strong afterglow signal, proving that the RHSO probe can specifically respond to the arthritis lesion microenvironment at the in vivo level.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pH / viscosity dual-lock activated persistent luminescence small molecule probe, characterized in that, The structural formula of the afterglow luminescent small-molecule probe is as follows: 。 2. The method for preparing the pH / viscosity dual-lock activated persistent luminescence small-molecule probe of claim 1, characterized in that, The steps are as follows: S1. 4-bromoacetophenone and SeO2 are added into mixed solvent I of dioxane and water, and intermediate 1 is prepared through reaction I; S2. Intermediate 1 and N,N-dimethylaniline are added into mixed solvent II of hexafluoroisopropanol and dichloromethane to react, and intermediate 2 is obtained; S3. Intermediate 2 and mercaptoethanol are reacted in the presence of trimethylsilyl chloride to prepare intermediate 3; intermediate 3 and pinacol diboron are prepared in the presence of a metal palladium complex catalyst and an alkaline environment to obtain intermediate 4; S4. 4-bromo-2-formylbenzoic acid, 3-diethylaminophenol and p-toluenesulfonamide are added into propionic acid, and intermediate 5 is obtained through reaction II; S5. Intermediate 4 and intermediate 5 are added into mixed solvent III of dioxane, water and anhydrous ethanol in the presence of a metal palladium complex catalyst and an alkaline environment, and the afterglow luminescent small-molecule probe is obtained through reaction III. The structural formulas of the intermediate 1, the intermediate 2, the intermediate 3, the intermediate 4 and the intermediate 5 are as follows, respectively: , , , , .

3. The method for preparing the pH / viscosity dual-lock activated afterglow luminescent small molecule probe according to claim 2, characterized in that, In step S1, the molar ratio of 4-bromoacetophenone to SeO2 is 1:1.2-1.5, and the volume ratio of dioxane to water in mixed solvent I is 30:0.5-1; in step S2, the molar ratio of intermediate 1 to N,N-dimethylaniline is 1.2-1.5:

1.

4. The method for preparing the pH / viscosity dual-lock activated afterglow luminescent small molecule probe according to claim 2, characterized in that, In step S1 and step S3, 2-3 mol of intermediate 2 is added per L of mercaptoethanol; the molar ratio of intermediate 3 to pinacol diboron is 1:2-2.

5.

5. The method for preparing the pH / viscosity dual-lock activated afterglow luminescent small molecule probe according to claim 2, characterized in that, In step S4, the molar ratio of 4-bromo-2-formylbenzoic acid, 3-diethylaminophenol and p-toluenesulfonamide is 1:2:0.2-0.5; in step S5, the molar ratio of intermediate 4 to intermediate 5 is 2:1-1.2; and the volume ratio of dioxane, water and anhydrous ethanol in mixed solvent III is 4:1:1-0.

5.

6. The application of the pH / viscosity double-lock activated afterglow luminescent small-molecule probe in claim 1 as an afterglow luminescent imaging agent for non-disease diagnosis and treatment purposes.

7. Use according to claim 5, characterized in that, The activation condition of the afterglow luminescence is pH or viscosity.

8. The use of the pH / viscosity dual-lock activated persistent luminescence small molecule probe of claim 1 as a dual-lock activated persistent luminescence optical imaging agent for non-disease diagnosis and treatment purposes, characterized in that, The activation condition of the double-lock activation is that the pH is lower than 7 and the viscosity is greater than 1.76 mPa·s.

9. The application of the pH / viscosity double-lock activated afterglow luminescent small-molecule probe in claim 1 in the preparation of an inflammatory disease imaging agent for non-disease diagnosis and treatment purposes.

10. Use according to claim 9, characterized in that, The inflammatory disease includes joint inflammation.

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