Activatable biological bionic probe with aggregation-induced emission and application

Abstract

The invention provides an activatable biological bionic probe with aggregation-induced emission and application, the bionic probe is obtained by incubating and combining H2O2-responsive aggregation-induced emission (AIE) molecules MTBPB and dextran particles (GPs), the bionic probe can specifically target macrophages, the infiltrated macrophages can generate a large amount of H2O2 under the stimulation of inflammatory cytokines, and the activity of the H2O2 is enhanced. Therefore, the fluorescent signal generated by the bionic probe is activated and can be used for early diagnosis of transplant rejection, and the accuracy is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, in particular to an activatable biomimetic probe with aggregation-induced emission and applications thereof. Background Art

[0002] Organ transplantation remains the most effective clinical intervention for patients with end-stage organ failure. However, despite significant advances in surgical techniques and the efficacy of immunosuppressive therapy, transplant rejection (TR) remains the main cause of transplant failure. The current gold standard in clinical practice is pathological biopsy. However, biopsy is invasive, increases the risk of infection in patients, and may produce false-negative results. Therefore, the development of non-invasive methods for early diagnosis of TR is crucial for the effective prevention and management of TR and has important clinical value.

[0003] Clinical studies have shown that the extent of macrophage infiltration in biopsy tissues of transplant patients is directly related to transplant prognosis. In the early stages of transplant rejection, the graft shows obvious macrophage infiltration, which directly leads to the rejection process through multiple pathways. Infiltrating macrophages release a variety of inflammatory cytokines, amplify the inflammatory response, recruit leukocytes, and induce donor-specific cytotoxic reactions. Recent studies have also found that macrophages can cause graft dysfunction through a unique mechanism known as "trained immunity." Therefore, real-time monitoring of macrophage infiltration in the graft is expected to diagnose transplant rejection at an early stage.

[0004] Several molecular imaging probes have been developed, most of which lack the ability to actively target macrophages. Ostroff et al. first reported on glucan particles (GPs) derived from yeast extract, demonstrating their ability to mimic the yeast intestinal infection pathway. After oral administration, GPs are transported by intestinal epithelial M cells to the gut-associated lymphoid tissue, where they specifically target macrophages. This pioneering study proposed a new strategy for disease diagnosis and treatment using oral GPs to target macrophages. Based on these findings, Hsing-Wen Sung et al. developed DOX-loaded glucan nanoparticles that can cross the intestinal epithelial barrier after oral administration. These nanoparticles are phagocytosed by macrophages and then transported across the blood-brain barrier by macrophages to deliver drugs to the glioblastoma site, achieving "gut-to-brain" targeted treatment of glioblastoma. Together, these studies highlight that GPs are an ideal biomimetic carrier with the potential to target oral macrophages, and therefore have the potential to be used to diagnose transplant rejection. However, it is still not feasible to directly connect target molecules to GPs, and positively charged nanoparticles must be prepared in advance, or polymer materials must be used to encapsulate the target molecules in GPs. This additional complexity poses a challenge to the construction of GPs-based biomimetic probes.

[0005] Our previous research found that cationic aggregation-induced emission (AIE) molecules can be stably bound to dextran particles (GPs) in aqueous solution. Based on this discovery, we successfully developed AIE-based biomimetic probes using dextran particles. After oral administration, these probes are specifically engulfed by macrophages and migrate with macrophages to the site of transplant rejection, thereby achieving non-invasive diagnosis of rejection. However, the fluorescence signal of the biomimetic probe remains in an "always on" state, causing macrophages to emit signals even in the absence of transplant rejection. This reduces the contrast of the graft signal during imaging and affects the accuracy of diagnosis. Summary of the invention

[0006] The purpose of the present invention is to propose an activatable biomimetic probe with aggregation-induced emission, which can specifically target macrophages. The infiltrating macrophages will produce a large amount of H2O2 under the stimulation of inflammatory cytokines, thereby activating the fluorescence signal generated by the biomimetic probe, which can be used for early diagnosis of transplant rejection reaction and improve the accuracy of diagnosis.

[0007] The technical solution of the present invention is achieved in this way: The first aspect of the present invention is to provide an aggregation-induced emission molecule responsive to H2O2, which is MTBPB and has a structural formula shown in Formula I: .

[0008] The second aspect of the present invention is to provide a method for preparing H2O2-responsive aggregation-induced emission molecules, comprising the following steps: S1. Pyridine-4-boronic acid and 4,7-dibromo-benzo[c]-[1,2,5]thiadiazole undergo coupling reaction in the presence of palladium catalyst to generate PBTBBr; S2. PBTBBr and [4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid undergo coupling reaction in the presence of palladium catalyst to generate TPABTBP; S3. TPABTBP and 4-bromomethylphenylboronic acid pinacol ester were refluxed under heating to obtain MTBPB; The reaction route of the preparation process of the MTBPB is as follows: .

[0009] In the above preparation scheme, in step S1: The palladium catalyst is Pd(PPh3)4; And / or, the reaction solvent is DMF; And / or, a basic salt is added during the reaction process; And / or, the reaction process is carried out under an inert atmosphere.

[0010] In the above preparation scheme, in step S2: The palladium catalyst is Pd(PPh3)4; And / or, the reaction solvent is DMF; And / or, a basic salt is added during the reaction process; And / or, the reaction process is carried out under an inert atmosphere.

[0011] In the above preparation scheme, in step S3: The reaction solvent is DMF; And / or, the molar ratio of TPABTBP to 4-bromomethylphenylboronic acid pinacol ester is 1:(1-1.5).

[0012] The third aspect of the present invention is to provide an activatable biomimetic probe with aggregation-induced emission, which is obtained by incubating the H2O2-responsive aggregation-induced emission molecules described in the first aspect or the H2O2-responsive aggregation-induced emission molecules obtained by the preparation method described in the second aspect with dextran particles.

[0013] The fourth aspect of the present invention is to propose the use of the activatable biomimetic probe with aggregation-induced emission described in the third aspect in the preparation of an M1 macrophage tracer.

[0014] Furthermore, the activatable biomimetic probe with aggregation-induced emission generates a fluorescent signal under the stimulation of inflammatory cytokines.

[0015] The fifth aspect of the present invention is to propose the use of the activatable biomimetic probe with aggregation-induced emission described in the third aspect in the preparation of a diagnostic reagent for transplant rejection.

[0016] Furthermore, the diagnostic reagent is used for early diagnosis of transplant rejection.

[0017] Furthermore, the diagnostic reagent is for oral administration.

[0018] Furthermore, the transplant rejection diagnosis process is: diagnosing the severity of the rejection based on the fluorescence intensity generated by the activatable biomimetic probe with aggregation-induced emission.

[0019] Compared with the prior art, the beneficial effects of the present invention are: The present invention proposes a H2O2-responsive aggregation-induced emission (AIE) molecule MTBPB, which contains a borate reaction site and can respond to H2O2 stimulation. Therefore, it can be used to prepare an activatable biomimetic probe with aggregation-induced emission.

[0020] The activatable biomimetic probe with aggregation-induced emission proposed in the present invention is obtained by incubating and combining the H2O2-responsive aggregation-induced emission (AIE) molecule MTBPB with dextran particles (GPs). The biomimetic probe can specifically target macrophages. The infiltrated macrophages will produce a large amount of H2O2 under the stimulation of inflammatory cytokines, thereby activating the fluorescence signal generated by the biomimetic probe, which can be used for early diagnosis of transplant rejection reactions.

[0021] When the activatable biomimetic probe with aggregation-induced emission provided by the present invention is used for early diagnosis of transplant rejection reaction, the severity of the rejection reaction is diagnosed based on the fluorescence intensity generated by the activatable biomimetic probe with aggregation-induced emission. Since the fluorescence intensity is caused by H2O2 produced by infiltrating macrophages under the stimulation of inflammatory cytokines, no fluorescence signal will be generated in the absence of transplant rejection reaction, thereby improving the accuracy of transplant rejection reaction diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 is the MTBPB in Example 1 1 H NMR spectrum.

[0024] Figure 2 is the MTBPB in Example 1 13 C NMR spectrum.

[0025] Figure 3 This is the HRMS spectrum of MTBPB in Example 1.

[0026] Figure 4 These are the characterization and performance evaluation results of MTBPB / GPs in Example 2.

[0027] Figure 5 This is the result of flow cytometry detection of the pro-inflammatory M1 macrophage phenotype after LPS stimulation for 24 hours in Example 3.

[0028] Figure 6 This is the secretion of inflammatory cytokines (TNF-α, IFN-γ) by macrophages after 24 hours of LPS stimulation in Example 3.

[0029] Figure 7These are the confocal visualization results and flow cytometry results of detecting intracellular ROS content in M1 macrophages after 24 hours of LPS stimulation in Example 3. DAPI and FITC are used to display cell nuclei and ROS staining, respectively. Scale bar: 10 μm.

[0030] Figure 8 These are the confocal display results and flow cytometry results of detecting the fluorescence intensity of MTBPB / GPs in macrophages after 24 hours of LPS stimulation in Example 4. DAPI shows nuclear staining, scale: 10 μm.

[0031] Fig. 9 These are the cytotoxic results of MTBPB / GPs on macrophages, liver and kidney cells in Example 5.

[0032] Fig.10 This is the migration result of MTBPB / GPs on macrophages in Example 5.

[0033] Fig.11 These are the results of fluorescence quantitative imaging of the allogeneic mouse skin transplant model in Example 6 at 0, 6, 12, 24, 36, and 48 hours after oral administration of MTBPB / GPs.

[0034] Fig.12 This is the test result of Example 6 for the ability to diagnose allogeneic transplant rejection at an early stage after oral administration of MTBPB / GPs.

[0035] Fig.13 This is an immunofluorescence co-localization image of the transplanted skin after oral administration of MTBPB / GPs in Example 6. DAPI, FITC, and Cy5 are used to show cell nuclei, and CD68 and iNOS staining are used, respectively. Scale bar: 20 μm.

[0036] Fig.14 These are the results of fluorescence quantitative imaging of allogeneic mouse skin transplantation in vivo after oral administration of MTBPB and MTBPB / GPs in Example 6. DETAILED DESCRIPTION

[0037] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1 Preparation of MTBPB 1) Synthesis of PBTBBr Reagents: pyridine-4-boronic acid (200 mg, 1.62 mmol), 4,7-dibromo-benzo[c][1,2,5]thiadiazole (472.8 mg, 1.62 mmol), potassium carbonate (250 mg), Pd(PPh3)4 (20 mg).

[0039] Solvent: 10 mL DMF, 2 mL water.

[0040] Reaction conditions: The mixture was stirred at 400 rpm under nitrogen atmosphere and heated under reflux to 80°C for 24 hours.

[0041] Post-treatment: After the reaction was cooled to 25°C, it was extracted with ethyl acetate under anhydrous conditions (extraction three times), the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure.

[0042] Purification: Silica gel column chromatography was used with a petroleum ether / ethyl acetate mixture (25:1, volume ratio) as the eluent.

[0043] Product: PBTBBr (226.2 mg, 48% yield).

[0044] The product spectral data are as follows: 1 H NMR (600 MHz, CDCl3), δ (ppm): 8.78-8.79 (m, 2H), 7.98-7.99 (d, 1H), 7.86-7.87 (d, 2H), 7.69-7.70 (d, 1H); 13 C NMR (150 MHz, CDCl3), delta (ppm): 153.96, 152.48, 150.22, 143.94, 132.12, 130.89, 128.92, 123.48, 115.42.

[0045] 2) Synthesis of TPABTBP Reactants: PBTBBr (55.1 mg, 0.19 mmol) and [4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid (66.3 mg, 0.19 mmol).

[0046] Solvent: 5 mL DMF.

[0047] Reaction conditions: The solution was stirred at 600 rpm at ℃. Then an aqueous solution containing potassium carbonate (50 mg) was added, and 10 mg of Pd(PPh3)4 was added as a catalyst. The mixture was refluxed at 80℃ under nitrogen atmosphere for 12 hours.

[0048] Confirmation of reaction completion: by thin layer chromatography (TLC).

[0049] Post-treatment: After the reaction was completed, the solvent was removed under reduced pressure (0.1 MPa).

[0050] Purification: Silica gel column chromatography was used with a petroleum ether / ethyl acetate mixture (25:1, volume ratio) as the eluent.

[0051] Product: TPABTBP (32.8% yield).

[0052] Product spectral data: 1 H NMR (600 MHz, CDCl3), (ppm): 8.92-8.93 (d, 2H), 8.58-8.57 (d, 2H), 8.07-8.09 (d, 1H), 7.88-7.90 (d, 2H), 7. 83-7.85 (d, 1H), 7.16-7.17 (m, 4H), 7.05-7.06 (d, 2H), 6.88-6.90 (d, 4H), 3.83 (s, 6H); 13 C NMR (150 MHz, CDCl3), (ppm): 156.63, 154.11, 153.19, 152.36, 150.13, 142.52, 139.92, 137.88, 130 .87, 130.26, 127.47, 127.02, 125.83, 125.29, 124.72, 118.92, 114.91, 55.53; HRMS (ESI+): calculated: C 31 H 25 N4O2S + [M+H] + : 517.1693, measured value: 517.1689.

[0053] 3) Synthesis of MTBPB Reactants: TPABTBP (82.6 mg, 0.16 mmol) and 4-bromomethylphenylboronic acid pinacol ester (62.9 mg, 0.20 mmol).

[0054] Solvent: 15 mL anhydrous DMF.

[0055] Reaction conditions: The mixture was heated to reflux and the reaction was continued for 8 hours.

[0056] Post-treatment: After the reaction mixture was cooled to room temperature, the precipitate was collected by filtration and washed thoroughly with acetone to obtain a red solid with a yield of 68.5%.

[0057] Product spectral data: 1 H NMR (600 MHz, CD3OD), (ppm): 9.05-9.07 (m, 2H), 8.96-8.97 (m, 2H), 8.40-8.42 (m, 1H), 7.96-7.99 (m, 3H), 7.85-7.86 (m, 1H), 7.73-7.74 (m, 1H), 7.5 0-7.54 (m, 2H), 7.10-7.12 (d, 4H), 6.91-6.97 (m, 6H), 5.85-5.87 (d, 2H), 3.80 (s, 6H), 1.20-1.34 (m, 11H), 0.88-0.99 (m, 1H); Figure 1 shown.

[0058] 13 C NMR (150 MHz, CD3OD), (ppm): 158.36, 155.28, 154.87, 154.83, 154.62, 151.70, 145.48, 145.43, 141.36, 13 9.68, 137.80, 136.92, 135.82, 133.73, 133.71, 131.67, 129.22, 129.12, 128.59, 128 .47, 127.90, 127.09, 124.56, 119.86, 117.95, 116.04, 85.52, 75.88, 64.90, 64.85, 56.01, 33.12, 30.82, 30.79, 30.64, 30.51, 30.36, 30.28, 25.22, 25.07, 23.78, 14.48; Figure 2 shown.

[0059] HRMS (ESI+): calculated: C 44 H 42 BN4O4S + [M-Br] + : 733.30, measured value: 733.3006. Figure 3 shown.

[0060] Example 2 Preparation and characterization of MTBPB / GPs 1) Thermal acid-base extraction of glucan particles (GPs) GPs were prepared by hot acid-base extraction: 13 g of Angel Yeast was accurately weighed, dispersed in 200 mL of 1 M NaOH using an ultrasonic cleaner, and placed in a water bath at 80°C for 1 hour. After the mixture was cooled to room temperature, it was centrifuged (2000 × g) for 5 minutes to separate the precipitate, and the obtained sample was resuspended in 200 mL of HCL solution pH = 4-5, and then stirred in a water bath at 60°C for 1 hour. The suspension was centrifuged at 2000 × g for 5 minutes to obtain a precipitate, which was washed 4 times with isopropanol and 2 times with acetone, and the precipitate was collected by centrifugation (2000 × g). Finally, GPs were obtained by drying at room temperature; 2) Preparation of MTBPB / GPs GPs (10 mg) were mixed in deionized water (1 mL). Then, 100 μL of MTBPB 1 mM ethanol solution was added to the above system, incubated in a shaker in the dark for 12 h, centrifuged (3000 rpm 2 min) to collect the precipitate, and washed with deionized water (3 times) to obtain the biomimetic dextran particle probe (MTBPB / GPs) with aggregation-induced emission effect, which was stored in the dark at 4 ° C for future use; 3) Characterization and evaluation of MTBPB / GPs The synthesized glucan particles (GPs) were characterized. SEM images showed that they were similar to the smooth yeast microcapsules ( Figure 4 A) Compared with GPs, the surface of GPs shows obvious wrinkles and pores, and the pore size is about 500-600 nm ( Figure 4 B). TEM image shows that untreated yeast microvesicles remain intact ( Figure 4 C), while obvious drug-loading space can be seen inside the extracted GPs ( Figure 4 D). TEM images of MTBPB / GPs further show that the interior of GPs is filled with MTBPB ( Figure 4 E).

[0061] The particle size and Zeta potential of yeast microcapsules, GPs and MTBPB / GPs were evaluated by dynamic light scattering (DLS) technology. The results showed that the particle size of yeast microcapsules was about 4.4 μm ( Figure 4 F), while the particle sizes of GPs and MTBPB / GPs after extraction were approximately 2.3 μm and 2.9 μm, respectively ( Figure 4 G, 4H), which is consistent with the morphological observation of the electron microscopy image above. In addition, due to its positive charge, MTBPB can stably bind to the dextran particles in aqueous solution and thus be successfully loaded into the particles ( Figure 4 I).

[0062] In the FT-IR spectra of GPs and MTBPB / GPs ( Figure 4 J), 1078 cm-1 The peak at 2919 cm -1 The peak at 3352 cm -1 The peaks at 2917 and 2949 cm-1 represent the stretching vibration of the -OH group. -1 The stretching vibration of alkyl CH appeared at 1504 cm -1 The C=C stretching vibration of the aromatic ring appears at 700-900cm -1 The peaks in the interval correspond to the CH bending vibration on the benzene ring. In the spectrum of MTBPB / GPs, the CH bending peak of the benzene ring and the C=C stretching vibration peak of the aromatic ring did not appear, indicating that MTBPB has been successfully encapsulated in the cavity of GPs. The XRD spectrum of MTBPB / GPs shows obvious crystallization peaks at 31.9° and 45.3°, further proving that MTBPB has formed a crystalline state in GPs ( Figure 4 K).

[0063] The UV absorption spectrum of MTBPB showed that MTBPB had obvious absorption at 310 and 540 nm wavelengths, and weak emission when excited at 520 nm. After the addition of H2O2 (5 μM), the absorption peak intensity at 540 nm decreased, and a new absorption peak appeared at 506 nm (Figure 4L). In addition, after the introduction of H2O2 (5 μM), the fluorescence emission spectrum showed that the fluorescence intensity of MTBPB at 670 nm increased significantly ( Figure 4 M). Fluorescence titration studies showed that the signal at 670 nm continued to amplify with the increase of H2O2 concentration ( Figure 4 N). In addition, the fluorescence intensity at 670 nm showed a linear relationship with the concentration of H2O2 (0-6 μM) (R2 = 0.97), confirming the high sensitivity of MTBPB to H2O2 ( Figure 4 O).

[0064] Adjust the pH value of PBS to 4-10 with dilute hydrochloric acid and sodium hydroxide, respectively (step 1). Take 10ul MTBPB (1mM) in 1ml PBS with different pH values. Use a fluorescence spectrometer (Ex=520nm) to measure the fluorescence spectrum, and choose the fluorescence intensity at 670nm as the comparison benchmark. Other analytes such as Glu, Cys, GSH, CH3COOH, ZnCl2, CuCl2, MgSO4, NaCl, KCl, H2O2, etc. are dissolved in PBS (10 mM, pH = 7.0). Detect the fluorescence reaction of MTBPB (10μM) with other species under the same parameters, such as Figure 4As shown in P, the fluorescence reaction confirmed the specificity of MTBPB for H2O2.

[0065] Example 3 Identification of pro-inflammatory M1 macrophages RAW 264.7 (1*10 per hole 5 ) were seeded in 6-well plates and divided into two groups-LPS group and control group. The LPS group was cultured with 1μg / ml LPS for 24 hours, and the control group was cultured with complete culture medium only. After 24 hours of LPS (1μg / ml) stimulation, the cells were collected into centrifuge tubes. Each centrifuge tube was incubated with 10μl of dead and live dye for 30 minutes, and then rinsed twice with PBS. Then, 2ul F4 / 80 and CD86 antibodies were added to each tube, incubated in the dark for 40 minutes, and then rinsed 3 times with PBS. RAW264.7 cells were resuspended in 200 μl PBS. The expression levels of F4 / 80 and CD86 were determined using a flow cytometer (BV421 and PE-Cy7 channels). RAW264.7 (8*10 per well) were plated in 6-well plates. 3 The cells were inoculated into 96-well plates, which was consistent with the above stimulation conditions. After 24 hours of LPS (1 μg / ml) stimulation, the cell supernatant was collected. The expression levels of TNF-α and IFN-γ in the cell supernatant were determined by Elisa.

[0066] RAW264.7 cells (1*10 per well 5 ) were seeded in 6-well plates or confocal culture dishes and stimulated with 1 μg / ml LPS for 24 hours. The cells were then incubated with diluted DCFH-DA (10 μM) at 37°C for 45 minutes and then washed 5 times with PBS. The cells were resuspended in 200 μl PBS for flow cytometry analysis to determine ROS levels (FITC channel). The cells in the confocal culture dishes were washed 3 times with PBS and then fixed with 4% paraformaldehyde. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence imaging was then performed using a laser scanning confocal microscope (LSCM), and the levels of intracellular ROS were quantitatively compared by flow cytometry.

[0067] The results showed that after LPS stimulation, the expression of CD86 on the cell membrane increased significantly, indicating that macrophages were polarized toward M1 type ( Figure 5 In addition, the expression of inflammatory cytokines TNF-α and IFN-γ secreted by macrophages increased significantly ( Figure 6 ). It was further confirmed that macrophages polarize toward a pro-inflammatory phenotype, and after polarization, M1 macrophages will produce a large amount of ROS ( Figure 7 ).

[0068] Example 4 ROS responsiveness of MTBPB / GPs in macrophages RAW264.7 cells (1*10 per well) 5 ) were seeded into 6-well plates or confocal culture dishes and stimulated with 1ug / ml LPS for 24 hours. Then, MTBPBs / GPs were added to RAW264.7 cells (1*10 5 The cells were incubated with PBS (control group) or PBS at 37°C for 24 hours. LSCM and flow cytometry were used to qualitatively and quantitatively observe the fluorescence intensity of the probe, respectively (Ex=480 nm, Em=620 nm).

[0069] Figure 8 The results showed that MTBPB / GPs in pro-inflammatory macrophages were significantly stronger than those in undifferentiated M0 macrophages, about 2.7 times stronger, confirming that MTBPB / GPs can target and trace pro-inflammatory M1 macrophages.

[0070] Example 5 Cytotoxicity of MTBPB / GPs Divided into negative control group and MTBPB / GPs group. The negative control group contained only DMEM medium. The ratio of macrophage number to MTBPB / GPs was 1:20, 1:50, 1:100 and 1:200, respectively. The viability of macrophages in each group was detected using CCK-8 kit. In the same way, the cytotoxicity of MTBPB / GP on cardiomyocytes (H29C2), hepatocytes (AML12) and kidney cells (293T) was detected using CCK-8 kit.

[0071] Fig. 9 The results showed that MTBPB / GPs had no significant cytotoxicity to macrophages after being co-incubated with macrophages for 24 hours ( Fig. 9 a). In addition, MTBPB / GPs had no obvious cytotoxicity to heart, liver, and kidney cells ( Fig. 9 bd). MTBPB / GPs have good biosafety and can be used in subsequent animal experiments.

[0072] Example 5 Effect of MTBPB / GPs on the migration function of macrophages RAW264.7 cells (1*10 per well) 5 ) were inoculated in 6 wells and cultured for 12 hours, and then MTBPB / GPs (1*10 5MTBPB / GPs) were cultured for another 12 h. Subsequently, these cells were seeded into the upper layer of the transwell chamber, and the lower layer of the chamber was filled with 20% culture medium containing 10 ng of monocyte chemoattractant protein-1 (MCP-1). After 12 h of cell culture, the cells were fixed with paraformaldehyde and stained with DAPI. Finally, the cells were observed under a fluorescence microscope, and the number of migrated cells was recorded.

[0073] Fig.10 The results showed that the migration ability of macrophages was not significantly affected after phagocytizing MTBPB / GPs.

[0074] Example 6 Fluorescence imaging of allogeneic transplantation after oral administration of MTBPB / GPs On the sixth day after surgery, allogeneic mice and syngeneic mice (5 mice in each group) were orally administered MTBPB / GPs (200 μl, 0.8 mg / kg MTBPB). In vivo imaging of small animals was performed at 0, 6, 12, 24, 36, and 48 h after oral administration (Perkin Elmer IVISLumina: Ex=480nm, Em=620nm).

[0075] The results showed that the fluorescence intensity value in the grafted skin increased at 6 hours. As time went on, the fluorescence intensity value of the grafted skin gradually increased and reached a peak at 36 hours after oral administration ( Fig.11 Therefore, 36h was chosen as the optimal imaging time point. Fig.12 The results showed that MTBPB / GPs can diagnose transplant rejection at an early stage.

[0076] First, this study successfully established a skin transplant rejection model ( Fig.12 a, 12b). Immunofluorescence results showed that the infiltration degree of M1 macrophages increased with the degree of allogeneic transplant rejection ( Fig.12 c, 12d). Small animal in vivo imaging was performed on days 1, 3, 5, and 7 after surgery. The results showed that MTBPB / GPs could be used to diagnose transplant rejection as early as day 3 after surgery (p<0.001) ( Fig.12 e, 12f). In summary, MTBPB / GPs can target and trace macrophages and migrate to the transplanted skin with macrophages, thereby diagnosing transplant rejection at an early stage. The fluorescence co-localization image of the transplanted skin also shows that MTBPB / GPs are engulfed by pro-inflammatory M1 macrophages in the transplanted skin ( Fig.13 ).

[0077] In addition, to verify the in vivo targeting of MTBPB / GPs, we orally administered MTBPB / GPs and MTBPB to allogeneic transplanted mice on the 6th day after surgery, and quantified the fluorescence intensity of the transplanted skin 36 hours later. The fluorescence intensity of the MTBPB / GPs group was approximately 1.67 times that of the MTBPB group, confirming that MTBPB was transported to the transplanted skin via GPs ( Fig.14 ).

[0078] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A H2O2-responsive aggregation-induced emission molecule, characterized in that: The molecule is MTBPB, having the structural formula shown in Formula I: 。 2. A method for preparing H2O2-responsive aggregation-induced emission molecules, characterized in that the steps include: S1. Pyridine-4-boronic acid and 4,7-dibromo-benzo[c]-[1,2,5]thiadiazole undergo coupling reaction in the presence of palladium catalyst to generate PBTBBr; S2. PBTBBr and [4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid undergo coupling reaction in the presence of palladium catalyst to generate TPABTBP; S3. TPABTBP and 4-bromomethylphenylboronic acid pinacol ester were refluxed under heating to obtain MTBPB; The reaction route of the preparation process of the MTBPB is as follows: 。 3. The preparation method according to claim 2, characterized in that: In step S1: The palladium catalyst is Pd(PPh3)4; And / or, the reaction solvent is DMF; And / or, a basic salt is added during the reaction process; And / or, the reaction process is carried out under an inert atmosphere.

4. The preparation method according to claim 2, characterized in that: In step S2: The palladium catalyst is Pd(PPh3)4; And / or, the reaction solvent is DMF; And / or, a basic salt is added during the reaction process; And / or, the reaction process is carried out under an inert atmosphere.

5. The preparation method according to claim 2, characterized in that: In step S3: The reaction solvent is DMF; And / or, the molar ratio of TPABTBP to 4-bromomethylphenylboronic acid pinacol ester is 1:(1-1.5).

6. An activatable biomimetic probe with aggregation-induced emission, characterized in that: The H2O2 responsive aggregation-induced emission molecule is obtained by incubating the H2O2 responsive aggregation-induced emission molecule described in claim 1 or the H2O2 responsive aggregation-induced emission molecule obtained by the preparation method described in any one of claims 2-5 with dextran particles.

7. Use of the activatable biomimetic probe according to claim 6 in the preparation of an M1 macrophage tracer.

8. The use according to claim 7, characterized in that The activatable biomimetic probe generates a fluorescent signal under the stimulation of inflammatory cytokines.

9. Use of the activatable biomimetic probe according to claim 6 in the preparation of a diagnostic reagent for transplant rejection.

10. The use according to claim 9, characterized in that The diagnostic reagent is used for early diagnosis of transplant rejection; and / or, the diagnostic reagent is for oral administration; And / or, the diagnosis of transplant rejection reaction is based on the fluorescence intensity generated by the activatable biomimetic probe to diagnose the severity of the rejection reaction.

Images