Activatable biomimetic probes with aggregation-induced emission and their applications

Abstract

The present invention proposes an activatable biomimetic probe with aggregation-induced emission and its application. The biomimetic probe 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. Infiltrating macrophages 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 and improve accuracy.

Description

Technical Field

[0001] The present 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 therapies, transplant rejection (TR) remains the leading cause of transplant failure. The current gold standard in clinical practice is biopsy. However, biopsy is invasive, increases the risk of infection, and can produce false-negative results. Therefore, the development of noninvasive methods for the early diagnosis of TR is crucial for its effective prevention and management and holds significant clinical value.

[0003] Clinical studies have shown that the extent of macrophage infiltration in transplant patient biopsies directly correlates with transplant outcome. In the early stages of transplant rejection, the graft exhibits significant macrophage infiltration, which directly contributes to the rejection process through multiple pathways. Infiltrating macrophages release a variety of inflammatory cytokines, amplifying the inflammatory response, recruiting leukocytes, and inducing donor-specific cytotoxic responses. Recent studies have also revealed that macrophages can cause graft dysfunction through a unique mechanism known as "trained immunity." Therefore, real-time monitoring of macrophage infiltration in grafts holds promise for early diagnosis of transplant rejection.

[0004] Several molecular imaging probes have been developed, but most 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 intestinal infection pathway of yeast. Following oral administration, GPs are transported by intestinal epithelial M cells to the gut-associated lymphoid tissue, where they specifically target macrophages. This groundbreaking study proposed a novel strategy for disease diagnosis and treatment using oral GPs to target macrophages. Building 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 to deliver the drug to glioblastoma sites, achieving "gut-to-brain" targeted therapy for glioblastoma. These studies collectively highlight GPs as an ideal biomimetic carrier with the potential to target oral macrophages, thus potentially being used for the diagnosis of transplant rejection. However, it is still not feasible to directly connect target molecules to GPs. Positively charged nanoparticles must be prepared in advance, or polymer materials must be used to encapsulate the target molecules within GPs. This additional complexity poses a challenge to the construction of GPs-based biomimetic probes.

[0005] Our previous research discovered that cationic aggregation-induced emission (AIE) molecules can stably bind to dextran particles (GPs) in aqueous solution. Building on this discovery, we successfully developed AIE-based biomimetic probes using dextran particles. Following oral administration, these probes are specifically phagocytosed by macrophages and migrate with them to sites of transplant rejection, enabling noninvasive diagnosis of transplant rejection. However, the fluorescence signal of the biomimetic probes remains "always on," causing macrophages to emit signals even in the absence of transplant rejection. This reduces the contrast of the transplant signal during imaging, compromising diagnostic accuracy. Summary of the Invention

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

[0007] The technical solution of the present invention is achieved as follows:

[0008] The first aspect of the present invention is to provide an H2O2-responsive aggregation-induced emission molecule, which is MTBPB and has the structural formula shown in Formula I:

[0009] .

[0010] The second aspect of the present invention is to provide a method for preparing an H2O2-responsive aggregation-induced emission molecule, comprising the following steps:

[0011] S1. Pyridine-4-boronic acid and 4,7-dibromo-benzo[c]-[1,2,5]thiadiazole undergo a coupling reaction in the presence of palladium catalyst to produce PBTBBr;

[0012] S2. PBTBBr reacts with [4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid in the presence of palladium catalyst to produce TPABTBP;

[0013] S3. TPABTBP and 4-bromomethylphenylboronic acid pinacol ester were reacted under heating and reflux to obtain MTBPB;

[0014] The reaction scheme of the preparation process of the MTBPB is as follows:

[0015] .

[0016] In the above preparation scheme, in step S1:

[0017] The palladium catalyst is Pd(PPh3)4;

[0018] And / or, the reaction solvent is DMF;

[0019] And / or, an alkaline salt is added during the reaction process;

[0020] And / or, the reaction process is carried out under an inert atmosphere.

[0021] In the above preparation scheme, in step S2:

[0022] The palladium catalyst is Pd(PPh3)4;

[0023] And / or, the reaction solvent is DMF;

[0024] And / or, an alkaline salt is added during the reaction process;

[0025] And / or, the reaction process is carried out under an inert atmosphere.

[0026] In the above preparation scheme, in step S3:

[0027] The reaction solvent is DMF;

[0028] And / or, the molar ratio of TPABTBP to 4-bromomethylphenylboronic acid pinacol ester is 1:(1-1.5).

[0029] The third aspect of the present invention is to propose an activatable biomimetic probe with aggregation-induced emission, which is obtained by incubating the H2O2-responsive aggregation-induced emission molecule described in the first aspect or the H2O2-responsive aggregation-induced emission molecule obtained by the preparation method described in the second aspect with dextran particles.

[0030] 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.

[0031] Furthermore, the activatable biomimetic probe with aggregation-induced emission generates a fluorescent signal under the stimulation of inflammatory cytokines.

[0032] 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.

[0033] Furthermore, the diagnostic reagent is used for early diagnosis of transplant rejection.

[0034] Furthermore, the diagnostic reagent is for oral administration.

[0035] 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.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 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.

[0038] 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. Infiltrating macrophages 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.

[0039] When the activatable biomimetic probe with aggregation-induced emission provided by the present invention is used for early diagnosis of transplant rejection, 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 fluorescent signal is generated in the absence of transplant rejection, thereby improving the accuracy of transplant rejection diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0041] Figure 1 is the MTBPB in Example 1 1 H NMR spectrum.

[0042] Figure 2 is the MTBPB in Example 1 13 C NMR spectrum.

[0043] Figure 3 This is the HRMS spectrum of MTBPB in Example 1.

[0044] Figure 4These are the characterization and performance evaluation results of MTBPB / GPs in Example 2.

[0045] Figure 5 This is the result of flow cytometry detection of the pro-inflammatory M1 macrophage phenotype after 24 hours of LPS stimulation in Example 3.

[0046] Figure 6 This is the secretion of inflammatory cytokines (TNF-α, IFN-γ) by macrophages after 24 hours of LPS stimulation in Example 3.

[0047] Figure 7 These are the confocal imaging 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 visualize cell nuclei and ROS staining, respectively. Scale bar: 10 μm.

[0048] Figure 8 These are the confocal microscopy and flow cytometry results of the fluorescence intensity of MTBPB / GPs detected in macrophages 24 hours after LPS stimulation in Example 4. DAPI indicates nuclear staining. Scale bar: 10 μm.

[0049] Figure 9 These are the cytotoxic results of MTBPB / GPs on macrophages, liver and kidney cells in Example 5.

[0050] Figure 10 This is the migration result of MTBPB / GPs on macrophages in Example 5.

[0051] Figure 11 These are the fluorescence quantitative results of in vivo imaging of the allogeneic mouse skin transplant model in Example 6 after oral administration of MTBPB / GPs at 0, 6, 12, 24, 36, and 48 hours.

[0052] Figure 12 This is the test result of the ability to diagnose allograft rejection in early stage after oral administration of MTBPB / GPs in Example 6.

[0053] Figure 13 This is an immunofluorescence colocalization image of the transplanted skin after oral administration of MTBPB / GPs in Example 6. DAPI, FITC, and Cy5 are used to visualize cell nuclei, and CD68 and iNOS staining, respectively. Scale bar: 20 μm.

[0054] Figure 14 These are the fluorescence quantitative results of in vivo imaging of allogeneic mouse skin transplants after oral administration of MTBPB and MTBPB / GPs in Example 6. DETAILED DESCRIPTION

[0055] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0056] Example 1 Preparation of MTBPB

[0057] 1) Synthesis of PBTBBr

[0058] 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).

[0059] Solvent: 10 mL DMF, 2 mL water.

[0060] Reaction conditions: The mixture was stirred at 400 rpm under nitrogen atmosphere and heated to reflux at 80°C for 24 hours.

[0061] Post-treatment: After the reaction was cooled to 25°C, it was extracted with ethyl acetate under anhydrous conditions (extraction three times), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure.

[0062] Purification: Silica gel column chromatography was performed using a petroleum ether / ethyl acetate mixture (25:1, volume ratio) as the eluent.

[0063] Product: PBTBBr (226.2 mg, 48% yield).

[0064] The product spectral data are as follows:

[0065] 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);

[0066] 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.

[0067] 2) Synthesis of TPABTBP

[0068] Reactants: PBTBBr (55.1 mg, 0.19 mmol) and [4-[bis(4-methoxyphenyl)amino]phenyl]boronic acid (66.3 mg, 0.19 mmol).

[0069] Solvent: 5 mL DMF.

[0070] Reaction conditions: The solution was stirred at 600 rpm at 1°C. An aqueous solution containing 50 mg of potassium carbonate and 10 mg of Pd(PPh3)4 as a catalyst was then added. The mixture was refluxed at 80°C under a nitrogen atmosphere for 12 hours.

[0071] Confirmation of reaction completion: by thin layer chromatography (TLC).

[0072] Post-treatment: After the reaction was completed, the solvent was removed under reduced pressure (0.1 MPa).

[0073] Purification: Silica gel column chromatography was performed using a petroleum ether / ethyl acetate mixture (25:1, volume ratio) as the eluent.

[0074] Product: TPABTBP (32.8% yield).

[0075] Product spectral data:

[0076] 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);

[0077] 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;

[0078] HRMS (ESI+): calculated: C 31 H 25 N4O2S +[M+H] + : 517.1693, measured value: 517.1689.

[0079] 3) Synthesis of MTBPB

[0080] Reactants: TPABTBP (82.6 mg, 0.16 mmol) and 4-bromomethylphenylboronic acid pinacol ester (62.9 mg, 0.20 mmol).

[0081] Solvent: 15 mL anhydrous DMF.

[0082] Reaction conditions: The mixture was heated to reflux and the reaction was continued for 8 hours.

[0083] 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%.

[0084] Product spectral data:

[0085] 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.

[0086] 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; e.g. Figure 2 shown.

[0087] HRMS (ESI+): calculated: C 44 H 42 BN4O4S + [M-Br] + : 733.30, measured value: 733.3006. Figure 3 shown.

[0088] Example 2 Preparation and Characterization of MTBPB / GPs

[0089] 1) Extraction of glucan particles (GPs) using hot acid-base method

[0090] GPs were prepared by hot acid-base extraction: 13 g of Angel Yeast was accurately weighed and dispersed in 200 mL of 1 M NaOH using an ultrasonic cleaner, and then 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. The obtained sample was resuspended in 200 mL of HCL solution at 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 then 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;

[0091] 2) Preparation of MTBPB / GPs

[0092] GPs (10 mg) were mixed in deionized water (1 mL). 100 μL of a 1 mM MTBPB ethanol solution was then added to the system. The mixture was incubated in a shaker in the dark for 12 h. The precipitate was collected by centrifugation (3000 rpm for 2 min) and washed three times with deionized water to obtain biomimetic glucan particle probes with aggregation-induced emission (MTBPB / GPs). The mixture was then stored in the dark at 4°C until use.

[0093] 3) Characterization and evaluation of MTBPB / GPs

[0094] The synthesized glucan particles (GPs) were characterized. SEM images showed that the GPs had a smooth surface and a Figure 4 A) Compared to GPs, the surface of GPs exhibits obvious wrinkles and pores, with pore sizes of approximately 500-600 nm ( Figure 4 B). TEM images show that untreated yeast microcapsules 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).

[0095] The particle size and Zeta potential of yeast microcapsules, GPs and MTBPB / GPs were evaluated by dynamic light scattering (DLS). The results showed that the particle size of yeast microcapsules was approximately 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 is successfully loaded into the particles ( Figure 4 I).

[0096] 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 is related to the vibration of -CH- group. -1 The peaks at 2917 and 2949 cm represent the stretching vibration of the -OH group. -1 The alkyl CH stretching vibration appears 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 vibrations on the benzene ring. In the spectrum of MTBPB / GPs, neither the CH bending peaks of the benzene ring nor the C=C stretching vibration peaks of the aromatic ring appear, indicating that MTBPB has been successfully encapsulated in the cavity of GPs. The XRD pattern 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).

[0097] The UV absorption spectrum of MTBPB showed that MTBPB had significant 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, while a new absorption peak appeared at 506 nm (Figure 4L). In addition, the fluorescence emission spectrum showed that the fluorescence intensity of MTBPB at 670 nm increased significantly after the introduction of H2O2 (5 μM). Figure 4 M). Fluorescence titration studies showed that the signal at 670 nm continued to amplify with increasing H2O2 concentration ( Figure 4 N). In addition, the fluorescence intensity at 670 nm was linearly correlated with the concentration of H2O2 (0-6 μM) (R2 = 0.97), confirming the high sensitivity of MTBPB to H2O2 ( Figure 4 O).

[0098] Adjust the pH of PBS to 4-10 with dilute hydrochloric acid and sodium hydroxide, respectively (step 1). Dissolve 10 μl of MTBPB (1 mM) in 1 ml of PBS with different pH values. Measure the fluorescence spectrum using a fluorescence spectrometer (Ex = 520 nm), and select the fluorescence intensity at 670 nm 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 (10 μM) with other species under the same parameters, such as Figure 4 As shown in P, the fluorescence reaction confirmed the specificity of MTBPB for H2O2.

[0099] Example 3 Identification of pro-inflammatory M1 macrophages

[0100] 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 dead dye for 30 minutes and then washed twice with PBS. Then, 2ul F4 / 80 and CD86 antibodies were added to each tube, incubated in the dark for 40 minutes, and then washed 3 times with PBS. RAW264.7 cells were resuspended in 200 μl PBS. The expression levels of F4 / 80 and CD86 were determined using flow cytometry (BV421 and PE-Cy7 channels). RAW264.7 (8*10 per well) were plated. 3Cells were seeded in 96-well plates using the same stimulation conditions as described above. After 24 hours of LPS stimulation (1 μg / ml), cell supernatants were collected. TNF-α and IFN-γ expression levels in the cell supernatants were measured using ELISA.

[0101] RAW264.7 cells (1*10 per well) 5 Cells were seeded in 6-well plates or confocal microplates and stimulated with 1 μg / ml LPS for 24 hours. Cells were then incubated with diluted DCFH-DA (10 μM) at 37°C for 45 minutes and rinsed five times with PBS. Cells were resuspended in 200 μl of PBS and analyzed by flow cytometry to determine ROS levels (FITC channel). Cells in confocal microplates were rinsed three times with PBS and fixed with 4% paraformaldehyde. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence imaging was performed using laser scanning confocal microscopy (LSCM), and intracellular ROS levels were quantified by flow cytometry.

[0102] The results showed that after LPS stimulation, the expression of CD86 on the cell membrane increased significantly, indicating that the macrophages were polarized toward the 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 produce a large amount of ROS ( Figure 7 ).

[0103] Example 4 ROS responsiveness of MTBPB / GPs in macrophages

[0104] 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. The fluorescence intensity of the probe was qualitatively and quantitatively analyzed using LSCM and flow cytometry (Ex = 480 nm, Em = 620 nm), respectively.

[0105] Figure 8 The results showed that MTBPB / GPs in pro-inflammatory macrophages were significantly stronger than those in undifferentiated M0 macrophages, approximately 2.7 times stronger, confirming that MTBPB / GPs can target and trace pro-inflammatory M1 macrophages.

[0106] Example 5 Cytotoxicity of MTBPB / GPs

[0107] The cells were divided into a negative control group and an MTBPB / GPs group. The negative control group contained only DMEM medium. The ratios of macrophage number to MTBPB / GPs were 1:20, 1:50, 1:100, and 1:200, respectively. Macrophage viability in each group was assessed using a CCK-8 assay. The cytotoxicity of MTBPB / GPs on cardiomyocytes (H29C2), hepatocytes (AML12), and renal cells (293T) was also assessed using a CCK-8 assay using the same method.

[0108] Figure 9 The results showed that MTBPB / GPs had no significant cytotoxicity to macrophages after being co-incubated with macrophages for 24 hours ( Figure 9 a). In addition, MTBPB / GPs had no obvious cytotoxicity to heart, liver, and kidney cells ( Figure 9 bd). MTBPB / GPs have good biosafety and can be used in subsequent animal experiments.

[0109] Example 5 Effect of MTBPB / GPs on the Migration Function of Macrophages

[0110] RAW264.7 cells (1*10 per well) 5 ) were inoculated into 6 wells and cultured for 12 hours, and then MTBPB / GPs (1*10 5 MTBPB / GPs) were cultured for an additional 12 hours. Subsequently, these cells were seeded onto the upper layer of a 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 hours of 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 counted.

[0111] Figure 10 The results showed that the migration ability of macrophages was not significantly affected after phagocytosis of MTBPB / GPs.

[0112] Example 6 Fluorescence Imaging of Allogeneic Transplantation after Oral Administration of MTBPB / GPs

[0113] On the sixth day after surgery, allogeneic and syngeneic mice (n=5 per group) were orally administered MTBPB / GPs (200 μl, 0.8 mg / kg MTBPB). In vivo imaging was performed at 0, 6, 12, 24, 36, and 48 hours after administration (Perkin Elmer IVISLumina: Ex = 480 nm, Em = 620 nm).

[0114] 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 ( Figure 11 Therefore, 36 h was selected as the optimal imaging time point. Figure 12 The results showed that MTBPB / GPs can diagnose transplant rejection at an early stage.

[0115] First, this study successfully established a skin transplant rejection model ( Figure 12 a, 12b). Immunofluorescence results showed that the infiltration degree of M1 macrophages increased with the degree of allograft rejection ( Figure 12 c, 12d). In vivo imaging of small animals 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) ( Figure 12 e, 12f). In summary, MTBPB / GPs can target and trace macrophages and migrate with them to the transplanted skin, thereby enabling early diagnosis of transplant rejection. Fluorescence colocalization images of transplanted skin also show that MTBPB / GPs are phagocytosed by pro-inflammatory M1 macrophages within the transplanted skin ( Figure 13 ).

[0116] In addition, to verify the in vivo targeting of MTBPB / GPs, we orally administered MTBPB / GPs and MTBPB to allografted mice on day 6 after surgery, and quantified the fluorescence intensity of the grafted 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 grafted skin via GPs ( Figure 14 ).

[0117] 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 in the scope of protection of the present invention.

Claims

1. An activatable biomimetic probe with aggregation-induced emission, characterized in that: The H2O2-responsive aggregation-induced emission molecule is incubated with dextran particles; the H2O2-responsive aggregation-induced emission molecule is MTBPB, having the structural formula shown in Formula I: 。 2. Use of the activatable biomimetic probe according to claim 1 in the preparation of an M1 macrophage tracer.

3. The use according to claim 2, characterized in that The activatable biomimetic probe generates a fluorescent signal under the stimulation of inflammatory cytokines.

4. Use of the activatable biomimetic probe according to claim 1 in the preparation of a diagnostic reagent for transplant rejection.

5. The use according to claim 4, 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.

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