Microenvironment activation probe for arthritis and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a microenvironment activation probe for arthritis and a preparation method thereof, the microenvironment activation probe for arthritis comprises bipyridine luminophor nanoparticles and IL-6R, and the IL-6R is connected to the surfaces of the bipyridine luminophor nanoparticles through a sulfydryl-maleimide addition reaction. The preparation method comprises the following steps: preparing a bipyridine illuminant; the preparation method comprises the following steps: carrying out nanocrystallization on a dipyridyl illuminant to prepare dipyridyl illuminant nanoparticles; the microenvironment activation probe for arthritis is prepared by modifying bipyridine luminophor nanoparticles with an anti-IL-6R antibody. The microenvironment activation probe provided by the invention remarkably inhibits release of proinflammatory factors, promotes repolarization of macrophages from M1 phenotype to M2 phenotype, and can remarkably relieve RA symptoms and joint damage. The preparation method provided by the invention is simple and easy to implement, convenient to operate and easy for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a microenvironment activation probe for arthritis and a preparation method thereof. Background Art

[0002] As a chronic inflammatory disease, rheumatoid arthritis is characterized by joint synovitis and irreversible cartilage and bone destruction. Therefore, the strategy of alleviating damage caused by inflammation has been included in the scope of RA treatment. A large amount of evidence shows that the excessive production of reactive oxygen species (ROS) plays a key role in the pathogenesis of RA. In the inflammatory RA cavity, infiltrating monocytes and macrophages continuously release high levels of ROS. The persistent oxidative stress not only induces cell apoptosis, but also activates immune cells, thereby triggering the release of proinflammatory cytokines and exacerbating joint inflammation. Elevated ROS levels in the joints provide a promising molecular target for the diagnosis and treatment of RA. Specifically, the use of ROS scavengers to target the removal of ROS has shown potential for intervention in RA. However, due to the multifactorial nature of RA pathology, a single ROS scavenging therapy is often insufficient to achieve effective relief.

[0003] Fluorescence (FL) is a noninvasive, nonradioactive imaging technique that excels in sensitivity, real-time feedback, and cost-effectiveness compared to conventional imaging techniques. Moreover, fluorescence imaging has the potential to provide precise pathophysiological insights into joints through FL-based detection of RA-specific biomarkers. Near-infrared II (NIR-II, 1000nm-1700nm) imaging can visualize deep tissue structures with higher resolution and contrast compared to conventional visible light and NIR-I (400nm-900nm) imaging due to reduced light scattering and tissue autofluorescence in the NIR-II window. Despite its potential, NIR-II imaging of RA-related bone diseases faces many challenges. The main problem is that most reported NIR-II luminophores are in an “always on” state with constant signals and lack of disease-specific activation, which weakens their effectiveness in selectively distinguishing between normal and pathological tissues. Therefore, there is an urgent need to design activatable probes that can emit NIR-II FL signals in response to specific pathological features to improve the sensitivity and accuracy of RA diagnosis. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a microenvironment activation probe for arthritis; the second object of the present invention is to provide a method for preparing a microenvironment activation probe for arthritis.

[0005] In order to achieve the first purpose, the technical solution adopted by the present invention is: A microenvironment activation probe for arthritis, comprising: Bipyridine luminescent nanoparticles and IL-6R, IL-6R is connected to the surface of bipyridine luminescent nanoparticles through thiol-maleimide addition reaction.

[0006] Furthermore, the bipyridine luminescent nanoparticles include a bipyridine luminescent body, and the bipyridine luminescent body includes an aggregation-induced luminescence core and a carbonyl manganese structure corresponding to active oxygen.

[0007] Furthermore, the aggregation-induced emission core is 4,4'-(dipyrido[3,2-a:2',3'-c][1,2,5]thiadiazolo[3,4-i]phenazine-10,14-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline), abbreviated as TT.

[0008] Furthermore, the bipyridine luminophore is 4,4'-(dipyrido[3,2-a:2',3'-c][1,2,5]thiadiazolo[3,4-i]phenazine-10,14-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline)tricarbonylbromomanganese (TTCO for short), and its structural formula is as follows: .

[0009] Furthermore, the bipyridyl luminescent nanoparticles are water-soluble nanoparticles.

[0010] Furthermore, the bipyridyl luminescent nanoparticles are water-soluble nanoparticles.

[0011] In order to achieve the second purpose, the technical solution adopted by the present invention is: A method for preparing a microenvironment activated probe for arthritis, for preparing any of the above-mentioned microenvironment activated probes, comprising the following steps: S100, using 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline as a raw material to prepare a bipyridine luminophore; S200, nano-forming the bipyridine luminescent body to prepare bipyridine luminescent body nanoparticles; S300. Modification of bipyridine luminescent nanoparticles with anti-IL-6R antibodies to prepare microenvironment-activated probes for arthritis.

[0012] Furthermore, the synthesis route of the bipyridine luminophore in S100 is as follows: .

[0013] Further, S200 includes the following steps: S201, dissolving a bipyridine luminophore and a lipid in an organic solvent to obtain a first mixture; The lipids are distearoylphosphatidylethanolamine-polyethylene glycol and 1,2-distearoyl-sn-glycerol-3-phosphoolamine-N-[maleimide (polyethylene glycol)], and the mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol to 1,2-distearoyl-sn-glycerol-3-phosphoolamine-N-[maleimide (polyethylene glycol)] is 1:1; S202, adding the first mixture into water and mixing evenly to obtain a second mixture; S203, dialyzing and filtering the second mixture to obtain a first nanoparticle solution.

[0014] Further, S300 includes the following steps: S301, adding IL-6R antibody to the first nanoparticle solution for reaction to obtain second nanoparticles; S302, centrifuging, purifying and concentrating the second nanoparticles to obtain bipyridine luminescent nanoparticles.

[0015] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides a microenvironment activation probe for arthritis, which includes bipyridine luminescent nanoparticles and IL-6R, and IL-6R is connected to the surface of bipyridine luminescent nanoparticles through a thiol-maleimide addition reaction. The bipyridine luminescent nanoparticles are self-assembled biocompatible nanoprobes, which are then combined with anti-interleukin-6 receptor (IL-6R) antibodies to achieve active targeting of rheumatoid arthritis. After intravenous injection into a collagen-induced arthritis mouse model (Rheumatoid arthritis, RA), the nanoprobe shows significant NIR-II fluorescence signal activation at the RA lesion, which can achieve high-sensitivity diagnosis and real-time treatment monitoring of RA. At the same time, the combination of ROS scavenging, CO gas released on demand and IL-6 signal blocking produces a powerful therapeutic effect and synergistic immunomodulatory effect. The microenvironment activation probe significantly inhibits the release of proinflammatory factors, promotes the repolarization of macrophages from M1 phenotype to M2 phenotype, and can significantly alleviate RA symptoms and joint damage. The present invention also provides a method for preparing a microenvironment activation probe for arthritis. The method is simple, easy to operate and easy to industrialize.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or 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 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.

[0018] Figure 1 This is the TTCO H NMR spectrum provided in Example 1 of the present invention.

[0019] Figure 2 This is the TTCO NMR carbon spectrum provided in Example 1 of the present invention.

[0020] Figure 3 This is a high-resolution mass spectrum of TTCO provided in Example 1 of the present invention.

[0021] Figure 4 This is a diagram of the molecular structure change of TTCO after being triggered by ROS provided in Example 1 of the present invention.

[0022] Figure 5It is a normalized absorption spectrum diagram of TTCO in tetrahydrofuran and the reaction of TTCO with ROS provided in Example 1 of the present invention.

[0023] Figure 6 It is a normalized fluorescence spectrum diagram of TTCO in tetrahydrofuran (THF) and the reaction of TTCO with ROS provided in Example 1 of the present invention.

[0024] Figure 7 The reaction of TTCO and TTCO with ROS in a mixed solvent of THF and water provided in Example 1 of the present invention is I / I 0 with water fraction (f w ) change graph.

[0025] Figure 8 It is a normalized absorption spectrum diagram of different groups before and after H2O2 treatment provided in Example 2 of the present invention.

[0026] Fig. 9 This is a fluorescence spectrum of TC@AI NPs before and after H2O2 treatment provided in Example 2 of the present invention.

[0027] Fig.10 This is the excitation-emission diagram of TC@AI NPs before and after H2O2 treatment provided in Example 2 of the present invention.

[0028] Fig.11 This is a curve diagram showing the relationship between the NIR-II fluorescence intensity of TC@AI NPs provided in Example 2 of the present invention and the H2O2 concentration.

[0029] Fig.12 This is a curve diagram showing the relationship between the NIR-II fluorescence signal and ROS concentration of TC@AI NPs provided in Example 2 of the present invention.

[0030] Fig.13 This is a curve diagram showing the relationship between H2O2 treatment time and CO release concentration provided in Example 2 of the present invention.

[0031] Fig.14 This is an image showing the effects of various interference factors on the CO concentration and fluorescence signal released by TC@AI NPs provided in Example 2 of the present invention.

[0032] Fig.15 These are representative CLSM images of non-activated and activated macrophages treated with DIO-labeled TC NPs or TC@AI NPs provided in Experimental Example 1 of the present invention.

[0033] Fig.16 This is an image of the uptake of DIO-labeled TC NPs or TC@AI NPs by non-activated and activated macrophages provided in Experimental Example 1 of the present invention by flow cytometry analysis.

[0034] Fig.17 This is a statistical graph of internalization analysis of TC NPs or TC@AI NPs in non-activated and activated macrophages based on flow cytometry data provided in Experimental Example 1 of the present invention.

[0035] Fig.18 It is a representative NIR-II fluorescence signal of TC NPs or TC@AI NPs provided in Experimental Example 2 of the present invention in non-activated and inflammatory macrophages.

[0036] Fig.19 This is a quantitative analysis statistical graph of the cell NIR-II fluorescence intensity provided in Experimental Example 2 of the present invention.

[0037] Fig. 20 These are representative CLSM images of macrophages stained with 2,7-dichlorofluorescein diacetate (2',7'-Dichlorodihydrofluorescein diacetate, DCFH-DA) treated with different nanoparticles provided in Experimental Example 2 of the present invention, representative CLSM images of non-activated and inflammatory macrophages treated with different nanoparticles, and images of different nanoparticles stained with CO probe.

[0038] Fig.21 This is a quantitative analysis statistical graph of the CO probe fluorescence intensity provided in Experimental Example 2 of the present invention.

[0039] Fig. 22 It is a representative CLSM image of the expression of iNOS (red fluorescence) and CD206 (green fluorescence) on RAW264.7 macrophages after being treated with different substances provided in Experimental Example 3 of the present invention.

[0040] Fig.23 It is an immunofluorescence staining image of HO-1 expression in unactivated and activated RAW264.7 macrophages after treatment with different substances provided in Experimental Example 3 of the present invention.

[0041] Fig.24 The Western blotting analysis provided in Experimental Example 3 of the present invention was to analyze the expression levels of HO-1 and Notch / Hes1 / Stat3 pathway-related proteins in RAW264.7 macrophages treated with different substances.

[0042] Fig.25 The Western blotting analysis provided in Experimental Example 3 of the present invention is to analyze the expression levels of proteins related to the p38 MAPK and NF-κB (p50 / p65) pathways in RAW264.7 macrophages after treatment with different substances.

[0043] Fig.26This is the NIR-II FL image of mice after TC@AI NPs were directly injected into the subcutaneous inflammatory site (right, LPS-induced) or the unaffected site (left, non-LPS-induced) provided in Experimental Example 4 of the present invention.

[0044] Fig. 27 It is the corresponding quantitative analysis graph of the NIR-II FL intensity of the inflammatory site or the unaffected side after administration of TC@AI NPs provided in Experimental Example 4 of the present invention.

[0045] Fig.28 These are representative NIR-II FL images of healthy or RA mice at different time points after intravenous injection of different substances provided in Experimental Example 4 of the present invention.

[0046] Fig.29 This is a corresponding quantitative analysis graph of the NIR-II FL intensity in the joints of healthy mice and RA mice treated with different substances provided in Experimental Example 4 of the present invention.

[0047] Fig.30 The arthritis scores of healthy mice and RA mice at different time points after receiving treatment with different substances provided in Experimental Example 5 of the present invention.

[0048] Fig.31 These are representative photos of the front and hind paws of healthy mice and RA mice treated with different substances provided in Experimental Example 5 of the present invention.

[0049] Fig.32 are representative micro-CT images of the hind paws of healthy mice and RA mice treated with different substances provided in Experimental Example 6 of the present invention.

[0050] Fig.33 This is a quantitative analysis statistical chart of the effects of different substances on mouse BV / TV and BV provided in Experimental Example 6 of the present invention.

[0051] Fig.34 These are representative in vivo NIR-II fluorescence images of the joints of mice in different treatment groups provided in Experimental Example 6 of the present invention.

[0052] Fig.35 This is a quantitative analysis statistical graph of the NIR-II FL signal intensity in the joints of mice in different treatment groups provided in Experimental Example 6 of the present invention.

[0053] Fig.36 These are H&E staining images of the paw joints of healthy mice and RA mice treated with different substances provided in Experimental Example 7 of the present invention.

[0054] Fig.37This is a statistical chart of H&E scores of healthy mice and mice treated with different substances provided in Experimental Example 7 of the present invention.

[0055] Fig.38 These are Safranin-O staining images of the paw joints of healthy mice and RA mice treated with different preparations provided in Experimental Example 7 of the present invention.

[0056] Fig.39 It is the Safranin-O score of the paw joints of healthy mice and RA mice treated with different preparations provided in Experimental Example 7 of the present invention.

[0057] Fig.40 This is the immunohistochemical staining of IL-6 expression in normal joints and inflamed joints of different treatment groups provided in Experimental Example 7 of the present invention.

[0058] Fig.41 This is a statistical graph of the intensity of IL-6 in normal joints and inflamed joints in different treatment groups provided in Experimental Example 7 of the present invention.

[0059] Fig.42 This is an immunohistochemical staining image of TNF-α expression in normal and inflamed joint sections after treatment with different treatment groups provided in Experimental Example 7 of the present invention.

[0060] Fig.43 It is a statistical graph of the relative staining intensity of immunohistochemical staining of TNF-α expression in normal and inflamed joint sections after different treatments provided in Experimental Example 8 of the present invention.

[0061] Fig.44 This is a statistical graph of the concentrations of IL-6 and TNF-α in joint tissue homogenates after different treatments using ELISA provided in Experimental Example 8 of the present invention.

[0062] Fig.45 The immunofluorescence imaging provided in Experimental Example 8 of the present invention shows the expression images of iNOS and CD206 in joint tissues after different treatments.

[0063] Fig.46 This is a quantitative analysis statistical graph of the fluorescence intensity of iNOS and CD206 in normal joints and inflammatory joints after different treatments provided in Experimental Example 8 of the present invention. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0065] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0066] Example 1 Preparation of TTCO.

[0067] 1. Preparation of 1-iodo-4-(octyloxy)benzene: 4-iodophenol (4.4 g, 20 mmol), 1-bromooctane (4.44 g, 23 mmol) and potassium carbonate (K2CO3) (8.98 g, 65 mmol) were mixed under argon atmosphere, and then anhydrous N,N-dimethylformamide (DMF) (90 ml) was added. After cooling to room temperature, water was added and extracted three times with dichloromethane (CH2Cl2). The collected organic phase was dried over magnesium sulfate and concentrated. The crude product was purified by silica gel column chromatography with CH2Cl2 / n-hexane (v / v 1:10 as the eluting solvent) to obtain colorless solid 1-iodo-4-(octyloxy)benzene with a yield of 83%.

[0068] 2. Preparation of 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline: Under argon atmosphere, 1-iodo-4-(octyloxy)benzene (9.96 g, 30 mmol), bromoaniline (2.06 g, 12 mmol), cuprous iodide (76 mg, 0.4 mmol), 1,10-phenanthroline (396 mg, 2.2 mmol) and potassium hydroxide (11.22 g, 200 mmol) were mixed, and then anhydrous toluene (100 ml) was added. After cooling to room temperature, water was added and extracted three times with CH2Cl2. The collected organic phase was dried over magnesium sulfate and concentrated. The crude product was purified by silica gel column chromatography with CH2Cl2 / n-hexane (v / v 1:6 as the eluting solvent) to obtain colorless solid 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline with a yield of 72%.

[0069] 3. Preparation of 4-(octyloxy)-N-(4-(octyloxy)phenyl)-N-(4-(tributyltin)phenyl)aniline: 4-Bromo-N,N-bis(4-(octyloxy)phenyl)aniline (3.48 g, 6 mmol) was placed in an argon atmosphere, and then anhydrous tetrahydrofuran (50 ml) was added. The reaction mixture was cooled to -78 o C and maintained for 30 minutes, then n-butyl lithium (2.5 M hexane solution, 2.4 mL, 6 mmol) was added. After stirring at this temperature for 2 hours, tributyltin chloride (1.63 mL, 6 mmol) was added, and the mixture was slowly warmed to room temperature and then stirred overnight. Subsequently, water was added to quench the reaction, and the resulting mixture was extracted three times with CH2Cl2. The collected organic phases were combined and dried over magnesium sulfate. The solvent was evaporated to obtain 4-(octyloxy)-N-(4-(octyloxy)phenyl)-N-(4-(tributyltin)phenyl)aniline.

[0070] 4. Preparation of 4,4'-(5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline): Under argon atmosphere, 4-(octyloxy)-N-(4-(octyloxy)phenyl)-N-(4-(tributyltin)phenyl)aniline (4.74 g, 6 mmol), 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole (0.96 g, 2.5 mmol) and tetrakis(triphenylphosphine)palladium (115.56 mg, 0.1 mmol) were mixed in argon atmosphere, and then anhydrous tetrahydrofuran (80 mL) was added. After cooling to room temperature, water was added and extracted three times with CH2Cl2. The collected organic phase was dried over MgSO4 and concentrated. The product was purified by column chromatography on silica gel with CH2Cl2 / n-hexane (v / v 1:2) as the eluting solvent to obtain 4,4'-(5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline) as a dark purple solid in a yield of 69%.

[0071] 5. Preparation of 4,7-bis(4-(bis(4-(octyloxy)phenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine: Under argon atmosphere, 4,4'-(5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline) (3.67 g, 3 mmol) and acetic acid (100 ml) were mixed, and then iron powder (5.03 g, 90 mmol) was added. After cooling to room temperature, water was added, and the resulting mixture was extracted three times with CH2Cl2. The collected organic phases were dried over MgSO4, and the solvent was evaporated to obtain 7-bis(4-(bis(4-(octyloxy)phenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine.

[0072] 6. Preparation of TT: 4,7-bis(4-(bis(4-(octyloxy)phenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine (1.17 g, 1 mmol), chloroform (30 ml) and acetic acid (30 ml) were mixed under argon atmosphere, and then 1,10-phenanthroline-5,6-dione (0.42 g, 2 mmol) was added. The mixture was heated to reflux and stirred for 12 hours. After cooling to room temperature, water was added and extracted three times with dichloromethane. The collected organic phases were dried over magnesium sulfate and concentrated. TT was purified by column chromatography on silica gel with CH2Cl2 / n-hexane (v / v 1:1) as the eluting solvent to obtain a dark green solid with a yield of 62%.

[0073] 7. Preparation of TTCO: TT (142 mg, 0.086 mmol) and Mn(CO)5Br (20 mg, 0.086 mmol) were dissolved in 25 ml of ether and heated to reflux for 3 hours under argon. After cooling to room temperature, the green precipitate was collected by vacuum filtration and washed with ether. TTCO was obtained as a dark green solid with a yield of 43%.

[0074] Characterization of TTCO molecules Figures 1 to 3 As shown, 1H NMR (400 MHz, chloroform-d) δ 8.93 (d, J = 4.5 Hz, 2H), 8.61 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.4 Hz, 4H), 7.30 (dd, J = 8.0, 4.5 Hz, 2H), 7.15 (d, J = 8.7 Hz, 8H), 6. 96 (d, J = 8.4 Hz, 4H), 6.83 (d, J = 8.7 Hz, 8H), 3.89 (t, J = 6.5 Hz, 8H), 1.73 (p, J = 6.8 Hz, 8H), 1.40 (p, J = 7.2 Hz, 8H), 1.28-1.19 (m, 32H), 0.82 (t, 12H). 13 C NMR (101 MHz, chloroform-d) δ 156.26, 154.91, 153.43, 150.20, 149.48, 139.97, 137.13, 135.22, 134.13, 130.18, 129.61, 127.62, 126.51, 125.88, 117.94, 115.48, 68.35, 31.85, 29.71, 29.40, 26.93, 22.68.

[0075] The molecular structure changes of TTCO after being triggered by ROS, such as Figure 4 shown.

[0076] Spectrum of TTCO before and after ROS triggering, such as Figures 5 to 7 shown.

[0077] in, Figure 7 Where I0 and I represent the maximum fluorescence intensity of pure THF and THF / water mixtures with different water fractions, respectively.

[0078] Example 2 Preparation of microenvironment activated probe for arthritis.

[0079] Material: Distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG for short); 1,2-Distearoyl-sn-glycero-3-phosphoolamine-N-[maleimide(polyethylene glycol)] (abbreviated as DSPE-PEG-MAL); Tetrahydrofuran (abbreviated as THF); Red fluorescent cell membrane dye (abbreviated as DiD); The preparation process is as follows: First, TTCO (1 mg), DSPE-PEG (2 mg), and DSPE-PEG-MAL (2 mg) were dissolved in 1 ml of THF. Then, the organic mixture was added dropwise to 10 ml of water and mixed thoroughly. The resulting solution was dialyzed using a 10 kDa membrane to remove free molecules and residual THF solvent. Then, the solution was further filtered using a 0.2 μm filter. Then, anti-IL-6R antibody (400 μg) was added to the nanoparticle solution (5 mL) and incubated at 4 o C for 12 h, and then centrifuged at 15,000 rpm for 15 min to remove the remaining agent and unreacted anti-IL-6R antibody in the solution. The mixture was then resuspended in PBS (10 mL) and the purification process was repeated twice. Finally, the suspension of anti-IL-6R antibody-modified 4,4'-(bipyridyl[3,2-a:2',3'-c][1,2,5]thiadiazole[3,4-i]phenazine-10,14-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline)tricarbonylbromomanganese nanoparticles (abbreviated as TC@AI NPs) was concentrated, and TC@AI NPs were microenvironment-activated probes for arthritis.

[0080] Preparation of fluorescently labeled nanoparticles for fluorescence imaging investigation. DiD was added to the mixed solution of TTCO, DSPE-PEG and DSPE-PEG-MAL to allow the dye to bind to TC@AI NPs, and the fluorescently labeled nanoparticles were prepared according to the above preparation process.

[0081] Figure 8 The results showed that TC@AI NPs showed a slight blue shift in the absorption spectrum after H2O2 treatment, which was attributed to the breakage of the electron-attracting manganese complexes.

[0082] Fig. 9 , Fig.10 The results showed that the NIR-II fluorescence signal of TC@AI NPs after H2O2 treatment was significantly enhanced, and a new emission peak appeared at about 1070 nm.

[0083] Fig.11 The results showed that the NIR-II fluorescence intensity of TC@AI NPs increased with the increase of H2O2 concentration; Fig.12 The results showed that the NIR-II fluorescence signal of TC@AI NPs was directly positively correlated with the ROS concentration.

[0084] After confirming the ROS-triggered NIR-II fluorescence on-state of TC@AI NPs, reduced hemoglobin was used as a probe to study the in situ generation of CO gas.

[0085] Fig.13 The results showed that the concentration of CO released from TC@AI NPs increased significantly with the increase of H2O2 treatment time.

[0086] Fig.14 The results showed that TC@AI NPs had a significant effect on the 2+ , Cu 2+ , Fe 3+ , K + , Mn 2+ 、Na + and Zn 2+ In the response to interfering ions, TC@AI NPs showed good selectivity for ROS.

[0087] In summary, TC@AI NPs can activate NIR-II fluorescence and produce therapeutic CO gas in situ under ROS stimulation.

[0088] Experimental Example 1 Investigation of the uptake of TC@AI NPs by macrophages (RAW264.7).

[0089] In order to evaluate the potential of TC@AI NPs to actively target inflammatory immune cells, the present invention pre-treated RAW264.7 mouse macrophages with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to induce inflammation, and tested the active binding of TC@AI NPs to immune cells. The specific process is as follows: TC@AI NPs labeled with a cell membrane green fluorescent probe (3,3′-dioctadecyloxacarbocyanine perchlorate, DIO) were incubated with inflammatory macrophages for 4 hours and imaged under a confocal laser scanning microscope (CLSM).

[0090] Fig.15The results showed that TC@AI NPs displayed strong fluorescence signals in activated macrophages, while cells treated with TC NPs without anti-IL-6R antibody modification showed weak fluorescence signals. These results indicate that the uptake of TC@AI NPs by activated macrophages was significantly increased. In normal macrophages that were not stimulated by proinflammatory cytokines, the incubation of TC@AI NPs produced a weak red fluorescence signal, in contrast to the strong signal in the treated activated macrophages. The above results indicate that TC@AI NPs have enhanced targeting ability for inflammatory-activated macrophages, which is due to the specific binding affinity of the antibody to IL-6R overexpressed on the surface of activated macrophages. Quantitative analysis of fluorescence intensity by flow cytometry, Fig.16 The results further verified that TC@AI NPs have enhanced targeting ability to inflammatory activated macrophages. In non-activated macrophages, the fluorescence intensity of each treatment group was low. The uptake of TC@AI NPs by inflammatory macrophages was about 3 times that of cells exposed to TCNPs, as shown in Figure 2. Fig.17 shown.

[0091] The above results indicate that TC@AI NPs can actively target M1 macrophages in an in vitro environment.

[0092] Experimental Example 2 Investigation of the sensitivity of ROS detection of TC@AI NPs in living cells.

[0093] RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) and IFN-γ to promote ROS generation and were subsequently incubated with various nanoparticles. As a control, normal macrophages without pro-inflammatory stimulation were also exposed to TC@AI NPs, and the intracellular fluorescence signal of the nanoprobes was visualized by NIR-II fluorescence microscopy after 6 hours of nanoparticle incubation.

[0094] like Fig.18 The results showed that the NIR-II fluorescence signal in activated macrophages treated with TC@AI NPs was significantly enhanced at an excitation wavelength of 808 nm and an emission wavelength of 1000 nm. In contrast, TC@AI NPs emitted only a weak NIR-II signal in normal macrophages. The NIR-II fluorescence signal intensity in cells exposed to TC@AI NPs after LPS + IFN-γ stimulation was 5.8 times higher than that in normal macrophages treated with TC@AI NPs, as shown in Figure 2. Fig.19 As shown, the results indicate that ROS induced the activation of NIR-II fluorescence signal.

[0095] After confirming the ROS-activated imaging ability of TC@AI NPs, the present invention continued to evaluate the ability of TC@AINPs to respond to and clear ROS in cells using DCFH-DA staining. DCFH-DA can be converted from a non-fluorescent compound to green fluorescent 2',7'-dichlorofluorescein (2',7'-Dichlorofluorescein, abbreviated as DCF) after reacting with ROS. Fig. 20 The results showed that RAW 264.7 cells showed a significant enhancement of ROS generation after LPS and IFN-γ stimulation, as manifested by strong DCF green fluorescence. Both TCNPs and TC@AI NPs showed effective ROS scavenging ability at the cellular level, especially TC@AI NPs, which showed a more significant ROS consumption effect due to its superior targeting ability. After subsequent treatment with TC NPs or TC@AI NPs, ROS levels were significantly reduced to 29.3% and 16.5% of the control level, respectively, as shown in Figure 2. Fig.21 In summary, the targeting ability of TC@AI NPs and the ROS-responsive NIR-II fluorescence turn-on characteristics were verified by in vitro macrophage activation experiments, which indicated the potential application of TC@AI NPs in the treatment of RA.

[0096] in, Fig.21 Non-activated refers to unactivated macrophages.

[0097] Experimental Example 3 Inflammation inhibition and immune regulation at the cellular level.

[0098] Immunofluorescence staining of TC@AI NPs showed that normal macrophages did not express iNOS and CD206 significantly. iNOS is a surface marker of M1 macrophages, and CD206 is a surface marker of M2 macrophages. This result indicates that they are mainly in an inactive state, such as Fig. 22 As shown. However, after LPS and IFN-γ stimulation, the ubiquitous expression of iNOS indicated the polarization of macrophages toward the M1 phenotype. After inflammatory RAW 264.7 cells were co-incubated with TC NPs, AI NPs, or TC@AI NPs, the number of iNOS-positive cells was significantly reduced, while the number of CD206-positive cells was significantly increased, and the effect was more obvious in cells treated with TC@AI NPs. The above results indicate that TC@AI NPs can serve as an effective regulator to promote the transition of macrophages from M1 to M2 phenotype.

[0099] Studies have shown that CO can activate the expression of HO-1, which is an endogenous antioxidant factor that maintains cell homeostasis under pathological conditions. The present invention evaluates the level of intracellular HO-1 by immunofluorescence staining. Fig.23As shown in Figure 2, TC@AINPs released CO on demand in activated macrophages, significantly increasing the expression of HO-1. The level of HO-1 was further evaluated by Western blot, which also confirmed that TC@AI NPs increased the expression of HO-1 in inflammatory macrophages, as shown in Figure 2. Fig.24 As shown. In addition, cells treated with TC@AI NPs significantly upregulated the protein expression levels of p-Stat3, Hes1 and Notch1. This result indicates that TC@AI NPs can activate the HO-1 and Notch / Hes1 / Stat3 signaling pathways, jointly promoting the resolution of inflammation and the formation of anti-inflammatory phenotypes of immune cells. The present invention further analyzed the ability of TC@AI NPs to reduce pro-inflammatory signals, and the ROS microenvironment can trigger pro-inflammatory pathways. After treatment with LPS and IFN-γ, the protein expression of p38 MAPK and NF-κB (p50 / p65) in macrophages increased significantly, as shown Fig.25 However, after incubation of inflammatory macrophages with TC@AI NPs, the expression of p38 and p50 / p65 was significantly downregulated. TC@AI NPs can interact with ROS and reduce intracellular ROS stress, thereby blocking the pro-inflammatory signaling pathway triggered by ROS.

[0100] in, Fig.24 and Fig.25 Non-activated refers to unactivated macrophages.

[0101] Experimental Example 4: NIR-II fluorescence imaging of TC@AI NPs activated in vivo.

[0102] In order to investigate the real-time activation of NIR-II fluorescence in the ROS overexpression inflammatory microenvironment, the present invention established a mouse right back local LPS subcutaneous inflammatory model. Six hours after LPS injection, TC@AI NPs were directly injected into the inflammatory lesion area. As a control, the same dose of TC@AI NPs was also subcutaneously injected into the healthy part of the left side of the mouse that was not induced by LPS. Two hours after the TC@AI NPs were injected, the mice were imaged using the NIR-II imaging system. Fig.26 As shown in the figure, the LPS-stimulated site emits a strong 1000 nm NIR-II fluorescence signal under 808 nm light irradiation, while the healthy site only shows a weak NIR-II signal. Quantitative analysis shows that two hours after the injection of TC@AI NPs, the NIR-II fluorescence signal intensity at the inflammatory site is 4.3 times that of the healthy control site, as shown in the figure. Fig. 27 This observation confirmed the real-time activation effect of TC@AI NPs in inflammatory lesions and indicated that this turn-on NIR-II imaging could facilitate accurate and sensitive imaging of inflammatory RA lesions using TC@AI NPs.

[0103] The present invention evaluates the ability of TC@AI NPs to target and activate imaging of RA in vivo in a CIA mouse model. The CIA mouse model exhibits immunological and pathological characteristics similar to those of human RA and is a widely used model for studying the pathogenesis of RA and evaluating new treatments. The CIA model was induced by primary immunization of DBA / 1 mice with bovine type II collagen (CII) emulsified with Freund's Adjuvant Complete (CFA) on day 0. A second booster immunization was performed on day 20 using type II collagen emulsified with incomplete Freund's adjuvant (IFA). After the second immunization, RA symptoms (such as redness and swelling of the joints) appeared, and the arthritis score was significantly higher than that of healthy mice, confirming that the RA model was successfully established.

[0104] RA mice were randomly divided into four groups and intravenously injected with PBS, TC NPs, and TC@AI NPs, respectively. Then, the images were taken using the NIR-II imaging system. Fig.28 As shown, there was almost no NIR-II fluorescence signal in the paws of mice injected with PBS, indicating low background noise in the NIR-II region. In contrast, mice injected with TC NPs showed moderate NIR-II fluorescence signals in RA joints. Due to the lack of targeting ligands, TC NPs may passively accumulate in RA lesions through the inflammation-mediated enhanced permeability and retention effect (EPR), a phenomenon that was initially observed in NPs delivery in tumors and has recently been found in certain inflammatory diseases. Mice injected with TC@AI NPs showed significantly brighter NIR-II fluorescence signals in the RA joints at all time points tested, and the fluorescence intensity 4 hours after injection was 1.7 times higher than that of mice injected with TC NPs, as shown in Figure 3. Fig.29 These results suggest that anti-IL-6R antibody modification can increase the accumulation of NPs in arthritic joints.

[0105] Experimental Example 5 The therapeutic effect of TC@AI NPs on RA mice was evaluated from a behavioral perspective.

[0106] The RA model was established using conventional technical means in the prior art, and mice showed obvious paw swelling and redness 28 days after the initial immunization.

[0107] The mice were randomly divided into four groups (5 mice in each group), namely, intravenous injection of PBS group, TC NPs group, AINPs group, and TC@AI NPs group. The treatment was repeated every three days for a total of three times, and the mice were killed on the 50th day. At the same time, healthy DBA / 1 mice without any intervention served as the control group. The joints of the mice were monitored every two days, and the severity of arthritis was assessed using a standard scoring method. The results are shown in Fig.30 As shown in Figure 5, injection of TC NPs or AI NPs partially alleviated arthritis symptoms. On day 50, the average arthritis scores of the TC NPs group (8.4 points) and the AI ​​NPs group (5.8 points) were lower than those of the PBS-treated mice (13.6 points). Significantly, TC@AI NPs treatment almost completely alleviated arthritis-induced paw swelling and redness, with an average arthritis score of 2.2 points. At the end of treatment, the paw joints of the mice were photographed, as shown in Figure 5. Fig.31 As shown, representative photos of the front and rear paws showed that the mice treated with TC@AI NPs had the least swelling and redness, and the paw condition was close to that of normal mice. The superior effect of TC@AI NPs in alleviating RA can be attributed to its multi-pathway synergistic therapeutic effect and enhanced targeting ability.

[0108] Experimental Example 6 The therapeutic effect of TC@AI NPs on RA mice was evaluated by micro-computed tomography (micro-CT) and NIR-II fluorescence imaging.

[0109] The bone morphology and microarchitecture of the paw joints of mice receiving different treatments were evaluated by micro-CT. Fig.32 As shown in the figure, the joints of RA model mice showed significant bone damage, characterized by rough surface, cartilage degradation and decreased bone density. Although the injection of TC NPs or AI NPs partially alleviated bone erosion, significant bone damage still existed. However, the TC@AI NPs treatment group showed significant restoration of bone structural integrity, characterized by clear bone boundaries and relatively smooth joint surfaces. This result indicates that TC@AI NPs can effectively reverse RA-induced bone erosion.

[0110] The micro-CT images were subjected to histomorphometric analysis to quantitatively evaluate the extent of bone damage in different experimental groups. Fig.33 As shown in the figure, the results showed that the BV / TV of RA mice treated with TC@AI NPs group increased significantly compared with the other treatment groups. The BV / TV value of the TC@AI NPs group (39.5%) was approximately 2.03 times that of the PBS-treated group (19.5%). By analyzing the bone volume of the affected bone area, the results showed a consistent pattern. These results indicate that the TC@AI NPs group has the potential to inhibit the progression of RA and improve bone degeneration and cartilage erosion.

[0111] in, Fig.33 In the figure, P represents the statistical significance relative to the TC@AI NPs group, BV is the abbreviation of bone volume, and TV is the abbreviation of tissue volume.

[0112] Given that TC@AI NPs can emit bright NIR-II fluorescence in response to elevated ROS levels in the inflammatory microenvironment, the present invention further explored the potential of TC@AI NPs to monitor therapeutic effects via activated NIR-II FL molecular imaging.

[0113] At the end of the treatment regimen, TC@AI NPs were intravenously injected into mice in different treatment groups. NIR-II FL imaging of the hind paws was performed 4 hours after injection. Fig.34 and Fig.35 As shown, the joints of RA mice treated with PBS exhibited strong NIR-II FL signals, attributed to elevated ROS levels in inflamed joints. The NIR-II FL signals in the joints of RA mice treated with TC NPs or AI NPs were attenuated. The joints of mice treated with the TC@AI NPs group showed the weakest NIR-II FL signals, highlighting the effective reduction in joint inflammation and ROS levels. This NIR-II FL imaging result was consistent with the observed paw scores and micro-CT analysis. In summary, TC@AI NPs not only effectively alleviate RA, but also can serve as a powerful imaging tool for accurate diagnosis of RA and tracking of treatment response.

[0114] Experimental Example 7 The therapeutic effect of TC@AI NPs on RA mice was evaluated by hematoxylin-eosin (H&E) staining, Safranin-O fast green and immunohistochemistry.

[0115] The joints of mice from different groups were sectioned and stained with hematoxylin and eosin (H&E) to evaluate joint pathology. Representative H&E images are shown in Figure 2. Fig.36 and Fig.37 As shown in Figure 2, the joints of CIA mice exhibited extensive inflammatory cell infiltration, severe bone erosion, and high activation of abnormal synovial fibroblasts—signature features of RA pathology. The joints of RA mice treated with TC NPs or AI NPs exhibited moderate immune cell infiltration and synovial hyperplasia. Notably, the joint cavity in the TC@AI NPs group almost returned to normal, characterized by clear bone boundaries, less immune cell infiltration, and limited bone deformity. Cartilage degradation and extracellular matrix changes were further assessed using safranin O-fast green staining, as shown in Figure 2. Fig.38 and Fig.39As shown in the figure, the articular cartilage of healthy mice is thick and proteoglycans are evenly distributed in the matrix. In contrast, the articular cartilage of RA mice degenerated significantly, and proteoglycans were significantly lost in the cartilage matrix. After the application of different nanoformulations, cartilage preservation was improved. The TC@AI NPs group showed a significant effect on alleviating cartilage degradation, characterized by an almost intact and smooth cartilage surface and evenly distributed proteoglycans in the bone cavity. The histological analysis was consistent with the arthritis score and micro-CT results, showing that TC@AI NPs had a significant therapeutic effect in the treatment of RA, which was attributed to its combined effects of scavenging ROS, releasing anti-inflammatory CO gas, and blocking the IL-6 pathway.

[0116] In addition, the present invention also analyzed the levels of proinflammatory cytokines in the joint microenvironment to evaluate the immunomodulatory effect of TC@AI NPs. Proinflammatory cytokines, such as IL-6 and TNF-α, are known to be overexpressed by activated macrophages in inflamed joints and promote the progression of RA. The expression of these cytokines in joint tissues was evaluated by immunohistochemical staining. The results are shown in Figure 2. Figure 40 to Figure 42 As shown, paw sections of RA mice showed significant upregulation of these inflammatory factors, while the expression was lower in healthy control mice. Administration of TC NPs or AI NPs suppressed the expression of inflammatory cytokines to some extent. These findings suggest that both the ROS-consuming and CO-releasing TTCO probe and the IL-6R-targeting AI NPs are able to suppress inflammatory responses. Through synergistic effects, the TC@AI NPs group had the most significant inhibitory effect on the expression of inflammatory cytokines among all groups, and the levels of pro-inflammatory cytokines were close to those of healthy mice.

[0117] Experimental Example 8 The therapeutic effect of TC@AI NPs on RA mice was evaluated by enzyme-linked immunosorbent assay and immunofluorescence staining.

[0118] Fig.43 The results showed that paw sections of RA mice showed significant upregulation of inflammatory factors TNF-ɑ and IL-6, while the expression was lower in healthy control mice.

[0119] The levels of IL-6 and TNF-α in joint tissue homogenates were further quantitatively evaluated by enzyme linked immunosorbent assay (ELISA). Fig.44 As shown, the levels of IL-6 and TNF-α in the joint homogenates of mice treated with TC@AI NPs group were significantly decreased, while the IL-6 levels in other groups were 6.53 to 2.18 times higher, while the TNF-α concentrations in other groups were 5.21 to 2.03 times higher.

[0120] The present invention further studies the polarization of macrophages M1 / M2 in the joint microenvironment. Immunofluorescence staining images, such as Fig.45As shown in Figure 2, the expression of M1 macrophage marker (iNOS) in the joints of mice treated with TC@AI NPs was significantly reduced, while the level of M2 macrophage marker (CD206) was increased. The expression of iNOS in the PBS group was 5.28 times higher than that in the TC@AI NPs group, and the expression of CD206 in the TC@AI NPs group was 10.70 times higher than that in the PBS group. Fig.46 This result indicates that TC@AI NPs promote the repolarization of M1 macrophages toward the anti-inflammatory M2 phenotype, which is consistent with the results of in vitro studies.

[0121] In summary, the above results demonstrated the synergistic efficacy of TC@AI NPs in the treatment of RA, attributed to their enhanced RA-targeting ability and multi-target anti-inflammatory and immunomodulatory effects.

[0122] In the figure, MFI is the abbreviation of Mean Fluorescence Intensity, and Healthy refers to healthy mice.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microenvironment activated probe for arthritis, characterized in that: include: Bipyridine luminescent nanoparticles and IL-6R, IL-6R is connected to the surface of bipyridine luminescent nanoparticles through thiol-maleimide addition reaction.

2. The microenvironment activated probe for arthritis according to claim 1, characterized in that: The bipyridine luminescent nanoparticles include a bipyridine luminescent body, and the bipyridine luminescent body includes an aggregation-induced luminescence core and a carbonyl manganese structure corresponding to active oxygen.

3. The microenvironment activated probe for arthritis according to claim 2, characterized in that: The aggregation-induced emission core is 4,4'-(bipyridyl[3,2-a:2',3'-c][1,2,5]thiadiazole[3,4-i]phenazine-10,14-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline).

4. The microenvironment activated probe for arthritis according to claim 3, characterized in that: The bipyridine luminophore is 4,4'-(bipyridyl[3,2-a:2',3'-c][1,2,5]thiadiazole[3,4-i]phenazine-10,14-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline)tricarbonyl manganese bromide, and its structural formula is as follows: 。 5. The microenvironment activated probe for arthritis according to claim 1, characterized in that: The bipyridyl luminescent nanoparticles are water-soluble nanoparticles.

6. The microenvironment activated probe for arthritis according to claim 1, characterized in that: The bipyridyl luminescent nanoparticles are lipid nanoparticles.

7. A method for preparing a microenvironment activated probe for arthritis, characterized in that: The method for preparing the microenvironment activation probe according to any one of claims 1 to 6 comprises the following steps: S100, using 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline as a raw material to prepare a bipyridine luminophore; S200, nano-forming the bipyridine luminescent body to prepare bipyridine luminescent body nanoparticles; S300. Modification of bipyridine luminescent nanoparticles with anti-IL-6R antibodies to prepare microenvironment-activated probes for arthritis.

8. The method for preparing a microenvironment activated probe for arthritis according to claim 7, characterized in that: The synthesis route of the bipyridine luminophore in S100 is as follows: 。 9. The method for preparing a microenvironment activated probe for arthritis according to claim 7, characterized in that: S200 includes the following steps: S201, dissolving a bipyridine luminophore and a lipid in an organic solvent to obtain a first mixture; The lipids are distearoylphosphatidylethanolamine-polyethylene glycol and 1,2-distearoyl-sn-glycerol-3-phosphoolamine-N-[maleimide (polyethylene glycol)], and the mass ratio of distearoylphosphatidylethanolamine-polyethylene glycol to 1,2-distearoyl-sn-glycerol-3-phosphoolamine-N-[maleimide (polyethylene glycol)] is 1:1; S202, adding the first mixture into water and mixing evenly to obtain a second mixture; S203, dialyzing and filtering the second mixture to obtain a first nanoparticle solution.

10. The method for preparing a microenvironment activated probe for arthritis according to claim 9, characterized in that: S300 includes the following steps: S301, adding IL-6R antibody to the first nanoparticle solution for reaction to obtain second nanoparticles; S302, centrifuging, purifying and concentrating the second nanoparticles to obtain bipyridine luminescent nanoparticles.

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