A microenvironment activation probe for arthritis and its preparation method

Abstract

The present invention relates to the technical field of biomedical materials, and particularly relates to a microenvironment-activated probe for arthritis and a preparation method thereof. The microenvironment-activated probe for arthritis includes bipyridine luminescent nanoparticles and IL-6R, and IL-6R is connected to the surface of the bipyridine luminescent nanoparticles through a thiol-maleimide addition reaction. The preparation method includes: preparing bipyridine luminescent bodies; nanosizing the bipyridine luminescent bodies to prepare bipyridine luminescent nanoparticles; modifying the bipyridine luminescent nanoparticles with anti-IL-6R antibodies to prepare a microenvironment-activated probe for arthritis. The microenvironment-activated probe provided by the present invention significantly inhibits the release of pro-inflammatory factors, promotes the repolarization of macrophages from the M1 phenotype to the M2 phenotype, and can significantly relieve RA symptoms and joint damage. The preparation method provided by the present invention is simple, easy to operate, and easy to industrialize production.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a microenvironment-activated 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 reducing the damage caused by inflammation has been incorporated into the scope of RA treatment. A large amount of evidence indicates that the overproduction 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 apoptosis but also activates immune cells, thereby triggering the release of pro-inflammatory cytokines and exacerbating joint inflammation. The elevated ROS level in the joint provides a promising molecular target for the diagnosis and treatment of RA. Specifically, the use of ROS scavengers to target and remove ROS has shown potential for RA intervention. However, due to the multi-factorial nature of the RA pathology, a single ROS scavenging therapy is often insufficient to achieve effective remission.

[0003] Fluorescence (FL) is a non-invasive and non-radioactive imaging technique. Compared with traditional imaging techniques, FL imaging performs excellently in terms of sensitivity, real-time feedback, and cost-effectiveness. Moreover, fluorescence imaging technology also has the potential to provide precise pathophysiological insights into joints by detecting FL-based RA-specific biomarkers. Near-infrared-II (NIR-II, 1000 nm - 1700 nm) imaging can visualize deep tissue structures with higher resolution and contrast compared to traditional visible light and NIR-I (400 nm - 900 nm) 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 the "always-on" state with a constant signal and lack 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 an on NIR-II FL signal in response to specific pathological features, thereby improving 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-activated probe for arthritis; the second object of the present invention is to provide a preparation method of a microenvironment-activated probe for arthritis.

[0005] To achieve the first object, the technical solution adopted by the present invention is as follows:

[0006] A microenvironment activation probe for arthritis, comprising:

[0007] A bipyridine luminophore nanoparticle and an IL-6R, and the IL-6R is connected to the surface of the bipyridine luminophore nanoparticle through a thiol-maleimide addition reaction.

[0008] Further, the bipyridine luminophore nanoparticle comprises a bipyridine luminophore, and the bipyridine luminophore comprises an aggregation-induced emission core and a carbonyl manganese structure corresponding to reactive oxygen species.

[0009] Further, 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) ((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)).

[0010] Further, 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) tricarbonyl bromomanganese (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, abbreviated as TTCO), and its structural formula is as follows:

[0011] .

[0012] Further, the bipyridine luminophore nanoparticle is a water-soluble nanoparticle.

[0013] Further, the bipyridine luminophore nanoparticle is a water-soluble nanoparticle.

[0014] To achieve the second object, the technical solution adopted by the present invention is as follows:

[0015] A preparation method of a microenvironment activation probe for arthritis, which is used to prepare the microenvironment activation probe described in any one of the above, includes the following steps:

[0016] S100: Using 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline as a raw material to prepare a bipyridine luminophore;

[0017] S200: Nanometerize the bipyridine luminophore to prepare bipyridine luminophore nanoparticles;

[0018] S300: Modify the bipyridine luminophore nanoparticles with an anti-IL-6R antibody to prepare a microenvironment activation probe for arthritis.

[0019] Further, the synthesis route of the bipyridine luminophore in S100 is as follows:

[0020] 。

[0021] Further, S200 includes the following steps:

[0022] S201: Dissolve the bipyridine luminophore and lipids in an organic solvent to obtain a first mixture;

[0023] Wherein, the lipid is distearoyl phosphatidylethanolamine-polyethylene glycol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)], and the mass ratio of distearoyl phosphatidylethanolamine-polyethylene glycol to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] is 1:1;

[0024] S202: Add the first mixture to water and mix evenly to obtain a second mixture;

[0025] S203: Dialyze and filter the second mixture to obtain a first nanoparticle solution.

[0026] Further, S300 includes the following steps:

[0027] S301: Add an IL-6R antibody to the first nanoparticle solution for reaction to obtain a second nanoparticle;

[0028] S302: Centrifuge, purify, and concentrate the second nanoparticle to obtain bipyridine luminophore nanoparticles.

[0029] One or more of the above technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0030] A microenvironment-activated probe for arthritis provided by the present invention, the probe comprising bipyridine luminescent nanoparticles and IL-6R, and the IL-6R is connected to the surface of the bipyridine luminescent nanoparticles through a thiol-maleimide addition reaction. The bipyridine luminescent nanoparticles are biocompatible nanoprobes self-assembled, and then combined with an anti-interleukin-6 receptor (IL-6R) antibody to achieve active targeting of rheumatoid arthritis. After intravenous injection into a collagen-induced arthritis mouse model (Rheumatoid arthritis, RA), the nanoprobe shows significant activation of NIR-II fluorescence signal at the RA lesion, enabling highly sensitive diagnosis and real-time treatment monitoring of RA. Meanwhile, the combination of ROS scavenging, on-demand release of CO gas, and IL-6 signal blockade produces a powerful therapeutic effect and a synergistic immunomodulatory effect. The microenvironment-activated probe significantly inhibits the release of pro-inflammatory factors, promotes the repolarization of macrophages from the M1 phenotype to the M2 phenotype, and can significantly relieve RA symptoms and joint damage. The present invention also provides a preparation method for the microenvironment-activated probe for arthritis, and the preparation method is simple, easy to operate, and easy to industrialize production.

[0031] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Brief Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0033] Figure 1 It is the 1H NMR spectrum of TTCO provided in Example 1 of the present invention.

[0034] Figure 2 It is the 13C NMR spectrum of TTCO provided in Example 1 of the present invention.

[0035] Figure 3 It is the high-resolution mass spectrum of TTCO provided in Example 1 of the present invention.

[0036] Figure 4 It is the molecular structure change diagram of TTCO after being triggered by ROS provided in Example 1 of the present invention.

[0037] Figure 5It is the normalized absorption spectrogram of TTCO and the product after the reaction of TTCO with ROS in tetrahydrofuran provided in Example 1 of the present invention.

[0038] Figure 6 It is the normalized fluorescence spectrogram of TTCO and the product after the reaction of TTCO with ROS in tetrahydrofuran (THF) provided in Example 1 of the present invention.

[0039] Figure 7 It is the I / I 0 variation diagram with respect to the water fraction (f w ).

[0040] Figure 8 It is the normalized absorption spectrogram of different groups before and after being treated with H 2 O 2 provided in Example 2 of the present invention.

[0041] Figure 9 It is the fluorescence spectrogram of TC@AI NPs before and after being treated with H 2 O 2 provided in Example 2 of the present invention.

[0042] Figure 10 It is the excitation-emission diagram of TC@AI NPs before and after being treated with H 2 O 2 provided in Example 2 of the present invention.

[0043] Figure 11 It is the relationship curve diagram between the NIR-II fluorescence intensity of TC@AI NPs and the concentration of H 2 O 2 provided in Example 2 of the present invention.

[0044] Figure 12 It is the relationship curve diagram between the NIR-II fluorescence signal of TC@AI NPs and the ROS concentration provided in Example 2 of the present invention.

[0045] Figure 13 It is the relationship curve diagram between the treatment time of H 2 O 2 and the CO release concentration provided in Example 2 of the present invention.

[0046] Figure 14 It is the image of the influence of various interfering factors on the CO release concentration and fluorescence signal of TC@AI NPs provided in Example 2 of the present invention.

[0047] Figure 15Representative 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.

[0048] Figure 16 Flow cytometry analysis images 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.

[0049] Figure 17 Statistical chart of quantitative analysis of the internalization 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.

[0050] Figure 18 Representative NIR-II fluorescence signals of TC NPs or TC@AI NPs in non-activated and inflamed macrophages provided in Experimental Example 2 of the present invention.

[0051] Figure 19 Statistical chart of quantitative analysis of the NIR-II fluorescence intensity of cells provided in Experimental Example 2 of the present invention.

[0052] Figure 20 Representative CLSM images of macrophages stained with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) after treatment with different nanoparticles, representative CLSM images of non-activated and inflamed macrophages treated with different nanoparticles, and images of different nanoparticles stained with a CO probe provided in Experimental Example 2 of the present invention.

[0053] Figure 21 Statistical chart of quantitative analysis of the fluorescence intensity of the CO probe provided in Experimental Example 2 of the present invention.

[0054] Figure 22 Representative CLSM images of the expression of iNOS (red fluorescence) and CD206 (green fluorescence) on RAW264.7 macrophages after treatment with different substances provided in Experimental Example 3 of the present invention.

[0055] Figure 23 Immunofluorescence staining images of the expression of HO-1 in non-activated and activated RAW264.7 macrophages after treatment with different substances provided in Experimental Example 3 of the present invention.

[0056] Figure 24 Western blotting analysis of the expression levels of HO-1 and proteins related to the Notch / Hes1 / Stat3 pathway in RAW264.7 macrophages treated with different substances provided in Experimental Example 3 of the present invention.

[0057] Figure 25 It is 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 provided in Experimental Example 3 of the present invention.

[0058] Figure 26 It is the NIR-II FL images of mice after directly injecting TC@AI NPs 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.

[0059] Figure 27 It is the corresponding quantitative analysis chart of the NIR-II FL intensity in the inflammatory site or the unaffected side after administration of TC@AI NPs provided in Experimental Example 4 of the present invention.

[0060] Figure 28 It is the representative NIR-II FL images in healthy or RA mice at different time points after intravenous injection of different substances provided in Experimental Example 4 of the present invention.

[0061] Figure 29 It is the corresponding quantitative analysis chart 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.

[0062] Figure 30 It is the arthritis scores of healthy mice and RA mice at different time points after treatment with different substances provided in Experimental Example 5 of the present invention.

[0063] Figure 31 It is the representative photos of the front and hind paws of healthy mice and RA mice after treatment with different substances provided in Experimental Example 5 of the present invention.

[0064] Figure 32 It is the representative micro-CT images of the hind paws of healthy mice and RA mice after treatment with different substances provided in Experimental Example 6 of the present invention.

[0065] Figure 33 It is the quantitative analysis statistical chart of the effects of different substances on BV / TV and BV in mice provided in Experimental Example 6 of the present invention.

[0066] Figure 34 It is the 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.

[0067] Figure 35 It is the quantitative analysis statistical chart 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.

[0068] Figure 36 These are H&E staining images of the paw joints of healthy mice provided in Experimental Example 7 of the present invention and RA mice after treatment with different substances.

[0069] Figure 37 These are statistical graphs of H&E scores of healthy mice provided in Experimental Example 7 of the present invention and those of RA mice after treatment with different substances.

[0070] Figure 38 These are Safranin-O staining images of the paw joints of healthy mice provided in Experimental Example 7 of the present invention and RA mice treated with different preparations.

[0071] Figure 39 These are Safranin-O scores of the paw joints of healthy mice provided in Experimental Example 7 of the present invention and those of RA mice treated with different preparations.

[0072] Figure 40 These are immunohistochemical stains of IL-6 expression in normal and inflamed joints of different treatment groups provided in Experimental Example 7 of the present invention.

[0073] Figure 41 These are statistical graphs of the intensity of IL-6 in normal and inflamed joints of different treatment groups provided in Experimental Example 7 of the present invention.

[0074] Figure 42 These are immunohistochemical staining images of TNF-α expression in normal and inflamed joint sections of different treatment groups provided in Experimental Example 7 of the present invention after treatment.

[0075] Figure 43 These are statistical graphs of the relative staining intensity of immunohistochemical stains of TNF-α expression in normal and inflamed joint sections after different treatments provided in Experimental Example 8 of the present invention.

[0076] Figure 44 These are statistical graphs of the concentrations of IL-6 and TNF-α in joint tissue homogenates after different treatments analyzed quantitatively using ELISA provided in Experimental Example 8 of the present invention.

[0077] Figure 45 These are images of the expression of iNOS and CD206 in joint tissues after different treatments shown by immunofluorescence imaging provided in Experimental Example 8 of the present invention.

[0078] Figure 46 These are statistical graphs of the quantitative analysis of the fluorescence intensities of iNOS and CD206 in normal and inflamed joints after different treatments provided in Experimental Example 8 of the present invention. Detailed implementation manners

[0079] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts fall 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.

[0080] In the following embodiments, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0081] Example 1 Preparation of TTCO.

[0082] I. Preparation of 1-iodo-4-(octyloxy)benzene:

[0083] Under an argon atmosphere, 4-iodophenol (4.4 g, 20 mmol), 1-bromooctane (4.44 g, 23 mmol) and potassium carbonate (K 2 CO 3 ) (8.98 g, 65 mmol) were mixed, and then anhydrous N,N-dimethylformamide (DMF) (90 ml) was added. After cooling to room temperature, water was added, and the mixture was extracted three times with dichloromethane (CH 2 Cl 2 ). The collected organic phase was dried over magnesium sulfate and concentrated. The crude product was purified by silica gel column chromatography, and the elution solvent was CH 2 Cl 2 / n-hexane (v / v 1:10) to obtain colorless solid 1-iodo-4-(octyloxy)benzene with a yield of 83%.

[0084] II. Preparation of 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline:

[0085] Under an argon atmosphere, 1-iodo-4-(octyloxy)benzene (9.96 g, 30 mmol), bromoaniline (2.06 g, 12 mmol), copper(I) 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 the mixture was extracted three times with CH 2 Cl 2 . The collected organic phase was dried over magnesium sulfate and concentrated. The crude product was purified by silica gel column chromatography, and the elution solvent was CH 2 Cl 2 / n-Hexane (v / v 1:6) to obtain a colorless solid, 4-bromo-N,N-bis(4-(octyloxy)phenyl)aniline, in 72% yield.

[0086] III. Preparation of 4-(octyloxy)-N-(4-(octyloxy)phenyl)-N-(4-(tributylstannyl)phenyl)aniline:

[0087] 4-Bromo-N,N-bis(4-(octyloxy)phenyl)aniline (3.48 g, 6 mmol) was placed under an argon atmosphere, and then anhydrous tetrahydrofuran (50 mL) was added. The reaction was cooled to -78 o °C and maintained for 30 minutes, and then n-butyllithium (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 allowed to warm slowly to room temperature and then stirred overnight. Subsequently, water was added to quench the reaction, and the mixture was extracted three times with CH 2 Cl 2 The resulting mixture was extracted three times. 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-(tributylstannyl)phenyl)aniline.

[0088] IV. Preparation of 4,4'-(5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N,N-bis(4-(octyloxy)phenyl)aniline):

[0089] Under an argon atmosphere, 4-(octyloxy)-N-(4-(octyloxy)phenyl)-N-(4-(tributylstannyl)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, and then anhydrous tetrahydrofuran (80 mL) was added. After cooling to room temperature, water was added, and the mixture was extracted three times with CH 2 Cl 2 The collected organic phases were dried over MgSO 4 and concentrated. Column chromatography purification on silica gel using CH 2 Cl 2 / n-Hexane (v / v 1:2) as the elution solvent gave 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 69% yield.

[0090] V. Preparation of 4,7-bis(4-(bis(4-(octyloxy)phenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine:

[0091] Under an 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 CH 2 Cl 2 . The collected organic phase was dried over MgSO 4 , 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.

[0092] VI. Preparation of TT:

[0093] Under an argon atmosphere, 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, and then 1,10-phenanthroline-5,6-dione (0.42 g, 2 mmol) was added. The mixture was heated to reflux and stirred continuously for 12 hours. After cooling to room temperature, water was added, and the mixture was extracted three times with dichloromethane. The collected organic phase was dried over magnesium sulfate and concentrated. Column chromatography purification was performed on silica gel using CH 2 Cl 2 / n-hexane (v / v 1:1) as the elution solvent to obtain TT as a dark green solid with a yield of 62%.

[0094] VII. Preparation of TTCO:

[0095] TT (142 mg, 0.086 mmol) and Mn(CO) 5 Br (20 mg, 0.086 mmol) were dissolved in 25 ml of diethyl ether, and the mixture was heated to reflux for 3 hours under an argon atmosphere. After cooling to room temperature, the green precipitate was collected by vacuum filtration and washed with diethyl ether. The resulting TTCO was a dark green solid with a yield of 43%.

[0096] The characterization of the TTCO molecule is as Figures 1 to 3 shown, 11H 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 13C 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.

[0097] The molecular structure change of TTCO after being triggered by ROS is as Figure 4 shown.

[0098] The spectrograms of TTCO before and after being triggered by ROS are as Figures 5 to 7 shown.

[0099] Among them, Figure 7 I 0 and I respectively represent the maximum fluorescence intensities of pure THF and THF / water mixtures with different water fractions.

[0100] Example 2 Preparation of a microenvironment-activated probe for arthritis.

[0101] Materials:

[0102] Distearoyl phosphatidylethanolamine - polyethylene glycol (abbreviated as DSPE-PEG);

[0103] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] (abbreviated as DSPE-PEG-MAL);

[0104] Tetrahydrofuran (abbreviated as THF);

[0105] Cell membrane red fluorescent dye (abbreviated as DiD);

[0106] The preparation process is as follows:

[0107] 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 dropped into 10 ml of water and mixed well. The resulting solution was dialyzed using a 10 kDa membrane to remove free molecules and residual THF solvent. Then, the solution was further filtered through a 0.2 μm filter. Then, anti-IL-6R antibody (400 μg) was added to the nanoparticle solution (5 mL), and the reaction was carried out at 4 o °C for 12 h, and then centrifuged at 15,000 rpm for 15 minutes to remove the remaining formulation and unreacted anti-IL-6R antibody in the solution. Subsequently, the mixture was resuspended in PBS (10 mL), and the purification process was repeated twice. Finally, the suspension of 4,4'-(bipyrrolo[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) tricarbonyl bromomanganese nanoparticles modified with anti-IL-6R antibody (abbreviated as TC@AI NPs) was concentrated, and TC@AI NPs is the microenvironment activation probe for arthritis.

[0108] Fluorescently labeled nanoparticles were prepared for fluorescence imaging investigation. DiD was added to the mixed solution of TTCO, DSPE-PEG, and DSPE-PEG-MAL to bind the dye to TC@AI NPs, and the fluorescently labeled nanoparticles were prepared according to the above preparation process.

[0109] Figure 8 The results showed that TC@AI NPs exhibited a slight blue shift in the absorption spectrum after H 2 O 2 treatment, which was due to the cleavage of the electron-withdrawing manganese complex.

[0110] Figure 9 、 Figure 10 The results showed that the NIR-II fluorescence signal of TC@AI NPs was significantly enhanced after H 2 O 2 treatment, and a new emission peak appeared at about 1070 nm.

[0111] Figure 11 The results showed that the NIR-II fluorescence intensity of TC@AI NPs increased with the increase of H 2 O 2 concentration;

[0112] Figure 12The results showed that the NIR-II fluorescence signal of TC@AI NPs was directly and positively correlated with the ROS concentration.

[0113] After confirming the ROS-triggered NIR-II fluorescence turn-on property of TC@AI NPs, reduced hemoglobin was used as a probe to study the in-situ generation of CO gas.

[0114] Figure 13 The results showed that with the increase of H 2 O 2 treatment time, the CO concentration released by TC@AI NPs increased significantly.

[0115] Figure 14 The results showed that among the responses to interfering ions such as Co 2+ , Cu 2+ , Fe 3+ , K + , Mn 2+ , Na + and Zn 2+ , TC@AI NPs had good selectivity for ROS.

[0116] In summary, TC@AI NPs can activate NIR-II fluorescence and generate therapeutic CO gas in-situ under ROS stimulation.

[0117] Experimental Example 1

[0118] Investigation of the uptake of TC@AI NPs by macrophages (RAW264.7).

[0119] To evaluate the potential of TC@AI NPs to actively target inflammatory immune cells, in this invention, RAW264.7 mouse macrophages were pretreated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to induce inflammation, and the active binding of TC@AI NPs to immune cells was tested. The specific process is as follows:

[0120] TC@AI NPs labeled with a cell membrane green fluorescent probe (3,3′-dioctadecyloxacarbocyanine perchlorate, DIO for short) were co-incubated with inflamed macrophages for 4 hours and imaged under a confocal laser scanning microscope (CLSM).

[0121] Figure 15The results showed that strong fluorescence signals were exhibited by TC@AI NPs in activated macrophages, while weak fluorescence signals were shown by cells treated with TC NPs without modification of anti-IL-6R antibody. These results indicated that the uptake of TC@AI NPs by activated macrophages was significantly increased. In normal macrophages not stimulated by pro-inflammatory cytokines, the incubation of TC@AI NPs produced weak red fluorescence signals, in contrast to the strong signals in the treated activated macrophages. The above results indicated that TC@AI NPs had enhanced targeting ability to inflammation-activated macrophages due to the specific binding affinity of the antibody to IL-6R overexpressed on the surface of activated macrophages. The quantitative analysis of fluorescence intensity by flow cytometry, Figure 16 as shown further verified that TC@AI NPs had enhanced targeting ability to inflammation-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 approximately 3 times that of cells exposed to TC NPs, as Figure 17 shown.

[0122] The above results indicated that TC@AI NPs could actively target M1 macrophages in the in vitro environment.

[0123] Experimental Example 2 Investigation of the sensitivity of TC@AI NPs for ROS detection in living cells.

[0124] RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) and IFN-γ to promote the generation of ROS, and then incubated with various nanoparticles. As a control, normal macrophages not subjected to pro-inflammatory stimulation were also exposed to TC@AI NPs. After 6 hours of nanoparticle incubation, the intracellular fluorescence signals of the nanoprobes were visualized by NIR-II fluorescence microscopy.

[0125] As Figure 18 the results showed, the NIR-II fluorescence signals in activated macrophages treated with TC@AI NPs were significantly enhanced at an excitation wavelength of 808 nm and an emission wavelength of 1000 nm. In contrast, TC@AI NPs emitted only weak NIR-II signals in normal macrophages. The intensity of the NIR-II fluorescence signal in cells exposed to TC@AI NPs after LPS + IFN-γ stimulation was 5.8 times higher than that of normal macrophages treated with TC@AI NPs, as Figure 19 shown, and this result indicated the activation of the NIR-II fluorescence signal induced by ROS.

[0126] After confirming the ROS-activated imaging ability of TC@AI NPs, the present invention continued to evaluate the ability of TC@AI NPs to respond to and scavenge intracellular ROS using DCFH-DA staining. DCFH-DA can be converted from a non-fluorescent compound to green fluorescent 2',7'-dichlorofluorescein (abbreviated as DCF) after reacting with ROS. Figure 20 The results showed that RAW 264.7 cells exhibited a significant enhancement in ROS generation after stimulation with LPS and IFN-γ, manifested as strong green fluorescence of DCF. Both TCNPs and TC@AI NPs showed effective ROS scavenging ability at the cellular level. In particular, TC@AI NPs showed a more significant ROS consumption effect due to their superior targeting ability. After treatment with TCNPs or TC@AI NPs, the ROS levels were significantly reduced to 29.3% and 16.5% of the control level, respectively, as Figure 21 shown. In summary, the targeting ability and ROS-responsive NIR-II fluorescence activation property of TC@AI NPs were verified through in vitro macrophage activation experiments, indicating the potential application of TC@AI NPs in RA treatment.

[0127] Among them, Figure 21 Non-activated refers to non-activated macrophages.

[0128] Experimental Example 3 Inflammatory inhibition and immunomodulation at the cellular level.

[0129] Immunofluorescence staining of TC@AI NPs showed that normal macrophages did not significantly express iNOS and CD206. iNOS is a surface marker of M1 macrophages, and CD206 is a surface marker of M2 macrophages, indicating that they are mainly in an inactive state, as Figure 22 shown. However, after stimulation with LPS and IFN-γ, the widespread expression of iNOS indicated the polarization of macrophages towards the M1 phenotype. After co-incubation of inflammatory RAW 264.7 cells with TCNPs, AI NPs, or TC@AI NPs, the number of iNOS-positive cells decreased significantly, while the number of CD206-positive cells increased significantly. The effect was more obvious in cells treated with TC@AI NPs. The above results showed that TC@AI NPs could act as effective regulators to promote the transformation of macrophages from the M1 to the M2 phenotype.

[0130] Studies have shown that CO can activate the expression of HO-1, which is an endogenous antioxidant factor that maintains cellular homeostasis under pathological conditions. The present invention evaluated the level of intracellular HO-1 through immunofluorescence staining. As Figure 23As shown, TC@AINPs release 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 Figure 24 shown. In addition, the protein expression levels of p-Stat3, Hes1, and Notch1 were significantly upregulated in cells treated with TC@AI NPs. The results indicate 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 an anti-inflammatory phenotype in immune cells. The present invention further analyzed the ability of TC@AI NPs to mitigate pro-inflammatory signals. 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 Figure 25 shown. 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, reducing intracellular ROS stress, thereby blocking the pro-inflammatory signaling pathway triggered by ROS.

[0131] Among them, Figure 24 and Figure 25 Non-activated in

[0132] refers to non-activated macrophages.

[0133] To investigate the real-time activation of NIR-II fluorescence in a ROS-overexpressing inflammatory microenvironment, the present invention established a subcutaneous inflammation model by locally injecting LPS into the right back of mice. Six hours after injecting LPS, 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 left part of mice not induced by LPS. Two hours after injecting TC@AI NPs, the mice were imaged using a NIR-II imaging system. As Figure 26 shown, the LPS-stimulated site emitted a strong 1000 nm NIR-II fluorescence signal under 808 nm light irradiation, while only a weak NIR-II signal was shown in the healthy part. Quantification showed that two hours after injecting TC@AI NPs, the intensity of the NIR-II fluorescence signal in the inflammatory site was 4.3 times that of the healthy control site, as Figure 27 shown. This observation confirmed the real-time activation effect of TC@AI NPs in inflammatory lesions and indicated that this turn-on type of NIR-II imaging can promote accurate and sensitive imaging of TC@AI NPs for inflammatory RA lesions.

[0134] This invention evaluated the ability of TC@AI NPs for in vivo RA-targeted and activatable imaging 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 treatment methods. The CIA model was induced by priming DBA / 1 mice with bovine type II collagen (CII) emulsified in complete Freund's adjuvant (CFA) on day 0. A second booster immunization was performed on day 20 with type II collagen emulsified in incomplete Freund's adjuvant (IFA). After the second immunization, RA symptoms such as joint redness and swelling appeared, and the arthritis score was significantly higher than that of healthy mice, confirming the successful establishment of the RA model.

[0135] RA mice were randomly divided into four groups and intravenously injected with PBS, TC NPs, and TC@AI NPs, respectively, and then imaged using a NIR-II imaging system. The results are as Figure 28 shown. Mice injected with PBS had almost no NIR-II fluorescence signal in the paws, indicating low background noise in the NIR-II region. In contrast, mice injected with TC NPs showed moderate NIR-II fluorescence signals in the 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), which was initially observed in the delivery of NPs in tumors and has recently also been found in some inflammatory diseases. Mice injected with TC@AI NPs showed significantly brighter NIR-II fluorescence signals in the RA joints at all tested time points, and the fluorescence intensity was 1.7 times higher than that of mice injected with TC NPs 4 hours after injection, as Figure 29 shown. These results indicate that anti-IL-6R antibody modification can increase the accumulation of NPs in arthritic joints.

[0136] Experimental Example 5 Evaluated the therapeutic effect of TC@AI NPs on RA mice from a behavioral perspective.

[0137] An RA model was established using conventional technical means of the prior art. Obvious paw swelling and redness appeared 28 days after the primary immunization of the mice.

[0138] Mice were randomly divided into four groups (5 mice per group), namely the 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 sacrificed on the 50th day. Meanwhile, healthy DBA / 1 mice without any intervention were used as the control group. The joints of the mice were monitored every two days, and the severity of arthritis was evaluated using a standard scoring method. The results are as Figure 30 shown. The injection of TC NPs or AI NPs partially alleviated the arthritis symptoms. On the 50th day, 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). Notably, the TC@AI NPs treatment almost completely alleviated the paw swelling and redness caused by arthritis, with an average arthritis score of 2.2 points. At the end of the treatment, the paw joints of the mice were photographed. As Figure 31 shown, the representative photos of the front and hind paws indicate that the mice treated with TC@AI NPs had the least swelling and redness, and the paw conditions were close to those of normal mice. The superior effect of TC@AI NPs in alleviating RA can be attributed to its multi-pathway synergistic treatment effect and enhanced targeting ability.

[0139] Experimental Example 6 The therapeutic effect of TC@AI NPs on RA mice was evaluated by micro-computed tomography (micro-CT) and near-infrared II (NIR-II) fluorescence imaging.

[0140] The bone morphology and microstructure of the paw joints of mice receiving different treatments were evaluated by micro-CT. As Figure 32 shown, the joints of the RA model mice showed significant bone damage, characterized by a rough surface, cartilage degeneration, and reduced bone density. Although the injection of TC NPs or AI NPs partially alleviated the bone erosion, obvious bone damage still remained. However, the TC@AI NPs treatment group showed a significant recovery of bone structure integrity, characterized by distinct bone boundaries and a relatively smooth joint surface. These results indicate that TC@AI NPs can effectively reverse the bone erosion caused by RA.

[0141] Histomorphological analysis was performed on the micro-CT images to quantitatively evaluate the degree of bone damage in different experimental groups. The results are as Figure 33 shown, and these results indicate that the bone volume fraction (BV / TV) of the RA mice treated with the TC@AI NPs group was significantly increased compared to that of 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 regions, consistent patterns were shown. 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.

[0142] Among them, Figure 33 in which, 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.

[0143] 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 explores the potential of TC@AI NPs to monitor the therapeutic effect through activated NIR-II FL molecular imaging.

[0144] At the end of the treatment protocol, 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, and the results are as Figure 34 and Figure 35 shown. The joints of RA mice treated with PBS exhibited strong NIR-II FL signals, attributed to the elevated ROS levels in the inflamed joints. The NIR-II FL signals of the joints of RA mice treated with TC NPs or AI NPs were weakened. The joints of mice treated with the TC@AI NPs group showed the weakest NIR-II FL signals, highlighting the effective reduction of joint inflammation and ROS levels. The results of this NIR-II FL imaging were consistent with the observed paw scores and micro-CT analysis. In summary, TC@AI NPs can not only effectively relieve RA but also serve as a powerful imaging tool for the accurate diagnosis of RA and the tracking of treatment responses.

[0145] Experimental Example 7 Investigation of the therapeutic effect of TC@AI NPs on RA mice was evaluated by hematoxylin-eosin (H&E) staining, Safranin-O fast green staining, and immunohistochemistry.

[0146] The joints of mice in different groups were sectioned and stained with hematoxylin-eosin (H&E) to evaluate joint pathology. Representative H&E images, as Figure 36 and Figure 37 shown. The joints of CIA mice exhibited extensive inflammatory cell infiltration, severe bone erosion, and highly activated abnormal synovial fibroblasts - hallmark features of RA pathology. The joints of RA mice treated with TC NPs or AI NPs showed moderate immune cell infiltration and synovial hyperplasia. Notably, the joint cavity of the TC@AI NPs group almost returned to normal, characterized by clear bone boundaries, less immune cell infiltration, and limited bone deformities. Further evaluation of cartilage degradation and extracellular matrix changes was performed using Safranin O-fast green staining, as Figure 38 and Figure 39As shown, the articular cartilage of healthy mice is thick, and proteoglycans are evenly distributed in the matrix. In contrast, the articular cartilage of RA mice is significantly degraded, and there is a significant loss of proteoglycans in the cartilage matrix. After applying different nanoformulations, cartilage preservation was improved. The TC@AI NPs group showed an obvious alleviating effect on cartilage degradation, characterized by an almost intact and smooth cartilage surface and evenly distributed proteoglycans in the bone cavity. 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 comprehensive effects of scavenging ROS, releasing anti-inflammatory CO gas, and blocking the IL-6 pathway.

[0147] In addition, the present invention also analyzed the levels of pro-inflammatory cytokines in the joint microenvironment to evaluate the immunomodulatory effect of TC@AI NPs. Pro-inflammatory 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 as Figures 40 to 42 shown. Paw sections of RA mice showed a significant upregulation of these inflammatory factors, while the expression in healthy control mice was low. Administration of TC NPs or AI NPs inhibited the expression of inflammatory cytokines to a certain extent. These findings indicate that the TTCO probe that consumes ROS and releases CO, as well as AI NPs targeting IL-6R, can both inhibit the inflammatory response. 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.

[0148] Experimental Example 8 Evaluated the therapeutic effect of TC@AI NPs on RA mice by enzyme-linked immunosorbent assay and immunofluorescence staining.

[0149] Figure 43 The results showed that paw sections of RA mice showed a significant upregulation of TNF-α and IL-6 inflammatory factors, while the expression in healthy control mice was low.

[0150] The levels of IL-6 and TNF-α in joint tissue homogenates were further quantitatively evaluated by enzyme-linked immunosorbent assay (ELISA). The results are as Figure 44 shown. The levels of IL-6 and TNF-α in the joint homogenates of mice treated with the TC@AI NPs group were significantly reduced. The IL-6 levels in other groups were 6.53 to 2.18 times higher, and the TNF-α concentrations in other groups were 5.21 to 2.03 times higher.

[0151] The present invention also further studied the M1 / M2 polarization of macrophages in the joint microenvironment. Immunofluorescence staining images, such as Figure 45As shown, the expression of the M1 macrophage marker (iNOS) in the joints of mice treated with the TC@AI NPs group was significantly reduced, while the level of the M2 macrophage marker (CD206) increased. The iNOS expression in the PBS group was 5.28 times higher than that in the TC@AI NPs group, and the CD206 expression in the TC@AI NPs group was 10.70 times higher than that in the PBS group, as Figure 46 shown. These results indicate that TC@AI NPs promoted the repolarization of M1 macrophages towards the anti-inflammatory M2 phenotype, which is consistent with the in vitro study results.

[0152] In summary, the above results demonstrated the synergistic efficacy of TC@AI NPs in the treatment of RA, attributed to its enhanced RA targeting ability and multi-target anti-inflammatory and immunomodulatory effects.

[0153] Among them, MFI in the figure is the abbreviation of Mean Fluorescence Intensity, and Healthy refers to healthy mice.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate 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 attached to the surface of bipyridine luminescent nanoparticles via thiol-maleimide addition reaction; 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; 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: 。 2. The microenvironment activated probe for arthritis according to claim 1, characterized in that: The bipyridyl luminescent nanoparticles are water-soluble nanoparticles.

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

4. 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 3 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.

5. The method for preparing a microenvironment activated probe for arthritis according to claim 4, characterized in that: The synthesis route of the bipyridine luminophore in S100 is as follows: 。 6. The method for preparing a microenvironment activated probe for arthritis according to claim 4, 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.

7. The method for preparing a microenvironment activated probe for arthritis according to claim 6, 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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