Multi-coordination polyphenol nanozyme and preparation method and application thereof

Abstract

The application discloses a kind of multi-coordination polyphenol nanozyme and its preparation method and application, belong to biological medicine field.The preparation method includes: tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde are dissolved in mixed solvent, and solvent thermal reaction is carried out under closed condition;Subsequently, add the mixed solvent of nickel (II) acetate tetrahydrate, DMF and water, continue to carry out solvent thermal reaction under closed condition, obtain multi-coordination polyphenol nanozyme.The Salen type N2O2 nickel coordination element and ortho polyphenol Ni-O4 bridging coordination element are integrated in the same coordination polymer in the application.The nanozyme has stable water dispersibility, good in vitro cell compatibility and blood compatibility, broad-spectrum ROS scavenging capacity, macrophage immune reprogramming capacity and vascular smooth muscle cell contraction phenotype maintenance capacity, and can be used for preparing drugs for preventing and / or treating aortic dissection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a multi-coordinated polyphenol nanozyme, its preparation method, and its application. Background Technology

[0002] Aortic dissection (AD) is a catastrophic vascular event characterized by the gradual separation of the aortic wall layers, often ending in fatal rupture. The fundamental pathological driver of AD is the catastrophic remodeling of the extracellular matrix (ECM) and the simultaneous phenotypic transformation of vascular smooth muscle cells (SMCs). Under chronic pathological stimulation, contractile SMCs, characterized by high expression of α-smooth muscle actin (α-SMA), transform into a synthetic state. Synthetic SMCs exhibit enhanced migration capacity and secrete large amounts of osteopontin (OPN), an ECM protein that further accelerates medial degeneration and aortic wall thinning. Despite this understanding, current AD treatment strategies remain primarily limited to blood pressure control and surgical intervention, neither of which can directly reverse the underlying SMC phenotypic regulatory dysregulation or restore aortic wall homeostasis.

[0003] Studies show that excessive accumulation of reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anions, and hydrogen peroxide in aortic lesions can induce macrophages to maintain a pro-inflammatory M1 phenotype, amplify local inflammatory responses, and promote the transformation of vascular smooth muscle cells (SMCs) to a synthetic phenotype through paracrine signals. Synthetic SMCs exhibit enhanced migration ability and secrete extracellular matrix-related proteins such as osteopontin, further exacerbating tunica media degeneration and aortic wall fragility. However, the precise molecular bridge connecting macrophage-derived signals with intracellular SMC signaling mechanisms remains insufficiently elucidated. Furthermore, existing pharmacological approaches attempting to modulate oxidative stress or inflammation often suffer from poor bioavailability, off-target toxicity, and difficulties in efficiently clearing ROS and reshaping the local immune microenvironment, leaving a critical gap in targeted therapy for Alzheimer's disease (AD).

[0004] Natural polyphenols possess inherent free radical scavenging capabilities, but their rapid metabolism, poor chemical stability, and limited bioavailability restrict their application in chronic vascular pathology. Furthermore, their monomeric properties cannot support multi-center synergistic catalysis and are difficult to integrate into well-defined therapeutic platforms. For nanozymes capable of targeting aortic macrophages to achieve intracellular ROS scavenging and immune reprogramming, several biophysical requirements must be met, including suitable hydrophilicity for stable dispersion in physiological media, good biointerface compatibility to promote phagocytosis and uptake, and low off-target toxicity to ensure biosafety. Traditional polyphenol-metal networks often aggregate in aqueous environments or exhibit poor membrane affinity, thus limiting their macrophage-targeting efficacy. Therefore, a nanozyme material with a stable coordination structure, low metal leakage, synergistic ROS scavenging capabilities, and immune microenvironment remodeling ability is needed for the preparation of drugs to prevent and treat aortic dissection. Summary of the Invention

[0005] To address the aforementioned limitations of existing technologies, the present invention aims to provide a multi-coordinated polyphenol nanozyme, its preparation method, and its applications. This invention uses tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde as building blocks, and constructs a multi-coordinated polyphenol nanozyme (CE-Salen-Ni) through Schiff base condensation and nickel ion coordination. The Salen coordination site and the adjacent polyphenol coordination site jointly participate in nickel ion coordination, forming a coordination polymer with multi-coordinate characteristics. This multi-coordinated polyphenol nanozyme solves the problems of existing antioxidant materials in simultaneously achieving stable water dispersion, good in vitro cell and blood compatibility, broad-spectrum ROS scavenging, macrophage immune reprogramming, and maintenance of vascular smooth muscle cell phenotype.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a multi-coordination polyphenol nanozyme (CE-Salen-Ni), comprising an amino-modified crown ether structural unit, a Ni-Salen layered framework formed by coordination of a polyphenol structural unit and a nickel ion; the coordination unit of the Ni-Salen layered framework is N2O2Ni; other adjacent oxygen-containing groups in the polyphenol structural unit coordinate with the nickel ion to form a bridging structure, thereby connecting the Ni-Salen layered framework; the coordination unit of the bridging structure is Ni-O4; and the nickel ion has a valence state of +2.

[0007] Preferably, the amino-modified crown ether structural unit contains four amino groups; the polyphenol structural unit contains four oxygen-containing groups, wherein the oxygen-containing groups are at least one of aldehyde or hydroxyl groups.

[0008] Preferably, the amino-modified crown ether structural unit is tetraamino-substituted dibenzo-18-crown-6; and the polyphenol structural unit is 2,4,5-trihydroxybenzaldehyde.

[0009] Preferably, the structural formula of the multi-coordinated polyphenol nanozyme is: .

[0010] A second aspect of the present invention provides a method for preparing multi-coordinated polyphenol nanozymes, wherein the preparation method comprises: Tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde were dissolved in a mixed solvent and subjected to a first solvothermal reaction under closed conditions. After the reaction was completed, nickel acetate, DMF and water were added and a second solvothermal reaction was carried out under closed conditions. After the reaction was completed, the mixture was cooled to room temperature, filtered and collected, and after washing and drying, a black solid was obtained, which is the multicoordinated polyphenol nanozyme.

[0011] Preferably, the molar ratio of the tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde is 1:4; the mixed solvent is obtained by mixing 1,4-dioxane, mesitylene and glacial acetic acid in a volume ratio of 5:5:2.

[0012] Preferably, the temperature of the first solvothermal reaction is 180°C and the time is 3 days.

[0013] Preferably, the molar ratio of nickel acetate to tetraamino-substituted dibenzo-18-crown-6 is 7:1; the second solvothermal reaction is carried out at a temperature of 85°C for 3 days.

[0014] A third aspect of the invention provides the use of multicoordinated polyphenol nanozymes in the preparation of medicaments for the prevention and / or treatment of aortic dissection.

[0015] Preferably, the application includes scavenging reactive oxygen species, regulating macrophage polarization, maintaining the contractile phenotype of vascular smooth muscle cells, or inhibiting oxidative stress-driven vascular wall remodeling.

[0016] The beneficial effects of this invention are: (1) This invention uses tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde as building blocks, and constructs a multi-coordinated polyphenol nanozyme CE-Salen-Ni through Schiff base condensation and nickel ion coordination reactions. Specifically, the Salen coordination site formed by the condensation of tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde coordinates with nickel ions. Simultaneously, adjacent phenolic hydroxyl groups further form bridging coordination structures with nickel ions, jointly constructing a stable multi-coordinated polymer. The CE-Salen-Ni constructed in this invention integrates two complementary nickel coordination units within the same coordination polymer. Multiple coordination sites collectively form a dense oxygen- and nitrogen-enriched hybrid coordination environment, tightly coating Ni with a high coordination ratio of oxygen and nitrogen coordinating atoms to nickel ions. 2+ The core structure endows the material with excellent synergistic ability to scavenge reactive oxygen species (ROS), giving it superoxide dismutase (SOD)-like activity, glutathione peroxidase (GPx)-like activity, and broad-spectrum free radical scavenging ability. At the same time, the macrocyclic effect of the crown ether enhances the stability of the overall coordination polymer and effectively inhibits nickel ion leaching, enabling the material to maintain good bioinertness before cell internalization, thus exhibiting excellent biosafety.

[0017] (2) The inherent amphiphilicity of the crown ether group in the CE-Salen-Ni constructed in this invention endows the nanozyme with stable aqueous dispersibility and good biocompatibility, enabling macrophages to phagocytose efficiently. The hierarchical pore layered structure increases the accessibility of active sites and substrate mass transfer channels. CE-Salen-Ni triggers macrophage M2 polarization through ROS scavenging, and then indirectly inhibits the ITGB1-AKT1-c-Jun-OPN axis of smooth muscle cells, restoring α-SMA expression, thus preventing the progression of aortic dissection from the level of immune-vascular crosstalk.

[0018] (3) This invention advances the modular design strategy of multicenter nanozymes, reveals an immune-mediated (non-cellular autonomous) AD treatment mechanism, and provides a precise nanomedical paradigm for oxidative stress-driven vascular diseases. It highlights how rationally designed polyphenol nanozymes reshape the immune-vascular interface and provide candidate materials for the prevention and treatment of aortic dissection. Attached Figure Description

[0019] Figure 1Morphology of CE-Salen-Ni; (a) Scanning electron microscope image of CE-Salen-Ni, scale bar 500 nm; (b) Scanning electron microscope image of CE-Salen-Ni, scale bar 100 nm; (c) Transmission electron microscope image of CE-Salen-Ni, scale bar 50 nm; (d) Transmission electron microscope image of CE-Salen-Ni, scale bar 100 nm; (e) Transmission electron microscope image of CE-Salen-Ni, scale bar 20 nm; (f) Corresponding lattice fringe analysis of CE-Salen-Ni, scale bar 5 nm; (g) High-resolution transmission electron microscope image and partial lattice fringes of CE-Salen-Ni, scale bar 5 nm. Figure 2 : Elemental distribution map of CE-Salen-Ni energy-dispersive X-ray spectrum, scale bar 1 μm; Figure 3 Fourier transform infrared spectra of tetraaminosubstituted dibenzo-18-crown-6,2,4,5-trihydroxybenzaldehyde and CE-Salen-Ni; Figure 4 Full X-ray photoelectron spectroscopy spectrum of CE-Salen-Ni; Figure 5 (a) Nickel 2p spectrum of CE-Salen-Ni; (b) Nitrogen 1s spectrum of CE-Salen-Ni; (c) Oxygen 1s spectrum of CE-Salen-Ni; (d) Carbon 1s X-ray photoelectron spectrum of CE-Salen-Ni. Figure 6 X-ray diffraction pattern of CE-Salen-Ni; Figure 7 (a) Dynamic light scattering intensity-weighted particle size distribution of CE-Salen-Ni. The bar chart represents experimental data, and the solid line represents Gaussian fitting with a coefficient of determination of 0.971. The peak center is 1085.6 ± 7.3 nm, and the full width at half maximum (FWHM) is 747.7 nm. (b) Nitrogen adsorption-desorption isotherms of CE-Salen-Ni. (c) Pore size distribution curve of CE-Salen-Ni. (d) Thermogravimetric analysis curve of CE-Salen-Ni. Figure 8 (a) Electron spin resonance spectrum of superoxide anion after CE-Salen-Ni (200 μg / mL) treatment; (b) Electron spin resonance spectrum of hydroxyl radical; (c) Electron spin resonance spectrum of DPPH radical. Figure 9(a) Evaluation of RAW264.7 cell viability using the Cell Counting Kit-8 assay; (b) MOVAS cell viability, with a sample size of 3 independent experiments; (c) Representative macroscopic images of hemolysis assay, with PBS as a negative control and H2O as a positive control; (d) Quantitative analysis of hemolysis rate, with a sample size of 3, and data are expressed as mean ± standard deviation.

[0020] Figure 10 (a) Representative fluorescence images of ROS levels in RAW264.7 cells after LPS stimulation, measured by DCFH-DA probe, scale bar: 75 μm; (b) Quantitative analysis of total cellular fluorescence intensity after DCFH-DA signal normalization correction; Statistical analysis was performed using one-way ANOVA and Dunnett multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 11 (a) Representative immunofluorescence images of CD86, CD206, and CD11b in RAW264.7 cells, scale bar 150 μm; (b) Quantitative analysis of mean corrected total cellular fluorescence intensity of CD86 expression; (c) Quantitative analysis of mean corrected total cellular fluorescence intensity of CD206 expression; (d) Quantitative analysis of the CD86 / CD206 ratio; Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 12 (a) Representative flow cytometry plots for assessing macrophage phenotypes; (b) Flow cytometry quantification of the percentage of CD86-positive cells; (c) Flow cytometry quantification of the percentage of CD206-positive cells; (d) Flow cytometry quantification of the CD86 / CD206 ratio, with a sample size of 3; Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 13(a) Western blot analysis of the expression levels of INOS, TNF-α, CD206, and IL-10 proteins; (b) quantification of INOS by Western blot using grayscale; (c) quantification of TNF-α by Western blot using grayscale; (d) quantification of CD206 by Western blot using grayscale; (e) quantification of IL-10 by Western blot using grayscale; (f) quantification of TNF-α by enzyme-linked immunosorbent assay (ELISA); (g) quantification of IL-10 secretion by ELISA; data are expressed as mean ± standard deviation; the sample size for both Western blot and ELISA experiments was 6 per group; statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 14 (a) Representative immunofluorescence images of α-smooth muscle actin and osteopontin in co-cultured MOVAS cells, scale bar 150 μm; (b) Quantitative analysis of mean corrected total cellular fluorescence intensity of OPN protein expression; (c) Quantitative analysis of mean corrected total cellular fluorescence intensity of α-SMA expression; (d) Quantitative analysis of the OPN / α-SMA ratio; Sample size: 6; Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 15 (a) Representative images of MOVAS cell scratch healing assays at 0 and 24 hours after co-culture, scale bar 650 μm; (b) Quantitative analysis of cell migration percentage; data are expressed as mean ± standard deviation; the scratch assay was performed in 3 independent experiments with a sample size of 3; statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 16(a) Western blot analysis of the proteins ITGB1, AKT1, p-AKT1, c-Jun, pc-Jun, OPN, α-SMA, and β-actin; (b) quantification of the ITGB1 / β-actin band grayscale; (c) quantification of the p-AKT1 / AKT1 band grayscale ratio, wherein the grayscale values ​​of both p-AKT1 and AKT1 bands were first normalized by β-actin; (d) quantification of the pc-Jun / c-Jun band grayscale ratio, wherein the grayscale values ​​of both pc-Jun and c-Jun bands were first normalized by β-actin; (e) quantification of the OPN / β-actin band grayscale; (f) quantification of the α-SMA / β-actin band grayscale; data are expressed as mean ± standard deviation, with a sample size of 6; statistical analysis was performed using one-way ANOVA and Dunnett's method. Multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 17 (a) Kaplan-Meier survival curves, with survival evaluated using the Log-rank (Mantel-Cox) test; (b) Incidence of aortic rupture, dissection, and healthy phenotype in each experimental group; Figure 18 Representative macroscopic images of the aorta from different groups; Figure 19 (a) Representative histological images of aortic sections stained with H&E, Masson trichrome, and EVG, with scale bars of 250 μm (low magnification) and 100 μm (high magnification), respectively; (b) Quantitative aortic injury scores based on H&E staining, with statistical differences analyzed using the Kruskal-Wallis test and Dunn multiple comparisons; (c) Quantitative masson positive area percentage, with statistical analysis using one-way ANOVA and Dunnett multiple comparisons; (d) EVG elastic fiber breakage scores, with statistical differences analyzed using the Kruskal-Wallis test and Dunn multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 20 (a) Representative image of ethidium dihydrofluorescence staining in aortic tissue, scale bar 50 μm; (b) Quantitative analysis of ethidium dihydrofluorescence intensity expressed as mean corrected total cellular fluorescence intensity; data are expressed as mean ± standard deviation, sample size 6; statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 21(a) Malondialdehyde level in aortic tissue; (b) Superoxide dismutase activity; (c) Glutathione peroxidase activity; (d) Catalase activity; Data are expressed as mean ± standard deviation, with a sample size of 6; Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 22 (a) Representative immunofluorescence images of CD86 and CD206 in aortic tissue, scale bar 50 μm; (b) Quantitative analysis of mean corrected total cellular fluorescence intensity of CD86 expression; (c) Quantitative analysis of mean corrected total cellular fluorescence intensity of CD206 expression; (d) Quantitative analysis of the CD86 / CD206 ratio; Data are expressed as mean ± standard deviation, sample size 6; Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 23 (a) Representative immunohistochemical images of ITGB1, p-AKT1, pc-Jun, OPN, and α-SMA in aortic tissue, scale bar 250 μm; (b) Quantitative percentage of ITGB1 immunohistochemical staining positive area; (c) Quantitative percentage of p-AKT1 immunohistochemical staining positive area; (d) Quantitative percentage of pc-Jun immunohistochemical staining positive area; (e) Quantitative percentage of OPN immunohistochemical staining positive area; (f) Quantitative percentage of α-SMA immunohistochemical staining positive area; Data are expressed as mean ± standard deviation, sample size 6; Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparisons; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] As introduced in the background section, although traditional metal-polyphenol networks can improve some antioxidant properties, they often suffer from problems such as aqueous phase aggregation, insufficient cell membrane interface compatibility, potential leakage of metal ions, and difficulty in simultaneously achieving the removal of reactive oxygen species outside and inside cells.

[0023] Based on this, the purpose of this invention is to provide a multi-coordinated polyphenol nanozyme, its preparation method, and its applications. This invention uses tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde as building blocks, and synergistically constructs the polymer CE-Salen-Ni through Schiff base condensation and nickel ion coordination reactions. Specifically, tetraamino-substituted dibenzo-18-crown-6 condenses with 2,4,5-trihydroxybenzaldehyde to form Salen-type N₂O₂ coordination sites, and coordinates with nickel ions to form Ni-Salen coordination units; simultaneously, the ortho-position polyphenol groups in 2,4,5-trihydroxybenzaldehyde further form Ni–O₄-type bridging coordination structures with nickel ions, thereby jointly constructing the multi-coordinated coordination polymer CE-Salen-Ni. The crown ether structure endows the material with good aqueous dispersibility and biocompatibility, which is beneficial for improving its stability in physiological environments and promoting cell internalization. The Ni-Salen coordination unit provides a planar N₂O₂ nickel coordination environment, exhibiting redox cycling capabilities similar to metalloenzymes; the ortho-polyphenol-nickel coordination unit provides reversible redox activity, which is beneficial for the scavenging of reactive oxygen species such as superoxide anions. By integrating the Salen nickel coordination site, the ortho-polyphenol nickel coordination site, and the crown ether structure into the same coordination polymer, CE-Salen-Ni achieves multi-active-site synergistic catalytic characteristics, thus demonstrating comprehensive antioxidant capacity superior to single-active-site nanozymes.

[0024] CE-Salen-Ni acts as a direct executor of extracellular and post-phagocytic intracellular ROS elimination. After internalization by macrophages, the material efficiently clears intracellular ROS, disrupting the ROS-driven inflammatory positive feedback that maintains the pro-inflammatory state of M1 cells. Consequently, macrophages polarize towards a reparative M2 phenotype, characterized by downregulation of M1 markers (CD86, inducible nitric oxide synthase (INOS), TNF-α) and upregulation of M2 markers (CD206, IL-10). This immune reprogramming creates a paracrine microenvironment, indirectly regulating adjacent stromal cells (SMCs). M2-derived anti-inflammatory signals inhibit the ITGB1-AKT1-c-Jun-OPN signaling axis in SMCs, leading to a decrease in OPN (a synthetic marker) and a restoration of α-SMA (a contractile marker) expression. Ultimately, the aortic wall structure is stabilized, extracellular matrix integrity is improved, and dissection progression is halted. By correcting the immune-redox imbalance and deactivating the ITGB1-driven signaling axis, CE-Salen-Ni provides a precise nanomedicine strategy for aortic wall stabilization and AD treatment, highlighting the unique advantages of synthetic artificial polyphenols: they simultaneously introduce multiple coordination sites (Ni-O4 and N2O2Ni) enhanced by coronary ethers to achieve synergistic, sequential, and broad-spectrum ROS scavenging.

[0025] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0026] The nickel acetate used in this invention is also known as nickel acetate tetrahydrate (II) or nickel acetate (II) tetrahydrate, CAS: 6018-89-9.

[0027] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0028] Example 1: Preparation of CE-Salen-Ni (1) Preparation of tetraamino-substituted dibenzo-18-crown-6 Nitric acid (65%, 15 mL) was added dropwise to a 200 mL solution of dibenzo-18-crown-6 (2.16 g, 6.0 mmol) in dichloromethane under stirring. After 10 min, sulfuric acid (95%, 7.5 mL) was gradually added. The mixture was stirred at room temperature for 7 days. After the reaction was complete, the precipitate was collected by filtration, washed thoroughly with deionized water, and dried under vacuum to obtain a crude tetranitrocrown ether product as a yellow powder. Subsequently, tetranitrocrown ether (0.50 g) and 10% Pd / C (0.25 g) were dispersed in degassed ethanol. The suspension was heated to 80 °C and purged with argon for 30 min. Under argon protection, hydrazine hydrate (15 mL) was added using a syringe, and the mixture was refluxed at 80 °C and stirred for 4 h under an argon atmosphere. After hot filtration, the filtrate was crystallized at 4 °C to obtain tetraamino-substituted dibenzo-18-crown-6.

[0029] (2) Preparation of 2,4,5-trihydroxybenzaldehyde 1,2,4-Phosphoryl 1,2,4-Benzene (0.90 g, 7.137 mmol) and triethyl orthoformate (5.64 mL, 33.9 mmol) were mixed and stirred in anhydrous tetrahydrofuran (25 mL) at 0 °C. Degassed aluminum chloride (1.428 g, 10.707 mmol) was then added, and the reaction mixture was brought to room temperature and stirred for 1 h. After quenching with 3 N hydrochloric acid, the mixture was extracted twice with 45 mL of diethyl ether each time, followed by two extractions with 45 mL of ethyl acetate each time. The mixture was washed twice with 70 mL of brine each time, dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by silica gel column chromatography using dichloromethane / methanol (10:1, v / v) as the eluent to give 0.57 g of a yellow solid, 2,4,5-trihydroxybenzaldehyde, in 52% yield.

[0030] (3) Synthesis of CE-Salen-Ni Tetraaminosubstituted dibenzo-18-crown-6 (42.0 mg, 0.10 mmol) and 2,4,5-trihydroxybenzaldehyde (61.6 mg, 0.40 mmol) were dissolved in a mixed solvent consisting of 1,4-dioxane (1.0 mL), mesitylene (1.0 mL), and glacial acetic acid (0.4 mL). The reaction system was sealed and heated at 180 °C for 3 days. Subsequently, nickel(II) acetate tetrahydrate (174.2 mg, 0.70 mmol) was added to the reaction system to allow Ni to react. 2+ The initial molar ratio of the tetraamino-substituted dibenzo-18-crown-6 to N,N-dimethylformamide (1.0 mL) and water (1.5 mL) was 7:1. The mixture was transferred to a 10 mL pressure-resistant glass reaction tube, sealed, and heated at 85 °C for 3 days. After the reaction was completed, the mixture was cooled to room temperature, and the resulting black solid was collected by filtration. The solid was washed successively with N,N-dimethylformamide, ethanol, and dichloromethane, and dried under vacuum to obtain the black solid product CE-Salen-Ni. The synthetic route is as follows: .

[0031] Example 2: Characterization (1) Microstructure and nanostructure of CE-Salen-Ni The ultrastructure of CE-Salen-Ni was evaluated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1 (a) and Figure 1 As shown in (b), CE-Salen-Ni exhibits a distinct layered nanosheet morphology with uniform size distribution and a rough surface. Notably, abundant macropores are clearly observed in this hierarchical structure, which originate from the gaps between the assembled nanosheets. Figure 1 (c)~ Figure 1 TEM imaging in (e) confirms this layered assembly and further reveals the presence of obvious mesopores. Meanwhile, clearly identifiable light-blocking nanoparticles, uniformly dispersed within the layered matrix, are also observed. Figure 1 (f) and Figure 1High-resolution TEM (HR-TEM) of (g) revealed the microporous structure through changes in light and dark contrast, confirming the coexistence of micropores, mesopores, and macropores. This multi-scale porous structure formed by the hierarchical stacking of nanosheets is the direct reason for the material's high surface area and enhanced mass transfer capacity, maximizing local ROS removal in bulk. In addition to the porous structure, HR-TEM also revealed the structural heterogeneity at the atomic level within the nanoparticles. The measured lattice spacing of 0.233 nm matched the (101) crystal plane of the main β-Ni(OH)2 framework. Meanwhile, discrete crystal domains with lattice spacings of 0.245 nm and 0.206 nm corresponded to the (111) and (200) crystal planes of in-situ generated NiO, respectively. A lattice spacing of 0.203 nm was also detected, corresponding to the (111) crystal plane of face-centered cubic (fcc) Ni, because hydroxyl groups have reducing properties and will reduce a small amount of Ni. 2+ Reduced to Ni 0 .

[0032] High-angle annular dark-field scanning TEM (HAADF-STEM) and corresponding elemental surface distribution maps ( Figure 2 This indicates that C, N, O, and Ni are spatially uniformly distributed.

[0033] (2) Structural, chemical and physical characterization of CE-Salen-Ni Using tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde as controls, the formation of the CE-Salen-Ni polyphenol skeleton was verified by Fourier transform infrared spectroscopy (FT-IR). Figure 3 Tetraamino-substituted dibenzo-18-crown-6 monomers at 3372 and 3305 cm⁻¹ -1 Characteristic NH stretching vibration bands are observed at 1209 and 1136 cm. -1 The presence of a strong COC stretching vibration band at this point confirms the presence of a primary amine group and a crown ether framework. For 2,4,5-trihydroxybenzaldehyde, the values ​​at 3383 and 3174 cm⁻¹ are also relevant. -1 The broad peak at 1631 cm⁻¹ is attributed to the stretching vibration of the phenolic hydroxyl group (OH). -1 The peak at [value] is attributed to the hydrogen-bonded formyl C=O stretching vibration. Simultaneously, the coordination between the hydroxyl group (-OH) and the Ni(II) ion can be determined by [value] at approximately 519 cm⁻¹. -1 The presence of a distinct Ni-O vibrational band indicates (corresponding to Ni-O4 coordinated nickel). Furthermore, at 618 cm⁻¹... -1The peak at the position originates from the Ni-N coordination bond within CE-Salen-Ni, supporting the formation of Salen-type Ni-N2O2 units. These two diagnostic coordination signals indicate that Ni(II) coordinates with the phenolic oxygen and imine nitrogen sites in the CE-Salen framework, supporting the successful formation of the CE-Salen-Ni complex.

[0034] XPS full spectrum of CE-Salen-Ni shows the presence of C, N, O and Ni elements on the material surface. Figure 4 Further peak fitting was performed on the high-resolution spectra of Ni 2p, N 1s, O 1s, and C 1s, as shown below. Figure 5 As shown in (a), the Ni 2p spectrum can be divided into four peaks. The two main peaks at 856.0 and 873.6 eV are attributed to Ni. 2+ Ni 2p 3 / 2 and Ni 2p 1 / 2 The spin-orbit splitting is approximately 17.6 eV. The additional peaks at 861.5 and 879.3 eV correspond to Ni. 2+ The shake-up satellite peaks confirmed that Ni in the product is in a divalent state. These results indicate that Ni is mainly coordinated to the Ni center in an N / O configuration. 2+ The environment exists. In Figure 5 In the N 1s spectrum of (b), the two fitted peaks at 398.4 and 400.0 eV belong to C=N and N-Ni species, respectively, indicating that imine nitrogen coordinates with Ni. Figure 5 (c) The O 1s spectrum shows three peaks at 531.0, 531.6, and 532.7 eV, corresponding to O-Ni, COC, and Ar-O species, respectively. The O-Ni component further supports the formation of Ni-O coordination in the product. Furthermore, Figure 5 The C 1s spectrum of (d) is fitted with three components at 284.7, 286.0, and 288.3 eV, which are assigned to CC, COC, and OC=C species, respectively. These binding spectra together demonstrate the stable integration of the metal nodes with the organic structural units.

[0035] Powder X-ray diffraction (PXRD) Figure 6 Characteristic peaks are observed at 2θ of 33.4° and 59.6°, corresponding to the (100) and (110) crystal planes of β-Ni(OH)2 coordination nodes, respectively, indicating that the nickel center is periodically embedded in the crystalline organic matrix.

[0036] Figure 7(a) Dynamic light scattering (DLS) analysis revealed a Z-mean particle size of 1214.5 ± 76.2 nm (mean ± SD, n = 3) and a polydispersity index (PDI) of 0.316 ± 0.02. The intensity-weighted distribution was unimodal. The peak value of the histogram after Gaussian fitting was located at 1086 nm (R0). 2 = 0.971). The average Z value is greater than the peak center, indicating a right-tailed distribution, consistent with moderate PDI. The Zeta potential is -26.92 ± 0.08 mV, and the conductivity is 0.1 mS / cm (RSD = 0.29%, n = 3), indicating that the material has a moderate negative surface charge. Figure 7 (b) Low-temperature N2 adsorption-desorption tests of CE-Salen-Ni showed typical Type IV isotherms, characterized by rapid adsorption in the low-pressure region and hysteresis loops in the desorption branches, confirming that its structure is mainly composed of micropores and mesopores. The rapid adsorption in the high-pressure region further indicates the presence of macropores. Figure 7 (c) The Brunauer-Emmett-Teller (BET) specific surface area was determined to be 91.9 m². 2 g -1 The total pore volume is 0.143 cm³. 3 g -1 The pore size distribution curves show that the pore size range extends from micropores to mesopores, with the main peak at 1.23 nm and two secondary peaks at 2.37 nm and 5.56 nm, respectively. This inherent hierarchical pore structure directly expands the accessible surface area, exposing a large number of Ni active sites, thereby promoting substrate mass transfer and maximizing in-vivo ROS scavenging. Figure 7 Thermogravimetric analysis (TGA) in (d) shows that CE-Salen-Ni exhibits staged weight loss. The minor desolvation below 100 °C (weight loss <10%) is attributed to the evaporation of physically adsorbed water. The total mass decreases slowly by 13% between 100 and 350 °C, attributed to the breakage of unstable groups. A residual mass of 47% is maintained up to 800 °C. These results confirm the excellent thermal stability of CE-Salen-Ni.

[0037] Example 3: Performance Testing of CE-Salen-Ni ESR spectroscopy was used to detect the effect of CE-Salen-Ni on O2. •- The effects of OH and DPPH radical signals were investigated. In the ESR test, the concentration of CE-Salen-Ni was 200 μg / mL, and its scavenging ability was evaluated by comparing the changes in signal intensity before and after its addition.

[0038] For O2 •-The detection showed that O2 was generated in pH 7.4 PBS using riboflavin (20 μM) and L-methionine (13 mM) under 4000 lux light. •- And record DMPO–O2 •- adduct signal. Figure 8 (a) The results showed that the signal was significantly weakened in the presence of CE-Salen-Ni, with an inhibition rate of 93.6%, confirming its efficient scavenging of superoxide radicals.

[0039] For the detection of •OH, a Fenton system was used to generate hydroxyl radicals. The system consisted of FeSO4 (1 mg / mL, 75 μL), H2O2 (30% w / w, 75 μL), DMPO (3% v / v, 20 μL), and PBS (pH 4.5, 600 μL), generating a DMPO–OH adduct signal. Figure 8 (b) The results showed that CE-Salen-Ni significantly reduced the •OH ESR signal, decreasing by 75.4% at 200 μg / mL.

[0040] For DPPH detection, changes in the ESR signal are monitored directly in the ethanol system. Figure 8 (c) The results showed that the DPPH radical signal was significantly quenched, with an attenuation rate of 97.8% at 200 μg / mL, indicating that it has strong overall antioxidant capacity.

[0041] Example 4: Evaluation of the biosafety and blood compatibility of CE-Salen-Ni (1) Cytotoxicity test The in vitro cytotoxicity of CE-Salen-Ni against RAW264.7 macrophages and MOVAS mouse vascular smooth muscle cells was evaluated using the CCK-8 assay. Both RAW264.7 and MOVAS cells were obtained from the Cell Bank of the Chinese Academy of Sciences. Cells were seeded at appropriate densities in 96-well plates and cultured for 24 h. Different concentrations of CE-Salen-Ni were then added to a final concentration ranging from 0 to 250 μg / mL, and incubation continued for another 24 h. Subsequently, CCK-8 reagent was added, and the cells were incubated at 37 ℃ for 2 h. Absorbance was measured at 450 nm. Cell viability was calculated using the following formula: Cell viability (%) = (A – A blank) / (A control – A blank) × 100%; Where A is the absorbance of the experimental well; A blank is the absorbance of the blank well (without CE-Salen-Ni); and A control is the absorbance of the control well without CE-Salen-Ni. Figure 9 (a) and Figure 9 (b) The results showed that the viability of RAW264.7 cells and MOVAS cells remained above 90% in all test concentration ranges from 0 to 250 μg / mL, indicating that CE-Salen-Ni had no significant cytotoxicity to either of the two cell types and had good in vitro cell compatibility.

[0042] (2) Hemolysis test The blood compatibility of CE-Salen-Ni was evaluated using a standard hemolysis test. Fresh blood was collected from healthy male mice purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The collected fresh blood was centrifuged at 1500 rpm for 20 min, and red blood cells were collected and washed three times with PBS to prepare a 5% (w / w) red blood cell suspension. The 5% red blood cell suspension was mixed with different concentrations of CE-Salen-Ni at a ratio of 1:9 (v / v) and incubated at 37℃ for 3 h. After incubation, the suspension was centrifuged at 12000 rpm for 20 min, and the supernatant was collected and its absorbance was measured at 540 nm. The PBS-treated group served as a negative control, and the distilled water-treated group served as a positive control. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = (A - An) / (Ap - An) × 100%; Where A is the absorbance of the red blood cell supernatant after the addition of CE-Salen-Ni; An is the absorbance of the PBS group, i.e., the absorbance of the negative control; and Ap is the absorbance of the distilled water group, i.e., the absorbance of the positive control. Figure 9 (c) and Figure 9 (d) The results showed that the PBS treatment group served as the negative control, and the distilled water treatment group served as the positive control. Compared with the distilled water positive control group, no obvious red blood cell lysis was observed in the CE-Salen-Ni treatment group; the hemolysis rate induced by CE-Salen-Ni was less than 5% in all tested concentration ranges, indicating that CE-Salen-Ni has good blood compatibility.

[0043] Example 5: In vitro antioxidant, macrophage polarization and smooth muscle cell phenotype regulation experiments of CE-Salen-Ni The experiment was divided into a normal control group, an LPS model group, a low-dose CE-Salen-Ni intervention group, and a high-dose CE-Salen-Ni intervention group. Before the experiment, RAW264.7 cells or MOVAS cells were seeded in appropriate culture plates or dishes and cultured at 37 ℃ with 5% CO2. Subsequent treatments were performed after the cells reached approximately 50% confluence. The normal control group received no LPS stimulation or CE-Salen-Ni treatment; the LPS model group received LPS (1 μg / mL) stimulation for 24 h; the low-dose and high-dose CE-Salen-Ni intervention groups received LPS stimulation plus CE-Salen-Ni (100 μg / mL) and CE-Salen-Ni (200 μg / mL), respectively, for 24 h. For MOVAS cell phenotypic regulation experiments, RAW264.7 cells and MOVAS cells treated with different methods were co-cultured non-contactly using a Transwell system. RAW264.7 cells were seeded in the upper chamber of a Transwell apparatus, and MOVAS cells were seeded in the lower chamber. The Transwell chambers had a pore size of 0.4 μm, and the seeding ratio was 1:1. After co-culturing for 24 h, MOVAS cells were collected for immunofluorescence, scratch assays, and Western blotting.

[0044] (1) Detection of intracellular ROS in RAW264.7 cells An inflammatory oxidative stress model was established using LPS stimulation of RAW264.7 macrophages, followed by CE-Salen-Ni intervention. Intracellular ROS levels were detected using the 2′,7′-dichlorodihydrofluorescein diacetate probe. DCFH-DA positive signals showed green fluorescence, and Hoechst counterstaining of the nuclei resulted in blue fluorescence. After acquiring fluorescence images, background subtraction was performed using ImageJ software, and the DCFH-DA signal was quantitatively analyzed using corrected total cellular fluorescence intensity. Figure 10 (a) The results showed that LPS stimulation significantly enhanced the green fluorescence signal of DCFH-DA in RAW264.7 cells, indicating an increase in intracellular ROS levels; after CE-Salen-Ni treatment, the green fluorescence signal was significantly weakened. Figure 10 (b) Normalized CTCF quantification results further confirmed the reduction in ROS levels. These results indicate that CE-Salen-Ni can effectively clear excess ROS in macrophages under inflammatory stimulation.

[0045] (2) Evaluation of macrophage polarization by cellular immunofluorescence To perform in vitro morphological characterization, immunofluorescence assays were conducted on RAW264.7 cells. Cultured RAW264.7 cells were fixed with 4% paraformaldehyde, permeabilized, and blocked, then incubated overnight with a primary antibody mixture. The primary antibody mixture included rat anti-CD86 antibody, goat anti-CD206 antibody, and rabbit anti-CD11b antibody (rat anti-CD86 antibody was purchased from Thermo Fisher, catalog number 14-0862-82, diluted 1:100; goat anti-CD206 antibody was purchased from Thermo Fisher, catalog number PA5-46994, diluted 1:100; rabbit anti-CD11b antibody was purchased from Thermo Fisher, catalog number PA5-90724, diluted 1:100). After washing, the cells were incubated with a species-matched fluorophore-conjugated secondary antibody mixture. The secondary antibody mixture included Alexa Fluor 488-labeled anti-rat IgG (H+L) secondary antibody, Alexa Fluor 568-labeled anti-goat IgG (H+L) secondary antibody, and Alexa Fluor 680-labeled anti-rabbit IgG (H+L) secondary antibody (Alexa Fluor 488-labeled anti-rat IgG (H+L) secondary antibody was purchased from Abcam, catalog number ab150153, at a dilution of 1:500; Alexa Fluor 568-labeled anti-goat IgG (H+L) secondary antibody was purchased from Abcam, catalog number ab175474, at a dilution of 1:500; Alexa Fluor 680-labeled anti-rabbit IgG (H+L) secondary antibody was purchased from Abcam, catalog number ab175775, at a dilution of 1:500). Cell nuclei were counterstained with DAPI before fluorescence imaging. Figure 11 (a) ~ Figure 11 (d) The results showed that after LPS stimulation, the CD86 positive fluorescence signal in RAW264.7 cells was enhanced, indicating that macrophages were polarized to the M1 pro-inflammatory phenotype. After CE-Salen-Ni treatment, the CD86 positive fluorescence signal decreased, the CD206 positive fluorescence signal increased, and the CD86 / CD206 ratio decreased, indicating that CE-Salen-Ni can promote the reprogramming of macrophages to the M2 anti-inflammatory phenotype.

[0046] (3) Flow cytometry evaluation of macrophage polarization The effect of CE-Salen-Ni on the polarization state of RAW264.7 macrophages was analyzed by flow cytometry using a commercial kit. After collecting RAW264.7 cells from each treatment group, they were first incubated with anti-CD16 / 32 to block Fc receptor-mediated non-specific binding, followed by surface labeling with FITC-anti-CD11b and PE-anti-CD86. For CD206 intracellular staining, cells were treated with a dedicated fixation and permeabilization buffer before incubation with APC-anti-CD206. After staining, data were collected by flow cytometry, and the proportions of CD86-positive cells, CD206-positive cells, and the CD86 / CD206 ratio were quantitatively analyzed. Figure 12 (a) ~ Figure 12 (d) The results showed that after CE-Salen-Ni treatment, the proportion of CD86 positive cells decreased, the proportion of CD206 positive cells increased, and the CD86 / CD206 ratio decreased, indicating that CE-Salen-Ni can promote the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type phenotype.

[0047] (4) Western blot and ELISA detection of macrophage polarization-related indicators Western blot was used to detect the protein expression of specific macrophage polarization markers in RAW264.7 cell lysates, and ELISA was used to quantitatively detect secreted cytokines, including TNF-α and IL-10, in the cell culture medium. Figure 13 (a) ~ Figure 13 The results in (e) showed that in RAW264.7 cells, the protein expression of M1-related markers INOS and TNF-α decreased after CE-Salen-Ni treatment, while the protein expression of M2-related markers CD206 and IL-10 increased. Figure 13 (f)~ Figure 13 ELISA results for (g) showed that the TNF-α secretion level was decreased and the IL-10 secretion level was increased in the cell culture supernatant of the CE-Salen-Ni treatment group. These results further indicate that CE-Salen-Ni can inhibit the polarization of pro-inflammatory macrophages and promote the transformation of macrophages to the anti-inflammatory M2 phenotype.

[0048] (5) Transwell indirect co-culture and MOVAS cell phenotype evaluation To evaluate whether macrophages regulated by CE-Salen-Ni could influence the phenotype of vascular smooth muscle cells through paracrine signaling, a Transwell indirect co-culture system of RAW264.7 cells and MOVAS cells was established. RAW264.7 cells treated with CE-Salen-Ni were placed in the upper chamber of the Transwell, and MOVAS cells were placed in the lower chamber, ensuring that the two cell types did not directly contact each other and interacted only through soluble paracrine factors. After co-culture, the expression changes of the synthetic phenotype marker OPN and the contractile phenotype marker α-SMA in MOVAS cells were detected by immunofluorescence. Specifically, MOVAS cells were fixed with 4% paraformaldehyde, permeabilized and blocked, and then incubated with rat anti-OPN and rabbit anti-α-SMA primary antibodies. After washing, they were incubated with the corresponding fluorescent secondary antibodies, and the cell nuclei were counterstained with DAPI. Figure 14(a)~ Figure 14 (d) The results showed that, compared with the inflammatory stimulation model group, co-culture of macrophages treated with CE-Salen-Ni reduced the OPN fluorescence signal, enhanced the α-SMA fluorescence signal, and decreased the OPN / α-SMA ratio in MOVAS cells, indicating that CE-Salen-Ni can indirectly promote the recovery of vascular smooth muscle cells to a contractile phenotype by regulating macrophage paracrine signals.

[0049] (6) Scratch healing assay to evaluate the migration ability of MOVAS cells To assess the migration ability of smooth muscle cells, a MOVAS cell scratch healing assay was performed. After MOVAS cells were cultured to a confluent monolayer, a standardized linear scratch was created in the center of each well using a sterile 200 μL pipette tip. After washing with PBS to remove detached cells and cell debris, the cells were maintained in low-serum medium under appropriate experimental conditions. MOVAS cell migration was monitored and imaged at 0 h and 24 h using an EVOS M7000 imaging system. Cell migration rate was quantitatively calculated using ImageJ software, determined by the percentage reduction in cell-free scratch area at 24 h relative to the initial scratch area at 0 h. Figure 15(a)~ Figure 15 (b) The results showed that, compared with the co-culture group under the model conditions, co-culture of macrophages treated with CE-Salen-Ni reduced the degree of scratch closure of MOVAS cells and decreased cell migration rate, indicating that CE-Salen-Ni can inhibit abnormal migration of vascular smooth muscle cells by regulating macrophage phenotype and paracrine signaling.

[0050] (7) Western blot detection of MOVAS cell phenotype and ITGB1-AKT1-c-Jun-OPN signal axis Western blotting was performed on MOVAS cells co-cultured in Transwell to evaluate smooth muscle cell phenotype and changes in the ITGB1-AKT1-c-Jun-OPN signaling axis. ImageJ software was used for quantitative analysis of protein band grayscale values. The grayscale values ​​of ITGB1, AKT1, p-AKT1, c-Jun, pc-Jun, OPN, and α-SMA bands were first normalized to the internal reference protein β-actin. For phosphorylated protein levels, the p-AKT1 / AKT1 and pc-Jun / c-Jun ratios were calculated using the corresponding total protein as a reference to reflect the phosphorylation activation levels of AKT1 and c-Jun. Figure 16 (a) ~ Figure 16 (f) The results showed that after co-culturing CE-Salen-Ni regulated macrophages with MOVAS cells, the ITGB1 / β-actin and OPN / β-actin ratios in MOVAS cells decreased, while the α-SMA / β-actin ratio increased; simultaneously, the p-AKT1 / AKT1 and pc-Jun / c-Jun ratios decreased. These results suggest that CE-Salen-Ni regulated macrophages can reduce the phosphorylation activation levels of AKT1 and c-Jun in MOVAS cells through paracrine signaling, inhibit the activation of the ITGB1-AKT1-c-Jun-OPN signaling axis, and help maintain the contractile phenotype of vascular smooth muscle cells.

[0051] Example 6: Therapeutic effect and mechanism verification of CE-Salen-Ni in an animal model of aortic dissection (1) Establishment of animal model of aortic dissection, administration and quantitative detection Three-week-old male C57BL / 6J mice, weighing 13±2 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. All mice were housed under specific pathogen-negative conditions at a temperature of 25±2℃ and a relative humidity of 50±10%. After acclimatization, the mice were randomly divided into five groups: The first group was the control group, consisting of 8 mice. They were given standard drinking water and intraperitoneal injections of 0.1 mL of physiological saline daily. The second group was the drug-only group, consisting of 8 mice. They were given standard drinking water and intraperitoneal injections of CE-Salen-Ni daily at a dose of 10 mg / kg and an injection volume of 0.1 mL. The third group was the aortic dissection model group, consisting of 12 mice. They drank water containing 4.5 g / L β-aminopropionitrile and received intraperitoneal injections of 0.1 mL of physiological saline daily. The fourth group was the low-dose intervention group, consisting of 11 mice. They drank water containing 4.5 g / L β-aminopropionitrile and received daily intraperitoneal injections of CE-Salen-Ni at a dose of 5 mg / kg and an injection volume of 0.1 mL. The fifth group was the high-dose intervention group, consisting of 11 mice. They drank water containing 4.5 g / L β-aminopropionitrile and received daily intraperitoneal injections of CE-Salen-Ni at a dose of 10 mg / kg and an injection volume of 0.1 mL.

[0052] All of the above-mentioned injectable solutions were prepared with physiological saline.

[0053] β-Aminopropionitrile (β-Aminopropionitrile) was administered into drinking water for 28 days. CE-Salen-Ni was administered starting from day 1 of β-Aminopropionitrile modeling and continued for 28 days. Therefore, the administration method in this embodiment was a simultaneous intervention to evaluate the inhibitory effect of CE-Salen-Ni on the occurrence and progression of β-Aminopropionitrile-induced aortic dissection.

[0054] During the experiment, the general condition, survival status, and time of death of the mice were observed daily. The time of death of all mice that died during the experiment was recorded and included in the Kaplan-Meier survival analysis.

[0055] On day 28, six surviving mice were randomly selected for endpoint sampling. Prior to sampling, the mice were deeply anesthetized by inhaling 2% isoflurane, and after confirming adequate anesthesia by no response to paw tweezers, they were euthanized by cervical dislocation. Aortic tissue and blood samples were then collected for histological, biochemical, and molecular biological analysis.

[0056] The criteria for determining aortic dissection include the presence of hematoma, dissecting separation, local dilation, and destruction of the aortic wall structure, or histological staining revealing separation of the aortic medial structure, rupture of elastic fibers, and blood components entering the aortic wall. The criteria for determining aortic rupture include discontinuity of the aortic wall accompanied by periaortic hemorrhage, blood clot formation, or pleural or peritoneal hemorrhage. For mice that die during the experiment, if aortic rupture-related manifestations can be observed through necropsy, it is counted as an aortic rupture event; if the cause of death cannot be clearly determined, they are only included in the survival analysis and not counted as definitive aortic rupture events.

[0057] The criteria for diagnosing aortic dissection include the presence of hematoma, dissecting separation, local dilation, and structural damage to the aortic wall, or histological staining revealing separation of the aortic medial structure, rupture of elastic fibers, and blood components entering the aortic wall. The criteria for diagnosing aortic rupture include discontinuity of the aortic wall accompanied by periaortic hematoma, blood clot formation, pleural hemorrhage, abdominal hemorrhage, or other significant internal bleeding. If autopsy reveals any of the above-mentioned aortic rupture-related manifestations, it is considered an aortic rupture event; if autopsy reveals dissecting changes but no clear rupture or massive hemorrhage, it is considered an aortic dissection event.

[0058] Histological damage scoring uses a semi-quantitative scoring standard of 0-4 points: 0 points indicates that the aortic wall structure is intact and no obvious pathological damage is seen; 1 point indicates mild medial structural disorder or focal mild elastic fiber rupture; 2 points indicate moderate medial damage, with obvious elastic fiber rupture or local inflammatory cell infiltration; 3 points indicate severe aortic wall structural destruction, with obvious medial separation, extensive elastic fiber rupture or obvious inflammatory infiltration; 4 points indicate very severe damage, with severe aortic wall structural destruction, dissection formation or rupture-related changes.

[0059] The degree of elastic fiber breakage was scored from 0 to 4: 0 indicates continuous and intact elastic fiber arrangement; 1 indicates focal mild breakage; 2 indicates multifocal breakage or disordered arrangement; 3 indicates extensive breakage with significant median structure destruction; and 4 indicates severe elastic fiber damage with intercalation or rupture-like changes. All histological scores were completed blinded by researchers who were unaware of the grouping information.

[0060] The survival of mice was continuously monitored throughout the experiment, and Kaplan-Meier survival analysis was used to evaluate the survival rate of mice in different treatment groups. At the end of the experiment, aortic tissue was separated, and healthy aortas, aortas with dissections, and ruptured aortas were classified, statistically analyzed, and quantitatively analyzed.

[0061] Figure 17 (a) ~ Figure 17 (b) Results showed that compared with the aortic dissection model group, the survival rate of mice in the CE-Salen-Ni intervention group was improving, and the incidence of aortic dissection and aortic rupture events was decreasing, with the trend being more pronounced in the high-dose intervention group. Macroscopic observation showed that the structural integrity of the aortic wall in the model group was disrupted, with visible dissection formation and local rupture. Figure 18 The results showed that after CE-Salen-Ni intervention, the overall structural integrity of the aorta was improved compared with the model group, and the degree of aortic wall damage was reduced. These results suggest that CE-Salen-Ni can inhibit the occurrence and progression of β-aminopropionitrile-induced aortic dissection in vivo and may reduce the risk of aortic rupture.

[0062] (2) Histological, morphological and imaging analysis of the aorta After isolating aortic tissues from each group of mice, histological and morphological evaluations were performed. Hematoxylin-eosin staining was used to evaluate the overall structure of the aortic wall and the degree of medial damage, with a semi-quantitative analysis using a 0–4 aortic damage score. Verhoeff-Van Gieson staining was used to evaluate the integrity of elastic fiber structure, with a 0–4 elastic fiber breakage grading for quantification. Masson's trichrome staining was used to evaluate collagen deposition, and the percentage of Masson-positive area was calculated. In vivo ROS levels were detected using ethidium dihydrogen iodide staining; DHE-positive signals showed red fluorescence, and cell nuclei showed blue fluorescence after DAPI counterstaining.

[0063] Figure 19 (a) ~ Figure 19 Histological staining results (d) showed that the aortic dissection model group exhibited significant medial degeneration, vessel wall structural disorder, elastic fiber rupture, and collagen structure damage, manifested as elevated H&E injury scores, increased EVG elastic fiber rupture grades, and a decrease in Masson-positive area. Compared with the model group, the CE-Salen-Ni intervention group showed reduced aortic medial structural damage, some preservation of collagen structure and extracellular matrix integrity, increased Masson-positive area, improved elastic fiber continuity, and reduced EVG elastic fiber rupture, with more significant improvements observed in the high-dose intervention group. Figure 20 (a) and Figure 20 (b) DHE staining results showed that the red fluorescence signal of DHE in the aortic tissue of the model group was significantly enhanced, indicating that the reactive oxygen species level in the aortic tissue was increased; after CE-Salen-Ni intervention, the DHE fluorescence signal decreased, indicating that CE-Salen-Ni can reduce the level of oxidative stress in the aortic tissue of aortic dissection model mice.

[0064] (3) Biochemical evaluation of oxidative stress in aortic tissue Aortic tissue from each group of mice was collected and homogenized. Oxidative stress-related indicators, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GDP), and catalase, were detected using commercially available reagent kits. (Figure 21(a)~) Figure 21 (d) The results showed that the level of MDA in the aortic tissue of the aortic dissection model group was increased, and the activities of antioxidant enzymes such as SOD, GPx and CAT were decreased, indicating that there was significant oxidative stress damage in the model group. After CE-Salen-Ni treatment, the level of MDA in the aortic tissue decreased, and the activities of SOD, GPx and CAT increased, indicating that CE-Salen-Ni can improve the oxidative stress state in the process of aortic dissection.

[0065] (4) Phenotypic analysis of local macrophages in the aorta Multiplex immunofluorescence assays were performed on paraffin-embedded aortic sections using a standard indirect immunofluorescence method to evaluate local macrophage phenotypic changes in the aortic tissue. After antigen retrieval and blocking, aortic tissue sections were incubated overnight with a primary antibody mixture, which included rat anti-CD86, goat anti-CD206, and rabbit anti-F4 / 80 (rat anti-CD86 antibody was purchased from Thermo Fisher, catalog number 14-0862-82, diluted 1:100; goat anti-CD206 antibody was purchased from Thermo Fisher, catalog number PA5-46994, diluted 1:100; rabbit anti-F4 / 80 antibody was purchased from Proteintech, catalog number 28463-1-AP, diluted 1:100). After washing, sections were incubated with a species-matched fluorophore-conjugated secondary antibody mixture, which included Alexa Fluor 488-labeled anti-rat IgG (H+L) secondary antibody, Alexa Fluor 568-labeled anti-goat IgG (H+L) secondary antibody, and Alexa Fluor 680-labeled anti-rabbit IgG (H+L) secondary antibody (Alexa Fluor 488-labeled anti-rat IgG (H+L) secondary antibody was purchased from Abcam, catalog number ab150153, at a dilution of 1:500; Alexa Fluor 568-labeled anti-goat IgG (H+L) secondary antibody was purchased from Abcam, catalog number ab175474, at a dilution of 1:500; Alexa Fluor 680-labeled anti-rabbit IgG (H+L) secondary antibody was purchased from Abcam, catalog number ab175775, at a dilution of 1:500). Cell nuclei were counterstained with DAPI before imaging. For both multiplex immunofluorescence and DHE image quantitative analysis, ImageJ software was used for background subtraction, and fluorescence intensity was measured using the average corrected total cellular fluorescence intensity. The corrected total cellular fluorescence intensity was calculated using the following formula: CTCF = Integrated Density - (selected cell area × background reading average fluorescence).

[0066] F4 / 80 staining was used to identify macrophage regions, and the fluorescence signals of CD86 and CD206 in F4 / 80-positive regions were quantitatively analyzed. Figure 22(a) and Figure 22(b) The results showed that the signal of CD86-positive M1-like macrophages was enhanced and the signal of CD206-positive M2-like macrophages was relatively reduced in the aortic tissue of the model group, suggesting the existence of a pro-inflammatory immune microenvironment in the aortic dissection. After CE-Salen-Ni treatment, the fluorescence signal of CD86 in the aortic tissue decreased, the fluorescence signal of CD206 increased, and the CD86 / CD206 ratio decreased, indicating that CE-Salen-Ni can promote the transformation of local aortic macrophages from a pro-inflammatory M1-like phenotype to an anti-inflammatory M2-like phenotype, thereby reshaping the local immune microenvironment of aortic dissection.

[0067] (5) Detection of ITGB1-AKT1-c-Jun-OPN signal axis in aortic tissue For immunohistochemical images, quantification was performed using the percentage of DAB-positive staining area. Specifically, ImageJ software was used to separate the DAB colorimetric channels, and a uniform threshold was applied to identify DAB-positive stained areas in each group of images. The percentage of DAB-positive stained area relative to the total area of ​​the selected tissue region was then calculated. The calculation formula is as follows: DAB positive area percentage (%) = DAB positive staining area / total area of ​​selected tissue region × 100%.

[0068] Three aortic tissue sections were selected from each animal, and three fields of view were randomly selected from each section for analysis. All image acquisition and quantitative analysis were performed under the same parameters, and the quantitative analysis was performed by researchers who were unaware of the grouping information.

[0069] Figure 23(a) shows the expression of ITGB1, p-AKT1, pc-Jun, OPN, and α-SMA in aortic tissue detected by immunohistochemistry, followed by DAB staining. Figure 23(b)~ Figure 23 (f) The results showed that the expression of ITGB1, p-AKT1, pc-Jun, and OPN was increased in the aortic tissue of the aortic dissection model group, while the expression of the smooth muscle contraction phenotype marker α-SMA was decreased. This suggests that the ITGB1-AKT1-c-Jun-OPN signaling axis is abnormally activated during the occurrence of aortic dissection, accompanied by the transformation of vascular smooth muscle cells from contractile to anabolic types. After CE-Salen-Ni treatment, the expression of ITGB1, p-AKT1, pc-Jun, and OPN decreased, while the expression of α-SMA recovered, indicating that CE-Salen-Ni can inhibit the overactivated ITGB1-AKT1-c-Jun-OPN signaling axis and maintain the contractile phenotype of vascular smooth muscle cells.

[0070] In summary, CE-Salen-Ni can improve survival rate, reduce the incidence of aortic dissection and rupture, alleviate aortic tissue damage and oxidative stress levels, promote the transformation of local aortic macrophages to the M2-like phenotype, and inhibit the abnormal activation of the ITGB1-AKT1-c-Jun-OPN signaling axis in a mouse aortic dissection model, thereby exerting an aortic protective effect.

[0071] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a multi-coordinated polyphenol nanozyme, characterized in that, The preparation method is as follows: Tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde were dissolved in a mixed solvent and subjected to a first solvothermal reaction under closed conditions. After the reaction was completed, nickel acetate, DMF and water were added and a second solvothermal reaction was carried out under closed conditions. After the reaction was completed, the mixture was cooled to room temperature, filtered and collected, and after washing and drying, a black solid was obtained, which is the multicoordinated polyphenol nanozyme.

2. The preparation method according to claim 1, characterized in that, The molar ratio of the tetraamino-substituted dibenzo-18-crown-6 and 2,4,5-trihydroxybenzaldehyde is 1:4; the mixed solvent is obtained by mixing 1,4-dioxane, mesitylene and glacial acetic acid in a volume ratio of 5:5:

2.

3. The preparation method according to claim 1, characterized in that, The temperature of the first solvothermal reaction was 180°C, and the reaction time was 3 days.

4. The preparation method according to claim 1, characterized in that, The molar ratio of nickel acetate to tetraamino-substituted dibenzo-18-crown-6 is 7:1; the second solvothermal reaction is carried out at 85°C for 3 days.

5. The use of the multicoordinated polyphenol nanozyme obtained by the preparation method according to any one of claims 1 to 4 in the preparation of a drug for preventing aortic dissection.

Images