A tin selenide-based nanozyme, its preparation method and applications

Abstract

This invention provides a tin selenide-based nanozyme, comprising tin selenide, with tannic acid and tetravalent cerium ions conjugated on the surface of the tin selenide. The invention also provides a method for preparing the above-mentioned nanozyme, comprising mixing tin selenide with anhydrous ethanol and ultrasonically dispersing the mixture; centrifuging the resulting mixture to obtain a supernatant, further centrifuging the supernatant to obtain a tin selenide nanosheet solution; adding the above tin selenide nanosheet solution and tannic acid solution together to deionized water; subsequently adding cerium nitrate solution and continuing stirring; then adding sodium hydroxide solution to adjust the pH to 8.0; after the reaction is complete, the tin selenide-based nanozyme is obtained. This invention also provides the application of the above-mentioned tin selenide-based nanozyme in the preparation of anti-atherosclerotic drugs. The nanozyme of this invention can effectively alleviate the progression of atherosclerosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to an artificial enzyme, specifically a SnSe-based nanozyme, its preparation method, and its uses. Background Technology

[0002] Atherosclerosis (AS) is an inflammatory disease characterized by lipid accumulation and plaque formation, leading to myocardial infarction, stroke, and other conditions. Cellular senescence is a stress response characterized by permanent growth arrest, and the development of atherosclerosis is highly associated with aging, accompanied by the secretion of bioactive molecules, known as the senescence-associated secretory phenotype (SASP). Recent evidence suggests that senescent endothelial cells are involved in AS progression, and the senescence of these cells is a key driver of atherosclerosis, playing a detrimental role in its pathogenesis. The accumulation of senescent endothelial cells triggers oxidative stress and inflammatory responses at plaque lesion sites, driving disease progression by increasing the expression of inflammatory cytokines and chemokines, thereby further impairing endothelial function, exacerbating atherosclerosis, inducing tissue remodeling, and ultimately increasing the risk of disease. Therefore, alleviating endothelial cell senescence is a potentially feasible strategy for treating AS. Senescent endothelial cells are highly pro-inflammatory secretors, and traditional anti-atherosclerotic drugs using antioxidant / anti-inflammatory treatment strategies have not been very effective, mainly due to the complex pathological microenvironment in AS.

[0003] In atherosclerotic plaque lesions, endothelial cells exhibit significant metabolic reprogramming, characterized by increased glycolytic activity. Increased glycolysis leads to increased lactate production, which is closely associated with various AS-related pathological processes, such as inflammation, endothelial dysfunction, and cellular senescence. Furthermore, the lactate-induced acidic microenvironment can lead to the loss of endothelial markers and trigger endothelial-mesenchymal transition (EndMT); this transition promotes the recruitment and infiltration of inflammation-associated immune cells, a key driver of endothelial dysfunction and AS. In addition, lactate can induce ROS production via the NOX1 pathway, driving endothelial cells towards a senescence-associated secretory phenotype, thereby exacerbating endothelial dysfunction. This metabolic shift represents a unique form of reprogramming in AS progression. Therefore, regulating lactate metabolism in senescent cells may be an effective strategy for treating AS. Summary of the Invention

[0004] In view of the above-mentioned technical problems in the prior art, the present invention provides a tin selenide-based nanozyme, its preparation method and uses, which aims to solve the technical problem of poor efficacy of drug treatment for atherosclerosis in the prior art.

[0005] This invention provides a tin selenide-based nanoenzyme, comprising tin selenide, with tannic acid and tetravalent cerium ions composited on the surface of the tin selenide.

[0006] This invention provides a method for preparing the above-mentioned tin selenide (SnSe)-based nanozyme, comprising the following steps:

[0007] 1) Weigh out tin selenide and anhydrous ethanol;

[0008] 2) Tin selenide is dispersed in anhydrous ethanol solution under ultrasonic oscillation, wherein the concentration of tin selenide in the anhydrous ethanol solution is 10~40 g / L. -1 After centrifugation at 3000-5000 rpm, the precipitate was discarded and the supernatant was collected. The supernatant was centrifuged again at 10000-15000 rpm and the precipitate was collected. The precipitate was washed with deionized water and mechanically stirred at room temperature to mix it evenly, thus obtaining a clear tin selenide nanosheet solution.

[0009] 3) Measure out the prepared tin selenide nanosheet solution, add tannic acid solution, and stir continuously at room temperature for 10-30 minutes to ensure thorough mixing of the tin selenide nanosheets and tannic acid. The concentration of tannic acid in the homogeneous solution should be 0.5-2 g / L. -1 ;

[0010] 4) Add cerium nitrate solution to the homogeneous solution obtained in step 3), the concentration of which is 0.5~2 g / L. -1 The volume ratio of the cerium nitrate solution to the tin selenide nanosheet solution is 1:(4~8). Stir to ensure thorough and uniform mixing.

[0011] 5) Add dilute sodium hydroxide solution to adjust the pH of the mixed reaction system to 8.0, and continue stirring at room temperature for 1-3 hours to complete the self-assembly of cerium-tannic acid on the surface of SnSe nanosheets;

[0012] 6) After the reaction is complete, wash with deionized water and remove unassembled free tannic acid, cerium ions and other impurities by centrifugation to obtain a SnSe-based nanozyme.

[0013] The present invention also provides the application of the above-mentioned tin selenide-based nanozyme in the preparation of drugs for treating atherosclerosis.

[0014] The present invention also provides the application of the above-mentioned SnSe-based nanozyme in the preparation of drugs for scavenging reactive oxygen species, anti-inflammation, scavenging lactic acid and anti-aging.

[0015] This invention, through rational design, introduces tannic acid and tetravalent cerium (Ce) onto the SnSe surface.4+ A metal-tannic acid network framework was formed on the SnSe surface, and the elemental composition and morphology of the nanomaterial were rationally controlled to successfully construct a two-dimensional SnSe@TC MEM nanozyme for the treatment of atherosclerosis (TC represents TA tannic acid, C represents Ce cerium ions, and MEM means enzyme mimic). The SnSe@TC MEM nanozyme provided by this invention can effectively scavenge ROS, downregulate the expression of inflammatory factors, clear lactate, and reduce the expression level of aging-related β-galactosidase (SA-β-Gal) in endothelial cells, thus exerting a synergistic anti-inflammatory and anti-aging effect. Furthermore, this nanozyme can further reverse the pro-inflammatory acidic microenvironment at plaque sites, effectively inhibit endothelial-mesenchymal transition, and improve endothelial dysfunction; thus synergistically exerting the best anti-AS therapeutic effect.

[0016] This invention demonstrates through in vitro and in vivo experiments that the anti-inflammatory / antioxidant therapy achieved by SnSe@TC MEM nanozymes has a synergistic effect with lactate regulation, providing a promising new therapeutic approach for the prevention and treatment of atherosclerosis.

[0017] Compared with existing technologies, the technical effects of this invention are positive and significant. This invention not only expands the biological application scope of SnSe-based nanozymes but also provides a high-performance therapeutic nanoplatform for combating atherosclerosis. The nanozyme of this invention (SnSe@TC MEM nanozyme) exhibits the effects of scavenging toxic reactive oxygen species (ROS), downregulating the expression of inflammation-related factors, anti-apoptosis, reversing the pro-inflammatory acidic microenvironment, and reducing plaque lactate content. By regulating lactate metabolism, it exerts an anti-aging effect to improve the progression of atherosclerosis, providing a new anti-aging therapeutic strategy for the prevention and treatment of atherosclerosis. Attached Figure Description

[0018] Figure 1 A schematic diagram of the SnSe@TC MEM nanozyme synthesis process and its application in anti-atherosclerotic nanotherapy are shown. Figure a illustrates the SnSe@TC MEM nanozyme synthesis process, including ultrasonic exfoliation and the tannic acid-cerium surface self-assembly process. Figure b illustrates the therapeutic process of the SnSe@TC MEM nanozyme, including its antioxidant / anti-inflammatory effects, lactic acid scavenging for anti-aging, and its role in reversing the endothelial-mesenchymal transition in human umbilical vein endothelial cells (HUVECs).

[0019] Figure 2The structure and composition characterization of SnSe nanozymes and SnSe@TC MEM nanozymes are shown. Image a is a transmission electron microscope (TEM) image of the SnSe nanozyme; scale bar: 100 nm. Image b is a high-resolution TEM image of the SnSe nanozyme; scale bar: 10 nm. Image c is a scanning electron diffraction (SEM) pattern of the SnSe nanozyme; scale bar: 50 nm. Image d is a TEM image of the SnSe@TC MEM nanozyme; scale bar: 100 nm. Image e is a SEM image of the SnSe@TC MEM nanozyme; scale bar: 100 nm. Image f is an elemental distribution map of the SnSe@TC MEM nanozyme; scale bar: 100 nm.

[0020] Figure 3 This is a valence state analysis diagram of SnSe@TC MEM nanozymes. In diagram a, the X-ray photoelectron spectrum of the SnSe@TC MEM nanozyme is shown. In diagram b and d, the high-resolution X-ray photoelectron spectra of tin 3d, selenium 3d, and cerium 3d are shown, respectively.

[0021] Figure 4 This is a structural analysis diagram of SnSe@TC MEM nanozyme crystals.

[0022] Figure 5 This study demonstrates the high catalytic efficiency of SnSe@TC MEM nanozymes and their potential applications in lactate elimination and anti-aging. Figure a shows the in vitro drug release kinetics of SnSe@TC MEM nanozymes under different pH conditions. Figure b shows the in vitro drug release kinetics of SnSe nanozymes and lactate dehydrogenase at different concentrations in the presence or absence of NAD+. + Enzyme activity when used as a coenzyme. Where c represents the effect of different lactate concentrations on the activities of SnSe nanozyme and lactate dehydrogenase.

[0023] Figure 6 It showed cellular phagocytosis ( Figure 6 a) and cell safety ( Figure 6 b) Evaluation.

[0024] Figure 7 The biosafety and biocompatibility of SnSe@TC MEM nanozymes were demonstrated. Section a shows HE-stained tissue sections of major mouse organs treated with different concentrations of SnSe@TC MEM nanozymes; scale bar: 50 μm. Section b shows the effects of different doses of SnSe@TC MEM nanozymes on mouse renal and liver function indicators. Section c shows the effects of different doses of SnSe@TC MEM nanozymes on mouse liver function indicators. Section d uses a heatmap to illustrate the changes in routine blood parameters in mice after treatment.

[0025] Figure 8The ROS elimination and anti-inflammatory properties of SnSe@TC MEM nanozymes are shown. Where 'a' represents the total ROS, H2O2, and O2 in human umbilical vein endothelial cells after different treatments. •- Confocal microscopy images; scale bar: 20 μm. Where bd represents total ROS, H2O2, and O2 in endothelial cells after different treatments, respectively. •- The fluorescence intensity.

[0026] Figure 9 This study demonstrates the anti-apoptotic effect of a two-dimensional SnSe@TC MEM nanozyme. The anti-apoptotic capacity of human umbilical vein endothelial cells after different treatments was also shown; scale bar: 40 μm; and quantitative statistical analysis of FITC fluorescence intensity in human umbilical vein endothelial cells after different treatments was also presented.

[0027] Figure 10 This study demonstrates the anti-inflammatory and anti-aging properties of a two-dimensional SnSe@TC MEM nanozyme. In diagram a, we show the changes in mitochondrial membrane potential in human umbilical vein endothelial cells under different treatments; unit length: 20 μm. In diagram b, we show the observation of DNA damage in human umbilical vein endothelial cells under different treatments; unit length: 40 μm. In diagram c, we show the quantitative fluorescence intensity analysis of γ-H2AX in human umbilical vein endothelial cells after different treatments.

[0028] Figure 11 The anti-inflammatory properties of the two-dimensional SnSe@TC MEM nanozyme were demonstrated. Under different treatment conditions, ICAM-1 in human umbilical vein endothelial cells induced by LPS (… Figure 11 a) and the adhesion factor level of VCAM-1 ( Figure 11 (b) in the middle.

[0029] Figure 12 This study demonstrates the anti-endothelial mesenchymal transition (EMT) ability of SnSe@TC MEM nanozymes and assesses their impact on endothelial cell barrier function. In Figure a, F-actin (red) expression is shown in human umbilical vein endothelial cells (HUVECs) under different treatment groups; scale bar: 20 μm. In Figure b, VE-cadherin (green) expression is shown in HUVECs under different treatment groups; the intercellular spaces marked by yellow triangles are induced by cytokines; scale bar: 20 μm. In Figure c, CD31 expression is shown in HUVECs after different treatments; scale bar: 20 μm. In Figure d, the fluorescence intensity of CD31 in HUVECs after different treatments is presented as a quantitative statistical graph. In Figure e, PAI-1 expression is shown in HUVECs after different treatments; scale bar: 20 μm. In Figure f, the fluorescence intensity of PAI-1 in HUVECs after different treatments is presented as a quantitative statistical graph.

[0030] Figure 13This study demonstrates the in vivo animal-level anti-AS effect of SnSe@TC MEM nanozymes. Figure a shows a schematic diagram of the in vivo treatment protocol using SnSe@TCMEM. Figure b shows ORO-stained aortic sections and corresponding quantitative analyses under different interventions; scale bar: 5 μm (n = 10). Figure cg shows representative images of ORO-stained frozen sections and corresponding quantitative analyses of the aortic root, aortic arch, and brachiocephalic artery; scale bar: 200 μm. Figure h shows images of aortic root sections stained with IL-6 and TNF-α antibodies using immunohistochemical staining. Figure i shows images of aortic arch and brachiocephalic artery sections stained with TNF-α antibodies using immunohistochemical staining; scale bar: 200 μm.

[0031] Figure 14 This study demonstrates the anti-apoptotic and anti-aging capabilities of a SnSe@TC MEM nanozyme. In section ab, the anti-AS effect after different treatments was assessed using HE staining and MASSON trichrome staining, respectively; scale bar: 400 μm. Section c shows fluorescence images of aortic root sections stained with anti-cysteine ​​protease-3 antibody after different treatments; scale bar: 100 μm. Section d shows fluorescence images of aortic root sections stained with anti-γ-H2AX antibody after different treatments; scale bar: 100 μm. Section eh shows the quantitative analysis of plaque area, collagen area ratio, Caspase-3 positive region ratio, and γ-H2AX positive region ratio after different treatments.

[0032] Figure 15 This section presents the anti-aging effects of SnSe@TC MEM nanozymes and their therapeutic effects against AS through immunomodulation after different treatments. Image a shows the fluorescence image of an aortic root section stained with anti-MMP antibody without treatment. Image b shows the quantitative analysis of MMP-9 area relative to total plaque area after different treatments. Image c shows double immunofluorescence staining images of SA-β-Gal (green) and ICAM-1 (red) in mice after different treatments. Image d shows the quantitative analysis of SA-β-Gal positive area relative to total plaque area. Image e shows the fluorescence of DAPI (blue), BODIPY (green), CD68 (red), and α-SMA (yellow) in aortic root sections of mice after different treatments using immunofluorescence detection; scale bar: 100 μm. Image fh shows the statistical graph of the quantitative analysis of the proportions of CD68 positive area, α-SMA positive area, and BODIPY positive area relative to total plaque area.

[0033] Figure 16 ApoE after different processing - / - A representative image of the aortic arch in a mouse. Detailed Implementation

[0034] The present invention will be further described in detail through the following embodiments in conjunction with the accompanying drawings. However, the technical content described in these embodiments is illustrative and not limiting, and should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1:

[0036] A method for preparing SnSe-based nanozymes (SnSe@TC MEM nanozymes) includes the following steps:

[0037] a) Synthesis of SnSe: First, 1 g of bulk SnSe material was mixed with 50 mL of anhydrous ethanol and dispersed by ultrasonic vibration in an ice bath for 24 hours (5 s on, 3 s off) using a 0.125-inch probe (60 W). Ultrasonic dispersion ensured complete exfoliation of the bulk SnSe. The mixture was centrifuged at 5000 rpm for 10 minutes at 22°C to obtain the supernatant. The supernatant was further centrifuged at 13000 rpm to obtain the precipitate. The precipitate was washed three times with deionized water and mechanically stirred at room temperature to obtain a clear SnSe nanosheet solution.

[0038] b) Synthesis of SnSe@TC MEM nanozyme: 10 ml of SnSe solution (100 μg / ml) was prepared. -1 ), 2 ml tannic acid solution (TA) (1.0 mg / ml) -1 Add to 50 ml of deionized water and stir for 2 minutes, then add 2 ml of Ce(NO3)4 solution (1 mg / ml). -1 Continue stirring for 3 minutes. Then add NaOH solution to adjust the pH of the solution to 8.0 and react for 2 hours, continuing stirring to allow tannic acid-cerium to self-assemble on the SnSe nanosheet surface. Collect by centrifugation and wash three times with deionized water to obtain the synthesized SnSe@TC MEM nanozyme.

[0039] Example 2:

[0040] The SnSe@TC MEM nanozyme prepared in Example 1 was observed using transmission electron microscopy.

[0041] Figure 1 This diagram illustrates the synthesis process of the SnSe@TC MEM nanozyme and the mechanism of anti-AS treatment using the constructed SnSe@TC MEM nanozyme, including antioxidant and anti-inflammatory effects, lactate scavenging and anti-aging, and the reversal of endothelial cell mesenchymal transition. Figure 2 As indicated by 'a' in the figure, the prepared SnSe nanozyme is approximately 100 nm in size. High-resolution transmission electron microscopy imaging and accompanying selected-area electron diffraction patterns reveal the SnSe nanozyme lattice, with lattice fringe lengths of 0.23 nm (see [reference needed]). Figure 2 (b and c in the original text). To further confirm the nanostructure of the synthesized SnSe@TC MEM nanozyme, transmission electron microscopy was performed on it (see [link to original text]). Figure 2 (d) and elemental scanning electron microscopy analysis (see d) Figure 2 (e, f in the text).

[0042] The results showed that the SnSe@TC MEM nanozyme retained the morphological and size characteristics of SnSe nanozymes, and the detection of O, Ce, Se, and Sn elements confirmed the successful synthesis of the SnSe@TC MEM nanozyme. Figure 3 It can be seen that, compared with SnSe nanozymes, SnSe@TC MEM nanozymes contain Ce (see Figure 3 The appearance of the characteristic peak in d) indicates that tannic acid-cerium has successfully self-assembled onto the surface of SnSe. Figure 4 The crystal structure of SnSe@TC MEM nanozyme is shown.

[0043] Example 3:

[0044] 100 μg mL -1 SnSe@TC MEM nanozymes were placed in culture media at pH 7.4 and 5.3. Supernatants were collected at different time points and analyzed using UV absorption spectroscopy. The release rate of SnSe@TC MEM nanozymes under simulated physiological conditions (pH 7.4) was 25%. In contrast, the release rate was 63% after 24 hours in an acidic environment (pH 5.3). SnSe nanozymes exhibited better catalytic efficiency than LDH at the same concentration in the concentration range of 50–200 μg mL⁻¹. To further understand the reaction kinetics of SnSe, we analyzed the reaction rate at different substrate concentrations. The results showed that SnSe nanozymes exhibited strong catalytic effects starting from 10 mM.

[0045] In summary, these results confirm the successful preparation of SnSe@TC MEM nanozymes. These nanozymes possess the ability to mimic lactate dehydrogenase, exhibiting high catalytic efficiency and demonstrating significant application potential in lactate elimination and anti-aging (e.g., ...). Figure 5 (As shown).

[0046] Example 4:

[0047] Figure 6 Figure 'a' shows the phagocytosis process of SnSe@TC MEM nanozymes by cells. SnSe@TC MEM nanozymes were observed entering cells at 15 minutes (black aggregates), mainly distributed in the cytoplasm with a relatively compact morphology. After 1 hour, the number and distribution of aggregates increased. After 2 hours, multiple distinct aggregates formed intracellularly, indicating that the uptake of the material by cells gradually accumulated over time.

[0048] Figure 6 Figure b shows the cytotoxicity of four materials (SnSe, TA, Ce(NO3)4, and SnSe@TC MEM) at different concentrations and time periods (12 hours and 24 hours). The results show that all four materials exhibit relatively good biocompatibility. These experiments demonstrate that SnSe@TC MEM nanozymes can be effectively internalized by cells in a time-dependent manner, exhibiting good biocompatibility and representing a promising biomedical nanomaterial.

[0049] Example 5:

[0050] The in vivo safety of SnSe@TC MEM nanozyme was assessed by intravenous injection into Kunming mice. Specifically, 15 healthy female Kunming mice (5-6 weeks old) were randomly divided into 3 groups (n=5 per group): a control group (treated with saline only), and experimental groups (treated with 10 mg / kg saline solution). -1 and 20 mg kg -1 The mice were intravenously injected with SnSe@TC MEM nanozyme at the prescribed dose. One month after treatment, blood and major organs were collected from the mice for comprehensive evaluation, including hematological, histological, and blood biochemical analyses. No significant behavioral changes were observed in the mice throughout the experiment. Histological examination of the major organs of the treated mice revealed no abnormal structures or significant pathological toxicity. Figure 7 (a) In addition, liver and kidney function tests and hematological indicators all fluctuated within the normal range (a). Figure 7 These results (bd in the text) further confirm the good biosafety and biocompatibility of SnSe@TC MEM nanozymes.

[0051] Example 6:

[0052] At the cellular level, total ROS (green fluorescence) levels were detected using DCFH-DA, and hydroxyl radicals were detected using APF. . OH (green fluorescence) levels and DHE detection of superoxide anion (O2) •- The results showed that the Control group had almost no fluorescence, while the H2O2 positive control group showed the strongest fluorescence from all three probes. The SnSe group showed significantly weaker fluorescence intensity than the H2O2 group, close to the Control group, indicating that SnSe itself hardly induces ROS production. The fluorescence intensity of the TA and Ce groups was similar, also much lower than that of the H2O2 group, suggesting that they had a weak inhibitory effect on ROS. In the SnSe@TC MEM group, the fluorescence of all three probes was the weakest, close to that of the Control group, indicating that this nanozyme can effectively remove intracellular ROS and exhibit significant antioxidant capacity (e.g., red fluorescence). Figure 8 (a) in the middle.

[0053] Subsequently, we evaluated the ROS production patterns of SnSe nanozymes and SnSe@TC MEM nanozymes in endothelial cells. The control group used fresh culture medium to culture cells, while the model group used H2O2 stimulation to stimulate endothelial cells. 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), aminophenylfluorescein (APF), and dihydroethidium (DHE) were used as fluorescent probes to detect total ROS in cells after different treatments. . OH and O2 •- Four hours after stimulation, the ROS levels in the model group significantly increased. In contrast, when stimulated HUVECs showed increases in TA and Ce... 4+ When SnSe@TC MEM nanozymes were co-incubated for 4 hours, the intensity of the DCFH-DA green fluorescence signal decreased, with the SnSe@TC MEM treatment group showing the largest decrease. This indicates that the SnSe@TC MEM nanozymes, after tannic acid-cerium self-assembly modification, possess strong antioxidant and anti-inflammatory effects. However, when stimulated human umbilical vein endothelial cells were co-incubated with SnSe for 4 hours, the green fluorescence signal intensity remained essentially unchanged. This may be because SnSe's affinity for hydrogen atoms hinders its ability to scavenge ROS. Consistent with these observations, APF (green fluorescence) and DHE (red fluorescence) staining further demonstrated that the SnSe@TC MEM nanozymes can significantly reduce the levels of ROS in human umbilical vein endothelial cells. . OH and O2 •- The generation of SnSe@TC MEM nanozymes was demonstrated. In summary, this study confirms that SnSe@TC MEM nanozymes can protect human umbilical vein endothelial cells from ROS-induced oxidative damage (such as ROS) by scavenging intracellular ROS. Figure 8 (bd in the middle).

[0054] Example 7:

[0055] Human umbilical vein endothelial cells were seeded into confocal glass culture dishes (cell density = 10⁻⁶). 5 Incubate in fresh culture medium (control), H2O2 (100 μM), and H2O2 (100 μM) + SnSe (20 μg / mL) for 24 hours, then incubate separately. -1 ), H2O2 (100 μM) + TA (20 μg mL -1 ), H2O2 (100 μM) + Ce(NO3)4 (20 μg mL -1 ) and H2O2 (100 μM) + SnSe@TC MEM (20 μg mL -1After incubation at 37 ℃ for 6 hours, the cells were incubated in the dark for 1 hour with 50 μl of Tunel assay solution. After washing twice with PBS, FITC expression was observed using CLSM. The results showed that SnSe@TC MEM nanozyme could reverse H2O2-induced apoptosis in human umbilical vein endothelial cells. This indicates that SnSe@TC MEM nanozyme has good anti-apoptotic ability, can effectively inhibit H2O2-induced DNA breaks, and restores the apoptosis level to near the normal control group. (See reference for details.) Figure 9 .

[0056] Example 8:

[0057] Human umbilical vein endothelial cells were seeded into confocal glass culture dishes (cell density = 10⁻⁶). 5 Incubate in fresh medium (control), H2O2 (100 μM), and H2O2 (100 μM) + SnSe (20 μg / mL) for 24 hours, then incubate separately. -1 ), H2O2 (100 μM) + TA (20 μg mL -1 ), H2O2 (100 μM) + Ce(NO3)4 (20 μg mL -1 ) and H2O2 (100 μM) + SnSe@TC MEM (20 μg mL -1 After incubation at 37°C for 6 hours, cells were fixed with 4% PFA for 5 minutes. The SnSe@TCMEM group showed the weakest green fluorescence and the strongest red fluorescence, indicating that the drug most effectively restores mitochondrial membrane potential and protects mitochondrial function.

[0058] γ-H2AX is a marker of DNA double-strand breaks, and senescent cells exhibit higher expression levels of γ-H2AX. The intensity of its green fluorescence is positively correlated with the degree of DNA damage. After endothelial cell senescence was induced, the expression levels of phosphorylated histone H2A family member X (γ-H2AX), a marker of DNA double-strand breaks, were observed using confocal laser scanning microscopy after different treatments. SnSe@TCMEM treatment significantly reduced γ-H2AX expression, with a greater reduction than that seen in SnSe, TA, and Ce. 4+ These results indicate that SnSe@TC MEM nanozymes can effectively protect DNA from damage.

[0059] Human umbilical vein endothelial cells were seeded into confocal glass culture dishes (cell density = 10⁻⁶). 5 Incubate in fresh medium (control), H2O2 (100 μM), and H2O2 (100 μM) + SnSe (20 μg / mL) for 24 hours, then incubate separately. -1), H2O2 (100 μM) + TA (20 μg mL -1 ), H2O2 (100 μM) + Ce(NO3)4 (20 μg mL -1 ) and H2O2 (100 μM) + SnSe@TC MEM (20 μg mL -1 After incubation at 37 ℃ for 6 hours, the cells were replaced with fresh DMEM containing 1 μM SPiDER-β-Gal and incubated for 30 minutes. The cells were washed twice with PBS, and SA-β-gal positive cells were observed using CLSM. Treatment with SnSe@TC MEM significantly reduced SA-β-Gal activity in endothelial cells, confirming that the SnSe@TC MEM nanozyme possesses strong anti-cellular senescence capabilities.

[0060] like Figure 10 As shown, the SnSe@TC MEM nanozyme exhibits properties that alleviate mitochondrial damage, DNA breakage, and cellular senescence.

[0061] Example 9:

[0062] Endothelial cells (1 × 10⁶ per well) 6 Cells were seeded in 12-well plates and incubated overnight. The experiment included a control group and a model group (10 ng / mL LPS). -1 LPS (10 ng mL) -1 ) + SnSe (20µg mL) -1 LPS (10 ng mL) -1 ) + TA (20µg mL) -1 LPS (10 ng mL) -1 ) + Ce 4+ (20µg mL) -1 ) and LPS (10 ng mL -1 ) + SnSe@TC MEM (20µg mL) -1 After 24 hours of treatment, ICAM-1 and VCAM-1 levels were detected using an ELISA kit. The anti-inflammatory capacity of SnSe@TC MEM was assessed.

[0063] like Figure 11As shown, SnSe@TC MEM significantly reduced ICAM-1 and VCAM-1 levels. Inflammation can induce cellular senescence and drive AS progression; therefore, we evaluated the anti-inflammatory properties of SnSe@TC MEM nanozymes. Using enzyme-linked immunosorbent assay (ELISA), we found that lipopolysaccharide (LPS) stimulated human umbilical vein endothelial cells to produce high levels of cell adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Treatment with SnSe@TC MEM nanozymes significantly reduced the levels of these cytokines, demonstrating their anti-inflammatory effects.

[0064] Example 10:

[0065] Human umbilical vein endothelial cells were seeded in confocal culture dishes (cell density = 10⁻⁶). 5 / dish), incubated for 24 hours. Then, the experimental groups included a control group, IL-1β (10 ng / mL) -1 ), IL-1β (10 ng mL) -1 ) + SnSe (20µg mL) -1 ), IL-1β (10 ng / mL) -1 ) + TA (20µg mL) -1 ), IL-1β (10 ng mL) -1 ) + Ce (20µg mL) -1 ) and IL-1β (10 ng mL) -1 ) + SnSe@TC MEM (20µg mL) -1Fixation and fluorescent staining were performed at 37 °C and 5% CO2 for 12 hours. Fluorescent staining with VE-cadherin and the cytoskeleton F-actin clearly showed that untreated human umbilical vein endothelial cells (HUVECs) formed an intact monolayer. Peripheral actin filaments along the cell boundaries were anchored by transmembrane cadherins, which together stabilized endothelial cell-cell contact and maintained barrier integrity. In contrast, the IL-1β-treated endothelial cell monolayer exhibited significant morphological changes, including discontinuous cell-cell adhesion, the appearance of intercellular spaces, and an increase in actin stress fibers along the cell body. After 6 hours of co-incubation of HUVECs with SnSe@TC MEM nanozyme and IL-1β, the endothelial layer maintained its structural integrity, without significant increase in intercellular spaces or actin stress fibers. This indicates that SnSe@TC MEM nanozyme can inhibit the destructive effects of cytokines on endothelial cells and suppress the transition of endothelial cells from a quiescent to an interstitial state. To further validate the effectiveness of the SnSe@TC MEM nanozyme, we observed the expression levels of CD31 and PAI-1 in endothelial cells using confocal microscopy. CD31 plays a crucial role in angiogenesis and is a typical marker of vascular endothelial cells. PAI-1, on the other hand, is a mesenchymal marker. IL-1β induced a decrease in CD31 expression and an increase in PAI-1 expression in endothelial cells, suggesting that endothelial cells initiate mesenchymal transition (MTT) under cytokine induction. However, the SnSe@TC MEM nanozyme significantly inhibited the initiation of MMT by endothelial cells. Figure 12 As shown, the evaluation of SnSe@TC MEM nanozyme's ability to alleviate cytokine-activated endothelial cell barrier function and resist endothelial-mesenchymal transition is presented.

[0066] Example 11:

[0067] ApoE - / - Mice (Jiangsu Jicui Co., Ltd.) were fed a high-fat diet for one month to induce the formation of atherosclerotic plaques. Fifty mice were randomly divided into a model group (treated with saline only) and a SnSe group (treated with 20 mg / kg of saline solution only). -1 ), TA group (20 mg / kg) -1 Ce 4+ Group (20 mg kg) -1 ), SnSe@TC MEM group (20 mg kg -1The treatment regimen consisted of intravascular injections twice weekly, with each injection consisting of 100 μL of drug. This continued for two months. After treatment, the aortas of the five groups were stained with Oil Red O (ORO). Quantitative results clearly showed that the average ORO-positive area was 18.7 ± 2.4% in the model group, 14.24 ± 3.75% in the SnSe group, 12.0 ± 2.2% in the TA group, and 13.5 ± 1.8% in the Ce group. The SnSe@TC MEM group had the smallest aortic plaque area (8.1 ± 1.8%), indicating that the SnSe@TC MEM nanozyme treatment was significantly effective. Frozen sections of the aortic root, aortic arch, and brachiocephalic trunk stained with ORO demonstrated that the SnSe@TC MEM nanozyme possessed effective anti-atherosclerotic capabilities. To verify the anti-inflammatory effects of the SnSe@TC MEM nanozyme observed in vitro, we performed immunostaining of aortic sinus sections from each group for the inflammatory factors IL-6 and TNF-α. Mice fed a high-fat diet for three consecutive months showed strong inflammation-related fluorescence intensity within plaques, while mice treated with SnSe@TC MEM nanozymes showed significantly reduced fluorescence intensity. Furthermore, the fluorescence intensity was significantly reduced when combined with SnSe, TA, or Ce. 4+ Compared to the control group, the SnSe@TC MEM nanozyme group showed a more significant decrease in IL-6 and TNF-α fluorescence intensity, confirming its synergistic anti-atherosclerotic and anti-inflammatory effects. Figure 13 As shown, SnSe@TCs nanozymes can significantly reduce the plaque area in the middle arteries of ApoE- / - mice, demonstrating a strong anti-inflammatory effect.

[0068] Example 12:

[0069] To elucidate the therapeutic mechanism of SnSe@TC MEM nanozymes, we performed histochemical analysis on aortic sinus plaque samples. H&E staining showed that SnSe@TC MEM nanozyme treatment significantly reduced the area of ​​necrotic portions within the plaque, thereby effectively inhibiting AS progression. MASSON and anti-MMP-9 staining indicated that SnSe@TC MEM nanozyme treatment reduced MMP-9 expression and activity while increasing intraplaque collagen content, enhancing plaque stability. Macrophage infiltration and MMP-9 expression were positively correlated with plaque susceptibility, while periplaque collagen deposition and smooth muscle cell (VSMC) proliferation contributed to plaque stabilization. Figure 14 As shown, compared with the model group, the SnSe@TC MEM nanozyme treatment of ApoE - / -The activation level of caspase-3 in the mouse aorta was also significantly downregulated. This confirms that SnSe@TC MEM nanozymes have a strong ability to inhibit the caspase-3-mediated apoptosis pathway. Given the potent anti-aging effects of SnSe@TC MEM nanozymes in vitro, we further evaluated their therapeutic effects in vivo. Immunohistochemical staining with γ-H2AX antibody showed that γ-H2AX expression in plaques treated with SnSe@TC MEM nanozymes was significantly reduced. (The text also mentions SnSe, TA, and Ce, but the connection to SnSe@TC MEM nanozymes is unclear and likely refers to a specific antibody or ingredient.) 4+ Compared to the control group, the γ-H2AX positive staining area in the SnSe@TC MEM treated group was much smaller, indicating that the SnSe@TC MEM nanozyme effectively protected DNA from ROS damage. Figure 14 As shown, SnSe@TCs nanozymes can effectively inhibit caspase-3-mediated apoptosis pathway and have good anti-aging effects.

[0070] Example 13:

[0071] Anti-MMP-9 staining showed that SnSe@TC MEM nanozyme treatment reduced MMP-9 expression levels while increasing collagen content in plaques and enhancing plaque stability. Analysis of the cellular senescence marker SA-β-Gal showed that SnSe@TC MEM nanozyme significantly reduced SA-β-Gal expression levels in plaque sites, with a significantly lower proportion of SA-β-Gal-positive cells in the SnSe@TC MEM group. ICAM-1 expression was also inhibited, indicating that SnSe@TC MEM can effectively alleviate vascular endothelial cell senescence, reduce inflammatory cell adhesion, and slow the progression of senescence in AS. α-SMA antibody staining showed a significant increase in vascular smooth muscle cells in plaques after SnSe@TC MEM nanozyme treatment. Anti-CD68 staining confirmed that the anti-inflammatory effect of SnSe@TC MEM nanozyme reduced macrophage accumulation in plaques. The total proportion of BODIPY-positive cells was lowest in the SnSe@TC MEM group, demonstrating a significant reduction in lipid deposition.

[0072] Figure 15 This study demonstrated the therapeutic effects of SnSe@TC MEM nanozymes on anti-inflammation, aging, and lipid metabolism in an ApoE- / - mouse atherosclerosis model. Results showed that SnSe@TC MEM nanozymes downregulated MMP-9 and ICAM-1 expression levels, alleviated the plaque inflammatory microenvironment, reduced SA-β-Gal positivity, mitigated endothelial cell senescence, reduced macrophage foaming, and decreased overall lipid deposition.

[0073] Example 14:

[0074] 40 ApoE - / -Mice were initially fed a high-fat diet for one month to induce the formation of atherosclerotic plaques. The mice were then divided into five groups of eight each: the model group received saline treatment, and the four experimental groups received 20 mg / kg of saline solution. -1 SnSe, TA, Ce(NO3)4, and SnSe@TC MEM were administered. In addition to a Western diet, all groups received weekly intravenous injections via the tail vein for two months. After two months of treatment, ApoE... - / - Mice were euthanized. Gross morphology of the aorta and a series of cross-sectional plaques showed that the SnSe@TC MEM nanozyme group had significantly the lightest plaque load, with only a small amount of scattered lipid deposition. The results indicate that the SnSe@TC MEM nanozyme formulation can significantly reduce the area of ​​lipid plaques in the aorta (…). Figure 16 ).

Claims

1. A tin selenide-based nanozyme, characterized in that, This includes tin selenide, on the surface of which tannic acid and tetravalent cerium ions are composited.

2. The method for preparing a tin selenide-based nanozyme according to claim 1, characterized in that... Includes the following steps: 1) Weigh out tin selenide and anhydrous ethanol; 2) Tin selenide is dispersed in anhydrous ethanol solution under ultrasonic oscillation, wherein the concentration of tin selenide in the anhydrous ethanol solution is 10~40 g / L. -1 After centrifugation at 3000-5000 rpm, the precipitate was discarded and the supernatant was collected. The supernatant was centrifuged again at 10000-15000 rpm and the precipitate was collected. The precipitate was washed with deionized water and mechanically stirred at room temperature to mix it evenly, thus obtaining a clear tin selenide nanosheet stock solution. 3) Measure out the prepared tin selenide nanosheet stock solution, add tannic acid solution, and stir continuously at room temperature for 10-30 minutes to ensure thorough premixing of the tin selenide nanosheets and tannic acid. The concentration of tannic acid in the homogeneous solution should be 0.5-2 g / L. -1 ; 4) Add cerium nitrate solution to the homogeneous solution obtained in step 3), the concentration of which is 0.5~2 g / L. -1 The volume ratio of the cerium nitrate solution to the tin selenide nanosheet stock solution is 1:(4~8). Stir to ensure that all components are in full contact. 5) Add dilute sodium hydroxide solution to adjust the pH of the mixed reaction system to 8.0, and let it stand at room temperature for 1-3 hours to complete the self-assembly modification of the metal-tannic acid framework on the surface of tin selenide nanosheets. 6) After the reaction is complete, wash with deionized water and remove unassembled free tannic acid, cerium ions and other impurities by centrifugation to obtain a tin selenide-based nanozyme.

3. The application of the tin selenide-based nanozyme according to claim 1 in the preparation of drugs for treating atherosclerosis.

4. The use of the tin selenide-based nanoenzyme of claim 1 in the preparation of drugs for scavenging reactive oxygen species, anti-inflammation, and scavenging lactic acid.

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