Copper / zinc-terephthalic acid nanosheet as well as preparation method and application thereof

Abstract

The invention discloses a copper / zinc-terephthalic acid nanosheet as well as a preparation method and application thereof. The preparation process comprises the following steps: dispersing zinc acetate in a polyvinylpyrrolidone aqueous solution; dispersing sodium hydroxide and terephthalic acid in water; mixing the two solutions, performing ultrasonic treatment, centrifuging, collecting supernate, performing ultrasonic treatment again to obtain a product, repeating the steps of ultrasonic treatment, centrifuging, collecting supernate and ultrasonic treatment for several times, and finally centrifuging and washing the product; and re-dispersing the product in distilled water, then adding a copper acetate solution, stirring, centrifuging the solution, and washing to obtain the product. The copper / zinc-terephthalic acid nanosheet can be co-coordinated with terephthalic acid through Cu < 2 + > and Zn < 2 + >, so that the copper / zinc-terephthalic acid nanosheet has the capability of catalyzing polymerization of dopamine, improving tyrosinase activity, superoxide dismutase-like activity, catalase-like activity and hydroxyl radical scavenging capability; therefore, the copper / zinc-terephthalic acid nanosheet is ensured to treat leucoderma through various mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine drug development, specifically relating to a copper / zinc-terephthalic acid nanosheet, its preparation method and application. Background Technology

[0002] Vitiligo is a global, chronic depigmenting skin disease clinically characterized by white patches that contrast sharply with the surrounding skin. Although vitiligo is often asymptomatic, its significant psychological and social impact can lead to a heavy burden and social isolation for patients in their daily lives. The pathogenesis of vitiligo is complex and is often associated with oxidative stress and abnormal melanin synthesis. Currently, treatments targeting a single mechanism are often ineffective in clinical practice. This application develops a multifunctional copper / zinc bimetallic organic framework nanosheet (Cu / Zn-TA) with the ability to catalyze dopamine polymerization, enhance tyrosinase (TYR) activity, and scavenge reactive oxygen species (ROS), which can be used to treat vitiligo. Due to Lewis acid sites induced by Zn²⁺ nodes and Jahn-Teller distortions induced by Cu²⁺ nodes, the copper / zinc-terephthalic acid nanosheet can coordinate with the phenolic hydroxyl groups of dopamine via Zn-O bonds, thereby catalyzing the oxidation and polymerization of dopamine, leading to an increase in exogenous melanin content. Simultaneously, Zn²⁺ nodes can enrich dopamine around the nanosheets through Lewis acid coordination, while Cu²⁺ nodes can promote electron transfer from the hydroxyl groups of dopamine to the binuclear copper center of tyrosinase via ligand-to-metal charge transfer (LMCT). Due to this synergistic effect, TYR activity can be enhanced, thereby upregulating endogenous melanin content. Furthermore, due to Cu… 2+ / Cu + Redox cycles and the interaction between terephthalic acid molecules and Zn 2+ Through coordination between the copper / zinc-terephthalic acid nanosheets, superoxide dismutase (SOD)-like activity, catalase (CAT)-like activity, and the ability to scavenge hydroxyl radicals (•OH) are observed. These properties help reduce oxidative stress at the lesion site, alleviate inflammation, and thus protect melanocytes. In summary, this application provides a two-dimensional bimetallic organic framework nanosheet that synergistically treats vitiligo through multiple mechanisms. Summary of the Invention

[0003] The purpose of this invention is to provide a copper / zinc-terephthalic acid nanosheet, its preparation method, and its applications. The copper / zinc-terephthalic acid nanosheets prepared by this invention can be processed using Cu... 2+ Replace part of Zn 2+ The two work synergistically to inhibit oxidative stress, reduce inflammation and protect melanocytes. At the same time, they catalyze dopamine polymerization to exogenously increase melanin content and increase tyrosinase activity to endogenously increase melanin content. This combination of mechanisms treats vitiligo.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing copper / zinc-terephthalic acid nanosheets includes the following steps: (1) Disperse zinc acetate in an aqueous solution of polyvinylpyrrolidone with a concentration of 1~10 mg / mL; (2) Disperse terephthalic acid and sodium hydroxide in distilled water, and then add the solution dropwise to the mixed solution in step (1) under ultrasonic conditions. Ultrasonicate for 0.5~1.5 h, then centrifuge the solution, collect the supernatant, and sonicate again for 0.5~1.5 h. Repeat the steps of ultrasonication-centrifugation-collecting supernatant-ultrasonication 2~4 times. Finally, centrifuge and wash the obtained product to obtain zinc-terephthalic acid nanosheets; wherein the molar ratio of zinc acetate, terephthalic acid and sodium hydroxide is (1.8~2.5):1:(2~3); (3) Redisperse 20~60 mg of zinc-terephthalic acid nanosheets from step (2) in distilled water, then add copper acetate solution with a concentration of 0.01~0.1 mmol / mL, stir for 0.5~2 h, and finally centrifuge and wash the solution to obtain copper / zinc-terephthalic acid nanosheets. The ratio of zinc-terephthalic acid nanosheets to copper acetate is (20~60) mg: (0.1~1) mmol.

[0005] Further, in step (1), the concentration of zinc acetate in the polyvinylpyrrolidone aqueous solution is 0.01~0.05 mmol / mL. Further, in step (2), the ultrasonic frequency is 20-25 KHz, the centrifugation speed is 500~1500 rpm, and the centrifugation time is 5~15 min. In step (2), the concentration of terephthalic acid in distilled water is 0.05~0.2 mmol / mL.

[0006] Furthermore, in step (3), the concentration of zinc-terephthalic acid nanosheets in distilled water is 1.5~3 mg / mL.

[0007] The copper / zinc-terephthalic acid nanosheets were prepared by the above method.

[0008] The above-mentioned application of copper / zinc-terephthalic acid nanosheets in the preparation of drugs for treating vitiligo, such as Figure 1 As shown, the copper / zinc-terephthalic acid nanosheets protect melanocytes by inhibiting oxidative stress and reducing inflammatory response, exogenously increase melanin content by catalyzing dopamine polymerization, and endogenously increase melanin content by increasing tyrosinase activity, thus synergistically treating vitiligo through multiple mechanisms.

[0009] Furthermore, the copper / zinc-terephthalic acid nanosheets are processed using Cu... 2+ Replace part of Zn 2+The two work synergistically to inhibit oxidative stress, reduce inflammation and protect melanocytes. At the same time, they catalyze dopamine polymerization to exogenously increase melanin content and increase tyrosinase activity to endogenously increase melanin content. This combination of mechanisms treats vitiligo.

[0010] This invention relates to copper / zinc-terephthalic acid nanosheets, which are two-dimensional sheet polymer materials with a metal-organic framework structure. 2+ Lewis acid activity and Cu 2+ The Jahn-Teller effect induced by this process can co-catalyze the polymerization of dopamine into polydopamine; Zn 2+ The Lewis acid activity enables dopamine to accumulate around copper / zinc-terephthalic acid nanosheets, combining with Cu 2+ Electron transfer between Cu and tyrosinase can enhance tyrosinase activity and promote the oxidation of dopamine to dopaquinone; + / Cu 2+ Circular transformation and Zn 2+ The coordination with terephthalic acid gives it superoxide dismutase-like activity, peroxidase-like activity, and hydroxyl radical scavenging ability.

[0011] The above-mentioned copper / zinc-terephthalic acid nanosheets are used in the preparation of anti-inflammatory or antioxidant drugs.

[0012] Compared with existing technologies, the copper / zinc-terephthalic acid nanosheets provided by this invention, as a therapeutic agent for vitiligo, can synergistically treat vitiligo by regulating redox balance, reducing inflammation, increasing tyrosinase activity, and promoting dopamine polymerization. The copper / zinc-terephthalic acid nanosheets are of suitable size, ensuring they can penetrate the basal layer of the epidermis and enter melanocytes, but cannot penetrate the entire dermis to avoid side effects. They exhibit good biocompatibility and application prospects. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the working principle of copper / zinc-terephthalic acid nanosheets prepared in Example 1 of the present invention and their application in the treatment of vitiligo. Figure 2 This is a scanning electron microscope image of the copper / zinc-terephthalic acid nanosheets prepared in Example 1 of the present invention; Figure 3 The X-ray diffraction pattern of the copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention; Figure 4 XPS image of copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention; Figure 5 XPS image of Zn 2p in copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention; Figure 6 XPS image of Cu 2p in the copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention; Figure 7 The content of Cu and Zn elements in the copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention; Figure 8 The UV-Vis absorption spectrum of oxWST-8 catalyzed by copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention; Figure 9 The graph shows the change in O2 content in the aqueous solution after the copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention catalyze the decomposition of H2O2. Figure 10 The UV-Vis absorption spectrum of methylene blue after catalytic removal of •OH by copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention; Figure 11 The UV-Vis absorption spectrum of the solution after dopamine polymerization catalyzed by copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention; Figure 12 The graph shows the change in UV-Vis absorption peak intensity at 475 nm over time of the solution prepared by copper / zinc-terephthalic acid nanosheets assisted by tyrosinase to catalyze the oxidation of dopamine in Example 1 of this invention. Figure 13 The cytotoxicity of copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention on mouse melanoma cells B16 and human immortalized melanoma cells MC-1; Figure 14 Fluorescence imaging of copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention after co-culturing with H2O2-treated cells; Figure 15 The level of inflammatory factors in cells after co-culturing copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention with H2O2-treated cells; Figure 16 The intracellular tyrosinase activity of copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention after co-culturing with cells; Figure 17 The content of intracellular melanin in cells after co-culturing copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention with cells; Figure 18 This is a diagram showing skin changes in a mouse model of vitiligo induced by H2O2-hydroquinone, obtained by treating a mouse with copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention. Figure 19The images show H&E staining (top) and melanin staining (bottom) of skin tissue from a mouse model of H2O2-hydroquinone-induced vitiligo treated with copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention. Figure 20 The levels of inflammatory factors in the skin tissue of mice with H2O2-hydroquinone-induced vitiligo model treated with copper / zinc-terephthalic acid nanosheets prepared in Example 1 of this invention.

[0014] Figures 15 to 17 as well as Figure 20 In the diagram, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001. Detailed Implementation

[0015] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but this is not intended to limit the scope of protection of the present invention.

[0016] Example 1 A method for preparing copper / zinc-terephthalic acid nanosheets (Cu / Zn-TA) is described below: Prepare a 40 mL solution of zinc acetate (Zn(OAc)2) with a concentration of 0.025 mmol / mL: At room temperature, disperse 0.3 g of polyvinylpyrrolidone (PVP) in 40 mL of distilled water and stir vigorously, then add 1 mmol of zinc acetate.

[0017] Prepare a 5 mL terephthalic acid solution: Disperse 50 mg of sodium hydroxide in 5 mL of distilled water, then add 0.5 mmol of terephthalic acid and stir until completely dissolved. Prepare zinc-terephthalic acid nanosheets: Under ultrasonic conditions (20 kHz), add the prepared 5 mL terephthalic acid solution dropwise to a prepared 40 mL zinc acetate solution and continue sonication for 1 h. Then centrifuge the product (1000 rpm, 10 min), collect the supernatant and sonicate it for 1 h (20 kHz), then centrifuge again (1000 rpm, 10 min), collect the supernatant and continue sonication. Repeat the sonication-centrifugation-collecting supernatant-sonication step 3 times. Finally, centrifuge the product (8000 r / min, 10 min, 3 times), wash 3 times with distilled water, and dry at 60 ℃ to obtain approximately 40 mg of sample.

[0018] Copper / zinc-terephthalic acid nanosheets were prepared by redispersing 40 mg of the prepared zinc-terephthalic acid nanosheets in 20 mL of distilled water. Then, 10 mL of 0.05 mol / L copper acetate solution was added dropwise under stirring, and the mixture was stirred continuously for 1 h. Finally, the product was centrifuged (8000 r / min, 10 min, 3 times), washed 3 times with distilled water, and dried at 60 ℃.

[0019] Scanning electron microscope images of the above products are as follows: Figure 2 As shown, by Figure 2 As can be seen, the product consists of irregular two-dimensional nanosheets with an average diameter of 266±58 nm. The X-ray diffraction pattern of the product is shown below. Figure 3 The results showed that the characteristic diffraction peaks of the product copper / zinc-terephthalic acid nanosheets were consistent with those of the product zinc-terephthalic acid nanosheets, and both matched the diffraction peaks of the previously reported metal-organic framework nanomaterial MOF-5 (CCDC 938392). However, due to the preferred orientation of the nanosheets, the order of diffraction peak intensities changed significantly compared with MOF-5.

[0020] Example 2 Detection of surface chemical properties of copper / zinc-terephthalic acid nanosheets The sample prepared according to Example 1 was subjected to X-ray photoelectron spectroscopy (XPS) analysis. Figure 4 It can be seen that both zinc-terephthalic acid nanosheets and copper / zinc-terephthalic acid nanosheets exhibit signals of C, O, and Zn elements. Compared with zinc-terephthalic acid nanosheets, the high-resolution X-ray photoelectron spectroscopy of Zn 2p in copper / zinc-terephthalic acid nanosheets shows a significant shift of its characteristic peak towards higher energies (0.4 eV). Figure 5 This shift is due to the electronegativity of Cu. 2+ Attracting electrons reduces Zn 2+ This is caused by the electron density. Furthermore, the high-resolution X-ray photoelectron spectroscopy of Cu 2p in copper / zinc-terephthalic acid nanosheets shows peaks at approximately 935 eV and 955 eV, which are attributed to Cu-O bonds rather than Cu-Cu bonds (932.5 eV and 952.4 eV), indicating that Cu... 2+ Coordination with terephthalic acid rather than simple adsorption on zinc-terephthalic acid nanosheets ( Figure 6 ).

[0021] Example 3 Detection of Cu and Zn content in copper / zinc-terephthalic acid nanosheets The sample prepared according to Example 1 was dispersed in a nitric acid solution (15 mol / L) to make the sample as clear and transparent as possible. The sample concentration was 0.5 mg / mL. The Cu and Zn content in the nanosheets was detected by inductively coupled plasma optical emission spectrometry (ICP-OES). The results are detailed in [link to relevant documentation]. Figure 7 .

[0022] Depend on Figure 7 It can be seen that the contents of Zn²⁺ and Cu²⁺ in the copper / zinc-terephthalic acid nanosheets are 20.7% (by weight) and 10.8% (by weight), respectively.

[0023] Example 4 Assay for superoxide dismutase-like activity of copper / zinc-terephthalic acid nanosheets 40 μL of the sample aqueous solution prepared as described in Example 1 was mixed with 320 μL of WST-8 / xanthine oxidase working solution; then, 40 μL of the working solution for initiating the reaction was added to the above system, resulting in final sample concentrations of 30, 40, 50, and 100 μg / mL. After incubation at 37 °C for 30 min, the absorbance of the solution at 450 nm was measured using a UV-Vis spectrophotometer. For detailed results, please refer to [link to relevant documentation]. Figure 8 .

[0024] Depend on Figure 8 It can be seen that the superoxide dismutase-like activity of the nanosheet increases with the increase of nanosheet concentration.

[0025] Example 5 Detection of catalase-like activity in copper / zinc-terephthalic acid nanosheets The sample aqueous solution (5 mL, 1 mg / mL) prepared as described in Example 1 was dispersed in 5 mL of 30 wt% hydrogen peroxide solution. The change in oxygen content in the solution was monitored in real time using a dissolved oxygen analyzer. For detailed results, please refer to [link to relevant documentation]. Figure 9 .

[0026] Depend on Figure 9 It was found that after co-incubation with copper / zinc-terephthalic acid nanosheets for 30 min, the O2 content in the hydrogen peroxide solution increased from 7.76 mg / mL to 35.4 mg / mL, indicating that the copper / zinc-terephthalic acid nanosheets have catalase activity.

[0027] Example 6 Determination of the hydroxyl radical scavenging activity of copper / zinc-terephthalic acid nanosheets The sample aqueous solution prepared according to Example 1 was added to a solution containing 40 μL MB (1 mg / mL), 80 μL FeSO4 (10 mM), and 180 μL H2O2 (10 mM). The final volume of the system was fixed to 4 mL with distilled water, and the final concentrations of the sample in the system were 25, 50, 100, 200, and 400 μg / mL, respectively. The mixed solution was allowed to stand for 3 min, and the remaining •OH was quantified by UV-Vis absorption spectroscopy. For detailed results, see [link to relevant documentation]. Figure 10 .

[0028] Depend on Figure 10 It can be seen that the hydroxyl radical scavenging activity of the nanosheet increases with the increase of nanosheet concentration.

[0029] Example 7 Determination of the ability of copper / zinc-terephthalic acid nanosheets to catalyze dopamine polymerization The sample aqueous solution prepared as described in Example 1 was added to 4 mL of dopamine hydrochloride aqueous solution (20 μg / mL), resulting in a final concentration of 100 μg / mL. After co-incubation for 2, 4, 6, and 8 h, the absorbance of the solution at 660 nm was measured using UV-Vis absorption spectroscopy. For detailed results, please refer to [link to relevant documentation]. Figure 11 .

[0030] Depend on Figure 11 It can be seen that the absorption peak of the solution at 660 nm showed a red shift and increased intensity with the extension of incubation time, indicating that copper / zinc-terephthalic acid nanosheets have the ability to catalyze the polymerization of dopamine.

[0031] Example 8 Detection of the ability of copper / zinc-terephthalic acid nanosheets to enhance tyrosinase activity The sample aqueous solution prepared as described in Example 1 was added to a solution containing 0.01 mL of dopamine hydrochloride (0.1 M) and 0.05 mL of tyrosinase (25 μg / mL). The final volume of the system was fixed with distilled water to 2 mL, and the final concentrations of the sample in the system were 10, 20, 30, 40, 60, 80, 100, and 200 μg / mL. The absorbance of the solution at 475 nm was then monitored in real time using UV-Vis absorption spectroscopy. For detailed results, please refer to [link to relevant documentation]. Figure 12 .

[0032] Depend on Figure 12 It can be seen that the initial oxidation rate of dopamine increases with increasing particle concentration.

[0033] Example 9 Cytotoxicity of copper / zinc-terephthalic acid nanosheets Mouse melanoma cells B16 and immortalized human melanocytes MC-1 were selected, and the cytotoxicity of copper / zinc-terephthalic acid nanosheets was studied using the MTT assay. B16 and MC-1 cells were grown in 96-well plates (Corning Glass Works, 1 × 10⁶ cells per well). 5 In each sample, when the cell density reached 50%, PBS solution prepared according to Example 1 was added for co-culturing (the final concentrations of the samples in fresh culture medium were 5, 10, 20, 50, and 100 μg / mL, respectively). After culturing the cells and particles for 24 hours, the culture medium was replaced with fresh culture medium (100 μL), and 10 μL of MTT solution was added for further 4 h of culturing. Then, dimethyl sulfoxide (DMSO) solution was added. Finally, the optical intensity of the derived CCK-8 solution (maximum absorption position at 490 nm) was measured using a microplate reader, indirectly reflecting the concentration of live cells. For detailed results, please refer to [link to relevant documentation]. Figure 13 .

[0034] Figure 13 The results showed that when the sample concentration prepared according to Example 1 was as high as 100 μg / mL, its cytotoxicity to B16 cells and MC-1 cells was negligible.

[0035] Example 10 Intracellular antioxidant properties of copper / zinc-terephthalic acid nanosheets The antioxidant capacity of the samples prepared according to Example 1 in cells was detected using fluorescence imaging technology. Intracellular ROS levels were detected using the fluorescent probe 2,7-dichlorodihydrofluorescein (DCFH-DA). MC-1 cells were seeded in 24-well plates (1 × 10⁶ cells per well). 5 Cells were cultured for 24 h, then the original culture medium was replaced with 1 mL of medium containing H2O2 (0.75 μmol / mL) and cultured for another 4 h. Next, the medium containing H2O2 was replaced with either the sample medium (prepared with PBS to a concentration of 200 μg / mL) or the control PBS buffer solution, and cultured for 6 h. The culture medium in each well was then aspirated, and 1 mL of DCFH-DA (10 μM) was added to each well. The cells were incubated at 37 °C for 20 min. Finally, the cells were washed three times with PBS and observed under an inverted fluorescence microscope. For detailed results, please refer to [link to relevant documentation]. Figure 14 .

[0036] Figure 14Compared to PBS-treated MC-1 cells or cells treated with the sample alone, H2O2 (0.75 mM)-treated cells showed a significant increase in green fluorescence signal due to the formation of oxidative stress. However, after sample treatment, this increased DCF fluorescence signal could be significantly reduced to a level similar to that of cells treated with PBS alone, indicating that the sample can maintain intracellular redox balance within a certain concentration of H2O2.

[0037] Example 11 Intracellular anti-inflammatory activity of copper / zinc-terephthalic acid nanosheets The anti-inflammatory activity of the sample prepared according to Example 1 in cells was detected by ELISA. B16 cells were seeded in 6-well plates (1 × 10⁶ cells per well). 5 Cells were cultured for 24 h, then the original culture medium was replaced with 2.5 mL of medium containing H2O2 (0.75 μmol / mL) and cultured for another 4 h. Next, the original culture medium was replaced with either the sample medium (prepared with PBS to a concentration of 200 μg / mL) or the control PBS buffer solution, and cultured for 6 h. Finally, the cell culture supernatant was collected, and the levels of inflammatory factors in the supernatant were detected using a commercially available mouse inflammatory factor kit for IL-10, TNF-α, and IL-1β. For detailed results, please refer to [link to results]. Figure 15 .

[0038] Figure 15 The results showed that in B16 cells treated with H2O2, the levels of pro-inflammatory factors TNF-α and IL-1β were significantly increased, while the level of anti-inflammatory factor IL-10 was significantly decreased. The addition of the sample could inhibit the increase of pro-inflammatory factors caused by H2O2, while promoting the increase of anti-inflammatory factor levels.

[0039] Example 12 The ability of copper / zinc-terephthalic acid nanosheets to enhance tyrosinase activity in cells The ability of the sample prepared according to Example 1 to increase tyrosinase activity in cells was detected using an enzyme-linked immunosorbent assay (ELISA) reader. B16 cells were seeded in 6-well plates (1 × 10⁶ cells per well). 5 Cells were cultured for 24 h, then replaced with 2.5 mL of medium containing the sample (dissolved in PBS and added to the medium, with a final sample concentration of 200 μg / mL), and cultured for another 6 h. Cells were then washed with PBS buffer and 50 μL of cell lysis buffer (Tratton X-100) was added. Cells were then placed in an ultra-low temperature freezer for 30 min and thawed. This process was repeated three times. Then, 50 μL of 0.25% levodopa solution was added to each well, and the cells were incubated at 37°C for 2 h on a shaker. Finally, the absorbance at 475 nm was recorded for each well using a microplate reader. See [link to microplate reader for details]. Figure 16 .

[0040] Figure 16 The results showed that the tyrosinase activity in cells treated with the sample was significantly higher than that in the control group cells.

[0041] Example 13 The ability of copper / zinc-terephthalic acid nanosheets to promote melanin production in cells The ability of the sample prepared according to Example 1 to promote melanin production in cells was detected using an ELISA reader. B16 cells were seeded in 6-well plates (1 × 10⁶ cells per well). 5 Each well was cultured for 24 h, then 2.5 mL of medium containing the sample (dissolved in PBS and added to the medium, with a final sample concentration of 200 μg / mL) was added to replace the original medium, and the culture was continued for another 6 h. Next, 100 μL of 0.2 M NaOH solution was added to each well, and the wells were incubated at 37 °C for 1 h in a shaker. Finally, the absorbance at 490 nm for each well was recorded using a microplate reader. For detailed results, please refer to [link to results]. Figure 17 .

[0042] Figure 17 The results showed that the melanin content in cells treated with the sample was significantly higher than that in the control group cells.

[0043] Example 14 The efficacy of copper / zinc-terephthalic acid nanosheets in treating vitiligo in a mouse model of vitiligo. (1) Add copper / zinc-terephthalic acid nanosheets or zinc-terephthalic acid nanosheets to ordinary skin care lotion to make the nanosheet concentration 2 mg / mL.

[0044] (2) Using female C57 mice as the model animal, a vitiligo animal model was established by combining hydrogen peroxide and hydroquinone. After anesthetizing the mice by intraperitoneal injection, the hair on the back was shaved off using an animal shaver, and 0.5 mL of 5% hydrogen peroxide was applied to the shaved skin area (4*4 cm) on the back of the mouse. 2 One hour later, rinse thoroughly with saline solution, then apply 0.5 mL of 5% hydroquinone solution to an area of ​​4 x 4 cm. 2The treatment was repeated twice daily for 37 consecutive days. Starting on day 31 of modeling, mice were randomly divided into three groups. Each group received one of the following treatments at the lesion site: 1) a standard skin lotion; 2) a standard skin lotion plus copper / zinc-terephthalic acid nanosheets (100 mg / kg); or 3) a standard skin lotion plus zinc-terephthalic acid nanosheets (100 mg / kg). The medication was administered two hours after each application of hydrogen peroxide and hydroquinone. The amount of lotion applied to each group was 1.25 mL, twice daily. A healthy control group received only the standard skin lotion. Skin color changes in the mice were photographed and recorded during the 7-day treatment period. On the last day of the experiment, all mice were euthanized, and the lesion skin was collected for analysis. The collected skin was fixed in 4% formalin solution for 48 h, then treated with ethanol and xylene, and finally embedded in paraffin. The samples were then sectioned, mounted on glass slides, and stained with hematoxylin and eosin (H&E) and Masson-Fontana. The results are detailed below. Figure 18-19 .

[0045] Figure 18 The results showed that mice treated with pure skin lotion developed obvious white patches on their backs, while mice treated with zinc-terephthalic acid nanosheets showed only a few white patches on their backs after 7 days of treatment, indicating a certain therapeutic effect on vitiligo. Mice treated with copper / zinc-terephthalic acid nanosheets showed almost no white patches throughout the entire treatment period, indicating a good therapeutic effect on vitiligo. Figure 19 H&E staining results showed that the dorsal skin of vitiligo mice treated with pure skin care lotion exhibited incomplete keratinization, epidermal cell nuclei disappearance and necrosis, extensive inflammatory infiltration in the dermis, and a reduction in the number of skin appendages. Mice treated with zinc-terephthalic acid nanosheets showed reduced inflammatory infiltration, and restoration of accessory structures such as hair follicles and sebaceous glands. Furthermore, the skin treated with copper / zinc-terephthalic acid nanosheets showed minimal changes compared to normal mice, further demonstrating its good therapeutic effect on vitiligo. Figure 19 Melanin staining results showed that, compared with normal skin, the melanocyte area ratio in the skin of vitiligo mice treated with pure skin care lotion decreased significantly from 2.75% to 0.29%. The melanin content in the skin of mice treated with zinc-terephthalic acid nanosheets recovered to 0.86%, while treatment with copper / zinc-terephthalic acid nanosheets restored the melanin content in the skin of mice to 1.13%.

[0046] Example 15 Skin samples from different mice in Example 14 were weighed and homogenized in physiological saline at 4 °C. The homogenate was then centrifuged at 2000 rpm for 20 min at 4 °C. The supernatant was collected, and the levels of anti-inflammatory factors (IL-10) and pro-inflammatory factors (TNF-α, IL-1β) were detected using a commercially available mouse IL-10, TNF-α, and IL-1β ELISA kit (Solarbio, China). The results are detailed below. Figure 20 .

[0047] Depend on Figure 20 It was found that, compared with vitiligo mice treated with pure skin care lotion, zinc-terephthalic acid nanosheets and copper / zinc-terephthalic acid nanosheets could reduce the levels of pro-inflammatory factors TNF-α and IL-1β, while increasing the level of anti-inflammatory factor IL-10, and copper / zinc-terephthalic acid nanosheets had a more significant effect on improving the level of inflammatory factors.

[0048] The above embodiments are only used to illustrate the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention within the scope of knowledge possessed by those skilled in the art should be considered within the scope of protection of this application.

[0049] The working principle of the copper / zinc-terephthalic acid nanosheets prepared in this invention for treating vitiligo is as follows: Figure 1 As shown, by Figure 1 It can be seen that the copper / zinc-terephthalic acid nanosheets prepared by this invention can pass through Cu 2+ and Zn 2+ Co-coordinated with terephthalic acid, it acquires the ability to catalyze dopamine polymerization, enhance tyrosinase activity, superoxide dismutase-like activity, catalase-like activity, and hydroxyl radical scavenging ability, thereby ensuring that copper / zinc-terephthalic acid nanosheets can treat vitiligo simultaneously through multiple mechanisms.

[0050] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing copper / zinc-terephthalic acid nanosheets, characterized in that, Includes the following steps: (1) Disperse zinc acetate in an aqueous solution of polyvinylpyrrolidone with a concentration of 1~10 mg / mL; (2) Disperse terephthalic acid and sodium hydroxide in distilled water, and then add the solution dropwise to the mixed solution in step (1) under ultrasonic conditions. Ultrasonicate for 0.5~1.5 h, then centrifuge the solution, collect the supernatant, and sonicate again for 0.5~1.5 h. Repeat the steps of ultrasonication-centrifugation-collecting supernatant-ultrasonication 2~4 times. Finally, centrifuge and wash the obtained product to obtain zinc-terephthalic acid nanosheets; wherein the molar ratio of zinc acetate, terephthalic acid and sodium hydroxide is (1.8~2.5):1:(2~3); (3) Redisperse 20~60 mg of zinc-terephthalic acid nanosheets from step (2) in distilled water, then add copper acetate solution with a concentration of 0.01~0.1 mmol / mL, stir for 0.5~2 h, and finally centrifuge and wash the solution to obtain copper / zinc-terephthalic acid nanosheets. The ratio of zinc-terephthalic acid nanosheets to copper acetate is (20~60) mg: (0.1~1) mmol.

2. The method for preparing copper / zinc-terephthalic acid nanosheets according to claim 1, characterized in that, In step (1), the concentration of zinc acetate in the polyvinylpyrrolidone aqueous solution is 0.01~0.05 mmol / mL.

3. The method for preparing copper / zinc-terephthalic acid nanosheets according to claim 1, characterized in that, In step (2), the concentration of terephthalic acid in distilled water is 0.05~0.2 mmol / mL.

4. The method for preparing copper / zinc-terephthalic acid nanosheets according to claim 1, characterized in that, In step (3), the concentration of zinc-terephthalic acid nanosheets in distilled water is 1.5~3 mg / mL.

5. Copper / zinc-terephthalic acid nanosheets prepared by any one of the preparation methods according to claims 1 to 4.

6. The use of the copper / zinc-terephthalic acid nanosheets according to claim 5 in the preparation of a drug for treating vitiligo, characterized in that, The copper / zinc-terephthalic acid nanosheets protect melanocytes by inhibiting oxidative stress and reducing inflammation, catalyze dopamine polymerization to exogenously increase melanin content, and increase tyrosinase activity to endogenously increase melanin content, thus synergistically treating vitiligo through multiple mechanisms.

7. The application according to claim 6, characterized in that, The copper / zinc-terephthalic acid nanosheets are made with Zn 2+ Lewis acid activity and Cu 2+ The Jahn-Teller effect induced by this process can catalyze the polymerization of dopamine into polydopamine, increasing the content of exogenous melanin.

8. The application according to claim 6, characterized in that, Through Zn 2+ The Lewis acid activity of Cu enriches dopamine around copper / zinc-terephthalic acid nanosheets and Cu 2+ Electron transfer between the enzyme and tyrosinase increases tyrosinase activity, promotes the oxidation of dopamine to dopaquinone, and increases endogenous melanin content.

9. The application according to claim 6, characterized in that, Through Cu + / Cu 2+ Circular transformation and Zn 2+ The coordination with terephthalic acid gives it superoxide dismutase-like activity, peroxidase-like activity, and hydroxyl radical scavenging ability, thus maintaining redox balance.

10. The use of the copper / zinc-terephthalic acid nanosheets of claim 5 in the preparation of anti-inflammatory or antioxidant drugs.

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