A two-dimensional metal polyphenol complex nanomaterial which can be enriched in ovaries, a preparation method and application thereof

Abstract

The present application relates to a kind of two-dimensional metal polyphenol complex nanomaterials that can be enriched in ovary, preparation method and application thereof, the nanomaterial can be enriched in ovary, including two-dimensional metal polyphenol complex as substrate, and follicle-stimulating hormone polypeptide grafted to the surface of the substrate;Wherein, the surface of the two-dimensional metal polyphenol complex grafts-COOH functional group and forms 2D MPC-COOH intermediate, the follicle-stimulating hormone polypeptide is covalently grafted with the-COOH on the surface of 2D MPC-COOH intermediate and obtains the two-dimensional metal polyphenol complex nanomaterial.The technical effect of the present application is that the preparation method of nanomaterial is simple, and nanoparticle dispersibility is good, and biosafety is high, and nanomaterial can physically absorb BRD7 target protein through its special structure, block BRD7 into cell nucleus, and then realize the safe down-regulation of KAT7 path and the effective reversal of granulosa cell stress aging.

Description

Technical Field

[0001] This invention relates to the field of medical nanobiomaterials technology, specifically to a two-dimensional metal polyphenol complex nanomaterial that can be enriched in the ovary, its preparation method, and its application. Background Technology

[0002] Premature ovarian failure (POF) refers to the decline or loss of ovarian function in women before the age of 40. It has become one of the important causes of female infertility, with an incidence rate of approximately 1-5%. Potential causes of POF mainly include infection, metabolic disorders, autoimmune diseases, and iatrogenic factors (such as ovarian surgery, radiotherapy, and chemotherapy). POF patients not only face infertility symptoms but also a series of menopausal syndromes and other complications, including psychological disorders, osteoporosis, ischemic heart disease, and even the risk of death. However, there are currently no effective strategies to protect and restore ovarian growth and hormone function. Therefore, it is urgent to clarify the relevant molecular mechanisms of POF pathology and develop new therapies to protect ovarian tissue in order to delay or even reverse the decline of ovarian function.

[0003] In the pathological evolution of premature ovarian failure, cellular senescence is considered a core driving mechanism. Studies have shown that the imbalance of ovarian microenvironment homeostasis is closely related to premature senescence of ovarian somatic cells (especially granulosa cells and stromal cells). Cellular senescence is not only a manifestation of tissue functional decline, but also a programmed process regulated by a sophisticated epigenetic network. Ovarian granulosa cells and stromal cells in premature ovarian failure undergo persistent cell cycle arrest, manifested by a significant upregulation of senescence-related markers (such as p16INKα and p21). Simultaneously, telomere depletion within the cells accelerates, and mitochondrial dysfunction leads to excessive accumulation of reactive oxygen species, thereby triggering an irreversible DNA damage response. More importantly, senescent cells are not metabolically silent, but rather release large amounts of pro-inflammatory cytokines (such as IL-6), chemokines, and matrix metalloproteinases into the extracellular matrix through the senescence-associated secretory phenotype (SASP), transmitting senescence signals to neighboring healthy follicles, inducing more cells to enter a state of stress-induced senescence, forming a persistent chronic inflammatory microenvironment that leads to accelerated depletion of the primordial follicle pool and irreversible decline in ovarian function.

[0004] Studies have shown that this "catalysis-recognition" molecular docking not only synergistically enhances the transcriptional output of the p53 signaling pathway by recruiting the chromatin remodeling complex, but also prompts cells to shift from a transient response to DNA damage to a permanent physiological aging process. Therefore, the KAT7 and BRD7 protein-protein interaction network is a core axis of "epigenetic-transcriptional stability" in the regulation of ovarian reproductive lifespan. Furthermore, the aging program mediated by KAT7 and BRD7 not only weakens the metabolic support and autocrine homeostasis of granulosa cells for oocytes, but also amplifies single-cell aging signals into an organ-scale chronic inflammatory microenvironment by promoting the release of senescence-associated secretory phenotypes (SASPs). This accelerates the depletion of primordial follicles and stromal fibrosis, ultimately leading to premature depletion of ovarian reproductive reserves.

[0005] The core objectives of current clinical management of postmenopausal ovarian failure (POF) are to alleviate symptoms of low estrogen, prevent long-term complications, and attempt fertility interventions. Because POF patients require more estrogen due to their vascular and bone health, higher doses of estrogen are typically recommended up to the average age of natural menopause. The main limitation of hormone replacement therapy is that it only prevents bone loss and cardiovascular degeneration, but is almost ineffective in restoring follicular reserve. For patients desiring fertility, the response rate to conventional ovulation-inducing drugs is extremely low due to near-depletion of ovarian reserve. Currently, the main method of fertility management still relies on donor egg insemination. Although existing protocols have played a role in improving symptoms, their lack of focus on functional recovery and addressing the root cause makes the development of innovative drug theories and formulations of urgent clinical significance. Given the current limited fertility options faced by POF patients, there is an urgent need to develop next-generation targeted drugs aimed at repairing damaged granulosa cells and even inducing quiescent follicles to return to a developmental trajectory, providing patients with the possibility of preserving their genetic offspring. Summary of the Invention

[0006] To overcome at least one problem in existing clinical ovarian protection technologies and address the shortcomings of current clinical drugs for premature ovarian failure, such as lack of ovarian specificity and low response rates, this invention provides a two-dimensional metal polyphenol complex nanomaterial that can accumulate in the ovary, its preparation method, and its applications. The preparation method is simple, the nanoparticles are well-dispersed, and the biocompatibility is high. Due to its ultrathin two-dimensional structure, high specific surface area, and abundant coordination unsaturated sites and polyhydroxy functional groups on the nanosheet surface, it provides ideal electrostatic adsorption and hydrogen bonding sites for the BRD7 protein. Through physical adsorption, it blocks BRD7 from entering the cell nucleus, thereby achieving safe downregulation of the KAT7 pathway and effective reversal of stress-induced senescence in granulosa cells. It epigenetically disrupts the transmission of the BRD7-KAT7 axial aging program, downregulates the secretion of the pro-aging phenotype SASP, and regulates the inflammatory microenvironment of lesions, providing a novel strategy for the precise treatment of premature ovarian failure.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention is a two-dimensional metal-polyphenol complex (2D MPC) nanomaterial that can be enriched in the ovary, denoted as 2D MPC-FSH, wherein the nanomaterial 2D MPC-FSH comprises a two-dimensional metal-polyphenol complex (MPC-COOH) as a substrate, and a follicle-stimulating hormone (FSH) polypeptide grafted onto the surface of the two-dimensional metal-polyphenol complex; In this process, the surface of the two-dimensional metal polyphenol complex is grafted with -COOH functional groups to form a 2D MPC-COOH intermediate, and the follicle-stimulating hormone polypeptide is covalently grafted with -COOH on the surface of the 2D MPC-COOH intermediate to obtain the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH that can be enriched in the ovary.

[0008] The two-dimensional metal polyphenol complex is surface-modified with Silane-PEG-COOH to graft easily modifiable -COOH functional groups onto its surface. The follicle-stimulating hormone peptide is grafted onto the surface of the two-dimensional metal polyphenol complex containing -COOH functional groups via an amide condensation reaction to target the ovary and achieve enrichment of the two-dimensional metal polyphenol complex in the ovarian region.

[0009] Furthermore, the surface of the two-dimensional metal polyphenol complex is modified with Silane-PEG-COOH by grafting -COOH, and the mass ratio of Silane-PEG-COOH to the two-dimensional metal polyphenol complex is 1:(50~200); preferably 1:50; the molecular weight of Silane-PEG-COOH is 2000~5000 Da; preferably 2000 Da.

[0010] Furthermore, the follicle-stimulating hormone polypeptide is covalently grafted with the -COOH group on the surface of the 2D MPC-COOH intermediate via an amide condensation reaction, and the mass ratio of the follicle-stimulating hormone polypeptide to the 2D MPC-COOH intermediate is 1:(100~400); preferably 1:200.

[0011] Furthermore, the average particle size of the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH is 200~400 nm.

[0012] Furthermore, the thickness of the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH is 1~5 nm.

[0013] Furthermore, the two-dimensional metal polyphenol complex is formed by coordination self-assembly of flavonoid polyphenol ligands and metal ions; The metal ions are selected from Al. 3+ Mg 2+ Cu 2+ Fe 3+ Zn 2+ 、Tb 3+ Eu 3+ Co 2+ Any one of them; The flavonoid polyphenol ligands are selected from any one of quercetin, luteolin, luteolin, 3-hydroxyflavone, and 5-hydroxyflavone.

[0014] In this invention, two-dimensional zinc lacquinone nanosheets (2D Zn-fis) in two-dimensional metal polyphenol complex nanomaterials are used as an example for illustration.

[0015] A second aspect of the present invention relates to a method for preparing two-dimensional metal polyphenol complex nanomaterials enrichable in the ovaries as described in the first aspect of the present invention, comprising the following steps: 1) The metal salt corresponding to the metal ion is fully dissolved in deionized water to form a metal salt solution; the flavonoid polyphenol is fully dissolved in an organic solvent to form a flavonoid polyphenol organic solution; the metal salt solution and the flavonoid polyphenol organic solution are added to a buffer system and stirred to carry out a molecular coordination reaction. After the reaction, the mixture is centrifuged and washed to obtain the two-dimensional metal polyphenol complex (2D MPC) powder. 2) Dissolve Silane-PEG-COOH in an ethanol solution, then add the two-dimensional metal polyphenol complex obtained in step 1) to carry out a silane coupling reaction, and prepare the 2D MPC-COOH intermediate by washing and centrifugation; 3) The 2D MPC-COOH intermediate obtained in step 2) is dispersed in a buffer solution, an activator is added and stirred to activate it, a follicle-stimulating hormone polypeptide solution is added and stirred at low temperature to carry out an amide condensation reaction, and after centrifugation and washing, the two-dimensional metal polyphenol complex nanomaterial is obtained, denoted as 2D MPC-FSH.

[0016] Further, in step 1), the metal salt is selected from any one of nitrates, chlorides, and sulfates; preferably, it is a nitrate.

[0017] Further, in step 1), the concentration of the metal salt solution is 20~100 mM; preferably 50 mM.

[0018] Further, in step 1), the concentration of the flavonoid polyphenol organic solution is 5~30 mM; preferably 15.8 mM.

[0019] Further, in step 1), the solvent of the flavonoid polyphenol organic solution is selected from any one of methanol, ethanol, isopropanol, n-butanol, acetic acid, and formic acid; preferably methanol.

[0020] Further, in step 1), the buffer system is either a phosphate buffer or a bicarbonate buffer; preferably a phosphate buffer.

[0021] Further, in step 1), the pH value of the buffer system is 7-8 and the concentration is 10-20 mM; preferably, it is a phosphate buffer with pH=7.4 and a concentration of 10 mM.

[0022] Further, in step 1), the volume ratio of the metal salt aqueous solution, the flavonoid polyphenol organic solution and the buffer system is (5~10):3:(50~200); preferably 8:3:100.

[0023] Further, in step 1), the conditions for the molecular coordination reaction are stirring at 800~1200 rpm for 10~60 min; preferably, stirring at 1100 rpm for 30 min.

[0024] Further, in step 1), the two-dimensional metal polyphenol complex is obtained by centrifugation and washing after the reaction, and the centrifugation and washing speed is 6000~10000 rpm and the time is 5~10 min; preferably, after centrifugation at 8000 rpm for 5 min, it is washed 3 times with phosphate buffer, the supernatant is discarded and then freeze-dried to obtain the powdered two-dimensional metal polyphenol complex.

[0025] Further, in step 2), the volume ratio of ethanol to water in the ethanol solution is (7~9):1; preferably 9:1. The dissolution of Silane-PEG-COOH in the ethanol solution is achieved by stirring for 0.5~2 hours, preferably 2 hours.

[0026] Further, in step 2), the concentration of Silane-PEG-COOH is 0.01~1 mg / mL; preferably 0.02 mg / mL.

[0027] Further, in step 2), the conditions for the silane coupling reaction are: stirring at room temperature and 400-500 rpm for 0.5-2 h, preferably at room temperature and 400 rpm for 2 h.

[0028] Further, in step 3), the activator is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(0.5~2); preferably 1:1.

[0029] Further, in step 3), the conditions for stirring activation are stirring and activating at 25~30 ℃ and 400~600 rpm for 10~30 min; preferably, stirring and activating at 25 ℃ and 500 rpm for 15 min.

[0030] Further, in step 3), the conditions for the amide condensation reaction are: stirring at a temperature of 2-10 °C and a rotation speed of 400-600 rpm for 12-15 hours; preferably, stirring at a temperature of 4 °C and a rotation speed of 400 rpm for 12 hours.

[0031] Furthermore, the centrifugation and washing process includes: centrifuging at 6000~10000 rpm for 5~10 min followed by washing with phosphate buffer 3~4 times; preferably, centrifuging at 8000 rpm for 5 min.

[0032] Furthermore, taking two-dimensional zinc laccase nanosheets (2D Zn-fis) in two-dimensional metal polyphenol complex nanomaterials as an example, the preparation of the two-dimensional zinc laccase complex nanomaterials that can be enriched in the ovary includes the following steps: dissolving zinc nitrate hexahydrate in deionized water to form solution A; dissolving laccase in methanol to form solution B; adding solutions A and B to phosphate buffer, stirring and reacting, centrifuging, washing, and cooling to obtain the product two-dimensional zinc laccase complex (Zn-fis); dissolving Silane-PEG-COOH in an ethanol / water mixture, stirring thoroughly, then adding the two-dimensional zinc laccase complex powder, reacting thoroughly, washing, centrifuging and collecting to obtain the sample (Zn-fis-COOH). The above product precipitate was added to phosphate buffer, and N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added respectively. The mixture was stirred and activated. The follicle-stimulating hormone peptide was dissolved in a small amount of phosphate buffer and added to the above system. The mixture was stirred at low temperature, washed, centrifuged, and freeze-dried to obtain two-dimensional zinc lactin complex nanomaterials that can be enriched in the ovary.

[0033] A third aspect of the present invention relates to a product containing a two-dimensional metallopolyphenol complex nanomaterial that can accumulate in the ovary, comprising the two-dimensional metallopolyphenol complex nanomaterial described in the first aspect of the present invention or the two-dimensional metallopolyphenol complex nanomaterial prepared by the preparation method described in the second aspect of the present invention, and a pharmaceutically acceptable carrier or excipient. The carrier or excipient is a substance conventionally used in the art to aid in formulation stability or to contribute to improved activity or bioavailability or to produce an acceptable taste or odor when taken orally.

[0034] Furthermore, the product is an injection solution prepared by dispersing the two-dimensional metal polyphenol complex nanomaterial in a biocompatible solution.

[0035] The biocompatible solution is physiological saline or phosphate buffer; specifically, the physiological saline contains NaCl at a concentration of 0.9 wt%; the phosphate buffer has a concentration of 10 mM and a pH of 7.4; the physiological saline and phosphate buffer can be prepared by oneself or obtained commercially.

[0036] The concentration of the two-dimensional metal polyphenol complex nanomaterial in the injection solution is 10~10000 μg / mL; preferably 3000~8000 μg / mL, more preferably 5000 μg / mL of 2D MPC-FSH. If the concentration is too low, the therapeutic effect on premature ovarian failure is not significant; the above concentration range is an optimized selection based on good biocompatibility.

[0037] Furthermore, the injection solution is an intravenous injection solution, and the dosage of the two-dimensional metal polyphenol complex nanomaterial is 5-50 mg / kg. Preferably, the dosage of the two-dimensional metal polyphenol complex nanomaterial is 10 mg / kg.

[0038] Furthermore, the preparation steps of the injection solution include: freeze-drying the above-mentioned 2D MPC-FSH that can be enriched in the ovary to obtain powdered 2D MPC-FSH, and weighing a certain mass of 2D MPC-FSH and dispersing it in a biocompatible solution.

[0039] The fourth aspect of the present invention is the use of the two-dimensional metal polyphenol complex nanomaterials described in the first aspect of the present invention, or the two-dimensional metal polyphenol complex nanomaterials prepared by the preparation method described in the second aspect of the present invention, or the products described in the third aspect of the present invention, in the preparation of medicaments for the prevention or treatment of premature ovarian failure and ovarian granulosa cell dysfunction.

[0040] Furthermore, the application is selected from at least one of the following applications: for the preparation of a medicine for the prevention or treatment of premature ovarian failure and ovarian granulosa cell dysfunction-related diseases induced by autoimmune disorders, exposure to environmental toxins or iatrogenic damage (such as chemotherapy or radiotherapy), specifically cellular senescence and its associated secretory phenotype (SASP) triggered by oxidative stress or DNA damage, which in turn leads to accelerated depletion of primordial follicle reserve and ovarian granulosa cell dysfunction-related diseases.

[0041] Furthermore, the aforementioned diseases related to accelerated depletion of primordial follicle reserves and ovarian granulosa cell dysfunction have at least one of the following cell biological characteristics: persistent cell cycle arrest in ovarian granulosa cells or interstitial cells; significantly upregulated expression levels of aging markers (p16, p21, etc.); accelerated telomere loss within cells; irreversible DNA damage caused by excessive accumulation of reactive oxygen species due to mitochondrial dysfunction; and senescent cells releasing large amounts of pro-inflammatory factors, chemokines, and matrix metalloproteinases into the extracellular matrix through senescence-related secretory phenotypes.

[0042] After the two-dimensional metal polyphenol complex nanomaterial targets and enters ovarian granulosa cells, it adsorbs the intracellular BRD7 protein with high affinity, blocks the translocation of BRD7 to the cell nucleus and its binding with acetyltransferase KAT7, downregulates the acetylation level of histone H3K14, thereby inhibiting irreversible cell cycle arrest and weakening the release of aging-related secretory pro-inflammatory factors.

[0043] Furthermore, the related diseases caused by autoimmune disorders include: premature ovarian failure caused by a chronic inflammatory environment triggered by autoantibody attack, which secretes a large number of pro-inflammatory cytokines and induces stress-induced cellular senescence in healthy granulosa cells through paracrine effects.

[0044] Furthermore, the related diseases caused by environmental toxins include: endocrine disruptors such as bisphenol A or heavy metals induce mitochondrial dysfunction and produce excessive reactive oxygen species, resulting in severe oxidative stress damage that disrupts telomere homeostasis, triggers telomere-dependent aging of ovarian granulosa cells, and thus induces premature ovarian failure.

[0045] Furthermore, the related diseases caused by the iatrogenic injury include premature ovarian failure and ovarian granulosa cell dysfunction caused by acute genotoxic stress from chemotherapy drugs such as cyclophosphamide and cisplatin. Specifically, acute genotoxic stress kills germ cells while forcing ovarian stromal cells into permanent senescence arrest, thereby losing the structural and metabolic support function for follicle development.

[0046] Preferably, the chemotherapeutic agent includes any one of cyclophosphamide, cisplatin, 5-fluorouracil, paclitaxel, and epirubicin. Cyclophosphamide is preferred.

[0047] Preferably, the two-dimensional metal polyphenol complex nanomaterial in the injection solution has at least one of the following functions: clearing inflammatory factors and adsorbing BRD7 protein.

[0048] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: (1) This invention utilizes the high specific surface area and abundant polyphenol sites on the surface of two-dimensional metal polyphenol complex nanomaterials to intercept the key aging scaffold protein BRD7 through high affinity adsorption. Through the "physical adsorption-driven molecular blocking" strategy, the binding and nuclear translocation of BRD7 to acetyltransferase KAT7 are effectively blocked, thereby blocking the expression of aging-related genes at the epigenetic landscape level. Its intervention depth is significantly better than that of conventional small molecule drugs.

[0049] (2) This invention utilizes two-dimensional metal polyphenol complex nanomaterials to block the BRD7-KAT7 axis, which not only induces granulosa cell cycle arrest but also inhibits their ability to release pro-inflammatory cytokines (IL-6, IL-8, TNFα, etc.) upstream. Its technical advantage lies in curbing the release of inflammatory factors at the source and reshaping the ovarian reproductive microenvironment.

[0050] (3) This invention integrates polyphenol molecules into two-dimensional nanosheets through metal-polyphenol coordination self-assembly technology, which significantly improves their dispersibility and stability in the physiological environment. This two-dimensional sheet structure not only achieves enrichment in damaged ovarian tissue through active targeted modification, but also enhances its anti-inflammatory effect and bioavailability through the synergistic effect of metal ions and polyphenol molecules, and greatly reduces toxic side effects. Attached Figure Description

[0051] Figure 1 This is a transmission electron microscope (TEM) image of two-dimensional metal polyphenol complex nanomaterial (Zn-fis-FSH) nanosheets prepared in one embodiment of the present invention. Figure 2 A color two-dimensional atomic force microscope (AFM) image of the two-dimensional metal polyphenol complex nanomaterial (Zn-fis-FSH) nanosheets prepared in one embodiment of the present invention. Figure 3 This is an AFM height profile of the two-dimensional metal polyphenol complex nanomaterial (Zn-fis-FSH) nanosheets prepared in one embodiment of the present invention. Figure 4 The X-ray photoelectron spectroscopy (XPS) full spectrum of the two-dimensional metal polyphenol complex nanomaterial (Zn-fis-FSH) nanosheets prepared in one embodiment of the present invention; Figure 5 for Figure 4High-resolution XPS spectrum of Zn 2p; Figure 6 for Figure 4 High-resolution XPS spectrum of O 1s; Figure 7 for Figure 4 High-resolution XPS spectrum of N 1s; Figure 8 This is a comparison of the Fourier transform infrared (FTIR) spectra of Zn-fis and Zn-fis-FSH in one embodiment of the present invention. Figure 9 This is a graph showing the CCK cell activity results after mouse granulocytes were incubated with different concentrations of two-dimensional metal polyphenol complex material (Zn-fis-FSH) for 48 hours in one embodiment of the present invention. Figure 10 This is a Western Blot image showing the effect of two-dimensional metal polyphenol complex nanomaterial (Zn-fis-FSH) on the expression level of BRD7 protein in granulocytes in one embodiment of the present invention. Figure 11 This is a Western Blot image showing the results of the verification of the interaction between two-dimensional metal polyphenol complex nanomaterials (Zn-fis-FSH) and the blocking of KAT7 protein expression level by BRD7-KAT7 in one embodiment of the present invention. Figure 12 These are microscopic images of senescence-related β-galactosidase (SA-β-gal) staining in granulocytes of different treatment groups in one embodiment of the present invention. Figure 13 This is a graph showing the pharmacokinetics of Zn-fis-COOH and Zn-fis-FSH in mice in vivo and the distribution curves of their enrichment in ovarian tissue over time in one embodiment of the present invention. Figure 14 This is a morphological evaluation image of H&E staining of mouse ovarian tissue from different treatment groups in one embodiment of the present invention; Figure 15 This is a graph showing the ELISA quantitative detection results of the expression levels of SASP-related factors (TNF-α, IL-6, MCP-1 and VEGF) in the serum of mice in different treatment groups in one embodiment of the present invention; Figure 16 This is an H&E stained tissue section of the main organs (heart, liver, spleen, lung, and kidney) of mice used for long-term in vivo safety evaluation of the two-dimensional metal polyphenol complex nanomaterial (Zn-fis-FSH) in one embodiment of the present invention. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0053] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0054] The 2D MPC surface described in this invention is rich in hydroxyl groups provided by polyphenol ligands and unsaturated metal sites. Silane-PEG-COOH, through a silane coupling reaction, allows its silanol groups to undergo dehydration condensation with the polyphenol hydroxyl groups on the 2D MPC surface, forming extremely stable Si-OC covalent bonds.

[0055] The receptor for FSH (FSHR) is specifically expressed in granulosa cells in the ovary, which are central to supporting primordial follicle development and inhibiting atresia. LH receptors, on the other hand, are more widely distributed in stromal cells and ovulatory follicles. For the design of this invention to reverse granulosa cell senescence, FSH offers stronger targeting specificity. In this invention, the FSH peptide is not only a guidance tool, but also, upon binding to its receptor, partially mimics endogenous signaling, promoting granulosa cell proliferation and inhibiting apoptosis. Therefore, in the premature ovarian failure model, FSH-mediated delivery more effectively maintains the homeostasis of the primordial follicle pool.

[0056] Two-dimensional metal-polyphenol complex nanomaterials that can accumulate in the ovary include two-dimensional metal-polyphenol complexes and follicle-stimulating hormone peptides grafted onto the surface of the two-dimensional metal-polyphenol complexes. In this invention, a two-dimensional flavin zinc complex is used as an example of a two-dimensional metal-polyphenol complex.

[0057] In one specific embodiment of the present invention, the obtained zinc flavin complex material Zn-fis-FSH has a flake-like morphology with an average particle size of 200-400 nm. It has excellent biocompatibility, dispersion stability and in vivo safety. It can be enriched in the ovary and has the ability to restore granulosa cell proliferation and support follicle development, while remodeling the ovarian reproductive microenvironment. The above-mentioned Zn-fis-FSH has a simple synthesis process, high yield, low cost, good therapeutic effect and high biosafety, and has excellent clinical application prospects in the biomedical field.

[0058] In one specific embodiment of the present invention, the method for preparing Zn-fis-FSH includes: (1) Dissolve zinc nitrate hexahydrate in deionized water to form solution A; dissolve lacquinone in methanol to form solution B; add solution A and solution B to phosphate buffer, stir and react, centrifuge, wash and dry to obtain the product two-dimensional lacquinone zinc complex (Zn-fis). (2) Dissolve Silane-PEG-COOH in an ethanol / water mixture, stir thoroughly, then add two-dimensional zinc lacquinone complex powder, react thoroughly, wash, centrifuge and collect to obtain the sample (Zn-fis-COOH). (3) The above product precipitate was added to phosphate buffer, and N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added respectively. The mixture was stirred and activated. The follicle-stimulating hormone polypeptide was dissolved in a small amount of phosphate buffer and added to the above system. The mixture was stirred at low temperature, washed, centrifuged and freeze-dried to obtain the two-dimensional metal polyphenol complex nanomaterial Zn-fis-FSH that can be enriched in the ovary.

[0059] The original 2D Zn-fis surface synthesized in this method mainly consists of zinc ions and hydroxyl groups from rutin molecules, lacking free carboxyl groups, thus preventing the grafting of FSH peptides via amide condensation. The Silane-PEG-COOH modified in this method not only introduces carboxyl groups, but its PEG content also reduces the steric hindrance between the FSH peptide and the inorganic sheet, improving the bioavailability and recognition flexibility of the targeted peptide. Compared to existing MOFs with carboxyl ligands, this method constructs a chemical modification platform on a two-dimensional inorganic surface that originally lacked bonding conditions through a silanization modification step.

[0060] The two-dimensional sheet-like structure of Zn-fis-FSH has extremely small longitudinal dimensions, facilitating deep penetration into the dense stroma of ovarian tissue. Furthermore, its two-dimensional structure provides a large contact area with granulosa cells.

[0061] In a specific embodiment of the present invention, the above-mentioned Zn-fis-FSH material can be used to prepare an injection solution, including the following steps: dispersing powdered Zn-fis-FSH in a biocompatible solution (physiological saline or phosphate buffer) to obtain a Zn-fis-FSH injection solution with adjustable concentration.

[0062] In one specific embodiment of the present invention, the above-mentioned Zn-fis-FSH material or injection can be used in drugs to prevent or treat cellular senescence and its related secretory phenotypes triggered by oxidative stress or DNA damage in women before the age of 40 due to autoimmune disorders, exposure to environmental toxins or iatrogenic damage (such as chemotherapy or radiotherapy), which leads to accelerated depletion of primordial follicle reserves and ovarian granulosa cell dysfunction. For example, it can be used for the prevention or treatment of premature ovarian failure caused by chemotherapy drugs such as cyclophosphamide.

[0063] The two-dimensional metal polyphenol complex nanomaterial (two-dimensional nanosheet), namely Zn-fis-FSH, prepared in this invention, targets senescent cells in prematurely aging ovaries. Granulosa cells and stromal cells in prematurely aging ovaries experience persistent cell cycle arrest and release large amounts of pro-inflammatory cytokines into the extracellular matrix via SASP, inducing other cells into a stress-induced senescent state and creating a persistent chronic inflammatory environment, leading to accelerated depletion of the follicle pool and irreversible decline in ovarian function. During this process, KAT7-mediated histone acetylation provides the physical basis for chromatin landscape remodeling during cellular senescence, while BRD7, through its bromodomain, achieves precise recognition and anchoring of these exogenous characteristics. Zn-fis-FSH, due to its unique "nano-protein" interface interaction, can efficiently intervene in the epigenetics of senescent cells. This two-dimensional metal polyphenol complex nanomaterial possesses highly coordinated unsaturated zinc sites and a polyphenolic structure rich in flavin, exhibiting a strong bioaffinity for the BRD7 protein. Zn-fis-FSH competitively adsorbs and blocks the translocation of BRD7 to the cell nucleus and its binding to the acetyltransferase KAT7. As a key bridge driving cellular senescence, the absence of BRD7 directly prevents KAT7 from being precisely recruited to the promoter regions of senescence-related genes (such as p16 and p21), thereby triggering an overall downregulation of histone H3K14 acetylation levels. This two-dimensional metal-polyphenol complex nanomaterial, through this epigenetic remodeling intervention, inhibits irreversible cell cycle arrest at its source, while significantly attenuating the release of pro-inflammatory factors (such as IL-6 and TNF-α) from SASP. Zn-fis-FSH nanosheets not only effectively reverse the senescent state of ovarian granulosa cells but also improve the microenvironment of premature ovarian failure and protect ovarian function.

[0064] The following is an illustrative description of the two-dimensional metal polyphenol complex nanomaterials that can be enriched in the ovaries provided by the present invention.

[0065] Example 1: Preparation of two-dimensional metal polyphenol complex nanomaterials This embodiment relates to a method for preparing 2D MPC-FSH, a two-dimensional metal polyphenol complex nanomaterial that can be enriched in the ovary, according to the present invention. Taking Zn-fis-FSH, a two-dimensional zinc laccase complex material that can be enriched in the ovary, as an example, the preparation process of 2D MPC-FSH is described in detail below: (1) Dissolve 300 mg of zinc nitrate hexahydrate in 20 mL of deionized water to form solution A; dissolve 45 mg of lacquer powder in 10 mL of methanol to form solution B; take 1.6 mL of solution A and 0.6 mL of solution B, add 20 mL of phosphate buffer (10 mM, pH=7.4), react at 1100 rpm for 30 minutes, then centrifuge at 8000 rpm for 5 minutes, wash 2-3 times with phosphate buffer, discard the supernatant, freeze dry to obtain Zn-fis powder; (2) Dissolve 2 mg of silane-PEG-COOH in 100 mL of ethanol / water mixture (90% ethanol and 10% water mixture), stir for 2 hours, add 100 mg of Zn-fis powder, stir at room temperature and 400 rpm for 2 hours, centrifuge at 8000 rpm for 5 minutes, wash twice with phosphate buffer, centrifuge, discard the supernatant, and collect the sample Zn-fis-COOH; (3) The above sample (100 mg) was resuspended in 50 mL of phosphate buffer, and 198 mg of N-hydroxysuccinimide and 350 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added. The mixture was stirred at 500 rpm for 15 minutes at 25 °C. 0.5 mg of follicle-stimulating hormone peptide was dissolved in 1 mL of phosphate buffer and added to the above reaction system. The mixture was stirred at 400 rpm overnight (12 h) at 4 °C. After centrifugation at 8000 rpm, the sample was washed three times with phosphate buffer, the supernatant was discarded, and the sample was freeze-dried. Zn-fis-FSH was then stored at -20 °C.

[0066] This embodiment also prepared a Zn-fis-FSH solution: powdered Zn-fis-FSH was dispersed in a biocompatible solution (10 mM, pH = 7.4 phosphate buffer was used here), and resuspended by sonication in a water bath for 10 minutes to obtain a Zn-fis-FSH injection solution with adjustable concentration (specifically, the concentration adjustment is 10~10000 μg / mL). When administering the injection, the concentration and volume should be prepared according to the actual usage. In the above injection solution, the optimal concentration of Zn-fis-FSH is 3~8 mg / mL (at which the material is most stable), specifically 5 mg / mL. The Zn-fis-FSH injection solution should be prepared and used immediately.

[0067] Example 2: Physicochemical characterization of two-dimensional metal polyphenol complex nanomaterials This embodiment relates to the physicochemical characterization of the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH prepared in Example 1. Specifically, it involves characterizing the physicochemical properties of Zn-fis, Zn-fis-COOH, and Zn-fis-FSH prepared in Example 1, including thickness measurement (AFM), elemental qualitative and quantitative analysis (XPS), Zn chemical valence state analysis (XPS), and functional group characterization (Fourier transform infrared spectroscopy). All the above measurement procedures are existing techniques and will not be described in detail here. The measurement results are as follows: Figures 1-8 As shown.

[0068] (1) Figure 1 TEM images of Zn-fis-FSH, by Figure 1 It can be seen that the Zn-fis-FSH prepared in Example 1 is a uniform two-dimensional sheet structure with slight folds or wrinkles, which is due to the extremely high surface free energy of the two-dimensional material. The nanosheets have high light transmittance, indicating that they are extremely thin, with an average lateral diameter of 200~300 nm, and clear edges. No obvious aggregation was observed.

[0069] (2) Figures 2-3 The images are respectively the AFM color 2D plot and height profile of Zn-fis-FSH, derived from... Figure 2 It can be seen that the Zn-fis-FSH prepared in Example 1 has a flake-like morphology. Through... Figure 3 The height profile measurements show that the average thickness of the nanosheets is 1.9 nm.

[0070] (3) Figures 4-7 The XPS full spectrum and high-resolution spectrum of Zn-fis-FSH are shown below. Figure 4 It can be seen that the Zn-fis-FSH prepared in Example 1 contains elements such as Zn, C, O, N, and Si. This can be determined by the high-resolution spectrum of Zn 2p (…). Figure 5It can be seen that the Zn element is based on Zn 2+ It exists in a form other than metallic Zn. In the high-resolution spectrum of O 1s (… Figure 6 In the N 1s spectrum, the peak at 531.8 eV is attributed to the Zn-O bond formed by the coordination of laccasein and zinc. Furthermore, the N 1s spectrum... Figure 7 The distinct signal at 400.1 eV corresponds to the peptide bonds and amino groups in the FSH protein, further verifying the success of the surface biofunctionalization modification. XPS measurements showed that the nitrogen content in the Zn-fis, Zn-fis-COOH, and Zn-fis-FSH samples was 0.19%, 0.98%, and 1.35%, respectively. Due to the two-dimensional ultrathin characteristics of 2D MPC, its density of exposed active sites is significantly higher than that of spherical particles of the same mass.

[0071] (4) Figure 8 The Fourier transform infrared spectra of Zn-fis and Zn-fis-FSH are compared. The general outlines of the spectral lines of Zn-fis and Zn-fis-FSH are similar, demonstrating that after complex surface chemical modifications such as silanization and peptide coupling, the two-dimensional core metal coordination framework of Zn-fis did not collapse or depolymerize, maintaining extremely high structural stability. The spectral lines of Zn-fis-FSH are concentrated at 1530 cm⁻¹. -1 A significant enhancement peak appeared at 1050 cm⁻¹, proving that the FSH peptide had been successfully coupled to the nanomaterial surface via covalent amide bonds through an NHS / EDC activation strategy. Furthermore, the Zn-fis-FSH spectrum showed a significant enhancement peak at 1050 cm⁻¹. -1 A strong, broad absorption peak is observed at this point, which is attributed to the typical asymmetric stretching vibration of the aliphatic ether bond (COC).

[0072] Example 3: Cytotoxicity Verification of Two-Dimensional Metal Polyphenol Complex Nanomaterials This embodiment relates to the toxicity verification of the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH, which can be enriched in the ovary, on ovarian granulosa cells. Taking the two-dimensional zinc laccase complex material Zn-fis-FSH as an example, in vitro cytotoxicity detection was performed as follows: (1) Experimental procedure: Mouse ovarian granulosa cells in the logarithmic growth phase were plated and adhered to the plate after 12 hours. They were then co-incubated with Zn-fis-FSH at different concentration gradients for 48 h, with concentrations of 0, 0.75, 1.5, 3, 6, 12, 25, 50 and 100 μg / mL, respectively. Cell viability was then detected by the CCK-8 assay.

[0073] (2) Experimental results: The results are as follows Figure 9As shown, after 48 hours of incubation, the cell viability at the corresponding concentrations was 100.0%, 98.5%, 100.9%, 100.4%, 106.0%, 101.5%, 92.2%, and 86.8%, respectively. The experimental data indicate that the drug does not cause significant toxicity to normal cells across a wide concentration range.

[0074] Example 4: Adsorption experiment of BRD7 protein by two-dimensional metal polyphenol complex nanomaterials This embodiment relates to the adsorption effect of the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH, which can be enriched in the ovary, on the target protein BRD7. Taking the two-dimensional zinc laccase complex material Zn-fis-FSH as an example, the adsorption and downregulation effect of the Zn-fis-FSH nanomaterial on the target protein BRD7 are verified. Details are as follows: (1) Experimental procedure: Mouse ovarian granulosa cells in the logarithmic growth phase were seeded into 6-well plates at a density of 2 × 10⁻⁶ cells / well. 5 Cells were cultured per well, and after adhesion, they were divided into a control group (Control) and an experimental group (Zn-fis-FSH). The control group received only complete culture medium, while the experimental group received complete culture medium containing 10 μg / mL Zn-fis-FSH for co-incubation. After 48 hours of incubation, the culture medium in the 6-well plate was discarded, and the plate was placed on ice. 1 mL of pre-chilled PBS was added to each well to gently wash the cells 2-3 times to thoroughly remove residual culture medium and nanomaterials that had not entered the cells. After aspirating the PBS, 100 μL of pre-chilled RIPA high-efficiency lysis buffer (containing a mixture of 1% PMSF protease inhibitor and phosphatase inhibitor) was added to each well. After lysis in the refrigerator for 5 minutes, adhered cells were scraped off using a sterile cell scraper and transferred to pre-chilled 1.5 mL centrifuge tubes. The centrifuge tubes were placed on ice for 30 minutes of lysis. The lysed samples were then centrifuged at 12,000 rpm for 15 minutes at 4 °C. The supernatant was then transferred to a new pre-chilled centrifuge tube. The absolute protein concentration of each group of samples was determined at 562 nm using the BCA protein assay kit, and the protein concentration of each group was adjusted to the same level using lysis buffer. 5× SDS-PAGE protein loading buffer was added to the supernatant at a volume ratio of 4:1, and the mixture was boiled in a metal bath at 95 °C–100 °C for 10 minutes to denature the protein. Subsequently, the expression level of intracellular BRD7 protein was detected by Western blotting (WB).

[0075] (2) Experimental results: combined with, for example Figure 10 As shown, compared with the blank control group, the expression level of BRD7 protein in granule cells co-incubated with Zn-fis-FSH was significantly reduced, confirming that the nanomaterial can efficiently adsorb and consume BRD7 protein in cells.

[0076] Example 5: Verification of the blocking effect of two-dimensional metal polyphenol complex nanomaterials on the BRD7-KAT7 interaction This embodiment relates to the verification of the blocking effect of the two-dimensional metal polyphenol complex nanomaterials enriched in the ovary of the present invention on KAT7 expression. Taking the two-dimensional zinc laccase complex material Zn-fis-FSH as an example, the cascade regulatory mechanism by which Zn-fis-FSH blocks KAT7 expression by downregulating BRD7 is verified as follows: (1) Experimental procedure: Mouse ovarian granulosa cells in the logarithmic growth phase were plated and divided into groups. The cell groups were set as blank control group (Control), negative control group (si-Control), BRD7 knockdown group (si-BRD7), and material intervention group (Zn-fis-FSH). Complete culture medium was added to the blank control group for further culture. Culture medium containing disordered control group siRNA was added to the si-Control negative control group (60 pmol of siRNA was mixed with 6 μL of siRNA-mateplus transfection reagent, incubated at room temperature for 15 minutes, and then added to a 6-well plate containing 1 mL of complete culture medium). Complete culture medium containing si-BRD7 was added to the si-BRD7 group (parameters as above). Complete culture medium containing Zn-fis-FSH (concentration of 10 μg / mL) was added to the material intervention group. After co-incubation for 48 hours, the cells were collected and total protein was extracted (protein extraction method is as described in Example 3). The expression level of KAT7 protein in the cells was then detected by Western blotting.

[0077] (2) Experimental results: The results are as follows Figure 11 As shown, combined with Figure 11 Western blot analysis revealed that the KAT7 expression levels in both the si-BRD7 group and the Zn-fis-FSH group were significantly lower than those in the control group. This result fully validates that Zn-fis-FSH can exert an upstream regulatory effect similar to si-BRD7, effectively blocking the BRD7-KAT7 interaction and thus downregulating the level of the aging driver factor KAT7.

[0078] Example 6: Cell senescence reversal experiment using two-dimensional metal polyphenol complex nanomaterials This embodiment relates to the effect of the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH, which can be enriched in the ovary, on reversing cellular senescence. Taking the two-dimensional zinc laccase complex material Zn-fis-FSH as an example, the preventive and therapeutic effects of Zn-fis-FSH on cyclophosphamide-induced premature senescence of granulosa cells were verified through co-culture, as detailed below: (1) Experimental procedure: Four groups were set up: blank control group, premature aging model group (incubated with complete medium containing 10 μg / ml cyclophosphamide for 48 h), prevention group (incubated with complete medium containing 10 μg / ml Zn-fis-FSH for 4 h, then cyclophosphamide was added to the culture system for continued incubation, with a total treatment time of 48 h (i.e., 44 h after drug addition), and the concentration of cyclophosphamide was 10 μg / mL), and treatment group (incubated with complete medium containing both cyclophosphamide and Zn-fis-FSH, both at a concentration of 10 μg / ml). After 48 hours of treatment, each group was stained with SA-β-gal kit, and the cell color was then observed under a light microscope.

[0079] (2) Experimental results: combined with Figure 12 Microscopic images revealed a large number of distinct deep blue senescent positive cells in the premature aging model group; the blue color was significantly reduced in the prevention and treatment groups, especially in the prevention group, where almost no blue cells were observed in the field of view, confirming that the material has excellent in vitro anti-aging activity.

[0080] Example 7: Verification of Ovarian Targeting Mediated by Two-Dimensional Metal Polyphenol Complex Nanomaterials This embodiment relates to the verification of the ovarian targeting effect mediated by the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH, which is enriched in the ovary, according to the present invention. Taking the two-dimensional zinc laccase complex material Zn-fis-FSH as an example, the targeting and promoting effect of FSH peptide modification on the ovarian tissue of live mice was verified by tissue distribution experiments, as follows: (1) Experimental Procedure: Healthy 8-week-old C57 mice were randomly divided into two groups (FSH peptide-modified group and non-FSH peptide-modified group). Unmodified Zn-fis-COOH (preparation method see Example 1) and targeted Zn-fis-FSH were injected via tail vein, respectively, at a dose of 20 mg / kg. Ovarian tissue was obtained by sacrifice at 4, 8, 12, and 24 hours after injection. To accurately quantify the enrichment of nanomedicines in the ovaries, inductively coupled plasma mass spectrometry (ICP-MS) was used to trace zinc levels. Specific experimental and calculation steps are as follows: The weighed ovarian tissue (dry weight recorded as...) W ovary To reduce weighing errors of small organs, ovarian samples from mice in the same group can be combined for processing. The organic matrix is ​​then thoroughly destroyed using aqua regia digestion, and the volume is adjusted to a final volume (the adjusted volume is denoted as...). V The total zinc concentration in the digestion solution was determined (denoted as ). C The quality of drug accumulation in the ovary ( ). M The final tissue distribution (%ID / g) is calculated using the following formula: A. Formula for calculating the absolute mass of nanomedicines in the ovary: M = C × V .

[0081] B. Formula for calculating tissue enrichment rate using double normalization: %ID / g = M / ( W ovary × Dose) × 100%. Where Dose is the total absolute dose of nanomedicine injected into the mouse via the tail vein.

[0082] (2) Experimental results: combined with Figure 13 The distribution curves show that the drug enrichment concentration and retention time in ovarian tissue of the Zn-fis-FSH group are significantly better than those of the Zn-fis-COOH group, confirming that FSH peptides can effectively cross the in vivo barrier and achieve precise targeting and long-term enrichment of nanomedicines in ovarian tissue.

[0083] Example 8: Verification of the effect of two-dimensional metal polyphenol complex nanomaterials on a mouse model of premature ovarian failure This embodiment relates to the role of the two-dimensional metal polyphenol complex nanomaterial 2D MPC-FSH, which can be enriched in the ovary, in a mouse model of premature ovarian failure. Taking the two-dimensional zinc laccase complex material Zn-fis-FSH as an example, it was injected into a mouse model of premature ovarian failure to verify the enrichment of the two-dimensional metal polyphenol complex nanomaterial prepared by this method in mouse ovarian tissue, as well as its repair and cytotoxic effects on the mouse model of premature ovarian failure. The details are as follows: (I) Establishment and treatment of a mouse model of premature ovarian failure Eight-week-old C57 mice were used in the experiment. The mice were divided into five groups: blank control group, premature aging model group, Zn-fis-FSH treatment group, prevention group, and treatment group. The blank control group was not treated at all during the modeling process and had normal diet and water. The premature aging model group, prevention group, and treatment group were modeled by intraperitoneal injection of cyclophosphamide. The dosage was 75 mg / kg (cyclophosphamide with a concentration of 15 mg / mL, administered in 100 μL) once a week for 4 consecutive weeks.

[0084] Concurrently, the Zn-fis-FSH treatment group did not receive cyclophosphamide injections, but only received Zn-fis-FSH via tail vein injection; the prevention group received Zn-fis-FSH via tail vein injection 12 hours before each cyclophosphamide injection; and the treatment group mice received targeted protective intervention concurrently with cyclophosphamide modeling. Zn-fis-FSH material was administered on days 1, 5, 8, 12, 15, 19, 22, and 26 of the experiment. At these time points, the Zn-fis-FSH nanomedicine was injected into mice via tail vein at a dose of 10 mg / kg.

[0085] In the prevention group, for the time points where modeling and treatment overlap (such as D1, D8, D15, D22), the procedure of first injecting targeted drugs and then injecting chemotherapy drugs after a certain interval was adopted to give full play to the pre-protective effect of nanomaterials.

[0086] The animal experiment lasted for 4 weeks. Three days after the last administration, the mice were euthanized and their tissues were harvested. Ovarian tissues, as well as heart, liver, spleen, lungs, and kidneys, were completely removed from each group, including the blank control group, the Zn-fis-FSH monotherapy group, the premature aging model group, the prevention group, and the treatment group, for further testing.

[0087] (II) Evaluation of ovarian function repair After mouse modeling was completed, ovarian tissues were completely harvested from each group, including the blank control group, the Zn-fis-FSH monotherapy group, the premature aging model group, the prevention group, and the treatment group. After fixation and sectioning, H&E staining was performed to observe the ovarian status. Results are as follows: Figure 14 As shown, the premature ovarian failure model group showed significant ovarian atrophy and a decrease in the number of follicles; while the ovarian morphology of the prevention group and the treatment group was significantly restored, demonstrating excellent treatment effects.

[0088] (III) Inhibition of SASP-related inflammatory factors After mouse modeling was completed, serum samples were collected from mice in each group, including the blank control group, the Zn-fis-FSH monotherapy group, the premature aging model group, the prevention group, and the treatment group. The expression levels of TNF-α, IL-6, MCP-1, and VEGF in the supernatant were quantitatively detected using an ELISA kit (purchased from Thermo Fisher Scientific). Combined with... Figure 15 As shown, the SASP factor in the blood of the premature aging model group increased significantly, while the values ​​in the treatment and prevention groups were significantly controlled, which confirms the effective improvement of the ovarian aging microenvironment at the molecular level.

[0089] (iv) In vivo safety evaluation of Zn-fis-FSH After the mouse model was established, a blank control group was taken ( Figure 16The winning designation is Control) and the Zn-fis-FSH treatment group ( Figure 16 The heart, liver, spleen, lungs, and kidneys of mice with a contracted concentration of 10 mg / kg were examined using H&E sections. Figure 16 Histological sections showed no abnormal damage to the major organs and functions of the experimental animals, confirming that the material has extremely high safety for in vivo application.

[0090] Example 9: Preparation of two-dimensional quercetin copper complex material Cu-que-FSH This embodiment relates to the preparation of the two-dimensional metal polyphenol complex nanomaterials of the present invention. Unlike Example 1, this embodiment prepares the ovarian-targeting two-dimensional quercetin copper complex material Cu-que-FSH, specifically including the following steps: (1) Dissolve 244 mg of copper nitrate trihydrate in 20 mL of deionized water to form solution A; dissolve 24 mg of quercetin powder in 10 mL of methanol to form solution B; add 1.6 mL of solution A and 0.6 mL of solution B to 20 mL of phosphate buffer (10 mM, pH=7.4), react at 1100 rpm for 1 h, then centrifuge at 8000 rpm for 5 minutes, wash 2-3 times with phosphate buffer, discard the supernatant and freeze dry to obtain Cu-que powder.

[0091] (2) Dissolve 2 mg of silane-PEG-COOH in 100 mL of ethanol / water mixture (80% ethanol and 20% water), stir for 2 hours, add 100 mg of Cu-que powder, stir at 400 rpm for 2 hours at room temperature, then centrifuge at 8000 rpm for 5 minutes, wash twice with phosphate buffer, centrifuge and discard the supernatant to collect the sample Cu-que-COOH. (3) Add the above sample to 50 mL of phosphate buffer, add 198 mg of N-hydroxysuccinimide and 350 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir at 500 rpm at room temperature for 15 minutes to activate. 0.5 mg of follicle-stimulating hormone peptide was dissolved in 1 mL of phosphate buffer and added to the above reaction system. The mixture was stirred at 400 rpm overnight at 4°C. After centrifugation at 8000 rpm, the supernatant was discarded after washing three times with phosphate buffer and then freeze-dried. The resulting Cu-que-FSH was stored at -20 °C.

[0092] Example 10: Preparation of two-dimensional luteolin magnesium complex material Mg-lut-FSH This embodiment relates to the preparation of the ovarian-targeted two-dimensional luteolin magnesium complex material Mg-lut-FSH of the present invention, specifically including the following steps: (1) Take 259 mg of magnesium nitrate hexahydrate and dissolve it in 20 mL of deionized water to form solution A; take 45 mg of luteolin powder and dissolve it in 10 mL of methanol to form solution B; take 1.6 mL of solution A and 0.6 mL of solution B, add them to 20 mL of phosphate buffer (10 mM, pH=7.4), react at 1100 rpm for 30 minutes, then centrifuge at 8000 rpm for 5 minutes, wash with phosphate buffer 2-3 times, discard the supernatant and freeze dry to obtain Mg-lut powder. (2) Dissolve 2 mg of silane-PEG-COOH in 100 mL of ethanol / water mixture (90% ethanol and 10% water), stir for 2 hours, add 100 mg of Mg-lut powder, stir at 400 rpm for 2 hours at room temperature, then centrifuge at 8000 rpm for 5 minutes, wash twice with phosphate buffer, centrifuge and discard the supernatant to collect the sample Mg-lut-COOH. (3) Add the above sample to 50 mL of phosphate buffer, add 198 mg of N-hydroxysuccinimide and 350 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir at 500 rpm for 15 minutes at room temperature to activate. 0.5 mg of follicle-stimulating hormone peptide was dissolved in 1 mL of phosphate buffer and added to the above reaction system. The mixture was stirred at 400 rpm overnight at 4 °C. Afterward, it was centrifuged at 8000 rpm, washed three times with phosphate buffer, and the supernatant was discarded. After freeze-drying, the obtained Mg-lut-FSH was stored at -20 °C.

[0093] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A two-dimensional metal polyphenol complex nanomaterial that can be enriched in the ovary, characterized in that, Includes a two-dimensional metal polyphenol complex and a follicle-stimulating hormone polypeptide grafted onto the surface of the two-dimensional metal polyphenol complex; Specifically, the surface of the two-dimensional metal polyphenol complex is grafted with -COOH functional groups to form a 2D MPC-COOH intermediate, and the follicle-stimulating hormone polypeptide is covalently grafted with -COOH groups on the surface of the 2D MPC-COOH intermediate to obtain the two-dimensional metal polyphenol complex nanomaterial.

2. The two-dimensional metal polyphenol complex nanomaterial that can be enriched in the ovary according to claim 1, characterized in that, The two-dimensional metal polyphenol complex is surface-modified with Silane-PEG-COOH grafted with COOH, and the mass ratio of Silane-PEG-COOH to the two-dimensional metal polyphenol complex is 1:(50~200), and the molecular weight of Silane-PEG-COOH is 2000~5000 Da; and / or The follicle-stimulating hormone peptide is covalently grafted via an amide condensation reaction between its amino group and the -COOH group on the surface of the 2D MPC-COOH intermediate, and the mass ratio of the follicle-stimulating hormone peptide to the 2D MPC-COOH intermediate is 1:(100~400); and / or The average particle size of the two-dimensional metal polyphenol complex nanomaterial is 200-400 nm; and / or The thickness of the two-dimensional metal polyphenol complex nanomaterial is 1~5 nm.

3. The two-dimensional metal polyphenol complex nanomaterial that can be enriched in the ovary according to claim 1, characterized in that, The two-dimensional metal polyphenol complex is formed by coordination self-assembly of flavonoid polyphenol ligands and metal ions; The metal ions are selected from Al. 3+ Mg 2+ Cu 2+ Fe 3+ Zn 2+ 、Tb 3+ Eu 3+ Co 2+ Any one of them; The flavonoid polyphenol ligands are selected from any one of quercetin, luteolin, luteolin, 3-hydroxyflavone, and 5-hydroxyflavone.

4. A method for preparing a two-dimensional metal polyphenol complex nanomaterial that can be enriched in the ovary as described in any one of claims 1-3, characterized in that, Includes the following steps: 1) A two-dimensional metal polyphenol complex was prepared by adding the metal salt solution corresponding to the metal ion and the flavonoid polyphenol organic solution into a buffer system for molecular coordination reaction. 2) Dissolve Silane-PEG-COOH in an ethanol solution, and then add the two-dimensional metal polyphenol complex obtained in step 1) to carry out a silane coupling reaction to prepare the 2D MPC-COOH intermediate; 3) Disperse the 2D MPC-COOH intermediate obtained in step 2) in a buffer solution, add an activator and stir to activate, add follicle-stimulating hormone polypeptide solution to carry out amide condensation reaction to obtain the two-dimensional metal polyphenol complex nanomaterial.

5. The preparation method according to claim 4, characterized in that, In step 1): The metal salt is selected from any one of nitrates, chlorides, and sulfates; and / or The concentration of the metal salt solution is 20~100 mM; and / or The concentration of the flavonoid polyphenol organic solution is 5-30 mM; and / or The solvent for the flavonoid polyphenol organic solution is selected from any one of methanol, ethanol, isopropanol, n-butanol, acetic acid, and formic acid; and / or The buffer system is either phosphate buffer or bicarbonate buffer; and / or The buffer solution system has a pH of 7-8 and a concentration of 10-20 mM; and / or The volume ratio of the metal salt aqueous solution, the flavonoid polyphenol organic solution, and the buffer solution is (5~10):3:(50~200); and / or The conditions for the molecular coordination reaction are stirring at 800-1200 rpm for 10-60 min; and / or In step 2): In the ethanol solution, the volume ratio of ethanol to water is (7~9):1; and / or The concentration of Silane-PEG-COOH is 0.01~1 mg / mL; and / or The conditions for the silane coupling reaction are: stirring at room temperature and 400 rpm for 0.5–2 h; and / or In step 3): The activator is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(0.5~2); and / or The activation conditions are: stirring at 25-30 °C and 400-600 rpm for 10-30 min; and / or The conditions for the amide condensation reaction are: stirring at a temperature of 2-10 °C and a rotation speed of 400-600 rpm for 12-15 hours.

6. A product containing a two-dimensional metal polyphenol complex nanomaterial that can be enriched in the ovary, characterized in that, It includes the two-dimensional metal polyphenol complex nanomaterials as described in any one of claims 1-3 or the two-dimensional metal polyphenol complex nanomaterials prepared by the preparation method described in any one of claims 4-5, as well as pharmaceutically acceptable carriers or excipients.

7. The product according to claim 6, characterized in that, The product is an injection solution prepared by dispersing the two-dimensional metal polyphenol complex nanomaterial in a biocompatible solution; Wherein, the biocompatible solution is physiological saline or phosphate buffer; and / or The concentration of the two-dimensional metal polyphenol complex nanomaterial in the injection solution is 10~10000 μg / mL; and / or The dosage of the two-dimensional metal polyphenol complex nanomaterial is 5~50 mg / kg.

8. The use of the two-dimensional metal polyphenol complex nanomaterial according to any one of claims 1-3, or the two-dimensional metal polyphenol complex nanomaterial prepared by the preparation method according to any one of claims 4-5, or the product according to any one of claims 6-7, in the preparation of drugs for premature ovarian failure and ovarian granulosa cell dysfunction-related diseases. in, After the two-dimensional metal polyphenol complex nanomaterial targets and enters ovarian granulosa cells, it adsorbs the intracellular BRD7 protein with high affinity, blocks the translocation of BRD7 to the cell nucleus and its binding with acetyltransferase KAT7, downregulates the acetylation level of histone H3K14, thereby inhibiting irreversible cell cycle arrest and weakening the release of aging-related secretory pro-inflammatory factors.

9. The application according to claim 8, characterized in that, The application is selected from at least one of the following applications: the use in the preparation of medicaments for the prevention or treatment of premature ovarian failure and diseases related to ovarian granulosa cell dysfunction caused by iatrogenic factors, infectious factors and environmental factors.

10. The application according to claim 9, characterized in that, The related diseases caused by the iatrogenic injury are: premature ovarian failure and ovarian granulosa cell dysfunction caused by acute genotoxic stress from chemotherapy drugs. The chemotherapy drugs include any one of cyclophosphamide, cisplatin, 5-fluorouracil, paclitaxel, and epirubicin.

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