A kind of nanocomposite based on ZIF-8 and its preparation method and application

Abstract

The application relates to the field of nanomedicine, in particular to a kind of nanocomposite based on ZIF-8 and its preparation method and application.The nanocomposite includes ZIF-8 nanosheet, PEG modification layer, Mg ion loading module and RGD polypeptide targeting module, the PEG modification layer is loaded on the surface of the ZIF-8 nanosheet, the Mg ion loading module is loaded in the ZIF-8 nanosheet carrier after PEG modification, and the RGD polypeptide targeting module is grafted to the PEG modification layer.In the application, endothelial targeting, Mg 2+ repair promotion, carrier stable release and organic integration are integrated in the same nanometer platform, and oxidative stress, inflammatory response and endothelial repair disorder are intervened synergistically, the system can efficiently protect damaged endothelium, inhibit abnormal proliferation of vascular smooth muscle and neointimal formation, and maintain AVF lumen patency.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to a ZIF-8-based nanocomposite material, its preparation method, and its application. Background Technology

[0002] Arteriovenous fistulas (AVFs) are the preferred vascular access for hemodialysis in patients with end-stage renal disease, and their long-term patency directly impacts patients' quality of life. Long-term patency after AVF surgery depends on rapid remodeling and functional recovery of endothelial cells at the anastomosis site and in the surrounding venous segment. However, after AVF surgery, the vascular endothelium is subjected to the combined effects of high shear stress, inflammatory cell infiltration, oxidative stress, and local mechanical damage, making it prone to endothelial cell shedding, barrier function disruption, and delayed repair. Insufficient endothelial repair can further induce vascular smooth muscle cell migration and abnormal proliferation, promoting neointimal formation. This is the core pathological mechanism leading to AVF maturation failure and subsequent stenosis.

[0003] Current treatment strategies for endothelial injury after AVF surgery mainly include anti-inflammatory drugs, antioxidants, and traditional growth factor delivery. However, existing technologies have significant limitations. On the one hand, most anti-inflammatory or antioxidant treatments rely on systemic administration, making it difficult for drugs to achieve effective concentrations locally in the AVF and lacking selective recognition of damaged endothelial areas, resulting in poor efficacy and potential systemic side effects. On the other hand, existing treatments often emphasize inhibiting damaging factors such as inflammation or oxidative stress, but their direct promoting effect on endothelial cell proliferation, migration, adhesion, and reendothelialization is limited, making it difficult to fundamentally accelerate barrier reconstruction of damaged blood vessels.

[0004] In recent years, ion therapy-based strategies have attracted attention. Mg 2+ Ions with endothelial repair-promoting functions have been shown to support endothelial cell proliferation, migration, and vascular function recovery. However, free Mg... 2+ Rapid distribution and metabolism in vivo make it difficult to maintain a stable and effective local concentration in AVF-damaged areas, limiting its therapeutic potential. ZIF-8, as a zinc-imidazolium metal-organic framework material, possesses high specific surface area, abundant pore structure, and pH-responsive degradation characteristics, and can be used as a Mg... 2+ ZIF-8 nanomaterials provide a loading and local release platform. However, unmodified ZIF-8 nanomaterials exhibit poor dispersion stability and insufficient blood compatibility in physiological environments, and lack targeted modification for endothelial cells, making it difficult to directly meet the in vivo application requirements of AVF. RGD peptides can bind to integrin receptors (such as αvβ3 and αvβ5) highly expressed in endothelial cells and neovascularization regions, promoting cell adhesion and enhancing the recognition ability of nanomaterials for endothelial injury areas.

[0005] Therefore, there is an urgent need to develop a method that can simultaneously target the endothelial region damaged by AVF and Mg 2+A multifunctional nanocomposite material for localized sustained release and endothelial repair promotion to facilitate AVF endothelial cell repair. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a ZIF-8-based nanocomposite material, its preparation method and application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A ZIF-8-based nanocomposite material includes ZIF-8 nanosheets, a PEG-modified layer, a Mg ion-supporting module, and an RGD peptide-targeting module; the PEG-modified layer is bonded to the surface of the ZIF-8 nanosheets, the Mg ion-supporting module is loaded in the PEG-modified ZIF-8 nanosheet carrier, and the RGD peptide-targeting module is grafted onto the PEG-modified layer.

[0008] This invention uses ZIF-8 nanosheets as the main carrier. ZIF-8 nanosheets have a high specific surface area, abundant porous structure, and pH-responsive degradation characteristics, which can responsively degrade and release Mg in the weakly acidic inflammatory microenvironment of the arteriovenous fistula injury area. 2+ This achieves localized sustained release of ions; the PEG-modified layer improves the colloidal dispersibility and blood compatibility of ZIF-8 nanosheets in aqueous systems, while the carboxyl groups at the ends of PEG-COOH serve as active sites for RGD peptide coupling, and the PEG chains act as flexible spacers to ensure effective exposure of RGD targeting groups; Mg 2+ As a component promoting endothelial repair, it exerts anti-inflammatory, antioxidant, and endothelial cell proliferation and migration effects through the Nrf2 signaling pathway. The RGD peptide can specifically recognize integrin receptors such as αvβ3 and αvβ5, which are highly expressed in damaged vascular endothelial cells and neointima regions after AVF surgery. This guides the nanocarrier to be precisely enriched at the inflammatory damage site of AVF, mediating the adhesion and spread of endothelial cells on the material surface, and providing structural support for re-endothelialization.

[0009] This invention also provides a method for preparing the ZIF-8-based nanocomposite material, comprising the following steps: S1. Dissolve zinc source and 2-methylimidazole in an aqueous system or a water-alcohol mixture, add morphology modifier, and react to obtain ZIF-8 NSs; S2. Disperse ZIF-8 NSs in buffer solution, add PEG-COOH, and after stirring or sonication, obtain ZIF-8-PEG; S3. Disperse ZIF-8-PEG in deionized water, add Mg source, mix and incubate to obtain Mg@ZIF-8-PEG; S4. The PEG-COOH end groups on the surface of Mg@ZIF-8-PEG were activated by EDC / NHS, and amino-modified RGD peptides were added. After the reaction, RGD-Mg@ZIF-8-PEG was obtained.

[0010] Specifically, 2-methylimidazole, as an organic ligand, interacts with Zn. 2+ ZIF-8 crystals with a zeolite topology are formed through coordination bond self-assembly. Morphology modifiers are used to restrict crystal growth in the thickness direction, promoting the formation of a two-dimensional plate-like morphology. Then, PEG-COOH is coated onto the ZIF-8 surface through coordination or physical adsorption. Mg... 2+ The Mg@ZIF-8-PEG nanosheets diffuse into the pores driven by the concentration gradient, and coordinate with the nitrogen-containing heterocycles in the ZIF-8 framework or the oxygen atoms in the PEG chain to achieve efficient loading. The remaining PEG terminal carboxyl groups on the surface of the Mg@ZIF-8-PEG are activated by EDC / NHS, and an amino-modified RGD peptide is added for coupling reaction to obtain RGD-Mg@ZIF-8-PEG.

[0011] Based on the above technical solutions, preferably, in step S1, the zinc source is zinc nitrate or zinc acetate, and the molar ratio of the zinc source to 2-methylimidazole is 1:8-20.

[0012] Based on the above technical solutions, preferably, in step S1, the morphology modifier is polyvinylpyrrolidone or triethylamine, and the mass ratio of the morphology modifier to the zinc source is 1-5:1.

[0013] Based on the above technical solution, preferably, in step S2, the buffer solution is a MES buffer solution with a pH of 6.0-6.5.

[0014] Based on the above technical solutions, preferably, in step S3, the Mg source is MgCl2 or Mg(OAc)2, and the mass ratio of ZIF-8-PEG to the Mg source is 1:0.5-2.

[0015] Based on the above technical solutions, preferably, in step S3, the temperature of the mixed incubation is 20-40℃ and the time is 12-24h.

[0016] Based on the above technical solution, preferably, in step S4, the mass ratio of Mg@ZIF-8-PEG to amino-modified RGD polypeptide is 1:0.003-0.02.

[0017] Based on the above technical solutions, preferably, in step S4, the reaction temperature is 20-40℃ and the reaction time is 2-6h.

[0018] The present invention also provides the application of ZIF-8-based nanocomposite materials in the preparation of drugs for treating arteriovenous fistulas.

[0019] Based on the above technical solutions, preferably, the ZIF-8-based nanocomposite material can be used for AVF endothelial injury repair via intravenous administration. During intravenous administration, a suspension of the ZIF-8-based nanocomposite material can be injected via the tail vein, allowing it to reach the inflammatory damage area of ​​the AVF through blood circulation.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves synergistic effect of three functions through modular preparation design: First, two-dimensional ZIF-8 nanosheets are used as the main framework, and Mg is loaded by utilizing their high specific surface area and rich pore structure. 2+ And achieve Mg degradation through the pH-responsive degradation characteristics of ZIF-8 2+ The design incorporates on-demand sustained release within the weakly acidic inflammatory microenvironment of AVF; secondly, surface modification of ZIF-8 nanosheets with PEG-COOH enhances the material's colloidal stability and biocompatibility, while also providing activatable carboxyl coupling sites for the RGD peptide; finally, amino-modified RGD peptides are covalently grafted onto the PEG ends via amidation to form a stable targeting modification layer. This design enables RGD-mediated endothelial targeting adhesion and ZIF-8 carrier-mediated pH-responsive Mg2+. 2+ Release, Mg 2 + The anti-inflammatory, antioxidant, and endothelial repair-promoting functions are organically integrated into the same nanoplatform, synergistically intervening in oxidative stress, inflammatory response, and endothelial repair disorders. This system can more effectively protect damaged endothelium, inhibit abnormal proliferation of vascular smooth muscle and formation of new intima, thereby maintaining AVF lumen patency.

[0021] (2) This invention achieves endothelial targeted enrichment and adhesion promotion through RGD peptides. RGD specifically recognizes integrin receptors (such as αvβ3 and αvβ5) highly expressed in damaged vascular endothelial cells and newly formed intima areas after AVF surgery. It can guide ZIF-8 nanocarriers to accurately enrich at AVF anastomosis sites and inflammatory damage sites, overcoming the defects of traditional drugs that diffuse throughout the body and have insufficient local concentration. It can also directly mediate the adhesion and spread of endothelial cells on the material surface, providing "anchor point" support for re-endothelialization and accelerating the repair of the damaged barrier from the cellular behavior level.

[0022] (3) This invention optimizes the physicochemical and application properties of the carrier through PEG modification. PEG modification can reduce the aggregation of ZIF-8 nanosheets and the adsorption of non-specific proteins, prolong the in vivo circulation time, and improve the blood compatibility and stability as a vascular endothelial repair material. The modified nanosheets are not easy to aggregate in the physiological environment and are quickly cleared by the mononuclear phagocytic system. Moreover, the material can be flexibly administered via the tail vein, making it more widely applicable in clinical translation. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Transmission electron microscope image of RGD-Mg@ZIF-8-PEG prepared in Example 1; Figure 2 The FTIR spectra of RGD-Mg@ZIF-8-PEG, ZIF-8 NSs, ZIF-8-PEG and Mg@ZIF-8-PEG prepared in Example 1 and Comparative Examples 1-3 are shown. Figure 3 XPS spectra of RGD-Mg@ZIF-8-PEG, ZIF-8 NSs, ZIF-8-PEG and Mg@ZIF-8-PEG prepared in Example 1 and Comparative Examples 1-3; Figure 4 The Zeta potential changes of RGD-Mg@ZIF-8-PEG, ZIF-8 NSs, ZIF-8-PEG and Mg@ZIF-8-PEG prepared in Example 1 and Comparative Examples 1-3 are shown in the figure. Figure 5 The image shows the coagulation safety evaluation results of RGD-Mg@ZIF-8-PEG prepared in Example 1; Figure 6 The graph shows the effects of Example 1, Comparative Example 2, and Comparative Example 3 on oxidative stress, inflammatory response, and liver function safety in AVF model mice. Figure 7 The image shows a comparison of the effects of RGD-Mg@ZIF-8-PEG prepared in Example 1 on the improvement of vascular tissue pathological damage and neointimal hyperplasia in AVF model mice. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels or synthesized from raw materials purchased through commercial channels.

[0027] Example 1 This embodiment provides a method for preparing RGD-Mg@ZIF-8-PEG, the specific steps of which are as follows: Preparation of S1 and ZIF-8 nanosheets Weigh 0.297 g of Zn(NO3)2·6H2O and dissolve it in 20 mL of deionized water to prepare a zinc source solution. Separately weigh 1.15 g of 2-methylimidazole and 0.891 g of polyvinylpyrrolidone (PVP, molecular weight approximately 40,000) and dissolve them in 80 mL of methanol to prepare a 2-methylimidazole-PVP mixed solution.

[0028] At room temperature, the zinc source solution was slowly added to a 2-methylimidazole-PVP mixed solution, and the mixture was stirred to ensure homogeneity. The reaction was then carried out at 25°C for 12 h. After the reaction, the reaction solution was centrifuged at 12000 rpm for 10 min to collect the precipitate. The supernatant was discarded, and the precipitate was washed three times with anhydrous ethanol. The washed precipitate was then vacuum-dried at 60°C for 2 h to obtain ZIF-8 nanosheets, i.e., ZIF-8 NSs.

[0029] Preparation of S2 and ZIF-8-PEG Take 50 mg of ZIF-8 NSs prepared in S1, disperse it in 50 mL of MES buffer (0.1 M, pH 6.0), and sonicate for 10 min until homogeneous. Then add 100 mg of carboxyl-terminated PEG (COOH-PEG-COOH, molecular weight 2000 Da), and stir at room temperature for 4 h. After the reaction is complete, collect the precipitate by centrifugation at 12000 rpm for 10 min, wash it three times with deionized water to remove unbound PEG, and obtain ZIF-8-PEG.

[0030] Preparation of S3, Mg@ZIF-8-PEG Take 40 mg of ZIF-8-PEG prepared by S2, disperse it in 20 mL of deionized water, add 50 mg of MgCl2·6H2O, and stir and incubate at 30℃ for 18 h to allow Mg to precipitate.2+ Loaded in ZIF-8-PEG.

[0031] After incubation, the precipitate was collected by centrifugation at 12,000 rpm for 10 min, and washed three times with deionized water to remove free Mg. 2+ Mg@ZIF-8-PEG was obtained.

[0032] Preparation of S4 and RGD-Mg@ZIF-8-PEG Take 30 mg of Mg@ZIF-8-PEG prepared in S3 and disperse it in 15 mL of MES buffer (0.1 M, pH 6.0). Add 3.0 mg of EDC·HCl and 1.5 mg of NHS sequentially, and activate at room temperature for 30 min. After activation, centrifuge at 12000 rpm for 5 min to remove the supernatant. Wash the precipitate once with MES buffer (0.1 M, pH 6.0) and resuspend it in 15 mL of MES buffer (0.1 M, pH 6.0) to obtain the activated Mg@ZIF-8-PEG dispersion.

[0033] The amino-modified RGD peptide was prepared into a 1 mg / mL RGD peptide stock solution using MES buffer (0.1 M, pH 6.0). 350 μL of the RGD peptide stock solution (350 μg of amino-modified RGD peptide) was added to the activated Mg@ZIF-8-PEG dispersion. The mixture was stirred at 30°C for 4 h in the dark. After the reaction, the precipitate was collected by centrifugation at 12000 rpm for 10 min. The precipitate was washed three times with MES buffer (0.1 M, pH 6.0) to remove uncoupled RGD peptides, yielding RGD-Mg@ZIF-8-PEG.

[0034] Example 2 This embodiment provides a method for preparing RGD-Mg@ZIF-8-PEG, the specific steps of which are as follows: Preparation of S1 and ZIF-8 nanosheets Weigh 0.297 g of Zn(NO3)2·6H2O and dissolve it in 20 mL of deionized water to prepare a zinc source solution. Separately weigh 0.657 g of 2-methylimidazole and 0.297 g of polyvinylpyrrolidone (PVP, molecular weight approximately 40,000), dissolve them in 80 mL of methanol to prepare a 2-methylimidazole-PVP mixed solution.

[0035] At room temperature, the zinc source solution was slowly added to a 2-methylimidazole-PVP mixed solution, and the mixture was stirred to ensure homogeneity. The reaction was then carried out at 25°C for 12 h. After the reaction, the reaction solution was centrifuged at 12000 rpm for 10 min to collect the precipitate. The supernatant was discarded, and the precipitate was washed three times with anhydrous ethanol. The washed precipitate was then vacuum-dried at 60°C for 2 h to obtain ZIF-8 nanosheets, i.e., ZIF-8 NSs.

[0036] Preparation of S2 and ZIF-8-PEG Take 50 mg of ZIF-8 NSs prepared in S1, disperse it in 50 mL of MES buffer (0.1 M, pH 6.0), and sonicate for 10 min until homogeneous. Then add 50 mg of carboxyl-terminated PEG (COOH-PEG-COOH, molecular weight 2000 Da), and stir at room temperature for 4 h. After the reaction is complete, collect the precipitate by centrifugation at 12000 rpm for 10 min, wash it three times with deionized water to remove unbound PEG, and obtain ZIF-8-PEG.

[0037] Preparation of S3, Mg@ZIF-8-PEG Take 40 mg of ZIF-8-PEG prepared by S2, disperse it in 20 mL of deionized water, add 20 mg of MgCl2·6H2O, and stir and incubate at 20℃ for 24 h to allow Mg to precipitate. 2+ Loaded in ZIF-8-PEG.

[0038] After incubation, the precipitate was collected by centrifugation at 12,000 rpm for 10 min, and washed three times with deionized water to remove free Mg. 2+ Mg@ZIF-8-PEG was obtained.

[0039] Preparation of S4 and RGD-Mg@ZIF-8-PEG Take 30 mg of Mg@ZIF-8-PEG prepared in S3 and disperse it in 15 mL of MES buffer (0.1 M, pH 6.0). Add 2.0 mg of EDC·HCl and 1.0 mg of NHS sequentially, and activate at room temperature for 30 min. After activation, centrifuge at 12000 rpm for 5 min to remove the supernatant. Wash the precipitate once with MES buffer (0.1 M, pH 6.0) and resuspend it in 15 mL of MES buffer (0.1 M, pH 6.0) to obtain the activated Mg@ZIF-8-PEG dispersion.

[0040] The amino-modified RGD peptide was prepared into a 1 mg / mL RGD peptide stock solution using MES buffer (0.1 M, pH 6.0). 90 μL of the RGD peptide stock solution (90 μg of amino-modified RGD peptide) was added to the activated Mg@ZIF-8-PEG dispersion. The mixture was stirred at 20 °C for 6 h in the dark. After the reaction, the precipitate was collected by centrifugation at 12000 rpm for 10 min. The precipitate was washed three times with MES buffer (0.1 M, pH 6.0) to remove uncoupled RGD peptides, yielding RGD-Mg@ZIF-8-PEG.

[0041] Example 3 This embodiment provides a method for preparing RGD-Mg@ZIF-8-PEG, the specific steps of which are as follows: Preparation of S1 and ZIF-8 nanosheets Weigh 0.297 g of Zn(NO3)2·6H2O and dissolve it in 20 mL of deionized water to prepare a zinc source solution. Separately weigh 1.64 g of 2-methylimidazole and 1.485 g of polyvinylpyrrolidone (PVP, molecular weight approximately 40,000) and dissolve them in 80 mL of methanol to prepare a 2-methylimidazole-PVP mixed solution.

[0042] At room temperature, the zinc source solution was slowly added to a 2-methylimidazole-PVP mixed solution, and the mixture was stirred to ensure homogeneity. The reaction was then carried out at 25°C for 12 h. After the reaction, the reaction solution was centrifuged at 12000 rpm for 10 min to collect the precipitate. The supernatant was discarded, and the precipitate was washed three times with anhydrous ethanol. The washed precipitate was then vacuum-dried at 60°C for 2 h to obtain ZIF-8 nanosheets, i.e., ZIF-8 NSs.

[0043] Preparation of S2 and ZIF-8-PEG Take 50 mg of ZIF-8 NSs prepared in S1, disperse it in 50 mL of MES buffer (0.1 M, pH 6.0), and sonicate for 10 min until homogeneous. Then add 200 mg of carboxyl-terminated PEG (COOH-PEG-COOH, molecular weight 2000 Da), and stir at room temperature for 4 h. After the reaction is complete, collect the precipitate by centrifugation at 12000 rpm for 10 min, wash it three times with deionized water to remove unbound PEG, and obtain ZIF-8-PEG.

[0044] Preparation of S3, Mg@ZIF-8-PEG Take 40 mg of ZIF-8-PEG prepared by S2, disperse it in 20 mL of deionized water, add 80 mg of MgCl2·6H2O, and stir and incubate at 40℃ for 12 h to allow Mg to precipitate. 2+ Loaded in ZIF-8-PEG.

[0045] After incubation, the precipitate was collected by centrifugation at 12,000 rpm for 10 min, and washed three times with deionized water to remove free Mg. 2+ Mg@ZIF-8-PEG was obtained.

[0046] Preparation of S4 and RGD-Mg@ZIF-8-PEG Take 30 mg of Mg@ZIF-8-PEG prepared in S3 and disperse it in 15 mL of MES buffer (0.1 M, pH 6.0). Add 4.0 mg of EDC·HCl and 2.0 mg of NHS sequentially, and activate at room temperature for 30 min. After activation, centrifuge at 12000 rpm for 5 min to remove the supernatant. Wash the precipitate once with MES buffer (0.1 M, pH 6.0) and resuspend it in 15 mL of MES buffer (0.1 M, pH 6.0) to obtain the activated Mg@ZIF-8-PEG dispersion.

[0047] The amino-modified RGD peptide was prepared into a 1 mg / mL RGD peptide stock solution using MES buffer (0.1 M, pH 6.0). 600 μL of the RGD peptide stock solution (600 μg of amino-modified RGD peptide) was added to the activated Mg@ZIF-8-PEG dispersion. The mixture was stirred at 40 °C for 2 h in the dark. After the reaction, the precipitate was collected by centrifugation at 12000 rpm for 10 min. The precipitate was washed three times with MES buffer (0.1 M, pH 6.0) to remove uncoupled RGD peptides, yielding RGD-Mg@ZIF-8-PEG.

[0048] Comparative Example 1 This comparative example provides a method for preparing ZIF-8 NSs, the specific steps of which are as follows: Weigh 0.297 g of Zn(NO3)2·6H2O and dissolve it in 20 mL of deionized water to prepare a zinc source solution. Separately weigh 1.15 g of 2-methylimidazole and 0.891 g of polyvinylpyrrolidone (PVP, molecular weight approximately 40,000) and dissolve them in 80 mL of methanol to prepare a 2-methylimidazole-PVP mixed solution.

[0049] At room temperature, the zinc source solution was slowly added to a 2-methylimidazole-PVP mixed solution, and the mixture was stirred to ensure homogeneity. The reaction was then carried out at 25°C for 12 h. After the reaction, the reaction solution was centrifuged at 12000 rpm for 10 min to collect the precipitate. The supernatant was discarded, and the precipitate was washed three times with anhydrous ethanol. The washed precipitate was then vacuum-dried at 60°C for 2 h to obtain ZIF-8 nanosheets, i.e., ZIF-8 NSs.

[0050] Comparative Example 2 This comparative example provides a method for preparing ZIF-8-PEG, the specific steps of which are as follows: Preparation of S1 and ZIF-8 nanosheets Weigh 0.297 g of Zn(NO3)2·6H2O and dissolve it in 20 mL of deionized water to prepare a zinc source solution. Separately weigh 1.15 g of 2-methylimidazole and 0.891 g of polyvinylpyrrolidone (PVP, molecular weight approximately 40,000) and dissolve them in 80 mL of methanol to prepare a 2-methylimidazole-PVP mixed solution.

[0051] At room temperature, the zinc source solution was slowly added to a 2-methylimidazole-PVP mixed solution, and the mixture was stirred to ensure homogeneity. The reaction was then carried out at 25°C for 12 h. After the reaction, the reaction solution was centrifuged at 12000 rpm for 10 min to collect the precipitate. The supernatant was discarded, and the precipitate was washed three times with anhydrous ethanol. The washed precipitate was then vacuum-dried at 60°C for 2 h to obtain ZIF-8 nanosheets, i.e., ZIF-8 NSs.

[0052] Preparation of S2 and ZIF-8-PEG Take 50 mg of ZIF-8 NSs prepared in S1, disperse it in 50 mL of MES buffer (0.1 M, pH 6.0), and sonicate for 10 min until homogeneous. Then add 100 mg of carboxyl-terminated PEG (COOH-PEG-COOH, molecular weight 2000 Da), and stir at room temperature for 4 h. After the reaction is complete, collect the precipitate by centrifugation at 12000 rpm for 10 min, wash it three times with deionized water to remove unbound PEG, and obtain ZIF-8-PEG.

[0053] Comparative Example 3 This comparative example provides a method for preparing Mg@ZIF-8-PEG, and the specific steps are as follows: Preparation of S1 and ZIF-8 nanosheets Weigh 0.297 g of Zn(NO3)2·6H2O and dissolve it in 20 mL of deionized water to prepare a zinc source solution. Separately weigh 1.15 g of 2-methylimidazole and 0.891 g of polyvinylpyrrolidone (PVP, molecular weight approximately 40,000) and dissolve them in 80 mL of methanol to prepare a 2-methylimidazole-PVP mixed solution.

[0054] At room temperature, the zinc source solution was slowly added to a 2-methylimidazole-PVP mixed solution, and the mixture was stirred to ensure homogeneity. The reaction was then carried out at 25°C for 12 h. After the reaction, the reaction solution was centrifuged at 12000 rpm for 10 min to collect the precipitate. The supernatant was discarded, and the precipitate was washed three times with anhydrous ethanol. The washed precipitate was then vacuum-dried at 60°C for 2 h to obtain ZIF-8 nanosheets, i.e., ZIF-8 NSs.

[0055] Preparation of S2 and ZIF-8-PEG Take 50 mg of ZIF-8 NSs prepared in S1, disperse it in 50 mL of MES buffer (0.1 M, pH 6.0), and sonicate for 10 min until homogeneous. Then add 100 mg of carboxyl-terminated PEG (COOH-PEG-COOH, molecular weight 2000 Da), and stir at room temperature for 4 h. After the reaction is complete, collect the precipitate by centrifugation at 12000 rpm for 10 min, wash it three times with deionized water to remove unbound PEG, and obtain ZIF-8-PEG.

[0056] Preparation of S3, Mg@ZIF-8-PEG Take 40 mg of ZIF-8-PEG prepared by S2, disperse it in 20 mL of deionized water, add 50 mg of MgCl2·6H2O, and stir and incubate at 30℃ for 18 h to allow Mg to precipitate. 2+ Loaded in ZIF-8-PEG.

[0057] After incubation, the precipitate was collected by centrifugation at 12,000 rpm for 10 min, and washed three times with deionized water to remove free Mg. 2+ Mg@ZIF-8-PEG was obtained.

[0058] Materials Characterization and Quality Control The microstructure of RGD-Mg@ZIF-8-PEG prepared in Example 1 was observed using transmission electron microscopy (TEM).

[0059] The surface potential changes of ZIF-8 nanosheets (Comparative Example 1), ZIF-8-PEG (Comparative Example 2), Mg@ZIF-8-PEG (Comparative Example 3) and RGD-Mg@ZIF-8-PEG (Example 1) were detected using a Zeta potential analyzer to help evaluate the changes in surface charge of materials after PEG modification, Mg²⁺ loading and RGD peptide coupling.

[0060] Fourier transform infrared spectroscopy (FTIR) was used to detect the characteristic absorption peaks and peak position changes of the samples prepared in Example 1 and Comparative Examples 1-3. This was used to analyze the ZIF-8 backbone, PEG-related functional groups, and amide bond-related signals of the RGD peptide, and combined with Mg... 2+ Analysis of peak shape and position changes before and after loading Mg 2+ Interaction between the material and the material.

[0061] X-ray photoelectron spectroscopy (XPS) was used to detect the composition and relative signal changes of Zn, N, C, O and Mg in the samples prepared in Example 1 and Comparative Examples 1-3, in order to verify the PEG modification, Mg element introduction and RGD peptide coupling.

[0062] C57BL / 6J mouse AVF model and in vivo validation An AVF model was established using 8-week-old male C57BL / 6J mice, with a carotid artery-jugular vein microanastomosis model selected. Mice were placed in an anesthesia induction chamber and anesthesia was induced with 3% isoflurane and 0.8 L / min oxygen. After the righting reflex disappeared, the mice were removed and fixed in a supine position, with continuous inhalation of 2%–2.5% isoflurane via a face mask to maintain anesthesia. The depth of anesthesia was assessed by the toe-clamping reflex, and the mice were placed on a constant-temperature heating pad to maintain their body temperature at 36–38°C during the operation.

[0063] After anesthesia, the mouse neck was prepared and routinely disinfected. A 1 cm incision was made along the midline of the neck, and the left external jugular vein and common carotid artery were bluntly dissected and exposed under a surgical microscope. The distal end of the external jugular vein was ligated and the vessel was severed. Blood flow to the common carotid artery was temporarily blocked using a microvascular clamp. An anastomosis was created on the lateral wall of the common carotid artery, and the severed end of the external jugular vein was anastomosed end-to-side to the lateral wall of the common carotid artery. After the anastomosis was completed, the microvascular clamp was released, and the filling and pulsation of the external jugular vein and the presence of bleeding from the anastomosis were observed to confirm patency. The incision was then closed layer by layer.

[0064] To alleviate perioperative pain, buprenorphine 0.1 mg / kg was administered subcutaneously 30 minutes before surgery; buprenorphine 0.1 mg / kg was administered subcutaneously every 12 hours postoperatively for 48 hours. Mice were placed in a warm environment postoperatively and returned to their cages after regaining spontaneous activity. Their mental state, feeding, wound healing, and pain response were observed daily.

[0065] The sham surgery group received the same anesthesia, neck incision, vascular dissection and postoperative management, but did not undergo vascular transection and arteriovenous anastomosis.

[0066] The experimental groups included: Sham group (blank control), AVF+PBS group (phosphate buffer solution, pH 7.4), AVF+ZIF-8 NSs group (comparative example 1), AVF+ZIF-8-PEG group (comparative example 2), AVF+Mg@ZIF-8-PEG group (comparative example 3), and AVF+RGD-Mg@ZIF-8-PEG group (Example 1).

[0067] The samples prepared in Examples 1 and Comparative Examples 1-3 were reconstituted into drug dispersions using sterile PBS before administration. These dispersions were vortexed and briefly sonicated before immediate use. Administration to each group began on postoperative day 1 via tail vein injection. Before administration, the weight of each mouse was measured, and the required sample mass per mouse was calculated based on a total sample mass and a dosage of 5 mg / kg. The corresponding sample mass was then reconstituted into a 100 μL drug dispersion using sterile PBS. Each mouse received a single 100 μL tail vein injection, administered every 3 days for a total of 4 administrations on postoperative days 1, 4, 7, and 10. The AVF model control group received 100 μL of sterile PBS at the same administration time and volume.

[0068] Blood was collected from mice on the 28th day after surgery, and AVF anastomosis and outflow tract vein tissue were also collected.

[0069] Anticoagulated blood was centrifuged to prepare plasma, and the levels of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) were measured to evaluate the effects of RGD-Mg@ZIF-8-PEG on coagulation function indicators.

[0070] Part of the AVF vascular tissue was fixed, embedded in paraffin, and sectioned. Hematoxylin-eosin (HE) staining was used to observe the vascular wall structure, lumen morphology, and neointimal formation to evaluate the pathological remodeling of AVF vessels.

[0071] The remaining AVF vascular tissue was homogenized, and malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and tumor necrosis factor-α (TNF-α) level were detected to evaluate local oxidative stress and inflammatory response.

[0072] Serum was prepared by centrifuging non-anticoagulated blood, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to evaluate their impact on liver function indicators.

[0073] Material characterization and experimental results are attached. Figure 1-7 As shown.

[0074] Figure 1 Transmission electron microscopy was used to observe the microstructure of RGD-Mg@ZIF-8-PEG prepared in Example 1. The results showed that RGD-Mg@ZIF-8-PEG exhibited a typical sheet-like structure with a relatively intact overall morphology. Thin sheet outlines were visible at the edges, indicating that the ZIF-8 nanosheet main structure remained stable after Mg ion loading, PEG modification, and RGD peptide coupling. This nanosheet structure has a large surface area, which is beneficial for Mg... 2+ The loading and local release of RGD provide a structural basis for RGD-mediated adhesion and targeted enrichment in damaged endothelial regions. These results demonstrate that the RGD-Mg@ZIF-8-PEG prepared in this application possesses excellent nanosheet morphology and can serve as a functional nanodelivery platform for AVF-damaged endothelial repair.

[0075] Figure 2 The FTIR spectra of RGD-Mg@ZIF-8-PEG, ZIF-8 NSs, ZIF-8-PEG, and Mg@ZIF-8-PEG prepared in Example 1 and Comparative Examples 1-3 are shown. As can be seen from the figures, ZIF-8 NSs shows a wavelength of 1582 cm⁻¹. -1 At C=N and 420 cm -1 The characteristic peak of Zn-N appears; after PEG modification, a peak of 2885 cm⁻¹ appears. -1 (-CH2) and 1110 cm -1 (COC) peak; after Mg loading, a broad OH peak and Mg coordination-related signal appeared; RGD-Mg@ZIF-8-PEG showed Amide I and Amide II peaks, indicating that the RGD peptide was successfully coupled.

[0076] Figure 3 XPS spectra of RGD-Mg@ZIF-8-PEG, ZIF-8 NSs, ZIF-8-PEG, and Mg@ZIF-8-PEG prepared in Example 1 and Comparative Examples 1-3 are shown. In the figures, ZIF-8 NSs shows Zn 2p, N 1s, and C 1s signals; ZIF-8-PEG shows enhanced O 1s and C 1s signals; Mg@ZIF-8-PEG shows a newly added Mg 1s peak; RGD-Mg@ZIF-8-PEG retains the Mg 1s peak with enhanced N 1s signals, indicating successful coupling of Mg loading and RGD peptides.

[0077] Figure 4The figure shows the zeta potential changes of RGD-Mg@ZIF-8-PEG, ZIF-8 NSs, ZIF-8-PEG, and Mg@ZIF-8-PEG prepared in Example 1 and Comparative Examples 1-3. In the figure, ZIF-8 NSs is positively charged; the potential decreases after PEG modification; the positive charge recovers after Mg loading; and the surface charge shifts to negative values ​​after RGD coupling, demonstrating that the surface chemical composition of RGD-Mg@ZIF-8-PEG changes systematically with each step.

[0078] Figure 5 The image shows the coagulation safety evaluation results of RGD-Mg@ZIF-8-PEG prepared in Example 1. PT, APTT, and TT are expressed in seconds (s), and FIB is expressed in g / L. There were no significant differences in PT, APTT, TT, and FIB levels between the PBS group and the RGD-Mg@ZIF-8-PEG group, indicating that RGD-Mg@ZIF-8-PEG did not significantly interfere with coagulation function and has good blood compatibility and coagulation safety.

[0079] Figure 6 The effects of Examples 1, 2, and 3 on oxidative stress, inflammatory response, and liver function safety in AVF model mice are shown in the figure. The results showed that compared with the Sham group, the AVF model group had significantly increased MDA and TNF-α levels, while SOD activity was significantly decreased, indicating that local oxidative stress and inflammatory response were significantly enhanced after AVF modeling. After intervention with RGD-Mg@ZIF-8-PEG, ZIF-8-PEG, and Mg@ZIF-8-PEG, MDA and TNF-α levels were reduced to varying degrees, and SOD activity was restored. The RGD-Mg@ZIF-8-PEG group showed the most significant improvement, indicating that RGD targeted modification and Mg ion loading can synergistically alleviate local oxidative stress and inflammatory damage in AVF mice. Serum ALT and AST levels showed no statistically significant difference between the RGD-Mg@ZIF-8-PEG group and the sham-operated group under the conditions of a dose of 5 mg / kg, a single injection volume of 100 μL / animal, and a total of 4 administrations. This indicates that the material did not cause significant abnormalities in ALT and AST levels under the administration conditions of this experiment, demonstrating good in vivo biocompatibility. Data are expressed as mean ± SD, **P < 0.01, and ns indicates no statistically significant difference.

[0080] Figure 7 shows the effect of RGD-Mg@ZIF-8-PEG prepared in Example 1 on the improvement of vascular tissue pathological remodeling and neointimal hyperplasia in AVF model mice. HE staining was used to observe the pathological morphological changes of vascular tissue in the Sham group, AVF model group, and RGD-Mg@ZIF-8-PEG intervention group. The results showed that the vascular wall structure in the Sham group was relatively intact, the lumen morphology was relatively regular, and no obvious pathological thickening was observed. The AVF model group showed significant vascular wall thickening, with more pronounced local cell proliferation and neointimal formation, indicating a significant trend of vascular remodeling and lumen narrowing after AVF establishment. After RGD-Mg@ZIF-8-PEG intervention, vascular wall thickening and intimal hyperplasia were significantly reduced compared to the AVF group, and the lumen structure was relatively improved. The results indicate that RGD-Mg@ZIF-8-PEG can effectively alleviate vascular pathological remodeling after AVF surgery, promote the repair of damaged vascular endothelium, and help inhibit neointimal hyperplasia and maintain fistula structural stability.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ZIF-8-based nanocomposite material, characterized in that, It includes ZIF-8 nanosheets, a PEG-modified layer, a Mg ion loading module, and an RGD peptide targeting module; the PEG-modified layer is bound to the surface of the ZIF-8 nanosheets, the Mg ion loading module is loaded in the PEG-modified ZIF-8 nanosheet carrier, and the RGD peptide targeting module is grafted onto the PEG-modified layer.

2. A method for preparing a ZIF-8-based nanocomposite material according to claim 1, characterized in that, Includes the following steps: S1. Dissolve zinc source and 2-methylimidazole in an aqueous system or a water-alcohol mixture, add morphology modifier, and react to obtain ZIF-8 NSs; S2. Disperse ZIF-8 NSs in buffer solution, add PEG-COOH, stir and react to obtain ZIF-8-PEG; S3. Disperse ZIF-8-PEG in deionized water, add Mg source, mix and incubate to obtain Mg@ZIF-8-PEG; S4. The PEG-COOH end groups on the surface of Mg@ZIF-8-PEG were activated by EDC / NHS, and amino-modified RGD peptides were added. After the reaction, RGD-Mg@ZIF-8-PEG was obtained.

3. The method for preparing ZIF-8-based nanocomposite materials according to claim 2, characterized in that, In step S1, the zinc source is zinc nitrate or zinc acetate, and the molar ratio of the zinc source to 2-methylimidazole is 1:8-20.

4. The method for preparing ZIF-8-based nanocomposite materials according to claim 2, characterized in that, In step S1, the morphology modifier is polyvinylpyrrolidone or triethylamine, and the mass ratio of the morphology modifier to the zinc source is 1-5:

1.

5. The method for preparing ZIF-8-based nanocomposite materials according to claim 2, characterized in that, In step S2, the buffer solution is a MES buffer solution with a pH of 6.0-6.

5.

6. The method for preparing ZIF-8-based nanocomposite materials according to claim 2, characterized in that, In step S3, the Mg source is MgCl2 or Mg(OAc)2, and the mass ratio of ZIF-8-PEG to the Mg source is 1:0.5-2.

7. The method for preparing ZIF-8-based nanocomposite materials according to claim 2, characterized in that, In step S3, the temperature for the mixed incubation is 20-40℃, and the time is 12-24h.

8. The method for preparing ZIF-8-based nanocomposite materials according to claim 2, characterized in that, In step S4, the mass ratio of Mg@ZIF-8-PEG to amino-modified RGD polypeptide is 1:0.003-0.

02.

9. The method for preparing ZIF-8-based nanocomposite materials according to claim 2, characterized in that, In step S4, the reaction temperature is 20-40℃ and the reaction time is 2-6h.

10. The use of the ZIF-8-based nanocomposite material according to claim 1 in the preparation of a medicament for treating arteriovenous fistula.

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