Preparation method of metal-based nano-enzyme and application of metal-based nano-enzyme in treatment of periodontitis

Abstract

The invention discloses a preparation method of a metal-based nano-enzyme and application of the metal-based nano-enzyme in treatment of periodontitis, the nano-enzyme combines two metal bases, and the nano-enzyme exerts anti-inflammatory, vascularization and osteogenesis effects through advantage complementation to treat periodontitis. The nano-enzyme for treating periodontitis has excellent anti-oxidation / anti-inflammatory performance, exerts an ROS elimination function in a specific environment, responds to a periodontitis pathology microenvironment to release metal ions, exerts a growth factor-like function of the metal ions and realizes repair of blood vessels and bone tissues. The multifunctional nano material has the advantages of simple process, low cost, high activity and the like, and has a good treatment effect on periodontitis.

Description

Technical Field

[0001] The present invention belongs to the research and development field of periodontitis nanomedicines, and relates to a method for preparing a metal-based nanozyme and its application in the treatment of periodontitis, and specifically to a method for preparing a multifunctional metal-based nanozyme responsive to a pathological environment and its use in the treatment of periodontitis. Background Art

[0002] Periodontitis is the destruction of periodontal tissues caused by pathogenic bacteria activating the host's immune system. Local tissue defects can affect the function of the oral and maxillofacial system. The goal of periodontal treatment is usually to control local inflammation and obtain tissue healing and structural regeneration to restore function and aesthetics. Clinically, mechanical-based treatment is the main approach, which can effectively control plaque and reduce inflammation, thereby alleviating disease progression and achieving tissue regeneration and repair through the host itself. However, for patients with advanced periodontitis (large tissue defects), even if plaque biofilms are effectively removed to control inflammation, satisfactory tissue regeneration effects and functional reconstruction cannot be achieved. They often have to rely on periodontal regeneration such as bone grafting and guided tissue regeneration to maintain healthy physiological functions. However, the implementation of periodontal surgery will increase the patient's damage and also create a greater risk of aesthetic restoration.

[0003] Current strategies for the treatment of periodontitis focus on the development of anti-inflammatory properties. Although they can effectively control the progression of inflammation, they cannot achieve long-term and stable tissue regeneration effects. Many metal ions (such as copper, iron, zinc, magnesium, strontium, and calcium) have good biological functions. While controlling inflammation, they can play a growth factor-like function to promote the regeneration of blood vessels and bone tissue to achieve periodontal tissue reconstruction. However, the biosafety of these metal ions limits their use. Although high-dose use can ensure the control of inflammation, there is a corresponding cytotoxicity risk; low-dose use can play a growth factor-like role, but it is difficult to achieve a stable anti-inflammatory treatment goal. How to ensure the stability of anti-inflammatory, angiogenic and osteogenic functions in the treatment and regeneration of periodontitis has become a technical difficulty. Antioxidant nanozymes have stable catalytic properties and can efficiently eliminate reactive oxygen species (ROS) to achieve good anti-inflammatory effects. However, the functions of antioxidant nanozymes are relatively single, and it is difficult to achieve ideal tissue repair effects.

[0004] Therefore, in response to the needs of clinical disease diagnosis and treatment and taking into account the pathological microenvironment of periodontitis, we seek a new type of multifunctional nanozyme to better match the treatment needs of periodontitis. Summary of the invention

[0005] Purpose of the invention: In view of the shortcomings of current periodontitis treatment, the technical problem to be solved by the present invention is to provide a method for preparing metal-based nanozymes with good biosafety, integrating metal ions with growth factor-like activity with antioxidant nanozymes.

[0006] The technical problem that the present invention also aims to solve is that the prepared metal-based nanozyme has good anti-inflammatory, angiogenic and osteogenic functions when used in the treatment of periodontitis.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a method for preparing a metal-based nanozyme, wherein the metal-based nanozyme is prepared in situ using a nanozyme or a precursor of a nanozyme, wherein the precursor of the nanozyme comprises an inorganic metal salt, wherein the inorganic metal salt comprises a metal ion having growth factor-like activity, and the nanozyme or the precursor of the nanozyme is an antioxidant nanozyme.

[0008] Preferably, the metal-based nanozyme is a metal-based nanozyme with a core-shell structure.

[0009] Among them, the metal ions with growth factor-like activity include but are not limited to one or more of copper ions, zinc ions, iron ions, fluoride ions, strontium ions, magnesium ions or calcium ions; preferably, the metal ions are copper ions; the antioxidant nanozymes include but are not limited to one or more combinations of cerium-based nanozymes, manganese-based nanozymes, ruthenium-based nanozymes, platinum-based nanozymes, nickel-based nanozymes, zirconium-based nanozymes, and MOF-based nanozymes; preferably, the antioxidant nanozyme is a ruthenium-based nanozyme.

[0010] Wherein, the inorganic metal salt includes but is not limited to one or more of cerium nitrate, manganese nitrate, manganese chloride, zirconium oxynitrate, zirconium chloride, nickel chloride, nickel nitrate, copper chloride, copper nitrate, ruthenium chloride or potassium chloroplatinite; preferably, the inorganic metal salt includes one or both of copper nitrate and ruthenium chloride.

[0011] Preferably, the metal-based nanozyme described in the present invention is a bimetallic nanozyme.

[0012] Preferably, the bimetallic nanozyme of the present invention is a bimetallic nanozyme obtained by reacting a metal nanozyme with an inorganic metal salt.

[0013] Preferably, the bimetallic nanozyme is a bimetallic nanozyme with a core-shell structure, and the bimetallic nanozyme with a core-shell structure is obtained by adding acid to the bimetallic nanozyme to carry out an etching reaction.

[0014] Preferably, the acid includes but is not limited to nitric acid.

[0015] The preparation method includes but is not limited to one or more of hydrothermal treatment, physical blending treatment, and acid etching treatment.

[0016] Among them, the metal-based nanozyme has intelligent response characteristics to the pathological microenvironment, including but not limited to one or more of pH response, enzyme response, temperature response, light response, molecular response and ROS response; preferably, the nanozyme is dual-responsive to pH and ROS.

[0017] The present invention also includes the use of the metal-based nanozyme in the preparation of drugs for treating periodontitis.

[0018] Wherein, the drugs include drugs for anti-inflammatory or bone-promoting ability.

[0019] Wherein, the medicine includes a medicine that promotes tissue regeneration.

[0020] Wherein, the regenerated tissue includes gums, periodontal ligament, cementum, alveolar bone and blood vessels.

[0021] Wherein, the drug includes a drug for increasing the expression level of functional proteins in endothelial tissue.

[0022] The present invention combines metal ions with growth factor-like activity with antioxidant nanozymes to ensure biosafety and exert biological functions in response to the pathological microenvironment of periodontitis.

[0023] As an embodiment of the present invention, the copper ions come from copper-based nanozymes.

[0024] As an embodiment of the present invention, the preparation method of the copper-based nanozyme is: polyvinyl pyrrolidone and Cu(NO3)2·3H2O are mixed and dissolved in ethylene glycol, and the copper-based nanozyme is obtained after heating.

[0025] As an embodiment of the present invention, the preparation method of the copper ruthenium nanozyme of the present invention comprises the following steps: reacting the copper-based nanozyme with ruthenium trichloride hydrate, stirring evenly at room temperature, recovering the product by centrifugation, dispersing the obtained product in water and adjusting the pH of the solution, heating the solution, recovering the product by centrifugation, and washing the product with pure water to obtain the copper ruthenium nanozyme.

[0026] As an embodiment of the present invention, the preparation method of the core-shell structured copper ruthenium nanozyme of the present invention comprises the following steps: dissolving and dispersing the copper ruthenium nanozyme in deionized water, slowly adding concentrated HNO3 solution while stirring, incubating for 0.5 h to 1 h, and collecting the product by centrifugation to obtain a copper ruthenium-based nanozyme with a multifunctional core-shell structure.

[0027] The present invention also includes the preparation method for preparing a copper-ruthenium-based nanozyme with a core-shell structure.

[0028] The present invention also includes the use of the copper-ruthenium-based nanozyme or the copper-ruthenium-based nanozyme with a core-shell structure in the preparation of a drug for treating periodontitis.

[0029] The drugs include drugs that reduce the clearance of intracellular ROS in macrophages and reduce the gene expression level of pro-inflammatory factors; preferably, the pro-inflammatory factors include but are not limited to one or more of IL-1β and TNF-α.

[0030] The drug includes a drug that promotes the ability of vascular endothelial cells to form small tubes and improve cell migration.

[0031] Wherein, the medicine includes a medicine that promotes the osteogenic ability of periodontal ligament stem cells.

[0032] Wherein, the drug includes a drug that increases the expression level of functional proteins in endothelial tissue. The functional proteins include but are not limited to VEGF-A.

[0033] The mechanism of the present invention is as follows: when metal ions are released into tissues, they can regulate the secretion of various growth factors to participate in the process of angiogenesis and bone mineralization: by increasing the expression and transcription of vascular active factors such as VEGF in tissues, promoting the proliferation and migration of endothelial cells, and forming a tubular structure to extend to the site of injury to supply blood and nutrition, showing good angiogenesis potential; by enhancing the activity of ALP, the collagen fiber deposition and mechanical properties of the bone matrix are increased, and the stress conduction stimulation between tissues is increased, thereby promoting the generation of new mineralized nodules and accelerating bone formation. In addition, angiogenesis is the basic condition for bone defect repair. Through the coupling of angiogenesis-osteogenesis, blood vessels and bones are closely connected, which is more conducive to the osteogenic differentiation of stem cells, the proliferation and functional regulation of osteoblasts, thereby improving the quality of repair. The present invention is based on obtaining effective copper ion angiogenesis and osteogenic function, and controls the content of copper-based nanozymes by the length of acid etching time, and uses ruthenium-based antioxidant nanozymes to make up for the anti-inflammatory effect within the safe dose range. Too high a content of copper ions will bring toxicity to cells, and too low a content of copper ions will not achieve the effect, so its etching time is extremely critical. The copper-ruthenium-based nanozyme prepared by the present invention has an outer layer of ruthenium-based antioxidant nanozyme that can catalyze the elimination of excess ROS in the periodontitis microenvironment and provide environmental support for tissue regeneration; the inner layer of copper-based nanozyme is oxidized under oxidative stress and acidic conditions and releases copper ions through surface pores, exerting biological functions and having a good effect on the treatment of periodontitis.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention obtains a new type of bimetallic nanozyme through a simple synthesis method. The present invention forms a nanomedicine by co-assembling a material with catalytic function and a metal ion with biological activity, which shows a good effect of eliminating intracellular ROS and improving the angiogenesis / osteogenesis function of cells under inflammatory environment. After being applied to a rat periodontitis model, it shows good in vivo anti-inflammatory, angiogenesis and osteogenic effects. By changing the type of functional element with copper ion release and the type of element with catalytic function, it can be extended to the synergistic treatment of other diseases. The multifunctional nanomedicine of the present invention has the advantages of simple preparation process, green economy, environmental friendliness, low cost, simple process and mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a schematic diagram of the therapeutic effect of the metal-based nanozyme CRNC of the present invention on periodontitis.

[0036] Figure 2 This is the preparation process of the metal-based nanozyme of the present invention.

[0037] Figure 3 The biocompatibility of nanozymes. (a) Activity detection of different concentrations of Cu2O nanozymes and macrophages RAW264.7 after culturing for 1 day; Activity detection of macrophages RAW264.7 and CRNC nanozymes prepared by etching for (b) 0.5h and (c) 1h after culturing for 1 day; Cell activity detection after CRNC nanozymes prepared by etching for 1h were co-cultured with (d) vascular endothelial cells HUVEC (1, 3d) and (e) periodontal ligament stem cells PDLSC (1, 3, 5, 7d).

[0038] Figure 4 Characterization of CRNC nanozymes. Images of (a) scanning electron microscopy, (b) transmission electron microscopy, (c) high-resolution transmission electron microscopy, and (d) element distribution of CRNC nanozymes; spectra of (e) Cu 2p, (f) Ru 3p, and (g) O1s of CRNC nanozymes; (h) surface potential detection of Cu2O, RNC, and CRNC nanozymes, and (i) copper ion release detection of CRNC nanozymes.

[0039] Figure 5 The anti-inflammatory effect of CRNC nanozymes. (a) Detection of SOD-like and CAT catalytic activities of CRNC nanozymes; detection of intracellular ROS removal in macrophages RAW264.7 (bc) and gene expression of pro-inflammatory factors (d) IL-1β and (e) TNF-α under inflammatory conditions.

[0040] Figure 6CRNC nanozymes promote angiogenesis in vitro. After using CRNC nanozymes in an inflammatory environment, the (ab) tubule formation ability, (cd) vertical movement and (ef) horizontal migration ability of HUVEC were detected.

[0041] Figure 7 The effect of CRNC nanozymes on osteogenic differentiation of PDLSCs. (a) ALP activity, (b) Alizarin red staining images, and (c) expression of osteogenic-related proteins.

[0042] Figure 8 The biological effects of copper ions. Comparison of the gene expression levels of proinflammatory factors (a) IL-1β and (b) TNF-α in RAW264.7 cells, (c) HUVEC tubule formation ability, and (d) ALP activity in PDLSCs under the action of low-concentration CRNC nanozymes and high-concentration RNC nanozymes.

[0043] Fig. 9 The in vivo anti-inflammatory and osteogenesis effects of CRNC nanozymes. (a) In vivo imaging to observe the level of ROS in rat periodontal tissues and (b) micro-CT to detect periodontal bone regeneration in rats.

[0044] Fig.10 The culture and staining of vascular tissue. (a) Extraction and culture of abdominal aorta; (b) HE staining, (c) Masson staining and (d) immunohistochemical VEGF-A staining analysis of vascular tissue. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is described in detail below with reference to examples.

[0046] Example 1 Preparation of CRNC Nanozymes and RNC Nanozymes

[0047] 1. 600 mg of polyvinyl pyrrolidone and 2 mmol of Cu(NO3)2·3H2O were mixed into a reactor and dissolved in 50 mL of ethylene glycol. Subsequently, the mixed solution was heated to 185°C and maintained for 1 h under the action of a magnetic stirrer. After cooling naturally to room temperature, the product was collected by centrifugation. Finally, the obtained product was washed alternately with ethanol and distilled water three times to obtain Cu2O nanozyme (product 1).

[0048] 2. Take 50 mg of the above-synthesized Cu2O nanozyme product 1 and dissolve it in 10 mL of pure water, then add 1 mL of RuCl3.xH2O (Cas number 14898-67-0, purchased from Shanghai Aladdin, item number R109234, molecular weight 207.43) solution containing 75 mg / mL, and stir at room temperature for 30 min until the solution is evenly mixed. Subsequently, the product was recovered by centrifugation, the obtained product was dispersed in 20 mL of water and the pH of the solution was adjusted to 10.0, and the solution was heated at 80 ° C for 30 min to improve the coating quality of RuO2 on the Cu2O surface, and finally the product was recovered by centrifugation, and the product was washed three times with pure water to obtain a core-shell structure (core-shell) Cu2O@RuO2 nanozyme (product 2).

[0049] 3. Then, HNO3 was used to etch it. 30 mg of Cu2O@RuO2 nanozyme product 2 was dissolved and dispersed in 20 mL of deionized water, and 10 μL of 68% concentrated HNO3 solution was slowly added while stirring. In order to control the internal copper content, after stirring and incubating for 0.5 h and 1 h, the products were collected by centrifugation, and washed alternately with pure water and anhydrous ethanol three times to obtain core-shell structure (yolk-shell) Cu2O@RuO2 (CRNC) nanozyme (product 3) and Cu2O@RuO2 nanozyme (product 4).

[0050] 4. In order to completely remove the internal copper, 30 mg of Cu2O@RuO2 nanozyme product 2 was dissolved in 20 mL of deionized water, and 30 μL of 65% concentrated HNO3 solution was slowly added while stirring. After stirring and incubating for 1 h, the product was collected by centrifugation and washed alternately with pure water and anhydrous ethanol three times to obtain hollow RuO2 nanozyme (RNC) (product 5) as a control.

[0051] Example 2 Biocompatibility of CRNC Nanozymes

[0052] Macrophages RAW264.7, human umbilical vein endothelial cells (HUVEC) and periodontal ligament stem cells (PDLSC) were cultured at 5×10 3 / well inoculated in a 96-well plate, after the cells adhered to the wall, different final concentrations (0, 5, 10, 20, 50 μg / mL) of Cu2O nanozyme (product 1), CRNC nanozyme (product 3), and Cu2O@RuO2 nanozyme (product 4) were added and co-cultured with the three types of cells (RAW264.7 cells were cultured for 1 day; HUVECs were cultured for 1 and 3 days; PDLSCs were cultured for 1, 3, 5, and 7 days). CCK-8 solution was used to detect cell activity at a wavelength of 450 nm on an ELISA reader. The results of RAW264.7 cells cultured for 1 day are shown in Figure 2. Figure 3a, 10 μg / mL Cu2O nanozymes inhibited the proliferation of the three cells, showing strong toxicity; CRNC nanozymes prepared with different etching times also had different effects on the proliferation of macrophages, such as Figure 3 b shows the effect of CRNC nanozyme (product 3) on the proliferation of macrophage RAW264.7. Figure 3 c is the effect of Cu2O@RuO2 nanozyme (product 4) on the proliferation of macrophages RAW264.7. The Cu2O@RuO2 nanozyme product 3 prepared by etching for 0.5h inhibits the proliferation of macrophages at a concentration of 20μg / mL, while the Cu2O@RuO2 nanozyme product 4 prepared by etching for 1h shows the corresponding effect at 50μg / mL, proving that extending the etching time from 0.5h to 1h can help reduce the toxicity of CRNC nanozyme. Subsequently, the cell activity of HUVEC and PDLSC was detected using the Cu2O@RuO2 nanozyme (product 4) prepared by etching for 1h. The results showed that with the extension of culture time, HUVEC ( Figure 3 d) and PDLSC ( Figure 3 e) The number of cells was gradually increasing, while the Cu2O@RuO2 nanozyme (product 4) at a concentration of 20 μg / mL still had no effect on the activity of the two cells, showing good biocompatibility. Therefore, CRNC nanozyme product 4 was selected for subsequent experiments.

[0053] Example 3 Characterization of CRNC Nanozyme

[0054] The surface microstructure and element distribution of the Cu2O@RuO2 nanozyme (product 4) prepared in Example 1 were observed using SEM, TEM and HADDF-STEM; XPS energy spectrum, zeta surface potential and ICP detection of Cu 2+ Release. The result is Figure 4 , Figure 4 The results show that CRNC nanozyme was successfully synthesized with a particle size of about 190nm. After etching, the surface of CRNC became rougher and pores were formed. The Cu element was distributed in the inner layer of the material, while Ru could evenly cover the surface of the Cu element. The O element was distributed in the core-shell layer of the material in the form of two metal oxides, verifying the core-shell structure of the material. The high-resolution spectrum of Cu 2p can be analyzed to observe the Cu + The dominant peaks indicate that the copper in CRNC nanozyme exists mainly in the form of Cu2O. The analysis of Ru 3p and O1s spectra confirms that a certain amount of Ru exists on the surface of CRNC nanozyme. 3+ and oxygen vacancies, laying the foundation for its catalytic activity; Figure 4h It can be seen that the Zeta potential of Cu2O is -1.1mV. After being wrapped and etched by RuO2 shell, the Zeta potential of CRNC nanozyme changes from negative to 23.3mV. Figure 4 It can be seen that as the pH in the solution decreases, Cu2O decomposes responsively, and the extension of time will also enhance the 2+ In addition, the presence of H2O2 further accelerates the dissolution of Cu2O and the release of Cu 2+ The release process confirmed that the system had pH and oxidative stress responses.

[0055] Example 4 Anti-inflammatory effect of CRNC nanozyme

[0056] The WST-1 solution was used to detect the catalytic performance of Cu2O nanozyme, RNC nanozyme and CRNC nanozyme (Cu2O@RuO2 nanozyme (product 4)) in simulating SOD; the H2O2 probe was used to detect the catalytic performance of Cu2O nanozyme, RNC nanozyme and CRNC nanozyme in simulating CAT.

[0057] RAW264.7 cells were cultured at 1×10 5 / well were inoculated in a 6-well plate, and after the cells adhered to the wall, 200ng / mL of LPS was added and cultured for 12h. After washing with PBS (0.01M, pH=7.4, the same below), 20μg / mL of RNC nanozyme or CRNC nanozyme (product 4) was added. After culturing for 24h, it was washed with PBS and incubated with DCFH-DA probe at 37℃ for 20min. After washing with PBS three times, it was observed under a laser confocal microscope; at the same time, total cell RNA was extracted after the corresponding time, and RT-qPCR experiments were performed after reverse transcription to detect the levels of proinflammatory cytokines IL-1β and TNF-α in the cells. The results are shown in Figure 5 As shown, Figure 5 The results of a showed that CRNC nanozymes showed the best SOD-like and CAT activities. With the increase of concentration (5, 10, 20, 50 μg / mL), O2 ·- and H2O2 removal efficiency gradually increased; Figure 5 The results of bc showed that the level of intracellular ROS increased under the action of LPS, and the two nanozymes had a significant scavenging effect on intracellular ROS; Figure 5 de showed that nanozyme treatment can reduce the gene expression levels of IL-1β and TNF-α in macrophages and alleviate inflammatory responses. Overall, copper-containing CRNC nanozymes have better effects on eliminating ROS in macrophages and reducing intracellular inflammatory responses than RNC nanozymes.

[0058] Example 5 Pro-angiogenic effect of CRNC nanozymes

[0059] HUVECs were cultured at 1.5 × 10 5 / well were inoculated in a 6-well plate. After the cells adhered to the wall, LPS with a final concentration of 10 μg / mL and RNC nanozyme or CRNC nanozyme (Cu2O@RuO2 nanozyme product 4) with a final concentration of 20 μg / mL were added to each well. After culturing for 3 days, the cells were digested, centrifuged and collected for later use. Pre-cooled Matrigel matrix gel was added to a 96-well plate at a content of 50 μL / well and incubated at 37°C for 30 minutes. After the gel solidified, the concentration of the above collected cells was adjusted to 1.5×10 4 100 μL of cell suspension was added to each well and incubated at 37°C. After 6 h, the formation of cell tubular structures was observed under a microscope. Serum-free ECM medium was used to adjust the cell concentration to 1×10 5 / mL, add 200 μL of cell suspension to the upper chamber of a 24-well transwell plate, add 600 μL of ECM complete medium to the lower chamber, culture for 24 hours, fix, stain with 0.1% crystal violet, and take pictures under a microscope to observe the vertical migration of cells.

[0060] HUVEC were cultured at 2×10 5 / well were inoculated in a 6-well plate. After the cells adhered to the wall, LPS with a final concentration of 10μg / mL and RNC nanozyme or CRNC nanozyme (Cu2O@RuO2 nanozyme product 4) with a final concentration of 20μg / mL were added to each well and cultured for 3 days. A 200μL micropipette tip was used to make a straight scratch perpendicular to the bottom of the well plate, PBS was used to wash to remove cell debris, serum-free medium containing double antibodies was added to incubate for 24h, and the horizontal migration of cells was observed under a microscope.

[0061] The results are as follows Figure 6 As shown, Figure 6 The results of ab showed that the number of tubular structures formed by HUVEC was significantly reduced after LPS treatment, while the tubule formation ability of HUVEC was significantly restored after treatment with nanozymes; the excessive inflammation caused by LPS stimulation inhibited the vertical ( Figure 6 cd) and level ( Figure 6 ef) Mobility, the treatment of the two nanozymes reversed the inhibitory effect of LPS and showed a good effect of promoting cell migration. Overall, CRNC nanozymes have a better ability to promote HUVEC tubule formation and cell migration under inflammatory conditions than RNC nanozymes.

[0062] Example 6 Osteogenic effect of CRNC nanozyme

[0063] PDLSCs were cultured at 2.5 × 10 4 / well were inoculated in a 24-well plate. After the cells adhered to the wall, the medium was replaced with osteogenic induction medium, and LPS with a final concentration of 10 μg / mL and RNC nanozyme or CRNC nanozyme (Cu2O@RuO2 nanozyme product 4) with a final concentration of 10 μg / mL were added. The medium was changed every 3 days, and the total cell protein was extracted on the 7th day for western blot detection of the expression of osteogenic-related proteins; ALP staining was performed on the 14th day; ARS staining was performed on the 21st day.

[0064] The results are as follows Figure 7 As shown, the results showed that both nanozymes could promote the ALP activity of PDLSCs under inflammatory conditions ( Figure 7 a) and calcium nodule deposition ( Figure 7 b), and increased the expression levels of BMP2, OCN, and RUNX2 in PDLSCZ under inflammatory conditions ( Figure 7 c) Overall, CRNC showed a better osteogenic effect than RNC.

[0065] Example 7 Biological effects of copper ions

[0066] The relief of inflammation is beneficial to the regeneration of blood vessels and bone tissue, but CRNC has a better anti-inflammatory effect than RNC at the same concentration, and can more significantly eliminate ROS in LPS-induced macrophages and reduce the expression level of pro-inflammatory factors (see Example 4 for details). We increased the concentration of RNC to eliminate the anti-inflammatory advantage of CRNC at the same dose, so as to further explore the angiogenic and osteogenic activity of copper ions.

[0067] RAW264.7 cells were cultured at 1×10 5 / well inoculated in 12 or 24-well plates, after the cells adhered to the wall, 200 ng / mL LPS was added and cultured for 12 hours, and after washing with PBS, CRNC nanozymes (Cu2O@RuO2 nanozyme product 4) with a final concentration of 10 μg / mL or 20 μg / mL (or RNC nanozymes with a final concentration of 12.5 μg / mL or 25 μg / mL) were added respectively, and cultured for 24 hours and then washed with PBS, and total cell RNA was extracted, and the IL-1β and TNF-α gene levels were detected after reverse transcription. The results are shown in Figure 8 a and Figure 8 b. Example 4 has demonstrated that CRNC has better anti-inflammatory activity at the same concentration, while Figure 8 The results of ab show that this example proves that reducing the amount of CRNC nanozyme used can achieve the same anti-inflammatory effect as high-concentration RNC nanozyme, thus further proving that the addition of copper ions plays a synergistic role, which can achieve comparable effects against two inflammatory factors IL-1β and TNF-α at lower concentrations.

[0068] Furthermore, HUVECs were cultured at 1.5×10 5 / well were seeded in a 6-well plate. After the cells adhered to the wall, LPS at a final concentration of 10 μg / mL and 20 μg / mL of CRNC nanozyme (Cu2O@RuO2 nanozyme product 4) (or 25 μg / mL of RNC nanozyme) were added to each well. After 3 days of culture, the cells were digested, centrifuged and collected for tubule formation ability detection. The results are shown in Figure 8 c. Low concentration of CRNC nanozyme (20 μg / mL) still has a better ability to promote HUVEC tube formation than high concentration of RNC nanozyme (25 μg / mL).

[0069] Furthermore, the PDLSCs after different treatments were cultured at 2.5×10 4 / well were inoculated in a 24-well plate. After the cells adhered to the wall, the medium was replaced with osteogenic induction medium, and 10 μg / mL LPS and 10 μg / mL CRNC nanozyme (Cu2O@RuO2 nanozyme product 4) (or 12.5 μg / mL RNC nanozyme) were added. The medium was changed every 3 days, and ALP staining was performed after 7 days of induction culture. Results are shown in Figure 8 d, Low concentration of CRNC still has a better effect on increasing ALP activity in PDLSCs than high concentration of RNC.

[0070] In summary, this example illustrates that the Cu contained in CRNC nanozyme 2+ It can help CRNC to have better angiogenesis and osteogenesis, and has the effect of synergizing RNC enzymes.

[0071] Example 8 In vivo experiment

[0072] Male SD rats, SPF grade, weighing about 180-200 g, were used for the following experiments after adaptive feeding:

[0073] 1) Under local anesthesia, 50 μL of LPS solution (1 mg / mL) was injected into the gingival papilla of the mandibular incisor of rats once a day for 3 consecutive days to establish a periodontal inflammation model. Then on the 4th day, 50 μL of RNC or CRNC nanozymes (200 μg / mL) were injected into the corresponding area for treatment. After 24 hours, after isoflurane anesthesia, the DCFH-DA probe solution was injected in situ, and the local ROS level was observed by in vivo imaging. The experiment was divided into 4 groups, with 3 SD male rats in each group: ① Control group Ctrl: healthy male SD rats were injected with PBS for 4 days and injected with DCFH-DA probe solution on the 5th day; ② LPS group: male SD rats were injected with LPS for the first 3 days, PBS for the 4th day, and DCFH-DA probe solution for the 5th day; ③ LPS+RNC nanozyme group: male SD rats were injected with LPS for the first 3 days, RNC nanozyme for the 4th day, and DCFH-DA probe solution for the 5th day; ④ LPS+CRNC nanozyme group: male SD rats were injected with LPS for the first 3 days, CRNC nanozyme for the 4th day, and DCFH-DA probe solution for the 5th day; the results are shown in Fig. 9 a. Fig. 9 The results of a showed that a surge in ROS levels in rat gingival tissue could be observed after LPS injection, while treatment with CRNC nanozymes could significantly reduce the level of local ROS, which was beneficial to the relief of inflammation in periodontal tissue.

[0074] 2) Under local anesthesia, a 0.2 mm ligature was passed through the mesial and distal spaces of the maxillary second molar and ligated on the buccal side to establish a periodontitis model. The rats were observed after ligation for 1 week, and 25 μL of RNC or CRNC nanozyme (Cu2O@RuO2 nanozyme product 4) with a concentration of 200 μg / mL was injected on the palatal side of the second molar, once every 3 days; after 4 weeks, the rats were killed and the maxilla of the rats were collected for micro-CT detection and analysis of the mineralization density (BMD) of the alveolar bone. The experiment was divided into 5 groups, with 6 rats in each group: ① Control group Ctrl: healthy male SD rats were not treated; ② Sham group Sham: the ligature was removed immediately after ligation and PBS treatment was performed; ③ Periodontitis group PD: PBS treatment was performed after ligation; ④ Periodontitis (PD) + RNC group: RNC nanozyme treatment was performed after ligation; ⑤ Periodontitis + CRNC group: CRNC nanozyme (Cu2O@RuO2 nanozyme product 4) was treated after ligation. Results can be found in Fig. 9 The CT images and reconstructed images in b show (red arrows; red frames), excluding the effects of ligation surgery and drug injection process on bone tissue (Sham group), the ligation of orthodontic wires will cause a decrease in the mineralization density of the alveolar bone around the second molar, while the treatment with CRNC nanozymes can significantly improve the BMD parameters of the jaw, indicating that CRNC nanozymes improve the bone condition in periodontitis.

[0075] 3) 16 healthy male SD rats, weighing about 160-180g, were killed by cervical dislocation. The abdomen was cut open, the fascia and blood vessels were separated, and the abdominal aorta of the rats was collected and immersed in PBS. The rats were transferred to a clean bench and rinsed repeatedly 5 times with PBS containing 10% double antibody. Then, the rats were divided into 4 groups, each with 4 rats, and cultured in ECM complete culture medium. One group was used as the ctrl group without any treatment. The other 3 groups included: ②LPS group: only 20μg / mL of LPS was added; ③LPS+RNC nanozyme group: 20μg / mL of LPS and 40μg / mL of RNC nanozyme; ④LPS+CRNC nanozyme group: 20μg / mL of LPS and 40μg / mL of CRNC nanozyme (Cu2O@RuO2 nanozyme product 4). After 7 days of culture, the tissues were fixed with 4% paraformaldehyde for subsequent embedding, sectioning and staining. The results after culturing the rat vascular tissue are as follows. Fig.10 a, Compared with the control group, under long-term inflammatory stimulation, the vascular endothelium showed a loose structure, wrinkles, disappearance of surface villi, and enlarged tissue gaps ( Fig.10 b); and the collagen fiber content is reduced, the structure is disordered, and the elasticity of the blood vessel wall is reduced ( Fig.10 c). Under the treatment of RNC nanozymes or CRNC nanozymes, the endothelial tissue restored the state of tight junctions, the detached cell fragments and fibers were significantly reduced, and the number of collagen fibers was significantly increased. In addition, the expression of endothelial cell functional protein VEGF-A was significantly reduced under the action of LPS, while the treatment of CRNC nanozymes can help increase the expression level of VEGF-A in endothelial tissue ( Fig.10 de).

[0076] The results showed that CRNC nanozymes can help restore the morphology and function of vascular tissue under inflammatory conditions, thereby exerting the effect of promoting vascular tissue regeneration.

Claims

1. A method for preparing a metal-based nanozyme, characterized in that: The metal-based nanozyme is prepared in situ using a nanozyme or a nanozyme precursor, the nanozyme precursor includes an inorganic metal salt, the inorganic metal salt contains a metal ion with growth factor-like activity, the nanozyme or the nanozyme precursor is an antioxidant nanozyme, preferably, the metal-based nanozyme is a bimetallic nanozyme, preferably, the bimetallic nanozyme is a bimetallic nanozyme with a core-shell structure.

2. The method for preparing the metal-based nanozyme according to claim 1, characterized in that: The metal ions with growth factor-like activity include one or more of copper ions, zinc ions, iron ions, strontium ions, magnesium ions or calcium ions; preferably, the metal ions are copper ions; the antioxidant nanozymes include one or more combinations of cerium-based nanozymes, manganese-based nanozymes, ruthenium-based nanozymes, platinum-based nanozymes, nickel-based nanozymes, zirconium-based nanozymes, and MOF-based nanozymes; preferably, the antioxidant nanozyme is a ruthenium-based nanozyme.

3. The method for preparing the metal-based nanozyme according to claim 1, characterized in that: The inorganic metal salt includes one or more of cerium nitrate, manganese nitrate, manganese chloride, zirconium oxynitrate, zirconium chloride, nickel chloride, nickel nitrate, copper chloride, copper nitrate, ruthenium chloride or potassium chloroplatinite; preferably, the inorganic metal salt includes one or both of copper nitrate and ruthenium chloride.

4. The method for preparing the metal-based nanozyme according to any one of claims 1 to 3, characterized in that: The preparation method comprises one or more of hydrothermal treatment, physical blending treatment and acid etching treatment.

5. The metal-based nanozyme prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The metal-based nanozyme has intelligent response characteristics to pathological microenvironment, including one or more of pH response, enzyme response, temperature response, light response, molecular response and ROS response; preferably, the nanozyme is dual-responsive to pH and ROS.

6. Use of the metal-based nanozyme according to claim 5 in the preparation of a drug for treating periodontitis.

7. The use according to claim 6, characterized in that: The drugs include anti-inflammatory or bone-promoting drugs.

8. The use according to claim 6, characterized in that: The drugs include drugs that promote tissue regeneration.

9. The use according to claim 8, characterized in that: The regenerative tissue includes gingiva, periodontal ligament, cementum, alveolar bone and blood vessels.

10. The use according to claim 6, characterized in that: The drugs include drugs that increase the expression level of functional proteins in endothelial tissue.

Images