Hydrotalcite nano composite material as well as preparation method and application thereof

Abstract

The invention discloses a hydrotalcite nano composite material as well as a preparation method and application thereof, and belongs to the technical field of medical bone tissue regeneration materials. The hydrotalcite nano composite material comprises an etched zinc-copper-molybdenum hydrotalcite nanosheet, magnesium peroxide and a 45S5 bioactive glass bracket, magnesium peroxide is loaded on the etched zinc-copper-molybdenum hydrotalcite nanosheet, and is marked as a-ZnCuMo-LDH (at) MgO2; the a-ZnCuMo-LDH (at) MgO2 is attached to the surface of the 45S5 bioactive glass bracket, and the alpha-ZnCuMo-LDH (at) MgO2 is marked as a-ZnCuMo-LDH (at) MgO2-BG. The hydrotalcite nano composite material disclosed by the invention has sonodynamic performance, relatively low cytotoxicity and degradability, good biocompatibility, excellent anti-tumor performance and bone repair promotion capability, and has potential application prospects in related orthopedic surgeries.

Description

Technical Field

[0001] This invention belongs to the field of medical bone tissue regeneration materials technology, specifically relating to a hydrotalcite nanocomposite material, its preparation method, and its application. Background Technology

[0002] Malignant bone tumors, especially osteosarcoma, face the dual challenges of radical resection and functional reconstruction in clinical treatment. On the one hand, the highly invasive nature of tumors makes it difficult to completely remove micrometastases surgically, resulting in a high risk of postoperative recurrence. Traditional systemic chemotherapy is limited in its efficacy due to severe toxic side effects (such as cardiotoxicity and bone marrow suppression) and insufficient local drug concentrations in the tumor. On the other hand, the large, irregular bone defects left after tumor resection exceed the supply limit of autologous bone transplantation. Allogeneic bone carries the risk of immune rejection, while traditional bioceramic materials (such as hydroxyapatite and tricalcium phosphate) generally suffer from high brittleness, a mismatch between degradation and osteogenic rates, and a lack of bioactive factors, making it difficult to meet the comprehensive requirements of ideal bone repair materials in terms of biocompatibility, osteoconductivity, osteoinductive properties, biodegradability, and matching mechanical properties. Therefore, developing an integrated "treatment-repair" biomaterial that can simultaneously achieve highly effective local anti-tumor therapy and guide in-situ bone tissue regeneration has become an urgent research direction in the field of bone tumor surgery.

[0003] Sonodynamic therapy (SDT), as an emerging non-invasive tumor treatment method, has attracted extensive research due to its advantages such as high specificity, minimal damage to normal cells, deep tissue penetration, and high spatial precision. SDT utilizes ultrasound (US) to activate sonosensitizers, generating a large amount of reactive oxygen species (ROS) to induce tumor cell death. Compared with light-triggered therapy strategies such as photodynamic therapy (PDT) and photothermal therapy (PTT), ultrasound-mediated SDT has higher tissue penetration, which is beneficial for the controlled and non-invasive treatment of deep or large tumors. However, most reported inorganic sonosensitizers typically exhibit limited US absorption efficiency and unsatisfactory ROS yields. To address these challenges, several nanomaterial engineering strategies have been proposed, such as defect engineering, heteroatom doping, and the construction of heterostructures, to further optimize their performance in SDT. Although significant progress has been made through these strategies, there is still considerable room for further improvement in ROS production. Therefore, there is an urgent need to develop a simple and effective strategy to construct inorganic sonosensitizers with high ROS production efficiency for efficient SDT. Summary of the Invention

[0004] In order to solve the problems mentioned in the background art, the purpose of this invention is to provide a hydrotalcite nanocomposite material, its preparation method and application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On one hand, the present invention provides a hydrotalcite nanocomposite material, comprising etched zinc-copper-molybdenum hydrotalcite nanosheets (denoted as a-ZnCuMo-LDH), magnesium peroxide (MgO2), and a 45S5 bioactive glass scaffold (BG scaffold); the magnesium peroxide is loaded on the etched zinc-copper-molybdenum hydrotalcite nanosheets, denoted as a-ZnCuMo-LDH@MgO2; the a-ZnCuMo-LDH@MgO2 is attached to the surface of the 45S5 bioactive glass scaffold, denoted as a-ZnCuMo-LDH@MgO2-BG.

[0006] Furthermore, the molar ratio of zinc ions, copper ions, and molybdenum ions in the etched zinc-copper-molybdenum hydrotalcite nanosheets is 4:1:(24.5-25).

[0007] On the other hand, the present invention provides a method for preparing any of the above-described hydrotalcite nanocomposites, comprising the following steps:

[0008] (1) Zinc-copper-molybdenum hydrotalcite nanosheets were prepared by hydrothermal method and denoted as ZnCuMo-LDH;

[0009] (2) The zinc-copper-molybdenum hydrotalcite nanosheets were etched with alkali to obtain etched zinc-copper-molybdenum hydrotalcite nanosheets, denoted as a-ZnCuMo-LDH;

[0010] (3) Using etched zinc-copper-molybdenum hydrotalcite nanosheets as a carrier, magnesium peroxide was loaded onto the etched zinc-copper-molybdenum hydrotalcite nanosheets to obtain a-ZnCuMo-LDH@MgO2;

[0011] (4) Using a surface coating method, a-ZnCuMo-LDH@MgO2 was combined with a 45S5 bioactive glass scaffold to obtain a-ZnCuMo-LDH@MgO2-BG.

[0012] Further, step (1) specifically involves: mixing zinc salt, copper salt, molybdenum salt, and water to obtain a uniform solution; under a nitrogen atmosphere, adding sodium hydroxide solution dropwise to the solution to adjust the pH to 9.6-9.7, followed by stirring at room temperature; after stirring, placing the solution in a Teflon-lined container for hydrothermal reaction to obtain zinc-copper-molybdenum hydrotalcite nanosheets. This invention does not impose any special limitations on the mixing process of the zinc salt, copper salt, molybdenum salt, and water; the materials can be mixed uniformly according to a process well-known in the art. The dropwise addition method used in this invention ensures sufficient mixing of the metal salt solution and the alkaline solution, facilitating the nucleation process and forming a well-defined crystalline structure of hydrotalcite.

[0013] Furthermore, the zinc-copper-molybdenum hydrotalcite nanosheets have a size of 50-140 nm (nanosheet diameter observed by transmission electron microscopy) and a thickness of 14-17 nm.

[0014] Furthermore, the zinc salt includes at least one of zinc nitrate, zinc sulfate, and zinc chloride;

[0015] The copper salt includes at least one of copper nitrate, copper chloride, and copper sulfate;

[0016] The molybdenum salt is ammonium molybdate;

[0017] The molar ratio of zinc ions in the zinc salt, copper ions in the copper salt, and molybdenum ions in the molybdenum salt is 4:1:(24.5-25).

[0018] Furthermore, the stirring time is 20-30 minutes;

[0019] The hydrothermal reaction is carried out at a temperature of 75-85℃ for 20-24 hours.

[0020] Further, step (2) specifically involves: placing zinc-copper-molybdenum hydrotalcite nanosheets in a sodium hydroxide solution for alkaline etching to obtain etched zinc-copper-molybdenum hydrotalcite nanosheets;

[0021] The concentration of the sodium hydroxide solution is 0.6-0.65 mol / L;

[0022] The alkaline etching temperature is 115-125℃, and the alkaline etching time is 2-2.5h.

[0023] Further, step (3) specifically involves mixing and stirring magnesium peroxide, etched zinc-copper-molybdenum hydrotalcite nanosheets and water to obtain a-ZnCuMo-LDH@MgO2;

[0024] The mass ratio of the magnesium peroxide to the etched zinc-copper-molybdenum hydrotalcite nanosheets is (5-6):(10-12).

[0025] Furthermore, the stirring temperature is room temperature, the stirring speed is 600-800 rpm, and the stirring time is 6-8 hours.

[0026] Furthermore, the method for preparing magnesium peroxide is as follows: dissolving magnesium hydroxide in ethanol to obtain a magnesium hydroxide ethanol solution, and then adding hydrogen peroxide to the magnesium hydroxide ethanol solution and stirring to react and obtain magnesium peroxide.

[0027] Furthermore, the ratio of magnesium hydroxide, ethanol, and hydrogen peroxide used is (0.6-0.65g): 10mL: 10mL.

[0028] Furthermore, the temperature of the stirring reaction is room temperature, the stirring speed is 600-800 rpm, and the stirring time is 4-4.5 h.

[0029] Further, step (4) specifically involves: adding a-ZnCuMo-LDH@MgO2 to water to obtain an a-ZnCuMo-LDH@MgO2 suspension, adding a 45S5 bioactive glass scaffold to the a-ZnCuMo-LDH@MgO2 suspension, sonicating and drying, and repeating the above steps multiple times to obtain a-ZnCuMo-LDH@MgO2-BG;

[0030] The concentration of the a-ZnCuMo-LDH@MgO2 suspension is 6-6.5 mg / mL;

[0031] The power of the ultrasound is 3 W / cm. 2 The ultrasound session lasted 3-5 minutes.

[0032] Furthermore, the drying temperature is 60-70℃, and the drying time is 20-30 minutes. The present invention does not impose any particular limitation on the drying process; any process well-known in the art can be followed.

[0033] Furthermore, the repetition is performed three to five times.

[0034] On the other hand, this invention provides the application of any of the above-described hydrotalcite nanocomposites or hydrotalcite nanocomposites prepared by any of the above-described preparation methods in the preparation of drugs for treating bone tumors and drugs for bone regeneration and repair. This invention does not specifically limit the method of application; any method well-known in the art can be used.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The hydrotalcite nanocomposite material a-ZnCuMo-LDH@MgO2-BG of the present invention achieves simultaneous tumor clearance and bone defect repair through a triple mechanism of enhanced acoustic and dynamic properties, sequential release of metal ions and activation of osteogenic genes, providing a new strategy for postoperative treatment of bone tumors.

[0037] (2) The hydrotalcite nanocomposite material a-ZnCuMo-LDH@MgO2-BG of the present invention can rapidly release zinc and copper ions within the first week and reach a steady state after two weeks, which is conducive to effective bone healing and vascular recovery. Magnesium peroxide (MgO2) can interact with water (H2O) to form oxygen (O2), thereby alleviating hypoxia in the tumor microenvironment. This allows a-ZnCuMo-LDH@MgO2-BG to maintain a high efficiency of ROS generation under hypoxic conditions. ROS damages tumor cell DNA, upregulates the pro-apoptotic gene Bax and downregulates the anti-apoptotic gene Bcl-2, which can effectively achieve tumor ablation. It also significantly upregulates osteogenic genes such as RUNX 2 / BMP 2 / OCN, accelerating the repair of bone tissue defects.

[0038] (3) The hydrotalcite nanocomposite material a-ZnCuMo-LDH@MgO2-BG of the present invention has acoustic and dynamic properties, low cytotoxicity and degradability, good biocompatibility, and excellent anti-tumor properties and bone repair promotion ability, and has potential application prospects in related orthopedic surgeries.

[0039] (4) The preparation method of the present invention is simple, easy to operate, and the raw materials required are cheap and readily available. Attached Figure Description

[0040] Figure 1 This is a TEM image of the zinc-copper-molybdenum hydrotalcite nanosheets prepared in Example 1 of this invention;

[0041] Figure 2 In the image, 'a' represents the AFM image of the zinc-copper-molybdenum hydrotalcite nanosheets prepared in Example 1 of this invention. Figure 2 In Figure a, b represents the thickness analysis of the two zinc-copper-molybdenum hydrotalcite nanosheets.

[0042] Figure 3 The images show the XRD patterns of ZnCuMo-LDH, a-ZnCuMo-LDH, a-ZnCuMo-LDH@MgO2 and magnesium peroxide prepared in Example 1 of this invention, where JCPSD 19-0771 is the PDF card number of the International Center for Diffraction Data (ICDD) for magnesium peroxide.

[0043] Figure 4 In the figure, 'a' is a digital photograph of BG in Comparative Example 6. Figure 4 b in the figure is a digital photograph of BG+LDH@MgO2 prepared in Example 1 of this invention;

[0044] Figure 5 In the diagram, 'a' is the SEM image of BG in Comparative Example 6. Figure 5 b in the figure is the SEM image of BG+LDH@MgO2 prepared in Example 1 of this invention;

[0045] Figure 6 The image shows the EDX diagram of BG+LDH@MgO2 prepared in Example 1 of this invention.

[0046] Figure 7 In this diagram, 'a' represents the fluorescence spectrum of the ZnCuMo-LDH singlet oxygen green fluorescent probe prepared in Example 1 of this invention under ultrasonic irradiation under normal oxygen conditions. Figure 7 In this diagram, b represents the fluorescence spectrum of the a-ZnCuMo-LDH singlet oxygen green fluorescent probe prepared in Example 1 of this invention under ultrasonic irradiation under normal oxygen conditions. Figure 7 In this diagram, 'c' represents the fluorescence spectrum of the a-ZnCuMo-LDH@MgO2 singlet oxygen green fluorescent probe prepared in Example 1 of this invention under ultrasonic irradiation under normal oxygen conditions. Figure 7 In the figure, d represents the quantitative analysis of the fluorescence intensity of the singlet oxygen green fluorescent probes ZnCuMo-LDH, a-ZnCuMo-LDH, and a-ZnCuMo-LDH@MgO2 prepared in Example 1 of this invention under ultrasonic irradiation under normal oxygen conditions. Figure 7 In this diagram, 'e' represents the fluorescence spectrum of the α-ZnCuMo-LDH singlet oxygen green fluorescent probe prepared in Example 1 of this invention under anaerobic ultrasonic irradiation. Figure 7 f in the figure represents the fluorescence spectrum of the singlet oxygen green fluorescent probe a-ZnCuMo-LDH@MgO2 prepared in Example 1 of this invention under ultrasonic irradiation under hypoxic conditions.

[0047] Figure 8 The fluorescence intensity of the singlet oxygen green fluorescent probe prepared by BG+LDH@MgO2 under ultrasonic irradiation under normal oxygen conditions is shown in the figure below.

[0048] Figure 9 In this context, 'a' represents the normalized UV-vis spectrum of 1,3-diphenylisobenzofuran obtained from ZnCuMo-LDH, a-ZnCuMo-LDH, and a-ZnCuMo-LDH@MgO2 under normal oxygen conditions and ultrasonic irradiation, as prepared in Example 1 of this invention. Figure 9 In this paper, b represents the UV-vis spectrum of 1,3-diphenylisobenzofuran obtained by BG+LDH@MgO2 prepared in Example 1 of this invention under ultrasonic irradiation under normal oxygen conditions. Figure 9 In this text, 'c' represents the UV-vis spectrum of 1,3-diphenylisobenzofuran obtained from α-ZnCuMo-LDH prepared in Example 1 of this invention under anaerobic ultrasonic irradiation. Figure 9 In this context, d represents the UV-vis spectrum of 1,3-diphenylisobenzofuran obtained by preparing a-ZnCuMo-LDH@MgO2 under anaerobic ultrasonic irradiation.

[0049] Figure 10 The diagram shows the Zn, Cu, and Mo ion release kinetics of BG+LDH@MgO2 prepared in Example 1 of this invention.

[0050] Figure 11 In the image, 'a' represents a representative CLSM image of 143 B cells stained with calcein-AM / PI with or without sonication, obtained from BG+LDH@MgO2 prepared in Example 1, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7, with or without sonication treatment. Figure 11 In the image, b represents the representative CLSM images of ROS fluorescence intensity of 143 B cells prepared by BG+LDH@MgO2 in Example 1, BG in Comparative Example 6, and BG+LDH prepared by Comparative Example 7, with or without ultrasound treatment. The control group is untreated, and +US represents ultrasound treatment.

[0051] Figure 12 In the figure, 'a' represents 143 B cells co-cultured with BG+LDH@MgO2 prepared in Example 1 of this invention, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7, after sonication and Western blotting analysis. Figure 12 In the figure, b represents the expression results of the apoptosis-related gene Bcl-2 detected by qPCR after co-culturing 143 B cells with BG+LDH@MgO2 prepared in Example 1 of this invention, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7, followed by sonication treatment. Figure 12 In the figure, 'c' represents the expression results of the apoptosis-related gene Bax detected by qPCR after sonication of 143 B cells co-cultured with BG+LDH@MgO2 prepared in Example 1 of this invention, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7. Figure 12 In the diagram, 'd' represents the expression of the apoptosis-related protein Bcl-2 in 143 B cells co-cultured with BG+LDH@MgO2 prepared in Example 1, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7, after sonication treatment, as detected by Western blotting. Figure 12 In the figure, 'e' represents the expression map of apoptosis-related protein Bax detected by Western blotting after co-culturing 143 B cells with BG+LDH@MgO2 prepared in Example 1 of the present invention, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7, and after sonication treatment. The control group is untreated, and +US represents sonication treatment.

[0052] Figure 13 In the image, 'a' represents the in vivo fluorescence image of a mouse with a tumor. Figure 13In the graph, b represents the change in tumor volume in mice. Figure 13 In the graph, 'c' represents the change in mouse body weight. Figure 13 In the graph, d represents the mouse mortality rate statistics. Figure 13 In the image, 'e' represents a digital photograph of the tumor tissue. The control group was untreated, and +US represents ultrasound treatment.

[0053] Figure 14 In the image, 'a' represents the staining pattern of hBMSCs by alkaline phosphatase. Figure 14 In the image, b represents the staining pattern of hBMSCs with Alizarin Red S. Figure 14 In the figure, c represents the Western blotting image obtained after sonication of hBMSC cells co-cultured with BG+LDH@MgO2 prepared in Example 1 of this invention, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7. Figure 14 In the figure, d represents the osteogenic protein detection results of hBMSC cells co-cultured with BG+LDH@MgO2 prepared in Example 1 of this invention, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7 after sonication treatment. Figure 14 In the figure, 'e' represents the results of qPCR evaluation of osteogenic gene expression in hBMSC cells co-cultured with BG+LDH@MgO2 prepared in Example 1, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7 after sonication treatment. The control group is untreated, and +US represents sonication treatment. Detailed Implementation

[0054] To better understand the content of this invention, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments thereof. The described embodiments are merely some embodiments of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0055] Unless otherwise specified, all raw materials required in this invention are commercially available products well known to those skilled in the art.

[0056] Example 1

[0057] Preparation of zinc-copper-molybdenum hydrotalcite nanosheets (ZnCuMo-LDH): Zinc nitrate (Zn(NO3)2·6H2O) (0.8 mmol), copper nitrate (Cu(NO3)2·3H2O) (0.2 mmol), and ammonium molybdate ((NH4)6Mo7O) were prepared. 24·4H2O (0.714 mmol) and deionized water (70 mL) were mixed evenly to obtain a mixture; under a nitrogen atmosphere, sodium hydroxide solution (40 mg / mL) was added dropwise to the mixture to adjust the pH to 9.6, and then the mixture was stirred at room temperature for 30 min. After stirring, the mixture was placed in a Teflon-lined container and hydrothermally reacted at 80 °C for 24 h. After the reaction, the mixture was washed three times with deionized water to obtain zinc-copper-molybdenum hydrotalcite nanosheets.

[0058] Preparation of etched zinc-copper-molybdenum hydrotalcite nanosheets (a-ZnCuMo-LDH): Zinc-copper-molybdenum hydrotalcite nanosheets were placed in sodium hydroxide solution (5 mL, 0.6 mol / L) and kept at 120 °C for 2 h to obtain etched zinc-copper-molybdenum hydrotalcite nanosheets.

[0059] Preparation of magnesium peroxide (MgO2): 0.6 g of magnesium hydroxide was dissolved in 10 mL of ethanol to obtain a magnesium hydroxide ethanol solution. Then, 10 mL of hydrogen peroxide was added to the magnesium hydroxide ethanol solution. The mixture was stirred at 600 rpm for 4 h at room temperature. Magnesium peroxide was extracted by centrifugation at 6000 rpm for 5 min and washed three times with deionized water to obtain magnesium peroxide.

[0060] Preparation of a-ZnCuMo-LDH@MgO2: Magnesium peroxide (5 mg), etched zinc-copper-molybdenum hydrotalcite nanosheets (10 mg), and deionized water (10 mL) were mixed and stirred at 600 rpm for 6 h at room temperature, then centrifuged at 6000 rpm for 5 min, and then washed with deionized water to obtain a-ZnCuMo-LDH@MgO2.

[0061] Preparation of a-ZnCuMo-LDH@MgO2-BG: A 6 mg / mL a-ZnCuMo-LDH@MgO2 suspension was prepared by adding a-ZnCuMo-LDH@MgO2 to deionized water. A 45S5 bioactive glass scaffold (manufactured by Qiyu New Materials Technology (Foshan) Co., Ltd.) was added to the 6 mg / mL a-ZnCuMo-LDH@MgO2 suspension and heated at 3 w / cm². 2 The sample was sonicated at a certain power for 5 minutes, and then dried in an oven at 60°C for 20 minutes. The entire process was repeated three times to obtain a-ZnCuMo-LDH@MgO2-BG (abbreviated as BG+LDH@MgO2).

[0062] Comparative Example 1

[0063] Preparation of a-ZnCuMo-LDH@MgO2-BG: A 1 mg / mL a-ZnCuMo-LDH@MgO2 suspension was prepared by adding a-ZnCuMo-LDH@MgO2 to deionized water. A 45S5 bioactive glass scaffold was added to the 1 mg / mL a-ZnCuMo-LDH@MgO2 suspension and incubated at 3 w / cm². 2 The sample was sonicated at a certain power for 5 minutes, and then dried in an oven at 60°C for 20 minutes. The entire process was repeated three times to obtain a-ZnCuMo-LDH@MgO2-BG (abbreviated as BG+LDH@MgO2).

[0064] Comparative Example 2

[0065] Preparation of a-ZnCuMo-LDH@MgO2-BG: A 2 mg / mL a-ZnCuMo-LDH@MgO2 suspension was prepared by adding a-ZnCuMo-LDH@MgO2 to deionized water. A 45S5 bioactive glass scaffold was added to the 2 mg / mL a-ZnCuMo-LDH@MgO2 suspension and incubated at 3 w / cm². 2 The sample was sonicated at a certain power for 5 minutes, and then dried in an oven at 60°C for 20 minutes. The entire process was repeated three times to obtain a-ZnCuMo-LDH@MgO2-BG (abbreviated as BG+LDH@MgO2).

[0066] Comparative Example 3

[0067] Preparation of a-ZnCuMo-LDH@MgO2-BG: A 4 mg / mL a-ZnCuMo-LDH@MgO2 suspension was prepared by adding a-ZnCuMo-LDH@MgO2 to deionized water. A 45S5 bioactive glass scaffold was added to the 4 mg / mL a-ZnCuMo-LDH@MgO2 suspension and incubated at 3 w / cm². 2 The sample was sonicated at a certain power for 5 minutes, and then dried in an oven at 60°C for 20 minutes. The entire process was repeated three times to obtain a-ZnCuMo-LDH@MgO2-BG (abbreviated as BG+LDH@MgO2).

[0068] Comparative Example 4

[0069] Preparation of a-ZnCuMo-LDH@MgO2-BG: an 8 mg / mL a-ZnCuMo-LDH@MgO2 suspension was prepared by adding a-ZnCuMo-LDH@MgO2 to deionized water. A 45S5 bioactive glass scaffold was then added to the 8 mg / mL a-ZnCuMo-LDH@MgO2 suspension, and the mixture was heated at 3 w / cm². 2The sample was sonicated at a certain power for 5 minutes, and then dried in an oven at 60°C for 20 minutes. The entire process was repeated three times to obtain a-ZnCuMo-LDH@MgO2-BG (abbreviated as BG+LDH@MgO2).

[0070] Comparative Example 5

[0071] Preparation of a-ZnCuMo-LDH@MgO2-BG: A 10 mg / mL a-ZnCuMo-LDH@MgO2 suspension was prepared by adding a-ZnCuMo-LDH@MgO2 to deionized water. A 45S5 bioactive glass scaffold was added to the 10 mg / mL a-ZnCuMo-LDH@MgO2 suspension and incubated at 3 w / cm². 2 The sample was sonicated at a certain power for 5 minutes, and then dried in an oven at 60°C for 20 minutes. The entire process was repeated three times to obtain a-ZnCuMo-LDH@MgO2-BG (abbreviated as BG+LDH@MgO2).

[0072] Comparative Example 6

[0073] BG bracket, denoted as BG.

[0074] Comparative Example 7

[0075] Preparation of zinc-copper-molybdenum hydrotalcite nanosheets (ZnCuMo-LDH): Zinc nitrate (Zn(NO3)2·6H2O) (0.8 mmol), copper nitrate (Cu(NO3)2·3H2O) (0.2 mmol), and ammonium molybdate ((NH4)6Mo7O) were prepared. 24 ·4H2O (0.714 mmol) and deionized water (70 mL) were mixed evenly to obtain a mixture; under a nitrogen atmosphere, sodium hydroxide solution (40 mg / mL) was added dropwise to the mixture to adjust the pH of the mixture to 9.6. After stirring, the mixture was placed in a Teflon-lined container and hydrothermally reacted at 80 °C for 24 h. After the reaction, the mixture was washed three times with deionized water to obtain zinc-copper-molybdenum hydrotalcite nanosheets.

[0076] Preparation of etched zinc-copper-molybdenum hydrotalcite nanosheets (a-ZnCuMo-LDH): Zinc-copper-molybdenum hydrotalcite nanosheets were placed in sodium hydroxide solution (5 mL, 0.6 mol / L) and kept at 120 °C for 2 h to obtain etched zinc-copper-molybdenum hydrotalcite nanosheets, abbreviated as a-LDH.

[0077] Preparation of a-ZnCuMo-LDH-BG: A 6 mg / mL a-ZnCuMo-LDH suspension was prepared by adding a-ZnCuMo-LDH to deionized water. A 45S5 bioactive glass scaffold (manufactured by Qiyu New Materials Technology (Foshan) Co., Ltd.) was added to the 6 mg / mL a-ZnCuMo-LDH suspension and heated at 3 w / cm². 2 The sample was sonicated at a certain power for 5 minutes, and then dried in an oven at 60°C for 20 minutes. The entire process was repeated three times, and finally α-ZnCuMo-LDH-BG (abbreviated as BG+LDH) was obtained.

[0078] Characterization and testing:

[0079] Transmission electron microscopy (TEM) was performed on zinc-copper-molybdenum hydrotalcite nanosheets, and the results are as follows: Figure 1 As shown. From Figure 1 As can be seen, the zinc-copper-molybdenum hydrotalcite nanosheets exhibit a nanosheet-like structure with a size of 50-140 nm.

[0080] Atomic force microscopy (AFM) was used to analyze zinc-copper-molybdenum layered double hydroxide nanosheets, and the results are as follows: Figure 2 As shown. From Figure 2 As can be seen, the thickness of the zinc-copper-molybdenum hydrotalcite nanosheets is 14-17 nm.

[0081] X-ray diffraction (XRD) tests were performed on the ZnCuMo-LDH, a-ZnCuMo-LDH, and a-ZnCuMo-LDH@MgO2 prepared in Example 1. The results are as follows: Figure 3 As shown. From Figure 3 As can be seen from the data, ZnCuMo-LDH was successfully prepared; the a-ZnCuMo-LDH@MgO2 sample exhibited the same characteristic peaks as magnesium peroxide, indicating that the hydrotalcite material was successfully combined with magnesium peroxide.

[0082] The BG in Comparative Example 6 and the BG+LDH@MgO2 prepared in Example 1 were photographed, and the digital photos are as follows: Figure 4 As shown. From Figure 4 As can be seen, the surface of BG+LDH@MgO2 is yellow, while the original BG is white, indicating that the material has successfully adhered to the surface of the scaffold.

[0083] Scanning electron microscopy (SEM) was performed on BG in Comparative Example 6 and BG+LDH@MgO2 prepared in Example 1. The results are as follows: Figure 5 As shown. From Figure 5 As can be seen, BG+LDH@MgO2 exhibits a rougher surface compared to the original BG, which is beneficial for cell adhesion.

[0084] Energy-dispersive X-ray spectroscopy (EDX) was performed on the BG+LDH@MgO2 prepared in Example 1, and the results are as follows: Figure 6 As shown. From Figure 6 The elemental distribution image shows that Si, O, and P elements from BG, as well as Zn, Cu, and Mo elements from the etched zinc-copper-molybdenum layered double hydroxide nanosheets, are uniformly distributed in BG+LDH@MgO2, proving its successful preparation.

[0085] The singlet oxygen green fluorescent probe (SOSG) was used to test the ZnCuMo-LDH, a-ZnCuMo-LDH, and a-ZnCuMo-LDH@MgO2 prepared in Example 1 under normal oxygen conditions. 1 O2 generation capacity, results as follows Figure 7 As shown in ad in the figure. From Figure 7 As can be seen from the ad, compared with ZnCuMo-LDH, under ultrasonic (US) irradiation, α-ZnCuMo-LDH has... 1 The O2 fluorescence signal intensity increased significantly, with the most significant increase observed under US irradiation in a-ZnCuMo-LDH@MgO2. 1 The O2 fluorescence signal intensity indicates that the incorporation of magnesium peroxide significantly improved the O2 supply. 1 O2 generation was investigated using a singlet oxygen green fluorescent probe (SOSG) to test the oxygen generation of a-ZnCuMo-LDH and a-ZnCuMo-LDH@MgO2 prepared in Example 1 under hypoxic conditions. 1 O2 generation capacity, results as follows Figure 7 As shown in ef. From Figure 7 As can be seen from the ef data, α-ZnCuMo-LDH exhibits very low efficiencies under US irradiation. 1 O2 fluorescence signal intensity, while a-ZnCuMo-LDH@MgO2 showed significant fluorescence intensity under hypoxic conditions. 1 The intensity of the O2 fluorescence signal indicates that magnesium peroxide can enhance O2 production to counteract the hypoxic limitations in the tumor environment.

[0086] The singlet oxygen green fluorescent probe (SOSG) was used to test the BG+LDH@MgO2 prepared in Example 1 and Comparative Examples 1-5 under normal oxygen conditions. 1 O2 generation capacity was analyzed quantitatively, and the results are as follows: Figure 8 As shown. From Figure 8 As can be seen, increasing the concentration of the α-ZnCuMo-LDH@MgO2 suspension significantly increased the SOSG fluorescence signal intensity at 6 mg / mL. -1 It reaches its maximum value at that time.

[0087] The ZnCuMo-LDH, a-ZnCuMo-LDH, a-ZnCuMo-LDH@MgO2, and BG+LDH@MgO2 prepared in Example 1 under normal oxygen conditions were tested using a 1,3-diphenylisobenzofuran (DPBF) probe. 1 O2 production capacity, results as follows Figure 9 a and Figure 9 As shown in b in the diagram. From Figure 9 As can be seen from 'a', under US irradiation, ZnCuMo-LDH exhibits the smallest decrease in DPBF absorbance at 410 nm, while a significant decrease in absorbance is observed in a-ZnCuMo-LDH. In contrast, under US irradiation, the absorbance of DPBF further decreases in a-ZnCuMo-LDH@MgO2, demonstrating that the introduction of magnesium peroxide and the resulting O2 replenishment contribute to its absorption. 1 O2 generation capacity is most efficient. From Figure 9 As can be seen from b, significant DPBF degradation was observed in BG+LDH@MgO2 treated with US irradiation, confirming its... 1 O2 production capacity was not affected by the introduction of the scaffold. The oxygen production capacity of a-ZnCuMo-LDH and a-ZnCuMo-LDH@MgO2 prepared in Example 1 under hypoxic conditions was tested using a 1,3-diphenylisobenzofuran (DPBF) probe. 1 O2 production capacity, results as follows Figure 9 c and Figure 9 As shown by d in the figure. From Figure 9 c and Figure 9 As can be seen from d, the addition of magnesium peroxide significantly improved the performance of a-ZnCuMo-LDH nanosheets under anaerobic conditions. 1 O2 generation capability.

[0088] The ion release behavior of BG+LDH@MgO2 prepared in Example 1 of this invention was determined by ICP-AES, and the results are as follows: Figure 10 As shown. From Figure 10 As can be seen, Zn and Cu ions exhibit rapid release in the first week, followed by a significant decrease in the release rate, eventually reaching a steady state after two weeks, which is beneficial for effective bone healing and vascular recovery.

[0089] 143 B cells (5 × 10) 5Cells were seeded in 48-well plates with BG (Comparative Example 6), BG+LDH (Comparative Example 7), and BG+LDH (Example 1) @MgO2, respectively, and incubated for 24 h. Cells were then treated with or without sonication (3 W / cm²) for 5 min. After co-staining with calcein-AM / PI (15 μg / mL) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (10 μmol / L), cell fluorescence images were obtained using a confocal laser scanning microscope (CLSM). Figure 11 As shown, from Figure 11 As shown in 'a', no red fluorescence was observed in the control group, BG+US group, and BG+LDH group, while significant red fluorescence was observed in the BG+LDH+US and BG+LDH@MgO2+US groups, confirming that BG+LDH+US and BG+LDH@MgO2+US treatments can effectively induce sonodynamic therapy-related apoptosis. Figure 11 As shown in b, no 2',7'-dichlorofluorescein (DCF) fluorescence was observed in cells treated with the control group, BG+US group, and BG+LDH group. ROS green fluorescence was observed in the BG+LDH+US group, with a more significant enhancement in green fluorescence in the MgO2-loaded BG+LDH@MgO2+US group. This indicates that the ability of α-ZnCuMo-LDH and α-ZnCuMo-LDH@MgO2 to generate reactive oxygen species was significantly enhanced under ultrasonic irradiation.

[0090] 143 B cells co-cultured with BG from Comparative Example 6, BG+LDH prepared in Comparative Example 7, and BG+LDH@MgO2 prepared in Example 1 were subjected to sonication. The expression levels of apoptosis-regulating proteins were detected by Western blotting. The results are as follows: Figure 12 As shown in a, under the condition of using GAPDH as an internal control, compared with other groups, the expression level of the pro-apoptotic protein Bax was significantly upregulated in the BG+LDH@MgO2+US group, while the expression level of the anti-apoptotic protein Bcl-2 was significantly downregulated. Further quantitative analysis of protein expression was performed using ImageJ 1.52v software to calculate the grayscale values ​​of the above Western blot bands, as shown in Figure a. Figure 12 d and Figure 12 As shown in 'e', ​​the quantitative analysis of protein expression levels further visually illustrates the upregulation trend of Bax protein and the downregulation trend of Bcl-2 protein. These changes in protein levels are consistently confirmed at the gene transcription level. Quantitative polymerase chain reaction (qPCR) was used to detect apoptosis-related genes, and the results are shown below. Figure 12 b and Figure 12As shown in Figure c, qPCR results indicated that the relative mRNA expression level of the Bax gene in the BG+LDH@MgO2+US group was significantly increased compared to the control group; conversely, the relative mRNA expression level of the Bcl-2 gene was significantly decreased. These results demonstrate that BG+LDH@MgO2-mediated SDT can effectively activate the intrinsic apoptotic signaling pathway of bone tumor cells by significantly upregulating the expression of the pro-apoptotic factor Bax and simultaneously downregulating the expression of the anti-apoptotic factor Bcl-2, acting synergistically at both the gene transcription and protein translation levels. This provides direct evidence for elucidating the molecular biological mechanism by which it inhibits tumor growth and achieves therapeutic effects in bone tumors.

[0091] In vivo antitumor experiments were conducted on BG from Comparative Example 6, BG+LDH prepared in Comparative Example 7, and BG+LDH@MgO2 prepared in Example 1. Five mice were used in each group. The tumor-clearing efficacy of BG+LDH@MgO2 was evaluated using a nude mouse orthotopic osteosarcoma model. The experiment simulated a clinical surgical procedure. Surgical resection was performed when the primary tumor volume reached approximately 200 mm³. Subsequently, BG, BG+LDH, or BG+LDH@MgO2 was implanted at the resection site, followed by treatment with or without US ultrasound irradiation. In vivo fluorescence imaging was performed on mice every four days for 16 consecutive days. The results are as follows: Figure 13 As shown in a. During the 16-day treatment period, the size and weight of the mouse tumors were recorded every two days, and the results are shown in Figure a. Figure 13 b and Figure 13 As shown in c in the figure. The tissue was dissected and photographed 16 days later, and the results are as follows. Figure 13 As shown in e in the diagram. From Figure 13 As shown in figures a, b, c, and e, tumor growth was significant in the control group and the BG+US group. Implantation of BG+LDH and BG+LDH@MgO2 had a weak inhibitory effect on tumor growth in mice, while BG+LDH+US showed a better inhibitory effect. In contrast, mice implanted with BG+LDH@MgO2 showed complete tumor regression under US irradiation and had good postoperative survival. These results confirm the significant inhibitory effect of the combination of BG+LDH@MgO2 and ultrasound radiation on tumor growth. The survival time of mice in different groups was recorded, and the results are shown below. Figure 13 As shown in d, from Figure 13 As can be seen from d, all mice in the BG+LDH@MgO2+US group survived for more than 40 days.

[0092] In vitro osteogenic performance test: The osteogenic activity of BG+LDH@MgO2 prepared in Example 1, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7 on hBMSCs was detected by alkaline phosphatase (ALP) and Alizarin Red S staining method. The results are as follows: Figure 14 a and Figure 14 As shown in b in the figure, compared with other groups, after 14 days of culture, the BG+LDH@MgO2 group showed more obvious ALP staining and Alizarin Red S staining of obvious mineralized nodules, indicating better osteogenic effect. hBMSCs co-cultured with BG+LDH@MgO2 prepared in Example 1 of this invention, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7 were subjected to Western blotting after sonication. The results are shown in the figure. Figure 14 As shown in c, the grayscale values ​​of the Western blot bands were calculated using ImageJ 1.52v software, and the quantitative analysis results of osteogenic protein expression are as follows. Figure 14 As shown by d in the figure. From Figure 14 c and Figure 14 As can be seen from d, osteogenic proteins were detected in both the BG+LDH+US and BG+LDH@MgO2+US groups. Furthermore, the expression levels of osteogenic proteins Col1, BMP2, and RUNX2 in the BG+LDH@MgO2+US group were 2.61, 2.54, and 2.45 times higher than those in the control group, respectively. hBMSCs co-cultured with BG+LDH@MgO2 prepared in Example 1, BG in Comparative Example 6, and BG+LDH prepared in Comparative Example 7 were subjected to sonication and then quantitative polymerase chain reaction (qPCR) was used to assess osteogenic gene expression. The results are as follows: Figure 14 As shown in e in the diagram. From Figure 14 As can be seen from 'e', ​​in the BG+LDH@MgO2+US group, the expression level of OCN gene was 3 times that of the control group, BMP 2 was 2.1 times that of the control group, and RUNX 2 was 2.2 times that of the control group. This indicates that BG+LDH@MgO2-mediated SDT has significant osteogenic properties and can upregulate the expression of osteogenic genes and proteins.

[0093] The above results indicate that the hydrotalcite nanocomposite material a-ZnCuMo-LDH@MgO2-BG mediated SDT provided by this invention has good application effects in the treatment of bone tumors and bone regeneration and repair, and has broad application prospects. Magnesium peroxide effectively alleviates the limitations of tumor microenvironment hypoxia on treatment, promotes the generation of ROS, effectively ablates tumors, and significantly upregulates the expression of osteogenic genes and proteins, promoting the repair and regeneration of bone tissue.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hydrotalcite nanocomposite material, characterized in that, The invention comprises etched zinc-copper-molybdenum layered double hydroxide nanosheets, magnesium peroxide, and a 45S5 bioactive glass scaffold. The magnesium peroxide is loaded on the etched zinc-copper-molybdenum layered double hydroxide nanosheets and is designated as a-ZnCuMo-LDH@MgO2. The a-ZnCuMo-LDH@MgO2 is attached to the surface of the 45S5 bioactive glass scaffold and is designated as a-ZnCuMo-LDH@MgO2-BG.

2. The hydrotalcite nanocomposite material according to claim 1, characterized in that, The molar ratio of zinc ions, copper ions, and molybdenum ions in the etched zinc-copper-molybdenum hydrotalcite nanosheets is 4:1:(24.5-25).

3. The method for preparing the hydrotalcite nanocomposite material according to claim 1 or 2, characterized in that, Includes the following steps: (1) Zinc-copper-molybdenum hydrotalcite nanosheets were prepared by hydrothermal method; (2) The zinc-copper-molybdenum hydrotalcite nanosheets were etched with alkali to obtain etched zinc-copper-molybdenum hydrotalcite nanosheets; (3) Using etched zinc-copper-molybdenum hydrotalcite nanosheets as a carrier, magnesium peroxide was loaded onto the etched zinc-copper-molybdenum hydrotalcite nanosheets to obtain a-ZnCuMo-LDH@MgO2; (4) Using a surface coating method, a-ZnCuMo-LDH@MgO2 was combined with a 45S5 bioactive glass scaffold to obtain a-ZnCuMo-LDH@MgO2-BG.

4. The preparation method according to claim 3, characterized in that, Step (1) is as follows: Zinc salt, copper salt, molybdenum salt and water are mixed evenly to obtain a mixed solution; under a nitrogen atmosphere, sodium hydroxide solution is added dropwise to the mixed solution to adjust the pH of the mixed solution to 9.6-9.7, and then stirred at room temperature; after stirring, it is placed in a Teflon-lined container for hydrothermal reaction to obtain zinc-copper-molybdenum hydrotalcite nanosheets.

5. The preparation method according to claim 4, characterized in that, The zinc salt includes at least one of zinc nitrate, zinc sulfate, and zinc chloride; The copper salt includes at least one of copper nitrate, copper chloride, and copper sulfate; The molybdenum salt is ammonium molybdate; The molar ratio of zinc ions in the zinc salt, copper ions in the copper salt, and molybdenum ions in the molybdenum salt is 4:1:(24.5-25).

6. The preparation method according to claim 4, characterized in that, The stirring time is 20-30 minutes; The hydrothermal reaction is carried out at a temperature of 75-85℃ for 20-24 hours.

7. The preparation method according to claim 3, characterized in that, Step (2) specifically involves placing zinc-copper-molybdenum hydrotalcite nanosheets in a sodium hydroxide solution for alkaline etching to obtain etched zinc-copper-molybdenum hydrotalcite nanosheets. The concentration of the sodium hydroxide solution is 0.6-0.65 mol / L; The alkaline etching temperature is 115-125℃, and the alkaline etching time is 2-2.5h.

8. The preparation method according to claim 3, characterized in that, Step (3) specifically involves mixing and stirring magnesium peroxide, etched zinc-copper-molybdenum hydrotalcite nanosheets, and water to obtain a-ZnCuMo-LDH@MgO2; The mass ratio of the magnesium peroxide to the etched zinc-copper-molybdenum hydrotalcite nanosheets is (5-6):(10-12).

9. The preparation method according to claim 3, characterized in that, Step (4) is as follows: a-ZnCuMo-LDH@MgO2 is added to water to obtain a-ZnCuMo-LDH@MgO2 suspension. 45S5 bioactive glass scaffold is added to a-ZnCuMo-LDH@MgO2 suspension, ultrasonicated and dried. The above steps are repeated multiple times to obtain a-ZnCuMo-LDH@MgO2-BG. The concentration of the a-ZnCuMo-LDH@MgO2 suspension is 6-6.5 mg / mL; The power of the ultrasound is 3 W / cm. 2 The ultrasound session lasted 3-5 minutes.

10. The use of the hydrotalcite nanocomposite material according to any one of claims 1-2 or the hydrotalcite nanocomposite material prepared by any one of claims 3-9 in the preparation of drugs for treating bone tumors and drugs for bone regeneration and repair.

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