A size-controllable hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedron and its preparation method and application

Abstract

The present invention discloses a size-controllable hyaluronic acid functionalized copper-cerium bimetallic peroxide nano-octahedron and its preparation method and application, belonging to the field of biomedicine technology. The present invention forms a bimetallic precursor solution with an aqueous solution of a metal salt of cerium (IV), a metal salt of copper (I) and polyvinyl pyrrolidone, adds sodium hydroxide and hydrogen peroxide to react, controls the size of the product by adjusting the molecular weight of polyvinyl pyrrolidone, adjusts the feeding concentration of sodium hydroxide to form an octahedral structure, obtains copper-cerium bimetallic peroxide nano-octahedron, which is subsequently coated with hyaluronic acid, and the obtained hyaluronic acid functionalized copper-cerium bimetallic peroxide nano-octahedron is used for anti-tumor treatment with high specificity, and combined immunotherapy can also form a complementary therapy of chemodynamic therapy-immunotherapy, which not only gives full play to the advantages of each single therapy, but also makes up for their respective shortcomings, thereby efficiently inhibiting the growth of primary tumors and metastatic tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a size-controllable hyaluronic acid-functionalized copper-cerium bimetallic peroxide nano-octahedron, and a preparation method and application thereof. Background Art

[0002] The mortality rate caused by malignant tumors has been increasing year by year and has become a key public health issue affecting human life and health. At present, the clinical treatment method for tumors is still the traditional treatment method based on radiotherapy and chemotherapy. However, due to the existence of the tumor microenvironment, the complexity and heterogeneity of tumors limit the further development and application of traditional radiotherapy and chemotherapy technologies. For example, tumor cells metabolize glucose through glycolysis, causing the pH value of the tumor microenvironment to be maintained between 6.5 and 6.8, activating drug resistance mediated by multiple signaling pathways. At the same time, non-specific radiotherapy and chemotherapy technologies can cause strong toxic side effects on normal tissues and organs. Therefore, there is an urgent need to develop new functional materials with good biocompatibility and new technologies that specifically respond to tumors / tumor microenvironments to treat malignant tumors.

[0003] Chemodynamic therapy, based on nanoparticles that specifically catalyze the Fenton reaction in situ, responds to the acidic conditions of the tumor microenvironment and in situ catalyzes hydrogen peroxide in the tumor microenvironment to generate hydroxyl radicals that kill tumor cells. This technology effectively avoids the toxic side effects of chemotherapy and radiotherapy, making it a promising new cancer treatment technology. Due to the enhanced permeability and retention (EPR) effect in tumor tissue, only nanoparticles of a specific size can penetrate the interstitial space of tumor vasculature and accumulate in tumor tissue. However, the weak acidity of the tumor microenvironment, low hydrogen peroxide concentration, and the mismatch between particle size and tumor vascular space make Fe-, Cu-, and Mn-based nanoparticles commonly used in chemodynamic therapy difficult to eradicate tumors.

[0004] In view of the application defects of the above-mentioned nanomedicines, the development of bimetallic nanomedicines has received widespread attention from researchers. For example, the Chinese invention patent with publication number CN118558325A discloses a bimetallic nanomedicine and its preparation method and application. The bimetallic nanomedicine includes a copper-cerium peroxide nanoparticle carrier and a drug carried thereon, and the drug contains doxorubicin, glucose oxidase and 1-methyl-tryptophan. The invention is based on the characteristics of copper-cerium bimetallic peroxide nanoparticles that exhibit excellent reactive oxygen species (ROS) and oxygen generation capabilities, and uses them as drug delivery carriers. Its nanostructure is used to enhance the anti-tumor effect of the drug, and its oxygen-producing properties are used to alleviate the hypoxic environment of the tumor, thereby improving the tumor microenvironment. In principle, the spherical or quasi-spherical nanoparticles decompose under the acidic microenvironment of the tumor, releasing H2O2, Cu 2+ , cerium ions and drug molecules. Among them, H2O2 and Cu 2+ The interaction generates a large amount of toxic ROS, enhancing the therapeutic effect of CDT. Similarly, Chinese invention patent publication number CN118558325A provides a method for preparing a CeO2 / CuO2 composite nanomaterial with synergistic catalytic properties, as well as the resulting products and applications. This method combines the inorganic nanomaterial CeO2 with CuO2, then loads it with the chemotherapy drug doxorubicin (DOX) and further modifies hyaluronic acid (HA) to create a CeO2 / CuO2 composite nanomaterial with synergistic catalytic properties. This invention utilizes the principle that CeO2 has excellent catalase activity. In the acidic environment of tumors, it catalyzes the decomposition of H2O2 in cells to produce a large amount of O2, thereby alleviating tumor hypoxia and enhancing the chemotherapeutic effect of DOX. The CeO2 / CuO2 composite nanomaterial can solve the problem of insufficient H2O2 content and enhance the catalytic effect of Fenton-like reactions. CuO2 nanoparticles loaded on CeO2 nanospheres decompose and release H2O2 in the slightly acidic environment of tumor cells, which can increase the H2O2 content in tumor tissues, and then increase the concentration of ·OH through the Fenton reaction, thereby enhancing the effect of chemodynamic therapy (CDT).

[0005] The above bimetallic nanomedicines still have room for optimization in application. First of all, in terms of particle structure, the existing copper-cerium bimetallic nanoparticles are spherical or quasi-spherical. Since the surface of the sphere is completely symmetrical or approximately symmetrical, the distance from all points to the center is consistent. It has the greatest symmetry and the smallest surface area among all types of geometric shapes, and has the smallest ratio of surface area to volume. Therefore, the surface energy of the sphere tends to be minimized, which makes it difficult to perform various functionalizations on its surface through positive and negative electrical adsorption. At the same time, the size adjustment of nanomedicines in the prior art is complex and difficult to meet different usage requirements. In addition, in terms of the catalytic effect of the reaction, the prior art adopts Cu 2+ and Ce 3+ The formed peroxide nanoparticles may contain Ce 4+ With Cu 2+ The composite nanomaterials have limited catalytic activity towards H2O2 and reaction activity with glutathione (GSH), and the bimetallic synergistic catalytic ability and therapeutic effect need to be improved.

[0006] Therefore, designing and developing new nanomedicines that are activated by the weak acid environment of tumors, self-supply hydrogen peroxide, and have controllable size for specific chemodynamic therapy technology, and provide combined treatment points with other tumor treatment technologies, is an urgent and significant task in the field of biomedical technology. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, in a first aspect of the present invention, a method for preparing hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra with simple process and controllable size is provided, comprising the following steps:

[0008] (1) A cerium (IV) metal salt and a copper (I) metal salt are dispersed in an aqueous solution of polyvinylpyrrolidone (PVP) to form a bimetallic precursor solution; sodium hydroxide and hydrogen peroxide are then added to react to obtain copper-cerium bimetallic peroxide nanooctahedra;

[0009] The size of the product is controlled by adjusting the molecular weight of polyvinyl pyrrolidone, and the octahedral structure is formed by adjusting the feeding concentration of sodium hydroxide.

[0010] (2) The obtained copper-cerium bimetallic peroxide nanooctahedron is coated with hyaluronic acid to obtain hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron.

[0011] Preferably, in step (1), the metal salt of cerium (IV) includes one of cerium sulfate and cerium chloride; the metal salt of copper (I) includes one of cuprous sulfate, cuprous chloride and cuprous nitrate.

[0012] Preferably, in step (1), the molar ratio of the cerium (IV) metal salt, the copper (I) metal salt and the water in the bimetallic precursor solution is 1-10:1-10:55.5.

[0013] Preferably, the step (1) adopts a rate-controlled precipitation method, which specifically comprises the following steps: completely dissolving a metal salt of cerium (IV) and a metal salt of copper (I) in an aqueous solution of polyvinyl pyrrolidone to form a bimetallic precursor solution; then adding an aqueous sodium hydroxide solution and an aqueous hydrogen peroxide solution to the bimetallic precursor solution at a rate of 5-20 mL / h to react, and after the reaction is completed, recovering and purifying the product to obtain copper-cerium bimetallic peroxide nanooctahedrons.

[0014] Further preferably, the volume ratio of the sodium hydroxide aqueous solution to the bimetallic precursor solution is 1:1-50; the volume ratio of the hydrogen peroxide aqueous solution to the bimetallic precursor solution is 1:1-25.

[0015] Further preferably, the concentration of the hydrogen peroxide solution is 0.39-9.79 mol / L.

[0016] The concentration of the sodium hydroxide solution must be controlled within an appropriate range. Too low a concentration can hinder the complete reaction and prevent the formation of an octahedral structure. Excessively high concentrations can increase costs and increase the number of washes during the purification process, leading to significant product loss and reduced yield.

[0017] Further preferably, in the process of forming the octahedral structure by adjusting the feeding concentration of sodium hydroxide, the concentration of the sodium hydroxide aqueous solution is 1-20 mmol / L.

[0018] The smaller the PVP molecular weight, the larger the size of the resulting copper-cerium bimetallic peroxide nanooctahedra; conversely, the larger the PVP molecular weight, the smaller the size of the resulting copper-cerium bimetallic peroxide nanooctahedra. Therefore, the particle size can be easily adjusted by the molecular weight of PVP.

[0019] Preferably, in the step (1), the molecular weight of polyvinyl pyrrolidone in the aqueous solution is 8000-58000; and the mass ratio of polyvinyl pyrrolidone to water is 1:5-20.

[0020] Preferably, in step (1), the reaction is carried out at room temperature and the reaction time is 1-12 h.

[0021] Preferably, the step (2) specifically comprises the following steps: dispersing the copper-cerium bimetallic peroxide nano-octahedron in water to obtain a dispersion; adding an aqueous solution of hyaluronic acid or sodium hyaluronate to the dispersion to react; after the reaction is completed, recovering and purifying the product to obtain hyaluronic acid-functionalized copper-cerium bimetallic peroxide nano-octahedron.

[0022] More preferably, the volume ratio of the dispersion to the aqueous solution of hyaluronic acid or sodium hyaluronate is 1:1-10.

[0023] Further preferably, in the dispersion, the mass ratio of copper-cerium bimetallic peroxide nanooctahedron to water is 1:500-1000; in the aqueous solution of hyaluronic acid or sodium hyaluronate, the mass ratio of hyaluronic acid or sodium hyaluronate to water is 1:500-1000.

[0024] More preferably, the reaction is carried out at room temperature for 4-24 h.

[0025] In the second aspect of the present invention, a size-adjustable, strong ability to actively target tumor tissue, high stability, good bimetallic synergistic catalytic ability and therapeutic effect hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedron is provided, which is prepared by the preparation method of the first aspect of the present invention.

[0026] In the third aspect of the present invention, there is provided an application of the hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron according to the second aspect of the present invention, specifically an application as an anti-tumor drug in tumor treatment.

[0027] Based on the above technical solutions, the design concept and principle of the present invention are as follows:

[0028] The process of this method is simple and convenient. The target octahedral structure is obtained by adjusting the feed concentration of sodium hydroxide, and the molecular weight of PVP is adjusted to achieve the purpose of size control. The required size and morphology can be selected according to the specific application. The size-controllable hyaluronic acid functionalized copper-cerium bimetallic peroxide prepared is more stable and more convenient to store than other metal peroxides due to the protective effect of hyaluronic acid on its surface. In addition, compared with spheres or spheroids, octahedral nanoparticles have some unique advantages in positive and negative electrical adsorption. First, the octahedral structure provides more surface active sites, which means that they can provide more adsorption sites, thereby improving adsorption efficiency. Second, the geometric shape of the octahedral nanoparticles leads to higher surface energy, which helps to enhance the interaction force with the adsorbed molecules. In the process of targeting tumors, hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedra take advantage of their controllable size and passively target tumor tissue using the EPR effect. This positively charged nano-octahedron can be functionalized through positive and negative adsorption, and can be adsorbed with negatively charged functional polymers such as hyaluronic acid to produce functionalized nano-octahedrons with negative surface charge. Hyaluronic acid functionalization can enhance the ability of the active octahedron to target tumor tissue.

[0029] According to the Nernst equation, under acidic conditions, E φ (Ce 4+ / Cu + ) = +1.567, and E φ (Cu 2+ / Ce 3+ ) = -1.567. This indicates that monovalent copper can reduce tetravalent cerium to trivalent cerium in a weakly acidic environment, while trivalent cerium cannot reduce divalent copper to monovalent copper. Therefore, when the elemental valence state composition of the copper-cerium bimetallic peroxide nanooctahedron is monovalent copper and the ratio of tetravalent cerium to trivalent cerium, the bimetallic synergistic catalytic ability and therapeutic efficacy of the copper-cerium bimetallic peroxide nanooctahedron can be greatly enhanced. During tumor treatment, in response to the relatively high pH value in the tumor microenvironment, a series of cascade reactions are initiated, including: the release of large amounts of tetravalent cerium ions, monovalent copper ions, and hydrogen peroxide through a weak acid response; the released tetravalent cerium ions are reduced to trivalent cerium ions with Fenton activity by monovalent copper ions and glutathione in the tumor microenvironment; the trivalent cerium ions and monovalent copper ions further undergo a Fenton-like reaction, converting hydrogen peroxide into highly cytotoxic hydroxyl radicals, achieving tumor-specific chemodynamic therapy.

[0030] During tumor treatment, these bimetallic peroxide nanooctahedra, with their bimetallic synergistic and cascade-like functions, can generate a variety of reactive oxygen species within tumor cells and the tumor microenvironment through the aforementioned cascade reaction, thereby enhancing oxidative stress in tumor cells and releasing a large number of tumor antigens and damage-associated molecular patterns. This promotes dendritic cell maturation, induces M1 polarization of tumor-associated macrophages, activates effector T cells, and enhances the anti-tumor immune response. This complements immunotherapies mediated by anti-programmed death protein-1 (αPD-1) antibodies and anti-programmed death receptor ligand-1 (αPD-L1) antibodies, effectively addressing the ineffectiveness of single-agent therapies. In combination with anti-αPD-1-mediated immunotherapy, it can effectively inhibit primary tumor growth, prevent the development of metastases, and significantly enhance the efficacy of chemodynamic immunotherapy.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] The present invention provides a preparation method of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedrons, which has the advantages of simple process, controllable size and wide source of raw materials.

[0033] The present invention provides a hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedron. The particle has adjustable size and has the characteristics of strong ability to actively target tumor tissue, high stability, bimetallic synergistic catalytic ability and good therapeutic effect.

[0034] The present invention provides an application of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nano-octahedron, which has broad application prospects in tumor treatment as an anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Flow chart of the preparation method of hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron;

[0036] Figure 2 The transmission electron microscope (TEM) images of copper-cerium bimetallic peroxide nano-octahedrons obtained at different sodium hydroxide feed concentrations are shown in FIG. Figure 2 A is the TEM image of copper-cerium bimetallic peroxide nanooctahedron obtained by feeding 1 mM sodium hydroxide. Figure 2 B is the TEM image of copper-cerium bimetallic peroxide nanooctahedron obtained by adding 5 mM sodium hydroxide. Figure 2C is the TEM image of copper-cerium bimetallic peroxide nanooctahedron obtained by feeding 10mM sodium hydroxide. Figure 2 D is the TEM image of copper-cerium bimetallic peroxide nanooctahedron obtained by feeding at a concentration of 20 mM sodium hydroxide;

[0037] Figure 3 TEM images of copper-cerium bimetallic peroxide nanooctahedra of different sizes obtained by feeding PVP with different molecular weights; Figure 3 A is the TEM image of copper-cerium bimetallic peroxide nanooctahedron obtained by adding 10000 MW molecular weight PVP. Figure 3 B is the TEM image of copper-cerium bimetallic peroxide nanooctahedron obtained by adding 24000 MW molecular weight PVP. Figure 3 C is the TEM image of copper-cerium bimetallic peroxide nanooctahedron obtained by feeding PVP with a molecular weight of 48000 MW;

[0038] Figure 4 Characterization of hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedra; Figure 4 A and 4B are TEM images of copper-cerium bimetallic peroxide nanooctahedra (CBPNs) and hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra (CBPNs@HA). Figure 4 C is a scanning transmission electron microscopy (STEM) image of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedrons. Figure 4 D is the element distribution diagram, Figure 4 E is the particle size test result, Figure 4 F is the X-ray photoelectron spectroscopy (XPS) test result of Ce3d orbital, Figure 4 G is the XPS test result of Cu2p orbital, Figure 4 H is the XPS test result of O1s orbital, Figure 4 I is the test result of producing H2O2 under different pH conditions, Figure 4 J is the potential test result;

[0039] Figure 5 The cytotoxicity of CBPNs@HA and its ability to disrupt the intracellular redox balance; Figure 5 A is the toxicity results of different materials on B16 tumor cells, Figure 5 B is the toxicity results of different materials on CT26 tumor cells. Figure 5 C is the toxicity results of different materials on 4T1 tumor cells, Figure 5D is the apoptosis and necrosis of tumor cells determined by flow cytometry;

[0040] Figure 6 is the in vivo anti-tumor effect of CBPNs@HA; Figure 6 A is the change in tumor size. Figure 6 B is the survival curve of tumor mice; Figure 6 C is the weight change result;

[0041] Figure 7 The construction of CBPNs@HA and its anti-tumor mechanism in combination with αPD-1. DETAILED DESCRIPTION

[0042] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It will be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, but not for limiting the present invention. Throughout the specification, unless otherwise specifically stated, the terms used herein are to be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. If there is a conflict, this specification takes precedence. Unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment etc. used in the present invention can be purchased from the market or can be obtained by existing methods.

[0043] The embodiment of the present invention provides a hyaluronic acid functionalized copper-cerium bimetallic peroxide nano-octahedron, such as Figure 7 As shown, the overall idea is as follows:

[0044] According to another typical embodiment of the present invention, a method for preparing hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra is provided, the method comprising:

[0045] (1) Dispersing cerium (IV) metal salt and copper (I) metal salt in a PVP aqueous solution, adding sodium hydroxide and hydrogen peroxide to obtain copper-cerium bimetallic peroxide nanooctahedrons;

[0046] As an optional embodiment, the copper-cerium bimetallic peroxide nano-octahedron is synthesized by a controlled rate precipitation method, specifically comprising:

[0047] A cerium (IV) metal salt and a copper (I) metal salt were completely dissolved in an aqueous solution of PVP to prepare a bimetallic precursor solution. A sodium hydroxide aqueous solution and a hydrogen peroxide solution were then added to the bimetallic precursor solution via a syringe pump through a pipeline at a rate of 20 mL / h to react. The reaction solution was removed and centrifuged and washed (8000-12000 rpm, 8-15 min) to obtain copper-cerium bimetallic peroxide nanooctahedra.

[0048] Wherein, in the bimetallic precursor solution, the molar ratio of the metal salt of cerium (IV) and the metal salt of copper (I) is 1:1;

[0049] The bimetallic precursor solution can be a cerium sulfate / cuprous sulfate aqueous solution, a cerium sulfate / cuprous chloride aqueous solution, a cerium sulfate / cuprous nitrate aqueous solution, a cerium chloride / cuprous chloride aqueous solution, a cerium chloride / cuprous nitrate aqueous solution, or a cerium nitrate / cuprous nitrate aqueous solution;

[0050] The volume ratio of sodium hydroxide aqueous solution to bimetallic precursor solution is 1:1; the volume ratio of hydrogen peroxide aqueous solution to bimetallic precursor solution is 1:25; the mass volume ratio of PVP to water in the PVP aqueous solution is 1 g:4 mL;

[0051] The concentration of sodium hydroxide is adjusted to obtain copper-cerium bimetallic peroxide nano-octahedrons;

[0052] By changing the concentration of sodium hydroxide, the completeness of the reaction was changed, and copper-cerium bimetallic peroxide nano-octahedra with regular and uniform morphology were obtained.

[0053] This step specifically includes:

[0054] The concentration of the sodium hydroxide aqueous solution is adjusted, and then the remaining steps are performed to obtain copper-cerium bimetallic peroxide nano-octahedrons with gradually complete morphologies;

[0055] The feeding concentration of sodium hydroxide aqueous solution is 1-20 mM;

[0056] The molecular weight of PVP in the PVP aqueous solution is adjusted to obtain size-controlled copper-cerium bimetallic peroxide nanooctahedrons; PVP with different molecular weights is dissolved in water to obtain PVP aqueous solutions with different molecular weights, and then the subsequent steps are carried out;

[0057] This step specifically includes:

[0058] The molecular weight of PVP in the PVP aqueous solution is adjusted to obtain PVP aqueous solutions with different molecular weights, and then the remaining steps are performed to obtain size-controlled copper-cerium bimetallic peroxide nanooctahedra;

[0059] Among them, the molecular weight of PVP in aqueous solutions of different molecular weights is 10,000-48,000 Mw;

[0060] (2) wrapping hyaluronic acid on the obtained copper-cerium bimetallic peroxide nanooctahedron to obtain hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron;

[0061] This step specifically includes:

[0062] Ultrasonic dispersion of copper-cerium bimetallic peroxide nanooctahedra in deionized water to obtain a dispersion; subsequently, a hyaluronic acid aqueous solution is added to the dispersion, and after sufficient reaction, centrifugation and washing are performed to obtain size-controllable hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra;

[0063] The mass volume ratio of copper-cerium bimetallic peroxide nanooctahedron to deionized water in the dispersion is 1-10 mg:10 mL;

[0064] The mass volume ratio of hyaluronic acid to deionized water in the hyaluronic acid aqueous solution is 1-10 mg:10 mL;

[0065] The volume ratio of the dispersion to the hyaluronic acid aqueous solution was 1:1.

[0066] According to another typical embodiment of the present invention, provided are size-controllable hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra prepared by the method.

[0067] The hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedron of the present application will be described in detail below with reference to the examples and experimental data.

[0068] Example 1

[0069] In this example, the preparation method of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra was used to prepare copper-cerium bimetallic peroxide nanooctahedra for studying their properties. The steps are as follows:

[0070] A bimetallic precursor solution was prepared by dissolving 1 mM cerium sulfate and 1 mM cuprous sulfate in deionized water containing 5 g of PVP to obtain a bimetallic precursor solution and a 1 mM sodium hydroxide solution. The sodium hydroxide solution was then added to the reactor containing the bimetallic precursor solution via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. Subsequently, 1 mL of a 30 wt.% H2O2 aqueous solution was added to the reactor via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. The mixture was then centrifuged multiple times (10,000 rpm, 10 min) and washed to obtain copper-cerium bimetallic peroxide nanooctahedra.

[0071] Example 2

[0072] In this example, the preparation method of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra was used to prepare copper-cerium bimetallic peroxide nanooctahedra for studying their properties. The steps are as follows:

[0073] A bimetallic precursor solution was prepared by dissolving 1 mM cerium sulfate and 1 mM cuprous sulfate in deionized water containing 5 g of PVP to obtain a bimetallic precursor solution and a 5 mM sodium hydroxide solution. The sodium hydroxide solution was then added to the reactor containing the bimetallic precursor solution via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. Subsequently, 1 mL of a 30 wt.% H2O2 aqueous solution was added to the reactor via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. The mixture was then centrifuged multiple times (10,000 rpm, 10 min) and washed to obtain copper-cerium bimetallic peroxide nanooctahedra.

[0074] Example 3

[0075] In this example, the preparation method of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra was used to prepare copper-cerium bimetallic peroxide nanooctahedra for studying their properties. The steps are as follows:

[0076] A bimetallic precursor solution was prepared by dissolving 1 mM cerium sulfate and 1 mM cuprous sulfate in deionized water containing 5 g of PVP to obtain a bimetallic precursor solution and a 10 mM sodium hydroxide solution. The sodium hydroxide solution was then added to the reactor containing the bimetallic precursor solution via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. Subsequently, 1 mL of a 30 wt.% H2O2 aqueous solution was added to the reactor via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. The mixture was then centrifuged multiple times (10,000 rpm, 10 min) and washed to obtain copper-cerium bimetallic peroxide nanooctahedra.

[0077] Example 4

[0078] In this example, the preparation method of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra was used to prepare copper-cerium bimetallic peroxide nanooctahedra for studying their properties. The steps are as follows:

[0079] A bimetallic precursor solution was prepared by dissolving 1 mM cerium sulfate and 1 mM cuprous sulfate in deionized water containing 5 g of PVP to obtain a bimetallic precursor solution and a 20 mM sodium hydroxide solution. The sodium hydroxide solution was then added to the reactor containing the bimetallic precursor solution via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. Subsequently, 1 mL of a 30 wt.% H2O2 aqueous solution was added to the reactor via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. The mixture was then centrifuged multiple times (10,000 rpm, 10 min) and washed to obtain copper-cerium bimetallic peroxide nanooctahedra.

[0080] In this example, PVP was set to 10,000 Mw, 24,000 Mw, and 48,000 Mw to prepare corresponding copper-cerium bimetallic peroxide nanooctahedra for studying the structural characteristics at different molecular weights.

[0081] Example 5

[0082] In this embodiment, the surface of copper-cerium bimetallic peroxide nano-octahedron is coated with sodium hyaluronate to complete the functionalization, and hyaluronic acid functionalized copper-cerium bimetallic peroxide nano-octahedron is obtained to study its performance, such as Figure 1 As shown, the steps are as follows:

[0083] (1) Prepare a bimetallic precursor solution: 1 mM cerium sulfate and 1 mM cuprous sulfate were dissolved in deionized water containing 5 g of PVP to prepare a bimetallic precursor solution and a 20 mM sodium hydroxide solution. The sodium hydroxide solution was then added to the reactor containing the bimetallic precursor solution via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. Subsequently, 1 mL of a 30 wt.% H2O2 aqueous solution was added to the reactor via a syringe pump through an independent pipe at a rate of 20 mL / h, and the mixture was stirred at room temperature for 1 h. The mixture was then centrifuged multiple times (10,000 rpm, 10 min) and washed to obtain copper-cerium bimetallic peroxide nanooctahedra (CBPNs).

[0084] (2) 10 mg of CBPNs were dispersed in 20 mL of deionized water and ultrasonicated for 5 min to obtain a dispersion. 5 mg of hyaluronic acid was dispersed in 20 mL of deionized water to prepare a hyaluronic acid aqueous solution. The above dispersion was added to the hyaluronic acid aqueous solution through an independent pipe by a syringe pump at a rate of 20 mL / h. The mixture was stirred and reacted at room temperature for 4 h. After sufficient reaction, the mixture was centrifuged and washed to obtain hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra (denoted as CBPNs@HA).

[0085] Example 6

[0086] In this embodiment, the surface of copper-cerium bimetallic peroxide nano-octahedron is coated with sodium hyaluronate to complete the functionalization, and hyaluronic acid functionalized copper-cerium bimetallic peroxide nano-octahedron is obtained to study its performance, such as Figure 1 As shown, the steps are as follows:

[0087] (1) Prepare a bimetallic precursor solution, 1 mM cerium sulfate and 1 mM cuprous sulfate dissolved in deionized water containing 5 g PVP to prepare a bimetallic precursor solution, and 20 mM sodium hydroxide solution, then add the above sodium hydroxide solution to the reactor containing the bimetallic precursor solution by a syringe pump through an independent pipe at 20 mL / h, and react at room temperature for 1 h; then add 1 mL of 0.39 mol / L H2O2 aqueous solution to the above reactor by a syringe pump through an independent pipe at 20 mL / h, and react at room temperature for 1 h, and then centrifuge several times (10000 rpm, 10 min) and wash to obtain copper-cerium bimetallic peroxide nanooctahedron;

[0088] (2) Disperse 10 mg of copper-cerium bimetallic peroxide nanooctahedra in 20 mL of deionized water and ultrasonicate for 5 min to uniformly disperse them to obtain a dispersion; disperse 20 mg of hyaluronic acid in 20 mL of deionized water to prepare a hyaluronic acid aqueous solution; add the above dispersion to the hyaluronic acid aqueous solution through an independent pipe by a syringe pump at a rate of 20 mL / h, stir and react at room temperature for 24 h, centrifuge and wash after sufficient reaction to obtain hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra.

[0089] Example 7

[0090] (1) Prepare a bimetallic precursor solution, 1 mM cerium sulfate and 10 mM cuprous sulfate dissolved in deionized water containing 5 g of PVP to prepare a bimetallic precursor solution, and 20 mM sodium hydroxide solution, then add the above sodium hydroxide solution to the reactor containing the bimetallic precursor solution by a syringe pump through an independent pipe at 5 mL / h, and react at room temperature for 1 h; then add 1 mL of 9.79 mol / L H2O2 aqueous solution to the above reactor by a syringe pump through an independent pipe at 5 mL / h, and react at room temperature for 1 h, and then centrifuge several times (10000 rpm, 10 min) and wash to obtain copper-cerium bimetallic peroxide nanooctahedron;

[0091] (2) Disperse 10 mg of copper-cerium bimetallic peroxide nanooctahedra in 10 mL of deionized water and ultrasonicate for 5 min to uniformly disperse them to obtain a dispersion; disperse 20 mg of hyaluronic acid or sodium hyaluronate in 10 mL of deionized water to prepare a hyaluronic acid or sodium hyaluronate aqueous solution; add the above dispersion to the hyaluronic acid aqueous solution through an independent pipe by a syringe pump at a rate of 5 mL / h, stir and react at room temperature for 4 h, centrifuge and wash after sufficient reaction to obtain hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra.

[0092] Example 8

[0093] (1) Prepare a bimetallic precursor solution, 1 mM cerium sulfate and 5 mM cuprous sulfate dissolved in deionized water containing 5 g PVP to prepare a bimetallic precursor solution, and 20 mM sodium hydroxide solution, then add the above sodium hydroxide solution to the reactor containing the bimetallic precursor solution by a syringe pump through an independent pipe at 10 mL / h, and react at room temperature for 1 h; then add 1 mL of 4.7 mol / L H2O2 aqueous solution to the above reactor by a syringe pump through an independent pipe at 10 mL / h, and react at room temperature for 1 h, and then centrifuge several times (10000 rpm, 10 min) and wash to obtain copper-cerium bimetallic peroxide nanooctahedron;

[0094] (2) Disperse 10 mg of copper-cerium bimetallic peroxide nanooctahedra in 15 mL of deionized water and ultrasonicate for 5 min to uniformly disperse them to obtain a dispersion; disperse 5 mg of hyaluronic acid or sodium hyaluronate in 15 mL of deionized water to prepare a hyaluronic acid or sodium hyaluronate aqueous solution; add the above dispersion to the hyaluronic acid aqueous solution through an independent pipe by a syringe pump at a rate of 10 mL / h, stir and react at room temperature for 14 h, centrifuge and wash after sufficient reaction to obtain hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra.

[0095] Test Example 1

[0096] Structural characterization of copper-cerium bimetallic peroxide nanooctahedra:

[0097] This test example uses transmission electron microscopy to characterize the morphology and size of the copper-cerium bimetallic peroxides of different morphologies in Examples 1-4. Figure 2 As shown in Figure 2, under different sodium hydroxide feed concentrations, copper-cerium bimetallic peroxides exhibit different morphologies; Figure 2 As shown in A, at a feed concentration of 1 mM sodium hydroxide, the copper-cerium bimetallic peroxide exhibits an irregular granular structure; Figure 2As shown in Figure B, at a feed concentration of 5 mM sodium hydroxide, a small amount of octahedral structure appeared in the copper-cerium bimetallic peroxide; Figure 2 As shown in C, at a feed concentration of 10 mM sodium hydroxide, there are a large number of octahedral structures in the copper-cerium bimetallic peroxide, with only a small amount of broken particles; Figure 2 As shown in Figure D, at a sodium hydroxide feed concentration of 20 mM, the copper-cerium bimetallic peroxide exhibits a nano-octahedral shape with regular structure and uniform size.

[0098] Test Example 2

[0099] Structural characterization of size-controlled copper-cerium bimetallic peroxide nanooctahedra:

[0100] This test example uses transmission electron microscopy to characterize the morphology and size of the copper-cerium bimetallic peroxide nanooctahedra prepared with different PVP molecular weights in Example 4. Figure 3 As shown in Figure 2, when using PVP with different molecular weights, the copper-cerium bimetallic peroxide nanooctahedra exhibit different sizes; Figure 3 As shown in Figure A, when using 10000 Mw of PVP, the size of the copper-cerium bimetallic peroxide nanooctahedron is about 180 nm; Figure 3 As shown in Figure B, when using 24000 Mw of PVP, the size of the copper-cerium bimetallic peroxide nanooctahedron is about 100 nm; Figure 3 As shown in Figure C, when using 48000 MW of PVP, the size of the Cu-Ce bimetallic peroxide nanooctahedron is about 50 nm.

[0101] Test Example 3

[0102] Structure and property characterization of hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedra:

[0103] The morphology and size of CBPNs@HA were characterized by transmission electron microscopy, scanning transmission electron microscopy and dynamic scattering. Figure 4 As shown in Figures 4A, 4B, and 4C, copper-cerium bimetallic peroxide and hyaluronic acid-functionalized copper-cerium bimetallic peroxide also exhibit uniform nano-octahedral shapes; Figure 4 As shown in D, in the hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron, Cu, Ce, O, and N elements are evenly distributed on the surface of each nanooctahedron; Figure 4 As shown in Figure E, the particle sizes of the obtained copper-cerium bimetallic peroxide nanooctahedron and hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron are ~180 nm and ~190 nm, respectively.

[0104] The physical and chemical properties of the prepared hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedra were measured using X-ray photoelectron spectroscopy and ultraviolet-visible spectrometry to determine whether the surface modification was successful, whether the properties of the material were affected, and the composition of the elements in the material; Figure 4 As shown in F and 4G, the valence distribution of Ce element in hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron is Ce 4+ :Ce 3+ = 61.68%:31.21%, the valence state of Cu element is Cu + ;like Figure 4 As shown in H, there are obvious peroxide bonds in the hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron, indicating the presence of peroxide groups in the material; Figure 4 As shown in FIG1 , the ability of copper-cerium bimetallic peroxide nanooctahedron and hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron to produce H 2 O 2 under acidic conditions is similar, proving that the function of copper-cerium bimetallic peroxide nanooctahedron will not be affected during the functionalization process; Figure 4 As shown in Figure 5, the surface of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedron is negatively charged, while the surface of copper-cerium bimetallic peroxide nanooctahedron is positively charged, which proves the successful preparation of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedron.

[0105] Application Example 1

[0106] In vitro experiments:

[0107] In vitro co-culture experiments were carried out with B16, CT26 and 4T1 tumor cells and the prepared CBPNs@HA to systematically investigate the in vitro anti-tumor properties and mechanisms of the material.

[0108] Including: Tumor cytotoxicity. First, B16 / CT26 / 4T1 cells were seeded in 96-well plates (1×10 5 cells / well) were cultured in complete RPMI medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for 24 hours. To verify that CBPNs@HA responds to the weakly acidic pH of the tumor microenvironment, hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra were co-cultured with B16 / CT26 / 4T1 tumor cells in medium with pH 6.8 and 7.2 for 24 hours. Then, 10% CCK8 solution was added and incubated for 1.5 hours. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader. Figure 5As shown in Figures AC, in a culture medium with a pH of 6.8, a large number of tumor cells were killed by CBPNs and CBPNs@HA materials, while when the culture medium pH was 7.2, only a portion of the tumor cells were killed by CBPNs and CBPNs@HA materials. At the same time, CBPNs@HA was more toxic to tumor cells, indicating that CBPNs@HA can further enhance the uptake of tumor cells, selectively respond to the pH of the culture environment, and enhance its toxicity to tumor cells.

[0109] Application Example 2

[0110] In vivo antitumor properties of hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra and enhanced αPD-1-mediated immunotherapy:

[0111] 6-8 week old BALB / c mice were used as model animals. 4T1 tumor-bearing mouse models were established by subcutaneous injection of 4T1 tumor cells. The efficacy of CBPNs@HA in treating tumors and the efficacy of combined therapy with αPD-1 after tail vein injection were systematically studied. 3 Tumor-bearing mice were randomly divided into six groups and injected via the tail vein on days 8 and 9, days 11 and 12, days 14 and 15, and days 17 and 18, respectively. Changes in body weight and tumor size were recorded every other day, and tumor volume was calculated (Still formula: V = W² × L / 2, where W is the short diameter of the tumor and L is the long diameter of the tumor).

[0112] like Figure 6 As shown, compared to other control materials (PBS, αPD-1, and CBPNs), treatment with hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedrons (CBPNs@HA) significantly inhibited tumor growth in tumor-bearing mice, and the tumor volume was significantly smaller than that of the other materials, demonstrating that CBPNs@HA has a strong inhibitory effect on tumor growth. After synergistic treatment with αPD-1, tumor growth in tumor-bearing mice was further suppressed, with a more significant reduction in tumor volume compared to the CBPNs@HA group, and no recurrence occurred after the end of treatment.

[0113] αPD-1 alone was ineffective. CBPNs, which were unfunctionalized copper-cerium bimetallic peroxide nanooctahedra, also showed poor therapeutic effects, with tumor recurrence occurring after treatment. Hyaluronic acid-functionalized CBPNs@HA demonstrated a good therapeutic effect on tumors in tumor-bearing mice during treatment, but tumors recurred during the post-treatment observation period. The combined αPD-1 and CBPNs@HA treatment group demonstrated a good tumor-suppressing effect during and after treatment, with no recurrence. This suggests that CBPNs@HA can actively target tumors and, when combined with αPD-1, significantly enhance the body's anti-tumor immunity, strengthening αPD-1-mediated anti-tumor immunotherapy.

[0114] In summary, the size-controllable hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron prepared by the present invention utilizes the active tumor targeting ability provided by hyaluronic acid to improve the enrichment effect of nanooctahedron in tumors. After targeting the tumor site, the hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron can initiate a series of cascade reactions, effectively causing a Fenton-like reaction, thereby amplifying oxidative stress and effectively inducing immunogenic cell death in tumor cells, thereby enhancing the immunotherapy effect. In this cascade reaction, the nanooctahedron decomposes at a relatively high pH in the tumor microenvironment to produce H2O2, Cu + and Ce 4+ , Ce 4+ Cu + and glutathione in tumors is reduced to Ce 3+ , Ce 3+ / Cu + It reacts with H2O2 in the tumor and self-supplied H2O2 in a Fenton-like reaction to produce highly cytotoxic ·OH. ·OH induces immunogenic death by enhancing oxidative stress, thereby releasing a large number of tumor antigens and damage-associated molecular patterns, thereby promoting the maturation of dendritic cells, inducing tumor-associated macrophages to polarize to the M1 type, activating effector T cells, and enhancing anti-tumor immune effects. Figure 7 As shown, the design and development of this copper-cerium bimetallic peroxide nanooctahedron provides a new strategy for efficient chemodynamic therapy and a new idea for the combined treatment of chemodynamic-immunotherapy.

[0115] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for preparing hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedrons, characterized in that: The steps include: (1) Completely dissolving a cerium (IV) metal salt and a copper (I) metal salt in an aqueous solution of polyvinyl pyrrolidone to form a bimetallic precursor solution; then, adding a sodium hydroxide aqueous solution and a hydrogen peroxide aqueous solution to the bimetallic precursor solution at a rate of 5-20 mL / h to react, and after the reaction is completed, recovering and purifying the product to obtain copper-cerium bimetallic peroxide nanooctahedrons; The metal salt of cerium (IV) includes one of cerium sulfate and cerium chloride; the metal salt of copper (I) includes one of cuprous sulfate, cuprous chloride, and cuprous nitrate; the molar ratio of the metal salt of cerium (IV), the metal salt of copper (I), and water in the bimetallic precursor solution is 1-10:1-10:55.5; The volume ratio of the sodium hydroxide aqueous solution to the bimetallic precursor solution is 1:1-50; the volume ratio of the hydrogen peroxide aqueous solution to the bimetallic precursor solution is 1:1-25; the concentration of the hydrogen peroxide aqueous solution is 0.39-9.79 mol / L; in the process of forming the octahedral structure by adjusting the feeding concentration of sodium hydroxide, the concentration of the sodium hydroxide aqueous solution is 1-20 mmol / L; In the aqueous solution of polyvinyl pyrrolidone, the molecular weight of polyvinyl pyrrolidone is 8000-58000; the mass ratio of polyvinyl pyrrolidone to water is 1:5-20; The reaction is carried out at room temperature and the reaction time is 1-12 h; The size of the product is controlled by adjusting the molecular weight of polyvinyl pyrrolidone, and the octahedral structure is formed by adjusting the feeding concentration of sodium hydroxide. (2) The obtained copper-cerium bimetallic peroxide nanooctahedron is coated with hyaluronic acid to obtain hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron.

2. The method for preparing hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra according to claim 1, characterized in that: The step (2) specifically comprises the following steps: dispersing the copper-cerium bimetallic peroxide nano-octahedron in water to obtain a dispersion; adding an aqueous solution of hyaluronic acid or sodium hyaluronate to the dispersion to carry out a reaction; and recovering and purifying the product after the reaction is completed to obtain hyaluronic acid-functionalized copper-cerium bimetallic peroxide nano-octahedron.

3. The method for preparing hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedra according to claim 2, characterized in that: The volume ratio of the dispersion to the aqueous solution of hyaluronic acid or sodium hyaluronate is 1:1-10; in the dispersion, the mass ratio of copper-cerium bimetallic peroxide nanooctahedra to water is 1:500-1000; in the aqueous solution of hyaluronic acid or sodium hyaluronate, the mass ratio of hyaluronic acid or sodium hyaluronate to water is 1:500-1000; the reaction is carried out at room temperature for 4-24 hours.

4. A hyaluronic acid-functionalized copper-cerium bimetallic peroxide nanooctahedron, characterized by: The invention is prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the hyaluronic acid functionalized copper-cerium bimetallic peroxide nanooctahedron as claimed in claim 4 in the preparation of anti-tumor drugs; wherein, The tumor type is one of melanoma, colon cancer, and breast cancer.

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