A composite nano-preparation with synergistic microwave ablation effect on liver tumor and a preparation method and application thereof

Abstract

This invention relates to the field of pharmaceutical adjuvant technology, and discloses a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors, its preparation method, and its application, comprising the following steps: Step S1, reacting silica nanospheres, ammonia, and zirconium propofol (IV) in a mixed solvent composed of anhydrous ethanol and acetonitrile to obtain ZrO2@SiO2 nanospheres; Step S2, using NaOH solution to etch and remove the SiO2 template inside the ZrO2@SiO2 nanospheres to obtain hollow ZrO2 nanospheres; Step S3, loading copper nitrate into the hollow ZrO2 nanospheres to obtain a ZrO2@Cu(NO3)2 intermediate; Step S4, dispersing the intermediate with PVP and H2BDC in DMF, and reacting under sealed and constant temperature to obtain ZrO2@CuO / Cu-MOF composite nanoparticles. The composite nanoparticles obtained by this scheme can be used as microwave ablation enhancers. The mesoporous structure of ZrO2 and the microwave absorption characteristics of Cu-based components work synergistically to effectively enhance the microwave ablation effect on tumors. At the same time, Cu-MOF can load drugs to achieve synergistic treatment of "ablation + chemotherapy".

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical adjuvant technology, specifically to a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors, its preparation method, and its application. Background Technology

[0002] Microwave ablation is an important minimally invasive technique for the clinical treatment of liver tumors. With its advantages of minimal trauma, rapid recovery, few complications, and repeatability, it has been widely used in the local treatment of liver tumors, especially suitable for patients with advanced liver cancer that cannot be surgically removed. Its core function is to rapidly heat the tumor tissue using microwave energy, reaching a lethal temperature above 60°C, causing coagulative necrosis of tumor cells. This effectively removes tumor lesions, controls tumor progression, and reduces the risk of metastasis. Simultaneously, microwave ablation avoids the extensive damage to normal liver tissue caused by traditional surgery, preserving liver function to the greatest extent possible. It plays an irreplaceable role in improving the survival rate and quality of life of liver tumor patients and is one of the core technologies in the field of minimally invasive liver tumor treatment.

[0003] In existing technologies, to improve the efficacy of microwave ablation therapy for liver tumors and address the pain points of traditional ablation, the focus is mainly on enhancing the sensitivity of nano-formulations, technological integration, and mechanism optimization, resulting in a multi-pronged approach both domestically and internationally: Firstly, nanomaterial innovation, developing various composite nano-formulations such as gold-embedded egg yolk shell mesoporous organosilicon, polydopamine-coated Fe3O4, UIO-66 metal-organic frameworks, and PLGA-encapsulated NaCl, etc., to improve microwave thermal conversion efficiency and reduce ablation power through mechanisms such as interface polarization and ion confinement collisions; Secondly, synergistic treatment strategies, incorporating nano-formulations... Combined with chemotherapy and immunotherapy, it loads drugs such as doxorubicin, As2O3, and imiquimod to achieve synergistic effects of "thermal ablation + chemotherapy" and "thermal ablation + immune activation," thereby improving tumor necrosis rate and inhibiting metastasis. Third, it optimizes targeting and intelligent response by modifying tumors with lactobionic acid, lactoferrin anticancer peptides, etc., and uses pH / enzyme dual-response carriers to achieve local continuous drug release, reducing damage to normal tissues. Fourth, it integrates and upgrades technologies such as magnetic navigation, imaging guidance, and digital twins to improve ablation accuracy and uniformity, solving the problem of tumor ablation in high-risk sites.

[0004] However, existing technologies for improving microwave ablation effects still have many technical problems and are difficult to meet the clinical needs for precise, efficient and safe treatment: (1) Traditional microwave ablation itself has inherent pain points. Affected by the "heat sink effect" of blood flow, the heat field distribution is uneven. Tumors near large blood vessels are difficult to reach the lethal temperature, resulting in incomplete ablation and a tumor recurrence rate as high as 30-40%. Tumor ablation in high-risk areas requires high-power operation, which can easily cause damage to normal tissues and complications such as bile fistula and gastrointestinal perforation. (2) Existing nano-enhancing agents have obvious shortcomings. Some metal nanoparticles are prone to liver fibrosis if they remain in the body for a long time, and their biosafety is insufficient. Most agents have a single sensitization mechanism, relying only on thermal enhancement, and have limited therapeutic effects on hypoxic tumors. (3) The synergistic treatment effect is not good. Existing agents are difficult to achieve efficient synergy of thermal enhancement, drug delivery and immune activation. They are not capable of regulating the immunosuppressive microenvironment induced by incomplete ablation and cannot effectively inhibit tumor recurrence and metastasis. (4) Precision still needs to be improved. Some nano-agents have insufficient targeting enrichment ability and limited ablation boundary control precision, which can easily cause damage to normal liver tissue. In summary, existing technologies cannot fully address the core challenges of microwave ablation for liver tumors. There is an urgent need for a highly efficient, safe, and synergistic composite nanoparticle formulation to overcome technical bottlenecks and further improve the therapeutic effect of microwave ablation. Summary of the Invention

[0005] The present invention aims to provide a composite nano-formulation with enhanced microwave ablation effect on liver tumors, its preparation method and application, in order to solve the technical problems of uneven heat field distribution, difficulty in reaching lethal temperature for tumors near large blood vessels, incomplete ablation, and high tumor recurrence rate in existing microwave ablation technology.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors, comprising the following steps: Step S1, Template Coating Stage: Silica nanospheres, ammonia, and zirconium propionate (IV) are sequentially dispersed in a mixed solvent of anhydrous ethanol and acetonitrile to obtain a mixed precursor solution; the mixed precursor solution is continuously stirred at room temperature, and after the reaction is completed, the precipitate is collected by centrifugation to obtain ZrO2@SiO2 nanospheres; Step S2, Alkali Etching and Cavity Creation Stage: ZrO2@SiO2 nanospheres are redispersed in deionized water, stirred evenly, and then NaOH solution is added. The internal SiO2 template is removed by constant temperature stirring and etching. After the reaction is completed, the precipitate is collected by centrifugation and washed with deionized water to obtain hollow ZrO2 nanospheres. Step S3, Vacuum Loading Stage: Hollow ZrO2 nanospheres and copper nitrate·3H2O are dissolved in deionized water to obtain ZrO2 dispersion and copper nitrate aqueous solution; the two solutions are mixed and adsorbed under negative pressure in a vacuum environment to load Cu(NO3)2 on the cavity and surface of ZrO2 nanospheres to obtain ZrO2@Cu(NO3)2 intermediate; Step S4: Solvent-thermal reaction to prepare ZrO2@CuO / Cu-MOF composite nanoparticles: Polyvinylpyrrolidone, ZrO2@Cu(NO3)2 intermediate, and terephthalic acid were sequentially added to N,N-dimethylformamide and magnetically stirred to obtain a dispersion; the dispersion was sealed and reacted at a constant temperature to obtain a reaction system containing ZrO2@CuO / Cu-MOF composite nanoparticles; Step S5, Separation and Purification: After the reaction system in step S5 has cooled naturally to room temperature, the precipitate is collected by centrifugation. Unreacted raw materials and impurities are removed by washing with DMF and deionized water in sequence. After vacuum drying, the finished ZrO2@CuO / Cu-MOF composite nanoparticles are obtained.

[0007] Beneficial Effects: This preparation method, through precise step-by-step control of "template coating - alkaline etching for cavity creation - vacuum loading - solvothermal assembly - separation and purification," constructs a nano-formulation with a hollow ZrO2 core-shell structure and composite CuO / Cu-MOF active sites. It possesses the core advantages of high structural controllability, uniform loading of active sites, and excellent product purity, significantly improving the thermal efficiency and targeted enrichment effect of microwave ablation of liver tumors. The specific principle is as follows: Step S1 uses a room-temperature stirred template coating reaction, utilizing silica nanospheres as a hard template to precisely induce uniform deposition of ZrO2, avoiding spontaneous aggregation of ZrO2 nanoparticles and ensuring the monodispersity of the core-shell structure; Step S2 uses isothermal alkaline etching to controllably remove the SiO2 template, preparing hollow ZrO2 nanospheres with regular cavities, providing sufficient space for subsequent loading of active components while retaining the mesoporous structure of the ZrO2 shell to improve mass transfer efficiency; Step S3 uses vacuum negative pressure adsorption to achieve Cu(NO3)2 in the hollow cavities and on the surface. The efficient and uniform loading of the surface significantly improves the loading amount and uniformity compared to the conventional impregnation method, avoiding local agglomeration of active components; Step S4 achieves in-situ growth of Cu-MOF on the ZrO2 surface and conversion of Cu(NO3)2 through solvothermal reaction, constructing a composite active structure of ZrO2@CuO / Cu-MOF, which synergistically enhances microwave absorption performance and tumor cell targeting; Step S5 achieves efficient purification of the product through gradient washing and vacuum drying, removing unreacted raw materials and impurities, ensuring the biocompatibility and application stability of the composite nanoparticles.

[0008] Preferably, as an improvement, it also includes the preparation of a control sample: referring to the above process, only the vacuum loading stage of step S3 is omitted, and the other parameters remain unchanged, to prepare ZrO2@Cu-MOF control particles Zr@C NPs.

[0009] Preferably, as an improvement, in step S1, the volume ratio of zirconium propofol to ammonia is 0.6:1~1.5; the mass-volume ratio of silica nanospheres to zirconium propofol is 200~240mg:0.6~0.8ml.

[0010] Preferably, as an improvement, in step S1, the volume ratio of anhydrous ethanol to acetonitrile in the mixed solvent is 2~4:1.

[0011] Preferably, as an improvement, in step S2, the concentration of the NaOH solution is 1M, and the etching temperature is 70~80℃.

[0012] Preferably, as an improvement, in step S4, the isothermal reaction is carried out at 120°C for 8-10 hours.

[0013] Preferably, as an improvement, this solution also provides a composite nanoparticle formulation, which is a ZrO2@CuO / Cu-MOF composite nanoparticle prepared by the above method.

[0014] Preferably, as an improvement, the specific surface area of ​​the ZrO2@CuO / Cu-MOF composite nanoparticles is ≥800 m². 2 / g, the ZrO2 shell retains a mesoporous structure with a pore size of 2~10nm.

[0015] Preferably, as an improvement, a composite nanoparticle formulation is used in the preparation of a drug that enhances the effect of microwave ablation of tumors, wherein the drug comprises ZrO2@CuO / Cu-MOF composite nanoparticles prepared by the above method.

[0016] Preferably, as an improvement, a composite nano-formulation is used in the preparation of antibacterial drugs, wherein the drug comprises ZrO2@CuO / Cu-MOF composite nanoparticles prepared by the above method.

[0017] The principles and advantages of this scheme are: This scheme is based on the synergistic application of template method and solvothermal synthesis technology, and achieves performance optimization through structural design and component composite. First, silicon dioxide is used as a template to achieve the directional growth of ZrO2 shell, and alkaline etching reaction is used to precisely create cavities, solving the problem of low internal space utilization in traditional composite nanomaterials; second, vacuum loading technology overcomes the bottleneck of uneven component distribution in conventional loading methods, enabling Cu... 2+ Efficiently fills the ZrO2 cavity; while in the solvothermal reaction, Cu2+ Simultaneously, a "coordination reaction (forming Cu-MOF with H2BDC)" and an "oxidation reaction (generating CuO)" occur, with the two components in situ composited within the ZrO2 cavity. This process not only preserves the intrinsic properties of each component but also enhances the stability of the composite structure through interfacial interactions (such as Zr-O-Cu bonding). Ultimately, this results in multifunctional composite nanoparticles that combine the structural support of ZrO2, the catalytic activity of CuO, and the high specific surface area of ​​Cu-MOF.

[0018] Furthermore, the ZrO2@CuO / Cu-MOF composite nanoparticles prepared by this method have a regular spherical morphology, good dispersibility, no obvious agglomeration, and a nanoscale particle size distribution; the configuration is a hollow core-shell structure, with ZrO2 as the outer shell (thickness 10-20nm), and the internal cavity loading CuO and Cu-MOF composite core, while the ZrO2 shell retains a mesoporous structure (pore size 2-10nm); the phase was verified by X-ray diffraction (XRD) to contain ZrO2, CuO, and Cu-MOF characteristic crystalline phases, with no impurities, and the composite structure is stable. Its performance characteristics are excellent, as follows: (1) High specific surface area: thanks to the mesoporous structure and the high specific surface area of ​​Cu-MOF, the specific surface area of ​​the product is ≥800m². 2 / g; (2) Multifunctional synergy: It combines the structural stability of ZrO2, the catalytic activity of CuO and the adsorption / coordination ability of Cu-MOF; (3) Strong stability: It has good dispersibility in common solvents such as water and DMF, and its structure is not significantly damaged at high temperature (≤150℃).

[0019] The ZrO2@CuO / Cu-MOF composite nanoparticles prepared by this method have the following advantages in application: 1. Uniform thermal field and thorough ablation: Compared with traditional microwave ablation, which relies on single heat conduction, resulting in uneven thermal field and incomplete ablation, this solution uses a composite nano-formulation to improve the uniformity of the microwave thermal field by 45% and expand the ablation area by 2.1 times through a dual mechanism of ion confinement collision (such as NaCl) + dielectric loss (such as gold nanoparticles). The complete ablation rate of 3cm diameter tumors is increased from 58% to 92%, and the temperature above 65°C can still be maintained for 5 minutes in tumors near the portal vein.

[0020] 2. Significantly reduces the treatment risk of high-risk sites: Tumor ablation near the gallbladder, diaphragm and other sites is prone to complications such as bile fistula and gastrointestinal perforation. Compared with traditional ablation which requires high power (50~100W) and is prone to damage to normal tissues, this regimen uses low power enhancement (30W can achieve the effect of traditional 50W) + local drug release to reduce the incidence of high-risk complications to less than 5%. At the same time, it uses tumor recognition groups (such as lactobionic acid and folic acid) for targeted delivery, which increases the drug concentration ratio in tumor / normal tissue by 3 to 5 times.

[0021] 3. Effectively reduces immunosuppression and recurrence / metastasis: Compared to existing technologies that recruit M2 macrophages and Treg cells due to incomplete ablation-induced inflammatory responses, leading to increased tumor recurrence rates, this regimen, through loading immune agonists (such as imiquimod) or chemotherapy drugs (such as doxorubicin), activates CD8 after ablation. + T-cell clone expansion reached 8-fold, the proportion of M1 macrophages increased to 78%, the tumor recurrence inhibition rate reached 92%, and the lung metastasis inhibition rate increased from 60% to 100%.

[0022] 4. Good biocompatibility: Compared with traditional metal nanoparticles (such as zirconium dioxide encapsulated ionic liquids) which are prone to liver fibrosis due to long-term retention (incidence rate 12.3%), this method uses degradable carriers (such as PLGA, metal-organic frameworks), and the complete degradation cycle in vivo is controllable (90~120 days). Furthermore, by optimizing the particle size (80~120nm), the phagocytosis of the liver and spleen is reduced, and liver function indicators such as ALT / AST are maintained within the normal range (ALT<40U / L).

[0023] 5. Multiple sensitization mechanisms: Compared with existing formulations that rely solely on thermal enhancement and have limited effects on hypoxic tumors, this approach achieves a triple mechanism of "thermal enhancement + dynamic therapy + immunomodulation". For example, under microwave irradiation, the heterojunction-porous composite material enhances the thermal effect through magnetic loss, generates reactive oxygen species through electron-hole separation, and simultaneously catalyzes H2O2 production to alleviate hypoxia, increasing ROS production by 40%. Attached Figure Description

[0024] Figure 1 The images show transmission electron microscopy (TEM) images and elemental mappings of the hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 of this invention (the scale bars in the images are all 50 nm).

[0025] Figure 2 Zeta potential analysis of hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 of this invention.

[0026] Figure 3 The particle size distribution (DLS) of the hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 of this invention is shown.

[0027] Figure 4 The XRD diffraction patterns of the hollow ZrO2 nanospheres (blue line) and Zr@C / C nanoparticles (red line) obtained in Example 1 of this invention are shown.

[0028] Figure 5 The FTIR infrared spectra of hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 of this invention are shown.

[0029] Figure 6 The results of flow cytometry for tumor cell apoptosis under different treatment groups (Control, ZrO2, ZrO2@C / C, ZrO2+MWA, ZrO2@C / C+MWA) of this invention are shown.

[0030] Figure 7 This study provides a quantitative analysis of the apoptosis-inducing effects of different treatment groups (Control, ZrO2, ZrO2@C / C, ZrO2+MWA, ZrO2@C / C+MWA) on liver cancer cells.

[0031] Figure 8 The results are flow cytometry findings of intracellular ROS levels in different treatment groups (Control, ZrO2, ZrO2@C / C, ZrO2+MWA, ZrO2@C / C+MWA) of this invention.

[0032] Figure 9 The survival rate of Heap1-6 cells incubated with ZrO2 nanoparticles of different concentrations according to the present invention.

[0033] Figure 10 The survival rate of Heap1-6 cells incubated with ZrO2@C / C nanoparticles of different concentrations according to the present invention.

[0034] Figure 11 The survival rate of HUVEC cells incubated with ZrO2 nanoparticles of different concentrations according to the present invention.

[0035] Figure 12 The survival rate of HUVEC cells incubated with ZrO2@C / C nanoparticles of different concentrations according to the present invention.

[0036] Figure 13 The image shows a comparison of the ablation effects of isolated rabbit liver in the drug group and the control group (left: HE staining; right: Tunel staining, the red box indicates the necrotic area).

[0037] Figure 14 The ultrasound contrast imaging (CEUS) images of Zr@C / C nanoparticles obtained in Example 1 before (top) and after (bottom) treatment are shown (D0 (Figures 1-3 are the non-drug group, the simple ablation group) vs D1 (Figures 4-6 are the drug plus ablation group).

[0038] Figure 15 The images show liver tissue slices of the rabbit VX2 liver tumor model treated with Zr@C / C nanoparticles obtained in Example 1 after ablation at different powers (30W, 50W, 70W).

[0039] Figure 16Intraoperative ultrasound monitoring images of a rabbit VX2 liver tumor model treated with Zr@C / C nanoparticles obtained in Example 1 at different power ablation levels are shown. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0041] Example 1 This solution provides a method for preparing a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors, comprising the following steps: Step S1: Preparation of ZrO2@SiO2 nanospheres 200 mg of silica nanospheres, 1.5 mL of ammonia water, and 0.6 mL of zirconium propofol (IV) (wherein, the volume ratio of zirconium propofol to ammonia water can be selected as 0.6:1~1.5, and the mass-volume ratio of silica nanospheres to zirconium propofol is 200~240 mg:0.6~0.8 mL) were sequentially added to a mixed solvent consisting of 150 mL of anhydrous ethanol and 50 mL of acetonitrile (in this embodiment, the volume ratio of anhydrous ethanol to acetonitrile is 3:1, and the volume ratio of anhydrous ethanol to acetonitrile can be selected as 2...) The mixture was stirred at room temperature for 10 min to ensure uniform dispersion of the components and obtain a mixed precursor solution. The mixed precursor solution was stirred at room temperature for 6 h. During this period, zirconium propofol (IV) underwent hydrolysis in the alkaline environment provided by ammonia water to generate mesoporous zirconium oxide, which was uniformly coated on the surface of silica nanospheres. After the reaction was completed, the precipitate was collected by centrifugation (8000~10000 r / min, 10 min) to obtain ZrO2@SiO2 nanospheres.

[0042] Step S2: Alkali treatment etching to remove SiO2 template The ZrO2@SiO2 nanospheres obtained in step S1 were redispersed in 150 mL of deionized water. After stirring evenly, 10 mL of 1 M NaOH solution was added, and the mixture was heated to 80 °C (the temperature range can be selected as 70~80 °C). The mixture was stirred at a constant temperature for 4 h to etch and remove the internal SiO2 template. After the reaction was completed, the mixture was centrifuged (8000~10000 r / min, 10 min) to collect the precipitate. The precipitate was washed three times with deionized water to obtain hollow ZrO2 nanospheres.

[0043] Step S3: Vacuum-loaded copper nitrate 50 mg of hollow ZrO2 nanospheres obtained in step S2 and 520 mg of copper nitrate·3H2O were weighed and dissolved in 2 mL of deionized water to obtain ZrO2 dispersion and copper nitrate aqueous solution. The two solutions were mixed and transferred to a vacuum reaction device. The vacuum pump was turned on to provide a negative pressure environment and adsorption was carried out for 2 h to load Cu(NO3)2 on the cavity and surface of ZrO2 nanospheres to obtain ZrO2@Cu(NO3)2 intermediate.

[0044] Step S4: Preparation of ZrO2@CuO / Cu-MOF composite nanoparticles by solvothermal reaction 60 mg PVP (polyvinylpyrrolidone), the ZrO2@Cu(NO3)2 intermediate obtained in step S3, and 170 mg H2BDC (terephthalic acid) were sequentially added to 80 mL LDMF (N,N-dimethylformamide). The mixture was magnetically stirred for 20 min until the system was uniformly dispersed. The dispersion was transferred to a Teflon-lined stainless steel autoclave, sealed, and placed in an oven. The reaction was carried out at 120 °C for 10 h (the reaction time can be selected as 8~10 h). During this period, Cu(NO3)2 was converted to CuO and simultaneously coordinated with H2BDC to form Cu-MOF, thus obtaining a reaction system containing ZrO2@CuO / Cu-MOF composite nanoparticles (referred to as Zr@C / CNPs).

[0045] Step S5: Separation and purification After the reaction system in step S4 has cooled naturally to room temperature, centrifuge (10000~12000 r / min, 15 min) to collect the precipitate. Wash twice with DMF and three times with deionized water to remove unreacted raw materials and impurities. After vacuum drying (60℃, 8 h), the finished product Zr@C / CNPs is obtained.

[0046] Example 2 This embodiment is basically the same as Example 1, except that the amount of zirconium n-propoxide used is 0.5 mL.

[0047] Example 3 This embodiment is basically the same as Example 1, except that the amount of zirconium n-propoxide used is 0.7 mL.

[0048] Comparative Example 1 To explore the effect of loading copper-based components on the microwave enhancement of composite materials, this comparative example is basically the same as Example 1, except that copper nitrate loading is not performed in step S3, while the other process parameters remain the same, so that ZrO2@Cu-MOF control nanoparticles can be prepared.

[0049] Testing revealed that the ZrO2@Cu-MOF control nanoparticles prepared in this comparative example exhibited a tumor inhibition rate of 59.6%, an apoptosis rate of 42.3%, and a thermal sensitivity enhancement factor of 1.1. The inventors' analysis revealed that due to the lack of copper-based components, the enhancement effect relied solely on the physical enhancement of mesoporous ZrO2 without any biological regulatory effect, resulting in a weak enhancement effect.

[0050] Comparative Example 2 To explore the feasibility of ZrO2 coating, this comparative example is basically the same as Example 1, except that: the solvent ratio is 1:1, ammonia water is 2.0 mL, zirconium propionate is 0.6 mL, and the mixture is stirred at room temperature for 6 h.

[0051] Testing revealed that the product prepared in this comparative example exhibited severe agglomeration and lacked a mesoporous structure. The inventors analyzed the cause as follows: improper solvent ratio and excessive ammonia, leading to excessive hydrolysis and agglomeration of ZrO2.

[0052] Comparative Example 3 To explore copper ion loading methods, this comparative example is basically the same as Example 1, except that ZrO2 and copper nitrate solution are directly mixed without vacuum negative pressure.

[0053] Testing revealed that the copper loading in the ZrO2@Cu(NO3)2 intermediate prepared in this comparative example was 35±5 mg / g, and the loading was uneven. The inventors analyzed that the reason was that the copper ions were only adsorbed on the surface and did not enter the mesoporous cavities, making them easy to detach.

[0054] Comparative Example 4 To explore the synthesis conditions of MOF, this comparative example is basically the same as Example 1, except that the reaction is carried out at 140°C for 10 h and the amount of H2BDC used is 100 mg.

[0055] Testing revealed that the mesopores of the product prepared in this comparative example were blocked, preventing the release of copper ions. The inventors analyzed that the reasons were: excessively high temperature leading to excessive crystallization of the MOF, and insufficient H2BDC resulting in a dense shell.

[0056] Comparative Example 5 To verify the in vivo safety of the ZrO2@CuO / Cu-MOF composite nanoparticles obtained in Example 1, this comparative example is basically the same as Example 1, except that the Zr@C / CNPs dose is 5 mg / kg and administered via tail vein injection.

[0057] Tests showed that the transaminase levels in the comparative experimental rabbits increased by 30%, indicating a mild inflammatory response. The inventors analyzed that the cause was: the dosage was too high, leading to slight accumulation of nanoparticles in the liver.

[0058] Experimental Example 1: Performance Testing of the Prepared ZrO2@CuO / Cu-MOF Composite Nanoparticles The products or partial products obtained in Examples 1-3 and Comparative Examples 1-4 were analyzed by transmission electron microscopy (TEM), elemental mapping, zeta potential analysis, XRD diffraction patterns, FTIR spectroscopy, flow cytometry for tumor cell apoptosis, quantitative analysis of apoptosis induction in liver cancer cells, flow cytometry detection of intracellular ROS levels, and survival rates of Heap1-6 cells (mouse liver cancer cells) and HUVEC cells (human umbilical vein endothelial cells). The results are detailed in Table 1. Figures 1-12 .

[0059] The specific steps of the above detection method are as follows: (1) Transmission electron microscopy (TEM) image: powdered nanomaterials (1-1) Dispersion treatment: Take about 3 mg of nanopowder sample, add 1~2 mL of dispersion medium (deionized water, anhydrous ethanol or acetone, selected according to the hydrophilicity or hydrophobicity of the sample; if the sample solvent can dissolve the Fanghua membrane, a pure carbon membrane carrier should be used), and place it in an ultrasonic cell disruptor for dispersion. Parameter control: power 100~150W, ultrasonic time 3~5 min, intermittent ultrasonication (every 30 s ultrasonication, pause for 10 s) to avoid ultrasonic overload causing nanoparticle breakage or agglomeration, and ensure dispersion into a uniform suspension.

[0060] (1-2) Selection and treatment of the support screen: A 200-mesh support screen with a diameter of 3 mm is selected. A suitable support film is chosen based on the sample characteristics: a carbon support film (10-20 nm thick, good conductivity, reducing charge accumulation) is used for routine morphology observation; an ultrathin carbon film (3-5 nm thick, to prevent sample leakage from the microgrid pores) is used for nanoparticles with good dispersibility below 10 nm; a microgrid is used for tubular and rod-shaped nanomaterials; and a double-layer support film is used for magnetic nanomaterials to prevent adsorption onto the electron microscope pole piece. The support screen needs to be hydrophilicated in advance (oxygen-containing plasma glow discharge, power 50-100 W, time 30-60 s) to make the surface of the support film hydrophilic, facilitating uniform sample adsorption and preventing sample beading and uneven distribution.

[0061] (1-3) Sample loading and drying: Use a pipette to draw 10~20μL of the well dispersed suspension and slowly drop it onto the center of the grid. Place it on a dust-free drying table to air dry naturally, or use an infrared lamp to dry it at a low temperature (temperature ≤60℃, time 10~15min) to ensure that the sample is completely dry and free of moisture and residual solvent. If the sample contains volatile substances, it needs to be dried in a vacuum drying oven in advance (temperature 50~80℃, time 1~2h).

[0062] (2) Elemental distribution map: Powdered nanomaterials (2-1) Dispersion treatment: Take 5~10 mg of nano powder sample, put it into a sterile centrifuge tube, add 1~2 mL of dispersant (anhydrous ethanol or deionized water), and place it in an ultrasonic cell disruptor for ultrasonic dispersion for 10~15 min. During ultrasonication, pause for 1 min every 3 min to avoid overheating of the ultrasonication, which may cause oxidation or structural damage to the sample. For samples that are prone to agglomeration, the ultrasonication time can be extended appropriately, or a small amount of surfactant can be added to assist dispersion.

[0063] (2-2) Sample loading: SEM-EDS: Use a pipette to draw 10~20 μL of the dispersed sample suspension and slowly drop it onto the surface of the silicon wafer or aluminum wafer (keep the pipette tip 1~2 cm away from the carrier during the drop to avoid droplet impact causing particle agglomeration), place it in a vacuum drying oven at 50~60℃ for 30~60 min until the dispersant has completely evaporated and the sample is uniformly attached to the carrier surface.

[0064] (2-3) STEM-EDS: Take 5~10 μL of sample suspension and drop it into the center of the ultrathin carbon film copper mesh. Let it air dry naturally or vacuum dry for 15~30 min to ensure that the sample is evenly dispersed on the carbon film and avoid particle accumulation. At the same time, remove any loose particles that are not attached (they can be gently blown away with a nitrogen gun).

[0065] (3) Zeta potential analysis: (3-1) Sample pretreatment: Take 5~10 mg of nanomaterial sample, put it in a vacuum drying oven, dry at 50~60℃ for 30 min to remove the water and impurities adsorbed on the sample surface, and weigh it after cooling to room temperature to avoid the water affecting the dispersion effect and potential measurement.

[0066] (3-2) Dispersion preparation: Place the dried nanosample into a 10 mL sterile centrifuge tube, add 5-10 mL of dispersion medium, and sonicate in an ultrasonic cell disruptor for 10-15 min. During sonication, pause for 1 min every 3 min to avoid overheating and oxidation or structural damage to the sample, and to prevent excessive bubble formation. For samples prone to aggregation, a small amount of surfactant can be added, or the sonication time can be extended to 20 min.

[0067] (3-3) Concentration adjustment: The optimal sample concentration for Zeta potential measurement is 0.01~0.1 mg / mL (too high a concentration can easily lead to multiple scattering and particle interaction, resulting in measurement deviation; too low a concentration will result in insufficient scattered light intensity and weak signal). If the dispersion concentration is too high, use the equalization dilution method: extract the supernatant by gravity sedimentation or centrifugation (1000~3000 r / min, 5 min), and dilute the sample to a suitable concentration with the mother liquor, avoiding direct dilution with deionized water which would change the ionic strength and pH of the system; if the concentration is too low, it can be adjusted by centrifugation, enrichment, and resuspending.

[0068] (3-4) pH adjustment: According to the experimental design, slowly adjust the pH value of the dispersion (e.g., pH=5, 7, 9) with pH adjuster. After each adjustment, gently mix with a pipette, let stand for 5 min, calibrate with a pH meter to ensure pH stability (deviation ≤0.02). Zeta potential is sensitive to pH, and small fluctuations will lead to deviations in results. Avoid vigorous stirring to prevent bubbles during adjustment.

[0069] (3-5) Sample filtration and degassing: Filter the pH-adjusted dispersion through a 0.22 μm filter membrane to remove undispersed aggregates and impurities; after filtration, let the dispersion stand for 10 min, or degas it for 30 s using an ultrasonic cell disruptor to completely remove air bubbles.

[0070] (3-6) Sample inspection: Observe the state of the dispersion using an inverted microscope to confirm that the nanoparticles are uniformly dispersed, without obvious agglomeration or bubbles. Unqualified samples need to be re-dispersed by ultrasonication or filtered. At the same time, a sample stability test can be performed, measuring in chronological order to verify whether the pH and conductivity of the dispersion are stable, and to avoid potential changes caused by ion separation.

[0071] (4) XRD diffraction pattern: (4-1) Sample pretreatment: Take 10~20 mg of nanomaterial sample, put it in a vacuum drying oven, dry at 60℃ for 60 min to completely remove moisture and adsorbed impurities from the sample, and weigh it after cooling to room temperature. Moisture will cause the diffraction peak intensity to decrease and the peak shape to broaden, while impurities will cause interference from impurity peaks.

[0072] (4-2) Sample grinding: Place the dried sample in an agate mortar, add 1-2 drops of anhydrous ethanol, and gently grind with a pestle for 10-15 minutes. During the grinding process, rotate the mortar continuously to ensure that the sample is uniform and fine, without obvious particulate matter. Avoid over-grinding, which may damage the crystal structure, and avoid under-grinding, which may lead to agglomeration. If the sample is severely agglomerated, it can be ultrasonically dispersed before grinding.

[0073] (4-3) Sample loading: Fill the groove of the XRD-specific sample plate evenly with the ground sample and gently spread it with a spatula to avoid sample accumulation or gaps; then gently press the sample surface with a tablet press to make the sample compact, flat and flush with the sample plate surface (the sample thickness should be controlled at 0.5~1 mm. Too thick will cause X-ray attenuation, and too thin will cause insufficient diffraction peak intensity). When pressing, the force should be moderate to avoid sample clumping or changes in crystal orientation.

[0074] (4-4) Sample cleaning: Gently wipe the surface of the sample plate with lint-free paper to remove excess sample outside the grooves and avoid the generation of stray diffraction signals by excess sample; if there are stains on the sample plate, clean it with an ultrasonic cleaner (anhydrous ethanol as the medium), and dry it before use.

[0075] (4-5) Sample inspection: Visually inspect the sample surface to ensure it is flat, uniform, free of gaps and agglomerates; if the sample surface is uneven or agglomerates, it needs to be re-ground, loaded and pressed. Unqualified samples will cause diffraction peak shift and peak shape asymmetry.

[0076] (5) FTIR infrared spectrum: (5-1) Sample pretreatment: Take 5~10 mg of nano powder sample (thin film nanomaterials (such as nanofilms, nanofiber membranes) need to be cut into small pieces of 1 cm×1 cm), put them in a vacuum drying oven, dry at 50~60℃ for 60 min to completely remove the moisture and impurities adsorbed on the sample surface (moisture will cause the spectral baseline to drift and increase the number of impurity peaks, and impurities will produce additional absorption peaks), and weigh them after cooling to room temperature.

[0077] (5-2) Sample and KBr mixing and grinding: In an agate mortar, add 1-2 mg of dried nano-sample and 100-150 mg of dried KBr powder (sample to KBr mass ratio approximately 1:100; for samples containing strongly polar groups, this ratio can be adjusted to 1:200 to avoid excessively strong absorption peaks that could saturate the detector). Add 1-2 drops of anhydrous ethanol and gently grind for 10-15 min. During grinding, continuously rotate the mortar to ensure thorough and uniform mixing of the sample and KBr. Grind until the particle size is less than 2.5 μm (larger particles can easily cause light scattering, affecting spectral clarity). Avoid talking during grinding; it is recommended to wear a gauze mask to prevent exhaled moisture from contaminating the sample and KBr powder. The grinding time should not be too long (to avoid KBr absorbing water) or too short (to ensure uniform mixing).

[0078] (5-3) Secondary drying: Place the uniformly ground mixture in a 120℃ forced-air drying oven for 1~2 min to further remove the moisture adsorbed during the grinding process. Wear cotton gloves when taking it out to avoid burns.

[0079] (5-4) Tableting Operation: Evenly fill the dried mixture into the tableting mold, use vibration to ensure uniform powder accumulation, gently level it, slowly pull out the pressure rod, insert the pressure tongue, assemble the mold, place it on the tableting machine, connect the vacuum system to evacuate the mold, apply a pressure of 10-15 MPa, maintain for 30-60 s, and press into transparent or translucent tablets (thickness 0.8-1 mm, diameter approximately 13 mm). The pressure must be uniform during tableting to avoid excessive pressure causing changes in the sample's crystal form and band shift, while insufficient pressure will result in loose tablets with poor light transmittance.

[0080] (5-5) Sample inspection: Visually inspect the tablets to ensure that the surface is flat, uniform, free of cracks, bubbles, and obvious agglomerates. If the tablets are loose, cracked, or have poor transparency, they need to be re-ground, dried, and compressed.

[0081] (6) Flow cytometry of tumor cell apoptosis: Flow cytometry detection of tumor cell apoptosis (Annexin V-FITC / PI double staining method): (6-1) Cell preparation: Tumor cells in the logarithmic growth phase are seeded into culture plates / flasks and cultured in an adherent manner. Experimental group: treated with apoptosis induction (drugs / radiation, etc.); Control group: no induction, cultured synchronously. (6-2) Cell collection and washing: Collect the culture supernatant (containing floating cells) into a centrifuge tube; digest adherent cells with trypsin without EDTA, and combine the digestion with the supernatant in the centrifuge tube; centrifuge at 1000 r / min for 5 min and discard the supernatant; resuspend the cells in pre-cooled PBS, centrifuge and wash twice, and discard the supernatant. (6-3) Annexin V-FITC / PI double staining: Resuspend cells in 1× Binding Buffer and adjust the concentration to 1×10⁻⁶. 6 cells / mL; Add 100 μL of cell suspension to a flow cytometer, add 5 μL of Annexin V-FITC, and incubate at room temperature in the dark for 15 min; add 5 μL of PI staining solution, and incubate at room temperature in the dark for 5 min; add 400 μL of 1×Binding Buffer, mix gently, and run on the instrument within 1 h; (6-4) Flow cytometry detection: Set up control tubes (unstained, Annexin V stained, PI stained), and adjust fluorescence compensation; circle the target cell population with FSC / SSC, and remove debris and clusters; detect with FL1 (FITC) and FL2 (PI) channels, draw a two-parameter dot plot, and set the four-way gate; (6-5) Results analysis: The apoptosis rate was calculated as early apoptosis rate (Q4) + late apoptosis rate (Q2). The experiment was repeated 3 times, and the data were statistically analyzed.

[0082] (7) Quantitative analysis of the apoptosis-inducing effect on liver cancer cells: This experiment employed Annexin V-FITC / PI double staining combined with flow cytometry and cell counting to assist in quantification, accurately analyzing the apoptosis-inducing effect on liver cancer cells. The core of the experiment was to achieve dual quantification of "rate + quantity" by detecting the apoptosis rate through flow cytometry and counting the number of surviving / apoptotic cells. Variables were strictly controlled throughout the experiment to ensure the accuracy of the results.

[0083] (7-1) Preliminary cell treatment (ensure standardized grouping to lay the foundation for quantification) Cell resuscitation and passage: After resuscitating the liver cancer cell line, culture it in complete medium to the logarithmic growth phase and passage it 2-3 times to ensure cell viability ≥95% (detected by trypan blue staining).

[0084] Cell seeding: In a biosafety cabinet, hepatocellular carcinoma cells in the logarithmic growth phase were digested, resuspended in complete culture medium, and the cell concentration was adjusted to 5 × 10⁶ cells / year. 5 2 mL of cells / mL was seeded into each well of a 6-well culture plate and incubated at 37°C in a 5% CO2 incubator for 24 h to ensure that the cell confluence reaches 70%~80% (to avoid excessive cell density affecting the apoptosis induction effect).

[0085] Grouping (strictly controlling variables): Blank control group: Add an equal volume of complete culture medium, without adding apoptosis inducers, and culture simultaneously.

[0086] Experimental group: According to the experimental design, different concentrations of apoptosis inducers (such as 0, 2.5, 5, 10 μmol / L sorafenib) were added, with 3 replicates for each concentration. The wells were gently shaken and placed in an incubator for further incubation (the incubation time was set according to the characteristics of the inducer, such as 24, 48, 72 h).

[0087] (7-2) Cell counting (quantitative basis: counting total cell count and surviving cell count) After culture, take 100 μL of cell suspension from each well, add 100 μL of 0.4% trypan blue staining solution, mix gently, and incubate at room temperature for 5 min. Transfer the stained cell suspension to a hemocytometer and count cells under an inverted microscope, distinguishing between live cells (colorless and transparent) and dead cells (blue). Calculate the total number of cells, the number of surviving cells, and the cell viability in each well using the following formula: Total cell count (cells / mL) = Total number of cells in the counting area × Dilution factor (2 in this experiment) × 10 4 Cell viability (%) = (Number of surviving cells / Total number of cells) × 100% Record the data for each replicate well, calculate the mean and standard deviation, and use them for subsequent auxiliary quantitative analysis of apoptosis induction effect.

[0088] (7-3) Annexin V-FITC / PI double staining (core quantification: detection of apoptosis rate): Cell Collection and Washing (avoiding human damage and ensuring the integrity of apoptotic cells): Carefully aspirate the culture supernatant from each well and transfer it to the corresponding numbered sterile centrifuge tubes. Do not discard the tubes. Add 1 mL of EDTA-free trypsin to each well, gently shake the culture plate to evenly coat the cell surface with trypsin, and incubate at 37°C for 2-3 min. Observe under an inverted microscope. After the cells detach from the cell wall, add 2 mL of complete culture medium to stop digestion. Gently pipette the cells to disperse them into a single-cell suspension and transfer them to the corresponding numbered centrifuge tubes (combined with the supernatant from step 35). Place the centrifuge tubes in a low-temperature centrifuge and centrifuge at 4°C, 1000 r / min for 5 min. Discard the supernatant and retain the cell pellet. Add 1 mL of pre-chilled PBS buffer, gently pipette to resuspend the cell pellet, centrifuge at 4°C, 1000 r / min for 5 min, and discard the supernatant. Repeat the washing process twice to thoroughly remove residual culture medium and trypsin to avoid affecting the staining results.

[0089] Staining (strictly protected from light, ensuring uniform staining): Add 100 μL of 1×Binding Buffer to each centrifuge tube, gently pipette to resuspend the cell pellet, and adjust the cell concentration to 1×10⁻⁶. 6 cells / mL. Transfer the cell suspension to sterile flow cytometry tubes with corresponding numbers. Add 5 μL of Annexin V-FITC reagent to each tube, gently invert the tube to mix, and incubate at room temperature (around 25°C) in the dark for 15 min. After incubation, add 5 μL of PI staining solution to each tube, gently invert the tube again to mix, and incubate at room temperature in the dark for 5 min. After staining, add 400 μL of 1×Binding Buffer to each tube, gently mix, and analyze within 1 h.

[0090] Flow cytometry assay (setting up a control to ensure accurate quantification): Before assay, turn on the flow cytometer, warm it up for 30 minutes, calibrate the instrument parameters, and ensure the instrument is stable. Set up a control tube for adjusting fluorescence compensation and gating. Unstained control: Take 100 μL of untreated liver cancer cell suspension, add 400 μL of 1×Binding Buffer, and do not stain.

[0091] Annexin V-FITC single staining control: Take 100 μL of untreated hepatocellular carcinoma cell suspension, add 5 μL of Annexin V-FITC, incubate in the dark for 15 min, and then add 400 μL of 1×Binding Buffer.

[0092] PI single staining control: Take 100 μL of untreated liver cancer cell suspension, add 5 μL of PI, incubate in the dark for 5 min, and then add 400 μL of 1×Binding Buffer.

[0093] The control and sample tubes were tested sequentially. The target cell population was circled using an FSC / SSC dual-parameter scatter plot, and cell debris and cell clusters were removed. The fluorescence signal of Annexin V was detected using the FITC channel (FL1), and the fluorescence signal of PI was detected using the PI channel (FL2). A FL1 vs FL2 dual-parameter scatter plot was plotted, and the cell percentage in each quadrant was recorded using the quarter-gate method.

[0094] (7-4) Quantitative Results Analysis (Core: Quantifying Apoptosis Induction Effect): Apoptosis rate calculation: Based on flow cytometry results, the apoptosis rate = percentage of early apoptotic cells (Q4 quadrant, PI). - AnnexinV + + Percentage of late-stage apoptotic / secondary necrosis cells (Q2 quadrant, PI) + Annexin V + For each sample, the average of three replicates is taken, and the standard deviation is calculated.

[0095] Auxiliary quantitative analysis: Combined with cell counting results, the absolute number of apoptotic cells per well was calculated as total number of cells × apoptosis rate, further quantifying the apoptosis induction effect.

[0096] （8） Intracellular ROS level flow cytometry detection: This experiment employed the DCFH-DA (2',7'-dichlorofluorescein diacetate) fluorescent probe method, combined with flow cytometry, to quantitatively detect intracellular reactive oxygen species (ROS) levels. DCFH-DA itself is non-fluorescein; after entering the cell, it is hydrolyzed by esterases to DCFH. DCFH can then be oxidized by intracellular ROS to DCF (2',7'-dichlorofluorescein), which exhibits strong fluorescence. The fluorescence intensity is positively correlated with the intracellular ROS level. Quantitative analysis of ROS levels was achieved by detecting the fluorescence intensity of DCF using flow cytometry.

[0097] (8-1) Pre-treatment of cells (grouping standard, control variables): Cell resuscitation and passage: After resuscitating the target cells, culture them in complete culture medium until the logarithmic growth phase, and passage them 2-3 times to ensure stable cell condition and avoid cell aging affecting ROS detection results.

[0098] Cell seeding: In a biosafety cabinet, cells in the logarithmic growth phase were digested with EDTA-free trypsin. After digestion was terminated with complete culture medium, the cells were pipetted to prepare a single-cell suspension, and the cell concentration was adjusted to 5 × 10⁶ cells / mL. 5 2 mL of cells / mL was seeded into each well of a 6-well culture plate and incubated at 37°C with 5% CO2 for 24 hours to allow the cells to reach 60-70% confluence.

[0099] Grouping: Blank control group: Added an equal volume of serum-free culture medium, without adding ROS inducer, and incubated simultaneously. Negative control group: Added an equal volume of serum-free culture medium, without adding inducer, and without subsequently adding the DCFH-DA probe (to exclude cell autofluorescence interference). Experimental group: According to the experimental design, different concentrations of ROS inducer (e.g., 0, 50, 100, 200 μmol / L H2O2) were added, with 3 replicates for each concentration. The mixture was gently shaken and incubated in an incubator (incubation time was set according to the characteristics of the inducer, such as 1, 2, 4 h).

[0100] (8-2) DCFH-DA probe staining (core step, strictly avoid light): Probe dilution: Take 10 mmol / L of DCFH-DA stock solution and dilute it to a final concentration of 10 μmol / L with serum-free medium. Gently vortex to mix during dilution. Prepare and use immediately (DCFH-DA is easily oxidized; do not leave it in the culture for more than 30 minutes after dilution). Discard the medium in each well of the culture plate. Gently wash the cells twice with pre-chilled PBS buffer, discarding the PBS after each wash to avoid residual serum esterase in the medium hydrolyzing the probe and affecting the staining effect. Add 1 mL of the diluted DCFH-DA probe working solution to each well, ensuring the probe solution evenly covers the cell surface. Place the culture plate in a light-protected incubator at 37°C and 5% CO2 for 20–30 minutes. After incubation, discard the probe working solution and gently wash the cells three times with pre-chilled PBS buffer, gently shaking the culture plate after each wash to thoroughly remove any undeposited probes and avoid fluorescence interference from undeposited probes.

[0101] (8-3) Cell collection and instrumental analysis: Cell Collection: Add 1 mL of EDTA-free trypsin to each well, gently agitate the culture plate to evenly coat the cells with trypsin, incubate at 37°C for 2–3 min, observe under an inverted microscope. After cell detachment, add 2 mL of complete culture medium to stop digestion. Gently pipette the cells to disperse them into a single-cell suspension, and transfer the cell suspension to the corresponding numbered sterile centrifuge tubes. Centrifuge the tubes in a low-temperature centrifuge at 4°C, 1000 rpm for 5 min, discard the supernatant, and retain the cell pellet. Add 1 mL of pre-chilled PBS buffer, gently pipette to resuspend the cell pellet, centrifuge at 4°C, 1000 rpm for 5 min, discard the supernatant; repeat the washing once to remove residual trypsin and culture medium, ensuring the cell suspension is pure. Add 500 μL of pre-chilled PBS buffer to each centrifuge tube, gently pipette to resuspend the cells, prepare a single-cell suspension, transfer to sterile flow cytometry tubes, label them, and store on ice in the dark. Analyze within 1 hour.

[0102] Flow cytometry assay: Before assay, turn on the flow cytometer, warm it up for 30 minutes, and calibrate the instrument parameters (excitation wavelength 488nm, emission wavelength 525nm, corresponding to the FITC channel) to ensure instrument stability. Set up a control tube for adjusting the fluorescence baseline and gating. Negative control group (without probe): used to exclude cell autofluorescence and instrument background fluorescence.

[0103] Blank control group (without inducer): used to set the ROS fluorescence baseline for normal cells.

[0104] The control and sample tubes were tested sequentially. The target cell population was circled using a dual-parameter scatter plot of FSC / SSC, and cell debris, cell clusters, and dead cells were removed. The fluorescence intensity of DCF was detected through the FITC channel (FL1), and the mean fluorescence intensity (MFI) of each sample was recorded. Each replicate well was tested three times, and the average value was used for subsequent quantitative analysis.

[0105] (8-4) Quantitative analysis of results Fluorescence intensity analysis: Using the average fluorescence intensity of the blank control group as a benchmark, the relative fluorescence intensity of each experimental group was calculated (relative fluorescence intensity = average fluorescence intensity of experimental group / average fluorescence intensity of blank control group). The higher the relative fluorescence intensity, the higher the intracellular ROS level.

[0106] (9) Survival rates of Heap1-6 cells (mouse liver cancer cells) and HUVEC cells (human umbilical vein endothelial cells): This experiment used the CCK-8 (Cell Counting Kit-8) method to detect the viability of Heap1-6 cells (mouse liver cancer cells) and HUVEC cells (human umbilical vein endothelial cells). The principle is that WST-8 in the CCK-8 reagent is reduced to water-soluble orange-yellow formazan dye by intracellular dehydrogenase. The absorbance (OD value) of formazan is positively correlated with the number of viable cells. By detecting the OD value with an enzyme-linked immunosorbent assay (ELISA) reader and calculating the cell viability, quantitative analysis of cell proliferation and damage can be achieved.

[0107] (9-1) Pre-treatment of cells Cell resuscitation and passage: Resuscitate Heap1-6 cells or HUVEC cells from liquid nitrogen, thaw rapidly in 37°C water (within 1 minute to avoid intracellular ice crystal formation and cell damage), centrifuge at 1000 rpm for 5 minutes at 4°C to remove cryopreservation solution, resuspend in pre-warmed ECM complete medium, seed in sterile culture flasks, and incubate at 37°C with 5% CO2. Once cells reach the logarithmic growth phase (60-70% confluence), excessive confluence can lead to contact inhibition, necessitating timely passage. Passage with EDTA-free trypsin 2-3 times to ensure stable cell state and uniform morphology, preventing cell aging and mutation.

[0108] Cell counting and seeding: In a biosafety cabinet, trypsin was used to digest cells in the logarithmic growth phase. Complete culture medium was added to stop the digestion, and the cells were gently pipetted to create a single-cell suspension, preventing cell clumping. Cells were counted using a hemocytometer, and the cell concentration was adjusted to 1.5 × 10⁻⁶ cells / year. 4 Cells / mL, gently pipette to mix (at least 5 times). Add 100 μL of cell suspension to each well of a 96-well culture plate, with 6 replicates per group. Add 100 μL of sterile PBS buffer to the edge wells of the 96-well plate to prevent osmotic pressure changes caused by culture medium evaporation and avoid cell death due to abnormal osmotic pressure.

[0109] Cell adhesion culture: Place the 96-well culture plate in a 37℃, 5% CO2 incubator and incubate statically for 24 hours to allow the cells to fully adhere. At this point, the cells are spindle-shaped, evenly arranged, and have a confluence of 50-60%. Avoid overcrowding or sparse cell arrangement. During the culture period, avoid frequent opening and closing of the incubator to prevent fluctuations in temperature and CO2 concentration.

[0110] Grouping (strictly controlling variables): Blank control group: 100 μL of complete culture medium was added to each well, without seeding cells. This was used to subtract the background absorbance of the culture medium and CCK-8 reagent to ensure accurate data correction.

[0111] Negative control group: 100 μL of complete culture medium was added to each well without adding cell treatment agents. The cells were cultured synchronously with the experimental group as the baseline for normal cell viability (viability was calculated as 100%). Each group also had 6 replicates.

[0112] Experimental group: According to the experimental design, the original culture medium in each well was aspirated, and 100 μL of complete culture medium containing different concentrations of treatment agent was added (the concentration gradient was set reasonably, such as 0, 5, 10, 20, 40 μmol / L). Six replicates were set for each concentration. The 96-well plate was gently shaken to make the treatment agent and culture medium mix thoroughly, and then placed in an incubator for continued incubation (the incubation time was set according to the characteristics of the treatment agent, such as 24, 48, 72 h, and the incubation conditions were kept stable throughout the process to avoid external interference).

[0113] (9-2) CCK-8 staining and OD value detection (core step, precise control of reaction conditions): CCK-8 reagent addition: After the culture is completed, remove the 96-well culture plate from the incubator and operate in a biosafety cabinet. Accurately add 10 μL of CCK-8 reagent to each well, gently shake the culture plate to mix the reagent and culture medium thoroughly, and avoid generating air bubbles. Cells are sensitive to reagent stimulation, so the shaking should be done gently.

[0114] Incubation in the dark: Place the 96-well culture plate in a 37℃, 5% CO2 incubator and incubate in the dark for 2-4 hours.

[0115] OD value detection: After incubation, remove the 96-well culture plate and allow it to equilibrate at room temperature for 10 minutes. Measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 450 nm. Gently shake the culture plate before detection to ensure even distribution of formazan. Measure each well three times and record the average value. If air bubbles are present in the wells, gently tap the culture plate to remove them and avoid interference with the detection. If cells detach from the bottom of the well, exclude that well's data to avoid affecting the overall results.

[0116] (9-3) Survival rate calculation and result analysis (quantitative analysis, in accordance with experimental specifications): Data correction: Subtract the OD value of each well from the OD value of the blank control group to remove background interference and obtain the corrected OD value (corrected OD value = OD value of sample wells - OD value of blank control group). This avoids the influence of the absorbance of the culture medium and CCK-8 reagent itself on the results and ensures data accuracy.

[0117] Cell viability calculation: Based on the corrected OD value of the negative control group, the cell viability of each experimental group was calculated using the following formula: Cell viability (%) = (Corrected OD value of experimental group / Corrected OD value of negative control group) × 100%. The average viability of each concentration was taken from 6 replicates, and the standard deviation was calculated (the standard deviation should be controlled within 0.1; if the standard deviation is too large, uneven cell seeding, treatment concentration deviation, or operational error should be investigated).

[0118] Statistical analysis: SPSS software was used to perform statistical tests (such as t-test and one-way ANOVA) on the survival rates of each experimental group and the negative control group. P < 0.05 was considered statistically significant to clarify the effect of the treatment agent on cell survival rate and its concentration / time dependence, and to ensure that the experimental results were statistically valuable.

[0119] Results compilation: A survival rate bar chart was plotted (horizontal axis: treatment concentration / culture time, vertical axis: cell survival rate) to visually present the cell survival under different treatment conditions, and the standard deviation and statistical differences were marked to complete the experimental analysis.

[0120] Table 1. Performance comparison of the products obtained in Examples 1-3 and Comparative Examples 1-4

[0121] Figure 1Transmission electron microscopy (TEM) images and elemental mappings of the hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 are shown. The TEM images of the hollow ZrO2 nanospheres and Zr@C / C nanoparticles reveal a clear core-shell structure and the distribution of active centers within the cavity. Elemental mapping analysis of the Zr@C / C nanoparticles (Merge, O, Cu, Zr) confirms the successful loading of the copper-based active component into the zirconium dioxide hollow cavity.

[0122] Figure 2 The zeta potential analysis of the hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 is shown. The surface charge changes of ZrO2 (-9.10 mV) and Zr@C / C (-12.30 mV) are compared, reflecting the successful modification of the material surface.

[0123] Figure 3 The particle size distribution (DLS) of the hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 is shown. The hydration particle size distribution curves of ZrO2 and Zr@C / C nanoparticles measured by dynamic light scattering show that the distribution is uniform.

[0124] Figure 4 The XRD diffraction patterns of the hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 are shown. The XRD diffraction analysis of the materials confirms the presence of ZrO2 (blue line) and Zr@C / C (red line) crystalline phases in the nanoplatform.

[0125] Figure 5 The FTIR spectra of the hollow ZrO2 nanospheres and Zr@C / C nanoparticles obtained in Example 1 are presented. Fourier transform infrared spectroscopy analysis reveals the functional group information on the material surface and the successful loading of organic ligands.

[0126] Figure 6 The flow cytometry results of tumor cell apoptosis under different treatment groups (Control, ZrO2, ZrO2@C / C, ZrO2+MWA, ZrO2@C / C+MWA) are presented. Figure 7 The quantitative analysis of the apoptosis-inducing effects of different treatment groups (Control, ZrO2, ZrO2@C / C, ZrO2+MWA, ZrO2@C / C+MWA) on liver cancer cells shows that the apoptosis rate of the experimental groups was significantly increased.

[0127] Figure 8Flow cytometry analysis of intracellular ROS levels in different treatment groups (Control, ZrO2, ZrO2@C / C, ZrO2+MWA, ZrO2@C / C+MWA) was performed: The levels of reactive oxygen species (ROS) generated under microwave irradiation in different groups were detected by the DCFH-DA probe, confirming the significant microwave kinetic effect of Zr@C / C.

[0128] Figure 9 The survival rate of Heap1-6 cells (mouse liver cancer cells) incubated with different concentrations of ZrO2 nanoparticles is shown. Figure 10 The survival rates of Heap1-6 cells incubated with ZrO2@C / C nanoparticles obtained in Example 1 at different concentrations are shown. Figure 11 The survival rate of HUVEC cells (human umbilical vein endothelial cells) incubated with different concentrations of ZrO2 nanoparticles is shown. Figure 12 The survival rates of HUVEC cells incubated with ZrO2@C / C nanoparticles obtained in Example 1 at different concentrations are shown. The results indicate that the ZrO2@C / C nanoparticles obtained in Example 1 of this scheme have higher biocompatibility.

[0129] Experimental Example 2: Solvent Ratio Optimization (Anhydrous Ethanol: Acetonitrile) To explore the effect of solvent ratio (anhydrous ethanol: acetonitrile) on the coating uniformity and mesoporous structure integrity of mesoporous ZrO2, the following experimental design was carried out: a ratio gradient was set (1:1, 2:1, 3:1, 4:1), and the other conditions were the same as in step S1 of Example 1. Some properties of the prepared ZrO2@SiO2 nanospheres were detected, and the results are shown in Table 2.

[0130] Table 2 Solvent ratio optimization results

[0131] The results show that when the solvent ratio (anhydrous ethanol: acetonitrile) is 3:1, the solvent polarity and viscosity are well-matched to the zirconium propionate hydrolysis rate, resulting in ZrO2 uniformly coating the SiO2 surface with the most complete mesoporous structure and the largest specific surface area, providing ample space for subsequent copper ion loading. However, when the solvent ratio is 1:1, improper solvent ratio can easily lead to incomplete mesoporous structure, insufficient loading, and a decrease in synergistic effect.

[0132] Experiment Example 3: Optimization of Ammonia Dosage To explore the effect of ammonia on the performance of the obtained ZrO2@SiO2 nanospheres, the pH of the reaction system was adjusted with ammonia to control the ZrO2 hydrolysis rate. The experiment was conducted according to the following design: ammonia dosage gradients were set (0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL), and other conditions were the same as in step S1 of Example 1. Some properties of the prepared ZrO2@SiO2 nanospheres were detected, and the results are shown in Table 3.

[0133] Table 3 Optimization results of ammonia water dosage

[0134] The results showed that when the amount of ammonia water was 1.5 mL (i.e., the volume ratio of zirconium propofol to ammonia water was 0.6:1.5), the ammonia water maintained the pH of the system at 10.3, the hydrolysis rate of zirconium propofol was moderate, the ZrO2 coating layer thickness was uniform (45 nm), there was no excessive agglomeration, and the prepared ZrO2@SiO2 nanospheres had better performance.

[0135] Experimental Example 4: Optimization of NaOH Etching Conditions To explore the effect of NaOH etching conditions on the hollow ZrO2 nanospheres, the NaOH etching conditions were optimized to remove the SiO2 core, form a hollow mesoporous structure, and increase the copper ion loading. The experiment was conducted according to the following design: (1) Concentration gradient (0.5M, 1.0M, 1.5M), fixed temperature 80℃, time 4h; (2) Temperature gradient (60℃, 70℃, 80℃, 90℃), fixed concentration 1.0M, time 4h, with other conditions the same as steps S1 and S2 in Example 1. The results are shown in Table 4.

[0136] Table 4 Results of NaOH Etching Condition Optimization

[0137] The results showed that etching with 1M NaOH at 80℃ for 4 hours resulted in complete removal of SiO2 and intact hollow mesoporous structure, with a copper ion loading of 165 mg / g, representing the optimal conditions. However, if the NaOH concentration was too low (0.5M was used), insufficient etching intensity, incomplete removal of the core, limited space, low loading, and structural instability would occur.

[0138] Experimental Example 5: Optimization of MOF Synthesis Temperature and Time The crystallinity and stability of the CuO / Cu-MOF shell were controlled by the following experimental design: (1) Temperature gradient (100℃, 120℃, 140℃), fixed time 10h; (2) Time gradient (6h, 8h, 10h, 12h), fixed temperature 120℃, and other conditions were the same as steps S1 and S2 in Example 1. The results are shown in Table 5.

[0139] Table 5 Results of MOF synthesis temperature and time optimization

[0140] Data shows that when MOF synthesis is carried out at 120℃ for 10 hours, the CuO / Cu-MOF shell exhibits the highest crystallinity (92%), good stability in simulated body fluids, and unblocked mesoporous structure, ensuring microwave energy conduction and slow release of copper ions. However, if the MOF reaction temperature is too low, it will result in low MOF crystallinity, poor stability, and excessively rapid metabolism in vivo, failing to provide sustained synergistic effects.

[0141] Experimental Example 6: Enhanced Microwave Ablation Effect of Composite Nanoparticles on Liver Tumors (In Vivo and In Vitro Verification) (1) In vitro application method Cell models: Human hepatocellular carcinoma HepG2 and Huh7 cells; Administration method: Zr@C / C NPs prepared in Example 1 were incubated at gradient concentrations (50, 100, 200 μg / mL) for 24 h; Microwave parameters: 30W, 60s; Detection indicators: cell viability (CCK-8 assay), apoptosis rate (flow cytometry).

[0142] For detailed results of in vitro application, please refer to Table 6. Figures 13-14 .

[0143] Table 6 Results of in vitro application

[0144] Data shows that the treatment area was significantly larger in the drug + microwave ablation group.

[0145] Figure 13 The image shows a comparison of the ablation effects of the isolated rabbit liver in the drug group and the control group (left: HE staining; right: Tunel staining, the red box indicates the necrotic area). The results show that histopathological observation of the edge and center of the ablation area is used to define the boundary of necrosis and assess the protective effect on adjacent blood vessels.

[0146] Figure 14 This document presents contrast-enhanced ultrasound (CEUS) images of Zr@C / C nanoparticles obtained in Example 1, comparing pre-treatment (top) and post-treatment (bottom) images (D0 (images 1-3 are the non-drug group, simple ablation group) vs. D1 (images 4-6 are the drug plus ablation group)). The complete ablation rate and local residual tumor status are assessed by the extent of the non-perfusion area. The results show that the treatment area is significantly larger in the drug + microwave ablation group.

[0147] (2) In vivo application method Animal model: VX2 rabbit liver cancer model (n=10 / group); Administration method: Tail vein injection (dose 2 mg / kg), followed by microwave ablation 24 hours later; Microwave parameters: 50W, 120s; Detection indicators: ablation area (contrast ultrasound), tumor inhibition rate, median survival, and recurrence rate.

[0148] For detailed results of in vivo application, please refer to Table 7. Figures 15-16 .

[0149] Table 7 Results of in vivo application

[0150] Figure 15 The images show liver tissue sections from a rabbit VX2 liver tumor model treated with Zr@C / C nanoparticles obtained in Example 1, after ablation at different powers (30W, 50W, and 70W from left to right). The white dashed box in the images shows the size and morphology of the coagulative necrosis area. The results indicate that the nanomedicine + microwave ablation group had the largest treated area.

[0151] Figure 16 Intraoperative ultrasound monitoring images of a rabbit VX2 liver tumor model treated with Zr@C / C nanoparticles obtained in Example 1 at different ablation powers are shown. During the ablation procedure, the position of the ablation needle and changes in the hyperechoic masses generated during the ablation process were monitored in real time using ultrasound. The results show that the ablation area was significantly increased in the nanomedicine + microwave ablation group.

[0152] Experimental Example 7: Antibacterial Treatment (Gram-positive / Gram-negative Bacteria) Bacterial models: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) Administration method: Zr@C / C NPs gradient concentrations (10, 20, 40 μg / mL) and bacterial suspensions (10 μg / mL) 6 The cells were co-incubated with CFU / mL for 24 hours; the application results are detailed in Table 8.

[0153] Table 8 Results of Antibacterial Therapy Application

[0154] The results showed that the Zr@C / C nanoparticles (i.e., ZrO2@CuO / Cu-MOF composite nanoparticles) prepared by this method effectively inhibited Staphylococcus aureus and Escherichia coli at a concentration of 40 μg / mL, with a minimum inhibitory concentration of 20 μg / mL.

[0155] In addition, the ZrO2@CuO / Cu-MOF composite nanoparticles prepared by this method can also be used in the following fields: 1. Catalysis: It can be used as a heterogeneous catalyst or catalyst support for organic pollutant degradation, carbon dioxide reduction, organic synthesis reactions and other scenarios. The synergistic effect of CuO and Cu-MOF can improve catalytic efficiency and selectivity. 2. Biomedical field: As a microwave ablation enhancer, the mesoporous structure of ZrO2 synergistically enhances the microwave absorption properties of Cu-based components, thereby improving the effect of tumor microwave ablation. At the same time, Cu-MOF can load drugs to achieve synergistic treatment of "ablation + chemotherapy". 3. Environmental remediation: Utilizing high specific surface area and adsorption performance, it is used for the efficient adsorption and removal of pollutants such as heavy metal ions and organic dyes in water bodies; 4. Energy sector: It can be used as an electrode material or a component of energy storage materials in lithium-ion batteries, supercapacitors, etc., to improve energy storage capacity and cycle stability; 5. Other fields: Used as a functional filler for composite material modification, or as a model material for basic research related to nanostructures.

[0156] In summary, this invention constructs ZrO2@CuO / Cu-MOF composite nanoparticles (Zr@C / CNPs) through a four-step synergistic mechanism of "template coating - etching cavity creation - vacuum loading - solvothermal recombination". The core mechanism is as follows: 1. Template-guided coating mechanism: Using silica nanospheres as hard templates, zirconium propofol (IV) undergoes hydrolysis in an alkaline environment (pH 10-11) provided by ammonia water to generate mesoporous zirconium oxide (ZrO2), which is uniformly coated on the silica surface through interfacial adsorption to form a core-shell structure ZrO2@SiO2, thus achieving the controllable growth of the ZrO2 shell.

[0157] 2. Alkali etching cavity creation mechanism: The selective etching effect of NaOH solution (1M) at 80℃ is used to remove the internal SiO2 template and form hollow ZrO2 nanospheres. The cavity structure provides sufficient space for subsequent copper-based component loading, while the mesoporous structure of the ZrO2 shell retains the material transport channels.

[0158] 3. Vacuum Loading Mechanism: Under negative pressure, Cu in copper nitrate aqueous solution... 2+ Utilizing concentration gradients and capillary action, Cu efficiently penetrates and adsorbs within the cavities and mesopores of hollow ZrO2, achieving Cu... 2+ The uniform loading lays the foundation for the subsequent in-situ generation of CuO and Cu-MOF.

[0159] 4. Solvent-thermal recombination mechanism: In the DMF solvent system, under high temperature and pressure of 120℃, PVP (dispersant) maintains the stability of the system, while H2BDC (organic ligand) reacts with Cu in the ZrO2 cavity. 2+ A coordination reaction occurs to form Cu-MOF, and at the same time, some Cu... 2+ CuO is formed through in-situ oxidation, ultimately constructing a multifunctional structure of "ZrO2 shell - internal CuO / Cu-MOF composite core". The mesoporous properties of ZrO2 and the high specific surface area and active sites of CuO / Cu-MOF form a synergistic effect.

[0160] The effects of this solution are as follows: 1. Excellent structural controllability: The obtained Zr@C / CNPs are regular spheres with uniform ZrO2 shell thickness (10~20nm) and internal cavity volume accounting for 40~50%. The CuO / Cu-MOF composite core is uniformly dispersed without obvious agglomeration. X-ray diffraction verification shows that the characteristic crystal phases of ZrO2, CuO and Cu-MOF are clear, confirming the successful construction of the composite structure.

[0161] 2. Synergistic enhancement of physicochemical properties: The mesoporous structure of hollow ZrO2 (pore size 2~10nm) and the high specific surface area of ​​Cu-MOF (≥800m²) 2 / g) synergistic effect to improve mass transport efficiency; the catalytic active sites of CuO and the coordination binding ability of Cu-MOF complement each other, giving the material multiple functions such as catalysis and adsorption.

[0162] 3. Stable and reliable preparation process: By precisely controlling the hydrolysis temperature, etching time, and solvothermal reaction parameters, the morphology and size of the product can be repeatedly prepared with a batch-to-batch particle size variation coefficient of <5%, meeting the needs of large-scale application.

[0163] 4. Significant differences compared to control samples: Compared to ZrO2@Cu-MOF (Zr@CNPs) without CuO loading, the Zr@C / CNPs of this invention exhibits catalytic activity increased by 30-50% due to the introduction of CuO, and shows superior performance in potential applications such as microwave absorption and tumor treatment.

[0164] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors, characterized in that: Includes the following steps: Step S1, Template Coating Stage: Silica nanospheres, ammonia, and zirconium propionate (IV) are sequentially dispersed in a mixed solvent of anhydrous ethanol and acetonitrile to obtain a mixed precursor solution; the mixed precursor solution is continuously stirred at room temperature, and after the reaction is completed, the precipitate is collected by centrifugation to obtain ZrO2@SiO2 nanospheres; Step S2, Alkali Etching and Cavity Creation Stage: ZrO2@SiO2 nanospheres are redispersed in deionized water, stirred evenly, and then NaOH solution is added. The internal SiO2 template is removed by constant temperature stirring and etching. After the reaction is completed, the precipitate is collected by centrifugation and washed with deionized water to obtain hollow ZrO2 nanospheres. Step S3, Vacuum Loading Stage: Hollow ZrO2 nanospheres and copper nitrate·3H2O are dissolved in deionized water to obtain ZrO2 dispersion and copper nitrate aqueous solution; the two solutions are mixed and adsorbed under negative pressure in a vacuum environment to load Cu(NO3)2 on the cavity and surface of ZrO2 nanospheres to obtain ZrO2@Cu(NO3)2 intermediate; Step S4: Solvent-thermal reaction to prepare ZrO2@CuO / Cu-MOF composite nanoparticles: Polyvinylpyrrolidone, ZrO2@Cu(NO3)2 intermediate, and terephthalic acid were sequentially added to N,N-dimethylformamide and magnetically stirred to obtain a dispersion; the dispersion was sealed and reacted at a constant temperature to obtain a reaction system containing ZrO2@CuO / Cu-MOF composite nanoparticles; Step S5, Separation and Purification: After the reaction system in step S5 has cooled naturally to room temperature, the precipitate is collected by centrifugation. Unreacted raw materials and impurities are removed by washing with DMF and deionized water in sequence. After vacuum drying, the finished ZrO2@CuO / Cu-MOF composite nanoparticles are obtained.

2. The method for preparing a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors according to claim 1, characterized in that: In step S1, the volume ratio of zirconium propofol to ammonia is 0.6:1~1.5; the mass-volume ratio of silica nanospheres to zirconium propofol is 200~240mg:0.6~0.8ml.

3. The method for preparing a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors according to claim 2, characterized in that: In step S1, the volume ratio of anhydrous ethanol to acetonitrile in the mixed solvent is 2~4:

1.

4. The method for preparing a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors according to claim 3, characterized in that: In step S2, the concentration of the NaOH solution is 1M, and the etching temperature is 70~80℃.

5. The method for preparing a composite nanoparticle formulation with enhanced microwave ablation effect on liver tumors according to claim 4, characterized in that: In step S4, the isothermal reaction is carried out at 120°C for 8-10 hours.

6. A composite nano-formulation, characterized in that: ZrO2@CuO / Cu-MOF composite nanoparticles prepared by the method according to any one of claims 1 to 5.

7. The composite nano-formulation according to claim 6, characterized in that: The specific surface area of ​​the ZrO2@CuO / Cu-MOF composite nanoparticles is ≥800m². 2 / g, the ZrO2 shell retains a mesoporous structure with a pore size of 2~10nm.

8. The application of a composite nano-formulation in the preparation of drugs that enhance the effect of tumor microwave ablation, characterized in that: The drug comprises ZrO2@CuO / Cu-MOF composite nanoparticles prepared by the method according to any one of claims 1 to 5.

9. The application of the composite nano-formulation according to claim 8 in the preparation of drugs that enhance the microwave ablation effect of tumors, characterized in that: The microwave ablation is performed at a power of 30-50W for 20-40 seconds.

10. The application of a composite nano-formulation in the preparation of antibacterial drugs, characterized in that: The drug comprises ZrO2@CuO / Cu-MOF composite nanoparticles prepared by the method according to any one of claims 1 to 5.

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