Nano-enzyme sonosensitizer Co3O4 (at) PAO (at) BSA (CPB) and preparation method and application thereof

Abstract

The invention relates to the field of medical materials, in particular to a nano-enzyme sonosensitizer Co3O4 (at) PAO (at) BSA (CPB) and a preparation method and application thereof. According to the invention, the Co3O4 (at) PAO (at) BSA (CPB) nano sound-sensitive agent is synthesized and regulated, a US enhanced enzyme kinetics strategy using Co3O4 CPHs as a substrate, the introduction of PAO consumes polyamine in tumors, and a carbonyl stress strategy of polyamine consumption successfully overcomes the defect of oxidative damage treatment, and the effect of Co ion mediated SDT / CDT induced oxidative damage treatment is enhanced through a nano enzyme cascade catalytic effect.

Description

Technical Field

[0001] This invention relates to the field of medical materials, and in particular to the nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB), its preparation method, and its application. Background Technology

[0002] Prostate, lung, and colorectal cancers account for nearly half (49%) of all male cancer cases. For women, breast, lung, and colorectal cancers account for 52% of all new diagnoses, with breast cancer alone accounting for 31% of female cancers, ranking first. Traditional treatments for breast cancer include surgery, chemotherapy, and radiotherapy. Surgery is highly invasive, while chemotherapy and radiotherapy lack high specificity, inevitably producing severe toxic side effects and drug resistance while treating the tumor, significantly impacting treatment outcomes. In recent years, immunotherapy and targeted therapy for breast cancer have achieved some clinical success. However, for some special types of breast cancer, such as triple-negative breast cancer, which are insensitive to conventional endocrine and targeted therapies and exhibit significant drug resistance, treatment outcomes are poor, overall survival is low, early recurrence and distant metastases to the lungs and brain are common, and mortality remains high. Therefore, there is an urgent need to develop new and highly effective treatment methods, modalities, and strategies, hoping to achieve not only highly effective treatment of breast cancer and other malignant tumors but also high treatment safety, ensuring patients' survival period and quality of life.

[0003] In recent years, the development of ultrasound diagnostic and therapeutic technologies has provided broader opportunities for the advancement of modern medicine. Tumor kinetic therapy, due to its advantages such as high treatment efficiency, low toxicity, and strong controllability, has become a hot topic in clinical and basic research. Chemokinetic therapy (CDT) is a type of therapy based on the transformation reaction of endogenous chemical products in tumors (Fenton reaction: through Fe...). 2+This novel tumor oxidative damage therapy, which mediates the catalysis of H2O2 into toxic •OH, differs from other methods in that it also utilizes an external field to excite active substances, generating cytotoxic reactive oxygen species (ROS) to kill tumor cells. Because ROS generation is limited to the range of external field excitation, its impact and damage to normal tissues and cells are smaller, resulting in lower toxicity. Currently, this field-based tumor kinetic therapy is mainly divided into two categories: photodynamic therapy (PDT) under light excitation and sonodynamic therapy (SDT) under ultrasound excitation. In recent years, PDT research has made significant progress and has been successfully applied to the clinical treatment of some tumors. However, due to the low tissue penetration depth of light, it is difficult to effectively treat tumors in deep organs and tissues. Ultrasound, with its advantages of being non-invasive, radiation-free, and having deep tissue penetration, has become a new source of excitation for tumor kinetic therapy. SDT is an emerging treatment method for oxidative damage, which mainly includes ROS-induced cell membrane lipid peroxidation (LPO) and DNA damage. Unlike traditional high-intensity focused ultrasound (HIFU) that utilizes thermal effects to treat tumors, SDT uses low-intensity ultrasound as the excitation source. Its penetration depth in soft tissue can reach over ten centimeters, allowing for more efficient focusing on tumor tissue for dynamic therapy. It offers higher treatment safety and has a wider range of applications and greater potential for clinical translation compared to PDT.

[0004] The tumor microenvironment (TME) refers to the internal environment upon which tumors depend for survival during growth. Tumor cells can alter and maintain their own survival and development conditions through autocrine and paracrine processes, thus promoting tumor growth and development. The TME is characterized by relatively high concentrations of hydrogen peroxide (H2O2), a slightly acidic pH, hypoxia, and overexpression of intracellular glutathione (GSH). Utilizing the complex microstructure of tumor tissue and the TME for tumor therapy has been a hot research topic both domestically and internationally. Reactive oxygen species (ROS) are cellular components of the TME produced by tumor cells and are typical TME species controlling tumor cell fate, including hydroxyl radicals (•OH), singlet oxygen (•OH), and reactive oxygen species (•OH). 1 O2) and superoxide anions (•O) 2-The physiological effects of ROS are closely related to their concentration. High concentrations of ROS can damage cell membranes, inhibit mitochondrial function, damage DNA causing gene mutations, leading to energy metabolism disorders, calcium overload, and other irreversible oxidative damage to tumor cells, resulting in apoptosis and programmed necrosis. Conversely, lower concentrations of ROS can regulate cell growth, proliferation, and differentiation, promoting tumor cell growth. Therefore, selectively altering the concentration of ROS within the tumor microenvironment (TME) can increase its anti-tumor activity. In recent years, enhancing tumor treatment efficacy through TME regulation has received increasing attention.

[0005] With the rapid development of nanomaterials science and technology, both domestically and internationally, specific responsive therapeutic nanoplatforms targeting functional small molecule nanocarriers, enzyme nanocarriers, and nanoparticles with special functions that regulate the tumor microenvironment (TME) have been developed and utilized, becoming a promising strategy for tumor treatment. Among them, enzyme nanocarriers utilize the enzyme-mimicking behavior of nanomaterials to generate reactive oxygen species in the TME, showing great promise in tumor treatment. Enzymes, as biocatalysts, possess high specificity and efficiency, mediating various reactions in biological processes such as signal transduction, metabolism, and digestion. However, natural enzymes suffer from high production costs, susceptibility to denaturation and inactivation, and low yields, hindering their widespread application. To address these issues, researchers have gradually developed various nanoscale nanozymes with enzyme-mimicking capabilities. As a novel artificial enzyme, nanozymes possess high enzyme-like catalytic activity, capable of regulating biochemical reactions. Furthermore, they inherit the characteristics of nanomaterials, exhibiting good stability, low production costs, and simple preparation / purification steps, thus overcoming the shortcomings of natural enzymes and representing a promising alternative. In TME, nanozymes with peroxidase-like (POD-like) activity can decompose H2O2 in situ into hydroxyl radicals (•OH), while nanozymes with oxidase-like (OXD-like) activity can catalyze O2 to generate superoxide radicals (•O). 2− Nanozymes with catalase-like activity can directly induce tumor cell apoptosis and necrosis and can catalyze the decomposition of H2O2 into O2. 2， O2 can be rapidly used as a substrate to generate •O through OXD-like activity. 2−To kill tumors, numerous nanomaterials have been shown to mimic the functions of natural enzymes such as POD, OXD, and CAT. These nanozymes have significant advantages in biomedical applications, particularly in the treatment of malignant tumors. Despite these significant advances in malignant tumor therapy, the limited H2O2 concentration at tumor sites and the low affinity of nanozymes for their substrate H2O2 often result in insufficient hydroxyl radicals (•OH) to effectively treat tumors. Furthermore, the inherent properties of nanomaterials, such as diverse surface configurations, varying crystal structures, and inhomogeneous elemental composition, lead to lower activity and specificity compared to natural enzymes, all of which reduce their efficacy in treating malignant tumors. Therefore, the rational design of highly efficient nanozymes that better mimic the active sites and spatial configurations of enzymes for nanocatalytic tumor therapy remains a challenge.

[0006] The high levels of reactive oxygen species (ROS) generated by sonosensitive agents in SDT therapy and the continuous oxygen consumption during treatment both contribute to further tumor hypoxia, a fatal weakness that severely hinders the full effect of SDT. This results in insufficient LPO levels induced by SDT, making the tumor more susceptible to reduction by antioxidant enzymes in tumor tissue, such as glutathione peroxidase 4 (GPx4). Damaged DNA is also easily repaired by intracellular DNA repair proteins, limiting the oxidative damage caused by SDT alone and resulting in a lack of significant therapeutic efficacy. Therefore, improving ROS levels while simultaneously alleviating SDT-induced hypoxia is crucial for enhancing its effectiveness in treating malignant tumors. In recent years, scholars both domestically and internationally have investigated various effective methods to synergistically enhance SDT therapy for malignant tumors, with numerous research reports emerging. However, to date, there is still significant room for research and potential in this field, and exploring superior methods to synergistically enhance SDT remains a hot topic and focus of research both domestically and internationally. Summary of the Invention

[0007] In view of this, the present invention provides a nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB), its preparation method and application.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing the nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB), comprising the following steps:

[0010] Step 1: Prepare and obtain Co3O4 CPHs;

[0011] Step 2: Load PAO to prepare Co3O4@PAO;

[0012] Step 3: Take the Co3O4@PAO obtained in Step 2 and modify it with BSA to obtain the nanoenzyme sound sensitizer Co3O4@PAO@BSA (CPB).

[0013] In some specific embodiments of the present invention, the following steps are included:

[0014] Step 1-1, Preparation of Co-MOFs: Mix cobalt nitrate hexahydrate aqueous solution with 2-methylimidazole aqueous solution, stir at 25℃±5℃ for 6h, centrifuge to collect purple precipitate, wash with ethanol, dry, and collect purple solid product as Co-MOFs;

[0015] Step 1-2, Preparation of Co3O4 CPHs: Take the Co-MOFs obtained in step 1-1, calcine them, and collect the black product, which is Co3O4 CPHs.

[0016] In some specific embodiments of the present invention, step 2 specifically involves: mixing the PAO with the Co3O4 CPHs, stirring at 25℃±5℃ for 12 hours, centrifuging to remove excess PAO, and collecting the black precipitate as Co3O4@PAO.

[0017] In some specific embodiments of the present invention, step 3 specifically involves: mixing BSA with the Co3O4@PAO obtained in step 2, stirring, centrifuging to remove the supernatant, and collecting the solid to obtain the nanoenzyme sonication agent Co3O4@PAO@BSA (CPB).

[0018] In some specific embodiments of the present invention, in step 1-1, the molar ratio of cobalt nitrate hexahydrate in the cobalt nitrate hexahydrate aqueous solution to 2-methylimidazole in the 2-methylimidazole aqueous solution is 1.56:67.2;

[0019] Preferably, the concentration of the cobalt nitrate hexahydrate aqueous solution is 0.52 M; and the concentration of the 2-methylimidazole aqueous solution is 3.36 M.

[0020] Preferably, the volume ratio of the cobalt nitrate hexahydrate aqueous solution to the 2-methylimidazole aqueous solution is 3:20.

[0021] In some specific embodiments of the present invention, in step 1-1, the ethanol washing is performed at least once; preferably, the ethanol washing is performed three times.

[0022] The drying process involves drying at 80°C for 24 hours.

[0023] In some specific embodiments of the present invention, in steps 1-2, the calcination is performed at 300°C for 3 hours.

[0024] In some specific embodiments of the present invention, in step 2, the mass ratio of PAO to Co3O4 CPHs is 1:1;

[0025] Preferably, the centrifugation is performed at 8000 rpm for 10 minutes.

[0026] Preferably, in step 3, the mass ratio of BSA to Co3O4@PAO is 2:1.

[0027] Secondly, the present invention also provides a nanoenzyme sonication agent Co3O4@PAO@BSA (CPB) prepared by the aforementioned preparation method.

[0028] Thirdly, the present invention also provides the use of the nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB) in any one or more of the following:

[0029] (I) Mimicking enzyme activity; preferably, the enzyme includes one or more of oxidase-like enzymes, peroxidase-like enzymes, catalase-like enzymes, or glutathione peroxidase;

[0030] (II) Reduces cell membrane stability;

[0031] (III) To prepare drugs or formulations that reduce oxidative damage during treatment;

[0032] (IV) Preparation of drugs for synergistic treatment with sonodynamic therapy / chemodynamic therapy;

[0033] (V) Preparation of drugs for treating malignant tumors;

[0034] Preferably, the nanoenzyme sonosensitive agent Co3O4@PAO@BSA (CPB) reverses the hypoxic microenvironment, consumes endogenous glutathione and ROS output, or promotes the valence state conversion of Co ions under exogenous ultrasound (US) excitation, thereby increasing ROS production and killing tumor cells.

[0035] This invention utilizes a strategy of synthesizing and regulating Co3O4@PAO@BSA (CPB) nano-sound sensitizers, using Co3O4CPHs as substrates to enhance enzyme kinetics. The introduction of PAO consumes polyamines in tumors, and the carbonyl stress strategy of polyamine consumption successfully overcomes the shortcomings of oxidative damage therapy. Furthermore, the nanozyme cascade catalytic effect enhances the efficacy of Co ion-mediated SDT / CDT-induced oxidative damage therapy. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figure 1 The results are shown in the scanning electron microscope (SEM) image.

[0038] Figure 2 The results are shown in the transmission electron microscope (TEM) image.

[0039] Figure 3 The X-ray diffraction (XRD) results are shown.

[0040] Figure 4 The results are shown in the X-ray photoelectron spectroscopy (XPS) diagram.

[0041] Figure 5 Show the result of element mapping;

[0042] Figure 6(A) shows the UV-Vis absorption spectra of PAO at different concentrations; Figure 6(B) shows the concentration-dependent standard curve of PAO.

[0043] Figure 7 The UV-Vis absorption spectra of PAO 12 hours before (red line) and after (blue line) the addition of Co3O4CPHs are shown.

[0044] Figure 8(A) shows the absorbance changes at 652 nm under different pH values; Figure 8(B) shows the absorbance changes at 652 nm under different treatment groups due to TMB oxidation.

[0045] Figure 9 DMPO capture O with different processing methods 2- ESR spectrum;

[0046] Figure 10(A) shows the absorbance changes at 652 nm under different treatment groups; Figure 10(B) shows the absorbance changes at 652 nm under different pH values; Figure 10(C) shows the change in the concentration-dependent absorbance of Co3O4 CPHs at 652 nm due to TMB oxidation; Figure 10(D) shows the change in the concentration-dependent absorbance of H2O2 at 652 nm due to TMB oxidation.

[0047] Figure 11(A) shows that oxygen production increases with increasing H2O2 concentration; Figure 11(B) shows that oxygen production increases with increasing Co3O4CPHs concentration.

[0048] Figure 12 The effect of different Co3O4CPHs concentrations on GSH consumption is shown.

[0049] Figure 13 This demonstrates that CPB nanomaterials possess reliable biocompatibility;

[0050] Figure 14 Intracellular ROS levels were shown in different treatment groups;

[0051] Figure 15 The relative viability of 4T1 cells with and without ultrasound was shown under different concentrations of Co3O4CPHs and CPB.

[0052] Figure 16 Fluorescence images of 4T1 cells stained with calcein-AM (green) and PI (red) after different treatments are shown.

[0053] Figure 17 The tumor change curves of mice in different treatment groups are shown, revealing the differences in treatment effects among the groups;

[0054] Figure 18 Representative images of tumors resected on day 14 after treatment are shown in different treatment groups;

[0055] Figure 19 The curves showing the changes in body weight of tumor-bearing mice in different treatment groups are shown. Detailed Implementation

[0056] This invention discloses a nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB), its preparation method, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0057] The following problems urgently needed to be solved during the research and development of this application:

[0058] (1) How to achieve the controllable synthesis of Co3O4@PAO@BSA (CPB) nanozyme sonication agent, thereby improving the generation efficiency of ROS under ultrasonic excitation?

[0059] To prepare high-performance sonosensitive agents and target the synergistic sonodynamic / chemodynamic therapy of breast cancer, this scientific problem must be solved first, and it is also the foundation for subsequent research.

[0060] Porous Co3O4 CPHs (concave polyhedrons, CPHs) mimic the activities of four enzymes: oxooxidase (OXD), peroxidase (POD), catalase (CAT), and glutathione peroxidase (GPx). They undertake primary therapeutic tasks through cascade catalytic reactions, including reversing the hypoxic microenvironment, depleting endogenous glutathione, and continuously producing reactive oxygen species (ROS). Exogenous ultrasound (US) excitation promotes the valence state transition of Co ions, which is beneficial for increasing ROS production and thus killing tumor cells. However, excessive production of polyamines in tumor tissue inhibits cell membrane lipid peroxidation (LPO) and promotes DNA repair, resulting in limited oxidative damage effects. The porous structure of Co3O4 CPHs allows for the loading of plasma amine oxidase (PAO). PAO loading consumes polyamines at the tumor site, producing large amounts of highly toxic acrolein and hydrogen peroxide. The generated hydrogen peroxide increases the efficiency of Co3O4 CPHs-driven chemodynamic therapy (CDT), promoting cell membrane and DNA damage. Furthermore, the depletion of polyamines further reduces the stability of cell membranes and DNA. The raw materials and steps for material synthesis include: preparing Co-MOFs by mixing cobalt nitrate hexahydrate and 2-methylimidazole, calcining them in a muffle furnace at low temperature for 3 hours (300 degrees Celsius) to obtain porous Co3O4 CPHs, then loading PAO onto them, and finally further modifying them with bovine serum albumin (BSA) to prepare Co3O4@PAO@BSA (CPB). By adjusting various parameters such as the ratio of cobalt nitrate hexahydrate to 2-methylimidazole, calcination time and temperature, and the ratio of Co3O4 CPHs to PAO, the size, morphology, and PAO loading of CPB NPs were controlled to meet the requirements of this project, such as exhibiting acoustic and chemical dynamic effects.

[0061] (2) How to utilize Co3O4@PAO@BSA (CPB) to achieve synergistic sonodynamic / chemodynamic therapy, thereby solving the problem of incomplete tumor treatment by sonodynamic monotherapy? Single SDT treatment easily leads to residual tumor tissue and cells, resulting in tumor recurrence and metastasis. This is a key scientific problem that must be solved when using SDT for tumor treatment. To address this issue, this project synthesizes and regulates Co3O4@PAO@BSA (CPB) nanosonic sensitizers, uses Co3O4 CPHs as substrates to enhance enzyme kinetics, introduces PAO to consume polyamines in the tumor, and the carbonyl stress strategy of polyamine consumption successfully overcomes the shortcomings of oxidative damage therapy. The effect of Co ion-mediated SDT / CDT-induced oxidative damage therapy is enhanced through the nanozyme cascade catalytic effect.

[0062] Based on the above theoretical foundations and current research status, sonodynamic therapy (SDT) remains a novel treatment method for malignant tumors with greater application potential and clinical translation prospects. However, given its limitations, there is an urgent need to develop a more innovative strategy to synergistically enhance the oxidative damage effect of SDT on malignant tumors, thereby significantly improving its therapeutic efficacy. Extensive literature review revealed that various novel nanozyme materials have been successfully applied to synergistic photodynamic therapy (PDT) for the treatment of malignant tumors. In particular, significant progress has been made in engineered composite nanozymes in recent years. These nanozymes integrate the properties of various nanomaterials and can provide a multifunctional nanoplatform combining photodynamic therapy (PDT), photothermal therapy (PTT), chemodynamic therapy (CDT), and sonodynamic therapy (SDT). This platform exhibits cascade catalysis and synergistic effects, significantly improving its therapeutic efficacy against malignant tumors by enhancing the pro-oxidative activity used for treatment. These findings provide an effective approach for designing next-generation nanomedicines to achieve highly efficient treatment of malignant tumors. Cobalt (Co) is an essential trace element found in vitamin B12 (cobalamin). Its central cobalt ion is coordinated with various cobalt-based nanozymes by a tetrapyrrole ring, including metal nanoparticles (such as PtCo), metal oxide nanoparticles (such as Co3O4), and Co-containing nanocomposites. A review of extensive literature revealed few reports on exploratory studies regarding the catalytic enhancement of sonodynamic therapy (SDT) using Co-based nanosynthetic enzymes for tumor treatment. Therefore, this project aims to prepare Co3O4 nanozymes to catalyze and enhance SDT for the treatment of malignant tumors.

[0063] Furthermore, relevant literature reports that polyamine levels in tumor tissues are often higher than in normal tissues. Polyamines possess various pro-tumor mechanisms, including promoting the repair of damaged DNA through inducing molecular condensation and aggregation, and protecting DNA molecules from free radical attacks. In addition, due to their cationic nature, polyamines can interact with acidic phospholipids in the cell membrane, maintaining cell membrane stability, inhibiting cell membrane peroxidation, and providing a defense mechanism against oxidative damage. However, studies have reported that polyamines can be catalyzed by plasma amine oxidase (PAO) to produce large amounts of H2O2 and highly toxic acrolein (ACR), exacerbating oxidative damage. Reactive carbonyl species (RCS) are active biomolecules, most containing 3-9 carbons and one or more carbonyl groups. Acrolein (ACR) is an RCS with high electrophilic activity. It can cross-link with proteins and DNA through addition reactions (carbonyl stress reaction), thereby efficiently causing glutathione peroxidase 4 (GPx4) and DNA repair proteins to lose their activity, leading to DNA damage. A review of numerous documents revealed no reports on exploratory studies of this polyamine-activated carbonyl stress strategy in synergistic enhancement of sonodynamic therapy (SDT) for the treatment of malignant tumors.

[0064] Therefore, this application proposes to adopt this strategy to compensate for the shortcomings of sonodynamic therapy (SDT) by inducing rapid consumption of polyamines and large-scale production of acrolein through plasma amine oxidase (PAO), thereby improving the sensitivity and efficacy of oxidative damage treatment for malignant tumors.

[0065] The nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB) provided by this invention, as well as its preparation method and the raw materials and reagents used in its application, are all commercially available.

[0066] The present invention will be further illustrated below with reference to the embodiments:

[0067] Example 1: Preparation and characterization of Co3O4@PAO@BSA (CPB)

[0068] Co3O4 CPHs were prepared by a low-temperature calcination topological transformation strategy, and then PAO was loaded into them. BSA was used for modification to finally obtain the target nanomaterial. The physicochemical properties of the nanomaterial, such as morphology, structure, particle size, potential, in vitro stability and PAO loading, were analyzed.

[0069] 1.1 The preparation process is as follows:

[0070] 1.1.1 Preparation of Co-MOFs: 3 mL of cobalt nitrate hexahydrate (Co(NO3)2·6H2O, 0.52 M) aqueous solution was mixed with 20 mL of 2-methylimidazole (3.36 M) aqueous solution. After stirring at room temperature (25℃±5℃) for 6 h, the purple precipitate was collected by centrifugation, washed three times with ethanol, and dried in an oven at 80℃ for 24 h. The purple solid product was then collected as Co-MOFs.

[0071] 1.1.2 Preparation of Co3O4 CPHs: The prepared Co-MOFs were calcined in a muffle furnace for 3 hours (300°C) to obtain the black product, which is Co3O4 CPHs.

[0072] 1.1.3 Preparation of CPB: 10 mg PAO was added to 2 ml of Co3O4 CPHs (5 mg / mL) solution, stirred at room temperature (25℃±5℃) for 12 h, and then centrifuged (8000 rpm for 10 min) to remove excess PAO, resulting in a black precipitate of Co3O4@PAO. Then, 10 mg BSA was added to 10 ml of Co3O4@PAO (0.5 mg / mL) solution and stirred for 3 h. Finally, the supernatant was removed by centrifugation to obtain the solid product Co3O4@PAO@BSA (CPB), which was then stored in a 4°C refrigerator for later use.

[0073] 1.2 Characterization of nanomaterials:

[0074] 1.2.1 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the prepared Co3O4 CPHs had a polyhedral structure with a rough and uneven surface, indicating that they had pores and were porous structures (e.g., Figure 1 , Figure 2 (As shown).

[0075] 1.2.2 X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and elemental mapping also showed that Co and O elements were uniformly distributed in Co3O4 CPHs (e.g., Figure 3 , Figure 4 , Figure 5 (As shown).

[0076] 1.2.3 Detect PAO load

[0077] The UV-Vis spectra of different concentrations of PAO (2.5 mg / mL, 1.25 mg / mL, 0.63 mg / mL, 0.31 mg / mL, 0.16 mg / mL, 0.08 mg / mL, 0.04 mg / mL) were detected, and PAO concentration standard curves were plotted, as shown in Figure 6(A) and Figure 6(B).

[0078] Table 1. UV-Vis absorption spectra of PAO at different concentrations

[0079]

[0080]

[0081]

[0082]

[0083] Table 2. PAO concentration-dependent standard curve

[0084]

[0085] 3 ml of Co3O4 CPHs solution (2.5 mg / mL) was mixed with 7.5 mg PAO. After stirring for 12 h, the supernatant was centrifuged and the UV-Vis absorption spectrum (250-400 nm) was measured, further confirming that the PAO content in Co3O4@PAO was approximately 23% (e.g., ...). Figure 7 (As shown).

[0086] Table 3. PAO before and after 12 hours of adding Co3O4 and CPHs

[0087]

[0088]

[0089]

[0090]

[0091] Example 2 Verification of ROS generation in aqueous solution of nanomaterials

[0092] In an in vitro simulated weakly acidic microenvironment of breast cancer, the study was divided into an ultrasound irradiation group and a non-ultrasound irradiation group to verify the activity of four enzymes and the generation of ROS by Co3O4 CPHs.

[0093] 2.1 Performance of OXD-like enzymes:

[0094] To verify the oxidase activity (OXD) of Co3O4CPHs, TMB was used as a substrate for colorimetric measurement. As shown in the figure below, TMB was oxidized in a weakly acidic environment (pH=6.0), and the maximum absorbance was observed at 652 nm. However, neither Co3O4CPHs nor TMB alone caused a colorimetric reaction. The experiment verified that Co3O4CPHs has oxidase-like activity, and its activity increases as the pH value decreases.

[0095] Procedure: Co3O4CPHs (1 mg / mL) solution was mixed with TMB (10 mM) in PBS (pH = 7.4, 6.0, 5.4, 4.5), and the absorbance at 652 nm was recorded using UV-Vis absorption spectroscopy. Similar procedures were followed for different groups (TMB+Co3O4CPHs group, Co3O4CPHs group, TMB group), as shown in Figures 8(A) and 8(B).

[0096] Table 4. Performance of OXD-like enzymes at different pH values

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Table 5. Different groups of OXD-like enzymes with different performance

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] Using 5,5-dimethyl-1-pyrrolidone-N-oxide (DMPO) as a trapping agent, electron spin resonance (ESR) spectroscopy was employed to detect ·O 2- The formation of [the substance / factor] was observed. Unlike the control, US and Co3O4CPHs exhibited negligible peaks, while the introduction of US (irradiation conditions: 1.5 W cm⁻¹) resulted in [the formation / deterioration]. -2 (Frequency 1.0MHz, irradiation time 1.5min) increased O 2- The generation, such as Figure 9 As shown.

[0109] 2.2 Performance of POD-like enzymes:

[0110] Similarly, to verify the activity (POD) of Co3O4CPHs-type peroxidases, TMB was used as the •OH detection probe. As shown in the figure below, after adding H2O2, the absorbance value at 652nm increased (Figure 10(A)). Furthermore, as the pH value decreased (Figure 10(B)), the concentration of Co3O4CPHs (Figure 10(C)) and the concentration of H2O2 (Figure 10(D)) increased, the absorbance also increased, indicating that the amount of •OH generated increased.

[0111] Procedure: Co3O4CPHs (1 mg / mL) solution was mixed with H2O2 (10 mM) and TMB (10 mM) in PBS (pH = 7.4, 6.0, 5.4, 4.5), and the absorbance at 652 nm was recorded using UV-Vis absorption spectroscopy. Similar procedures were followed for different groups (TMB+H2O2+Co3O4CPHs group, TMB+Co3O4CPHs group, H2O2+Co3O4CPHs group, TMB+H2O2 group).

[0112] Procedure: Co3O4CPHs solutions of different concentrations (1 mg / mL, 800 μg / mL, 600 μg / mL, 400 μg / mL, 200 μg / mL, 0 μg / mL) were mixed with H2O2 (10 mM) and TMB (10 mM) in PBS (pH=6.0), and the absorbance at 652 nm was recorded using an ELISA reader; H2O2 solutions of different concentrations (20 mM, 15 mM, 10 mM, 5 mM, 2.5 mM, 0 mM) were prepared using a similar procedure.

[0113] Table 6. Different groups of POD-like enzymes with different performance

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] Table 7. Performance of POD-like enzymes at different pH values

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] Table 8. Co3O4 CPHs Concentrations with Different POD-like Enzyme Performance

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] Table 9. Performance of POD-like enzymes at different H2O2 concentrations

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] 2.3 Performance of CAT-like enzymes:

[0138] The oxygen production was measured using a portable dissolved oxygen meter, as shown in Figures 11(A) and 11(B). The oxygen production was dependent on the concentrations of H2O2 and Co3O4CPHs, and the oxygen production increased with the increase of the concentrations of both.

[0139] Table 10. Performance of CAT-like enzymes at different H2O2 concentrations

[0140]

[0141] Table 11. Co3O4 CPHs Concentrations with Different CAT-like Enzyme Performance

[0142]

[0143] 2.4 Glutathione peroxidase (GPx)-like performance:

[0144] Different concentrations of Co3O4 CPHs (dissolved in bicarbonate buffer, pH=8.7) and glutathione (GSH) were shaken at 150 rpm for 4 h at room temperature (with GSH+H2O2 as a positive control). Then, DTNB solution diluted with Tri-HCl (pH=8.0) was added, and the supernatant was centrifuged at 1200 rpm to measure the absorbance at wavelengths of 380-500 nm. The results showed that at the same time point (4 h), the concentration of GSH decreased with increasing Co3O4 CPHs concentration, confirming that Co3O4 CPHs can effectively consume GSH (e.g., glutathione + H2O2). Figure 12 (As shown).

[0145] Table 12. Performance of glutathione peroxidase (GPx)

[0146]

[0147]

[0148] Example 3: Killing effect at the breast cancer cell level

[0149] 3.1 Biosafety evaluation of CPB

[0150] 3T3 fibroblasts, BV2 microglia, and HUVEC endothelial cells were cultured in vitro. The toxicity of CPB nanomaterials to normal cells was evaluated. Three 96-well culture plates were set up, each divided into 9 groups with 5 replicates per group. 3T3 fibroblasts were seeded in plate A, BV2 microglia in plate B, and HUVEC endothelial cells in plate C. After 24 h of culture and cell attachment, serially diluted concentrations of CPB nanomaterials (400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 0 μg / mL) were added, and the cells were cultured for another 24 h. Then, CCK-8 (Shanghai Beyotime, C0038) was added and incubated for 1 h. Cell viability in each well was measured, and absorbance at 450 nm was determined using a microplate reader. The cell viability rate of the control group was set at 100%, and the cell viability rate of each experimental group was calculated. The formula is as follows: Cell viability rate (%) = (OD experiment - OD blank group) / (OD control - OD blank group) × 100%.

[0151] With increasing CPB nanomaterial concentration, no significant toxic effects were observed on 3T3 fibroblasts, BV2 microglia, and HUVEC endothelial cells, indicating that CPB nanomaterials possess reliable biosafety (e.g., Figure 13 (As shown).

[0152] Table 13. Biosafety Evaluation of CPB

[0153]

[0154] Table 14. Biosafety Evaluation of CPB

[0155]

[0156]

[0157] 3.2 Detection of ROS at the cellular level using nanomaterials and nanomaterials combined with sonodynamics (both with ultrasound and no ultrasound groups)

[0158] 4T1 breast cancer cells were seeded into 6-well plates and incubated at 37°C and 5% CO2 for 24 h. The culture medium was discarded, and the cells were washed with PBS buffer. Then, complete DMEM medium containing CPB nanomaterials (basal DMEM medium (Gibco, C11995500BT), fetal bovine serum (Gibco, 10099141)) was added, and the cells were incubated for 12 h. The supernatant was discarded, and basal DMEM medium (Gibco, C11995500BT) containing DCFH-DA (Shanghai Beyotime, S0033S) was added, followed by incubation for another 20 min. The irradiated group received ultrasound irradiation of a certain intensity (irradiation conditions: 1.0 W cm⁻¹). -2 The frequency was 1.0 MHz, and the irradiation time was 3 min. The treated cells were washed with PBS to remove probes that were not taken up by the 4T1 cells, and observed using a fluorescence microscope.

[0159] like Figure 14 As shown in the figure, the CPB+US group exhibited the strongest green fluorescence signal, indicating that the CPB combined with ultrasound therapy group could generate a large amount of ROS.

[0160] 3.3 Killing effect of nanomaterials and nanomaterials combined with sonodynamics on breast cancer cells (ultrasound group and no ultrasound group were set up).

[0161] 3.3.1 Analysis of the toxic effects of CPB combined with sonodynamics on 4T1 breast cancer cells using the CCK-8 assay

[0162] 4T1 breast cancer cells were seeded in 96-well plates and incubated at 37°C. 0 After incubation at 5% CO2 for 24 h until cell adhesion, different concentrations of nanomaterials (200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 0 μg / mL) were added at serially diluted levels and cultured for another 24 h. (The ultrasound group was subjected to ultrasound irradiation of a certain intensity 1.0 W cm⁻¹ 12 h after drug administration.) -2 The cells were irradiated at a frequency of 1.0 MHz for 3 minutes, then incubated with CCK-8 for 1 hour. Cell viability in each well was then measured using a microplate reader at 450 nm. The control group had a cell viability of 100%. Cell viability in each experimental group was calculated using the following formula: Cell viability (%) = (OD experiment - OD blank group) / (OD control - OD blank group) × 100%. Results are as follows: Figure 15 As shown, the relative survival rate of 4T1 cells gradually decreased with increasing CPB concentration in a concentration-dependent manner, and the addition of ultrasound further increased the cytotoxicity of CPB.

[0163] Table 15. Killing effects of nanomaterials and combined nanomaterials with acoustic dynamics on breast cancer cells

[0164]

[0165] 3.3.2 Fluorescence detection of live and dead cells

[0166] 4T1 breast cancer cells were seeded into 6-well plates and incubated at 37°C. 0 After incubation at 5% CO2 for 24 h, culture medium containing nanomaterials was added. After 12 h of incubation, cells were sonicated according to experimental groups (Control group, PAO group, sonication only group, PAO + sonication group, Co3O4 CPHs group, CPB group, Co3O4 + sonication group, CPB + sonication group). After another 12 h of incubation, the culture medium was carefully aspirated, and then calcein-AM and pyridine iodide (PI) (Wuhan Servicebio, G1707-100T) were added for staining for 20 min. After washing gently with PBS, the cells were observed and photographed under a fluorescence microscope. Live cells showed green fluorescence, while dead cells showed red fluorescence.

[0167] like Figure 16 As shown, a moderate amount of red fluorescence signal was observed in the Co3O4+US group, and almost all cells in the CPB+US group showed red fluorescence, while other groups showed a large amount of green fluorescence and no obvious red fluorescence, which proves that the CPB+US group has the strongest anti-tumor effect.

[0168] Example 4: Verification of the therapeutic effect of the above nanomaterials on breast cancer in vivo tumors.

[0169] 4.1 Establishing a nude mouse animal model:

[0170] SPF-grade female BALB / c nude mice, 4-5 weeks old and weighing 15-20 g, were subcutaneously inoculated to model breast cancer. After inoculation, the mice were randomly placed in a prepared ventilated and clean cage IVC system and kept as usual. Tumor growth at the inoculation site was observed every 2 days, and the size of the tumor was measured and recorded using electronic calipers. When the tumor volume reached 50-70 mm... 3 This can then be used for subsequent experiments. The formula for calculating tumor volume is as follows: V = L × W 2 / 2 (where V is the tumor volume in the nude mouse, L is the longest diameter of the tumor, and W is the shortest diameter of the tumor.)

[0171] 4.2 In vivo antitumor experiments:

[0172] The control group received intratumoral administration of saline, the ultrasound group received ultrasound treatment alone, and the other groups received the corresponding preparations, namely the Control group, PAO group, US group, PAO+US group, Co3O4 CPHs group, CPB group, Co3O4+US group, and CPB+US group; ultrasound treatment was administered 4 hours after intratumoral administration (irradiation conditions: 1.5W cm⁻¹). -2 The irradiation frequency was 1.0 MHz, and the irradiation time was 3 min. Under normal feeding conditions, the mice were administered the drug once every 2 days for a total of 7 doses. After different treatments, the tumor volume and body weight of each group of mice were measured and recorded every 2 days. After treatment, the volume was calculated using the volume calculation formula, and a relative tumor volume change curve was plotted (e.g., ...). Figure 17 (As shown).

[0173] During the treatment period, the tumor growth curves of the Control group, PAO group, US group, and PAO+US group showed continuous and rapid growth. However, the tumor growth curves of the Co3O4 CPHs group, CPB group, and Co3O4+US group all showed varying degrees of slowed growth. Compared with the other groups, the CPB+US group had the most significant tumor inhibition effect and the slowest tumor volume growth (e.g., Figure 18 (As shown).

[0174] Table 16. Tumor volume in mice of different treatment groups

[0175]

[0176]

[0177] Table 17. Weight of mice

[0178]

[0179]

[0180] 4.3 In vivo safety assessment of nanomaterials

[0181] Tumor-bearing mice were administered drugs in different groups, and their general condition and weight curves were observed. The control group was injected with physiological saline.

[0182] During the treatment, the mice in all groups maintained a steady increase in body weight, with no significant weight loss or symptoms of toxicity. This result indicates that CPB nanomaterials have good biocompatibility and in vivo safety (e.g., Figure 19 (As shown).

[0183] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing the nanoenzyme sound-sensing agent Co3O4@PAO@BSA (CPB), characterized in that, Includes the following steps: Step 1: Prepare and obtain Co3O4 CPHs; Step 2: Load PAO to prepare Co3O4@PAO; Step 3: Take the Co3O4@PAO obtained in Step 2 and modify it with BSA to obtain the nanoenzyme sound sensitizer Co3O4@PAO@BSA (CPB).

2. The preparation method according to claim 1, characterized in that, Includes the following steps: Step 1-1, Preparation of Co-MOFs: Mix cobalt nitrate hexahydrate aqueous solution with 2-methylimidazole aqueous solution, stir at 25℃±5℃ for 6h, centrifuge to collect purple precipitate, wash with ethanol, dry, and collect purple solid product as Co-MOFs; Step 1-2, Preparation of Co3O4 CPHs: Take the Co-MOFs obtained in step 1-1, calcine them, and collect the black product, which is Co3O4 CPHs.

3. The preparation method according to claim 1 or 2, characterized in that, Step 2 specifically involves mixing the PAO with the Co3O4CPHs, stirring at 25℃±5℃ for 12 hours, centrifuging to remove excess PAO, and collecting the black precipitate as Co3O4@PAO.

4. The preparation method according to any one of claims 1 to 3, characterized in that, Step 3 specifically involves: mixing BSA with the Co3O4@PAO prepared in step 2, stirring, centrifuging to remove the supernatant, and collecting the solid to obtain the nanoenzyme sound sensitizer Co3O4@PAO@BSA (CPB).

5. The preparation method according to any one of claims 2 to 4, characterized in that, In step 1-1, the molar ratio of cobalt nitrate hexahydrate in the cobalt nitrate hexahydrate aqueous solution to 2-methylimidazole in the 2-methylimidazole aqueous solution is 1.56:67.2; Preferably, the concentration of the cobalt nitrate hexahydrate aqueous solution is 0.52 M; and the concentration of the 2-methylimidazole aqueous solution is 3.36 M. Preferably, the volume ratio of the cobalt nitrate hexahydrate aqueous solution to the 2-methylimidazole aqueous solution is 3:

20.

6. The preparation method according to any one of claims 2 to 5, characterized in that, In step 1-1, the ethanol washing is performed at least once; preferably, the ethanol washing is performed three times. The drying process involves drying at 80°C for 24 hours.

7. The preparation method according to any one of claims 2 to 6, characterized in that, In steps 1-2, the calcination is performed at 300°C for 3 hours.

8. The preparation method according to any one of claims 1 to 7, characterized in that, In step 2, the mass ratio of PAO to Co3O4CPHs is 1:1; Preferably, the centrifugation is performed at 8000 rpm for 10 minutes. Preferably, in step 3, the mass ratio of BSA to Co3O4@PAO is 2:

1.

9. The nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB) prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB) as described in claim 9 in any one or more of the following: (I) Mimicking enzyme activity; preferably, the enzyme includes one or more of oxidase-like enzymes, peroxidase-like enzymes, catalase-like enzymes, or glutathione peroxidase; (II) Reduces cell membrane stability; (III) To prepare drugs or formulations that reduce oxidative damage during treatment; (IV) Preparation of drugs for synergistic treatment with sonodynamic therapy / chemodynamic therapy; (V) Preparation of drugs for treating malignant tumors; Preferably, the nanoenzyme acoustic sensitizer Co3O4@PAO@BSA (CPB) reverses the hypoxic microenvironment, consumes endogenous glutathione and ROS output, or promotes the valence state transformation of Co ions under exogenous ultrasound excitation, thereby increasing ROS production and killing tumor cells.

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