Z-type heterojunction TiO2-CeO2 nano-enzyme material as well as preparation method and application thereof

Abstract

The invention discloses a Z-type heterojunction TiO2-CeO2 nano-enzyme material as well as a preparation method and application thereof, and belongs to the technical field of antitumor drug material development. The material is composed of a TiO2 (at) CeO2 compound and PEG (Polyethylene Glycol) modified on the surface of the compound, the TiO2 (at) CeO2 compound is formed by self-assembly of an aminated TiO2 hollow nanosphere and carboxylated CeO2; and the TiO < 2 > (at) CeO < 2 > compound has a Z-type heterojunction structure. According to the present invention, the compound is adopted as the sound-sensitive agent to prepare the antitumor drug, has high biological safety, can catalytically decompose high-concentration H2O2 under the ultrasonic effect, can produce O2 and ROS in TME so as to induce tumor cell apoptosis and ICD, and particularly has good effect on breast cancer cells.

Description

Technical Field

[0001] This invention relates to a Z-type heterojunction TiO2@CeO2 nanozyme material, its preparation method, and its application, particularly to a Z-type heterojunction TiO2@CeO2 nanozyme material, its preparation method, and its application in the preparation of tumor drugs, belonging to the field of antitumor drug material development technology. Background Technology

[0002] Breast cancer poses a serious threat to women's health worldwide. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. TNBC patients often suffer from residual lesions and recurrence after standard treatment, with a median survival typically less than one year. Given the limited efficacy and side effects of current treatments, exploring new strategies for treating TNBC is imperative. Recent advances in immunotherapy have provided a new treatment option for TNBC patients. However, the limited response to immunotherapy often weakens treatment efficacy due to the presence of the immunosuppressive tumor microenvironment (TME). Therefore, reshaping the immunosuppressive TME and developing novel treatment methods capable of breaking down high concentrations of H2O2, alleviating hypoxia, and generating large amounts of reactive oxygen species (ROS) to promote cancer immunogenic cell death (ICD) within the TME is of great significance for the treatment of TNBC.

[0003] Sonodynamic therapy (SDT) offers an alternative strategy for inducing tumor intracellular lesions (ICDs), boasting advantages such as non-invasiveness, high tissue penetration, and high spatiotemporal selectivity. Furthermore, mounting evidence suggests that reactive oxygen species (ROS) can induce ICDs in cancer cells. However, due to self-protective mechanisms within the tumor tissue environment (TME), existing single-SDT strategies produce limited ICD effects, failing to release sufficient tumor-associated antigens to effectively activate systemic anti-tumor immunity. Therefore, there is an urgent need to explore novel methods to improve the efficiency of sonodynamic therapy and overcome the protective mechanisms of the TME during SDT.

[0004] Nanozymes have attracted widespread attention in cancer treatment due to their unique properties, such as high catalytic activity, good stability, low cost, ease of modification, and non-immunogenicity. In particular, nanozymes can effectively target key cells and signaling molecules within the tumor microenvironment (TME) to enhance cancer therapy, thereby inhibiting tumor growth and enhancing the combined therapeutic effect of tumor-specific therapy (SDT). Titanium dioxide (TiO2), as an inorganic sonosensitive agent with high stability and biocompatibility, has attracted considerable attention in recent years. For example, the authors and colleagues of the article (C. Tang, H. Li, M. Sha, J. Song, X. Bai, K. Liu, Y. Liu, B. Yuan, J. Yan, J. Chang, J. Kang, A tumor microenvironment responsive titanium-based nanosonosensitizer for combination sonodynamic-immunotherapy with calcium ion overload, Chem. Eng. J., 475 (2023) 146054) reported a TME-responsive CaCO3@Pt-TiO2 nanocomposite material (CaPT). CaPT utilizes TiO2 as a sonosensitizer to amplify oxidative stress in cancer cells for sonodynamic immunotherapy, providing a novel approach for sonodynamic synergistic immunotherapy. However, the clinical translation of traditional TiO2-based sonosensitizers faces significant challenges because their rapid electron-hole recombination significantly reduces their quantum yield and SDT efficacy. Cerium dioxide (CeO2) nanoparticles exhibit catalase-like (CAT) activity, which can significantly alleviate hypoxia in the tumor mesodynamic environment (TME), and hold promise as a nanozyme material to be incorporated into TiO2 materials for the preparation of tumor drugs. However, there are currently no reports on the use of TiO2 / CeO2 nanozymes for cancer sonodynamic therapy. Summary of the Invention

[0005] To address the limitations of TiO2 as a sonic sensitizer in existing technologies and the hypoxia condition in TME, the first objective of this invention is to provide a Z-type heterojunction TiO2@CeO2 nanozyme material. This material utilizes the Z-type heterojunction structure to inhibit the recombination of intrinsic electrons and holes in the nanomaterial, and enhances the intensity of the material under fluorescence through PEG modification, thereby enhancing the endocytosis efficiency of the material and the ultrasonic activity of the nanozyme.

[0006] The second objective of this invention is to provide a method for preparing Z-type heterojunction TiO2@CeO2 nanoenzyme materials, which has advantages such as simple process flow and mild reaction conditions.

[0007] The third objective of this invention is to provide an application of Z-type heterojunction TiO2@CeO2 nanozyme material as a sonosensitive agent in the preparation of antitumor drugs. It has high biosafety and can catalyze the decomposition of high concentrations of H2O2 under ultrasound, generating O2 and ROS in the TME, thereby inducing tumor cell apoptosis and ICD, especially showing good effects on breast cancer cells.

[0008] To achieve the above-mentioned technical objectives, the present invention provides a Z-type heterojunction TiO2@CeO2 nanozyme material, which is composed of a TiO2@CeO2 composite and its surface-modified PEG; the TiO2@CeO2 composite is formed by the self-assembly of aminated TiO2 hollow nanospheres and carboxylated CeO2; and the TiO2@CeO2 composite has a Z-type heterojunction structure.

[0009] In the material of this invention, the hollow TiO2 nanospheres exhibit high stability and become positively charged after amination, while the selected CeO2 possesses catalase-like activity and becomes negatively charged after carboxylation. The Z-shaped heterojunction in the material retains the more positive valence band (VB) position of TiO2 and the more negative conduction band (CB) position of CeO2, thereby inhibiting the recombination of intrinsic electrons and holes in the nanomaterial and enhancing its ability to generate ROS. Furthermore, surface modification with PEG enhances the material's intensity under fluorescence, thereby increasing its endocytosis efficiency and enhancing the ultrasonic activity of the nanozyme. Therefore, this material can promote the separation of excited electrons and holes under acoustic activation and effectively regulate oxidative stress, thus improving SDT-induced ICD. In addition, the hollow structure of the TiO2 nanospheres exposes more surface active sites, forming a more effective heterojunction interface, and the hollow structure is more conducive to generating a synergistic resonance effect with ultrasound, thereby achieving more effective electron-hole pair excitation and separation and stronger ROS generation.

[0010] As a preferred embodiment, the mass ratio of the aminated TiO2 hollow nanospheres to the carboxylated CeO2 is (1~2):(1~2). Within this mass ratio range, it is beneficial to ensure effective matching of the surface charges of the two types of particles, thus providing a good foundation for subsequent PEG surface modification and enhancing fluorescence, endocytosis, and ultrasonic response performance. However, excessive aminated TiO2 will result in unoccupied positive charge sites, while excessive carboxylated CeO2 will result in unoccupied negative charge sites, failing to form an optimal, compact, uniform, and stable heterogeneous interface.

[0011] This invention also provides a method for preparing Z-type heterojunction TiO2@CeO2 nanozyme materials, comprising the following steps:

[0012] S1 modified TiO2 hollow nanospheres with an amino-containing silane coupling agent to obtain aminated TiO2 hollow nanospheres.

[0013] S2 sequentially silanizes and oxidizes CeO2 to obtain carboxylated CeO2;

[0014] S3 mixes and self-assembles a solution including aminated TiO2 hollow nanospheres, carboxylated CeO2, and PEG-NH2 to obtain PEG-modified TiO2@CeO2 nanozyme material.

[0015] In the preparation method of this invention, the electrostatic interaction between positively charged aminated TiO2 and negatively charged carboxylated CeO2 is used to form a self-assembled complex. The amino groups on PEG-NH2 can further electrostatically bind with the carboxyl groups on the complex, thereby achieving surface modification. This method has advantages such as simple process flow and mild reaction conditions.

[0016] As a preferred embodiment, the preparation process of the TiO2 hollow nanospheres is as follows: a solution containing triethanolamine-modified tetrabutyl titanate is added dropwise to a polystyrene nanosphere dispersion for reaction. The resulting reaction product is separated, dried, and then calcined. This invention utilizes a template-assisted sol-gel method combined with calcination decomposition to prepare TiO2 hollow nanospheres. First, triethanolamine acts as a chelating agent to form a stable complex with tetrabutyl titanate. During the dropwise addition process, the negatively charged surface of the polystyrene nanospheres attracts the positively charged triethanolamine-modified tetrabutyl titanate, simultaneously undergoing hydrolysis and condensation to form a core-shell structure with TiO2 as the shell and polystyrene nanospheres as the core. The core is then decomposed by calcination to obtain the final product.

[0017] In this invention, the triethanolamine-modified tetrabutyl titanate solution is achieved by adding triethanolamine and tetrabutyl titanate to an ethanol solution, wherein the mass ratio of triethanolamine to tetrabutyl titanate is 0.3:(1~2).

[0018] As a preferred embodiment, the reaction product is annealed before calcination to improve its crystallinity.

[0019] As a preferred embodiment, the calcination conditions are: 3~5°C min. -1 The heating rate is from room temperature to 400~500℃, held for 2~4 hours, and then increased by 3~5℃ per minute. -1 The rate at which it decreases to room temperature.

[0020] As a preferred embodiment, the amino-containing silane coupling agent includes at least one of 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, and 3-aminopropylmethyldimethoxysilane.

[0021] As a preferred embodiment, the conditions for the amination modification are: temperature of 60~70°C, time of 1~3h, and solid-liquid ratio of TiO2 hollow nanospheres to amino-containing silane coupling agent of (250~500) mg: (2.5~5) mL.

[0022] As a preferred embodiment, in S2, the surface silanization process is as follows: CeO2 is modified by adding CeO2 into an alcohol-water mixture of vinyltrimethoxysilane to obtain vinylated CeO2.

[0023] As a preferred embodiment, the oxidation is performed by adding potassium permanganate and NaNO3 in an acidic environment.

[0024] The modification process of carboxylated CeO2 in S2 is as follows: Vinyltrimethoxysilane is first hydrolyzed in an acidic environment, and then condensed in an alkaline environment to form an oligomer; the silanol groups on the oligomer condense with the hydroxyl groups on the CeO2 surface to introduce vinyl groups into CeO2, and then the vinyl groups are oxidized and broken to generate carboxyl groups.

[0025] As a preferred embodiment, the surface alkylation modification conditions are as follows: the mass ratio of CeO2 to vinyltrimethoxysilane is (1~2):(1~2), the temperature is 75~80°C, the time is 2~4h, and the pH is 9~11.

[0026] As a preferred embodiment, the oxidation temperature is 80~90°C and the time is 4~6 hours.

[0027] As a preferred embodiment, in step S3, the self-assembly conditions are: a time of 12-24 hours, and the amount of PEG-NH2 used is 0.5-10 wt% of the total amount of aminated TiO2 hollow nanospheres and carboxylated CeO2. In this invention, a longer self-assembly time allows for sufficient bonding between the aminated TiO2 hollow nanospheres and the carboxylated CeO2. However, a lower amount of PEG-NH2 is detrimental to improving the dispersibility of the material and also to improving its cellular endocytosis efficiency.

[0028] Finally, this invention also provides an application of Z-type heterojunction TiO2@CeO2 nanozyme material, which is used as a sonication agent in the preparation of antitumor drugs.

[0029] The mechanism of action of the antitumor drug prepared using the Z-shaped heterojunction TiO2@CeO2 nanozyme material provided in this invention is as follows: Because the formation of the Z-shaped heterojunction in this invention can retain the more positive valence band position of TiO2 and the more negative valence conduction band position of CeO2, and after PEG modification, it exhibits stronger fluorescence intensity, endocytosis efficiency, and higher sonication activity. Therefore, under sonication, this nanozyme can catalyze the decomposition of high concentrations of H2O2, generating O2 and ROS in the TME, thereby inducing tumor cell apoptosis and ICD. Mechanistic analysis shows that by polarizing tumor-associated macrophages from the M2 phenotype to the M1 phenotype, the immunosuppressive state of the TME can be regulated, which promotes the maturation of dendritic cells and enhances CD8... + T-cell infiltration activates the immune system to eliminate tumors and suppress distant metastases.

[0030] As a preferred embodiment, the antitumor drug includes drugs used to treat benign breast tumors or breast cancer. Experiments have shown that the antitumor drug of the present invention exhibits high selectivity for TNBC in breast cancer cells and has the potential to serve as an anti-TNBC drug.

[0031] As a preferred embodiment, the antitumor drug comprises Z-type heterojunction TiO2@CeO2 nanozyme material, physiological saline, and excipients. The excipients include those commonly used in injectable formulations, such as phosphate buffer, phenol, and glucose.

[0032] As a preferred embodiment, the drug is an injectable formulation, and more preferably a pharmaceutically acceptable local injectable formulation.

[0033] As a preferred embodiment, the Z-type heterojunction TiO2@CeO2 nanozyme material is not less than the pharmaceutically effective amount, and further, the drug dosage is 1 to 100 mg / kg; preferably, 10 to 12.5 mg / kg is applied to each drug-administered subject (e.g., mouse).

[0034] As a preferred embodiment, the ultrasound conditions are: power of 1~3W cm⁻¹. -2 The frequency is 20~50kHz, and the duration is 3 minutes. Furthermore, the power is 1.75~3W cm⁻¹. -2 .

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

[0036] (1) The nanozyme material provided by the present invention utilizes the Z-type heterojunction structure to inhibit the recombination of intrinsic electrons and holes in the nanomaterial, and enhances the intensity of the material under fluorescence through PEG modification, thereby enhancing the endocytosis efficiency of the material and enhancing the ultrasonic activity of the nanozyme.

[0037] (2) The Z-type heterojunction PEG / TiO2@CeO2 nanozyme established in this invention, combined with sonodynamics, can alleviate the hypoxia in the tumor microenvironment and generate reactive oxygen species, thereby reshaping the immunosuppressive state in the tumor microenvironment. Experimental results show that this combined therapy can not only directly kill tumor cells through reactive oxygen species-mediated cytotoxicity, but also effectively induce immunogenic cell death and significantly enhance the infiltration of immune cells in tumor tissue, especially showing significant effects on breast cancer.

[0038] (3) The preparation method of the present invention is simple, the reaction conditions are relatively mild, the cost is low, the application prospects are great, and it can be prepared on a large scale.

[0039] (4) The nanozyme material of the present invention exhibits good biosafety and excellent selectivity for TNBC when applied to the preparation of antitumor drugs. Attached Figure Description

[0040] Figure 1 This is a flowchart and schematic diagram illustrating the process of preparing Z-type heterojunction TiO2@CeO2 nanozymes and their application.

[0041] Figure 2 This is a material characterization diagram of the Z-type heterojunction TiO2@CeO2 nanozyme (PTC NZs) prepared in Example 1 of the present invention, wherein, Figure 2 Images (a) to (c) in the figure are TEM images of PTC NZs. Figure 2 (c) is the image of (a) at high magnification; Figure 2 (d) in the image represents the EDS distribution of PTC NZs; Figure 2 In the diagram, (e~f) represent the PXRD and XPS spectra of PTC NZs, respectively. Figure 2 (g) in the spectrum represents the high-resolution Ce 3d XPS spectrum of PTC NZs. Figure 2 In this context, (h) represents the UV-Vis diffuse reflectance spectrum of PTC NZs. Figure 2 In the diagram, (i) is the Tauc curve and (j) is the band structure diagram of TiO2 HNs and CeO2NPs.

[0042] Figure 3 The images show the electron spin resonance (ESR) spectra of different nanoparticles under different ultrasonic irradiation conditions, where (a) represents •OH and (b) represents... 1 O2 and (c) are •O2 - The generation of , the horizontal axis is the irradiation time, the unit is s, in the figure (1) PTC+US, (2) CeO2+US, (3) TiO2+US, (4) PTC, (5) CeO2 and (6) TiO2.

[0043] Figure 4 This is a comparison of the effects of different nanoparticles on 4T1 cells under hypoxic and normoxic conditions, as shown by hypoxia detection.

[0044] Figure 5 CRT exposure and HMGB-1 release on the surface of TNBC cells, scale bar 50 μm.

[0045] Figure 6 The images show flow cytometry data of cell uptake in the control group, TiO2@CeO2 group, and PTC group.

[0046] Figure 7 (a) shows fluorescence images of HUVECs (human umbilical vein endothelial cells) and (b) 4T1 cells after incubation with Hoechst 33342 and Cy5.5-PTC for 0, 1.0, 2.0 and 4.0 hours, with a scale bar of 25 μm.

[0047] Figure 8 In the table, (a) represents the average tumor volume (n=7) within 14 days after treatment in different groups, and (b) represents the survival rate of tumor-bearing mice within 14 days after treatment in different groups. Detailed Implementation

[0048] The following embodiments are only specific descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. For those skilled in the art, any improvements made without departing from the present invention should be considered within the scope of protection of the present invention.

[0049] Example 1

[0050] (1) Preparation of TiO2 hollow nanospheres

[0051] The specific synthesis method for preparing TiO2 hollow nanospheres (TiO2 HNs) is as follows: 95 mL of ethanol was added to a 250 mL three-necked flask, followed by the addition of 5 mL of NH3·H2O and 5 mL of polystyrene nanosphere (PS NSs) dispersion (containing 125 mg PS NSs) under stirring. The temperature of the reaction system was stabilized at 50 ± 2 °C using an oil bath. A triethanolamine (TEOA) modified tetrabutyl titanate (TBT) solution was prepared by adding 0.32 g TEOA and 1.6 g TBT to 9 mL of ethanol and stirring until clear. While stirring, the TEOA-modified TBT solution was added dropwise to the reaction system containing PS NSs, maintaining the temperature of the entire reaction system at 50 °C and continuing stirring for 4 h. The solid product was separated from the system by centrifugation and washed with EtOH at least three times. After drying in air, the TiO2 HNs precursor was obtained.

[0052] The precursor was annealed in a muffle furnace to improve its crystallinity. The muffle furnace was set at 5°C for 1 minute. -1 The heating rate was increased from room temperature to 500°C and held for 2 hours, followed by a further heating at 5°C for 1 minute. -1 The rate of melting is reduced to room temperature, thus obtaining TiO2HNs.

[0053] (2) Synthesis of CeO2 nanoparticles

[0054] Ce(NO3)3·6H2O (320 mg, 0.73 mmol) and polyvinyl alcohol (PVP, 100 mg) with a molecular weight of 58000 were dissolved in 20 mL of ethylene glycol, and then 0.5 mL of water was added. After stirring continuously for 20 min in a magnetic stirrer, the clear solution was transferred to a 15 mL high-pressure hydrothermal reactor lined with polytetrafluoroethylene, and then hydrothermally reacted overnight at 150 °C. After the reaction was completed, the reactor was cooled to room temperature, the reaction solution was removed, and centrifuged for 10 min. The precipitate was then washed three times with deionized water and anhydrous ethanol to obtain CeO2 nanoparticles, which were then stored in a refrigerator for later use.

[0055] (3) Assembly of TiO2@CeO2 and PEG modification

[0056] Amination modification of TiO2 surface: To enhance the interaction between TiO2 HNs and carboxylated CeO2 and facilitate the formation of a complex, TiO2 HNs need to be amination-modified to give their surface a positive charge. Specifically, 250 mg of TiO2 HNs was dispersed in 50 mL of a 3-aminopropyltriethoxysilane (APTES) / EtOH solution (APTES approximately 5 vol%) using an ultrasonic method. The dispersion was stirred at 70°C for 1 h and then naturally cooled to room temperature. The resulting amination-modified TiO2 HNs were collected by centrifugation, washed with EtOH at least three times, and dried in air to obtain the amination-modified TiO2 HNs.

[0057] Carboxylation modification of CeO2: First, 2.0 g of vinyltrimethoxysilane was dissolved in 200 mL of an alcohol-water solution with a volume ratio of 1:1. After ultrasonic dispersion for 15 min, the pH of the solution was adjusted to 4 with glacial acetic acid, and then adjusted to 10 with ammonia. Next, 2.0 g of CeO2 was added to the above solution, and the mixture was stirred at 75 °C for 2 h for modification. After modification, the mixture was cooled to room temperature, and the precipitate was washed repeatedly with deionized water and anhydrous ethanol until the filtrate was colorless, yielding vinylated CeO2. The precipitate was then dissolved in 25 mL of concentrated sulfuric acid and stirred at 10 °C for 20 min. Then, NaNO3 (120 mg) and KMnO4 (500 mg) were added, the temperature was raised to 80 °C, and the reaction was stirred for 5 h. After cooling to room temperature, the precipitate was washed with 0.1 M hydrochloric acid and deionized water until the pH was weakly acidic or neutral, yielding carboxylated CeO2.

[0058] PEG / TiO2@CeO2 Assembly: Aminated TiO2 HNs were dispersed in H2O, and a carboxylated CeO2 NPs dispersion (with the mass of CeO2 NPs equal to that of TiO2 HNs) was added. The mixture was stirred at room temperature for 12 h. Since the aminated TiO2 HNs are positively charged and the carboxylated CeO2 is negatively charged, they assemble into a TiO2@CeO2 composite through electrostatic interaction. Then, PEG-NH2 (100 μg / mL) was added. -1 10 mL), the amount of PEG-NH2 used was 1 wt% of the total amount of aminated TiO2 hollow nanospheres and carboxylated CeO2, stirred for 12 h, and finally obtained PEG surface modified TiO2@CeO2, namely Z-type heterojunction PEG / TiO2@CeO2 nanozyme (PTC NZs).

[0059] Morphological characterization, XRD, and elemental analysis of PTC NZs were performed, and the results are as follows: Figure 2 As shown. Figure 2 As shown in (a) and (c), PTC exhibits the morphology of hollow nanospheres with a shell thickness of approximately 15 nm. In the high-resolution TEM image (b), lattice fringes corresponding to the anatase TiO2 (101) and CeO2 (112) crystal planes are clearly visible. Furthermore, the EDS elemental distribution map shows that titanium is concentrated on the shell of the hollow nanospheres, while cerium is distributed around the shell (e.g., ...). Figure 2 (d) in the middle. For example Figure 2As shown in (e), the TiO2 HNs exhibit characteristic diffraction peaks corresponding to anatase TiO2 in the PXRD pattern. These characteristic peaks remain unchanged after amination, indicating that the amination process did not disrupt the crystal structure of the TiO2 HNs. After loading carboxylated CeO2 NPs onto amination-type TiO2 HNs via electrostatic assembly, PTC simultaneously exhibited characteristic PXRD diffraction peaks of both CeO2 NPs and TiO2 HNs, confirming the successful preparation of PTC. Zeta potential measurements also indicate that the expected materials were obtained at each synthesis stage. Meanwhile, Figure 2 XPS analysis of (f) further identified these elements. Moreover, the high-resolution Ce 3d XPS spectrum of PTC showed characteristic absorption peaks in two series, V and U, confirming that Ce... 3+ and Ce 4+ The existence of two different oxidation states ( Figure 2 (g)). The Ti 2p spectrum shows sharp peaks at 458.15 eV and 464 eV, corresponding to Ti 4+ Ti 2p 3 / 2 and Ti 2p 1 / 2 The O 1s spectrum shows two characteristic peaks at 531.45 eV and 532.95 eV, which are attributed to the Ti-O bonds within the crystal structure and the hydroxyl (-OH) groups adsorbed on the surface, respectively.

[0060] Furthermore, to investigate the effect of CeO2 NPs on the acoustic sensitization activity of TiO2 HNs, the band structure of PTC was further characterized, such as... Figure 2 As shown in (i~j), the band gaps of CeO2 NPs and TiO2 HNs were measured to be 2.92 eV and 3.06 eV, respectively, and the valence band values ​​of TiO2 HNs and CeO2 NPs were 2.48 eV and 1.82 eV, respectively. Using the band gaps and valence band values, the final band structure of PTC was deduced, as follows. Figure 2 As shown in (j), when TiO2 HNs and CeO2 NPs are excited by ultrasonic energy, excitons are generated and rapidly separate into electrons (e). - ) and holes (h + Electrons from TiO2 HNs migrate to the valence band of CeO2 NPs under the influence of the intrinsic electric field, where they recombine with holes in the CeO2 NPs. Experiments revealed that the formation of the Z-type heterojunction preserves the more positive valence band position of TiO2 and the more negative conduction band (CB) position of CeO2. This configuration suppresses the recombination of intrinsic electrons and holes within each semiconductor, thereby enhancing the nanozyme's ability to catalyze the generation of reactive oxygen species (ROS), and consequently improving the ultrasound-sensitized activity.

[0061] The PTC NZs, CeO2, and TiO2 prepared in Example 1 were tested under ultrasonic irradiation or without ultrasonic treatment to determine their production. 1 The abilities of O2 and •OH are named as follows: (1) PTC+US, (2) CeO2+US, (3) TiO2+US, (4) PTC, (5) CeO2 and (6) TiO2, where US indicates the addition of ultrasound.

[0062] 1 O2 generation test: 200 μg mL of nanoparticle dispersion was tested. -1 ) and DPBF solution in PBS (1 mg / mL) -1 Mix for approximately 2.5 hours. Then, heat the mixture at 50 kHz, 1.75 W / cm². -2 The sample was subjected to ultrasonic treatment, and the absorbance was measured at 410 nm using a UV-Vis spectrophotometer.

[0063] •OH generation test: Prepare a nanoparticle dispersion (200 μg mL) -1 Mix with TMB solution and at 50 kHz, 1.75 W cm⁻¹ -2 The mixture was then subjected to ultrasonic treatment. The absorbance of the mixture was measured at 652 nm using a UV-Vis spectrophotometer.

[0064] Electron spin resonance (ESR) spectroscopy was employed, using 2,2,6,6-tetramethylpiperidin-1-oxygen (TEMP) as the singlet oxygen ( 1 A specific scavenger of O2, using 5,5-dimethyl-1-pyrrolidone-N-oxide (DMPO) as the hydroxyl radical (•OH) and superoxide anion (•O2). - The trapping agent of PTC was used, and the ability of PTC to generate •OH under ultrasonic irradiation was studied using TMB reagent. The results are as follows: Figure 3 As shown in (a) to (c), the results indicate that for both the TiO2 HNs +US and PTC +US groups, the absorption intensity at 410 nm and 652 nm increases rapidly with increasing ultrasonic irradiation time. This suggests that both groups can generate large amounts of •OH and 1 O2. In contrast, the absorption intensity of the CeO2 NPs group did not change significantly, meaning that CeO2NPs lack acoustic dynamic activity, and the PTC+US group has the strongest •OH, 1 O2 and •O2 - The ESR signal indicates that the PTC NZs prepared in this invention have excellent ROS generation ability and ultrasonic-triggered •OH generation efficiency.

[0065] Hypoxia detection was performed on PBS, TiO2, CeO2, and PTC NZs prepared in Example 1, following the steps below:

[0066] 4T1 cells were fed at a dose of 1×10 4 Cells were seeded at a density of [insert density here] in 96-well plates and cultured overnight to allow adherence. Cells were then divided into two groups: a hypoxic group (PBS, TiO2 HNs, TiO2 HNs+US, CeO2 NPs, PTC, PTC+US) and a normoxic group (PBS, PTC, PTC+US). The appropriate solutions were added to the culture medium in the 96-well plates, and a layer of paraffin oil was placed on the surface of the medium to create a hypoxic environment. No paraffin oil was added to the normoxic group. After overnight incubation, the TiO2 HNs+US and PTC+US groups from the hypoxic and normoxic conditions were irradiated with ultrasound (50 kHz, 1.75 W cm⁻¹). -2 After sonication for 3 minutes, the culture medium was removed, and the cells were washed twice with PBS. The hypoxia probe [Ru(dpp)3]Cl2 (final concentration: 30 μM) was added, and the cells were incubated for 4 hours. Subsequently, the cells were stained with Hoechst 33342 dye at 37°C in the dark. After incubation, the cells were observed under a laser confocal microscope.

[0067] The results are as follows Figure 4 As shown, cells in the PBS and TiO2 HNs groups exhibited strong red fluorescence, indicating a hypoxic state. When treated with TiO2 HNs+US, CeO2 NPs, and PTC groups, the red fluorescence of the probes decreased, indicating partial relief of hypoxia. Furthermore, the red fluorescence intensity of the PTC+US group was comparable to that observed under normoxic conditions, suggesting that PTC+US treatment can effectively improve hypoxia.

[0068] Immunogenicity, cell death rate, and metastasis were determined using PBS, TiO2, CeO2, and PTC NZs prepared in Example 1. The specific steps are as follows:

[0069] First, ATP levels were measured. Type I rat tail collagen was diluted to 1.2 μg / mL with 6 μM acetic acid solution. -1 Collagen solution was evenly spread on culture dishes and dried overnight. 4T1 cells were seeded at a density of 5000 cells / well on collagen-coated culture dishes and incubated overnight. Cells were divided into the following groups: (1) PBS; (2) TiO2; (3) TiO2 + US; (4) CeO2; (5) PTC; (6) PTC + US; (7) US. The PTC + US group received ultrasound irradiation (50 kHz, 1.75 W cm⁻¹). -2Centrifuge the mixture in the petri dish for 3 minutes. Then centrifuge the mixture in the petri dish and collect the supernatant. Measure the ATP level using an ATP assay kit.

[0070] After ATP level measurement, the culture dishes were treated with 4% paraformaldehyde and fixed in a 37°C incubator for 15 minutes. Anti-CRT and anti-HMGB-1 antibodies were added to the culture dishes, and the cells were incubated overnight at 4°C. The next day, Cy3.5-labeled secondary antibody was added to the CRT-stained culture dishes. Cells were then stained with DAPI. AF647-labeled secondary antibody was added to the HMGB-1-stained culture dishes. Cells were stained with DAPI again. Finally, the cells were imaged using a laser confocal microscope.

[0071] The development of adaptive immunity requires immunogenic death of tumor cells, which leads to the release of damage-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP), calreticulin (CRT), and high-mobility group box 1 (HMGB-1). Experimental results are as follows... Figure 5 As shown, compared with the control group, PTC under ultrasound irradiation increased ATP secretion levels by approximately 4.3 times. Figure 5 As shown, the green fluorescence signal representing CRT was weak and uniformly distributed within the cytoplasm in the PBS, US, and TiO2 groups, indicating that most cells were non-immunogenic. In other groups, some green fluorescence was observed to aggregate in one area, indicating that intracellular CRT translocation occurred. Particularly in the PTC+US group, CRT showed more pronounced aggregation, with all CRT concentrated at one edge of the cell, indicating that CRT was fully exposed on the 4T1 cell surface. The red fluorescence signal (HMGB-1) and the blue fluorescence signal (nucleus) largely overlapped in the PBS, US, and TiO2 HNs groups, indicating that HMGB-1 remained within the nucleus. In the PTC group under US irradiation, more red fluorescence was observed outside the nucleus, indicating that HMGB-1 was released in large quantities from the nucleus. These findings demonstrate that PTC can act as an ICD inducer, promoting ICD effects in TNBC cells through a synergistic effect of sonodynamics and enzymatic catalysis.

[0072] The in vitro inhibitory effect of PTC NZs prepared in Example 1 on TNBC cells was studied.

[0073] First, a cell uptake assay was performed to assess its internalization efficiency. The cell uptake assay was conducted using the following steps: 4T1 cells were arranged at a density of 1×10⁶ cells / cells. 4Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight to allow them to adhere. After adhesion, cells were treated with PBS, unmodified TiO2@CeO2, and PTC for 4 hours, respectively. Flow cytometry analysis was used to detect the results. Figure 6 As shown. Meanwhile, HUVECs (human umbilical vein endothelial cells) and 4T1 cells were respectively arranged at a density of 1×10⁻⁶. 4 Cells were seeded at a density in 96-well plates and cultured overnight to allow them to adhere. After adhesion, the cells were incubated with Hoechst 33342 (red) and Cy5.5-PTC (blue) for 0, 1.0, 2.0, and 4.0 h. The process of PTC entry into the cells was observed using a fluorescence microscope. The results are as follows: Figure 7 As shown.

[0074] like Figure 6 As shown, PTC exhibits higher fluorescence intensity compared to unmodified TiO2@CeO2, indicating that PEG modification effectively enhances endocytosis efficiency. Furthermore, as... Figure 7 As shown, the intracellular fluorescence intensity of mouse-derived triple-negative breast cancer cells (4T1) was increased in a time-dependent manner compared with human umbilical vein endothelial cells (HUVECs), demonstrating that PTC has a superior intracellularization capacity in cancer cells.

[0075] Human umbilical vein endothelial cells (HUVEC), 4T1, and human triple-negative breast cancer cells (MDA-MB-231) were selected as samples to evaluate the toxicity and antitumor activity of PTC against different cell lines. The experimental procedures were as follows: ① HUVEC, 4T1, and MDA-MB-231 cells were seeded into 96-well plates (density: 1 × 10⁴ cells / well) and cultured overnight to allow adhesion; ② Different concentrations (5, 10, 20, 30, 50, 75, 100, 150, and 200 μg / mL) were added to the 96-well plates. −1 ) PTCNZs were incubated for 12h and 24h respectively; ③ After washing the cells with PBS, a mixture of CCK-8 and culture medium was added to each well at a ratio of 1:10, and incubation was continued for 2h; ④ The absorbance value of the cells at 450nm was detected using a multi-functional microplate reader, and the cell viability was calculated.

[0076] The results showed that with increasing PTC concentration in the cell culture medium, the viability of normal HUVECs remained above 90%, while the viability of 4T1 and MDA-MB-231 cells gradually decreased. When the PTC concentration increased to 200 μg / mL... -1At that time, the survival rate of 4T1 and MDA-MB-231 cells decreased to approximately 10%. These results indicate that PTC is highly selective for TNBC and has the potential as an anti-TNBC drug. The effects of PTC on ultrasound irradiation (50 kHz, 1.75 W / cm²) were also evaluated. -2 The cytotoxicity of TNBC4T1 and MDA-MB-231 cells was investigated under US irradiation (for 3 min). The PTC group showed a decrease in cell viability of approximately 40%. In particular, under US irradiation, the synergistic effect of sonodynamics and enzyme catalysis led to apoptosis in over 90% of TNBC cells. Furthermore, even with the introduction of US irradiation, the antitumor effect of TiO2 HNs was still less significant than that of the PTC+US group. This is mainly attributed to the lack of Z-shaped heterojunctions between TiO2 HNs and CeO2NPs, resulting in ineffective sonodynamic effects.

[0077] Animal experiments were conducted on TiO2 (i.e., TiO2 HNs), CeO2 (i.e., CeO2 NPs), and the PTC NZs prepared in Example 1.

[0078] Animal experiments were conducted in accordance with experimental procedures and ethical guidelines at the Animal Center Laboratory of Xiangya School of Medicine, Central South University.

[0079] Bilateral tumor models were established by subcutaneous injection of 4T1-Luc tumor cells into mice. When the primary tumor volume exceeded 100 mm², a bilateral tumor model was established. 3 Mice were divided into 5 groups (n=7 per group): (1) PBS; (2) TiO2 + US; (3) CeO2; (4) PTC; (5) PTC + US. All nanoparticles were administered at 12.5 mg / kg. -1 The dosage was prepared with saline and injected intratumorally into the primary tumor. 24 and 48 hours post-injection, the TiO2 HNs+US group and the PTC+US group received ultrasound irradiation (50 kHz, 1.75 W cm⁻¹). -2 5 minutes. Then calculate the tumor volume.

[0080] Short-term tumor growth record: Tumor size and mouse weight were recorded from day 0 to day 14. On day 14, mice were anesthetized with sodium pentobarbital and euthanized by cervical dislocation. Tumors were dissected and collected for hematoxylin-eosin (H&E) staining and TdT-mediated dUTP nick-end marker (TUNEL) staining.

[0081] Long-term growth monitoring: The survival status of mice was recorded until day 50, at which time all remaining mice were sacrificed. At the same time, major organs (liver, heart, spleen, lungs, and kidneys) were harvested and stained with H&E.

[0082] like Figure 8As shown in (a), the PBS group showed the most significant tumor proliferation. The TiO2+US group, CeO2 group and PTC group all showed tumor growth, but the growth rate of the PTC group was slower than the other two groups. Under ultrasound irradiation, tumor proliferation was significantly inhibited, and the PTC+US group showed obvious tumor growth inhibition.

[0083] like Figure 8 As shown in (b), over a 50-day period, the combined treatment group (PTC+US) significantly prolonged the lifespan of tumor-bearing mice and increased their survival rate to 80%, which was significantly superior to other treatment groups. Furthermore, the final tumor weights of the different groups indicated that the combined treatment effectively inhibited tumor growth. Moreover, no significant damage was observed to the major organs (heart, liver, spleen, lungs, and kidneys) during the treatment process.

[0084] Example 2

[0085] The only difference between this embodiment and Example 1 is that the mass ratio of aminated TiO2 hollow nanospheres to carboxylated CeO2 was changed to 1:2 during the PEG / TiO2@CeO2 assembly process, and the stirring time at room temperature was replaced with 6 hours. All other steps and conditions remained the same. The prepared TiO2@CeO2 nanozyme material had the same morphology as Example 1, also exhibiting a Z-shaped heterojunction structure. However, due to the shortened self-assembly time and the improved quality of the carboxylated CeO2, the nanozyme's performance in generating reactive oxygen species under the same conditions was slightly lower than that of Example 1.

[0086] In summary, the Z-shaped heterojunction PEG / TiO2@CeO2 nanozyme prepared in this invention, and the method of combining the Z-shaped heterojunction PEG / TiO2@CeO2 nanozyme with sonodynamics during application, can alleviate the hypoxic condition in the tumor microenvironment, generate reactive oxygen species, and thus reshape the immunosuppressive state in the tumor microenvironment. Experimental results show that this combined therapy can not only directly kill tumor cells through reactive oxygen species-mediated cytotoxicity, but also effectively induce immunogenic cell death and significantly enhance the infiltration of immune cells in tumor tissue, especially showing significant effects on breast cancer.

Claims

1. A Z-type heterojunction TiO2@CeO2 nanoenzyme material, characterized in that: The product is composed of a TiO2@CeO2 composite and its surface-modified PEG; the TiO2@CeO2 composite is formed by the self-assembly of aminated TiO2 hollow nanospheres and carboxylated CeO2; and the TiO2@CeO2 composite has a Z-shaped heterojunction structure.

2. The Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 1, characterized in that: The mass ratio of the aminated TiO2 hollow nanospheres to the carboxylated CeO2 is (1~2):(1~2).

3. The method for preparing a Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 1 or 2, characterized in that: Includes the following steps: S1 modified TiO2 hollow nanospheres with an amino-containing silane coupling agent to obtain aminated TiO2 hollow nanospheres. S2 sequentially silanizes and oxidizes CeO2 to obtain carboxylated CeO2; S3 mixes and self-assembles a solution including aminated TiO2 hollow nanospheres, carboxylated CeO2, and PEG-NH2 to obtain PEG-modified TiO2@CeO2 nanozyme material.

4. The method for preparing a Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 3, characterized in that: The preparation process of the TiO2 hollow nanospheres is as follows: a solution of tetrabutyl titanate modified with triethanolamine is added dropwise to a polystyrene nanosphere dispersion and reacted under alkaline catalysis. The resulting reaction product is separated, dried, and then calcined. and / or; The amino-containing silane coupling agent includes at least one of 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, and 3-aminopropylmethyldimethoxysilane.

5. The method for preparing a Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 3, characterized in that: The conditions for the amination modification are: temperature of 60~70°C, time of 1~3h, and solid-liquid ratio of TiO2 hollow nanospheres to amino-containing silane coupling agent of (250~500) mg: (2.5~5) mL.

6. The method for preparing a Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 4, characterized in that: In S2, the surface silanization process is as follows: CeO2 is added to an alcohol-water mixed solution of vinyltrimethoxysilane for modification to obtain vinylated CeO2; and / or; The oxidation process involves adding potassium permanganate and NaNO3 in an acidic environment.

7. The method for preparing a Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 6, characterized in that: The surface alkylation modification conditions are as follows: the mass ratio of CeO2 to vinyltrimethoxysilane is (1~2):(1~2), the temperature is 75~80°C, the time is 2~4h, and the pH is 9~11; and / or; The oxidation is carried out at a temperature of 80-90°C for 4-6 hours.

8. A method for preparing a Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 6 or 7, characterized in that: In S3, the self-assembly conditions are: time is 12~24h, and the amount of PEG-NH2 is 0.5~10wt% of the total amount of aminated TiO2 hollow nanospheres and carboxylated CeO2.

9. The application of the Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 1 or 2, characterized in that: It is used as a sound-sensitizing agent in the preparation of antitumor drugs.

10. The application of the Z-type heterojunction TiO2@CeO2 nanoenzyme material according to claim 9, characterized in that: The antitumor drugs include those used to treat benign breast tumors or breast cancer.

Images