A composite nanomaterial with glucose catalytic consumption ability and ultrasonic piezoelectric response performance, a preparation method and application thereof

Abstract

This invention discloses a composite nanomaterial with glucose catalytic consumption capability and ultrasonic piezoelectric response performance, its preparation method, and its application, belonging to the fields of biomedical nanomaterials and anti-tumor drugs. The material comprises an N-doped TiNbC nanosubstrate, and Au nanoparticles and BaTiO3 piezoelectric components loaded on its surface. This material possesses both glucose catalytic activity and ultrasonic piezoelectric response performance. The invention also provides a preparation method for the composite nanomaterial and its application in the preparation of drugs for ultrasound-assisted tumor therapy. Through a sequential treatment mode of "pre-metabolic pretreatment followed by ultrasound synergy," Au nanoparticles first consume glucose in the tumor microenvironment, inhibiting the tricarboxylic acid cycle and oxidative phosphorylation, causing tumor cells to enter a metabolically weak state; then, the piezoelectric components respond to ultrasound to generate a piezoelectric effect, synergistically inducing reactive oxygen species generation, activating mitochondrial autophagy and apoptosis pathways, achieving a highly efficient anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials and anti-tumor drugs, specifically relating to a composite nanomaterial with glucose catalytic consumption capability and ultrasonic piezoelectric response performance, its preparation method and application. Background Technology

[0002] Malignant tumors pose a serious threat to human life and health. Traditional treatments such as surgery, chemotherapy, and radiotherapy have many drawbacks, including large-scale trauma, strong toxic side effects, easy development of drug resistance, and insufficient targeting, which limit their clinical application. Therefore, developing new, efficient, and low-toxicity synergistic tumor treatment strategies is of great practical significance.

[0003] Ultrasound therapy, with its advantages of strong penetration, high safety, non-invasiveness, controllability, and ease of operation, has shown promising application prospects in the field of non-invasive tumor treatment. However, the killing effect of ultrasound stimulation alone on tumor cells is limited, making it difficult to achieve efficient tumor suppression. Therefore, there is an urgent need to combine ultrasound with functional nanomaterials to construct a synergistic treatment system to enhance anti-tumor efficacy.

[0004] Tumor cells possess unique metabolic reprogramming characteristics, and abnormal glucose metabolism is a key feature of malignant tumor proliferation, as they are highly dependent on glucose supply to maintain rapid proliferation and energy. Current anti-tumor research largely focuses on directly killing tumor cells, with less emphasis on regulating the inherent metabolic patterns of tumor cells to achieve tumor suppression. Effectively depleting glucose within the tumor microenvironment, causing glucose deprivation in tumor cells, can significantly disrupt normal cellular physiological metabolism, inhibiting core energy metabolism pathways such as the tricarboxylic acid cycle and oxidative phosphorylation, thus placing tumor cells in a metabolically vulnerable state.

[0005] Currently, some nanomaterials are used in ultrasound-assisted tumor therapy, but most of them rely solely on physical effects to achieve cell damage. They lack design ideas to intervene in the physiological state of tumor cells from a metabolic perspective, cannot combine the advantages of metabolic disorders to further amplify the ultrasound-mediated cell damage effect, and are difficult to initiate tumor cell apoptosis by inducing autophagy pathways. The treatment mechanism is singular, and the overall tumor suppression effect still needs to be improved.

[0006] Therefore, developing a composite nanomaterial that can efficiently consume glucose, induce metabolic abnormalities in tumor cells, and simultaneously respond to ultrasound stimulation to synergistically activate autophagy and induce tumor cell apoptosis can fill the gaps in existing technologies, enrich the non-invasive synergistic treatment system for tumors, and has broad clinical translational and application value. Summary of the Invention

[0007] To address the problems of existing technologies, this invention aims to provide a composite nanomaterial with glucose catalytic consumption capability and ultrasonic piezoelectric response performance, as well as its preparation method and application. This addresses the issues of existing technologies, such as the single tumor catalytic / physical therapy mode, reliance on natural enzymes, and lack of metabolic pretreatment sequence design. The goal is to achieve a stepwise, highly efficient anti-tumor effect by first inducing metabolic abnormalities through glucose consumption, followed by ultrasound-assisted apoptosis induction.

[0008] This invention is achieved through the following technical solution: First, this invention provides a composite nanomaterial possessing both glucose catalytic activity and ultrasonic piezoelectric response. The material comprises an N-doped TiNbC nanosubstrate, and Au nanoparticles and a BaTiO3 (BTO) piezoelectric component supported on the surface of the nanosubstrate. The Au nanoparticles and the BTO piezoelectric component form a heterojunction structure on the surface of the N-doped TiNbC nanosubstrate. This composite nanomaterial exhibits both glucose catalytic activity and ultrasonic piezoelectric response.

[0009] In this composite nanomaterial, the N-doped TiNbC nanosubstrate serves as a carrier, providing a loading platform for Au nanoparticles and BTO piezoelectric components. The Au nanoparticles, as a glucose catalytic component, can catalyze the consumption of glucose in the tumor microenvironment. The BTO, as a piezoelectric component, can generate a piezoelectric effect under ultrasonic stimulation and synergistically enhance the overall therapeutic effect with the Au nanoparticles.

[0010] Secondly, the present invention also provides a method for preparing the above-mentioned composite nanomaterial, comprising the following steps: (1) Synthesis of BTO: Barium hydroxide octahydrate was mixed with titanium dioxide and subjected to hydrothermal reaction. After washing and drying, BTO powder was obtained. (2) Synthesis of N-TiNbC: TiNbAlC MAX phase powder was added to a mixed etching acid solution composed of hydrofluoric acid and concentrated hydrochloric acid, etched, washed and dried to obtain layered TiNbC; then the layered TiNbC was mixed with tetramethylammonium hydroxide solution, stirred at room temperature for intercalation and exfoliation, washed and dried to obtain TiNbC nanosheets; the TiNbC nanosheets were mixed with urea, reacted with solvent heat, washed and dried to obtain N-TiNbC powder.

[0011] (3) Synthesis of Au-N-TiNbC: N-TiNbC powder was dispersed in deionized water, and HAuCl4 solution was added under nitrogen protection. After stirring, ascorbic acid was added for reduction, and Au-N-TiNbC was obtained by centrifugation. (4) Synthesis of Au-N-TiNbC-BTO: BTO powder is mixed with Au-N-TiNbC suspension, stirred under nitrogen protection, and dried and annealed to obtain Au-N-TiNbC / BTO composite nanomaterials.

[0012] Preferably, in step (1), the molar ratio of barium hydroxide octahydrate to titanium dioxide is 2.5~3.5:1, the hydrothermal reaction temperature is 180~220℃, and the reaction time is 36~60 hours.

[0013] Preferably, in step (2), the etching acid solution is prepared by mixing 47%–49% hydrofluoric acid and 11.5–12.5M concentrated hydrochloric acid in a volume ratio of 5–7:1, the etching temperature is 70–90°C, and the etching time is 36–60 hours. The concentration of the tetramethylammonium hydroxide solution used for intercalation stripping is 0.5–2M, and the stirring time is 10–20 hours.

[0014] Preferably, in step (2), the mass ratio of TiNbC nanosheets to urea is 1:5 to 1:10, the solvothermal reaction temperature is 160 to 200°C, and the reaction time is 6 to 24 hours.

[0015] Preferably, in step (3), 200-400 μL of HAuCl4 solution with a concentration of 50-150 mM is added for every 50-80 mg N-TiNbC, and the concentration of the reducing agent ascorbic acid is 5-20 mM.

[0016] Preferably, in step (4), the mass-to-volume ratio of BTO powder to Au-N-TiNbC suspension is 0.5-2 mL of Au-N-TiNbC suspension with a concentration of 15-25 mg / mL for every 60-100 mg of BTO.

[0017] Finally, the present invention also provides the application of the above-mentioned composite nanomaterials in the preparation of drugs for ultrasound-assisted tumor treatment, particularly in the treatment of oral cancer.

[0018] The drug is used in combination with ultrasound stimulation to achieve an anti-tumor effect through a two-stage approach: first, it consumes local glucose in the tumor to induce metabolic abnormalities, and then it works in conjunction with ultrasound stimulation to induce tumor cell apoptosis.

[0019] Specifically, after administration, the drug consumes glucose in the tumor microenvironment through the glucose oxidase-like catalytic activity of the Au nanoparticles, thereby inhibiting the tricarboxylic acid cycle and / or oxidative phosphorylation in tumor cells. Following this glucose depletion leading to impaired tumor cell metabolism, ultrasound stimulation is then applied. The ultrasound, in conjunction with the BTO piezoelectric component, synergistically activates tumor cell autophagy, regulates the mitochondrial apoptosis pathway, upregulates pro-apoptotic protein expression, reduces anti-apoptotic protein expression, and activates apoptosis-executing proteins, thereby inducing tumor cell apoptosis.

[0020] Furthermore, the drug acts on the tumor lesion through local administration or targeted delivery.

[0021] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention marks the first time a three-in-one heterojunction structure integrating "support + catalysis + piezoelectricity" has been constructed: For the first time, an N-doped dual-transition metal TiNbC nanosubstrate, an Au nanozyme catalytic component, and a BTO piezoelectric response component are integrated into a single heterojunction structure through chemical reduction and in-situ growth strategies. This structure achieves synergistic cooperation among the three functions of "support, catalysis, and piezoelectricity," laying a solid material foundation for subsequent multimodal therapy.

[0023] Achieving efficient glucose depletion in the tumor microenvironment while mitigating the risks associated with natural enzymes: The composite nanomaterials of this invention utilize the glucose oxidase-like catalytic activity of Au nanoparticles to efficiently deplete local glucose in the tumor without the need for external natural enzymes, thus cutting off the energy supply to tumor cells at its source. Compared to methods relying on natural glucose oxidases, this effectively avoids the risks of immunogenicity and enzyme inactivation, improving the stability and safety of treatment.

[0024] Significantly inhibiting core energy metabolism pathways and inducing a stable state of energy starvation: Under glucose deficiency, the material of this invention can significantly inhibit the tricarboxylic acid cycle and oxidative phosphorylation process in tumor cells, forming a stable metabolic abnormality and energy starvation state. This greatly reduces the physiological tolerance of tumor cells, creating extremely favorable conditions for subsequent physical intervention.

[0025] This invention proposes and validates a step-by-step treatment strategy of "pre-treatment followed by ultrasound stimulation": A sequential treatment model is proposed, in which tumor cells are first brought into a metabolically weakened state through glucose depletion, followed by ultrasound stimulation to further aggravate damage on top of the metabolic impairment. Experiments have demonstrated that this step-by-step strategy of "pre-treatment followed by ultrasound stimulation" has a significantly better synergistic effect than single therapy or simultaneous combined therapy.

[0026] Orderly regulation of autophagy and apoptosis processes to achieve efficient induction of tumor cell death: Experimental verification shows that the material of this invention can effectively activate cell autophagy and simultaneously initiate the mitochondrial apoptosis pathway, thereby achieving efficient apoptosis induction of tumor cells.

[0027] High biocompatibility and wide applicability: This material exhibits excellent biocompatibility and low toxicity to normal tissue cells. Combined with non-invasive ultrasound therapy, it offers advantages such as minimal trauma and ease of operation, making it widely applicable in the treatment of various solid tumors.

[0028] The mechanism of action is clear and reproducible: This invention achieves tumor suppression by regulating three major physiological states of cells: metabolism, autophagy, and apoptosis. The logic of action is intuitive and clear, and the experiment is easy to repeat, which is conducive to subsequent mechanism research and clinical translation development. Attached Figure Description

[0029] Figure 1 The morphology, structure, and piezoelectric properties of Au-N-TiNbC-BTO composite nanomaterials are characterized. A shows transmission electron microscope images of different products; B shows the elemental distribution of the Au-N-TiNbC-BTO composite particles using energy-dispersive X-ray spectroscopy, indicating the uniform distribution of each element within the composite particles; C shows the X-ray diffraction patterns of different products; and D shows the full X-ray photoelectron spectrum of Au-N-TiNbC-BTO.

[0030] Figure 2 shows the piezoelectric properties and piezoelectric generation capacity characterization of the Au-N-TiNbC-BTO piezoelectric nanocomposite. In the figure, A is the three-dimensional morphology of the BTO substrate obtained by atomic force microscopy; B is the amplitude-voltage loop of the piezoelectric response force microscopy of the BTO material; C is the three-dimensional morphology of the Au-N-TiNbC-BTO composite nanomaterial obtained by atomic force microscopy; D is the amplitude-voltage loop of the piezoelectric response force microscopy of the Au-N-TiNbC-BTO composite nanomaterial; and E is the piezoelectric coefficient d of BTO and Au-N-TiNbC-BTO. 33 Quantitative statistical bar chart; F is the curve of piezoelectric output current density of BTO and Au-N-TiNbC-BTO under external field stimulation as a function of time.

[0031] Figure 3 The catalytic activity of Au-N-TiNbC-BTO composite nanomaterials under ultrasound-induced conditions was characterized. In this study, A represents the singlet oxygen (¹O₂) generation detection results of Au-N-TiNbC-BTO under different treatment conditions; B represents the total reactive oxygen species (ROS) generation detection results of Au-N-TiNbC-BTO; C represents the nitric oxide (NO) generation of Au-N-TiNbC-BTO with and without ultrasound; and D represents the peroxidase-like activity of Au-N-TiNbC-BTO with and without ultrasound.

[0032] Figure 4 This study illustrates the effects of Au-N-TiNbC-BTO composite nanomaterials on the metabolism of tumor cells and the influence of autophagy-related signals. Specifically, A represents a metabolic volcano map of tumor cells treated with Au-N-TiNbC-BTO; B represents a transcriptome volcano map of differentially expressed genes in tumor cells treated with Au-N-TiNbC-BTO; C represents enriched signals in metabolic pathways after treatment with Au-N-TiNbC-BTO; D represents enriched KEGG signals after treatment with Au-N-TiNbC-BTO; E represents GSEA analysis of autophagy signals in the transcriptome; F represents GSEA analysis of apoptosis signals in the transcriptome; and G represents intracellular glucose fluorescence in tumor cells treated with Au-N-TiNbC-BTO.

[0033] Figure 5 shows the transcriptome, pathway enrichment, cell oxidation and mitochondrial morphology, proliferation and autophagy apoptosis protein validation results of tumor cells after Au-N-TiNbC-BTO combined with ultrasound intervention. Among them, A is the differential gene volcano plot between the Au-N-TiNbC-BTO + ultrasound group and the control group; B is the top 10 enrichment bubble plot of the upregulated gene KEGG in the combined treatment group; C is the GSEA enrichment curve of apoptosis and mitophagy pathways; D is the mitochondrial membrane potential fluorescence and ROS reactive oxygen species fluorescence imaging of JC-1 cells in the control group, ultrasound group alone, and Au-N-TiNbC-BTO + ultrasound group; E is the mitochondrial morphology observation of the three groups of cells by transmission electron microscopy (TEM); F is the quantitative bar chart of MOC-1 cell proliferation rate after 24 h and 36 h of different treatments; G is the Western blot protein blot of autophagy marker P62 and apoptosis antiinhibition protein Bcl-2 in the gradient treatment group.

[0034] Figure 6 shows the in vivo tumor suppression effect of Au-N-TiNbC-BTO combined with ultrasound and the results of tumor tissue pathology and immunological characterization. In this figure, A is an in vitro tumor photograph; B is an HE pathological section (upper half) and Ki-67 proliferation marker immunohistochemical staining (lower half) of the tumor tissue of each group; C is a quantitative bar chart of tumor volume of each group at the treatment endpoint; D is a quantitative bar chart of tumor mass of each group at the treatment endpoint; and E is the dynamic change curve of tumor volume growth of each group during the 21-day dosing cycle.

[0035] Figure 7 shows the in vitro biosafety and in vivo organ biocompatibility evaluation results of Au-N-TiNbC-BTO nanomaterials. Among them, A is a bar chart of cell proliferation rate after treatment of RAW297.4 macrophages and HUVEC human umbilical vein endothelial cells with different concentrations of materials; B is a bar chart of quantitative detection of hemolysis rate of gradient concentration materials; C is an actual image of the main ex vivo organs of mice in the PBS control group and the Au-N-TiNbC-BTO material group; D is an HE pathological section of the heart, liver, spleen, lung and kidney tissues of the two groups of mice.

[0036] Figure 8 A schematic diagram illustrating the mechanism of action of Au-N-TiNbC-BTO combined with ultrasound in the treatment of oral cancer in mice. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0038] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0039] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0040] Table 1 below shows the source and specifications of the main raw materials and reagents in the examples.

[0041] Table 1. Sources and specifications of main raw materials and reagents in the embodiments.

[0042] Example 1: Preparation of Au-N-TiNbC / BTO composite nanomaterials Step (1): Synthesis of BTO Barium hydroxide octahydrate (Ba(OH)₂·8H₂O) and titanium dioxide (TiO₂) were accurately weighed at a molar ratio of 3:1. The mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene (PTFE), ensuring the filling volume did not exceed 70% of the autoclave's internal volume. A hydrothermal reaction was carried out at 200°C for 48 hours. After the reaction, the mixture was allowed to cool naturally to room temperature. The product was then removed and repeatedly washed with 0.2 mol / L hydrochloric acid solution, followed by washing with deionized water until the washings were neutral (pH≈7). The product was dried at 60°C for 12 hours to obtain a white BTO powder.

[0043] Step (2): Synthesis of N-TiNbC TiNbAlC MAX phase powder was slowly added to an etching acid solution consisting of 48% hydrofluoric acid and 12M concentrated hydrochloric acid at a volume ratio of 6:1, and etched at a constant temperature of 80°C for 48 hours. After etching, the mixture was centrifuged at 5000 rpm for 5 minutes, repeatedly washed with deionized water until the pH of the supernatant was approximately 6, and then freeze-dried under vacuum to obtain layered TiNbC material.

[0044] The above-mentioned layered TiNbC material was mixed with 1M TMAOH solution at a ratio of 1g:20mL and stirred at room temperature for 15 hours for intercalation exfoliation. After exfoliation, the mixture was centrifuged at 10,000 rpm for 10 minutes, washed several times with deionized water, and freeze-dried under vacuum to obtain TiNbC nanosheets. TEM showed that a typical, flat, and clean two-dimensional ultrathin nanosheet structure was successfully constructed. XDR showed that the N-TiNbC diffraction pattern after exfoliation exhibited a generally flat baseline, with characteristic peaks significantly broadened or even disappearing. This phenomenon fully confirms that the dense long-range ordered structure in the precursor MAX phase has been successfully broken, resulting in a two-dimensional flexible nanosheet layer with ultrathin properties. The TiNbC nanosheets and urea were dispersed in anhydrous ethanol at a mass ratio of 1:8, ultrasonically mixed for 30 minutes, and then transferred to a polytetrafluoroethylene-lined autoclave for solvothermal reaction at 180℃ for 12 hours. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 10 minutes, washed alternately with deionized water and anhydrous ethanol until the pH was near neutral, and dried at 60°C to obtain N-doped TiNbC (N-TiNbC) powder.

[0045] Step (3): Preparation of Au-N-TiNbC The N-TiNbC (60 mg) obtained in step (2) was dispersed in 60 mL of deionized water and sonicated for 15 minutes. Under nitrogen protection, 300 μL of 100 mM HAuCl4 solution was added, and the mixture was stirred for 5 hours. Then, 10 mM ascorbic acid was added and the mixture was stirred for another 15 minutes. The mixture was centrifuged at 20,000 rpm for 20 minutes to obtain Au-N-TiNbC, which was redispersed in deionized water (concentration 20 mg / mL) and stored at 4 °C.

[0046] Step (4): Preparation of Au-N-TiNbC-BTO The BTO powder (80 mg) obtained in step (1) was mixed with the Au-N-TiNbC suspension (1 mL, 20 mg / mL) obtained in step (3) and magnetically stirred for 24 hours under nitrogen protection. After vacuum drying (60 °C, 12 hours), it was annealed at 300 °C for 2 hours under nitrogen atmosphere and naturally cooled to room temperature to obtain Au-N-TiNbC / BTO composite nanomaterials.

[0047] Example 2: Morphology, structure, and piezoelectric properties characterization of composite nanomaterials To verify the structural integrity and functional properties of the prepared Au-N-TiNbC-BTO composite nanomaterials, the nanomaterials prepared in Example 1 were systematically characterized as follows.

[0048] (1) Transmission electron microscopy (TEM) characterization Take appropriate amounts of BTO powder, N-TiNbC, Au-N-TiNbC, and Au-N-TiNbC-BTO composite nanomaterial samples prepared in Example 1, disperse them in anhydrous ethanol, and sonicate for 10 minutes to ensure uniform dispersion. Take a small amount of the suspension and drop it onto the surface of a carbon film copper mesh. After natural drying, observe the morphological characteristics of different products under a transmission electron microscope.

[0049] Results: TEM images show (see...) Figure 1 In the first part, N-TiNbC exhibits a typical thin-layer nanosheet structure with a smooth surface; Au nanoparticles (approximately 5-20 nm in diameter) are uniformly and densely deposited on the surface of N-TiNbC without obvious agglomeration; BTO nanoparticles (approximately 50-100 nm in diameter) are further loaded onto the surface of Au-N-TiNbC, and the three form a tightly contacted heterojunction structure, indicating that the composite of each component was successfully achieved through a chemical reduction and in-situ growth strategy.

[0050] (2) Energy-dispersive X-ray spectroscopy (EDS) analysis The sample dispersed on the copper grid was taken and its elemental distribution was analyzed under the EDS detector attached to the transmission electron microscope. The scanning mode was high-angle annular dark field (HAADF) mode.

[0051] Results: EDS elemental distribution plot shown (see...) Figure 1 Elements such as B, C, Ti, Nb, N, Au, Ba, Ti, and O are uniformly distributed in the composite particles, with highly overlapping elemental profiles and no obvious elemental segregation. This result further confirms that Au nanoparticles and BTO piezoelectric components have been successfully and uniformly loaded onto the surface of an N-TiNbC substrate, forming a structurally complete integrated heterojunction structure.

[0052] (3) X-ray diffraction (XRD) analysis Crystalline phase characterization was performed using X-ray diffraction with Cu Kα rays (λ = 0.15406 nm). The scanning range was 2θ = 10°–90°; the scanning rate was 5 ° / min; the operating voltage was 40 kV, and the operating current was 40 mA; the step size was 0.02°. 2θ-diffraction intensity curves were acquired for each sample, and the spectra were compared with standard PDF cards to determine the crystalline phase.

[0053] Results: X-ray diffraction (XRD) showed (see...) Figure 1The diffraction patterns of Au-N-TiNbC (C) largely maintained the flat trend of the two-dimensional substrate. However, due to the extremely small grain size of the gold nanoparticles, its characteristic diffraction signals were relatively weak. The final composite system, Au-N-TiNbC-BTO, perfectly and completely reproduced all the characteristic diffraction peaks of pure BaTiO3, and the peak shapes still maintained extremely high sharpness. (4) X-ray photoelectron spectroscopy (XPS) characterization An appropriate amount of Au-N-TiNbC-BTO powder sample was taken, pressed into a sheet, and then placed in an X-ray photoelectron spectrometer for full-spectrum scanning and high-resolution narrow-spectrum scanning. The excitation source was monochromatic Al Kα rays (hv = 1486.6 eV), and C 1s (284.8 eV) was used as the charge correction reference.

[0054] Results: XPS full-spectrum scan results show (see...) Figure 1 The characteristic peaks of Ba, Ti, O, Nb, C, N and Au elements were clearly detected in the system, which intuitively confirmed the successful construction of the target composite system from a macroscopic perspective.

[0055] (5) Piezoelectric microscopy (PFM) characterization An alternating bias voltage is applied using an atomic force microscope (AFM) probe to induce localized minute deformations in the sample using the inverse piezoelectric effect, and the probe simultaneously acquires deformation response signals; then, the charge polarization reversal behavior of the sample is detected using the direct piezoelectric effect.

[0056] Result: See Figure 2 In the study, PFM was used to demonstrate that the Au-N-TiNbC-BTO composite material has significant ferroelectric polarization reversal capability and piezoelectric properties: sensor images revealed that the material has a microscopic three-dimensional morphology with nanoscale undulations; the amplitude curve exhibits a "butterfly-shaped" symmetry feature, which intuitively reflects the typical piezoelectric strain response generated by the material under electric field driving; and the phase curve shows a hysteresis loop jump of about 180° with the switching of bias voltage, which indicates that the ferroelectric domains inside the material have achieved complete polarization reversal under the action of external electric field.

[0057] (6) The piezoelectric constant (d) of the material as determined by the quasi-static testing instrument 33 Quantitative The longitudinal piezoelectric coefficient d of the sample was quantitatively determined using a quasi-static piezoelectric constant tester. 33 Conductive electrodes were prepared by double-sided silver plating of sintered ceramic sheets. The tests were repeated 5 times under fixed and uniform mechanical excitation conditions, and the average value was taken to compare the piezoelectric properties of pure BTO and Au-N-TiNbC-BTO.

[0058] See results Figure 2In the case of pure-phase BTO, the piezoelectric constant is relatively low. However, after combining it with two-dimensional nitrogen-doped N-TiNb and Au, the macroscopic piezoelectric response of the Au-N-TiNbC-BTO heterojunction system is significantly enhanced, and its d 33 The value jumped significantly to 16.9.

[0059] (7) Detection of material-triggered piezoelectric current density response Sintered silver-plated BTO and Au-N-TiNbC-BTO piezoelectric ceramic sheets were used as working electrodes. Conductive silver paste was connected to copper wires to lead out the electrodes. The samples were insulated and encapsulated, retaining only the stress-bearing area to avoid leakage interference with the current signal. A periodic mechanical vibration device was used to simulate ultrasonic mechanical stress, applying cyclic pressure stimulation of constant frequency and uniform amplitude to the samples to trigger the piezoelectric effect of the materials and generate piezoelectric charges. An electrochemical workstation was used to acquire the output current signal in real time and record the current density-time curve.

[0060] Result: See Figure 2 By applying periodic ultrasonic (US) pulses under zero bias conditions, the transient piezoelectric current response of BTO and Au-N-TiNbC-BTO heterojunctions was evaluated. Both materials generated sharp current pulses at the moment of ultrasonic (US) triggering, confirming their excellent piezoelectric conversion performance.

[0061] Example 3: Preparation of Au-N-TiNbC / BTO composite nanomaterials Referring to the method in Example 1, some raw material ratios and preparation process parameters were adjusted as follows: Step (1): The molar ratio of barium hydroxide octahydrate to titanium dioxide is 2.5:1, the hydrothermal reaction temperature is 180℃, and the reaction time is 36 hours.

[0062] Step (2): The etching acid solution is a mixture of 47% hydrofluoric acid and 11.5M concentrated hydrochloric acid in a volume ratio of 5:1. The etching temperature is 70℃ and the etching time is 36 hours. The concentration of TMAOH solution used for intercalation and stripping is 0.5M, and the stirring time is 10 hours at room temperature. The mass ratio of TiNbC nanosheets to urea is 1:5. The solvothermal reaction temperature is 160℃ and the reaction time is 6 hours.

[0063] Step (3): 50 mg of N-TiNbC was added to 200 μL of 50 mM HAuCl4 solution; the concentration of ascorbic acid was 5 mM.

[0064] Step (4): The amount of BTO powder used is 60 mg, and the amount of Au-N-TiNbC suspension used is 0.5 mL (concentration 15 mg / mL).

[0065] The remaining operating steps and conditions are the same as in Example 1. The obtained product was confirmed by XRD, SEM and TEM, and Au-N-TiNbC / BTO composite nanomaterials with a typical core-shell composite structure were successfully obtained, indicating that the reaction steps can still proceed effectively under the lower limit of parameters, and the target product can be obtained.

[0066] Example 4: Preparation of Au-N-TiNbC / BTO composite nanomaterials Referring to the method in Example 1, some raw material ratios and preparation process parameters were adjusted as follows: Step (1): The molar ratio of barium hydroxide octahydrate to titanium dioxide is 3.5:1, the hydrothermal reaction temperature is 220℃, and the reaction time is 60 hours.

[0067] Step (2): The etching acid solution is a mixture of 49% hydrofluoric acid and 12.5M concentrated hydrochloric acid in a volume ratio of 7:1. The etching temperature is 90℃ and the etching time is 60 hours. The TMAOH solution used for intercalation and stripping has a concentration of 2M and a stirring time of 20 hours at room temperature. The mass ratio of TiNbC nanosheets to urea is 1:10. The solvothermal reaction temperature is 200℃ and the reaction time is 24 hours.

[0068] Step (3): 80 mg of N-TiNbC was added to 400 μL of 150 mM HAuCl4 solution; the concentration of ascorbic acid was 20 mM.

[0069] Step (4): The amount of BTO powder used is 100 mg, and the amount of Au-N-TiNbC suspension used is 2 mL (concentration 25 mg / mL).

[0070] The remaining operating steps and conditions are the same as in Example 1. The obtained product was confirmed by XRD, SEM and TEM, and Au-N-TiNbC / BTO composite nanomaterials with a typical core-shell composite structure were successfully obtained. This shows that under the upper limit of parameters, the reaction of each step can still proceed effectively, and there are no problems such as excessive oxidation of MXene or severe agglomeration of Au nanoparticles, thus obtaining the target product.

[0071] Example 5: Preparation of composite materials with different mass ratios of BTO and Au-N-TiNbC Referring to the method of Example 1, only the amount of BaTiO3 powder in step (4) was changed, and composite materials were prepared according to the mass ratio of BTO:Au-N-TiNbC of 2:1, 4:1, 6:1 and 8:1 respectively, while keeping the other conditions unchanged, to obtain four different composition ratios of Au-N-TiNbC / BTO composite nanomaterials.

[0072] Example 6: In vitro catalytic capacity detection To verify the ability of the Au-N-TiNbC-BTO composite nanomaterial of this invention to generate ROS under ultrasonic stimulation, the following methods were used for detection: 1. Singlet oxygen (¹O2) detection The yield of singlet oxygen (¹O2) in the system was detected using a DPBF probe. Au-N-TiNbC-BTO prepared in Example 1 was mixed with a DPBF ethanol solution. An ultrasonic control was set up, and the characteristic absorbance of DPBF at 410 nm was collected periodically. The ¹O2 generation efficiency was quantified by the absorbance decay rate. Simultaneously, the UV-Vis absorption spectra of 400–700 nm were scanned to visually compare the absorption peak intensities of the singlet oxygen oxidation products in each group.

[0073] The results are as follows Figure 3 As shown in Figure A, the synergistic effect of the Au-N-TiNbC-BTO heterojunction system in generating singlet oxygen was strongly confirmed by DPBF degradation kinetics: within an 8-minute monitoring period, the Au-N-TiNbC-BTO group exhibited the most significant decrease in absorbance (up to 30%), and its efficiency in generating singlet oxygen far exceeded that of the single-component or component-deficient control. This phenomenon demonstrates that the piezoelectric potential generated by BTO and the high conductivity of Au / MXene form a perfect charge-regulated coupling, greatly promoting the excitation and conversion of oxygen molecules.

[0074] 2. Total reactive oxygen species (ROS) detection Total reactive oxygen species (ROS) in the system were quantified using a DCFH-DA broad-spectrum probe. A blank control group, a materials-only group, and a materials-plus-ultrasound group were set up. UV-Vis absorption spectra at 500–800 nm were collected periodically. The total ROS generation rate and cumulative yield were dynamically monitored by observing changes in the characteristic absorbance of DCF at 650 nm, thus evaluating the overall performance of ultrasonically excited Au-N-TiNbC-BTO piezoelectric catalysis for ROS generation.

[0075] The results are as follows Figure 3 As shown in Figure B, the experimental results strongly confirm, through NBT absorption spectroscopy, that the Au-N-TiNbC-BTO composite material possesses excellent acoustic-dynamic catalytic performance, and its ROS generation efficiency depends on the piezoelectric synergistic effect of multiple components. Ultrasonic stimulation is a necessary condition for triggering a large-scale ROS generation, and the significant increase in absorbance peak confirms the material's highly efficient acoustic response.

[0076] 3. Detection of nitric oxide (NO) release The kinetics of nitric oxide (NO) release catalyzed by ultrasound (US) excitation was evaluated using the Griess colorimetric method. A blank control group, a simple Au-N-TiNbC-BTO group, and an Au-N-TiNbC-BTO group combined with ultrasound were set up. Supernatants were collected at different time points and reacted with Griess reagent in the dark. The cumulative NO concentration was quantified by the characteristic absorbance at 540 nm. Time-NO concentration kinetic curves were plotted to analyze the effect of ultrasound stimulation on the NO catalytic release efficiency of the materials.

[0077] Result: As Figure 3 As shown in Figure C, the Au-N-TiNbC-BTO composite system exhibits a more efficient NO generation capacity within the same test cycle. This fully demonstrates that the tight interfacial coupling between BTO and the two-dimensional N-TiNbC support constructs an excellent charge transport channel. Even in the absence of US stimulation, Au-N-TiNbC-BTO still maintains a moderate NO release rate, indicating that this material can achieve slow basal release under physiological conditions. However, the NO generation rate significantly increases upon the introduction of US.

[0078] Detection of 4-oxidase (POD-like) catalytic activity 3,3',5,5'-Tetramethylbenzidine (TMB) was used as the chromogenic substrate, and the oxidation kinetics of the substrate were monitored in real time using ultraviolet-visible absorption spectroscopy (UV-Vis). TMB oxidizes to generate a blue cationic radical, exhibiting a characteristic absorption peak at 652 nm. The absorbance value was positively correlated with the content of the oxidation product. A blank control group, a simple Au-N-TiNbC-BTO material group, and an Au-N-TiNbC-BTO combined with ultrasound (US) experimental group were set up, with equal volumes of TMB working solution added to each group. The ultrasound group was subjected to ultrasound stimulation with uniform parameters, while the other groups were simultaneously placed in the dark. The full UV-Vis spectrum in the 400–800 nm range was acquired at a preset time gradient, and the absorbance change at the characteristic wavelength of 652 nm was recorded. Time-absorbance kinetic curves were plotted to quantitatively compare the catalytic oxidation ability of different systems of TMB, reflecting the ultrasound-responsive catalytic activity of the materials.

[0079] The results are as follows Figure 3 As shown in Figure D, the Au-N-TiNbC-BTO composite material, upon ultrasonic excitation, exhibits a highly significant characteristic absorption peak at approximately 652 nm (corresponding to the characteristic product oxTMB generated by the oxidation of TMB). This strong signal demonstrates that the composite material possesses excellent POD nanozyme activity, capable of efficiently catalyzing the decomposition of H2O2 and releasing highly oxidizing reactive oxygen species (ROS, mainly hydroxyl radicals (·OH)). Example 7: Au-N-TiNbC-BTO Intervention in Tumor Cell Metabolism To verify that the Au-N-TiNbC-BTO composite nanomaterial of this invention induces abnormal intracellular metabolism due to cellular glucose consumption, the following methods were used for detection: 1. Cell preparation and grouping MOC-1 oral and tongue squamous cell carcinoma cells in the logarithmic growth phase were selected and subjected to a assay of 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of cells / well in culture plates and incubated overnight at 37°C with 5% CO2 until complete cell adhesion. Two groups were set up, with three biological replicates in each group. Blank control group: Cells were cultured in normal DMEM high-glucose complete medium without any additional treatment; Simple material intervention group: Au-N-TiNbC-BTO composite nanomaterials prepared in Example 1 were added to the culture medium, with a final concentration of 100 µg / mL, and incubated at 37℃ for 24 h.

[0080] 2. Metabolomics detection and analysis After cell incubation in each group, the culture medium was discarded, and the cell pellet was collected by trypsin digestion. All intracellular metabolites were extracted using a pre-cooled methanol / acetonitrile / water (2:2:1, v / v / v) mixture. The supernatant was collected after vortexing, low-temperature ultrasonic extraction, and high-speed centrifugation. Metabolite separation, qualitative and quantitative detection were performed using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Significantly differential metabolites were screened by multivariate statistical analysis, and KEGG pathway enrichment analysis was conducted on the differential metabolites.

[0081] Results: Volcano plot of differentially metabolized metabolites in the metabolome (see...) Figure 4 As shown in Figure A), compared with the blank control group, the expression levels of a large number of sugar metabolism and energy metabolism-related metabolites in cells were significantly altered after Au-N-TiNbC-BTO intervention alone. A total of 367 significantly downregulated metabolites and 56 significantly upregulated metabolites were screened.

[0082] Downregulated differential metabolite KEGG enrichment bubble diagram (see) Figure 4 In the case of C), material intervention significantly downregulated metabolites highly enriched in tumor-centric carbon metabolism and mitochondrial energy production pathways such as the tricarboxylic acid cycle (TCA cycle), oxidative phosphorylation, alanine / aspartate / glutamate amino acid metabolism, and glycoamino acid biosynthesis.

[0083] The results confirm that Au-N-TiNbC-BTO can directly inhibit aerobic glycolysis and mitochondrial energy supply in tumor cells, continuously consume intracellular glucose, induce tumor cells to enter a metabolically weak state of energy deficiency, and complete the metabolic sensitization pretreatment of tumor cells.

[0084] 3. Transcriptome RNA-seq sequencing analysis Cell pellets from each group were collected, and total RNA was extracted using TRIzol reagent. After RNA integrity and purity quality control, paired-end transcriptome sequencing was performed using the Illumina NovaSeq 6000 sequencing platform. Differentially expressed genes were screened using DESeq2 software, and KEGG pathway enrichment analysis was performed on significantly upregulated and significantly downregulated differentially expressed genes. GSEA gene set enrichment analysis was also performed on key pathways of mitophagy and apoptosis.

[0085] Result: As Figure 4 As shown in B and D, the transcriptome differential gene volcano plot (see Figure 1). Figure 4 As shown in Figure B, after simple material treatment, a large number of stress and death-related genes in tumor cells were significantly upregulated; the top 10 KEGG enrichment bubble chart of differentially regulated genes in cellular processes (see Figure B) Figure 4 The results showed that the gene concentrations of p53 signaling pathway, mitophagy, multi-species apoptosis, cellular senescence, and cell cycle arrest pathways were significantly upregulated, indicating that nanomaterial intervention alone can simultaneously initiate mitochondrial damage, autophagy clearance, and programmed cell death in tumor cells.

[0086] GSEA enrichment curve of the mitochondrial autophagy pathway (see) Figure 4 The normalized enrichment score (NES) for the apoptosis pathway was 1.8, with a nominal P-value of 0.003; the GSEA enrichment curve for the apoptosis pathway (see [link to data]). Figure 4 The normalized enrichment score (NES) was 1.22, with a nominal P of 0.01. This indicates that, based on overall gene expression, Au-N-TiNbC-BTO treatment alone only affected the activation of autophagy but did not affect the occurrence of apoptosis. This suggests that under the influence of Au-N-TiNbC-BTO, cells induce mitochondrial abnormalities by consuming cellular glucose, thereby initiating mitophagy.

[0087] 4. Intracellular glucose fluorescence imaging detection After cell treatment, the culture medium was discarded, and the cells were incubated with a glucose-specific fluorescent probe at 37°C in the dark for 20 min. The cells were washed three times with PBS, and fluorescence images were collected using an inverted fluorescence microscope to visually and qualitatively compare the intracellular glucose storage levels of the two groups of cells.

[0088] Results: Intracellular glucose fluorescence imaging (see...) Figure 4 As can be seen from the data (G), the glucose fluorescence signal of the blank control group cells was bright and the intensity was high; the fluorescence signal of the Au-N-TiNbC-BTO treatment group was significantly weakened, which directly proves that the intracellular glucose of the tumor cells was consumed in large quantities after the nanomaterial treatment, and the cells were in a glucose-deficient state, which is consistent with the metabolomics test results.

[0089] Example 8: In vitro experiment on ultrasound-induced autophagy and apoptosis To elucidate the molecular mechanism by which the Au-N-TiNbC-BTO composite nanomaterials of this invention induce tumor cell growth inhibition through mitophagy, the following systematic experiments were conducted.

[0090] 1. Cell treatment and grouping MOC-1 cells (oral squamous cell carcinoma cells) were selected and cultured using standard methods. The following experimental groups were set up: blank control group (PBS), ultrasound-only group (US, ultrasound irradiation only), and material + ultrasound combined treatment group (US + Au-N-TiNbC-BTO).

[0091] Au-N-TiNbC-BTO composite nanomaterials (final concentration 100 µg / mL) prepared in Example 1 were added to the cell culture medium of both the simple material group and the combined treatment group, and the cells were co-cultured at 37°C and 5% CO2 for 24 hours. Afterwards, the simple ultrasound group and the combined treatment group were subjected to in vitro ultrasound irradiation treatment with specific parameters (power density 0.5 W / cm², duty cycle 30%, duration 5 minutes).

[0092] 2. Transcriptome sequencing and enrichment analysis Cell pellets from each group were collected and RNA-seq transcriptome sequencing was performed using the Illumina platform. Differentially expressed genes were screened. KEGG pathway enrichment was performed on differentially expressed molecules in the combined treatment group, and GSEA enrichment analysis was performed on apoptosis and mitophagy pathways.

[0093] Result: As Figure 5 As shown in Figures A and B, the differentially expressed gene volcano plots between the combined treatment group and the blank control group (see Figures A and B). Figure 5 (A) shows that after ultrasound combined with material treatment, a large number of stress and death-related genes in tumor cells were significantly upregulated; the top 10 KEGG enrichment bubble plots of differentially regulated genes in cellular processes (see...) Figure 5 The results showed that the genes were significantly upregulated and concentrated in the pathways of mitophagy, apoptosis, and cell cycle arrest, indicating that ultrasound combined with material intervention can simultaneously initiate mitophagy and programmed cell death in tumor cells.

[0094] The normalized enrichment score (NES) of the GSEA enrichment curve for the apoptosis pathway was 1.3 (P = 0.035); the NES for the mitophagy pathway was 1.57 (P = 0.009). Both pathways showed significantly higher enrichment scores than the material-only group, confirming a synergistic effect of ultrasound and nanomaterials in pathway activation (see...). Figure 5 (C)

[0095] 3. Qualitative detection of mitochondrial membrane potential and ROS using dual fluorescence. JC-1 mitochondrial membrane potential probe and DCFH-DA reactive oxygen species fluorescent probe were used to label cells in each group, and the cells were incubated in the dark before being imaged by fluorescence microscopy. The red-green fluorescence ratio of JC-1 reflects the degree of mitochondrial membrane potential damage, and the green fluorescence intensity characterizes the level of intracellular ROS accumulation.

[0096] Result: As Figure 5 As shown in Figure D, the red and green fluorescence distributions were balanced in the blank control group and the ultrasound-only group, and the mitochondrial membrane potential remained intact. The green fluorescence in the material-only group increased slightly. In the material-ultrasound combined group, almost all cells exhibited green fluorescence, indicating severe depolarization damage to the mitochondrial membrane potential. Only the Au-N-TiNbC-BTO combined with ultrasound group showed extremely strong green fluorescence, indicating that the ultrasound-induced piezoelectric catalytic effect can significantly amplify the intracellular reactive oxygen species generation mediated by nanomaterials, synergistically enhancing oxidative stress damage.

[0097] 4. Transmission electron microscopy (TEM) observation of cell ultrastructure Cells in each group were prefixed with 2.5% glutaraldehyde, postfixed with 1% osmium tetroxide, dehydrated with graded ethanol, embedded in epoxy resin, and prepared as ultrathin sections. Transmission electron microscopy was then used to observe changes in mitochondrial morphology and ultrastructural changes in autophagic vesicles.

[0098] Result: As Figure 5 As shown in Figure E, the mitochondria in the control group were intact with clear internal cristae structures; the mitochondria in the ultrasound-only group showed only slight edema; the mitochondria in the material-only group showed a small number of damaged mitochondria; the mitochondria in the material-ultrasound combined group showed large-area shrinkage, internal cristae dissolution and breakage, and a large number of intracellular double-membrane autophagy vesicles wrapped the damaged mitochondria, showing typical ultramicroscopic features of mitochondrial autophagy. The degree of mitochondrial damage was significantly aggravated after synergistic treatment.

[0099] 5. Quantitative detection of CCK-8 cell proliferation MOC-1 cells were seeded in 96-well plates and treated for 24 h and 36 h according to four treatment protocols. 10 μL of CCK-8 reagent was added to each well and the cells were incubated at 37°C in the dark for 1–4 h. The absorbance was measured at 450 nm using a microplate reader, and the cell proliferation inhibition rate of each group was calculated.

[0100] Result: As Figure 5As shown in Figure F, at two time gradients of 24 h and 36 h, ultrasound alone and the material alone only slightly inhibited tumor cell proliferation; the cell proliferation rate of the Au-N-TiNbC-BTO combined with ultrasound treatment group decreased significantly, and the inhibitory effect was further enhanced with the extension of culture time, demonstrating a time-dependent synergistic tumor-suppressing effect.

[0101] 6. Western Blot detection of autophagy and apoptosis marker proteins Cells were collected from the PBS control group, the BTO group alone, the TiNbC group alone, the Au-N-TiNbC-BTO group alone, and the material + ultrasound combined treatment group. Total cell protein was extracted. After protein quantification, electrophoresis, membrane transfer, and blocking, cells were incubated with P62 autophagy marker and Bcl-2 anti-apoptotic protein primary antibody, respectively. β-Tubulin was used as an internal control protein. Chemiluminescence imaging was used to observe the differences in protein expression among the groups.

[0102] Result: As Figure 5 As shown in Figure G, compared with the PBS, BTO, N-TiNbC, and Au-N-TiNbC treatment groups, the expression level of the autophagy marker P62 protein in the Au-N-TiNbC-BTO combined with ultrasound intervention group was significantly downregulated, and the expression of the anti-apoptotic protein Bcl-2 was simultaneously significantly reduced. The above protein quantification results confirm at the molecular protein level that ultrasound can synergistically amplify the autophagy activation and apoptosis induction effects of Au-N-TiNbC-BTO in tumor cells.

[0103] Example 9: In vivo animal-level synergistic anti-tumor therapy experiment To evaluate the in vivo antitumor effect of the Au-N-TiNbC-BTO composite nanomaterial of the present invention, a tumor-bearing mouse model was constructed and systematically evaluated.

[0104] 1. Animal model establishment and experimental grouping Female C57 mice aged 4-6 weeks and weighing 18-22g were selected and subcutaneously injected with 1×10⁻⁶ mmol / L of the drug into the right axilla. 6 One cancer cell. When the tumor volume grew to approximately 100 mm³, the mice were randomly divided into 4 groups of 5 mice each: control group, ultrasound alone group (US), low-dose group of combined material and ultrasound therapy (US + Au-N-TiNbC-BTO: 3 mg / kg), medium-dose group of combined material and ultrasound therapy (US + Au-N-TiNbC-BTO: 6 mg / kg), and high-dose group of combined material and ultrasound therapy (US + Au-N-TiNbC-BTO: 9 mg / kg).

[0105] 2. Administration and Treatment Regimen (1) The Au-N-TiNbC-BTO composite nanomaterial prepared in Example 1 of this invention was administered via tail vein to mice in different dose groups of the material and combined treatment (dosage was 3 mg / kg, 6 mg / kg, and 9 mg / kg), while the control group was given an equal volume of PBS; (2) Twelve hours after administration, the tumor sites of mice in the combined treatment group were subjected to targeted in vitro ultrasound stimulation (ultrasound parameters: power density 0.5W / cm², frequency 50 MHz, duty cycle 30%, duration 5 minutes). (3) Administer medication and perform ultrasound treatment once every 3 days, for a total of 4-5 treatments. During the treatment period, measure the tumor volume every 2 days (formula: tumor volume = long axis × short axis² / 2) and record the patient's weight.

[0106] A schematic diagram of its mechanism of action is shown below. Figure 8 .

[0107] 3. Analysis of tumor growth inhibition effect After treatment, tumor tissues were dissected from each group of mice and photographed. The tumor volume and weight of each group were statistically compared.

[0108] 4. Histopathological analysis Tumor tissues from each group were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (4 µm thick), and then subjected to the following staining analysis: (1) HE staining: The pathological changes of tumor tissue were observed after the sections were stained with hematoxylin and eosin.

[0109] (2) Ki67 immunohistochemical staining: Immunohistochemical staining was performed using anti-Ki67 antibody to assess the proliferative activity of tumor cells.

[0110] 5. Experimental Results (1) Tumor proliferation curve as shown Figure 6 As shown in Figure E, the tumor volume in the control group and the ultrasound-only group increased rapidly. In contrast, the tumor growth in the low, medium, and high dose groups treated with the combination of material and ultrasound was most significantly inhibited, and the tumor volume growth curves were significantly flatter, indicating that the combined treatment regimen had the best tumor-suppressing effect, which was dose-dependent; the tumor-suppressing effect increased with increasing material dosage.

[0111] (2) Tumor images, such as Figure 6 As shown in Figure A, the tumor tissue in the different dose combined treatment groups was significantly smaller than that in the control group and the ultrasound group, showing a significant inhibitory effect in both volume and morphology, with the high-dose group showing the best effect.

[0112] (3) Tumor volume statistics, such as Figure 6As shown in Figure C, the average tumor volume of the different dose combination therapy groups was significantly smaller than that of the control group and the ultrasound-only group, and the difference was statistically significant.

[0113] (4) Tumor quality statistics: After treatment, the weight of tumor tissue in each group was measured. For example... Figure 6 As shown in Figure D, the average tumor mass of the combined treatment groups with different doses was significantly lower than that of the control group and the ultrasound-only group, further quantitatively confirming the superiority of the combined treatment regimen.

[0114] (5) HE staining, such as Figure 6 As shown in the upper part (B), the tumor tissue in the control group exhibits a high density of tumor cells with large, deeply stained nuclei and a high nucleocytoplasmic ratio; while the tumor tissue in the combined treatment group shows large areas of necrosis, a significantly reduced tumor cell density, and clear tissue destruction characteristics. Ki67 immunohistochemical staining is shown below. Figure 6 As shown in the lower half of Figure B, the proportion of Ki67-positive cells in the tumor tissue of the control group was relatively high, indicating that the tumor cells were actively proliferating; while the proportion of Ki67-positive cells in the combined treatment group was significantly reduced, indicating that the material combined with ultrasound treatment effectively inhibited the proliferation of tumor cells.

[0115] Example 10 Safety Assessment Experiment To evaluate the safety of the Au-N-TiNbC-BTO composite nanomaterial of this invention, a systematic assessment was conducted by detecting the proliferation of macrophages (RAW297.4) and venous endothelial cells (HUWEC), performing hemolysis experiments, and staining important mouse organs with hematoxylin and eosin (HE).

[0116] (1) CCK-8 cell proliferation inhibition experiment Cells were seeded at a density of 5 × 10³ cells per well in 96-well plates. Different concentrations (25, 50, 100, 200, 400 μg / ml) of Au-N-TiNbC-BTO composite nanomaterials prepared in Example 1 were added to each well. An equal volume of PBS was added to the control group. 10 µL of CCK-8 reagent was added to each well. The plates were incubated at 37°C in the dark for 1–4 hours. The absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated.

[0117] like Figure 7 As shown in Figure A, compared with the control group, the inhibition rate of different concentrations of the material on macrophages (RAW297.4) and venous endothelial cells (HUWEC) was consistently below 15%, indicating that the material is safe for normal cells.

[0118] (2) Hemolysis test Fresh mouse venous blood was collected, centrifuged at low temperature to remove plasma, and red blood cells were repeatedly washed with pre-cooled physiological saline until the supernatant was colorless, preparing a 2% (v / v) red blood cell suspension for later use. The positive control group (distilled water, completely hemolyzed), the negative control group (physiological saline, no hemolysis), and different concentration gradients (25, 50, 100, 200, 400 μg / ml) of Au-N-TiNbC-BTO material prepared in Example 1 were used. In each system, the red blood cell suspension and material dispersion were mixed in equal volumes and incubated at 37 ℃ for 4 h. After incubation, the supernatant was collected by centrifugation, and the characteristic absorbance of hemoglobin at 540 nm was measured using a UV-Vis spectrophotometer.

[0119] The results are as follows Figure 7 As shown in Figure B, Au-N-TiNbC-BTO did not induce significant red blood cell rupture across the entire concentration range, and the hemolysis rate remained within a safe range. This indicates that the piezoelectric nanomaterial does not cause damage to blood cells and has reliable blood biosafety, making it suitable for intravenous administration in tumor sonodynamic therapy scenarios.

[0120] (3) Safety evaluation experiment in mice Animals and grouping: SPF-grade female C57BL / 6 mice aged 4-6 weeks, weighing 18-22 g, were selected and randomly divided into a control group (PBS) and a material group (Au-N-TiNbC-BTO), with 5 mice in each group.

[0121] Dosage regimen: The material group received a single intravenous injection of Au-N-TiNbC-BTO composite nanomaterials (dose 9 mg / kg) via tail vein, while the control group received an equal volume of PBS. Mice were observed for 21 consecutive days after administration, during which their general condition was monitored.

[0122] Tissue sampling and appearance observation: Mice were sacrificed 21 days after administration, and important organs such as heart, liver, spleen, lung, and kidney were quickly separated. The shape, color, size, and presence of any abnormal lesions of each organ were observed and photographed.

[0123] Histopathological examination: The above-mentioned organ tissues were fixed in 4% paraformaldehyde solution for 48 h, routinely dehydrated, and embedded in paraffin to prepare continuous sections with a thickness of 4 μm. After staining with hematoxylin and eosin (HE), the tissue morphology and structure were observed under an optical microscope to assess the presence of pathological changes such as cell necrosis, inflammatory cell infiltration, edema, or organic damage.

[0124] See the photograph of mouse tissue morphology. Figure 7 In the middle C group, no significant differences in morphology were observed between the material group and the control group. Organ HE section results (see...) Figure 7The results showed that the heart, liver, spleen, lung, and kidney tissues of the control group and the Au-N-TiNbC-BTO group were intact and clear, with regular cell morphology. No obvious pathological changes such as cell necrosis, inflammatory infiltration, tissue edema, or organ damage were observed, proving that the composite nanomaterial has no obvious organ toxicity at therapeutic doses and has good biosafety.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite nanomaterial possessing glucose catalytic consumption capability and ultrasonic piezoelectric response properties, characterized in that, include: N-doped TiNbC nanosubstrate; Au nanoparticles loaded on the surface of the N-doped TiNbC nanosubstrate; BaTiO3 piezoelectric component loaded on the surface of the N-doped TiNbC nanosubstrate; The Au nanoparticles and the BaTiO3 piezoelectric component form a heterojunction structure on the surface of the N-doped TiNbC nanosubstrate; The composite nanomaterial possesses both glucose catalytic activity and ultrasonic piezoelectric response properties.

2. A method for preparing the composite nanomaterial according to claim 1, characterized in that, Includes the following steps: (1) Synthesis of BaTiO3: Barium hydroxide octahydrate was mixed with titanium dioxide, subjected to hydrothermal reaction, washed and dried to obtain BaTiO3 powder; (2) Synthesis of N-TiNbC: TiNbAlC MAX phase powder was added to a mixed etching acid solution composed of hydrofluoric acid and concentrated hydrochloric acid, etched, washed and dried to obtain layered TiNbC, which was mixed with tetramethylammonium hydroxide solution, stirred at room temperature for intercalation and exfoliation, washed and dried to obtain high-purity TiNbC nanosheets, and then the TiNbC nanosheets were mixed with urea, reacted with solvothermal reaction, washed and dried to obtain N-TiNbC powder; (3) Synthesis of Au-N-TiNbC: N-TiNbC powder was dispersed in deionized water, and HAuCl4 solution was added under nitrogen protection. After stirring, ascorbic acid was added for reduction, and Au-N-TiNbC was obtained by centrifugation. (4) Synthesis of Au-N-TiNbC-BTO: BaTiO3 powder was mixed with Au-N-TiNbC suspension and stirred under nitrogen protection. After drying and annealing, Au-N-TiNbC / BTO composite nanomaterials were obtained.

3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of barium hydroxide octahydrate to titanium dioxide is 2.5~3.5:1; the hydrothermal reaction temperature is 180~220℃, and the reaction time is 36~60 hours.

4. The preparation method according to claim 2, characterized in that, In step (2), the etching acid solution is a mixture of 47% to 49% hydrofluoric acid and 11.5 to 12.5M concentrated hydrochloric acid in a volume ratio of 5 to 7:1; the etching reaction temperature is 70 to 90°C and the etching time is 36 to 60 hours.

5. The preparation method according to claim 2, characterized in that, In step (2), the concentration of the tetramethylammonium hydroxide solution is 0.5~2M, and the stirring time is 10~20 hours.

6. The preparation method according to claim 2, characterized in that, In step (2), the mass ratio of TiNbC nanosheets to urea is 1:5 to 1:10; the solvothermal reaction temperature is 160 to 200°C, and the reaction time is 6 to 24 hours.

7. The preparation method according to claim 2, characterized in that, In step (3), the ratio of N-TiNbC to HAuCl4 is 200-400 μL of HAuCl4 solution with a concentration of 50-150 mM for every 50-80 mg N-TiNbC; the reducing agent is ascorbic acid with a concentration of 5-20 mM.

8. The preparation method according to claim 2, characterized in that, In step (4), the concentration of the Au-N-TiNbC suspension is 15-25 mg / mL, and the mass-volume ratio of BaTiO3 powder to Au-N-TiNbC suspension is 0.5-2 mL of Au-N-TiNbC suspension for every 60-100 mg.

9. The use of the composite nanomaterial according to claim 1 or the composite nanomaterial prepared by any one of claims 2-8 in the preparation of a drug for ultrasound-assisted tumor treatment.

10. The application according to claim 9, characterized in that, The tumor is oral cancer.

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