Strontium titanate nanoparticles, composite scaffold, preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine, and specifically to strontium titanate nanoparticles, composite scaffolds, and their preparation methods and applications. The strontium titanate nanoparticles include mesoporous piezoelectric SrTiO3 nanoparticles loaded with epigallocatechin gallate. The nanoparticles described herein can be used in PANoptosis-mediated ultrasound immunotherapy, achieving specificity for PANoptosis within tumors, improving the efficiency of sonochemical reactions and ROS generation, enhancing tumor cell sensitization, and enhancing the efficiency of PANoptosis activation.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to strontium titanate nanoparticles, a composite stent, and a preparation method and application thereof. Background Art

[0002] PANoptosis, a form of programmed cell death encompassing pyroptosis, apoptosis, and necroptosis, is being recognized as a revolutionary pillar of cancer therapy and is receiving increasing attention. The integration of multiple programmed cell death (PCD) pathways can directly damage tumor cells and release danger-associated molecular patterns (DAMPs), triggering immunogenic cell death (ICD) and activating a robust and sustained tumor immune response, demonstrating the potential of PANoptosis to enhance anti-tumor immunity. However, currently available PANoptosis therapies are still limited by the major drawbacks of uncontrolled inflammatory responses and systemic toxicity.

[0003] Ultrasound (US), a typical exogenous energy source, can activate sonosensitizers to generate reactive oxygen species (ROS), also known as the sonodynamic effect, allowing for spatiotemporally and temporally controlled ROS generation. The generated ROS leads to phosphorylation of the mixed lineage kinase domain-like (MLKL) protein (p-MLKL) and expression of caspase family proteins, effectively inducing necroptosis, apoptosis, and / or pyroptosis in cancer cells. Therefore, US-mediated on-demand accumulation of ROS can serve as an exogenous input signal for in situ activation of panoptosis within tumors. Piezoelectric materials, capable of generating internal electric fields under mechanical force, have emerged as emerging sonosensitizers capable of generating ROS under US irradiation. However, the limited ROS quantum yield of existing piezoelectric materials has hindered their widespread application in remote clinical settings. Although various engineering approaches, such as heterostructure construction, oxygen vacancy introduction, and metal ion doping, have been developed to enhance ROS generation, their mass production and widespread application remain limited by complex manufacturing processes.

[0004] In addition to ultrasound-mediated ROS generation, the sensitivity of tumor cells to oxidative stress-induced PANoptosis also significantly affects the efficiency of ultrasound-mediated PANoptosis. Notably, PANoptosis has key features of pyroptosis, apoptosis, and necroptosis, but cannot be attributed solely to any one of these processes. In particular, as an inflammatory PCD, pyroptosis plays an irreplaceable role in PANoptosis, stimulating strong and sustained anti-tumor immunity. Although studies have confirmed that ROS can induce pyroptosis in cancer cells, the silencing state of the gas conductor (GSDM) family of proteins in tumors significantly limits their activation efficiency. Summary of the Invention

[0005] The present invention aims to provide strontium titanate nanoparticles, composite scaffolds, and preparation methods and applications thereof for PANoptosis-mediated ultrasound immunotherapy, thereby achieving the specificity of PANoptosis occurring in tumors, improving the efficiency of sonochemical reactions and ROS generation, increasing the sensitization of tumor cells, and enhancing the efficiency of PANoptosis activation.

[0006] To achieve the above object, the present invention adopts the following technical solution: a strontium titanate nanoparticle, including mesoporous piezoelectric SrTiO3 nanoparticles, and the mesoporous piezoelectric SrTiO3 nanoparticles are loaded with epigallocatechin gallate.

[0007] The principles and advantages of the present application are as follows: In the present application, by loading epigallocatechin gallate on mesoporous piezoelectric SrTiO3 nanoparticles, a PANoptosis activation strategy driven by an "external ultrasonic piezoelectric and internal epigenetic regulation" logic gate is designed to perform ultrasound immunotherapy of tumors by synergistically inducing apoptosis, necroptosis and inflammatory necrosis of tumor cells. Specifically, by utilizing the high acoustic dynamic efficiency of mesoporous piezoelectric SrTiO3 nanoparticles and using ultrasonic irradiation as the "external" input signal, the mesoporous piezoelectric SrTiO3 nanoparticles are activated to generate a large amount of ROS, thereby improving the efficiency of sonochemical reactions and ROS generation. In addition, the epigallocatechin gallate loaded on the mesoporous piezoelectric SrTiO3 nanoparticles acts as an "internal" switch to alleviate DNA methylation, improve the sensitization of tumor cells and enhance the efficiency of PANoptosis activation, further improving the pyroptosis of cancer cells. Therefore, the "external and internal" logic gates in the present application can be specifically activated to achieve piezoelectric PANoptosis.

[0008] Therefore, the strontium titanate nanoparticles in this application have the function of exogenously / endogenously activating PANoptosis and can be used for the treatment of tumors.

[0009] Preferably, as an improvement, the pore size of the mesoporous piezoelectric SrTiO3 nanoparticles is 11-13 nanometers. In the prior art, mesoporous piezoelectric SrTiO3 nanoparticles are mostly used in the field of catalysis, but not in the field of biomedicine. The main reason is that the pore size of the mesoporous piezoelectric SrTiO3 nanoparticles is too large. However, through the preparation method of the present application, the pore size of the mesoporous piezoelectric SrTiO3 nanoparticles in the present application is 11-13 nanometers, making the pore size of the mesoporous piezoelectric SrTiO3 nanoparticles larger and capable of being used in the field of biomedicine. The pore size of the mesoporous piezoelectric SrTiO3 nanoparticles is set to the above range, which is conducive to improving the ultrasonic piezoelectric efficiency.

[0010] Preferably, as an improvement, the specific surface area of ​​the mesoporous piezoelectric SrTiO3 nanoparticles is 34-37 m² / g. The inventors have discovered that increasing the specific surface area of ​​the mesoporous piezoelectric SrTiO3 nanoparticles promotes the active sites of the piezoelectric material under ultrasonic irradiation, thereby improving the efficiency of sonochemical reactions and ROS generation. Therefore, the specific surface area of ​​the mesoporous piezoelectric SrTiO3 nanoparticles is 34-37 m² / g, which is beneficial for improving the efficiency of sonochemical reactions and ROS generation.

[0011] Preferably, as an improvement, the loading amount of epigallocatechin gallate is 75-77%. Thus, the loading amount of epigallocatechin gallate on the nanoparticles is high, and the mesoporous piezoelectric SrTiO3 nanoparticles have a good encapsulation capacity for epigallocatechin gallate.

[0012] To achieve the above object, the present invention also adopts the following technical solution: a composite scaffold comprising a biological scaffold and any one of the above-mentioned strontium titanate nanoparticles hybridized on the biological scaffold.

[0013] Thus, a composite scaffold is obtained by hybridizing the biological scaffold and the strontium titanate nanoparticles of the present application. The composite scaffold of the present application can be used for tumor treatment, such as osteosarcoma.

[0014] In the current state of the art, simply eliminating malignant tissue is insufficient to achieve comprehensive treatment goals in the clinical management of this aggressive tumor. Infection is a significant complication during surgical procedures for implantation of implants, particularly in the setting of deep-seated tumors such as osteosarcoma. Infections caused by drug-resistant bacteria, which are persistently resistant to conventional antibiotics, further complicate clinical management. Therefore, the development of biomaterials with both antimicrobial and osteogenic properties, in addition to tumor-killing capabilities, is of great significance in the field of orthopedics.

[0015] This application provides a composite scaffold that can not only be used for PANoptosis immunotherapy of osteosarcoma, but also utilizes nanoparticles to generate abundant ROS, which can disrupt the selective permeability of bacterial membranes, effectively killing bacteria and significantly reducing the incidence of drug-resistant bacteria after osteosarcoma scaffold implantation. Furthermore, the bioactivity of the composite scaffold and the Sr element can promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) after implantation of the composite scaffold in bone defects, thereby enhancing bone regeneration. Therefore, the composite scaffold described in this application is not only suitable for osteosarcoma immunotherapy but also possesses satisfactory antibacterial and osteogenic properties, facilitating bone regeneration associated with bone tumors.

[0016] Preferably, as an improvement, the biological scaffold is a bioactive glass scaffold.

[0017] To achieve the above object, the present invention also adopts the following technical solution: a method for preparing a composite scaffold, wherein the biological scaffold is immersed in acidic anhydrous ethanol in a strontium titanate nanoparticle solution, then taken out and air-dried at room temperature, and the process is repeated multiple times.

[0018] To achieve the above object, the present invention also adopts the following technical solution: a method for preparing strontium titanate nanoparticles, comprising the following steps:

[0019] S1. dissolving tetrabutyl titanate in ethanol to form a mixed solution; then, adding an ammonia solution to the mixed solution to obtain a precipitated hydroxide; filtering and washing the hydroxide multiple times with distilled water;

[0020] S2. Then, Sr(NO)2, KOH microspheres and PVA solution were added, stirred vigorously and dispersed in distilled water to form a suspension;

[0021] S3, transferring the suspension to an autoclave and heating it to 200°C for 20 hours, and then cooling it back to room temperature; filtering the obtained product, and washing it with dilute nitric acid and distilled water, thereby preparing mesoporous piezoelectric SrTiO3 nanoparticles;

[0022] S4. Mixing the mesoporous piezoelectric SrTiO3 nanoparticles and epigallocatechin gallate in an aqueous solution and dispersing them in acidic deionized water; stirring the mixture for 24 hours; and then purifying the obtained mixture with acidic deionized water and dispersing it in acidic deionized water.

[0023] Thus, the above-described preparation method achieves the preparation of strontium titanate nanoparticles. By introducing PVA and extending the hydrothermal treatment time (specifically, to 20 hours), this preparation method can produce nanoparticles with larger pore sizes. These nanoparticles can be used in the biomedical field and help improve ultrasonic piezoelectric efficiency.

[0024] To achieve the above-mentioned purpose, the present invention also adopts the following technical solution: application of the composite scaffold as an osteosarcoma transplant.

[0025] To achieve the above-mentioned purpose, the present invention also adopts the following technical solution: application of strontium titanate nanoparticles as piezoelectric acoustic sensor.

[0026] To achieve the above-mentioned purpose, the present invention also adopts the following technical solution: application of strontium titanate nanoparticles as PANoptosis-mediated ultrasound immunotherapy drugs.

[0027] Preferably, as an improvement, strontium titanate nanoparticles are used as PANoptosis immunotherapy drugs for osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1a is the transmission electron microscopy (TEM) image of MeST nanoparticles, the scale bar is 200 nm.

[0029] Figure 1 b Elemental mapping of MeST nanoparticles, the scale bar is 200 nm.

[0030] Figure 2 Transmission electron microscopy images of NpST, MiST, and MeST NPs. Scale bar: 50 nm.

[0031] Figure 3 A shows the particle size of MeST in DLS; Figure 3 B shows the hydrated particle size of MeST dissolved in PBS over 7 days. n = 3.

[0032] Figure 4 Schematic diagram of the PMF characterization and amplitude butterfly loop of MeST nanoparticles, showing the phase and displacement responses.

[0033] Figure 5 Schematic diagram of the phase lag loop and amplitude butterfly loop of MeST, MiST and NpST under piezoelectric force microscopy.

[0034] Figure 6 The MeST, MiST, and NpST impedance curves are shown.

[0035] Figure 7 Schematic diagram of the acoustoelectric effect of ST nanoparticles.

[0036] Figure 8 i is the electron spin resonance (ESR) curve, which detects the piezoelectric effect. 1 O2.

[0037] Figure 8 j is the electron spin resonance (ESR) curve, which detects ·OH generated by the piezoelectric effect.

[0038] Figure 8 k shows the time-dependent consumption of DPBF by ST nanoparticles under ultrasound irradiation.

[0039] Figure 8 l Schematic diagram of the time-dependent consumption of MB by ST nanoparticles under ultrasound irradiation.

[0040] Figure 9 The time-dependent depletion of DPBF is shown, indicating that the depletion of DPBF by H2O, NpST, MiST, and MeST under US irradiation is induced. 1 O2 is generated.

[0041] Figure 10Schematic diagram of the temporal reduction of MB induced by H2O, NpST, MiST, and MeST under US irradiation, indicating the generation of ·OH.

[0042] Figure 11 The absorbance changes of EGCG characteristic peaks at different pH levels and time intervals are shown.

[0043] Figure 12 Schematic diagram of CLSM imaging showing ROS generation on BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US scaffolds, and the performance of MNNG / HOS Cl cells. Scale bar is 400 μm.

[0044] Figure 13 Figure 1. Schematic diagram of ROS production and FCM quantification in MNNG / HOS Cl cells treated with A (BG), B (BG + US), C (STE-BG), D (ST-BG + US), and E (STE-BG + US). N = 3.

[0045] Figure 14 h shows the FCM determination of apoptosis levels and quantitative data of MNNG / HOS Cl cells treated with BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US.

[0046] Figure 14 i Schematic diagram of the changes in mitochondrial membrane potential of MNNG / HOSCl cells treated with BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US observed by FCM.

[0047] Figure 15 CLSM images of MNNG / HOS Cl cells stained with JC-1 monomer (green channel) on scaffolds treated with BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US. Scale bar: 400 μm.

[0048] Figure 16 The expression of total MLKL and MLKL (phosphorylated) proteins in MNNG / HOS Cl cells after treatment with A (BG), B (BG + US), C (STE-BG), D (ST-BG + US), and E (STE-BG + US) are shown.

[0049] Figure 17 The figure shows the FCM analysis of the necroptosis levels of MNNG / HOSCl cells treated with BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US.

[0050] Figure 18 k Schematic diagram of CLSM imaging showing immunofluorescence staining of 5-hmC in MNNG / HOS Cl cells treated with BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US. Scale bar: 400 µm.

[0051] Figure 18 l Schematic representation of the differential regulation of cytochrome C (cyto C) expression in the BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US groups, as shown by immunofluorescence staining. Scale bar, 400 µm.

[0052] Figure 19 e shows CLSM images of MNNG / HOS Cl cells treated with different scaffolds and stained with Calcein-AM (green channel) and PI (red channel). The scale bar is 400 μm.

[0053] Figure 19 g shows the CRT (green channel) in the scaffolds of the BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US groups as shown by CLSM imaging. The scale bar is 400 µm.

[0054] Figure 20 i is a schematic diagram of the Transwell assay protocol. The diagram is from BioRender.com.

[0055] Figure 20 j Schematic diagram of the secretion of IFN-γ inflammatory factors in the BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US groups after different treatments.

[0056] Figure 20 k shows the secretion of IL-6 inflammatory factor in BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US groups after different treatments.

[0057] Figure 20 l Schematic diagram of the secretion of TNF-α inflammatory factor in BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US groups after different treatments.

[0058] Figure 20 m shows the ratio of mature dendritic cells (CD80+CD86+ in CD11c+ cells) in FCM analysis and Figure 20Quantitative analysis of each group (n = 3). Statistical significance was calculated by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0059] Figure 21 a Schematic diagram of the in vivo treatment protocol.

[0060] Figure 21 b Schematic diagram of the average body weight (n=5) of tumor-bearing mice in different groups (BG, BG+US, STE-BG, ST-BG+US, STE-BG+US).

[0061] Figure 21 c Tumor weight is shown.

[0062] Figure 21 d Relative tumor volume (RTV) is shown.

[0063] Figure 21 e shows the tumor inhibition rate (n = 5) of BG, BG + US, STE-BG, ST-BG + US, and STE-BG + US treatments, the results after 14 days of treatment.

[0064] Figure 21 f shows H&E, TUNEL, and PCNA staining images of tumors in mice in the BG, BG+US, STE-BG, ST-BG+US, and STE-BG+US groups. Scale bar represents 50 μm.

[0065] Figure 21 g shows the changes in tumor volume of the corresponding groups during treatment and the photos of tumors in different treatment groups (n=5).

[0066] Figure 21 h Schematic diagram of mature dendritic cells (ratio of CD80+ CD86+ to CD11c+ cells) in different groups in FCM analysis (n=3).

[0067] Figure 21 i Schematic representation of representative FCM images showing the distribution of CD8+ T cells among CD3+ cells in tumors and the corresponding quantitative analysis (n=3).

[0068] Figure 21 j shows representative FCM images showing the distribution of CD45+ T cells in tumors and corresponding quantitative analysis (n=3).

[0069] Figure 22The immunofluorescence staining of cleaved caspase 3, CRT, and HMGB1 in tumors of different treatment groups (BG, BG + US, STE-BG, ST-BG + US, and STE-BG + US) is shown. The scale bar is 50 μm.

[0070] Figure 23 Schematic diagram of the flow cytometry gating strategy. FCM gating strategy for different mature DCs populations.

[0071] Figure 24 is the proportion of mature DCs in each group (CD11c + CD80 in cells + CD86 + ) corresponds to quantitative analysis (n = 3).

[0072] Figure 25 a Representative images of the spread of MRSA colonies on agar plates after treatment in different experimental groups.

[0073] Figure 25 b Fluorescence microscopy images of MRSA cells stained with NucGreen (green channel) and EthD-III (red channel) after treatment with Control, Control + US, STE, and STE + US. Scale bar: 100 μm.

[0074] Figure 25 c Schematic diagram showing the wound healing process of mice from different experimental groups.

[0075] Figure 25 d shows H&E, Masson's, and CD31 immunohistochemical staining images of skin tissue at the wound site in different mouse groups. Scale bars: 600 μm, 80 μm, 1000 μm, 80 μm, and 50 μm.

[0076] Figure 25 e is the Alizarin red staining image of BMSCs in different groups after 21 days / 14 days of culture.

[0077] Figure 25 f: ALP staining images of BMSCs in different groups after 21 days and 14 days of culture. Scale bar: 5 mm.

[0078] Figure 25 g: Fluorescence staining image of the cytoskeleton of BMSCs adhered to the STE-BG scaffold. Scale bar: 50 μm.

[0079] Figure 25 h Schematic diagram of the cell viability of BMSCs after incubation with BG and STE-BG scaffolds.

[0080] Figure 25 i Schematic diagram of the qPCR analysis of osteogenic gene expression (ALP, OSTERIX, OPG, RUNX-2) of BMSCs in different groups (control, BG, STE-BG) after 7 days of in vitro culture.

[0081] Figure 25 j shows the ALP OD values ​​of the control group, BG group, and STE-BG group measured by UV-Vis spectroscopy.

[0082] Figure 25 k is a simplified schematic diagram of the brain defect model. The defect area was implanted with a STE-BG scaffold (right) and a control group (left) for comparison.

[0083] Figure 25 l is a Micro-CT image of SD rats after implantation at 4 and 12 weeks. The scale bar is 2.5 mm.

[0084] Figure 25 m shows the expression of BMCs in different groups (control, STE-BG) at different time points (4 weeks and 12 weeks).

[0085] Figure 25 nShows the expression of BMD in different groups (control, STE-BG) at different time points (4 weeks and 12 weeks).

[0086] Figure 26 a Schematic diagram of H&E and Masson staining of the skull defect area in each group at 12 weeks (control group and STE-BG). Scale bar, 200 μm.

[0087] Figure 26 b: MicroCT imaging of the skull after STE-BG stent implantation. Scale bar: 2 mm. DETAILED DESCRIPTION

[0088] The following is further described in detail through specific implementation methods:

[0089] This example discloses strontium titanate nanoparticles, including mesoporous piezoelectric SrTiO3 nanoparticles (MeSTNPs), loaded with epigallocatechin gallate (EGCG). The pore size of the mesoporous piezoelectric SrTiO3 nanoparticles is 11-13 nanometers, specifically 12 nanometers. The specific surface area of ​​the mesoporous piezoelectric SrTiO3 nanoparticles is 34-37 m² / g, specifically 35.8 m² / g. The epigallocatechin gallate loading in this example is 75-77%, specifically 76.10%.

[0090] This embodiment also discloses a method for preparing strontium titanate nanoparticles (denoted as STE), comprising the following steps:

[0091] S1. Dissolve 1.94 mL of tetrabutyl titanate in 50 mL of ethanol to form a mixed solution; then, add 0.15 M ammonia solution (0.2625 g in 0.288 mL of aqueous ammonia) to the mixed solution to obtain a precipitated hydroxide; filter and wash the hydroxide six times with distilled water;

[0092] S2. Then, Sr(NO)2 (3.17 g), KOH microspheres (1.4 g), and PVA solution (0.2 g) were added to the hydroxide, and then vigorously stirred and dispersed in 50 mL of distilled water to form a suspension. In the final suspension state, the Ti concentration was 0.1 M, the Sr / Ti ratio was 3:1, and the PVA concentration was 4 g / L;

[0093] S3, transferring the suspension into a 50 mL stainless steel polytetrafluoroethylene-lined autoclave and heating it to 200°C for 20 h, and then cooling it back to room temperature; filtering the obtained product, and washing it with dilute nitric acid and distilled water, thereby preparing mesoporous piezoelectric SrTiO3 nanoparticles;

[0094] S4. Mixing mesoporous piezoelectric SrTiO3 nanoparticles and epigallocatechin gallate in an aqueous solution and dispersing them in acidic deionized water; gently stirring the mixture without any light interference for 24 hours; then, purifying the obtained mixture with acidic deionized water and dispersing it in acidic deionized water for further use.

[0095] In the above preparation method, the hydrothermal reaction time in S3 is 20 hours, thus preparing mesoporous piezoelectric SrTiO3 nanoparticles. When it is necessary to prepare microporous piezoelectric SrTiO3 nanoparticles (MiST), the above preparation method can be used to shorten the hydrothermal reaction time to 6 hours. The prepared microporous piezoelectric SrTiO3 nanoparticles have a pore size of approximately 3 nanometers and a specific surface area of ​​11.2 m² / g. When it is necessary to prepare non-porous piezoelectric SrTiO3 nanoparticles (NpST), it is sufficient to omit the PVA solution in the above preparation method of MeST NPs. The specific surface area of ​​the prepared NpST is 6.6 m² / g.

[0096] This embodiment also discloses a composite scaffold comprising a biological scaffold (specifically, a bioactive glass scaffold (BG scaffold)) and strontium titanate nanoparticles hybridized on the biological scaffold. Specifically, the BG scaffold is immersed in an acidic anhydrous ethanol solution containing strontium titanate nanoparticles (400 ppm) for 15 minutes, followed by air drying at room temperature. This step is repeated three times to obtain the final composite scaffold (STE-BG scaffold). This composite scaffold can be used as a composite scaffold for osteosarcoma treatment. Furthermore, the ST-BG scaffold described below is prepared by immersing the BG scaffold in an acidic anhydrous ethanol solution containing MeST (400 ppm) for 15 minutes, followed by air drying at room temperature. This step is repeated three times to obtain the final ST-BG scaffold.

[0097] The strontium titanate nanoparticles in this embodiment can be used as piezoelectric acoustic sensors in practical applications.

[0098] In practical applications, the strontium titanate nanoparticles in this embodiment can be used as PANoptosis-mediated ultrasound immunotherapy drugs, specifically as PANoptosis immunotherapy drugs for osteosarcoma.

[0099] The properties, effects, and application effects of the strontium titanate nanoparticles in this application are demonstrated and explained through a series of experiments.

[0100] 1. Study on the properties of SrTiO3 nanoparticles

[0101] Combine Figure 1 As shown in Figure a, it can be seen from the transmission electron microscope (TEM) image that the MeST formed in this embodiment has a uniform spherical morphology with a diameter of about 200 nm, and the pore size gradually increases from NpST to MeST (as shown in Figure 4). Figure 2 In addition, combined with Figure 1 As shown in Figure b, through elemental mapping, MeST contains strontium, titanium and oxygen, which shows that the SrTiO3 nanoparticles were successfully prepared. Dynamic light scattering (DLS) measurement showed that the average particle sizes of NpST, MiST and MeST were 216.9 nm, 218.7 nm and 218.0 nm, respectively, indicating that the pore size changes of the three particles have almost no effect on the particle size of ST NPs. Figure 3As shown, MeST showed minimal change in hydrodynamic diameter after 7 days of dispersion in phosphate-buffered saline (PBS), demonstrating its excellent long-term stability. The pore size of ST NPs was evaluated using the Barrett-Joyner-Halenda (BJH) model, revealing that MeST exhibits a relatively large mesopore size of approximately 12 nanometers, four times the pore size of MiST (3 nanometers). The Brunauer-Emmett-Teller (BET) specific surface area of ​​MeST (35.8 m² / g) is also higher than that of MiST (11.2 m² / g) and NpST (6.6 m² / g).

[0102] Considering that phase / crystal structure is an important determinant of piezoelectric performance, the inventors used piezoelectric force microscopy (PFM) to evaluate the characteristics of three samples: MeST, MiST, and NpST. Figure 4 and Figure 5 As shown in Figure 2, the phase image of MeST shows a clear phase difference, and the image contrast is opposite to the amplitude signal. It is worth noting that the hysteresis amplitude-voltage curve of MeST presents a butterfly-shaped ring, which is consistent with the typical characteristics of piezoelectric materials. In addition, combined with Figure 6 As shown in the figure, MeST exhibits a slightly smaller radius in the electrochemical impedance curve compared to the other two samples, indicating that it has a faster electron-hole pair separation rate and exhibits more superior piezoelectric performance.

[0103] Band gap is a key parameter for evaluating the ability of piezoelectric photosensitive materials to generate ROS. According to the Kubelka-Munk transformation results, the band gap of MeST is calculated to be 3.02 eV. At the same time, its valence band position is measured by XPS analysis to be approximately 2.26 eV, which shows a more positive potential than the water / ·OH oxygen reduction potential (2.10 eV vs RHE). Based on the difference between the band gap and the valence band, the conduction band position of MeST is determined to be -0.76 eV, which is more negative than the O2 / ·O2- oxygen reduction potential (-0.33 eV vs RHE). Mechanistically, as Figure 7 As shown in Figure 1, piezoelectric materials are polarized under external mechanical stimulation of ultrasound at a specific frequency, forming an intrinsic electric field. Under the guidance of the continuously updated built-in electric field, the separated electrons (e-) and holes (h+) migrate to opposite surfaces respectively. It is worth noting that the surface redox reaction of STNPs is conducive to the generation of OH and O2- (which can be further oxidized by holes to 1 O2), ultimately ensuring the ultrasonic piezoelectric effect of MeST.

[0104] The classical electron spin resonance (ESR) experiment was conducted using 2,2,6,6-tetramethylpiperidine (TEMP) as 1 The generation of ROS was studied by using O2 species capture agent. Figure 8 As shown in i, MeST showed the strongest triplet signal compared with MiST and NpST groups, with an intensity ratio of 1:1:1. 1 O2 production was also evaluated using 1,3-diphenylisobenzofuran (DPBF) (the specific evaluation method was as follows: 100 µL of DPBF (1 mg / mL, DMSO) was mixed with 2900 µL of sample aqueous suspension (diluted to 100 μg / mL). The mixture was then exposed to ultrasonic (US) irradiation (1 MHz, 1.0 W / cm2, 50% duty cycle, 5 minutes, US irradiation for 1 minute, followed by a 30-second pause). The change in DPBF concentration was calculated based on the absorption intensity at 416 nm in the UV-vis absorption spectrum). The time series curves showed that in MeST+US, the combination of Figure 8 k and Figure 9 As shown in Figure 2, the characteristic absorption peak of DPBF at 416 nm decreased significantly with the extension of ultrasound irradiation time, and the decrease was most obvious in the MeST+US group. Figure 8 As shown in Figure j, a quadruple signal was also observed in the ESR (with an intensity ratio of 1:2:2:1), proving the generation of ·OH during ultrasound stimulation. In addition, the generation of ·OH was verified using methylene blue (MB) (the specific detection method is: using methylene blue (MB) as a probe, the formation of ·OH is detected by oxidizing MB to colorless MB-OH. To detect the generation of ·OH, 5 uL of 1 mg / mL MB was added to 3 mL of sample (100 μg / mL), and then US irradiation was performed in the dark for 0-5 minutes, and then the concentration of MB in the solution was measured using UV-vis). Figure 8 l and Figure 10 As shown, the MB absorption peak at 665 nm in MeST decreases significantly with increasing irradiation time, with the most pronounced decrease in the MeST+US group. Quantitative analysis indicates a decrease of approximately 70% compared to the initial value. Together, these data confirm that increasing the pore size of MeST NPs significantly improves their ultrasonic piezoelectric efficiency compared to MiST and NpST.

[0105] 2. Study on the properties of EGCG

[0106] The stability of EGCG under different pH conditions was studied by UV-visible spectroscopy. Figure 11 As shown in the results, when exposed to systems with pH values ​​of 7 and 9, EGCG degraded by approximately 38.92% and 69.82% within 24 hours, respectively, while it remained stable in acetate buffer (pH = 5) without significant content changes. Therefore, the good stability of EGCG in acidic environments can serve as an acid-responsive "logic gate" in the tumor microenvironment.

[0107] The inventors also investigated the EGCG loading capacity of different ST NPs. UV-vis calculations revealed that the EGCG loading in NpST, MiST, and MeST was approximately 9.90%, 26.05%, and 76.10%, respectively. Therefore, MeST, due to its superior drug loading capacity, was further used to encapsulate EGCG to form STEs. Furthermore, ultrasound irradiation of the nanoparticles increased the EGCG release rate to approximately 77.8% within 24 hours, attributed to the mechanical or cavitation effect induced by ultrasound, which facilitated the dissociation of EGCG from MeST.

[0108] 3. Study on the activation of PANoptosis by STE-BG scaffold in vitro

[0109] From a logic gate perspective, the nanoparticles of the present application use ultrasound irradiation as an "external" input signal to generate ROS, while the bioactive drug EGCG acts as an "internal" input signal to achieve epigenetic demethylation regulation, thereby strongly triggering tumor PANoptosis.

[0110] In this study, MNNG / HOS Cl cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. They were then maintained in a humidified incubator at 37°C with 5% CO₂. Subsequently, a predetermined number of cells were seeded onto BG / ST-BG / STE-BG scaffolds (Ø8 mm x 2 mm) in 48-well plates and incubated for 24 hours until fully attached and proliferated. Subsequently, cells were treated with US (1 W / cm², 5 minutes) or not.

[0111] The inventors evaluated five groups for flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) analysis: A: BG, B: BG + US, C: STE-BG, D: ST-BG + US, and E: STE-BG + US. For FCM analysis, cells on the scaffolds were detached with trypsin and stained appropriately before FCM. During CLSM analysis, cells on the scaffolds were washed multiple times with PBS to achieve adequate staining. The old culture medium was discarded, and the scaffolds were transferred to a dedicated culture medium for CLSM observation and documentation. Live cells were stained with Calcein-AM (green fluorescence), and dead cells were stained with PI (red fluorescence).

[0112] The inventors used 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) probe to evaluate the generation of intracellular ROS. Confocal laser scanning microscopy (CLSM) images showed that under ultrasound stimulation, osteosarcoma cells seeded on ST-BG / STE-BG scaffolds emitted strong green fluorescence, indicating that a large amount of ROS was generated ( Figure 12At the same time, due to the superior piezoelectric properties of MeST, the fluorescence intensity of the ST-BG+US and STE-BG+US groups shown by flow cytometry (FCM) was significantly higher than that of the other groups ( Figure 13 shown).

[0113] The continuous generation and accumulation of intracellular ROS may lead to the deterioration of mitochondrial function, thereby promoting the widespread activation of apoptosis. The apoptosis rate detected by FCM showed corresponding results ( Figure 14 h). In addition, the difference between ST-BG and STE-BG in the figure is attributed to the enhanced pro-apoptotic efficiency of EGCG. The mitochondrial membrane potential of the different treatment groups was evaluated using a mitochondrial membrane potential detection kit (JC-1). FCM results confirmed that the proportion of mitochondrial membrane negative potential was higher in the STE-BG plus ultrasound irradiation group (54.07±0.58%) ( Figure 14 i). In addition, JC-1 staining images showed that the combination Figure 15 As shown in the figure, the cells on the scaffolds treated with STE-BG+US or ST-BG+US showed more obvious green fluorescence compared with the control group, indicating that the ultrasound-piezoelectric effect had a significant damaging effect on mitochondria.

[0114] Recent studies have revealed that excessive production of intracellular ROS may trigger PANoptosis, a process of cell necroptosis, in which the key executor, PANoptosome, releases damage-associated complexes to promote the expression of p-MLKL. To this end, under ultrasound irradiation in the ST-BG / STE-BG group, upregulation of phosphorylated MLKL protein was detected by Western blot, while the total MLKL protein level did not change significantly ( Figure 16 To further demonstrate the ROS-induced necroptosis of osteosarcoma cells triggered by ultrasound-piezoelectric effect, the inventors performed YO-PRO-1 (YP-1) / PI staining, an indicator of necroptotic cells, and examined the necroptosis status using FCM ( Figure 17 The results showed that PI and YP-1 positive necrotic cells were almost absent in the BG, BG+US, and STE-BG groups, while those in the ST-BG+US and STE-BG+US groups were 28.27% and 30.11%, respectively.

[0115] Inhibiting GSDME gene methylation with DNMT inhibitors can remove hypermethylation, thereby triggering the pyroptosis pathway. As a key mediator of pyroptosis, GSDME expression is significantly lower in various tumor cells than in normal cells, primarily due to the promotion of methylation. Furthermore, as an "internal" input signal for PANoptosis activation, EGCG released by STEs is expected to upregulate GSDME expression, a necessary prerequisite for pyroptosis.

[0116] To this end, the inventors further used immunofluorescence (IF) staining to detect the intracellular 5-hydroxymethylcytosine (5hmC) level, which is a downstream product of GSDME demethylation transcription. From the captured images, obvious green fluorescence (5hmC) was observed only in the STE-BG and STE-BG+US samples ( Figure 18 k), combined with Figure 18 l, Western blot experiments also showed the same trend, thus verifying that the loaded EGCG alleviated the methylation status of GSDME and then upregulated the transcription and expression of GSDME.

[0117] These results indicate that the combined effect of STE-BG and ultrasound can activate PANoptosis in osteosarcoma cells by upregulating the expression of demethylated genes, providing a potential target for subsequent tumor treatment.

[0118] IV. Study on the Anti-tumor Effect and Immune Response of PANoptosis Induced by STE Embedded Scaffolds

[0119] Based on the mechanism by which STE-embedded scaffolds activate PANoptosis, the inventors further evaluated its anti-tumor effects and immune responses in vitro. Specifically, activation of the "inside-outside" logic gate triggered PANoptosis, disrupting the cell barrier and releasing DAMPs, thereby initiating an immune response, promoting the maturation of dendritic cells (DCs), and enhancing anti-tumor immunity. Therefore, cell-based experiments were conducted to explore the feasibility of this "logic gate" in PANoptosis activation and tumor cell death.

[0120] First, osteosarcoma cells (MNNG / HOS Cl cells) were seeded on the STE-BG scaffold to evaluate cell compatibility and therapeutic potential. CCK8 assay (cell counting kit) showed that the cell viability of the STE-BG scaffold was not affected after 24 hours of co-culture with osteosarcoma cells. In contrast, after ultrasound irradiation, the cell mortality rate of the STE-BG scaffold exceeded 90%. In addition, MNNG / HOS Cl cells were stained with Calcein-AM and propidium iodide (PI) to visually observe the anti-tumor effect. Figure 19 As shown in Figure e, the BG, BG+US, and STE-BG groups mainly showed green spots, while the ST-BG and STE-BG groups clearly showed red fluorescence under ultrasound irradiation, proving that ultrasound stimulated the anti-tumor effect of PANoptosis treatment.

[0121] PANoptosis is characterized by cell membrane rupture, which can release a large number of pro-inflammatory molecules to reshape the microenvironment and activate the immune response. The inventors detected the concentration of adenosine triphosphate (ATP) in cells after different treatments. In the samples treated with STE-BG+US, the intracellular ATP level was approximately 70% of that in the control group. Calreticulin (CRT) is a protein located in the endoplasmic reticulum and is exposed to the cell membrane surface in the early stages of immune cell death (ICD). CLSM images showed that under ultrasound irradiation, bright green fluorescence ( Figure 19 g), demonstrating that PANoptosis treatment induced CRT translocation. Similarly, the content of high-mobility group protein 1 (HMGB1) in the cell supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Quantitative results showed that secretion levels in the STE-BG + US group were significantly increased, 2.02-fold and 1.32-fold higher than those in the control and ST-BG + US groups, respectively. These studies suggest that activation of PANoptosis contributes to the generation of DAMPs, creating an inflammatory immune microenvironment and thereby enhancing the efficacy of ultrasound immunotherapy.

[0122] In addition, the ability of STE-BG+US in inducing DCs maturation was investigated ( Figure 20 i) MNNG / HOS Cl cells were seeded on scaffolds (Φ8 mm × 2 mm) of different groups (BG, BG+US, STE-BG, ST-BG+US, STE-BG+US) and placed on the upper layer of the transwell plate co-culture system. After corresponding US or non-US stimulation, MNNG / HOS Cl cells were co-cultured with immature dendritic cells in the lower layer for 24 hours. Dendritic cells were stained with anti-CD45+-PerCP, anti-CD11c-+FITC, anti-CD80+-PE, and anti-CD86+-APC antibodies and analyzed by FCM. In addition, the concentrations of IFN-γ, TNF-α, and IL-6 in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The results showed that after STE-BG treatment, ultrasound irradiation significantly increased the levels of proinflammatory cytokines (IFN-γ, IL-6, and TNF-α) ( Figure 20 j-20l). Combined Figure 20 m、 Figure 20 n, STE-BG+US treatment can significantly promote the maturation of DCs, reaching 67.9% (the ratio of CD80+CD86+ in CD11c+ DCs), which is related to the presence of EGCG promoting cell membrane rupture and leakage of inflammatory components.

[0123] Taken together, these findings provide strong evidence that the "external ultrasound piezoelectric AND internal demethylation regulation" logic-gated PANoptosis approach can effectively reprogram tumor cells, converting the immune quiescent state into an activated ultrasound immunotherapy.

[0124] 5. In vivo anti-tumor evaluation of the STE-BG scaffold

[0125] The inventors of this study further investigated its tumor clearance effect in mice. Twenty-five 6-week-old Balb / c nude mice were randomly divided into five groups: G1: BG, G2: BG+US, G3: STE-BG, G4: ST-BG+US, and G5: STE-BG+US. MNNG / HOS Cl cells (5×106 cells / site) suspended in PBS were injected into the right thigh root of the mice to establish an osteosarcoma model. After feeding and growth, the average tumor volume reached approximately 65 mm. 2 Afterwards, the corresponding stents were implanted into the subcutaneous transplanted tumor in the right leg of anesthetized mice, and ultrasound (1 W / cm 2 , 5 min) treatment (e.g. Figure 21 (a). Related thermal imaging results showed that skin temperature increased by only 2.2°C after ultrasound treatment. Furthermore, analysis of nude mouse skin tissue by photography and hematoxylin-eosin (H&E) staining revealed no local damage in the ultrasound group. Therefore, these results indicate that SDT treatment parameters are safe and reliable. During the 14-day treatment period, there were no significant differences in body weight changes among all groups (e.g., Figure 21 To evaluate the in vivo biosafety of the scaffold, H&E staining was performed on vital organs, including the heart, liver, spleen, lungs, and kidneys, after treatment. The results showed minimal pathological changes, confirming the excellent tissue compatibility of the composite scaffold. Furthermore, the average tumor weight in the STE-BG+US group was approximately 0.118 g, only 16.2% of that in the BG group (see Figure 2). Figure 21 c). The tumor volume growth curve showed that the tumor growth rate in the STE-BG+US group was significantly slower than that in the other groups ( Figure 21 d and Figure 21 g). It is noteworthy that on day 14, the relative tumor volume of the STE-BG+US group was only 24.6% of that of the BG group. The tumor inhibition rate of the STE-BG+US group was 73.47±5.2%, showing a significant reduction in tumor volume ( Figure 21 d and Figure 21 e). Analysis of H&E-stained tumor sections showed that the STE-BG+US group showed the most obvious cell and tissue pathological damage ( Figure 21f). In addition, compared with the other groups, the tumor cell apoptosis rate in the STE-BG+US group was the highest and the cell proliferation ability was the lowest, which was reflected in the terminal deoxynucleotidyl transferase (TUNEL) staining and proliferating cell nuclear antigen (PCNA) staining images ( Figure 21 f), which indicated that ultrasound amplified injury had a good therapeutic effect.

[0126] In addition, the inventors further verified the ability of the composite STE-BG scaffold to induce an immune response in vivo. To assess whether ICD was induced after treatment, the inventors analyzed tumor sections by immunofluorescence (IF) staining to detect cleaved caspase-3 (green), CRT (red), and HMGB1 (red). Notably, a significant positive fluorescence signal was observed in the STE-BG+US group, indicating that the STE-BG scaffold successfully induced ICD in vivo ( Figure 22 The above-mentioned specific antigens are recognized by tumor antigen-presenting cells (mainly DCs), which in turn promote the maturation of DCs and present tumor-associated antigens to T cells. Specifically, flow cytometry (FCM) data collected from the corresponding solid tumor groups showed that the proportion of mature DCs (CD80+ CD86+ in CD11c+ DCs) increased significantly to 34.4% after STE-BG+US treatment, an increase of 3.37 times compared with the BG group ( Figure 21 h, Figure 23-24 As shown). It is well known that the infiltration of T cells in tumors is crucial for the optimal effect of cancer immunotherapy. Flow cytometry showed that the proportion of CD8+ T cells in the STE-BG+US group was approximately 2.6 times that of the control group, while the proportion of CD4+ T cells was approximately 1.8 times ( Figure 21 i).

[0127] These experimental results indicate that the STE-BG+US composite scaffold can activate immune response, induce ICD, and significantly enhance anti-tumor immune response.

[0128] VI. Evaluation of the Antibacterial and Osteogenic Potential of STE-BG Scaffolds Under Ultrasound Irradiation

[0129] Infection around implanted grafts is a significant complication in implant therapy, especially in the setting of deep tumors such as osteosarcoma, which can lead to poor bone repair. Therefore, it is crucial to incorporate antimicrobial strategies into osteosarcoma treatment. In this study, methicillin-resistant Staphylococcus aureus (MRSA) was selected to evaluate the anti-infective effect of the STE + US composite through its ultrasonic piezoelectric properties. As can be seen from the standard colony count images, the STE + US group effectively inhibited the growth of MRSA ( Figure 25a). At the same time, live / dead staining showed that almost all bacteria died after STE+US treatment, while most bacteria in other groups were still alive ( Figure 25 b), confirming its anti-infective potential.

[0130] The inventors further studied the antibacterial potential of the STE+US group in vivo. The corresponding materials were topically applied to a mouse model of MRSA-infected skin wounds. The wound repair results at different time points were as follows: Figure 25 c. On day 11, the wound area in the STE+US group (10.69%) was significantly smaller than that in the control group (22.9%), the control+US group (19.33%), and the STE group (19.37%), indicating that ROS generated by ultrasonic piezoelectricity have a strong bactericidal ability.

[0131] To further explore the healing process of infected wounds, the inventors performed comprehensive histological analysis on day 11 using H&E staining, Masson staining, and CD31 immunohistochemical staining ( Figure 25 d). H&E staining revealed narrower granulation tissue and thicker re-epithelial tissue in the STE + US group, indicating enhanced antibacterial efficacy and improved skin tissue regeneration. Furthermore, Masson staining revealed a higher density of ordered collagen structures at the wound site in the STE + US group. Consistent with epithelial regeneration, CD31 immunohistochemical staining also revealed accelerated neovascularization in the STE + US group, indicating that the treatment promoted angiogenesis and vascular perfusion.

[0132] In addition to its excellent anti-infection ability after surgery, the composite STE-BG scaffold also has great potential for bone defect repair due to the excellent bioactivity of BG and the osteogenic ability of the Sr element in MeSTNPs. The inventors first used aluminum red dye, which forms an orange-red complex with calcium salts to evaluate calcium deposition and mineralization levels. Figure 25 In Figure e, compared with the control and BG groups, BMSCs cultured on the STE-BG scaffolds showed significant aluminum red precipitation in a dose-dependent manner. This is due to the osteoinductive properties of the composite STE-BG scaffold, which promoted the upregulation of osteogenic calcium nodules. Alkaline phosphatase (ALP) is a key enzyme in osteogenesis and an early marker of osteoblast differentiation. ALP staining results showed that in the STE-BG group, the intensity of the blue-purple precipitation increased in a dose-dependent manner after the addition of STE ( Figure 25 f), indicating enhanced osteogenic differentiation activity. In addition, on days 1, 3, and 5 after BMSCs were seeded on the scaffold, intercellular adhesion of BMSCs was clearly observed by cytoskeleton fluorescence staining ( Figure 25g). These results clearly demonstrate that BMSCs cultured on STE-BG scaffolds have a strong proliferation capacity and are able to promote osteoblast differentiation. In addition, after co-culturing BMSCs with BG and STE-BG scaffolds, CCK-8 assays showed no significant decrease in cell viability ( Figure 25 h). At the mRNA gene level, the inventors used quantitative polymerase chain reaction (qPCR) technology to quantitatively detect the expression levels of typical osteogenic gene markers (ALP, OSTERIX, OPG, RUNX-2) in BMSCs. The results showed that compared with the control group, the expression levels of the corresponding genes in the STE-BG group were upregulated by 307%, 323%, 265% and 262%, respectively ( Figure 25 i), indicating that the STE-BG scaffold has superior osteogenic properties. The quantitative results of UV-Vis spectrophotometry also showed a similar upward trend, indicating that the ALP activity of cells in the STE-BG group was enhanced ( Figure 25 j).

[0133] The inventors used Sprague-Dawley (SD) rats to establish a skull defect model to evaluate the in vivo osteogenesis of the STE-BG scaffold. Figure 25 As shown in Figure k, a scalpel was used to make an incision along the midline of the skull, and a 5 mm bone drill was used for drilling. Defects were created on the left and right sides of each rat, and the STE-BG scaffold was implanted in the right skull defect. The left side served as a blank negative control group. Notably, after 12 weeks, a larger area of ​​new bone tissue was observed in the microCT images of the STE-BG group ( Figure 25 l). H&E and Masson staining further demonstrated that the newly formed mineralized bone was slightly more in the STE-BG scaffold group compared with the control group, which was consistent with the microCT analysis ( Figure 26 a). In addition, micro-CT imaging showed that the STE-BG scaffold deformed after implantation and slightly fused with the newly formed bone tissue around the defect (Figure S26b). Quantitative analysis showed that at 12 weeks, the bone mineral density (BMC) of the STE-BG group increased by 1.4 times, and the bone surface density (BMD) reached 1.5 times that of the control group ( Figure 25 m and 25n), these changes indicate that the STE-BG scaffold has good osteogenic potential. In summary, these findings confirm that the composite STE-BG scaffold can effectively promote the proliferation and differentiation of BMSCs, thereby accelerating the bone regeneration process.

[0134] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A strontium titanate nanoparticle, characterized in that: The invention comprises mesoporous piezoelectric SrTiO3 nanoparticles, wherein the mesoporous piezoelectric SrTiO3 nanoparticles are loaded with epigallocatechin gallate; The method for preparing strontium titanate nanoparticles comprises the following steps: S1. dissolving tetrabutyl titanate in ethanol to form a mixed solution; then, adding an ammonia solution to the mixed solution to obtain a precipitated hydroxide; filtering and washing the hydroxide multiple times with distilled water; S2. Then, after adding Sr(NO)2, KOH and PVA solution, vigorously stirred and dispersed in distilled water to form a suspension; S3, transferring the suspension to an autoclave and heating it to 200°C for 20 hours, and then cooling it back to room temperature; filtering the obtained product, and washing it with dilute nitric acid and distilled water, thereby preparing mesoporous piezoelectric SrTiO3 nanoparticles; S4. Mixing the mesoporous piezoelectric SrTiO3 nanoparticles and epigallocatechin gallate in an aqueous solution and dispersing them in acidic deionized water; stirring the mixture for 24 hours; and then purifying the obtained mixture with acidic deionized water and dispersing it in acidic deionized water.

2. The strontium titanate nanoparticle according to claim 1, characterized in that: The pore size of the mesoporous piezoelectric SrTiO3 nanoparticles is 11-13 nanometers.

3. The strontium titanate nanoparticles according to claim 1, wherein: The specific surface area of ​​mesoporous piezoelectric SrTiO3 nanoparticles is 34-37m² / g.

4. The strontium titanate nanoparticles according to claim 1, characterized in that: The loading of epigallocatechin gallate was 75-77%.

5. Composite bracket, characterized by: The invention comprises a biological scaffold and strontium titanate nanoparticles according to any one of claims 1 to 4 hybridized on the biological scaffold.

6. The method for preparing a composite stent according to claim 5, characterized in that: The bioscaffold was immersed in acidic anhydrous ethanol in a solution of strontium titanate nanoparticles, then removed and air-dried at room temperature, and this was repeated multiple times.

7. A method for preparing strontium titanate nanoparticles, characterized in that: The following steps are involved: S1. dissolving tetrabutyl titanate in ethanol to form a mixed solution; then, adding an ammonia solution to the mixed solution to obtain a precipitated hydroxide; filtering and washing the hydroxide multiple times with distilled water; S2. Then, after adding Sr(NO)2, KOH and PVA solution, vigorously stirred and dispersed in distilled water to form a suspension; S3, transfer the suspension to an autoclave and heat to 200°C for 20 h, then cool back to room temperature; The product obtained by filtration was washed with dilute nitric acid and distilled water to prepare mesoporous piezoelectric SrTiO3 nanoparticles; S4. Mixing the mesoporous piezoelectric SrTiO3 nanoparticles and epigallocatechin gallate in an aqueous solution and dispersing them in acidic deionized water; stirring the mixture for 24 hours; and then purifying the obtained mixture with acidic deionized water and dispersing it in acidic deionized water.

8. Use of the composite scaffold according to claim 5 in preparing a graft for treating osteosarcoma.

9. Use of the strontium titanate nanoparticles according to any one of claims 1 to 4 in the preparation of a drug for PANoptosis-mediated ultrasound immunotherapy of osteosarcoma.

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