Nanoscale sonosensitizer and its preparation nanocomposite hydrogel in enhancing the effect of acoustic dynamic therapy of melanoma

Abstract

The present application relates to the technical field of nanomaterials, and in particular to the application of a nano-sound sensitizer and a nano-composite hydrogel prepared therefrom in enhancing the effect of sound dynamic therapy for melanoma. The present application provides the application of monopersulfate in the preparation of a nano-sound sensitizer, and provides a nano-sound sensitizer in which monopersulfate is loaded on the surface of zinc oxide nanosheet microspheres. The present application also provides a nano-composite hydrogel prepared using the nano-sound sensitizer, and optimizes the preparation process of the nano-sound sensitizer and the nano-composite hydrogel. In addition, the present application provides the application of the nano-sound sensitizer and the nano-composite hydrogel in enhancing the effect of sound dynamic therapy for melanoma. The nano-sound sensitizer and the nano-composite hydrogel provided by the present application are stable in structure, simple in preparation process, and have good therapeutic effect when applied in sound dynamic therapy for melanoma.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to the application of nano-acoustic agents and their prepared nanocomposite hydrogels in enhancing the sonodynamic therapeutic effect of melanoma. Background Technology

[0002] Melanoma is highly invasive, drug-resistant, and heterogeneous. With the rapid development of nanobiomedicine, sonodynamic therapy (SDT), an emerging minimally invasive nanotherapy modality for tumors, is developed based on photodynamic therapy. It utilizes low-intensity ultrasound to stimulate sonosensitive agents accumulated in tumors, exerting physical (e.g., mechanical and cavitation) and biochemical (e.g., reactive oxygen species) effects, inducing corresponding biological effects (e.g., apoptosis, necrosis, pyroptosis, ferroptosis, autophagy), thereby effectively killing tumor cells and showing great promise for application. 1 O2), superoxide radicals (O2) - ), hydroxyl radicals ( OH) and sulfate radicals (SO4) - ROS (Reactive Oxygen Species), a newly emerging form of reactive oxygen species (ROS), plays a crucial role in the cell life cycle. At low concentrations, ROS acts as a key intracellular signaling molecule, regulating cell growth, proliferation, and differentiation. Excessive ROS can damage cell membranes, inhibit mitochondrial function, damage DNA leading to gene mutations, cause energy metabolism disorders, and result in irreversible oxidative damage to cells, leading to apoptosis and programmed necrosis. Therefore, selectively increasing ROS yield can effectively induce tumor cell death. SDT (Single-Directed Therapy), as an emerging tumor treatment method, has lower toxicity and is less likely to induce drug resistance. Compared with radiotherapy and chemotherapy, SDT has advantages such as non-invasiveness, no radiation, precision, and controllability. Compared with phototherapy, SDT has superior tissue penetration (penetrating depth up to 10 cm), enabling the treatment of deep tumors.

[0003] SDT (Sonic Therapy) utilizes sonosensitive agents to generate reactive oxygen species (ROS) under ultrasonic stimulation based on acoustic aperture, sonochemistry, and sonoluminescence. An effective sonosensitive agent is a crucial prerequisite for SDT. While SDT is a superior strategy with excellent application prospects, the generated ROS is limited by insufficient O2 concentration in the tumor microenvironment, leading to reduced ROS yield and decreased SDT therapeutic efficacy. This is a key scientific problem that must be solved for SDT in tumor treatment. Organic sonosensitive agents, mainly including porphyrins and their derivatives, are early-used and often derived from photosensitizers. They suffer from poor hydrophilicity, limited bioavailability, and persistent phototoxicity, and are easily metabolized rapidly in vivo, failing to form effective accumulation at the tumor site, thus failing to achieve the desired sonodynamic effect. Compared to organic sonosensitive agents, inorganic sonosensitive agents possess enhanced physicochemical stability, reduced phototoxicity, and certain unique physicochemical properties, making them easier to circulate in the blood. These mainly include zinc oxide, titanium dioxide, and iron oxide sonosensitive agents. As a typical semiconductor nanomaterial, zinc oxide sonosensitive agents have advantages such as good chemical stability and strong redox ability, achieving electron (electron) emission under ultrasonic excitation. - ) and holes (h + The separation of ROS (reactive oxygen species) and the generation of ROS to kill tumor cells have broad application prospects. However, problems such as the wide band gap, the easy recombination of electrons and holes generated by ultrasonic excitation, and the easy aggregation of nanoparticles can lead to a decrease in the generation efficiency of ROS. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a nano-sound-sensitizing agent and the nanocomposite hydrogel prepared therefrom in enhancing the sonodynamic treatment effect of melanoma. The nano-sound-sensitizing agent and nanocomposite hydrogel provided by the present invention have stable structures, simple preparation processes, and good therapeutic effects when applied to the sonodynamic treatment of melanoma.

[0005] This invention provides the application of peroxide monosulfate in the preparation of nano-acoustic agents.

[0006] Due to insufficient O2 concentration in the tumor microenvironment, the yield of ROS remains low, reducing the efficacy of SDT (suppressive tumescent drug therapy). This invention addresses this issue by electrostatically adsorbing peroxymonosulfate (PMS) onto the surface of H-ZnO nanosheets / microspheres. Electron excitation generated by the piezoelectric catalysis of H-ZnO activates the PMS, initiating a cascade catalytic reaction to generate various ROS (reactive oxygen species). OH, SO4 1 O2 and O2 - It relies less on O2 in the tumor microenvironment to achieve multiple amplifications of tumor oxidative stress, while SO4 As a novel ROS, it has stronger toxicity, longer half-life, faster reaction speed and higher selectivity, thus effectively killing tumor cells and solving the problems of SDT treatment being limited by insufficient O2 in the tumor microenvironment and poor tumor treatment effect caused by single ROS.

[0007] In some embodiments, the persulfate includes potassium persulfate.

[0008] This invention also provides a nano-acoustic sensitizer, which is a porous H-ZnO nanosheet microsphere loaded with the aforementioned peroxymonosulfate, wherein: The mass ratio of the H-ZnO porous nanosheets to the peroxymonosulfate is (8~12):(1~1.5). The raw materials for the H-ZnO porous nanosheets and microspheres include zinc acetate, hexamethylenetetramine, and trisodium citrate in a mass ratio of (2~3):(2~3):(0.2~0.5).

[0009] Compared with other nano-acoustic agent raw materials, the nano-acoustic agent provided by this invention has a close combination of components, thereby obtaining a more accurate technical effect.

[0010] In some specific embodiments, the peroxymonosulfate is potassium peroxymonosulfate.

[0011] Preferably, the mass ratio of the H-ZnO porous nanosheets to the peroxymonosulfate is 1:0.113; The raw materials for the H-ZnO porous nanosheets and microspheres include zinc acetate, hexamethylenetetramine, and trisodium citrate in a mass ratio of 2.8:2.1:0.3.

[0012] Experiments show that the nano-sound-sensitizing agents prepared from the components at the above mass ratios are most effective in enhancing the sonodynamic treatment of melanoma, thus achieving more accurate technical results.

[0013] The present invention also provides a method for preparing the aforementioned nano-acoustic sensor, comprising the following steps: Step 1: After dissolving and incubating the zinc acetate, hexamethylenetetramine and trisodium citrate, the resulting precipitate is dried, heated and calcined to obtain H-ZnO porous nanosheet microspheres; Step 2: Dissolve, react, and centrifuge the H-ZnO porous nanosheets and the peroxide monosulfate to obtain the nano-acoustic agent.

[0014] In some embodiments, step 1, the calcination includes calcination at 300°C for 1 hour in a mixed gas of 3 vol% H2 and 97 vol% Ar.

[0015] Experiments have verified that the H-ZnO porous nanosheets and microspheres obtained by calcining with the calcined mixed gas and air have stable crystal structures, and at the same time, they have higher oxygen vacancy concentrations and more effective US generation carrier separation capabilities, thus achieving more accurate technical results.

[0016] In some embodiments, in step 1, the incubation includes incubation in a 90°C water bath for 3 hours, and the heating includes heating in air at 300°C for 1 hour.

[0017] In some embodiments, in step 2, the reaction time is 2 hours, and the centrifugation includes centrifugation at 3000 rpm for 3 minutes.

[0018] The present invention provides a nanocomposite hydrogel, the raw materials of which include chitosan, β-glycerol phosphate and the nanoacoustic agent described above or prepared by the preparation method described above in a mass ratio of (1~2):(6~7):(0.01~0.05).

[0019] In some specific embodiments, the raw materials include chitosan, β-glycerol phosphate, and the aforementioned nano-acoustic sensitizer or the nano-acoustic sensitizer prepared by the aforementioned method, in a mass ratio of 1:6:0.02464.

[0020] This invention provides a method for preparing the aforementioned nanocomposite hydrogel, wherein the chitosan is dissolved in an acetic acid solution, the β-glycerophosphate is dissolved in water, the aqueous solution of the β-glycerophosphate is mixed with the acetic acid solution of the chitosan, and then mixed with the aforementioned nanoacoustic sensitizer or the nanoacoustic sensitizer prepared by the aforementioned method to obtain the nanocomposite hydrogel.

[0021] This invention provides the use of at least one of the following (1) to (4) in the preparation of a sonodynamic therapy agent for cancer cells and / or a medicament for treating cancer: (1) The aforementioned nano-acoustic sensitizer; (2) The nano-acoustic sensor prepared by the above preparation method; (3) The aforementioned nanocomposite hydrogel; (4) The nanocomposite hydrogel prepared by the above preparation method.

[0022] This invention provides a sonodynamic therapy reagent for cancer cells and / or a drug for treating cancer, comprising at least one of the following ① to ④: ① The aforementioned nano-acoustic sensor; ② The nano-acoustic sensor prepared by the aforementioned method; ③ The aforementioned nanocomposite hydrogel; ④ The nanocomposite hydrogel prepared by the aforementioned method.

[0023] In some embodiments, the cancer cells are melanoma cells.

[0024] This invention provides a treatment method for melanoma, comprising: administering the said cancer cells a sonodynamic therapy agent and / or a drug for treating cancer.

[0025] This invention provides the application of peroxide monosulfate in the preparation of nano-acoustic agents, and provides nano-acoustic agents with peroxide monosulfate loaded on the surface of zinc oxide nanosheets / microspheres. A nanocomposite hydrogel prepared using the aforementioned nano-acoustic agent is also provided. The preparation process of the nano-acoustic agent and the nanocomposite hydrogel is optimized. Furthermore, the invention provides the application of the nano-acoustic agent and the nanocomposite hydrogel in enhancing the sonodynamic treatment effect of melanoma. Compared with existing nano-acoustic agents: 1. This invention synthesizes ZnO via a hydrothermal method and calcines it in a mixture of hydrogen and argon to prepare H-ZnO porous nanosheet microspheres. The nanosheet microspheres can avoid agglomeration, and the polygonal pores formed on the surface significantly increase the specific surface area. In addition, the concentration of oxygen vacancies and majority carriers increases after calcination in a mixture of hydrogen and argon, which ultimately significantly improves the piezoelectric catalytic performance, enhances the ROS generation efficiency, and achieves effective killing of tumor cells. 2. Because peroxymonosulfate (PMS) is further loaded onto the surface of H-ZnO nanosheets via electrostatic adsorption, the electron excitation generated by the piezoelectric catalysis of H-ZnO activates the PMS, leading to a cascade catalytic reaction that generates various ROS (reactive oxygen species). OH, SO4 1 O2 and O2 - This approach, independent of O2 in the tumor microenvironment, further addresses the issue of low ROS yields due to insufficient O2 concentration in the tumor microenvironment, achieving multiple amplifications of tumor oxidative stress, while simultaneously reducing SO4 levels. As a novel ROS, it has stronger toxicity, longer half-life, faster reaction speed and higher selectivity, thus effectively killing tumor cells and solving the problem of insufficient O2 in tumor microenvironment in SDT treatment. H-ZnO continuously replenishes the consumed electrons through piezoelectric catalysis under ultrasonic excitation, ensuring that tumor SDT treatment can be carried out efficiently and continuously, realizing highly efficient SDT treatment for melanoma and providing another effective treatment mode for melanoma treatment. 3. The nanocomposite hydrogel prepared using nanoacoustic sensitizers can effectively enhance the accumulation of nanoacoustic sensitizers in tumor cells, further enhancing the efficacy of nanoacoustic sensitizers in SDT treatment of melanoma. Attached Figure Description

[0026] Figure 1Comparison of the crystal structures of H-ZnO (calcined in hydrogen) and A-ZnO (calcined in air) observed by X-ray diffraction (XRD); Figure 2 The results of solid-state ultraviolet spectroscopy and Mott-Schottky diagram analysis of the potential of H-ZnO and A-ZnO are shown in the figure. In the figure, figure a is the band gap of H-ZnO and A-ZnO obtained by ultraviolet diffuse reflectance test, and figures b and c are the Mott-Schottky diagrams measured by electrochemical workstation. Figure 3 In Example 3, DPBF was used as a chemical probe to detect ultrasonic triggering. 1 O2 generation results are shown in the figure. In the figure, a, b, c and d represent the control group, A-ZnO+US group, H-ZnO+US group and H-ZnO-PMS+US group respectively, and e is the statistical results. Figure 4 The degradation test results of methylene blue (MB) in Example 3 are shown. In the figure, a, b, c and d represent the control group, A-ZnO+US group, H-ZnO+US group and H-ZnO-PMS+US group, respectively, and e is the statistical result. Figure 5 The results of ESR assessment of toxic free radical generation in Example 3 are shown, where Figures a and b represent verification for different groups. 1 The results of O2 production are shown in Figure c, which verifies the O2 production. - The results are shown in Figure d, which verifies SO4. ·- and OH is produced as a result; Figure 6 The results of CCK8 assay for cell viability in Example 4 are shown in the figure. In the figure, a, b, c and d represent the effects of different experimental groups, different H-ZnO-PMS concentrations, different ultrasound durations and different ultrasound power densities on cell viability, respectively. Figure 7 The results of cell-level verification of the performance of the nano-acoustic sensitizer SDT in Example 4 are shown. In Figure a, the ROS production capacity at the cellular level is tested by 2',7'-dichlorofluorescein diacetate (DCFH-DA). Figure b shows the staining results of live cells and dead cells by Calcein-AM and Propidium Iodide, respectively. Figure 8 This diagram illustrates the volume of melanomas in different groups in Example 6. Figure 9 The statistical results of the tumor-inhibiting effect of the nano-acoustic sensitizer in Example 6 are shown. In the figure, a, b, c and d represent the changes in tumor weight, tumor inhibition rate, tumor volume and relative tumor volume in different groups, respectively. Detailed Implementation

[0027] This invention provides the application of nano-sound-sensitizing agents and their prepared nanocomposite hydrogels in enhancing the sonodynamic treatment effect of melanoma. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0028] Melanoma is the most common malignant skin tumor, highly malignant and with extremely poor prognosis from traditional treatments. With the rapid development of nanobiomedicine, sonodynamic therapy (SDT), as an emerging treatment approach, utilizes low-intensity ultrasound (US) combined with nano-sound-sensitizing agents to generate toxic reactive oxygen species (ROS) for precise tumor treatment, showing great promise. Despite its superior strategy, insufficient O2 content in the tumor microenvironment (TME) prevents the generation of enough ROS to effectively eradicate the tumor. Therefore, developing safe and efficient nano-sound-sensitizing agents with high ROS yields to address the hypoxia problem in the tumor microenvironment is crucial for effective SDT treatment. This study, based on the development of high-performance inorganic nano-sound-sensitizing agents and overcoming the hypoxia problem in the tumor microenvironment, designed a novel ROS-based composite nanoplatform, H-ZnO-PMS, which generates various toxic ROS (SO4) under low-frequency ultrasound irradiation. OH、 1 O2, O2 - This study addresses the challenge of hypoxia in TME (tumor-mediated endothelial growth factor) and significantly improves ROS yield, achieving highly efficient nano-SDT (sound-assisted therapeutic therapy) for melanoma. This research will systematically investigate the composition / structure and function relationship of H-ZnO-PMS nano-sound-sensitizers. It will systematically analyze the molecular mechanism by which ultrasound-excited piezoelectric catalytic activation of PMS amplifies tumor oxidative stress, achieving highly efficient SDT therapy for melanoma, both at the in vitro cellular level and in vivo animal tumor model level. The study will also analyze the biosafety and clinical translation prospects of this nano-sound-sensitizer, providing a solid theoretical and experimental foundation for its clinical application in tumor sonodynamic synergistic diagnosis and treatment. This research project will be of great significance to the development of clinical tumor treatment and the advancement of nano-ultrasound medicine, and is expected to promote the clinical trials of novel nanobiomaterials and technologies. It also aligns with my country's economic and social development and people's important health needs.

[0029] A good sonosensitive agent is a prerequisite for efficient sonodynamic therapy; therefore, the preparation of high-performance sonosensitive agents is the primary scientific problem to be solved and the foundation for subsequent research. ZnO nanoparticles can establish a dynamic piezoelectric built-in electric field under ultrasonic excitation to drive carrier migration, causing electrons and holes to separate, and further react with surrounding water and oxygen molecules to produce cytotoxic ROS (singlet oxygen). 1 O2), thereby promoting tumor cell apoptosis and necrosis. However, its wide band gap, the easy recombination of electrons and holes generated by ultrasonic excitation, and the tendency of nanoparticles to aggregate lead to a decrease in ROS generation efficiency. Based on this, this project proposes a hydrothermal method to synthesize ZnO and finally prepare H-ZnO porous nanosheet microspheres by calcination in a mixture of hydrogen and argon. Since the nanosheet microspheres can avoid aggregation and the polygonal pores formed on the surface significantly increase the specific surface area, coupled with the increased concentration of oxygen vacancies and majority carriers (electrons) after calcination in a mixture of hydrogen and argon, the piezoelectric catalytic performance is significantly improved, enhancing the ROS generation efficiency. Preparation of H-ZnO nano-sound sensor: Using a hydrothermal method, 2.8g Zn(CH3COO)2 and 2.1g C6H 12 N4 was dissolved in deionized water. 0.3 g of Na3C6H5O72H2O was added to the above solution and magnetically stirred for 15 minutes. The solution was then transferred to a 100 ml water bath and incubated at 90 °C for 3 hours. The resulting white precipitate was centrifuged and washed several times with deionized water and ethanol, then dried at 60 °C. The obtained powder was placed in a muffle furnace and heated in air at 300 °C for 1 hour to remove the surfactant. Finally, the sample was calcined in a tube furnace under a hydrogen atmosphere (3 vol% H2 + 97 vol% Ar mixed atmosphere) at 300 °C for 1 hour to prepare H-ZnO nanosheets / microspheres with stable material properties, good dispersibility, and superior acoustic dynamic effects. This project, focusing on the regulation of oxygen vacancy concentration and ROS generation efficiency of H-ZnO nanoacoustic agents, provides theoretical and experimental technical guidance for further optimization to obtain nanoacoustic agents more suitable for tumor acoustic dynamic therapy.

[0030] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0031] Example 1: Preparation of Nano-Sound Sensitive Agents and Nanocomposite Hydrogels

[0032] 1. Preparation of H-ZnO porous nanosheet microspheres

[0033] The hydrothermal method was used, specifically as follows: 2.8g Zn(CH3COO)2 and 2.1g C6H 12N4 was dissolved in deionized water. 0.3 g of Na3C6H5O72H2O was added to the above solution and magnetically stirred for 15 minutes. The solution was then transferred to a 100 ml water bath and incubated at 90°C for 3 hours. The resulting white precipitate was centrifuged and washed several times with deionized water and ethanol, then dried at 60°C. The obtained powder was placed in a muffle furnace and heated in air at 300°C for 1 hour to remove the surfactant. Finally, the sample was calcined in a tube furnace under a hydrogen atmosphere (3 vol% H2 + 97 vol% Ar mixed atmosphere) at 300 °C for 1 hour to prepare H-ZnO porous nanosheet microspheres. Morphology was observed and characterized using transmission electron microscopy (TEM) and scanning electron microscopy (SEM), crystal structure was observed using X-ray diffraction (XRD), elemental valence states were analyzed using XPS, surface zeta potential and particle size were analyzed using a nanoparticle size and zeta potential analyzer (DLS), specific surface area was analyzed using a fully automated specific surface area and porosity analyzer (BET), piezoelectricity was analyzed using atomic force microscopy (AFM), and band gap was analyzed using ultraviolet / visible / near-infrared diffuse reflectance testing.

[0034] 2. Preparation of H-ZnO-PMS (nano-sound-sensitizing agent)

[0035] The prepared H-ZnO solution and PMS solution (peroxymonosulfate, specifically potassium peroxymonosulfate, with the molecular formula KHSO5·0.5KHSO4·0.5K2SO4) were stirred at room temperature for 2 hours in a 1:1, w / w ratio. After centrifugation at 3000 rpm for 3 minutes and washing three times with deionized water, the product was collected to obtain H-ZnO-PMS nanomaterials, which were dispersed in deionized water and stored at 4°C for subsequent experiments. The potential of the zinc oxide nanosheets / microspheres was determined using a nanoparticle size and Zeta potential analyzer; the potential was positive, while the potential of peroxymonosulfate (PMS) was negative. PMS can be adsorbed onto the pores of the zinc oxide nanosheets / microspheres through electrostatic interaction.

[0036] The morphology of H-ZnO-PMS nanomaterials was characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), and the elemental composition was quantified by inductively coupled plasma (ICP).

[0037] The peroxymonosulfate loaded on zinc oxide nanosheets was determined by a spectrophotometric method modified from iodometric titration. First, H-ZnO nanosheets and peroxymonosulfate (PMS) were dissolved in deionized water at a mass ratio of 1:1 to prepare a 1 mg / mL mixed solution (e.g., 5 mg of H-ZnO nanosheets and 5 mg of peroxymonosulfate could be dissolved together in 5 mL of deionized water to obtain a 1 mg / mL H-ZnO solution and a 1 mg / mL PMS solution). Then, the mixed solution was stirred to allow PMS to be loaded into the pores on the surface of the H-ZnO nanosheets through electrostatic adsorption. After stirring for about 3 hours, wash the mixed solution with deionized water and centrifuge 2-3 times to remove free PMS that is not loaded onto the H-ZnO nanosheets. Add deionized water to the precipitate after washing and centrifugation to prepare a 1 mg / mL solution (for example, if 5 mL of deionized water is added above, then add another 5 mL of deionized water to the remaining precipitate after washing and centrifugation). Then, measure the concentration of peroxymonosulfate actually loaded on the zinc oxide nanosheets using a spectrophotometric method modified by iodometric titration of the obtained 1 mg / mL solution, which is 113 ug / mL.

[0038] 3. Preparation of H-ZnO-PMS-Gel (nanocomposite hydrogel)

[0039] 100 mg of chitosan (degree of deacetylation: 75–85, viscosity: 0.2–0.8 Pa·s, 25 °C, Sigma-Aldrich) was gradually dissolved in 4 mL of acetic acid (0.1 M) with continuous stirring at room temperature until the solution became clear, yielding a chitosan solution. Then, 600 mg of β-GP (β-glycerophosphate, Sigma-Aldrich) powder was dissolved in 1 mL of deionized water and sterilized using a 0.22 μm syringe filter. Both solutions were cooled to 4 °C for 20 minutes before being mixed together. Subsequently, the β-GP solution was carefully added dropwise to the chitosan solution with gentle stirring in an ice bath to obtain a homogeneous liquid solution. The final solution contained 2% (w / v) chitosan and 12% (w / v) β-GP. Injectable H-ZnO-PMS-Gel was prepared by adding an H-ZnO-PMS nanoparticle solution (concentration 4–5 mg / mL) to a blank chitosan hydrogel at a volume ratio of 1:9. Mix the mixture thoroughly using a vortex mixer for 2 minutes. Store the final solution at 4°C until used.

[0040] Example 2: Investigation of Preparation Conditions for Nanoscale Acoustic Sensitive Agents

[0041] In Example 1, during the preparation of H-ZnO porous nanosheet microspheres, the sample was calcined in air at 300 °C for 1 hour to obtain H-ZnO porous nanosheet microspheres; the remaining processes remained unchanged. The crystal structure was observed using X-ray diffraction (XRD). Figure 1 ), solid-state ultraviolet spectra and Mott-Schottky diagrams ( Figure 2 The differences between the porous nanosheets prepared in Example 1 and those prepared in Example 2 were investigated.

[0042] The results show that Figure 1 The strong peaks on the (100) and (101) crystal planes and the weak peak on the (002) crystal plane suggest that the morphology of ZnO may be nanosheets. In addition, the diffraction peaks of zinc oxide calcined in air or a mixture of hydrogen and argon did not show significant shifts, which means that the atmosphere treatment did not change the crystal form of ZnO.

[0043] The band arrangement was analyzed using solid-state UV spectroscopy and Mott-Schottky diagrams to elucidate the potential SDT mechanism of H-ZnO. Figure 2 As shown in figure a, the band gap of H-ZnO, calculated from the Tauc plot of the modified Kubelka-Munk function, is 2.96 eV, narrower than that of A-ZnO (zinc oxide calcined in air) at 3.06 eV. Furthermore, the flat band potentials of H-ZnO and A-ZnO relative to the conventional hydrogen electrode (NHE) are -0.63 and -0.61 V, respectively. Figure 2 (b, 2c). Since the conduction band (CB) of both catalysts is less than -0.33V, e- can transfer from the valence band (VB) to CB, reducing O2 to ·O2. - Due to O2 - The unstable properties of O2 - It will be oxidized immediately. 1 O2. Furthermore, the CB energy of the H-ZnO system is higher than that of A-ZnO, thus exhibiting stronger reducing power and more efficient US generation carrier separation capability. Moreover, the VB potential of both H-ZnO and A-ZnO is higher than... The redox potential of OH / OH- (1.99V) Figure 2 c), it can be seen that OH- is directly oxidized to OH.

[0044] Example 3: SDT Performance Evaluation of Nano-Sound Sensitive Agents

[0045] To further evaluate the SDT performance of the nano-acoustic sensor, 1,3-diphenylisobenzofuran (DPBF) was used as a chemical probe to detect ultrasonic triggering. 1 O2 generation (results as follows) Figure 3 As shown), a degradation test using methylene blue (MB) was conducted (results are shown in the figure). Figure 4As shown), the generation of toxic free radicals (SO4) is assessed by ESR. ·- , 1 O 2， OH,·O2 - ), using 5,5-dimethyl-1-pyridine N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) as free radical scavengers (results as follows). Figure 5 (As shown).

[0046] Figure 3 The results showed that the typical absorption peak of DPBF gradually decreased with increasing ultrasonic irradiation time, indicating that the nano-acoustic sensor can continuously generate... 1 O2 ( Figure 3 a, 3b, 3c, and 3d represent the control group, A-ZnO+US group, H-ZnO+US group, and H-ZnO-PMS+US group, respectively (US refers to ultrasonic treatment). Through inter-group comparison, H-ZnO-PMS+US showed a significantly higher DPBF oxidation rate, indicating that SDT performance was the strongest.

[0047] Figure 4 The results showed that the H-ZnO-PMS+US group had the strongest MB degradation ability. Figure 4 a, 4b, 4c, and 4d represent the control group, the A-ZnO+US group, the H-ZnO+US group, and the H-ZnO-PMS+US group, respectively.

[0048] Figure 5 The results showed that H-ZnO-PMS+US can generate a variety of toxic free radicals (SO4). ·- , 1 O 2， OH,·O2 - ).

[0049] Example 4: Evaluation of the efficacy of H-ZnO-PMS sonodynamic therapy for melanoma cells

[0050] Different experimental groups were used, including: a control group (cells received no treatment); a low-intensity ultrasound group (cells were irradiated with therapeutic ultrasound through the bottom of a culture plate); a PMS group (cells were irradiated with the same concentration of PMS); a low-intensity ultrasound + PMS group (cells were irradiated with therapeutic ultrasound through the bottom of a culture plate with the same concentration of PMS solution); a H-ZnO group (cells were irradiated with the same concentration of H-ZnO nanoparticles); and a low-intensity ultrasound + H-ZnO group. - Add H-ZnO nanoparticles of the same concentration as H-ZnO, and irradiate the cells to be treated with therapeutic ultrasound through the bottom of the culture plate; H-ZnO-PMS group - Add H-ZnO-PMS nanoparticles of the same concentration as PMS and H-ZnO; Low-intensity ultrasound + H-ZnO-PMS group - The drug addition method is the same as the H-ZnO-PMS group, and the cells to be treated are irradiated with therapeutic ultrasound through the bottom of the culture plate. Ultrasound irradiation conditions: frequency 1MHz, duty cycle 50%, sound intensity: 1.5W / cm². 2 Irradiate with ultrasound for 2 minutes, and then conduct subsequent experiments.

[0051] The experiment was conducted using melanoma cells, with 100 μL added to each well at a density of 1 × 10⁻⁶ cells / well. 4 Melanoma cells were cultured for 24 hours until cell adhesion was achieved. The effects of different experimental groups, different H-ZnO-PMS concentrations, different sonication durations, and different sonication power densities on cell viability were evaluated using CCK-8 assay. Cells were cultured for another 24 hours according to the above groups, followed by 1 hour of co-incubation with CCK-8. Cell viability in each well was then measured, and absorbance at 450 nm was determined using a microplate reader. The results are as follows: Figure 6 As shown.

[0052] At the cellular level, the ROS production efficiency of the different experimental groups was observed using the ROS fluorescent probe DCFH-DA (2,7-Dichlorofluorescindiacetate). The results are as follows: Figure 7 As shown in a.

[0053] To further analyze the cytotoxic effect of synergistic therapy on melanoma cells, cells from each group were cultured in an incubator for 1 hour after different treatments. Live and dead cells were stained with Calcein-AM and Propidium Iodide, respectively, and cell viability was observed under a fluorescence microscope. To further quantify the apoptotic state, cells were digested and collected after different treatments, stained, and then quantitatively analyzed for apoptosis by measuring the fluorescence intensity of the dyes using flow cytometry. The results are as follows: Figure 7 As shown in b.

[0054] Figure 6The results further confirmed that the SDT performance of the H-ZnO-PMS+US group was the strongest and that the SDT performance of the H-ZnO-PMS+US group increased with increasing concentration, ultrasonic time and ultrasonic intensity.

[0055] Figure 7 As shown, experiments at the cell level further confirmed that the SDT performance of the H-ZnO-PMS+US group was the strongest. Figure 7 a represents the ability of cells to produce ROS at the cellular level as measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA), with the H-Zn-PMS+US group exhibiting the strongest ROS production capacity; Figure 7 b) The typical Calcein-AM (green) and propidium iodide (red) double staining method was used to further verify that H-ZnO-PMS+US had the strongest anti-tumor ability.

[0056] Example 5: Biosafety evaluation and in vivo metabolism of H-ZnO-PMS-Gel

[0057] B16F10 melanoma cells and HUVEC human venous endothelial cells were cultured in vitro. The toxicity of H-ZnO-PMS nanoparticles to tumor cells and normal cells was evaluated. Two 96-well culture plates were set up, each plate divided into 5 groups, with 6 replicates per group. In plate A, 100 μL of H-ZnO-PMS nanoparticles at a density of 1 × 10⁻⁶ μL were added to each well. 4 Melanoma cells were added to each well of a B plate at a density of 5 × 10⁻⁶ cells. 3 Human venous endothelial cells were cultured for 24 hours until the cells adhered to the well. Then, H-ZnO-PMS nanoparticles of different concentrations were added at equal dilutions and cultured for another 24 hours. CCK-8 was then added and incubated for about 1 hour. The cell viability in each well was detected, and the absorbance at 450 nm was measured using a microplate reader.

[0058] 4.2 Twenty ICR mice were randomly divided into four groups of five each, and subcutaneously injected with different doses of H-ZnO-PMS-Gel nanoparticles. The biosafety assessment included the following aspects: ① General condition of ICR mice: Observe their mental state, diet, body temperature, etc. daily.

[0059] ② Changes in major organs and hematological indicators: After the treatment course was completed, blood was collected from the retroocular vein of the four groups of ICR mice for hematological indicator testing. After blood collection, the mice were sacrificed and the major organs (heart, liver, spleen, lung, and kidney) were collected. After fixation with 4% paraformaldehyde, the gross tissue specimens were stained and the histological structure of the major organs was observed.

[0060] Example 6: Evaluation of the efficacy of H-ZnO-PMS sonodynamic therapy for melanoma in nude mice

[0061] Subcutaneous injection of 5×10⁻⁶ mg / L into nude mice 5 After establishing a nude mouse melanoma xenograft model using melanoma cells, the maximum and minimum diameters of the tumors were measured to calculate tumor volume. All tumor-bearing mice were randomly divided into 7 groups of 7 mice each. The injection dose of nanoparticles was 1 mg / kg. The ultrasound-related treatment groups received ultrasound irradiation 1 hour after each nanoparticle injection, with treatment occurring every other day. Ultrasound irradiation conditions: frequency 1 MHz, duty cycle 50%, sound intensity: 1.5 W / cm². 2 Ultrasound irradiation for 2 minutes. Experimental groups and corresponding treatments: Control group—tumor-bearing mice received no treatment; Gel group—tumor-bearing mice received a local injection of blank gel into the tumor; Gel+US group—tumor-bearing mice received a local injection of blank gel into the tumor, followed by therapeutic ultrasound irradiation 1 hour later; PMS-Gel+US group—tumor-bearing mice received a local injection of PMS-Gel into the tumor, followed by therapeutic ultrasound irradiation 1 hour later; H-ZnO-Gel+US group—tumor-bearing mice received a local injection of H-ZnO-Gel into the tumor, followed by therapeutic ultrasound irradiation 1 hour later; H-ZnO-PMS-Gel group—tumor-bearing mice received a local injection of H-ZnO-PMS-Gel into the tumor; H-ZnO-PMS-Gel+US group—tumor-bearing mice received a local injection of H-ZnO-PMS-Gel into the tumor, followed by therapeutic ultrasound irradiation 1 hour later.

[0062] The observation of treatment effects includes the following aspects: 1. General condition of tumor-bearing rats: Observe their mental state, diet, weight changes, etc. daily.

[0063] 2. Tumor growth curve: The longest diameter (L) and shortest diameter (W) of the tumor were measured every other day using calipers. The growth curve was calculated using the formula V = L·W. 2 / 2 Calculate tumor volume until the end of the entire treatment course, and plot the tumor growth curve. Tumor inhibition rate (IR) = (Tumor volume in control group - Tumor volume in treatment group) / Tumor volume in control group × 100%.

[0064] 3. Post-treatment tumor pathological changes: After the 15-day treatment course, tumor-bearing mice were sacrificed and tumor specimens were collected. The volume of the tumor tissue was weighed and recorded. The tumor tissue was fixed with 4% paraformaldehyde for further gross tissue section staining and pathological examination.

[0065] Figure 8This diagram illustrates the size of melanomas in different groups obtained from animal experiments (Groups I-VII represent the control group, Gel group, Gel+US group, H-ZnO-PMS+Gel group, PMS+Gel+US group, H-ZnO+Gel+US group, and H-ZnO-PMS+Gel+US group, respectively). The results show that the tumor volume in the H-ZnO-PMS+Gel+US group is the smallest, indicating that the H-ZnO-PMS+Gel+US group has the strongest tumor-suppressing effect.

[0066] Figure 9 Animal experiments further demonstrated that the H-ZnO-PMS+Gel+US group had the strongest tumor-suppressing effect. Figure 9 a, 9b, 9c, and 9d represent the changes in tumor weight, tumor inhibition rate, tumor volume, and relative tumor volume in different groups, respectively.

[0067] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nano-sound-sensitizing agent, characterized in that, It consists of H-ZnO porous nanosheets microspheres loaded with peroxymonosulfate, wherein: The mass ratio of the H-ZnO porous nanosheets to the peroxymonosulfate is 1:0.113; The raw materials for the H-ZnO porous nanosheets and microspheres consist of zinc acetate, hexamethylenetetramine, and trisodium citrate in a mass ratio of 2.8:2.1:0.

3. The H-ZnO porous nanosheets and microspheres were prepared by hydrothermal synthesis of ZnO and calcination in a mixture of hydrogen and argon.

2. The preparation method of the nano-acoustic sensor according to claim 1, characterized in that, Includes the following steps: Step 1: After dissolving and incubating the zinc acetate, hexamethylenetetramine and trisodium citrate, the resulting precipitate is dried, heated and calcined to obtain the H-ZnO porous nanosheet microspheres; Step 2: Dissolve, react, and centrifuge the H-ZnO porous nanosheets and the peroxide monosulfate to obtain the nano-acoustic agent.

3. The preparation method according to claim 2, characterized in that, In step 1, the calcination includes calcination at 300°C in a mixed gas of 3 vol% H2 and 97 vol% Ar for 1 hour.

4. The preparation method according to claim 2, characterized in that, In step 1, the incubation includes incubation in a 90°C water bath for 3 hours, and the heating includes heating in air at 300°C for 1 hour.

5. The preparation method according to claim 2, characterized in that, In step 2, the reaction time is 2 hours, and the centrifugation includes centrifugation at 3000 rpm for 3 minutes.

6. A nanocomposite hydrogel, characterized in that, The raw materials include chitosan, β-glycerophosphate, and the nano-acoustic agent as described in claim 1 or prepared by the method described in any one of claims 2 to 5, in a mass ratio of (1~2):(6~7):(0.01~0.05).

7. The method for preparing the nanocomposite hydrogel according to claim 6, characterized in that, The chitosan is dissolved in an acetic acid solution, and the β-glycerol phosphate is dissolved in water. The aqueous solution of the β-glycerol phosphate is mixed with the acetic acid solution of the chitosan, and then mixed with the nano-acoustic sensitizer according to claim 1 or the nano-acoustic sensitizer prepared by any one of claims 2 to 5 to obtain the nanocomposite hydrogel.

8. The use of at least one of the following (1) to (4) in the preparation of a drug for treating cancer: (1) The nano-acoustic sensitizer according to claim 1; (2) The nano-acoustic sensor prepared by the preparation method according to any one of claims 2 to 5; (3) The nanocomposite hydrogel according to claim 6; (4) The nanocomposite hydrogel prepared by the preparation method described in claim 7.

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