A pH-responsive bismuth-based nanodrug, a preparation method thereof and pharmaceutical use thereof

Abstract

The application discloses a pH-responsive bismuth-based nanodrug and a preparation method and pharmaceutical application thereof. The nanodrug is hyaluronic acid modified cuprous oxide / iodine bismuth oxide heterojunction nanoparticles (Cu2O@BiOI-HA), the cuprous oxide has a double-layer hollow mesoporous structure, the iodine bismuth oxide is loaded on the surface of the cuprous oxide in a nanolayer to form a heterojunction, the particle size is 200-700 nm, and the band gap is 1.46-1.49 eV. The nanodrug produces active oxygen by promoting the separation of electron-hole pairs through band bending under the excitation of ultrasound, releases Cu + ions under acidic conditions, generates hydroxyl radicals by means of a Fenton-like reaction, realizes the synergistic treatment of sonodynamic and chemical dynamics, and simultaneously, the Cu + ions induce copper death, the active oxygen activates the pyroptosis pathway, the multimodal cell death synergistically triggers immunogenic cell death, and the anti-tumor immune response is activated. The application establishes a structure-activity relationship between synthesis parameters and sonodynamic performance, and has application value in the treatment of breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine material preparation technology, specifically relating to a hyaluronic acid-modified cuprous oxide / bismuth iodide heterostructure nanomedicine, its preparation method, and its pharmaceutical applications. Background Technology

[0002] Against the backdrop of a continuously rising global incidence of malignant tumors, breast cancer, as one of the most common malignant tumors in women, requires significant innovation in its treatment. Currently, traditional treatment modalities for breast cancer mainly include surgical resection, radiotherapy, and chemotherapy. However, these treatments still face inherent limitations in inhibiting tumor metastasis, reducing systemic toxicity, and overcoming acquired drug resistance. Surgical resection is difficult to completely eliminate micrometastases and carries a high risk of postoperative recurrence; radiotherapy is limited by the radiation dose's potential damage to normal tissues; and chemotherapy generally suffers from poor targeting, significant toxic side effects, and the tendency for tumor cells to develop multidrug resistance. Therefore, there is an urgent clinical need to explore novel treatment strategies that combine high efficacy and safety.

[0003] Sonodynamic therapy, as an emerging tumor treatment method, offers a unique technological platform for precision tumor treatment due to its excellent deep tissue penetration, non-invasive operation, and spatiotemporal controllability. This technology activates nano-sound-sensitizers through external ultrasound irradiation, generating reactive oxygen species (ROS) with strong oxidative activity via energy conversion, thereby inducing oxidative damage in tumor cells. By rationally designing the composition, morphology, and heterogeneous interface structure of the nano-sound-sensitizers, charge separation efficiency can be optimized to improve ROS quantum yield. Simultaneously, multiple functions such as active targeted delivery, tumor microenvironment-responsive release, and integrated diagnostic and therapeutic functions can be achieved within a single nanoplatform, thus expanding the clinical application potential of sonodynamic therapy.

[0004] Bismuth-based nanomaterials, due to their low cytotoxicity, good biocompatibility, and narrow band gap, have become an important candidate material system for constructing high-performance sonosensitive agents. Their unique layered crystal structure and tunable electronic properties endow them with excellent energy coupling capabilities. However, existing bismuth-based sonosensitive agents generally suffer from insufficient sonocatalytic activity when applied in vivo, specifically manifested as a high electron-hole recombination rate, leading to a low reactive oxygen species quantum yield and failing to achieve the desired tumor cell killing effect. Furthermore, single-component bismuth-based sonosensitive agents have relatively limited functions, making it difficult to meet the needs of multi-mechanism synergistic therapy in the complex tumor microenvironment. The tumor microenvironment is characterized by acidity, high glutathione levels, and high hydrogen peroxide concentrations; single treatment modalities often fail to effectively reverse the immunosuppressive microenvironment, thus limiting anti-tumor efficacy.

[0005] To further enhance the antitumor effects of bismuth-based nano-sound-sensitizers, existing technologies attempt to utilize the narrow bandgap characteristics of bismuth oxyhalides by constructing heterojunctions to regulate the band structure, thereby promoting rapid electron migration and inhibiting electron-hole recombination, thus increasing reactive oxygen species (ROS) production. Simultaneously, copper-based metal oxides are introduced into the heterojunction components, leveraging their ability to release copper ions at tumor sites to specifically consume high levels of glutathione, disrupting intracellular redox balance and enhancing the oxidative damage effect of ROS. However, the influence of key parameters such as particle size and morphology of bismuth-based heterojunctions on ROS generation efficiency remains unclear in existing technologies, and a systematic method for optimizing synthesis conditions is lacking, making it difficult to controllably regulate the sound-sensitizing properties of nanomedicines. Furthermore, the synergistic mechanism between ROS and copper ions is insufficiently studied in existing technologies, failing to effectively achieve the cascade activation of multiple cell death pathways and the systematic regulation of antitumor immune responses, thus limiting the full realization of the synergistic therapeutic effects of sonodynamics-chemodynamics-immunotherapy.

[0006] Based on the aforementioned problems, there is an urgent need in this field to develop novel bismuth-based nano-sound-sensitizers that combine high acoustic catalytic activity, tumor microenvironment-responsive release capability, and multimodal synergistic therapeutic function, and to clarify the structure-activity relationship between their structural parameters and acoustic-sensitivity properties, in order to enhance the anti-tumor effect of acoustic-dynamic synergistic immunotherapy. Simultaneously, it is necessary to establish a systematic method for optimizing synthesis conditions to obtain nanomedicines with controllable particle size, regular morphology, and optimal acoustic-sensitivity activity, and further endow them with good biocompatibility and targeted delivery capability through surface engineering modification, thereby significantly reducing toxic side effects on normal tissues while improving the efficacy of anti-breast cancer therapy. Summary of the Invention

[0007] For this purpose, the main objective of the present invention is to provide a pH-responsive bismuth-based nanomedicine, wherein the nanomedicine is a hyaluronic acid-modified cuprous oxide / bismuth iodide heterojunction nanoparticle, denoted as Cu2O@BiOI-HA, wherein the cuprous oxide has a bilayer hollow mesoporous structure, the bismuth iodide is loaded on the surface of the cuprous oxide in the form of a nanolayer to form a heterojunction, and the hyaluronic acid is coated on the surface of the heterojunction through electrostatic adsorption or chemical bonding. The nanomedicine has a particle size of 200-700 nm and a band gap of 1.46-1.49 eV.

[0008] Furthermore, the nanomedicine releases Cu in response to pH conditions of 5.0-6.5. + ions, the Cu + Ions passing through Cu + / Cu 2+ The valence transition consumes hydrogen peroxide and reduced glutathione, and generates hydroxyl radicals through a Fenton-like reaction.

[0009] Furthermore, the nanomedicine operates at an ultrasonic power density of 0.5-2.0 W / cm². 2Under the conditions of 20 kHz-3 MHz, electron-hole pair separation is promoted by the bending of the heterojunction band and the narrowing of the band gap, generating reactive oxygen species.

[0010] Further, a method for preparing a pH-responsive bismuth-based nanomedicine is included, comprising the following steps: (1) dissolving hexadecyltrimethylammonium bromide in water, and adding Cu... 2+ (1) Add ascorbic acid to the aqueous solution and heat to 50-70℃, keep for 15-30 min, add sodium hydroxide aqueous solution dropwise, and centrifuge and wash to obtain Cu2O nanoparticles with a double-layer hollow mesoporous structure; (2) Disperse bismuth nitrate pentahydrate and polyvinylpyrrolidone in anhydrous ethanol-water mixed solution, add Cu2O nanoparticles and stir for 30-60 min, add potassium iodide solution dropwise, continue stirring for 20-40 min, transfer to microwave reactor and heat to 160-200℃ for 1.5-2.5 h, centrifuge and wash to obtain Cu2O@BiOI heterojunction, wherein by adjusting the volume ratio of anhydrous ethanol to water and the amount of polyvinylpyrrolidone, the particle size of the obtained heterojunction is 200-700 nm and the band gap is 1.46-1.49 eV; (3) Hyaluronic acid and Cu2O@BiOI heterojunction are mixed in water, dispersed by ultrasound, stirred for 20-30 h, and centrifuged and washed to obtain the pH-responsive bismuth-based nanomedicine.

[0011] Furthermore, in step (2), the volume ratio of anhydrous ethanol to water is 16:1, the amount of polyvinylpyrrolidone used is 10-20 mg, the particle size of the resulting heterojunction is 200-250 nm, and the band gap is 1.45-1.47 eV.

[0012] Furthermore, in step (2), the volume ratio of anhydrous ethanol to water is 16:1, the amount of polyvinylpyrrolidone used is 3-8 mg, the particle size of the resulting heterojunction is 300-350 nm, and the band gap is 1.47-1.49 eV.

[0013] Furthermore, in step (2), the volume ratio of anhydrous ethanol to water is 1:16, the amount of polyvinylpyrrolidone used is 3-8 mg, the particle size of the resulting heterojunction is 600-700 nm, and the band gap is 1.48-1.50 eV.

[0014] Furthermore, in step (2), the amide group in the polyvinylpyrrolidone molecule reacts with Bi... 3+ Coordination occurs, causing Bi 3+ Uniformly anchored on the Cu2O surface, under the guidance and steric hindrance protection of polyvinylpyrrolidone, I - with Bi 3+An in-situ reaction occurs and a BiOI nanolayer is nucleated and grown on the Cu2O surface, with a thickness of 5-20 nm.

[0015] It also includes the use of a pH-responsive bismuth-based nanomedicine in the preparation of a sonodynamic-chemodynamic synergistic antitumor drug.

[0016] Furthermore, the antitumor drug is an injectable nanomedicine formulation, which is used in conjunction with external ultrasound irradiation after administration, wherein the power density of the ultrasound irradiation is 0.5-2.0 W / cm². 2 The irradiation time is 3-10 minutes.

[0017] The beneficial effects of this invention are: Compared with existing technologies, this invention establishes the structure-activity relationship between the structural parameters and acoustic properties of bismuth-based heterojunction nanomedicines by controlling the volume ratio of anhydrous ethanol to water and the amount of polyvinylpyrrolidone in the synthesis system. This yields nanomedicines with controllable particle size, regular morphology, and optimal acoustic activity, overcoming the shortcomings of insufficient acoustic catalytic activity and unclear structure-performance correlation in existing bismuth-based acoustic sensitizers. Under ultrasonic excitation, this nanomedicine establishes a highly efficient charge transport channel through its heterostructure, inhibiting electron-hole pair recombination and significantly improving reactive oxygen species yield. Simultaneously, it intelligently responds to the acidic tumor microenvironment by releasing Cu. + Ions, through Cu + / Cu 2+ The continuous consumption of endogenous hydrogen peroxide and reduced glutathione through valence state transitions enhances the chemodynamic therapeutic effect through a Fenton-like reaction, achieving a synergistic amplification of sonodynamics and chemodynamics.

[0018] This invention further utilizes the released Cu + Ions bind to lipid-acylated tricarboxylic acid cycle proteins, inducing copper death. Simultaneously, excess reactive oxygen species activate the Caspase-1 / GSDMD-dependent pyroptosis pathway, synergistically triggering immunogenic cell death through a multimodal cell death mechanism. This promotes dendritic cell maturation and enhances T cell infiltration into tumor tissues, effectively reversing the immunosuppressive microenvironment and achieving a systematic synergy of sonodynamic therapy, chemodynamic therapy, and immune activation. This method features a simple preparation process, readily available and inexpensive raw materials, and surface hyaluronic acid modification endows the nanomedicine with good biocompatibility and targeting properties. While enhancing the efficacy of anti-breast cancer therapy, it significantly reduces toxic side effects on normal tissues, demonstrating high potential for clinical application and industrialization. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the synthesis and treatment mechanism of CBH.

[0020] Figure 2 This is a transmission electron microscope image of Cu2O.

[0021] Figure 3 Transmission electron microscopy image of CB-1 (ethanol and water volume ratio of 16:1, polyvinylpyrrolidone content of 15 mg).

[0022] Figure 4 The hydrated particle size of CB-1 is given.

[0023] Figure 5 Transmission electron microscopy image of CB-2 (ethanol and water volume ratio of 16:1, polyvinylpyrrolidone content of 5 mg).

[0024] Figure 6 The hydrated particle size of CB-2 is given.

[0025] Figure 7 Transmission electron microscopy image of CB-3 (ethanol and water volume ratio of 1:16, polyvinylpyrrolidone content of 5 mg).

[0026] Figure 8 The hydrated particle size of CB-3.

[0027] Figure 9 This is a transmission electron microscope image of CBH-1.

[0028] Figure 10 The hydrated particle size of CBH-1 is given.

[0029] Figure 11 XPS valence band spectra of three CBHs.

[0030] Figure 12 These are the solid-state UV diffuse reflectance spectra of three CBHs.

[0031] Figure 13 The relative absorbance of the three CBHs at the characteristic peak of 1,3-diphenylisobenzofuran (DPBF) at 426 nm changes over time.

[0032] Figure 14 The value represents the amount of hydroxyl radicals (·OH) generated in CBH-1 solution over time at a pH of 5.7.

[0033] Figure 15 The tumor volume growth curves of tumor-bearing mice under different treatment conditions are shown.

[0034] Figure 16 Immunohistochemical staining images of calreticulin and high-mobility cluster protein B1 in tumor sections of tumor-bearing mice under different treatment conditions; immunofluorescence staining images of helper lymphocytes 4 and 8 in tumor tissue under different treatment conditions.

[0035] Figure 17Immunofluorescence staining images of caspase 3, ferricopentin 1, glutathione peroxidase 4, desmopressin terminal fragment and caspase 1 in tumor tissue of tumor-bearing mice under different treatment conditions. Detailed Implementation

[0036] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0037] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to the figures and specific embodiments.

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and should not be construed as limiting the invention.

[0039] A pH-responsive bismuth-based nanomedicine, denoted as Cu2O@BiOI-HA, comprises hyaluronic acid-modified cuprous oxide / bismuth iodide heterojunction nanoparticles. The cuprous oxide has a bilayered hollow mesoporous structure, and bismuth iodide is loaded onto the cuprous oxide surface as a nanolayer to form a heterojunction. Hyaluronic acid is coated onto the heterojunction surface via electrostatic adsorption or chemical bonding. The nanomedicine has a particle size of 200-700 nm and a band gap of 1.46-1.49 eV. This technique improves the biocompatibility and tumor targeting of the nanomedicine through hyaluronic acid modification and achieves efficient charge separation using the heterojunction structure, making it suitable for sonodynamic-chemodynamic synergistic antitumor therapy.

[0040] Preferably, the nanomedicine releases Cu in response to pH 5.0-6.5 conditions. + ions, the Cu + Ions passing through Cu + / Cu 2+ The valence state transition consumes hydrogen peroxide and reduced glutathione, generating hydroxyl radicals via a Fenton-like reaction. The effectiveness of this technology lies in leveraging the weakly acidic nature of the tumor microenvironment to achieve intelligent drug release. It disrupts the redox balance by consuming high concentrations of glutathione within tumor cells, while simultaneously generating highly oxidizing hydroxyl radicals through a Fenton-like reaction, thereby enhancing oxidative damage to tumor cells. In practice, the nanomedicine is dispersed in a phosphate buffer solution at pH 5.7, and a TMB probe and hydrogen peroxide are added. The generation of hydroxyl radicals is verified by detecting changes in the absorbance of the system.

[0041] Preferably, the nanomedicine has an ultrasonic power density of 0.5-2.0 W / cm². 2 Under frequencies of 20 kHz to 3 MHz, electron-hole pair separation is promoted through band bending and band gap narrowing in heterojunctions, generating reactive oxygen species (ROS). The effectiveness of this technology lies in using external ultrasound to excite heterojunction nanomedicines and utilizing band structure modulation to inhibit electron-hole pair recombination, significantly improving the quantum yield of ROS, thereby achieving non-invasive and precise treatment of deep tissues. During implementation, a nanoparticle solution is mixed with a DPBF probe at a power of 1.0 W / cm². 2 Under ultrasonic conditions, the absorbance change at 426 nm was recorded every minute for 5 minutes, and the reactive oxygen species generation efficiency was evaluated by the degree of absorbance decay.

[0042] A method for preparing a pH-responsive bismuth-based nanomedicine includes the following steps: (1) dissolving hexadecyltrimethylammonium bromide in water, and adding Cu... 2+ (1) Add ascorbic acid to an aqueous solution and heat to 50-70℃, maintain for 15-30 min, add sodium hydroxide aqueous solution dropwise, centrifuge and wash to obtain Cu2O nanoparticles with a double-layer hollow mesoporous structure; (2) Disperse bismuth nitrate pentahydrate and polyvinylpyrrolidone in anhydrous ethanol-water mixed solution, add Cu2O nanoparticles and stir for 30-60 min, add potassium iodide solution dropwise, continue stirring for 20-40 min, transfer to microwave reactor and heat to 160-200℃ for 1.5-2.5 h, centrifuge and wash to obtain Cu2O@BiOI heterojunction, wherein by adjusting the volume ratio of anhydrous ethanol to water and the amount of polyvinylpyrrolidone, the particle size of the obtained heterojunction is 200-700 nm and the band gap is 1.46-1.49 eV; (3) Mix hyaluronic acid and Cu2O@BiOI heterojunction in water, disperse by ultrasonication and stir for 20-30 min. h, centrifugation and washing yielded the pH-responsive bismuth-based nanomedicine. The advantage of this technique lies in the controllable preparation of heterojunction nanomedicines through the combined use of soft template method and microwave-assisted synthesis, establishing the structure-property relationship between synthesis parameters and material properties, and providing process guidance for performance optimization of similar bismuth-based nanomedicines. In practice, CTAB and Cu... 2+ The assembled bilayer vesicles served as a soft template, and ascorbic acid reduced Cu. 2+ Cu2O is formed, and BiOI is then loaded onto the Cu2O surface in situ through PVP-assisted microwave synthesis. Finally, hyaluronic acid is surface-coated by ultrasonic dispersion.

[0043] Preferably, in step (2), the volume ratio of anhydrous ethanol to water is 16:1, the amount of polyvinylpyrrolidone is 10-20 mg, and the resulting heterojunction has a particle size of 200-250 nm and a band gap of 1.45-1.47 eV. The effect of this technology is to obtain heterojunction nanomedicines with small particle size and narrow band gap, which is beneficial to improve their penetration ability and reactive oxygen species yield in tumor tissues. In practice, 5 mg of bismuth nitrate pentahydrate and 15 mg of polyvinylpyrrolidone are dispersed in 8 mL of anhydrous ethanol-water mixed solution with a volume ratio of 15:1. 15 mg of Cu2O nanoparticles are added and stirred for 40 min. Anhydrous ethanol solution of 1.5 mM potassium iodide is added dropwise and stirred for another 30 min. After being transferred to a microwave tube, anhydrous ethanol-water solution is added to make the final volume ratio 16:1. The microwave reactor is heated to 180°C and maintained for 2 h.

[0044] Preferably, in step (2), the volume ratio of anhydrous ethanol to water is 16:1, the amount of polyvinylpyrrolidone is 3-8 mg, and the resulting heterojunction has a particle size of 300-350 nm and a band gap of 1.47-1.49 eV. The advantage of this technique is that by reducing the amount of polyvinylpyrrolidone, a medium-sized heterojunction nanomedicine can be obtained, achieving a balance between reactive oxygen species yield and material stability. In practice, 5 mg of bismuth nitrate pentahydrate and 5 mg of polyvinylpyrrolidone are dispersed in 8 mL of anhydrous ethanol-water mixed solution with a volume ratio of 15:1. The remaining operations are the same as above. The morphology and size of the final product are verified by transmission electron microscopy and hydration particle size determination.

[0045] Preferably, in step (2), the volume ratio of anhydrous ethanol to water is 1:16, the amount of polyvinylpyrrolidone is 3-8 mg, and the resulting heterojunction has a particle size of 600-700 nm and a band gap of 1.48-1.50 eV. The advantage of this technology is that it can obtain heterojunction nanomedicines with larger particle sizes, which is suitable for tumor models with low penetration depth requirements or applications that require prolonged in vivo circulation time. In practice, 5 mg of bismuth nitrate pentahydrate and 5 mg of polyvinylpyrrolidone are dispersed in 8 mL of anhydrous ethanol-water mixed solution with a volume ratio of 1:15. 15 mg of Cu2O nanoparticles are added and stirred for 40 min. A 1.5 mM potassium iodide aqueous solution is added dropwise, and stirring is continued for 30 min. After being transferred to a microwave tube, anhydrous ethanol-water solution is added to make the final volume ratio 1:16. The microwave reactor is heated to 180°C and maintained for 2 h.

[0046] Preferably, in step (2), the amide group in the polyvinylpyrrolidone molecule reacts with Bi... 3+ Coordination occurs, causing Bi 3 + Uniformly anchored on the Cu2O surface, under the guidance and steric hindrance protection of polyvinylpyrrolidone, I- with Bi 3+ An in-situ reaction occurs, and BiOI nanolayers are nucleated and grown on the Cu2O surface, with a thickness of 5-20 nm. The advantage of this technique lies in utilizing the dual effects of polyvinylpyrrolidone (PVP) to achieve uniform loading and controllable growth of BiOI on the Cu2O surface, forming a tight heterojunction that facilitates the rapid migration and separation of photogenerated carriers. In practice, the concentration of PPVP is controlled to regulate the growth of BiOI. 3+ The enrichment density on the Cu2O surface was used to adjust the thickness and coverage of the BiOI nanolayer, and the changes in valence band potential and band gap were verified by X-ray photoelectron spectroscopy and solid-state ultraviolet diffuse reflectance spectroscopy.

[0047] The application of a pH-responsive bismuth-based nanomedicine in the preparation of a sonodynamic-chemodynamic synergistic antitumor drug. The advantage of this technology lies in using the nanomedicine in the preparation of antitumor drugs, achieving multi-mechanism and multi-pathway tumor cell killing through the synergistic effect of sonodynamic and chemodynamic therapy, overcoming the drug resistance problem of single-treatment modalities. In practice, the nanomedicine is mixed with pharmaceutically acceptable excipients to prepare an injectable nanomedicine formulation for tumor treatment.

[0048] Preferably, the antitumor drug is an injectable nanomedicine formulation, which is used in conjunction with external ultrasound irradiation after administration, wherein the power density of the ultrasound irradiation is 0.5-2.0 W / cm². 2 The irradiation time is 3-10 minutes. The effect of this technology lies in achieving drug enrichment and in-situ activation at the tumor site through intratumoral injection combined with external ultrasound irradiation, improving treatment precision and reducing systemic toxicity. In practice, 100 μg / mL of the nanomedicine formulation is injected intratumorally, and 6 hours later, the tumor site is irradiated with a power density of 1.0 W / cm². 2 The mice were subjected to ultrasound irradiation for 5 minutes, and the administration and irradiation were repeated every 7 days for a two-week treatment cycle. During the treatment, the tumor volume and mouse weight were measured regularly. After the treatment, the tumor tissue was taken for immunohistochemistry and immunofluorescence staining analysis.

[0049] Example 1: Preparation of Cu2O@BiOI heterojunction nanomedicine (CB) according to Figure 1 As shown in the diagram, the overall synthesis route and sonodynamic-chemodynamic synergistic therapeutic mechanism of the pH-responsive bismuth-based nanomedicine of this invention clearly demonstrate the design concept and therapeutic principle of the nanomedicine. Figure 1 The synthesis process of hyaluronic acid-modified cuprous oxide / bismuth iodide heterojunction nanoparticles (Cu2O@BiOI-HA, denoted as CBH) is schematically depicted, as well as their generation of reactive oxygen species under ultrasonic excitation and release of Cu in response to the acidic tumor microenvironment.+ A complete therapeutic mechanism that integrates multiple cell death pathways, including ions, triggered copper death, and pyroptosis, and synergistically activates anti-tumor immune responses.

[0050] The preparation of the bismuth-based nanomedicine CB used in this invention includes the following steps: 2.98 g of CTAB was dissolved in 47 mL of H2O. While stirring rapidly, 7.8 mL of an aqueous solution containing 80 mg of copper nitrate trihydrate (Cu(NO3)2·3H2O) was added, followed by 0.3 g of ascorbic acid (AA). The mixture was stirred and heated to 60 °C, maintained for 20 min, and then 15 mL of an aqueous solution containing 100 mg of sodium hydroxide (NaOH) was added dropwise. The resulting solution was then centrifuged at high speed (10000 rpm, 10 min) and washed three times with ethanol (EtOH) and H2O. The resulting Cu2O nanoparticles were dispersed in H2O for later use. Figure 2 As shown, the prepared Cu2O nanoparticles have a double-layer hollow mesoporous structure, which is beneficial for the uniform loading of the subsequent BiOI nanolayer.

[0051] 5 mg of bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) and 15 mg of PVP were dispersed in 8 mL of a 15:1 volume ratio EtOH-H2O mixed solution. 15 mg of the aforementioned Cu2O nanoparticles were then added, and the mixture was stirred continuously for 40 min. Next, a 1.5 mM potassium iodide (KI) solution of EtOH was added dropwise, and the mixture was stirred for another 30 min. After transferring the mixture to a microwave tube, a 1:1 EtOH-H2O solution was added to ensure that the final volume ratio of EtOH to H2O in the mixture was 16:1. Figure 3 As shown, CB-1 prepared under these conditions exhibits a regular core-shell structure. According to... Figure 4 The hydrated particle size analysis results show that the particle size of CB-1 is approximately 230 nm.

[0052] 5 mg Bi(NO3)3·5H2O and 5 mg PVP were dispersed in 8 mL of a 15:1 EtOH-H2O mixed solution. 15 mg Cu2O nanoparticles were added, and the mixture was stirred continuously for 40 min. Then, a 1.5 mM KI solution of EtOH was added dropwise, and the mixture was stirred for another 30 min. After transferring the mixture to a microwave tube, a 1:1 EtOH-H2O solution was added to ensure that the final mixture had a 16:1 volume ratio of EtOH to H2O. Figure 5 As shown, the particle size of CB-2 prepared by reducing the amount of PVP increased. According to... Figure 6 As shown, the hydrated particle size of CB-2 is approximately 315 nm.

[0053] 5 mg Bi(NO3)3·5H2O and 5 mg PVP were dispersed in 8 mL of a 1:15 volume ratio EtOH-H2O mixed solution. 15 mg Cu2O nanoparticles were added, and the mixture was stirred continuously for 40 min. Then, a 1.5 mM KI H2O solution was added dropwise, and the mixture was stirred for another 30 min. After transferring the mixture to a microwave tube, a 1:1 EtOH-H2O solution was added to ensure that the final volume ratio of EtOH to H2O in the mixture was 1:16. Figure 7 As shown, when the solvent ratio was adjusted to be mainly aqueous, the particle size of the prepared CB-3 increased significantly. According to... Figure 8 As shown, the hydrated particle size of CB-3 is approximately 660 nm.

[0054] The three solutions were separately placed in a microwave reactor, and the temperature was programmed to rise to 180 °C and held for 2 hours. The resulting solutions were centrifuged at 10,000 rpm for 10 minutes and washed three times with EtOH and H2O. The final product was redispersed in H2O and stored at 4 °C.

[0055] according to Figure 9 As shown, transmission electron microscopy images of CBH-1 (i.e., CB-1 modified with hyaluronic acid) reveal that a uniform polymer layer forms on the surface of the nanoparticles after hyaluronic acid coating, while maintaining the overall morphology. According to... Figure 10 As shown, the hydrated particle size of CBH-1 is slightly increased compared to CB-1, indicating that hyaluronic acid was successfully coated on the surface of the nanoparticles.

[0056] The core materials described in this invention include CB-1, CB-2, and CB-3, with particle sizes of 230 nm, 315 nm, and 660 nm, respectively. According to... Figure 11 The XPS valence band spectra of the three CBHs shown indicate that the valence band potentials of CB-1, CB-2, and CB-3 change sequentially, reflecting the differences in the interface band structure of the heterojunction under different synthesis conditions. Based on... Figure 12 The solid-state UV diffuse reflectance spectroscopic analysis results of the three CBHs shown indicate that the band gaps of CB-1, CB-2, and CB-3 are 1.46 eV, 1.48 eV, and 1.49 eV, respectively, with the reactive oxygen species yield decreasing sequentially.

[0057] Example 2: Preparation of hyaluronic acid-modified Cu2O@BiOI heterojunction nanomedicine (CBH) according to Figure 1 As shown, hyaluronic acid modification is a key step in endowing nanomedicines with tumor targeting and biocompatibility. The method involves linking hyaluronic acid (HA) to CB and coating it onto the material surface, and includes the following steps: Dissolve 3 mg CB (CB-1, CB-2 or CB-3 respectively) and 15 mg HA in 20 mL of deionized water and sonicate (US) for 10 min to ensure that the components are fully dispersed.

[0058] The solution was stirred at 500 rpm for 24 hours to allow hyaluronic acid to be coated onto the surface of the heterojunction via electrostatic adsorption or chemical bonding. The mixture was then washed three times by centrifugation with deionized water, and the resulting products were finally redispersed in deionized water to obtain CBH-1, CBH-2, and CBH-3, respectively.

[0059] Example 3: Establishment and Performance Testing of a CBH-Based Treatment System according to Figure 1 As shown, the nanomedicine of this invention promotes electron-hole pair separation and generates reactive oxygen species through heterojunction band bending and band gap narrowing under ultrasonic excitation, while simultaneously releasing Cu in response to the acidic tumor microenvironment. + Ions generate hydroxyl radicals through a Fenton-like reaction, achieving synergistic therapy through sonodynamics and chemodynamics.

[0060] The ability to generate reactive oxygen species (ROS) under ultrasound (US) was evaluated using the reactive oxygen species probe DPBF. Different concentrations (0, 25, 50, 75, 100, 150 μg / mL) were used. -1 Take 3 mL of each of the nanoparticle solutions, add 40 μL of DPBF (80 μM), mix well, and heat at a power of 1 W / cm². -2 Under UV-Vis spectrometry, the absorbance change of DPBF at 426 nm was recorded every 1 minute using a UV-Vis spectrometer for 5 minutes. Figure 13 As shown, the relative absorbance of the three CBHs at the characteristic peak of 1,3-diphenylisobenzofuran (DPBF) at 426 nm over time indicates that CBH-1 has the highest reactive oxygen species yield, followed by CBH-2, and CBH-3 has the lowest. This is consistent with... Figure 12 The band gap variation patterns shown are consistent, that is, the narrower the band gap, the higher the reactive oxygen product yield.

[0061] The absorbance at 426 nm was normalized using the formula A = At ​​min / A0 min, where A0 min is the initial absorbance before US treatment, and At min is the absorbance after t minutes of US irradiation. Figure 13 As shown, CBH-1 exhibits the most significant decrease in the relative absorbance of DPBF within 5 minutes, indicating that it possesses the best acoustic dynamic performance.

[0062] The amount of hydroxyl radicals (·OH) generated by CBH-1 over time was detected using a TMB probe as an indicator. Different concentrations (0, 25, 50, 75, 100, 150 μg / mL) were used. -1 CBH-1 nanoparticles were dispersed in 3 mL of PBS (pH = 5.7 or pH = 7.4), and TMB (30 μL, 80 μM) and H2O2 (30 μL, 10 mM) were added. The absorbance of the reaction system was recorded as a function of time or concentration. Figure 14 As shown, CBH-1 can continuously generate hydroxyl radicals under weakly acidic conditions of pH = 5.7, and the amount generated increases with time, verifying the chemical kinetic response characteristics of this nanomedicine under acidic conditions in the tumor microenvironment.

[0063] Example 4: Animal experiments to verify anti-tumor effects according to Figure 1 As shown, the nanomedicine of this invention triggers multimodal cell death pathways such as copper death and pyroptosis through the synergistic effect of sonodynamics and chemodynamics, and synergistically activates anti-tumor immune responses to eliminate breast cancer cells.

[0064] Using 4T1 breast cancer cells as the tumor cell source, 6-week-old female Balb / c mice were selected to construct an animal model. The 4T1 tumor-bearing mice were randomly divided into 4 groups of 4 mice each to minimize the difference in tumor size. The groups were as follows: (1) Control group (200 μL PBS), (2) Ultrasound group (US), (3) CBH-1 (200 μL, 100 μg / mL), (4) CBH-1 + US (200 μL, 100 μg / mL). PBS or CBH was administered via intratumoral injection for two weeks, on day 0 and day 7, respectively. Six hours after the nanomedicine injection, the tumor sites in groups 3 and 4 were irradiated with US (1.0 W cm⁻¹). -2 (5 min).

[0065] Tumor size was measured using calipers on days 0, 2, 4, 6, 8, 10, 12, and 14, and mouse weight was measured using an analytical balance. According to... Figure 15 As shown in the figure, the tumor volume growth curves of tumor-bearing mice under different treatment conditions indicate that tumor growth in the CBH-1 + US group was significantly inhibited, with a significantly better tumor-suppressing effect than the CBH-1 alone group and the US alone group, while the tumors in the control group continued to grow rapidly. These results validate the superiority of the synergistic treatment of sonodynamics and chemodynamics.

[0066] After treatment, blood was collected via the eyeballs and refrigerated. Mice were then euthanized by cervical dislocation, and tumors, heart, lungs, liver, spleen, and kidneys were dissected and fixed in 4% paraformaldehyde fixative. All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee.

[0067] according to Figure 16 As shown, immunohistochemical staining images of calreticulin and high-mobility cluster protein B1 in tumor sections of tumor-bearing mice under different treatment conditions revealed significantly increased expression levels of calreticulin and high-mobility cluster protein B1 in the material and ultrasound (CBH-1 + US) groups, indicating that this treatment can effectively induce immunogenic cell death (ICD) and promote the release of damage-associated molecular patterns (DAMPs). Figure 16 As shown in the immunofluorescence staining images of helper lymphocytes 4 and 8 in tumor tissue under different treatment conditions, the number of tumor-infiltrating T cells in the CBH-1 + US group was significantly increased, indicating that this synergistic treatment can effectively activate the anti-tumor immune response and reverse the immunosuppressive microenvironment.

[0068] according to Figure 17 As shown, immunofluorescence staining images of caspase 3, ferricyanide 1, glutathione peroxidase 4, desmoplastic protein terminal fragment, and caspase 1 in tumor tissues of tumor-bearing mice under different treatment conditions indicate that: Figure 17 Immunofluorescence staining images of caspase 3 showed that the expression of activated caspase 3 was significantly upregulated in the CBH-1 + US group, suggesting activation of the apoptosis pathway; according to Figure 17 Immunofluorescence staining images of ferroredoxin 1 (FDX1) show that FDX1 expression levels were significantly increased in the CBH-1 + US group, combined with... Figure 1 The mechanism shown indicates that Cu + The successful release of ions induced copper death (Cuproptosis); according to Figure 17 Immunofluorescence staining images of glutathione peroxidase 4 (GPX4) showed downregulated GPX4 expression, suggesting that ferroptosis-related pathways may be involved in synergistic antitumor effects; according to Figure 17 Immunofluorescence staining images of the terminal fragment of gentamicin D protein and caspase 1 show that the expression of the N-terminal fragment of gentamicin D protein (GSDMD-N) and activated caspase 1 (c-Caspase 1) was significantly enhanced in the CBH-1 + US group, confirming that the Caspase-1 / GSDMD-dependent pyroptosis pathway was effectively activated.

[0069] according to Figures 1 to 17A comprehensive analysis of the experimental results shows that the pH-responsive bismuth-based nanomedicine CBH-1 described in this invention achieves highly efficient reactive oxygen species generation through heterojunction band structure optimization under ultrasonic excitation, while simultaneously releasing Cu in response to the acidic tumor microenvironment. + Ions enhance oxidative damage through a Fenton-like reaction and trigger immunogenic cell death through multimodal cell death pathways such as copper death and pyroptosis, ultimately achieving a systemic synergy of sonodynamic therapy, chemodynamic therapy, and immune activation, effectively inhibiting breast cancer growth and activating anti-tumor immune responses.

[0070] The study investigated the consistency between the therapeutic effect in a tumor-bearing mouse model and the reactive oxygen species generation effect of the nanomedicine. Based on... Figure 13 and Figure 15 The comparative analysis shown indicates that CBH-1 exhibits the best reactive oxygen species yield in vitro and the best tumor inhibition effect in animal experiments, with good consistency between the two, verifying the reliability of the structure-activity relationship of this nanomedicine.

[0071] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes and modifications made to the above-disclosed embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the protection scope of the technical solution of the present invention.

[0072] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A pH-responsive bismuth-based nanomedicine, characterized in that, The nanomedicine is a hyaluronic acid-modified cuprous oxide / bismuth iodide heterojunction nanoparticle, denoted as Cu2O@BiOI-HA. The cuprous oxide has a double-layer hollow mesoporous structure, and the bismuth iodide is loaded on the surface of the cuprous oxide in the form of a nanolayer to form a heterojunction. The hyaluronic acid is coated on the surface of the heterojunction through electrostatic adsorption or chemical bonding. The nanomedicine has a particle size of 200-700 nm and a band gap of 1.46-1.49 eV.

2. A pH-responsive bismuth-based nanomedicine as described above, characterized in that, The nanomedicine releases Cu in response to pH 5.0-6.5 conditions. + ions, the Cu + Ions passing through Cu + / Cu 2+ The valence transition consumes hydrogen peroxide and reduced glutathione, and generates hydroxyl radicals through a Fenton-like reaction.

3. A pH-responsive bismuth-based nanomedicine as described above, characterized in that, The nanomedicine operates at an ultrasonic power density of 0.5-2.0 W / cm². 2 Under the conditions of 20 kHz-3 MHz, electron-hole pair separation is promoted by the bending of the heterojunction band and the narrowing of the band gap, generating reactive oxygen species.

4. A method for preparing a pH-responsive bismuth-based nanomedicine as described above, characterized in that, Includes the following steps: (1) Dissolve hexadecyltrimethylammonium bromide in water, and add Cu-containing... 2+ (1) Add ascorbic acid to an aqueous solution and heat to 50-70℃, keep for 15-30 min, add sodium hydroxide aqueous solution dropwise, and centrifuge and wash to obtain Cu2O nanoparticles with a double-layer hollow mesoporous structure; (2) Disperse bismuth nitrate pentahydrate and polyvinylpyrrolidone in anhydrous ethanol-water mixed solution, add Cu2O nanoparticles and stir for 30-60 min, add potassium iodide solution dropwise, continue stirring for 20-40 min, transfer to microwave reactor and heat to 160-200℃ for 1.5-2.5 h, centrifuge and wash to obtain Cu2O@BiOI heterojunction, wherein by adjusting the volume ratio of anhydrous ethanol to water and the amount of polyvinylpyrrolidone, the particle size of the obtained heterojunction is 200-700 nm and the band gap is 1.46-1.49 eV; (3) Mix hyaluronic acid and Cu2O@BiOI heterojunction in water, disperse by ultrasonication and stir for 20-30 min. h, centrifugation and washing yielded the pH-responsive bismuth-based nanomedicine.

5. A preparation method as described in claim 4, characterized in that, In step (2), the volume ratio of anhydrous ethanol to water is 16:1, the amount of polyvinylpyrrolidone used is 10-20 mg, the particle size of the resulting heterojunction is 200-250 nm, and the band gap is 1.45-1.47 eV.

6. A preparation method as described in claim 4, characterized in that, In step (2), the volume ratio of anhydrous ethanol to water is 16:1, the amount of polyvinylpyrrolidone used is 3-8 mg, the particle size of the resulting heterojunction is 300-350 nm, and the band gap is 1.47-1.49 eV.

7. A preparation method as described in claim 4, characterized in that, In step (2), the volume ratio of anhydrous ethanol to water is 1:16, the amount of polyvinylpyrrolidone used is 3-8 mg, the particle size of the resulting heterojunction is 600-700 nm, and the band gap is 1.48-1.50 eV.

8. A preparation method as described in claim 4, characterized in that, In step (2), the amide group in the polyvinylpyrrolidone molecule reacts with Bi... 3+ Coordination occurs, causing Bi 3+ Uniformly anchored on the Cu2O surface, under the guidance and steric hindrance protection of polyvinylpyrrolidone, I - with Bi 3+ An in-situ reaction occurs and a BiOI nanolayer is nucleated and grown on the Cu2O surface, with a thickness of 5-20 nm.

9. The use of a pH-responsive bismuth-based nanomedicine as described in any one of claims 1-3 in the preparation of a sonodynamic-chemodynamic synergistic antitumor drug.

10. The use as described in claim 9, characterized in that, The antitumor drug is an injectable nanomedicine formulation, which is used in conjunction with external ultrasound irradiation after administration. The power density of the ultrasound irradiation is 0.5-2.0 W / cm². 2 The irradiation time is 3-10 minutes.

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