Preparation method of injectable BiOCl (at) CuOF127 (at) rGO thermosensitive hydrogel for superficial tumor treatment

Abstract

The invention relates to the technical field of medical treatment, in particular to a preparation method of injectable BiOCl (at) CuOF127 (at) rGO thermosensitive hydrogel for superficial tumor treatment, which comprises the following steps: pretreating raw materials, preparing BiOCl nanosheets by a solvothermal method, washing and drying for later use; according to the manufacturing method of the injectable BiOCl (at) CuOF127 (at) rGO thermosensitive hydrogel for superficial tumor treatment, the injectable BiOCl (at) CuOF127 (at) rGO thermosensitive hydrogel is embedded into injectable, conductive and thermosensitive hydrogel composed of PluronicF127 (F127) and reduced graphene oxide (rGO), the injectable BiOCl (at) CuOF127 (at) rGO thermosensitive hydrogel is used for combined treatment of breast cancer, remarkable ROS outbreak and effective breast cancer cell ablation are induced through in-vitro and synergistic therapy, toxicity to normal cells is minimum, and the injectable BiOCl (at) CuOF127 (at) rGO thermosensitive hydrogel can be applied to in-vivo and in-vivo treatment of breast cancer. In-situ breast tumors treated with US + NIR show obvious fading, proliferation and angiogenesis reduction, apoptosis and immunogen cell death pathway activation and good biocompatibility, an accurate and minimally invasive strategy is established, piezoelectric catalysis and photo-thermal conversion are combined, and the in-situ breast tumors are used for effective breast cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of medical technology, specifically to a method for manufacturing an injectable BiOCl@CuOF127@rGO thermosensitive hydrogel for the treatment of superficial tumors. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, posing a serious threat to their health. While traditional treatments such as surgery, chemotherapy, and radiotherapy are widely used, their effectiveness is often hampered by issues such as tumor recurrence and metastasis, treatment resistance, and unavoidable systemic toxicity. In recent years, piezoelectric catalytic therapy (PCT), as an emerging mechanically driven treatment approach, has shown considerable potential in cancer treatment. PCT utilizes piezoelectric nanomaterials to drive electron-hole separation under ultrasonic (US) stimulation, reacting with water and oxygen to generate reactive oxygen species (ROS), such as hydroxyl radicals (·OH) and singlet oxygen (1O2). These ROS cause oxidative damage to cancer cells, leading to cell death.

[0003] However, the effectiveness of PCT is limited by insufficient electron-hole pair separation efficiency and rapid carrier recombination under US excitation.

[0004] Based on this, the present invention provides a method for manufacturing an injectable BiOCl@CuOF127@rGO thermosensitive hydrogel for the treatment of superficial tumors, in order to solve the technical problems mentioned above. Summary of the Invention

[0005] The purpose of this invention is to provide a method for manufacturing an injectable BiOCl@CuOF127@rGO thermosensitive hydrogel for the treatment of superficial tumors, thereby solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention proposes a method for manufacturing an injectable BiOCl@CuOF127@rGO thermosensitive hydrogel for the treatment of superficial tumors, comprising the following steps:

[0008] S1. Pre-treat the raw materials and prepare BiOCl nanosheets by solvothermal method, then wash and dry them for later use;

[0009] S2. The BiOCl prepared in S1 was reacted with copper acetate in an organic solvent, and BiOCl@CuO heterostructure nanosheets were prepared by centrifugation, washing and drying.

[0010] S3. Disperse reduced graphene oxide in deionized water, add Pluronic F127 powder, and stir at low temperature to prepare F127@rGO composite solution;

[0011] S4. The BiOCl@CuO heterostructured nanosheets prepared in S2 were added to the F127@rGO composite solution obtained in S3 and stored at low temperature to obtain an injectable BiOCl@CuO / F127@rGO thermosensitive hydrogel.

[0012] S5. The hydrogel obtained in S4 was characterized in terms of performance. Based on the characterization results, the process parameters were adjusted to ensure that its sol-gel transition, conductivity and biocompatibility met the requirements for breast cancer treatment applications.

[0013] S6. Treat the waste liquid and waste generated during the preparation process in a harmless manner to ensure that the discharge meets the standards.

[0014] Preferably, the implementation process of step S1 is as follows: select Bi(NO3)3·5H2O with a purity ≥99%, mannitol, and NaCl as raw materials; dissolve 0.486g of bismuth nitrate pentahydrate in 25mL of 0.1mol / L mannitol solution and stir continuously for 30 minutes; then add 5mL of saturated NaCl solution dropwise and continue stirring for 30 minutes; transfer the mixed solution to a stainless steel autoclave with a Teflon liner and react at 160℃ for 3 hours; after the reaction is completed, collect the precipitate, wash it three times alternately with ethanol and deionized water, dry it at 60℃ for 4 hours, grind it into fine powder, and obtain BiOCl nanosheets for later use.

[0015] Preferably, the implementation process of step S2 is as follows: take 0.65g of BiOCl nanosheets prepared in S1 and 0.05g of Cu(CH3COO)2·H2O, add them to a conical flask containing 50.0mL of N,N-dimethylformamide, and stir continuously for 15 minutes; transfer the conical flask to a constant temperature water bath at 90℃ and stir the reaction for 4 hours; after the reaction is completed, cool to room temperature, centrifuge the product at 9000rpm for 5 minutes, wash it 3 times with deionized water, and dry it at 60℃ to obtain BiOCl@CuO heterostructure nanosheets.

[0016] Preferably, the implementation process of step S3 is as follows: Reduced graphene oxide is dispersed in 8 mL of deionized water at a concentration of 5 mg / mL, and uniform dispersion is achieved by ultrasonic treatment; then 1.9 g of Pluronic F127 powder is added, and the mixture is magnetically stirred at 4 °C for 4 hours to obtain a uniform F127@rGO composite solution.

[0017] Preferably, the implementation process of step S4 is as follows: the BiOCl@CuO heterostructured nanosheets prepared in S2 are added to the F127@rGO composite solution obtained in S3 in a set ratio, stirred evenly, and stored at 4°C overnight to obtain an injectable BiOCl@CuO / F127@rGO thermosensitive hydrogel; the hydrogel is in a fluid sol state at room temperature and rapidly transforms into an elastic gel state at 23-24°C, and its injectability and tissue adhesion meet the requirements for in vivo application.

[0018] Preferably, the implementation process of step S5 is as follows: the microstructure of the hydrogel and nanosheets is characterized by scanning electron microscopy and transmission electron microscopy to ensure that the BiOCl@CuO nanosheets are uniformly dispersed in the hydrogel; the zeta potential of the BiOCl@CuO nanosheets is analyzed by zetasizer, and the formation of BiOCl@CuO heterojunctions is verified by XRD and XPS; the sol-gel transition temperature, oscillation strain performance and self-healing performance of the hydrogel are tested by rheometer; the conductivity of the hydrogel is detected by conductivity testing device; if the characterization results do not meet the standards, the ratio of BiOCl to copper acetate or the reaction time in S2, or the concentration of reduced graphene oxide in S3, are adjusted until the material meets the standards for breast cancer treatment.

[0019] Preferably, the implementation process of step S6 is as follows: collecting the washing waste liquid generated during the preparation process of S1 and S2, neutralizing it to about pH 7 before discharging it; collecting the small amount of waste gas generated during the ultrasonic dispersion process, treating it through an activated carbon adsorption device before discharging it; and disposing of the waste containers and consumables used in the preparation process in accordance with medical waste regulations after high-temperature sterilization.

[0020] Preferably, the amount of BiOCl@CuO heterostructured nanosheets added in step S4 needs to meet the following requirements: the concentration of BiOCl@CuO in the final hydrogel is 50-200 μg / mL, ensuring that it has efficient photothermal conversion performance under near-infrared irradiation and can effectively generate reactive oxygen species under ultrasonic stimulation.

[0021] Preferably, in step S5, the biocompatibility of the hydrogel is further verified by an in vitro cytotoxicity test: when the concentration of BiOCl nanosheets is lower than 12.5 μg / mL, the survival rate of mouse NIH-3T3 fibroblasts is maintained above 95%; if the survival rate is lower than 95%, the concentration of BiOCl@CuO added in S4 is adjusted until the biocompatibility requirements are met.

[0022] Preferably, in step S1, the product yield of the solvothermal reaction in S1 and the heterojunction formation efficiency in S2 are recorded. When the yield is less than 75%, the solvothermal reaction temperature in S1 is increased by 5-10°C. When the heterojunction formation efficiency is insufficient, the amount of copper acetate in S2 is increased. Based on the characterization results in S5, the ultrasonic dispersion time and stirring speed parameters are recalibrated after every 5 batches of production to achieve stable process cycling.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention discloses a method for manufacturing an injectable BiOCl@CuOF127@rGO thermosensitive hydrogel for the treatment of superficial tumors. This hydrogel is embedded within an injectable, conductive, and thermosensitive hydrogel composed of Pluronic F127 (F127) and reduced graphene oxide (rGO) for combined treatment of breast cancer. Under ultrasound (US) stimulation, BiOCl@CuO establishes a strong interfacial electric field, enhancing charge separation and accelerating the generation of O2 and ·OH. Simultaneously, near-infrared (NIR) irradiation activates BiOCl@CuO to convert light into heat, increasing the intratumoral temperature and further amplifying ROS-mediated cytotoxicity, thus achieving photothermal effects. The therapeutic approach (PTT), integrating BiOCl@CuO / F127@rGO hydrogel, exhibits rapid sol-gel transition, strong tissue adhesion, and sustained local retention at physiological temperatures. In vitro, the synergistic therapy induces significant ROS bursts and effective ablation of breast cancer cells with minimal toxicity to normal cells. In vivo, orthotopic breast tumors treated with US+NIR show significant regression, reduced proliferation and angiogenesis, activation of apoptotic and immunogenic cell death pathways, and good biocompatibility. This establishes a precise, minimally invasive strategy that combines piezoelectric catalysis with photothermal conversion for effective breast cancer treatment. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the characterization of the BiOCl@CuO nanosheets of the present invention;

[0026] Figure 2 This is a schematic diagram of the in vitro catalytic and photothermal properties of the BiOCl@CuO nanosheets of this invention;

[0027] Figure 3 This is a schematic diagram illustrating the characterization of the BiOCl@CuO / F127@rGO hydrogel of the present invention;

[0028] Figure 4 This is a schematic diagram illustrating the in vitro tumor treatment effects of the BiOCl@CuO / F127@rGO hydrogel of the present invention under NIR irradiation in the United States;

[0029] Figure 5 This is a schematic diagram illustrating the in vivo antitumor effect of the present invention on in situ breast cancer.

[0030] Figure 6 This is a schematic diagram illustrating the in vivo immune activation of in situ breast cancer according to the present invention. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] For examples, please refer to Figures 1 to 6 This invention proposes a method for manufacturing an injectable BiOCl@CuOF127@rGO thermosensitive hydrogel for the treatment of superficial tumors, comprising the following steps:

[0033] S1. Pre-treat the raw materials and prepare BiOCl nanosheets by solvothermal method, then wash and dry them for later use;

[0034] S2. The BiOCl prepared in S1 was reacted with copper acetate in an organic solvent, and BiOCl@CuO heterostructure nanosheets were prepared by centrifugation, washing and drying.

[0035] S3. Disperse reduced graphene oxide in deionized water, add Pluronic F127 powder, and stir at low temperature to prepare F127@rGO composite solution;

[0036] S4. The BiOCl@CuO heterostructured nanosheets prepared in S2 were added to the F127@rGO composite solution obtained in S3 and stored at low temperature to obtain an injectable BiOCl@CuO / F127@rGO thermosensitive hydrogel.

[0037] S5. The hydrogel obtained in S4 was characterized in terms of performance. Based on the characterization results, the process parameters were adjusted to ensure that its sol-gel transition, conductivity and biocompatibility met the requirements for breast cancer treatment applications.

[0038] S6. Treat the waste liquid and waste generated during the preparation process in a harmless manner to ensure that the discharge meets the standards.

[0039] In this embodiment, it should also be noted that the implementation process of step S1 is as follows: Bi(NO3)3·5H2O with a purity ≥99%, mannitol, and NaCl are selected as raw materials; 0.486g of bismuth nitrate pentahydrate is dissolved in 25mL of 0.1mol / L mannitol solution and stirred continuously for 30 minutes; then 5mL of saturated NaCl solution is added dropwise and stirred for another 30 minutes; the mixed solution is transferred to a stainless steel autoclave with a Teflon liner and reacted at 160℃ for 3 hours; after the reaction is completed, the precipitate is collected, washed three times alternately with ethanol and deionized water, dried at 60℃ for 4 hours, and ground into fine powder to obtain BiOCl nanosheets for later use.

[0040] In this embodiment, it should also be noted that the implementation process of step S2 is as follows: 0.65g of BiOCl nanosheets prepared in S1 and 0.05g of Cu(CH3COO)2·H2O are added to a conical flask containing 50.0mL of N,N-dimethylformamide and stirred continuously for 15 minutes; the conical flask is transferred to a constant temperature water bath at 90℃ and stirred for 4 hours; after the reaction is completed, the mixture is cooled to room temperature, the product is centrifuged at 9000rpm for 5 minutes, washed 3 times with deionized water, and dried at 60℃ to obtain BiOCl@CuO heterostructure nanosheets.

[0041] In this embodiment, it should also be noted that the implementation process of step S3 is as follows: Reduced graphene oxide is dispersed in 8 mL of deionized water at a concentration of 5 mg / mL and uniformly dispersed by ultrasonic treatment; then 1.9 g of Pluronic F127 powder is added and magnetically stirred at 4 °C for 4 hours to obtain a uniform F127@rGO composite solution.

[0042] In this embodiment, it should also be noted that the implementation process of step S4 is as follows: the BiOCl@CuO heterostructure nanosheets prepared in S2 are added to the F127@rGO composite solution obtained in S3 in a set ratio, stirred evenly, and stored at 4°C overnight to obtain an injectable BiOCl@CuO / F127@rGO thermosensitive hydrogel; the hydrogel is in a flowing sol state at room temperature and rapidly transforms into an elastic gel state at 23-24°C, and its injectability and tissue adhesion meet the requirements for in vivo application.

[0043] In this embodiment, it should also be noted that the implementation process of step S5 is as follows: the microstructure of the hydrogel and nanosheets is characterized using scanning electron microscopy and transmission electron microscopy to ensure that the BiOCl@CuO nanosheets are uniformly dispersed in the hydrogel; the zeta potential of the BiOCl@CuO nanosheets is analyzed by zetasizer, and the formation of BiOCl@CuO heterojunctions is verified by XRD and XPS; the sol-gel transition temperature, oscillatory strain properties and self-healing properties of the hydrogel are tested by rheometer; the conductivity of the hydrogel is detected by conductivity testing device; if the characterization results are not up to standard, the ratio of BiOCl to copper acetate or the reaction time in S2, or the concentration of reduced graphene oxide in S3, is adjusted until the material meets the standards for breast cancer treatment.

[0044] In this embodiment, it should also be noted that the implementation process of step S6 is as follows: the washing waste liquid generated during the preparation process of S1 and S2 is collected, neutralized to about pH 7 and then discharged in compliance with the standard; a small amount of waste gas generated during the ultrasonic dispersion process is collected, treated by an activated carbon adsorption device and then discharged; the waste containers and consumables used in the preparation process are disinfected at high temperature and then disposed of in accordance with the medical waste regulations.

[0045] In this embodiment, it should also be noted that the amount of BiOCl@CuO heterostructure nanosheets added in step S4 must meet the following requirements: the concentration of BiOCl@CuO in the final hydrogel is 50-200 μg / mL, ensuring that it has efficient photothermal conversion performance under near-infrared irradiation and can effectively generate reactive oxygen species under ultrasonic stimulation.

[0046] In this embodiment, it should also be noted that in step S5, the biocompatibility of the hydrogel needs to be verified by an in vitro cytotoxicity test: when the concentration of BiOCl nanosheets is lower than 12.5 μg / mL, the survival rate of mouse NIH-3T3 fibroblasts is maintained above 95%; if the survival rate is lower than 95%, the concentration of BiOCl@CuO added in S4 is adjusted until it meets the biocompatibility requirements.

[0047] In this embodiment, it should also be noted that in step S1, the product yield of the solvothermal reaction in S1 and the heterojunction formation efficiency in S2 are recorded. When the yield is less than 75%, the solvothermal reaction temperature in S1 is increased by 5-10°C. When the heterojunction formation efficiency is insufficient, the amount of copper acetate in S2 is increased. Based on the characterization results in S5, the ultrasonic dispersion time and stirring speed parameters are recalibrated after every 5 batches of production to achieve stable process cycling.

[0048] Please see Figure 1(AK), where A, B) SEM images of BiOCl (A) and BiOCl@CuO (B). C) TEM image of BiOCl@CuO. D) Representative EDS mapping of BiOCl@CuO; E) Size distribution of BiOCl@CuO. F) Zeta potentials of BiCl, CuO, and BiOCl@CuO. G) XRD pattern of BiOCl@CuO. H) XPS spectrum of BiOCl@CuO. 1) High-resolution XPS spectrum of Bi4f of BiOCl@CuO. J) High-resolution XPS spectrum of Cu2p for BiOCl@CuO. K) High-resolution XPS spectrum of O1s for BiOCl@CuO.

[0049] BiOCl nanosheets were synthesized by a solvothermal method, and then CuO was grown in situ on BiOCl to form a composite heterostructure (BiOCl@CuO) according to a published scheme. Figure 1 A and B are scanning electron microscope (SEM) images of BiOCl and BiOCl@CuO, respectively, showing CuO uniformly distributed on the nanosheets. Figure 1 Transmission electron microscopy and energy-dispersive X-ray spectroscopy (EDS) images of Bi, O, Cl, and Cu in C and D further confirm the successful synthesis of BiOCl@CuO. These nanosheets have a size distribution between 100-400 nm and an average diameter of 260 nm. Figure 1 E). Figure 1 F shows the zeta potential results for BiOCl (-20 mV), CuO (-15 mV), and BiOCl@CuO (-25 mV). The addition of CuO further enhances the surface charge of BiOCl@CuO, improving its stability and dispersibility. X-ray diffraction (XRD) results ( Figure 1 (G) indicates that the BiOCl@CuO nanosheets simultaneously possess characteristic peaks of both BiOCl and CuO. This further confirms the heterojunction of BiOCl and CuO. X-ray photoelectron spectroscopy (XPS) results reveal the chemical state of the BiOCl@CuO heterojunction. Figure 1 HK). Bi, O, Cl, and Cu were present in all samples. Calculations showed that the atomic ratios of Bi:O:Cl and Cu:O were 1:1:1 and 1:1, respectively. In BiOCl@CuO ( Figure 1 In H), the spin-orbit splitting photoelectron shifts of Bi(4f5 / 2) and Bi(4f7 / 2) reached 175.4 eV and 169.3 eV, respectively, indicating a strong interaction between CuO nanoparticles and BiOCl nanosheets at their interface. Figure 1 I). Figure 1J represents the high-resolution XPS spectrum of Cu2p. The Cu2p orbital splits into two main peaks, Cu2p1 / 2 and Cu2p3 / 2, with a separation of approximately 20 eV, a typical characteristic of copper. Furthermore, two distinct satellite peaks appear at higher binding energies of the main peaks, further confirming the presence of Cu2+. In the O1s spectrum, two distinct binding energy peaks were found. The lower binding energy peak is related to lattice oxygen, such as Cu-O and Bi-O bonds, while the higher binding energy peak is related to adsorbed oxygen, which is caused by the formation of oxygen vacancies. Figure 1 These results confirm the formation of the BiOCl@CuO heterojunction.

[0050] Please see Figure 2 (AG), where: A) ESR spectra of 10°C trapped by 2,2,6,6-tetramethylpiperidine (TEMP) and BiOCl@CuO nanosheets under ultrasonic treatment; B) ESR spectra of ·OH trapped by 5; absorbance of 5-dimethyl-1-pyridine n-oxide (DMPO) BiOCl@CuO nanosheets at different times under ultrasonic treatment; C) absorbance of BiOCl@CuO nanosheets at different times under ultrasonic treatment; E) UV absorbance of BiOCl, CuO, and BiOCl@CuO nanosheets; F) temperature of BiOCl@CuO nanosheets under different light intensities; G) temperature of BiOCl@CuO nanosheets of different concentrations under photostimulation; H) photothermal cycling test of BiOCl@CuO nanosheets.

[0051] The generation process of ROS under US influence was analyzed using electron spin resonance (ESR) spectroscopy. As shown in Figure A, the 1O2 signal in the H2O2 and H2O2+US groups is negligible, while the signal in the BiOCl@CuO+H2O2 group is slightly enhanced. In contrast, the BiOCl@CuO+H2O2+US group exhibits a significant increase in 1O2 signal. Figure 2 A). For example Figure 2 As shown in Figure B, compared with the H2O2, H2O2+US, and BiOCl@CuO+H2O2 groups, the BiOCl@CuO+H2O2+US group also exhibited a significant ·OH signal intensity. In conclusion, BiOCl@CuO can effectively generate ROS under ultrasonic irradiation. Figure 2 As shown in Figure C, the absorbance value of methylene blue (MB) gradually decreased from 0 to 10 min with increasing treatment time, indicating that the ·OH generated by BiOCl@CuO increased under ultrasonic treatment. Furthermore, Figure 2D showed a significant 3,3',5,5'-Tetramethylbenzidine (TMB) oxidation absorbance, which gradually increased with ultrasound treatment on BiOCl@CuO. From 0 to 10 min, the absorbance gradually increased with increasing treatment time. This indicates that BiOCl@CuO has a high ROS generation capacity under ultrasound treatment. Figure 2 E shows that BiOCl@CuO nanosheets exhibit stronger light absorption intensity than BiOCl and CuO in the 400-1000 nm wavelength range. Furthermore, with gradually increasing NIR intensities (0.5, 1, and 1.5 W / cm²), the temperature of the nanosheets gradually increases. Figure 2 F). The temperature change of PBS under NIR stimulation was negligible. In contrast, BiOCl@CuO nanosheets could effectively generate a thermal effect, and the temperature rise was greater with increasing concentration (50, 100, 200 μg / mL). Figure 2 G). These BiOCl@CuO nanosheets exhibit excellent photothermal cycling stability (G). Figure 2 H).

[0052] Then, a thermosensitive hydrogel was synthesized using F127 as a raw material. Subsequently, graphene oxide (GO) and BiOCl@CuO nanosheets were introduced and added to the F127 hydrogel through simple mixing to form the final hydrogel (BiOCl@CuO / F127@GO). Figure 3 As shown in Figure A, before and after the addition of BiOCl@CuO, the hydrogel remains fluid at room temperature and rapidly gels at body temperature. Furthermore, the BiOCl@CuO / F127@GO hydrogel exhibits strong injectability. Figure 3 SEM images of B show successful incorporation of BiOCl@CuO while maintaining the hydrogel structure. Figure 3 C shows the Fourier transform infrared (FTIR) spectra of F127, F127@GO, and BiOCl@CuO / F127@GO hydrogels. Characteristic peaks for F127 and F127@GO were observed in BiOCl@CuO / F127@GO, indicating successful synthesis of the composite hydrogel. The conductivity of the BiOCl@CuO / F127 hydrogel is approximately 3500 S·m⁻¹.

[0053] Please see Figure 3(AG), where A) Photograph of BiOCl@CuO / F127@rGO hydrogel. B) SEM image of FHG hydrogel. Red arrows indicate GO. C) FTIR spectrum of BiOCl@CuO / F127@rGO hydrogel. D) Electrical conductivity of BiOCl@CuO / F127 and BiOCl@CuO / F127@rGO hydrogels. E) Oscillatory strain test of BiOCl@CuO / F127@rGO hydrogel. F) Dynamic rheological test of BiOCl@CuO / F127@rGO hydrogel. The ordinate represents the value of storage modulus (G') / loss modulus (G”). G) Stepped strain rheological test of BiOCl@CuO / F127@rGO hydrogel.

[0054] Adding reduced graphene oxide to BiOCl@CuO / F127 hydrogel increased the conductivity to approximately 4250 S·m⁻¹. Figure 3 D). The results showed that the incorporation of reduced graphene oxide significantly improved the conductivity of the hydrogel. Oscillatory strain experiments indicated that the composite viscosity of both F127@rGO and BiOCl@CuO / F127@rGO hydrogels decreased as the oscillatory strain increased from 0% to 100%. Figure 3 E). This indicates that the hydrogel has been processed to have excellent injectability. Figure 3 F represents the temperature-dependent changes in G′ and G″ of the BiOCl@CuO / F127@rGO hydrogel. As the temperature gradually increases from 10℃ to 23℃, the changes in G′ and G″ values ​​of the BiOCl@CuO / F127@rGO hydrogel are significant. When the temperature approaches the 23℃-24℃ range, the storage modulus begins to increase rapidly, while the loss G″ also increases, albeit at a relatively small rate. When the temperature reaches a certain level, the value of G′ exceeds G′, and the gap gradually widens, indicating that the material gradually transitions from a predominantly viscous state to a predominantly elastic gel state. The results demonstrate that the BiOCl@CuO / F127@rGO hydrogel exhibits good temperature sensitivity. Figure 3 The G-distribution model shows that the composite viscosity initially remains at a relatively high level. As time approaches 100 seconds, the composite viscosity drops sharply to a lower value and remains at this low level until around 200 seconds. Subsequently, it recovers to a higher level after 200 seconds and remains stable. This trend indicates that the BiOCl@CuO / F127@rGO hydrogel possesses shear thinning and self-healing properties. This ensures the injectability of the hydrogel.

[0055] To further investigate the therapeutic effects of BiOCl@CuO / F127@rGO hydrogel on cancer under US and NIR irradiation conditions, a series of experiments were conducted. The cytotoxicity of BiOCl@CuO / F127@rGO hydrogel to normal cells was assessed using a Cell Counting Kit-8 (CCK-8) assay. Figure 4 As shown in Figure A, after 24 hours of treatment with BiOCl@CuO / F127@rGO, the survival rate of mouse NIH-3T3 fibroblasts remained above 95% when the BiOCl nanosheet concentration was below 12.5 μg / mL, with no statistically significant difference compared to the control group. When the BiOCl concentration exceeded 12.5 μg / mL, the cell survival rate of the BiOCl@CuO / F127@rGO+US+NIR group decreased slightly. This observation confirms that the BiOCl@CuO / F127@rGO hydrogel has good cell compatibility with normal cells and does not induce significant cytotoxicity under US and NIR irradiation.

[0056] Please see Figure 4 (AF), where: A) Cell viability of NIH-3T3 cells after treatment with BiOCl@CuO / F127@rGO and BiOCl@CuO / F127@rGO+US+NIR (n=3). B) Cell viability of HEPA1-6 cells after treatment with BiOCl@CuO / F127@rGO and BiOCl@CuO / F127@rGO+US+NIR (n=3). C) Live / dead staining results of HEPA1-6 cells after treatment with Control, F127@rGO, and BiOCl@CuO / F127@rGO, and BiOCl@CuO / F127@rGO+US+NIR, and BiOCl@CuO / F127@rGO+US+NIR. No experimental group served as a control. Live cells and dead cells were stained green and red, respectively. D) Corresponding statistical results showing the percentage of live cells (n=3). E) Fluorescence images of HEPA1-6 cells treated with control, F127@rGO, BiOCI@CuO / F127@rGO, BiOCI@CuO / F127@rGO+US+NIR, and BiOCI@CuO / F127@rGO+US+NIR. (n=3). Untreated cells served as the control group. Cell nuclei were stained blue. F) Corresponding statistical results show the relative mean fluorescence intensity in E (n=3). Data are expressed as mean SDs. p-values ​​were calculated using one-way ANOVA, “p>0.05 and **p<0.0001”.

[0057] Figure 4B shows the cell viability of 4T1 cells exposed to BiOCl@CuO / F127@rGO hydrogel under different concentrations of BiOCl nanosheets (0-400 μg / mL) in ultrasound and NIR treatment and no treatment. When the BiOCl nanosheet concentration was below 25 μg / mL, there was no significant difference in cell viability between the BiOCl@CuO / F127@rGO group and the BiOCl@CuO / F127@rGO+US+NIR group. Importantly, when the threshold concentration exceeded 25 μg / mL, the BiOCl@CuO / F127@rGO hydrogel+US+NIR group showed concentration-dependent enhancement of cytotoxicity, with significantly reduced cell viability compared to the BiOCl@CuO / F127@rGO control group. These significant differences indicate that BiOCl@CuO / F127@rGO hydrogel exhibits enhanced antitumor activity under US and NIR irradiation. Figure 4 Live / Dead staining results in C and D further validated the significant tumor cell killing ability of BiOCl@CuO / F127@rGO hydrogel under US and NIR assays. These results are consistent with the CCK-8 assay results. Figure 4 C,D). The changes in intracellular ROS levels in different groups of cells were studied using 2,7-dichlorofluorescein (DCFH-DA). Figure 4 (E, F). Among the Control, F127@rGO, BiOCl@CuO / F127@rGO, and BiOCl@CuO / F127@rGO+US+NIR groups, the BiOCl@CuO / F127@rGO+US+NIR group had the highest intracellular ROS level. In summary, these results indicate that under the influence of US and NIR, BiOCl@CuO / F127@rGO hydrogel can significantly promote the generation of free radicals, thereby inducing cellular oxidative stress and mediating tumor cell death.

[0058] Please see Figure 5 (AE), where (A) Schematic diagram of cancer treatment schedule. (B) Photograph of tumor tissue removed at the end of the treatment period (n=5). (C) Mean tumor weight after removal on day 14 (n=5). (D) Representative tumor growth curves of different groups of mice during the 14-day treatment period (n=5). (E) H&E staining; immunohistochemical staining of Caspase-3, Ki-67 and CD31 in tumor tissues of the control group, F127@rGO hydrogel+US+NIR group, BiOCI@CuO / F127@rGO hydrogel group and BiOCI@CuO / F127@rGO hydrogel+US+NIR group.

[0059] To evaluate the in vivo antitumor effect of BiOCI@CuO / F127@rGO hydrogel, a breast cancer mouse model was established in this invention, such as... Figure 5 As shown in Figure A. On day 12 post-inoculation, mice were randomly divided into a control group, an F127@rGO hydrogel + US + NIR group, a BiOCI@CuO / F127@rGO hydrogel group, and a BiOCI@CuO / F127@rGO hydrogel + US + NIR group. After 14 days of treatment, quantitative analysis showed that the BiOCI@CuO / F127@rGO hydrogel + US + NIR group had a stronger tumor growth inhibitory effect compared to the other groups. Figure 5 B). For example Figure 5 As shown in C and D, the tumor weight and volume were significantly reduced in the BiOCI@CuO / F127@rGO hydrogel + US + NIR group. Notably, tumor regression was observed in the BiOCI@CuO / F127@rGO hydrogel + US + NIR group, achieving an 80% tumor remission rate. This not only indicates that tumor growth has stopped but also that the tumor burden has been truly reduced.

[0060] Histopathological analysis using hematoxylin and eosin (H&E) staining confirmed a significant loss of malignant cells in the BiOCI@CuO / F127@rGO hydrogel + US + NIR group. Figure 5 E). Immunohistochemical analysis showed that, compared with the control group, the BiOCI@CuO / F127@rGO+US+NIR group exhibited significantly upregulated Caspase-3 expression, confirming robust activation of the tumor apoptosis pathway. Simultaneously, the decreased expression of Ki-67 and CD31 demonstrated effective inhibition of tumor growth and angiogenesis.

[0061] Please see Figure 6 (AE), where (A) is a representative FACS analysis image of CD86+CD80+ DCs after treatment. (B) is a representative FACS analysis image of the proportion of CD4+ and CD8+ T cells in T cells after treatment. (C) is a representative FACS analysis image of IFN-γ+CD8+ T cells after treatment. (D) ELISA detection of the secretion levels of cytokines TNF-α and IL-6 in mouse serum (n=3). (E) H&E staining of the heart, mammary gland, spleen, lung, and kidney of mice in the control group, F127@rGO hydrogel+US+NIR group, BiOCl@CuO / F127@rGO hydrogel group, and BiOCl@CuO / F127@rGO hydrogel+US+NIR group. Data are expressed as mean ± SDs. p-values ​​were calculated by one-way ANOVA, with nsp>0.05 and p<0.0001.

[0062] To systematically investigate the immunomodulatory effects of BiOCI@CuO / F127@rGO hydrogel under US and NIR irradiation, this invention further evaluated the activation of tumor-infiltrating immune cells. Flow cytometry confirmed that, under both US and NIR irradiation, the co-stimulatory molecules CD80 and CD86 of dendritic cells (DCs) were significantly upregulated in tumors treated with BiOCI@CuO / F127@rGO hydrogel. Figure 6 A) demonstrated effective activation of antigen-presenting cells. This combination therapy also significantly increased the proportion of CD8+ T cells and their IFN-γ secretion in tumor-infiltrating lymphocytes. Figure 6 B, C) indicates robust activation of the cytotoxic CD8+ T cell response. At the systemic immune level, this treatment regimen elicited a significant inflammatory response, characterized by significantly elevated serum levels of key cytokines (including TNF-α and IL-6), as measured by ELISA (…). Figure 6 D). The above results indicate that type I and type II immune responses were successfully initiated. Importantly, a comprehensive safety assessment based on histological analysis of major organs showed no signs of pathological changes or inflammatory infiltration. Figure 6 E) confirmed the excellent in vivo biocompatibility of the BiOCI@CuO / F127@rGO hydrogel. This BiOCI@CuO / F127@rGO hydrogel-mediated piezoelectric catalysis and thermotherapy combined therapy demonstrates that it can directly induce apoptosis of immunogenic tumor cells through piezoelectric catalysis and thermotherapy, while simultaneously activating DCs and CD8+ effector cytotoxic T cells, thereby producing a multimodal therapeutic effect and establishing a pro-inflammatory tumor microenvironment. The synergistic integration of locally efficient piezoelectric catalysis and photothermal effects with systemic immune activation makes the BiOCI@CuO / F127@rGO hydrogel a novel precision therapeutic platform for breast cancer, possessing the dual advantages of targeted tumor ablation and immune-mediated protection.

[0063] In summary, this invention overcomes the inherent limitations of PCT by developing a multifunctional therapeutic system based on BiOCl@CuO heterostructure nanosheets. This system embeds an injectable, conductive, and thermosensitive hydrogel formulated with F127 and graphene oxide. Under US irradiation, it generates a strong interfacial electric field, effectively inhibiting electron-hole recombination and accelerating the generation of tumor-lethal ROS. Simultaneously, NIR irradiation activates the photothermal conversion capacity of BiOCl@CuO, increasing intratumoral temperature, enhancing ROS-mediated cytotoxicity, and promoting PTT, achieving piezocatalysis-photothermal synergistic treatment. In vivo, tumors treated with combined US and NIR irradiation showed significant regression, while simultaneously inhibiting tumor cell proliferation and angiogenesis, and activating apoptosis and immunogenic cell death pathways. Importantly, the entire system maintains good biocompatibility. This study establishes a precise, minimally invasive, and spatiotemporally precise therapeutic strategy, innovatively combining piezocatalysis with photothermal conversion. This system overcomes the limitations of PCT, utilizing multimodal therapeutic synergy, and provides a promising and effective method for breast cancer treatment.

[0064] An experiment was conducted on the manufacturing method of the injectable BiOCl@CuOF127@rGO thermosensitive hydrogel for the treatment of superficial tumors according to the present invention. The steps are as follows:

[0065] 1. Prepare the following materials: bismuth nitrate pentahydrate (Bi(NO3)3·5h2o), mannitol, sodium chloride (NaCl), and copper acetate. Unless otherwise specified, all are commercially available.

[0066] 2. Monohydrate (Cu(CH3COO)2·h2O), N,N-dimethylformamide (C3H7NO, DMF), and anhydrous ethanol were all from Aladdin (China). Pluronic F127, reduced graphene oxide, tetramethylbenzidine (TMB), methylene blue (MB), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (USA). Phosphate-buffered saline (PBS), Cell Count Kit-8 (CCK-8), and calcixanthine / PI cell viability / cytotoxicity assay kit were purchased from Beyotime Biotechnology (China). NIH-3T3 mouse fibroblasts and 4T1 mouse breast cancer cells were obtained from the Procell system (China). Female C57BL / 6 mice were obtained from Charles River Laboratories (China). The protease inhibitor cocktail was purchased from USAT Hermo Scientific. All fluorescently labeled antibodies used in the experiments were purchased from Biolegend (USA). The TNF-α and IL-6 ELISA kits were purchased from ThermoScientific (USA).

[0067] 2. Characteristic Analysis:

[0068] Scanning electron microscopy (SEM) images were obtained using a field emission Magellan 400 microscope (FEI Corporation, USA). Transmission electron microscopy (TEM) images were obtained using a JEM-2100F electron microscope (200 kV). Zeta potentials were analyzed using zeta sizing nanosizing systems (zetaPlus, Brookhaven Instruments Corporation). X-ray diffraction (XRD) analysis was performed on a Rigaku D / MAX-2200PCXRD system. X-ray photoelectron spectroscopy (XPS) analysis was performed using an ESCAlab250 (Thermal Scientific, US). ESR signals were recorded on an ESR spectrometer (JEOL-fa200, JEOL, Japan). UV-Vis absorption spectra were recorded on a UV-3600 Shimadzu UV-Vis spectrometer. Temperature changes under 808 nm NIR irradiation were monitored in real time using a FLIRA 655sc infrared thermal imager. Confocal images were acquired using a Carl Zeiss LSM 900 confocal laser scanning microscope (CLSM). Flow cytometry analysis was performed on a BDL SFR Ortessa flow cytometer.

[0069] 3. Preparation of BiOCl@CuO:

[0070] BiOCl nanosheets were prepared via a one-pot solvothermal method. In short, 0.486 g of bismuth nitrate pentahydrate (Bi(NO3)3·5H2O) was dissolved in 25 mL of 0.1 mol / L mannitol solution and stirred continuously for 30 minutes. Then, 5 mL of saturated NaCl solution was added dropwise to the mixture, followed by further stirring for 30 minutes. The solution was then transferred to a Teflon-lined stainless steel autoclave and reacted at 160 °C for 3 hours.

[0071] In the solvothermal reaction, the precipitate formed was collected and washed alternately with ethanol and deionized water, three times with each solvent. The washed product was dried at 60°C for 4 hours and ground into a fine powder for subsequent use. To prepare BiOCl@CuO, 0.65 g of prepared BiOCl and 0.05 g of Cu(CH3COO)2·H2O were added to an Erlenmeyer flask containing 50.0 mL of LDM. The mixture was stirred continuously for 15 minutes, and then the Erlenmeyer flask was transferred to a constant temperature water bath at 90°C and stirred for an extended period (4 hours). When the solution cooled to room temperature, the BiOCl@CuO composite material was collected, centrifuged at 9000 rpm for 5 minutes, washed three times with deionized water, and dried at 60°C for subsequent experiments.

[0072] 4. Preparation of BiOCl@CuO / F127@rGO hydrogel:

[0073] First, reduced graphene oxide was dispersed at a concentration of 5 mg / mL in 8 mL of deionized water and uniformly dispersed by ultrasonic treatment. Then, 1.9 g of F127 powder was added to the reduced graphene oxide dispersion, and the mixture was magnetically stirred at 4 °C for 4 h to obtain an F127@rGO composite solution. Finally, the previously prepared BiOCl@CuO was added to the F127@rGO solution, and the BiOCl@CuO / F127@rGO solution was stored overnight at 4 °C.

[0074] The experiment details include:

[0075] Chemical kinetic properties:

[0076] To detect the generation of hydroxyl radicals (·OH) during the chemical kinetics of BiOCl@CuO in the presence of H2O2, MB and TMB were used as colorimetric probes. Specifically, 200 μL of BiOCl@CuO (7 mg / mL) suspension was added to 7 mL of MB solution (5 mg / mL). Subsequently, 10 μL of 30% H2O2 solution was added to the mixture. The mixture was irradiated with UV at different times (0, 1, 2, 5, 8, 9, and 10 min), then centrifuged, and the supernatant was analyzed by UV-Vis absorption spectroscopy. For TMB detection, 200 μL of BiOCl@CuO (7 mg / mL) was added to 7 mL of TMB solution (5 mg / mL). Subsequently, 10 μL of 30% H2O2 solution was added to the mixture. The mixture was irradiated with UV at different times (0, 2, 4, 6, 8, and 10 min), then centrifuged, and the supernatant was analyzed by UV-Vis absorption spectroscopy.

[0077] Photothermal properties of BiOCl@CuO:

[0078] Synthesized BiOCl@CuO was dispersed in deionized water to prepare a suspension containing α at a concentration of 0.2 mg / mL. The 0.2 mL suspension was transferred to a 1.5 mL sterile conical centrifuge tube. For photothermal performance evaluation, the suspension was irradiated with 808 nm NIR, and the real-time temperature change during irradiation was recorded using a thermal imager (FLIRA 655sc, USA). To investigate the effect of BiOCl@CuO concentration on its photothermal performance, the concentration of BiOCl@CuO was adjusted to 0, 50, 100, and 200 μg / mL, while other experimental conditions remained constant. The temperature rise curves of the suspension at each concentration were recorded. Similarly, to elucidate the effect of laser power density on the photothermal effect, the concentration of BiOCl@CuO was kept constant at 200 μg / mL, while the laser power density varied to 0.5, 1.0, and 1.5 W / cm². At each power density, the temperature evolution of the suspension was monitored to evaluate the photothermal response. The photothermal stability of BiOCl@CuO was tested using four consecutive heating-cooling curves at a concentration of 200 μg / mL and a power density of 1 W / cm². The temperatures were recorded every 30 seconds.

[0079] Rheological testing:

[0080] The rheological properties of the prepared BiOCl@CuO / F127@rGO hydrogels were determined using a Physical MCR301 rheometer (Anton Paar GmbH, Austria). To minimize volume shrinkage caused by water evaporation, the hydrogel samples were carefully deposited on the lower plate of the rheometer, and a thin layer of high-purity, low-density mineral oil was applied around the edges of the hydrogels to seal them. For dynamic strain scanning tests, the applied strain ranged from 0.1% to 100%. The heating rate was 1 °C / min, with the temperature increasing from 10 °C to 42 °C. Frequency, amplitude, and temperature scan measurements were performed within the linear viscoelastic region.

[0081] In vitro cell viability assay:

[0082] NIH-3T3 cells (1×10⁵) were cultured in 96-well plates and treated with BiOCl@CuO / F127@rGO hydrogels at concentrations of 0, 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL under US and NIR irradiation. After 24 h of incubation, cytotoxicity was determined using the CCK-8 assay. 100 μL of CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 60 min. The concentration of formaldehyde dye was measured at 450 nm using a MultiscanMK3 (Thermo, USA) microplate reader. Each data point was analyzed in triplicate.

[0083] Apoptosis analysis:

[0084] CCK-8 assay: 1×10⁵ 4T1 cells were cultured in 96-well plates and treated with BiOCl@CuO / F127@rGO hydrogels at concentrations of 0, 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL under US and NIR irradiation. After 24 h of incubation, cytotoxicity was evaluated using CCK-8. 100 μL of CCK-8 solution was added to each well and incubated at 37 °C for 60 min. The concentration of formaldehyde dye was measured at 450 nm using a MultiscanMK3 (Thermo, USA) microplate reader. Each data point was analyzed in triplicate. Live / dead cell staining: 4T1 cells (5 × 10⁵) were cultured in 96-well plates and divided into four groups: Control, F127@rGO hydrogel + US + NIR, BiOCl@CuO / F127@rGO hydrogel, and BiOCl@CuO / F127@rGO hydrogel + US + NIR. 300 μL of neural basal medium containing calcein-AM (0.5 μM) and PI (3 μM) was added, and the cells were cultured for 24 h, incubated at 37°C for 30 min, and observed using CLSM. ImageJ software was used to select three images from each group to quantify the number of live and dead cells to determine the survival rate.

[0085] Intracellular ROS level detection:

[0086] Five × 10⁵ HEPA1-6 cells were cultured in 48-well plates and divided into four groups: a control group, an F127@rGO hydrogel + US + NIR group, a BiOCl@CuO / F127@rGO hydrogel group, and a BiOCl@CuO / F127@rGO hydrogel + US + NIR group. After 24 h of incubation, intracellular ROS levels were detected by DCFH-DA. Fluorescence was observed using CLSM, and relative fluorescence intensity was quantified using ImageJ software on three selected images from each group.

[0087] Animal models:

[0088] Female C57BL / 6 mice were placed in isolation cages under a 12-hour light-dark cycle and fed with sterile food and sterile water using a standard mouse nutrition formula. A mammary gland cell model was established using 4T1 mouse cells. Mice were anesthetized by inhaling 2% isoflurane mixed with medical oxygen. Using a 29-gauge insulin syringe, 50 μL of cell suspension (containing 2 × 10⁶ cells) was injected into the fourth mammary fat pad of each mouse for in situ implantation.

[0089] Following injection, the mice were placed on a heating pad until the anesthesia was fully resolved. The study design, procedures, husbandry, sample size, animal allocation, and results of this animal experiment all followed the ARRIVE guidelines.

[0090] Histological analysis:

[0091] For H&E staining, tissue samples were fixed in 10% neutral buffered formalin for 24 hours, dehydrated, and embedded in paraffin. Sections with a thickness of 3-5 μm were prepared and examined under a microscope after staining with hematoxylin and eosin (H&E). Whole-section imaging was performed using a KFBIOKF-PRO-120 digital pathology scanner (China).

[0092] Flow cytometry:

[0093] To analyze live cells, ZombieDye staining was performed before surface labeling. For cytokine detection, cells were stimulated for 4 hours with a cell activation cocktail (containing PMA, iomycin, and brefeldina) at the manufacturer's recommended concentration to induce cytokine production while blocking protein transport. After stimulation, cells were fixed and permeabilized using appropriate buffer according to standard protocols.

[0094] ELISA:

[0095] Mouse blood samples were collected from the orbital venous plexus and placed into non-anticoagulant tubes. Serum was separated by centrifugation at 3000×g at 4℃ for 10 min, then re-introduced and stored at -80℃, avoiding repeated freeze-thaw cycles. Serum IL-6 and TNF-α concentrations were determined using a commercial ELISA kit. In short, all reagents and serum samples were equilibrated to room temperature for 30 min. A total of 100 μL of standard solution or diluted serum sample (diluted 1:2 with sample diluent) was added to the wells of a pre-coated microplate and incubated at 37℃ for 1 h. After discarding the contents of the wells, the plate was washed 3-4 times with washing buffer. Next, 100 μL of biotinylated detection antibody was added to each well, and the plate was incubated at 37℃ for 30 min. After another round of washing, 100 μL of streptavidin-horseradish peroxidase conjugate was added, and the plate was incubated at 37℃ for 15 min. After the final washing step, add 100 μL of TMB substrate solution to each well and incubate at room temperature in the dark for 10 min. Terminate the reaction by adding 50 μL of stop solution. Measure the absorbance at 450 nm using a microplate reader. Calculate the concentrations of IL-6 and TNF-α in the samples based on the standard curve generated from the standard solutions.

[0096] 5. Statistical Analysis:

[0097] Statistical analysis was performed using GraphPadPrism 9.0, with continuous variables expressed as mean ± SD. Two-tailed Student's test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Survival analysis was performed using Kaplan-Meier curves and the log-rank test to assess significance.

[0098] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0099] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for the manufacture of injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel for superficial tumor treatment, characterized by, The method comprises the following steps: S1. Preparing BiOCl nanosheets by a solvothermal method, and washing and drying the BiOCl nanosheets for standby; S2. Preparing BiOCl@CuO heterostructure nanosheets by reacting BiOCl prepared in S1 with copper acetate in an organic solvent, and centrifuging, washing and drying the BiOCl@CuO heterostructure nanosheets; S3. Preparing a F127@rGO composite solution by dispersing reduced graphene oxide in deionized water and adding Pluronic F127 powder and stirring at low temperature; S4. Preparing an injectable BiOCl@CuO / F127@rGO thermosensitive hydrogel by adding BiOCl@CuO heterostructure nanosheets prepared in S2 to the F127@rGO composite solution prepared in S3 and storing the mixture at low temperature; S5. Characterizing the hydrogel prepared in S4, and adjusting process parameters based on the characterization results to ensure that the sol-gel transition, electrical conductivity and biocompatibility of the hydrogel meet the requirements for breast cancer treatment applications; S6. Harmlessly treating waste liquid and waste generated in the preparation process to ensure that the discharge meets the standards.

2. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 1, characterized in that, The implementation procedure of the step S1 is as follows: 0.486 g of Bi(NO3)3·5H2O with a purity of ≥99%, mannitol and NaCl are selected as raw materials; 0.486 g of Bi(NO3)3·5H2O is dissolved in 25 mL of 0.1 mol / L mannitol solution, and continuous stirring is performed for 30 minutes; then 5 mL of saturated NaCl solution is added dropwise, and continuous stirring is performed for 30 minutes; the mixed solution is transferred to a stainless steel autoclave lined with Teflon, and reaction is performed at 160℃ for 3 hours; after the reaction is completed, the precipitate is collected, washed with ethanol and deionized water alternately for 3 times, dried at 60℃ for 4 hours, ground into fine powder, and the BiOCl nanosheets are obtained for standby.

3. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 2, characterized in that, The implementation procedure of the step S2 is as follows: 0.65 g of BiOCl nanosheets prepared in S1 and 0.05 g of Cu(CH3COO)2·H2O are added to a conical flask containing 50.0 mL of N,N-dimethylformamide, and continuous stirring is performed for 15 minutes; the conical flask is transferred to a constant-temperature water bath at 90℃, and stirring reaction is performed for 4 hours; after the reaction is completed, the product is centrifuged at 9000 rpm for 5 minutes, washed with deionized water for 3 times, and dried at 60℃, and the BiOCl@CuO heterostructure nanosheets are obtained.

4. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 3, characterized in that, The implementation procedure of the step S3 is as follows: reduced graphene oxide is dispersed in 8 mL of deionized water at a concentration of 5 mg / mL, and uniform dispersion is achieved by ultrasonic treatment; then 1.9 g of Pluronic F127 powder is added, and magnetic stirring is performed at 4℃ for 4 hours, and a uniform F127@rGO composite solution is obtained.

5. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 4, characterized in that, The implementation procedure of the step S4 is as follows: the BiOCl@CuO heterostructure nanosheets prepared in S2 are added to the F127@rGO composite solution prepared in S3 in a set proportion, stirred uniformly, and stored at 4℃ overnight, and an injectable BiOCl@CuO / F127@rGO thermosensitive hydrogel is obtained; the hydrogel is in a flowing sol state at room temperature, and is quickly converted into an elastic gel state at 23-24℃, and the injectability and tissue adhesion meet the requirements for in-vivo applications.

6. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 5, characterized in that, The implementation procedure of the step S5 is: using a scanning electron microscope and a transmission electron microscope to characterize the microstructure of the hydrogel and the nanosheet, to ensure that the BiOCl@CuO nanosheet is uniformly dispersed in the hydrogel; analyzing the zeta potential of the BiOCl@CuO nanosheet by using a Zetasizer, and verifying the formation of the BiOCl@CuO heterojunction by using XRD and XPS; testing the sol-gel transition temperature, oscillatory strain performance and self-healing performance of the hydrogel by using a rheometer; detecting the conductivity of the hydrogel by using a conductivity testing device; if the characterization results do not meet the standards, adjusting the ratio of BiOCl to copper acetate in S2 or the reaction time, or the concentration of the reduced graphene oxide in S3, until the material for breast cancer treatment meets the standards.

7. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 6, characterized in that, The implementation procedure of the step S6 is: collecting the washing waste liquid generated in the preparation process of S1 and S2, and discharging it after neutralization treatment to pH 7 or so; A small amount of waste gas generated in the ultrasonic dispersion process is collected and discharged after treatment by an activated carbon adsorption device; The waste containers and consumables used in the preparation process are disposed according to the medical waste specifications after high-temperature disinfection treatment.

8. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 7, characterized in that, The addition amount of the BiOCl@CuO heterostructure nanosheet in the step S4 needs to meet: the concentration of BiOCl@CuO in the final hydrogel is 50-200 μg / mL, to ensure that it has high efficient photo-thermal conversion performance under near-infrared irradiation, and can effectively produce reactive oxygen under ultrasonic stimulation.

9. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 8, characterized in that, The biocompatibility of the hydrogel also needs to be verified by in vitro cytotoxicity test in the step S5: when the concentration of BiOCl nanosheet is less than 12.5 μg / mL, the survival rate of mouse NIH-3T3 fibroblasts is maintained at more than 95%; if the survival rate is less than 95%, adjust the addition concentration of BiOCl@CuO in S4 until the biocompatibility requirements are met.

10. The injectable BiOCl@CuOF127@rGO thermo-sensitive hydrogel manufacturing method for superficial tumor treatment according to claim 9, characterized in that, The product yield of the S1 solvothermal reaction and the heterojunction formation efficiency are recorded in the step S1, when the yield is less than 75%, the solvothermal reaction temperature of S1 is increased by 5-10℃; when the heterojunction formation efficiency is insufficient, the amount of copper acetate in S2 is increased; based on the characterization results of S5, the ultrasonic dispersion time and stirring speed parameters are recalibrated every 5 batches of production, to realize stable process circulation.

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