A two-dimensional black phosphorus hybrid nanomedicine modified based on RC48 protein, its preparation method and application

Abstract

The present invention discloses a two-dimensional black phosphorus hybrid nanomedicine modified by RC48 protein, and its preparation method and application. The medicine uses a metabolizable two-dimensional black phosphorus quantum sheet as an inorganic carrier, and is modified by methoxypolyethylene glycol monoamine to improve the colloidal stability and biocompatibility, and enhance the antigen capture ability of antigen-presenting cells. Further conjugated with vedicitumumab to achieve active targeting of HER2-positive tumors, and at the same time loaded with adjuvant resiquimod to activate immune cells and induce macrophages to secrete M1-type cytokines to reverse tumor immunosuppression. This system realizes precise treatment through a "target recognition-photothermal ablation-immune activation" synergistic mechanism. Its innovation lies in the first integration of RC48 and R848 on a photosensitive black phosphorus platform, which significantly enhances the ability to regulate the tumor microenvironment while ensuring mild reaction conditions, providing a new strategy for targeted immunotherapy of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of nano-drugs, and particularly relates to a two-dimensional black phosphorus hybrid nano-drug modified with RC48 protein, and a preparation method and application thereof. Background Art

[0002] Breast cancer is one of the most common and deadly malignant tumors among women globally, with the incidence rate ranking first among all female malignant tumors. With the progress of early screening methods, early diagnosis and treatment of breast cancer have gradually achieved certain results, but advanced breast cancer remains a major public health challenge. Traditional treatment methods include surgical resection, radiotherapy, chemotherapy, targeted therapy, etc. However, when applied to certain advanced breast cancer cases, these treatment methods often face problems such as limited efficacy, high recurrence rate, and drug resistance.

[0003] Disadvantages of the prior art: 1. Inaccurate positioning: The in-vivo positioning accuracy of targeted drugs in tumor tissues is poor, resulting in reduced treatment efficiency and potential damage to normal tissues. 2. Single treatment method: It is difficult to solve the complexity of tumors by using photothermal therapy or immunotherapy alone. Especially for HER2-positive breast cancer, traditional treatment methods are difficult to overcome immune escape and recurrence problems. 3. Limitation of immunotherapy: There is an immunosuppressive mechanism in the tumor microenvironment, making it difficult for immunotherapy to completely eliminate tumors, and the immune response rate is slow. 4. Material problems: Most of the existing drug carrier materials are non-degradable materials, which may cause biological toxicity or long-term residue in the body.

[0004] Specific reasons for the disadvantages: 1. Insufficient targeting: Traditional nano-drug design fails to fully combine the targeting function for HER2-positive breast cancer. 2. Lack of combination of treatment methods: Single treatment methods lack synergistic effects and fail to effectively combine the advantages of photothermal, targeted, and immunotherapy. 3. Insufficient performance of carrier materials: Most traditional carrier materials are difficult to meet the requirements of high drug loading rate, photothermal conversion efficiency, and biocompatibility.

[0005] In recent years, immunotherapy, as an emerging treatment strategy, has shown great potential in the treatment of breast cancer. Immunotherapy activates the patient's own immune system to help immune cells recognize and attack tumor cells, and has become one of the important directions in tumor treatment.

[0006] However, despite the initial success of immunotherapy in some breast cancer patients, there are often some limitations when using immunotherapy alone. First of all, it is often difficult to completely eradicate local solid tumors when using immunotherapy alone, and there are problems such as a relatively low rate of immune response and a strong immune escape mechanism. Tumor cells often evade the recognition of the immune system through the immunosuppressive microenvironment, which makes the effect of single immunotherapy unsatisfactory in the treatment of advanced breast cancer. Therefore, there is an urgent need for a comprehensive treatment strategy that combines immunotherapy with other treatment methods to overcome the limitations of existing treatment methods.

[0007] An ideal treatment plan should rapidly eliminate local solid tumors through combination therapy and induce the production of tumor-specific antigens, thereby activating the body's immune response. Through this mechanism, immunotherapy can more effectively identify and eliminate the remaining tumor cells and prevent tumor recurrence. Among various treatment methods, Photothermal Therapy (PTT), as an emerging local treatment method, has been proven to be able to effectively induce tumor ablation and stimulate the host's immune response due to its non-invasive and highly targeted advantages. Through the photothermal effect, nanomaterials can rapidly heat up under specific light conditions, and local tumor tissues undergo thermal damage or necrosis at high temperatures, thereby promoting the release of tumor antigens and activating the immune system.

[0008] In recent years, based on the small-size effect and easy functionalization modification characteristics of nanomaterials, new possibilities have been provided for the combination of photothermal therapy and immunotherapy. Nanomaterials can not only enhance the photothermal effect but also carry immunomodulatory molecules through surface modification to help effectively regulate the immune response. Therefore, the design of nanodrugs has become a promising treatment method that can achieve the synergistic effects of targeted drug delivery, immunomodulation, and photothermal effect during the treatment process.

[0009] Searching the domestic and foreign literature and patents on hybrid nanodrugs shows that currently, there are no reports on the application aspects such as two-dimensional black hybrid nanodrugs targeting HER2-positive breast cancer modified with RC48 protein and their preparation methods. Summary of the Invention

[0010] The objective of the present invention is to solve the problems of inaccurate in vivo localization of existing targeted drugs, incomplete photothermal therapy, targeted therapy, and immunotherapy, and immunosuppression. The present invention aims to solve the key problems in the prior art by designing a novel nano-drug that organically combines photothermal therapy, targeted therapy, and immunotherapy to improve the treatment effect of HER2-positive breast cancer. The specific objectives are as follows: 1. Enhance targeting: Utilize the targeting function of the RC48 protein to enable the nano-drug to accurately recognize and act on HER2-positive breast cancer cells, thereby reducing damage to normal tissues and improving the precision of treatment. 2. Improve the treatment effect: Combine photothermal therapy and immunotherapy. Rapidly destroy tumor tissues through the photothermal effect, release tumor antigens, and further stimulate the immune system response, thereby overcoming the limitations of immunotherapy used alone and achieving an efficient anti-tumor effect. 3. Overcome the immunosuppressive microenvironment: By binding to the immune adjuvant R848, activate immune cells (such as dendritic cells and macrophages), enhance the immune response ability, and improve the immunosuppressive state of the tumor microenvironment. 4. Optimize the performance of the nano-carrier: Use two-dimensional black phosphorus as the carrier, endowing the drug with high photothermal conversion efficiency and complete metabolizability. At the same time, improve its colloidal stability and biocompatibility through mPEG-NH2 modification to ensure in vivo safety and degradability. 5. Achieve synergistic therapy: Innovatively integrate photothermal therapy, targeted therapy, and immunotherapy into a nano-drug platform, and significantly enhance the treatment effect through synergistic effects, reducing recurrence and drug resistance. Through the above design, the present invention provides a novel, precise, efficient, and safe treatment strategy for HER2-positive breast cancer, providing a new solution to the problem of treating advanced breast cancer.

[0011] To solve the above technical problems, the present invention adopts the following technical solutions:

[0012] A two-dimensional black phosphorus hybrid nano-drug modified with RC48 protein, comprising a carrier and an active ingredient loaded on the carrier; the carrier comprises two-dimensional black phosphorus nanosheets modified with mPEG-NH2; the active ingredient comprises a specific targeting antibody and an immune adjuvant.

[0013] Design of the novel two-dimensional black phosphorus hybrid nano-drug: 1. Material selection: Use two-dimensional black phosphorus (BP) as the nano-drug carrier, which has the following innovative features: high photothermal conversion efficiency, capable of rapidly heating up under near-infrared light irradiation; biodegradability, metabolized into non-toxic phosphates, reducing potential biological toxicity. 2. Surface modification: Improve the colloidal stability and biocompatibility of the nanosheets through mPEG-NH2 to ensure the stability of the drug in vivo and reduce the aggregation tendency of the drug in body fluids.

[0014] Preferably, the specific targeting antibody comprises a monoclonal antibody specifically targeting HER2.

[0015] The nano-drug platform designed by the present invention is not only applicable to HER2-positive breast cancer, but also has scalability and can be applied to different types of cancers by modifying other targeting molecules.

[0016] Preferably, the specific targeting antibody includes RC48; the immunoadjuvant includes R848.

[0017] Preferably, the active component is composed of the RC48 antibody targeting HER2 and the immunoadjuvant R848.

[0018] RC48 is formed by binding a monoclonal antibody with a cytotoxic drug (for example, a taxane-like drug) through a chemical linker. The monoclonal antibody specifically recognizes and binds to the HER2 antigen on the surface of cancer cells, and the cytotoxic drug directly acts on tumor cells under the guidance of the antibody, thereby achieving an efficient therapeutic effect. The target of RC48 is the HER2 receptor. HER2 is a protein widely expressed on the surface of certain types of cancer cells, especially breast cancer, gastric cancer, non-small cell lung cancer, etc. HER2-positive tumor cells are sensitive to this drug. Once RC48 binds to HER2-positive tumor cells, enzymes inside the cells will cause the drug to be released. The drug inhibits microtubule polymerization, prevents the division and proliferation of cancer cells, and ultimately leads to the death of cancer cells. RC48 is mainly used to treat HER2-positive cancers that are insensitive or resistant to traditional chemotherapy. In clinical trials, it has shown strong anti-tumor activity, especially against HER2-positive gastric cancer and breast cancer. In some trials, RC48 has shown more superior efficacy than traditional monoclonal antibody therapy (such as Herceptin). By modifying the RC48 protein (antibody-drug conjugate targeting HER2-positive breast cancer), the nano-drug of the present invention can efficiently recognize HER2-positive breast cancer cells and achieve precise targeting. The high specificity of RC48 significantly reduces the toxicity to normal tissues and reduces side effects; it overcomes the problems of uneven in vivo distribution and inaccurate localization of existing targeted drugs.

[0019] Two-dimensional black phosphorus nanosheets are a new type of two-dimensional inorganic nanomaterial with a high specific surface area and photothermal conversion efficiency, and can be completely metabolized into non-toxic phosphates and phosphonates in vivo. In this patent, two-dimensional black phosphorus is modified with methoxypolyethylene glycol amine (mPEG-NH2) and then loaded with vedicituximab RC48, and combined with the immune adjuvant resiquimod (R848) to prepare the drug BP-PEG-RC48-R848. The experimental results show the structure and properties of BP, as well as its potential in photothermal conversion efficiency. BP can be used as an effective carrier for photothermal therapy, with good biocompatibility and degradability, and is suitable for tumor photothermal therapy. R848 can activate immune cells such as promoting the activation of DC cells, and then promote macrophages to secrete M1-related cytokines, playing a role in tumor immunosuppression to achieve targeted photothermal immunotherapy for human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Using two-dimensional black phosphorus (BP) as a carrier, it has a high photothermal conversion efficiency and can quickly heat up to the temperature required for tumor ablation (>56°C). The stability of BP in photothermal therapy has been tested through multiple laser on / off cycles, and its performance remains stable. Traditional photothermal therapy materials such as gold nanorods and carbon-based materials have certain effects, but often have problems of biocompatibility or non-degradability. The BP carrier of the present invention is completely metabolizable, avoiding the biological toxicity caused by residues.

[0020] The cooperation principle of each component of the present invention: 1. The synergistic effect of BP-PEG-RC48-R848: BP provides a photothermal effect; RC48 is responsible for targeting HER2-positive tumor cells; R848 activates the immune system to achieve tumor ablation and recurrence prevention. 2. The structural characteristics of the nano-drug: Each component acts synergistically through functional modification to improve the therapeutic effect.

[0021] Most of the existing technologies are single treatment modes and cannot fully mobilize multiple anti-tumor mechanisms. The present invention overcomes the problem of single efficacy in traditional methods through multi-functional synergistic therapy. The present invention integrates photothermal therapy, targeted therapy and immunotherapy into a nano-drug platform for the first time. The three functions act synergistically to significantly improve the anti-tumor effect. The photothermal effect induces the release of tumor antigens, combined with the immune adjuvant R848 to activate the immune system, overcome tumor immune escape and prevent tumor recurrence.

[0022] A preparation method of the above-mentioned two-dimensional black phosphorus hybrid nano-drug modified based on the RC48 protein, comprising:

[0023] A. Dissolve the bulk black phosphorus crystal powder in an amide organic solvent, ultrasonically vibrate and break it, then centrifuge and wash it with water to obtain an aqueous solution of black phosphorus nanosheets, and store it at low temperature in the dark for later use;

[0024] B. Mix the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then perform ultrasonic centrifugation and water washing to obtain black phosphorus nanosheets modified with mPEG-NH2;

[0025] C. Mix the black phosphorus nanosheets modified with mPEG-NH2 with the aqueous solution of the specific targeting antibody, then stir, centrifuge and wash with water to obtain an intermediate product loaded with the specific targeting antibody;

[0026] D. Mix the intermediate product loaded with the specific targeting antibody and the alcoholic solution of the immune adjuvant, then stir, centrifuge and wash with water to obtain the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification.

[0027] Precise preparation method of a multifunctional drug platform: 1. Liquid-phase exfoliation method: Use NMP dissolution and ultrasonic centrifugation processes to prepare two-dimensional black phosphorus nanosheets with uniform particle sizes (100 - 200 nm). 2. Multi-step modification process: Gradually modify mPEG-NH2, RC48, and R848 by methods such as ultrasonic treatment and stirring to ensure the successful combination and functional synergy of each component.

[0028] In the prior art, the preparation process of multifunctional nanomedicines is complex and the reaction conditions are harsh, making it difficult to achieve industrial applications. In the present invention, the liquid-phase exfoliation method is adopted in the preparation of nanomaterials. The preparation process is simple and the conditions are mild, enabling large-scale production. The process flow is scientifically designed to ensure the functionality and stability of each component, laying a foundation for clinical transformation. While ensuring performance, the present invention optimizes the preparation process, having higher operability and promotion value.

[0029] Preferably, the mass-volume ratio of the bulk black phosphorus crystal powder to the amide organic solvent is 50 - 100 mg: 20 - 30 mL; the mass feeding ratio of the black phosphorus nanosheets, mPEG-NH2, the specific targeting antibody, and the immune adjuvant is 1: 8 - 10: 1.5 - 2.5: 1 - 1.5; the aqueous solution concentration of the black phosphorus nanosheets is 4 - 6 mg / mL; the aqueous solution concentration of mPEG-NH2 is 10 - 20 mg / mL; the aqueous solution concentration of the specific targeting antibody is 5 - 10 mg / mL; the alcoholic solution concentration of the immune adjuvant is 5 - 10 mg / mL.

[0030] Preferably, the black phosphorus nanosheets include two-dimensional black phosphorus nanosheets with a size of 100 - 200 nm; the amide organic solvent includes NMP; mPEG-NH2 includes mPEG-NH2 with a molecular weight of 5000; the alcoholic solution of the immune adjuvant includes the methanol solution of the immune adjuvant.

[0031] Preferably, it includes:

[0032] A. Dissolve the block-shaped black phosphorus crystal powder in the amide organic solvent, first use a probe ultrasonic oscillator to break it up, then perform ultrasonic oscillation cleaning in a low-temperature water bath. After that, centrifuge at 2500 - 3500 rpm for 10 - 15 minutes to remove large black phosphorus precipitates. Then centrifuge at 12000 - 13000 rpm and wash with deionized water to obtain an aqueous solution of the black phosphorus nanosheets with uniform dispersion, and store it in the dark at 4 - 8 °C for later use;

[0033] B. Mix the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then perform ultrasonic oscillation in a water bath at 10 - 15 °C in the dark for 2 - 4 hours, centrifuge at 12000 - 13000 rpm for 10 - 15 minutes and wash with deionized water, and collect all the precipitates to obtain the black phosphorus nanosheets modified with mPEG-NH2;

[0034] C. Mix the black phosphorus nanosheets modified with mPEG-NH2 with the aqueous solution of the specific targeting antibody, stir in the dark for 10 - 14 hours, centrifuge at 12000 - 13000 rpm for 10 - 15 minutes and wash with deionized water, and collect all the precipitates to obtain the intermediate product loaded with the specific targeting antibody;

[0035] D. Mix the intermediate product loaded with the specific targeting antibody and the alcoholic solution of the immune adjuvant, then stir with an open mouth in the dark for 12 - 16 hours, centrifuge at 12000 - 13000 rpm for 10 - 15 minutes and wash with deionized water, and collect all the precipitates to obtain the two-dimensional black phosphorus hybrid nanomedicine modified based on the RC48 protein.

[0036] Preferably, in step A, the operation of first using a probe ultrasonic oscillator to break it up and then performing ultrasonic oscillation cleaning in a low-temperature water bath includes: first use a cell crusher to perform probe ultrasonic oscillation to break it up, and use a 6 - type horn ultrasonic cell crusher to perform ice bath ultrasonic crushing at a power of 810 - 900 W and an ultrasonic frequency of on for 2.0 s and off for 1.0 s in a cycle for 60 - 80 minutes, repeat 3 times, and then perform ultrasonic cleaning in a water bath at 10 - 15 °C for 1 - 1.5 hours.

[0037] An application of the above two-dimensional black phosphorus hybrid nanomedicine modified based on the RC48 protein, in the application of preparing a cancer photothermal immunocomposite therapy targeted drug.

[0038] Preferably, the cancer is HER2 - positive breast cancer.

[0039] The present invention can be directly used for the photothermal immunocomposite therapy of cancer, or for preparing related drugs, especially for the photothermal immunocomposite therapy of in - situ breast cancer.

[0040] Implementing the present invention has the following beneficial effects:

[0041] 1. The preparation process of the present invention is simple and efficient. The liquid-phase exfoliation method is used to obtain two-dimensional black phosphorus quantum sheets with uniform size. The colloidal stability and biocompatibility are significantly improved by surface covalent modification with methoxypolyethylene glycol amine (mPEG-NH2), and the antigen capture ability of antigen-presenting cells is enhanced. Further, the anti-HER2 antibody RC48 is modified by the directional coupling technology to achieve tumor-specific targeting. At the same time, the immune adjuvant R848 is loaded to activate the TLR7 / 8 signaling pathway, forming a "photothermal-targeted-immune" triple synergistic treatment system. The whole process of this process is completed in an aqueous environment at 25 °C, and the reaction conditions are mild and controllable, with the potential for industrial production.

[0042] 2. The present invention innovatively integrates the RC48 antibody and the R848 adjuvant on a photosensitive black phosphorus platform, breaking through the limitations of traditional therapies. Among them, RC48 realizes the precise recognition and drug delivery of HER2-positive tumors through antigen-antibody specific binding, and directly kills tumor cells by combining with the high photothermal conversion efficiency of black phosphorus; R848 activates dendritic cells (DCs) and induces macrophages to secrete M1-type cytokines by inducing the secretion of TNF-α and IL-6, reversing the tumor immunosuppressive microenvironment. Experiments show that the drug loading rate of this system is high, and the synergistic effect of photothermal ablation and immune activation is significantly better than that of single therapies.

[0043] 3. The present invention uses metabolizable two-dimensional black phosphorus as the core carrier, which has both high biological safety and functional programmability. The pharmacokinetic properties are optimized by mPEG-NH2 modification, and the synergistic loading of RC48 and R848 realizes the full-chain treatment of "targeted recognition-local ablation-systemic immunity" for the first time. Utilizing the high photothermal conversion efficiency of BP, local heating of tumor tissues is induced by near-infrared light irradiation, causing thermal damage and antigen release of tumor cells. The anti-tumor ability of the immune system is activated by R848 to eliminate residual tumor cells and prevent recurrence. Compared with the prior art, its breakthrough lies in the modular integration of antibody-targeted therapy, photothermal ablation and immune regulation, providing a new strategy for the precise combined treatment of HER2-positive breast cancer, with broad clinical application prospects.

[0044] 4. The present invention provides a two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification for targeting HER2-positive breast cancer, which realizes the combined treatment of photothermal, targeted and immune therapies through the following technical steps: 1. Selection of nanocarrier: Two-dimensional black phosphorus (BP) is used as the drug carrier, which has high photothermal conversion efficiency and biodegradability. 2. Multifunctional modification: mPEG-NH2 (amino methoxypolyethylene glycol) is used to improve the colloidal stability and biocompatibility of BP; in combination with RC48 (vedicitumumab, a HER2-targeted antibody), precise targeting is achieved; and R848 (resiquimod, an immune adjuvant) is loaded to activate the immune system. 3. Comprehensive treatment: Integrating photothermal therapy (PTT) and immunotherapy to achieve the synergistic treatment of HER2-positive breast cancer. Through innovative nanomaterial design and multifunctional modification, the deficiencies in targeting and single treatment effect in the prior art are overcome, realizing the precise and efficient treatment of HER2-positive breast cancer, significantly improving the treatment effect and reducing side effects. This solution provides strong technical support and application prospects for the combined photothermal and immune therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is an analysis diagram of the substances prepared at different stages in Example 1 of the present invention, where: (a) is the TEM image of BP-PEG-RC48-R848; (b) is the AFM image of BP-PEG-RC48-R848; (c) is the elemental mapping analysis diagram of BP-PEG-RC48-R848.

[0046] Figure 2 It is a bar chart of the surface potential of BP-PEG-RC48-R848, its intermediate products and other components in Example 1 of the present invention.

[0047] Figure 3 It is the ultraviolet absorption spectrum in Example 1 of the present invention: (a) The ultraviolet absorption spectra of BP-PEG-RC48-R848 solutions with different concentrations of 100, 50, 25, 12.5, 6.25 μg / mL in the range of 220 - 280 nm; (b) The ultraviolet absorption spectra of R848, BP-PEG-RC48 and BP-PEG-RC48-R848 solutions in the range of 220 - 280 nm.

[0048] Figure 4 It is two temperature curve diagrams in Example 1 of the present invention: (a) The heating-up diagram of BP-PEG-RC48-R848 with different concentrations irradiated by an 808 nm laser at 1.0 W / cm 2 for 10 min; (b) 100 μg / mL of BP-PEG-RC48-R848 at 1.0 W / cm 2The heating-cooling curve under 808 nm laser irradiation.

[0049] Figure 5 The ultraviolet absorption spectrum of R848 immunoadjuvant in Example 1 of the present invention: (a) The ultraviolet absorption spectra of R848 solutions with different concentrations in the range of 220 - 300 nm; (b) The ultraviolet absorption standard curve of R848 solution at 247 nm.

[0050] Figure 6 Schematic diagram for evaluating the dark toxicity of cells in Example 2 of the present invention, where: The cell survival rate (a) and cell morphology diagram (c) of 4T1 cells after co-culturing with different concentrations of RC48 and BP-PEG-RC48-R848 for 24 h; The cell survival rate (b) and cell morphology diagram (d) of SKBR3 cells after co-culturing with different concentrations of RC48 and BP-PEG-RC48-R848 for 24 h.

[0051] Figure 7 Schematic diagram for evaluating the phototoxicity of cells in Example 2 of the present invention, where: (a) The cell survival rate of 4T1 cells after co-incubating with different concentrations of BP-PEG-RC48-R848 for 4 h with or without laser irradiation; (b) The cell survival rate of SKBR3 cells after co-incubating with different concentrations of BP-PEG-RC48-R848 for 4 h with or without laser irradiation (n = 3, *P<0.05,**P<0.01, ***P<0.001 vs. +L).

[0052] Figure 8 Schematic diagram of cell viability / cytotoxicity double staining for evaluating the phototoxicity of cells in Example 2 of the present invention, where: (a) The Calcein-AM / PI double-stained cell diagram of 4T1 cells after co-culturing with different concentrations of drugs for 4 h and then irradiated with 808 nm laser (L) with a power of 1.0 W / cm 2 for 10 min; (b) The Calcein-AM / PI double-stained cell diagram of SKBR3 cells after co-culturing with different concentrations of drugs for 4 h and then irradiated with 808 nm laser (L) with a power of 1.0 W / cm 2 for 10 min (scale bar is 100 μm).

[0053] Figure 9 Schematic diagram of cell targeting in Example 2 of the present invention: (a) Microscopic and DAPI staining diagrams of 4T1 cells after co-culturing with different concentrations of drugs; (b) Microscopic and DAPI staining diagrams of SKBR3 cells after co-culturing with different concentrations of drugs (scale bar is 50 μm).

[0054] Figure 10In Example 3 of the effects of the present invention, the contents of TNF-α (a) and IL-6 (b) cytokines secreted by BMDC after treatment with different samples (n = 3, ***P<0.001 vs. BP-PEG-RC48-R848).

[0055] Figure 11 In Example 3 of the effects of the present invention, mature DC (CD80 + CD86 + ) representative flow cytometry plots (a) and percentages (b) of BMDC after treatment with different samples (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. +BP-PEG-RC48-R848).

[0056] Figure 12 In Example 4 of the effects of the present invention, on day 3 after treatment, representative flow cytometry plots of CD3 + CD4 + T cells (a), CD3 + CD8 + T cells (b), and percentages of activated T cells (c, d) in the spleen (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. +BP-PEG-RC48-R848+L).

[0057] Figure 13 In Example 4 of the effects of the present invention, on day 3 after treatment, representative flow cytometry plots (a) and percentages (b) of DC maturation (CD80 + CD86 + ) in the lymph nodes (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. +BP-PEG-RC48-R848+L).

[0058] Figure 14 In Example 5 of the effects of the present invention, (a) is a comparison plot of fluorescence intensity monitoring of the tumor region at multiple time points after adding the drug and a quantitative analysis plot of fluorescence intensity for a HER2-positive mouse model; (c) is a comparison plot of drug fluorescence intensity in vivo 3 days after injection of the drug in HER2-negative and positive mice; (d) is an analysis plot of tumor fluorescence intensity in HER2-negative and positive mice 3 days after injection of the drug; (e) is a comparison plot of drug fluorescence intensity in vivo 24 h after injection of the drug in HER2-negative and positive mice; (f) is a comparison plot of the accumulation amount of fluorescence intensity in various organs in vivo 3 days after injection of the drug in HER2-negative and positive mice (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. HER2+).

[0059] Figure 15 In Example 6 of the effects of the present invention, (a) is a schematic diagram of the anti-tumor experiment process in mice; (b) is the temperature rise curve of the tumor site after laser irradiation (1.0 W / cm 2 ) 24 h after intravenous injection of the drug; (c) is the curve of the change in tumor volume of mice after different treatment methods over time; after sacrificing the mice on the 21st day, statistical analysis of the tumor weights of the mice treated by different methods (d) and comparison of the anatomical morphology of the tumor tissues in different treatment groups (e). Detailed implementation manners

[0060] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0061] Example 1

[0062] The preparation of the BP-PEG-RC48-R848 drug includes the following processes:

[0063] (1) The black phosphorus nanosheets are prepared by a liquid-phase exfoliation method. The powdered black phosphorus crystals are dissolved in NMP. First, the cell crusher probe is used for ultrasonic vibration and crushing. Under the condition of a cycle of turning on for 2.0 s and turning off for 1.0 s at a power of 810 W and an ultrasonic frequency, the ultrasonic cell crusher with a No. 6 amplitude transformer is used for ice-bath ultrasonic crushing for 60 min, and the process is repeated 3 times. Then, it is ultrasonically washed in a 10°C water bath for 1 h. After that, it is centrifuged at 3500 rpm for 15 min to remove the large black phosphorus precipitate, and then centrifuged at 13000 rpm for 12 min to remove the residual NMP, and washed with deionized water to obtain the uniformly dispersed aqueous solution of the black phosphorus nanosheets. Finally, the concentration of black phosphorus is quantified by a UV-visible spectrophotometer and stored at 4°C in the dark for standby.

[0064] (2) The aqueous solution of the black phosphorus nanosheets prepared above and the aqueous solution of mPEG-NH2 (20 mg / mL) are mixed and ultrasonically washed in a 10°C water bath in the dark for 4 h, and centrifuged at 13000 rpm for 12 min to remove the unbound excessive mPEG-NH2, and washed with deionized water, and all the precipitates are collected to obtain the BP-PEG aqueous solution.

[0065] (3) The BP-PEG aqueous solution prepared above and the RC48 aqueous solution (10 mg / mL) are mixed and stirred in the dark for 12 h, and centrifuged at 13000 rpm for 12 min to remove the unbound excessive RC48, and washed with deionized water, and all the precipitates are collected to obtain the BP-PEG-RC48 aqueous solution.

[0066] (4)Mix the prepared BP-PEG-RC48 aqueous solution and R848 methanol solution (10 mg / mL), stir it open to the air and protected from light for 12 h, centrifuge at 13000 rpm for 12 min to remove the unbound excessive R848, wash it with deionized water, collect all the precipitates, and obtain the BP-PEG-RC48-R848 aqueous solution.

[0067] (5)Mix the BP-PEG-RC48-R848 aqueous solution prepared in (4) of Example 1 and ICG-NHS alcohol solution (5 mg / mL), stir it open to the air and protected from light for 12 h, centrifuge at 13000 rpm for 12 min to remove the unbound excessive ICG-NHS, wash it with deionized water, collect all the precipitates, and obtain the BP-PEG-RC48-R848-ICG aqueous solution for in vivo targeting evaluation experiment in mice. ICG (indocyanine green) is a near-infrared fluorescent probe, and ICG staining can be used for in vivo imaging for easy observation.

[0068] Example 2

[0069] Preparation of the BP-PEG-RC48-R848 drug, including the following process:

[0070] (1)The black phosphorus nanosheets are prepared by a liquid-phase exfoliation method. Dissolve the powdered black phosphorus crystal in NMP, first use the probe of a cell crusher to ultrasonically oscillate and break it, use a 6th horn ultrasonic cell crusher to ultrasonically break it in an ice bath for 60 min under the condition of a cycle of 2.0 s on and 1.0 s off at a power of 900 W and an ultrasonic frequency, repeat it 3 times, and then ultrasonically wash it in a 15°C water bath for 1 h. Then centrifuge at 2500 rpm for 15 min to remove the large black phosphorus precipitate, then centrifuge at 12000 rpm for 15 min to remove the residual NMP, and wash it with deionized water to obtain the uniformly dispersed black phosphorus nanosheet aqueous solution. Finally, quantify the black phosphorus concentration with a UV-visible spectrophotometer and store it at 8°C protected from light for standby.

[0071] (2)Mix the prepared black phosphorus nanosheet aqueous solution and mPEG-NH2 aqueous solution (15 mg / mL), ultrasonically bath at 15°C protected from light for 2 h, centrifuge at 12000 rpm for 15 min to remove the unbound excessive mPEG-NH2, wash it with deionized water, collect all the precipitates, and obtain the BP-PEG aqueous solution.

[0072] (3)Mix the prepared BP-PEG aqueous solution and RC48 aqueous solution (7 mg / mL), stir it protected from light for 14 h, centrifuge at 12000 rpm for 15 min to remove the unbound excessive RC48, wash it with deionized water, collect all the precipitates, and obtain the BP-PEG-RC48 aqueous solution.

[0073] (4) After mixing the prepared BP-PEG-RC48 aqueous solution and R848 methanol solution (7 mg / mL), stir it open to the air and away from light for 16 h, centrifuge at 12000 rpm for 15 min to remove the unbound excess R848, wash it with deionized water, collect all the precipitates, and obtain the BP-PEG-RC48-R848 aqueous solution.

[0074] Example 3

[0075] Preparation of the BP-PEG-RC48-R848 drug, including the following process:

[0076] (1) The black phosphorus nanosheets are prepared by the liquid-phase exfoliation method. Dissolve the powdered black phosphorus crystals in NMP, first use the probe of the cell crusher to ultrasonically oscillate and break, use the ultrasonic cell crusher with a No. 6 amplitude transformer to ultrasonically break in an ice bath for 80 min under the condition of a cycle of 2.0 s on and 1.0 s off at a power of 810 W and an ultrasonic frequency, repeat 3 times, and then ultrasonically clean in a 12°C water bath for 1.5 h. Then centrifuge at 3500 rpm for 10 min to remove the large black phosphorus precipitate, then centrifuge at 12500 rpm for 10 min to remove the residual NMP, and wash it with deionized water to obtain the uniformly dispersed black phosphorus nanosheet aqueous solution. Finally, quantify the black phosphorus concentration with a UV-visible spectrophotometer and store it away from light at 6°C for standby.

[0077] (2) After mixing the prepared black phosphorus nanosheet aqueous solution and mPEG-NH2 aqueous solution (10 mg / mL), ultrasonically stir in a 12°C water bath away from light for 3 h, centrifuge at 12500 rpm for 10 min to remove the unbound excess mPEG-NH2, wash it with deionized water, collect all the precipitates, and obtain the BP-PEG aqueous solution.

[0078] (3) After mixing the prepared BP-PEG aqueous solution and RC48 aqueous solution (5 mg / mL), stir it away from light for 12 h, centrifuge at 12500 rpm for 10 min to remove the unbound excess RC48, wash it with deionized water, collect all the precipitates, and obtain the BP-PEG-RC48 aqueous solution.

[0079] (4) After mixing the prepared BP-PEG-RC48 aqueous solution and R848 methanol solution (5 mg / mL), stir it open to the air and away from light for 14 h, centrifuge at 12500 rpm for 10 min to remove the unbound excess R848, wash it with deionized water, collect all the precipitates, and obtain the BP-PEG-RC48-R848 aqueous solution.

[0080] Effect Example 1

[0081] Taking the sample prepared in Example 1 as an example, the transmission electron microscope characterization shows ( Figure 1a), The BP-PEG-RC48-R848 nanocomposite presented a typical layered topological structure with serrated fold features at the edges. The average particle size was about 100 - 200 nm. RC48 was attached to the surface of BPNSs, wrapping the BPNSs, which demonstrated the successful loading of RC48. Three-dimensional topography analysis by atomic force microscopy ( Figure 1 b) further verified that the cross-sectional height distribution showed a single-peak feature, with the thickest reaching 23.48 nm and the average thickness being 20.37 ± 3.17 nm, significantly lower than that of bare BP nanosheets. The energy-dispersive spectroscopy (Figure 1c) indicated that BP-PEG-RC48-R848 mainly consisted of element P, along with a large number of other elements such as carbon (C), nitrogen (N), and oxygen (O).

[0082] Zeta potential analysis revealed the stepwise assembly process of the hybrid nanosystem ( Figure 2 ). The original black phosphorus nanosheets (BPNSs) showed a significant negative charge (-30.87 ± 0.25 mV) due to the formation of PO4 3- groups on the surface by surface oxidation. BPNSs and mPEG-NH2 were prepared by electrostatic binding. After the negatively charged BPNSs were modified by mPEG-NH2 (1.70 ± 0.29 mV), the potential of the product BP-PEG increased significantly to (-14.39 ± 0.18 mV). This change was attributed to the electrostatic neutralization effect of the protonated amino group at the end of mPEG-NH2 and the negative charge on the BP surface. Since RC48 is a macromolecular antibody protein, it was electro-neutral (-0.62 ± 0.13 mV). Therefore, the potential of BP-PEG-RC48 (-14.12 ± 0.21 mV) only underwent a slight adjustment. R848 was electro-negative (-17.06 ± 1.37 mV). After the binding of BP-PEG-RC48 and R848, the potential of the final product BP-PEG-RC48-R848 decreased significantly to -18.38 ± 0.42 mV, which also demonstrated the successful loading of R848.

[0083] Ultraviolet-visible absorption spectroscopy analysis showed ( Figure 3 ) that BP, R848, BP-PEG-RC48, and BP-PEG-RC48-R848 all had spectral absorptions in the range of 220 - 280 nm. Among them, Figure 3 a showed the ultraviolet absorption characteristics of BP-PEG-RC48-R848 at concentration gradients of 100, 50, 25, 12.5, and 6.25 μg / mL. As Figure 3 shown in b, after loading R848, BP-PEG-RC48-R848 showed a characteristic absorption peak completely consistent with that of free R848 at 245 - 248 nm ( Figure 3As shown by the b arrow, the successful loading of R848 was further verified.

[0084] To evaluate the effects of the RC48 antibody and R848 loading on the photothermal properties of the material, this patent verified the therapeutic potential of the composite material through systematic photothermal heating experiments and cyclic stability tests. As Figure 4 shown in a, after irradiation with an 808 nm laser (1.0 W / cm 2 ) for 10 minutes, the temperature of the 100 μg / mL BP-PEG-RC48-R848 solution increased significantly to 56.2 ± 0.5 °C, and even when the concentration was reduced to 50 μg / mL, it could still reach 51.1 ± 0.3 °C, fully confirming that the composite system has a concentration-dependent photothermal conversion ability. More notably, after four heating / cooling cycles of the 100 μg / mL BP-PEG-RC48-R848 solution ( Figure 4 b), the maximum temperature of the material only decreased by about 2%, showing highly stable photothermal properties, which provides a key guarantee for repeated treatment in clinical translation.

[0085] To ensure the accuracy of drug quantitative analysis, this patent established a standard curve for R848 through ultraviolet-visible absorption spectroscopy ( Figure 5 a). Standard samples were prepared using the gradient dilution method (15 - 0.625 μg / mL, a total of 9 concentration points). Spectral analysis showed that R848 had a significant characteristic absorption peak at 247 nm. The standard curve calculated from the characteristic peak values ( Figure 5 b) showed excellent linearity (R 2 = 0.9997). Based on this, the loading rate of R848 in BP-PEG-RC48-R848 was calculated to be 23.04%, providing an accurate drug quantitative basis for subsequent efficacy experiments.

[0086] Effect Example 2

[0087] Taking the sample prepared in Example 1 as an example, the cytotoxicity of RC48 and BP-PEG-RC48-R848 was evaluated through CCK-8 experiments and cell morphology observations. As Figure 6 shown in a, in 4T1 cells with low HER2 expression, neither of the two drugs showed significant cytotoxicity in the concentration range of 0 - 50 μg / mL, and the cell survival rate remained above 85%. However, in SKBR3 breast cancer cells with high HER2 expression ( Figure 6b), both RC48 and BP-PEG-RC48-R848 showed dose-dependent killing effects: when the concentration reached 50 μg / mL, the cell viability of the free RC48 group was 67.37% ± 6.26%, while the viability of the BP-PEG-RC48-R848 group (black phosphorus concentration 50 μg / mL) was further reduced to 58.21% ± 3.25%, indicating that the nanocarrier might enhance the cytotoxicity by enhancing drug targeted delivery. The above results were further verified by cell morphology ( Figure 6 c). In 4T1 cells, even when the black phosphorus concentration of BP-PEG-RC48-R848 reached 50 μg / mL, compared with the PBS control group, the cells co-cultured for 24 hours still maintained a complete adherent morphology, with well-extended pseudopodia, no obvious membrane rupture or fragmentation, and its viability was 89.56% ± 6.45%, which was highly consistent with the CCK-8 data. Bright-field microscopic images ( Figure 6 d) further showed that SKBR3 cells presented typical apoptotic morphological features in the BP-PEG-RC48-R848 treatment group, including cell shrinkage, detachment and cytoplasmic vacuolization, while 4T1 cells maintained a normal morphology, further verifying the reliability of the CCK-8 experiment. The above results indicated that BP-PEG-RC48-R848 had good biocompatibility in HER2-negative cells, while showed specific killing effects on HER2-positive tumor cells, providing an experimental basis for the safety and effectiveness of subsequent targeted therapy.

[0088] To verify the synergistic enhancement effect of the photothermal conversion performance of black phosphorus on the drug killing effect, this study further evaluated the in vitro phototoxicity effects of BP-PEG-RC48-R848 on 4T1 (HER2-negative) and SKBR3 (HER2-positive) cells. In the experimental design, after co-culturing the cells with different concentrations of the nanodrug for 4 hours, 808 nm near-infrared laser (1.0 W / cm 2 ) was used to irradiate for 10 minutes, and the cell viability was quantified by the CCK-8 method. As Figure 7 shown, the photothermal synergistic effect significantly improved the cell killing efficiency of the nanodrug. For HER2-negative 4T1 cells ( Figure 7 a), under laser irradiation, the cytotoxicity of BP-PEG-RC48-R848 increased in a concentration-dependent manner. When the black phosphorus concentration reached 50 μg / mL, the cell viability decreased to 49.93% ± 4.55%, which was about 35% lower than that of the non-irradiated group (viability > 85%) (p < 0.01), indicating that the photothermal effect of black phosphorus could break through the non-targeting limitation of HER2-negative cells. For HER2-positive SKBR3 cells ( Figure 7b), at the same black phosphorus concentration (50 μg / mL), the cell survival rate further decreased to 25.17% ± 3.36%, which was approximately 50% lower than that of the 4T1 group (p < 0.01), confirming the synergistic mechanism of targeted delivery and photothermal effect. It shows that the local hyperthermia (ΔT ≈ 25°C) generated by black phosphorus nanosheets under near-infrared laser excitation can induce an increase in the permeability of the tumor cell membrane and promote the intracellular penetration of RC48; at the same time, HER2 targeting enables the drug to accumulate at a higher concentration inside SKBR3 cells. It should be noted that after the drug is loaded with R848, its killing effect on HER2-positive cells is significantly stronger than that of pure RC48, and it increases with the increase in concentration. The synergistic effect of the two leads to DNA damage and mitochondrial dysfunction, ultimately triggering an apoptotic cascade reaction.

[0089] To visually evaluate the photothermal synergistic killing effect of BP-PEG-RC48-R848, the present study used the Calcein-AM / PI double staining method for live and dead cells to quantitatively analyze HER2-negative 4T1 cells ( Figure 8 a) and HER2-positive SKBR3 cells ( Figure 8 b). In the experimental design, the cells were co-cultured with different concentrations of the nano-drug (0 - 50 μg / mL, calculated based on the black phosphorus concentration) for 4 hours, and then irradiated with an 808 nm near-infrared laser (power density 1.0 W / cm²) for 10 minutes. The survival status of the cells was observed and quantified by a fluorescence microscope. As the concentration of BP-PEG-RC48-R848 increased (0 - 50 μg / mL), the PI red fluorescence signal (marking dead cells) of the two cell lines increased significantly, while the Calcein-AM green fluorescence signal (marking live cells) decreased synchronously. The mortality rate of SKBR3 cells was higher than that of 4T1 cells, which was consistent with the analysis of the phototoxic survival rate. It should be noted that in the control group irradiated with PBS plus laser, a large number of live cells survived, confirming that pure photothermal stimulation has no significant toxicity, and the cell killing by BP-PEG-RC48-R848 depends on the synergistic effect of the drug and photothermal. By integrating targeted delivery and photothermal conversion characteristics, BP-PEG-RC48-R848 significantly enhances the selective killing of HER2-positive tumors, providing a key experimental basis for the development of photothermal-chemotherapy combined treatment regimens.

[0090] To verify the targeting specificity of BP-PEG-RC48-R848, the present study observed the aggregation behavior of the nano-drug around HER2-negative (4T1) and positive (SKBR3) breast cancer cells through fluorescence microscopy imaging technology. Figure 9 As shown in a, the drug was sparsely distributed in dots around 4T1 cells, indicating that its aggregation mainly depends on non-specific adsorption or passive penetration (such as the EPR effect). Figure 9In Figure b, the drug formed a dense aggregation on the surface and around the SKBR3 cells, and the aggregation effect far exceeded that of 4T1, confirming that the HER2-targeted binding mediated by RC48 significantly enhanced drug enrichment. As a fragment of HER2 monoclonal antibody, RC48 drives the nano-drug to anchor on the surface of SKBR3 cell membrane through antigen-antibody specific recognition and promotes receptor-mediated endocytosis; while 4T1 cells lack HER2 expression, and the drug internalization efficiency is limited, and only a small amount can be taken up through membrane adsorption or pinocytosis.

[0091] Effect Example 3

[0092] Taking the sample prepared in Example 1 as an example, to evaluate the immune activation effect of BP-PEG-RC48-R848 nanocomplex on bone marrow-derived dendritic cells (BMDCs), the secretion levels of pro-inflammatory cytokines TNF-α and IL-6 were quantitatively detected by ELISA in this study ( Figure 10 ). The experimental data showed that the nano-drug loaded with R848 exhibited significant immune activation ability: in terms of the secretion level of TNF-α ( Figure 10 a), the concentration of the BP-PEG-RC48-R848 group reached 277.52 ± 12.38 pg / mL, which was 14.4 times higher than that of the PBS control group (19.26 ± 1.47 pg / mL) (P<0.001), and was significantly higher than that of the BP-PEG-RC48 group without R848 loading (96.52 ± 1.33 pg / mL, P<0.001); in terms of the secretion level of IL-6 ( Figure 10 b), the concentration of the BP-PEG-RC48-R848 group was as high as 181.75 ± 6.35 pg / mL, which was about 7 times higher than that of the PBS group (17.54 ± 4.11 pg / mL) and the BP-PEG-RC48 group without R848 adjuvant loading (25.97 ± 2.76 pg / mL) (both P<0.001). Mechanistic studies have shown that the loaded adjuvant R848 drives the differentiation of BMDCs into a pro-inflammatory phenotype by activating the MyD88 / NF-κB signaling pathway mediated by the TLR7 / 8 receptor, and at the same time synergistically enhances the antigen presentation ability of the nanocarrier. This finding not only confirmed the immune activation function of the nanocomplex, but also revealed its potential application value in constructing a pro-inflammatory microenvironment and developing DC vaccines.

[0093] To further evaluate the effect of BP-PEG-RC48-R848 nanocomplex on the immune activation function of BMDCs, BMDCs were co-cultured with PBS, RC48, BP-PEG-RC48 and BP-PEG-RC48-R848 for 24 h in this study, and the expression levels of mature DCs markers (CD80+CD86+) were analyzed by flow cytometry. Representative flow cytometry plots ( Figure 11a) Results showed that the proportion of mature DCs in the BP-PEG-RC48-R848 group was significantly higher than that in other treatment groups. Quantitative analysis showed that ( Figure 11 b), compared with the PBS group (40.63 ± 2.12%), the RC48 group (25.83 ± 1.84%), and the BP-PEG-RC48 group (48.83 ± 2.80%), the proportion of mature DCs in the BP-PEG-RC48-R848 treatment group increased to 55.40 ± 4.68%. This indicated that BP-PEG-RC48-R848 loaded with the adjuvant R848 could activate BMDC cells, increase the proportion of mature BMDC cells, and further enhance the antigen presentation function of DCs by synergistically targeting the delivery of RC48 and the immune-stimulating effect of R848.

[0094] Effect Example 4

[0095] Taking the sample prepared in Example 1 as an example, a HER2-positive breast cancer xenograft model was constructed using 6-8-week-old female Balb / c mice. A suspension of HER2-positive breast cancer cells of EMT6 (100 - 200 μL, concentration 1 - 5×10 6 cells / mL) was injected subcutaneously on the right side. After the tumor volume reached 150 mm³, the mice were randomly grouped and treated by tail vein injection (100 - 120 μL / mouse). The experimental group was injected with the black phosphorus composite nanodrug (BP concentration 1 mg / mL), and the control group was given the equivalent loading amount of RC48 alone. The first administration was recorded as day 0. After 24 hours, the tumor area of the light irradiation group of mice was irradiated with 808 nm near-infrared laser (power density 1 W / cm², lasting for 10 min). On the 3rd day after treatment, the mice were sacrificed, and the tumor-draining lymph nodes and spleen tissues were aseptically isolated. The tissue samples were mechanically ground in 3 mL of PBS to prepare a single-cell suspension. After centrifugation at 1500 rpm for 5 min to remove the supernatant, pre-cooled erythrocyte lysate was added and incubated on ice for 3 min, and then centrifuged again to remove the lysis products. The obtained cell pellet was resuspended in PBS and filtered through a 30 μm cell strainer to finally obtain a high-purity single-cell suspension. 100 μL of the spleen cell suspension was added with CD3, CD4, and CD8 fluorescent antibodies respectively; 100 μL of the lymphocyte suspension was added with CD11c, CD80, and CD86 fluorescent antibodies respectively. After incubation in the dark, flow cytometry was used to quantitatively analyze the expression ratios of CD3⁺CD4⁺ and CD3⁺CD8⁺ in the spleen cell suspension and the expression ratio of CD80⁺CD86⁺ in the lymphocytes.

[0096] To evaluate the regulatory effect of BP-PEG-RC48-R848 combined with photothermal therapy on the immune system, first, the changes in the spleen T lymphocyte subsets of mice in different treatment groups were detected by flow cytometry ( Figure 12 a and 12b are CD3 respectively+ CD4 + and CD3 + CD8 + flow cytometry representative diagram). Figure 12 c shows that under the condition of 808 nm laser irradiation, in the BP-PEG-RC48-R848+L treatment group, the proportion of helper T cells (CD3 + CD4 + ) was significantly increased to 11.27% ± 0.42%, which was about 2.7 times higher than that of the PBS control group (4.21% ± 0.19%) (***p<0.001), and 1.6 times higher than that of the BP-PEG-RC48 group without R848 loading (7% ± 0.07%). It is worth noting that this proportion was higher than that of the non-irradiated drug administration group (BP-PEG-RC48-R848: 7.84% ± 0.07%); similarly, as Figure 12 shown in d, the proportion of cytotoxic T lymphocytes (CD3 + CD8 + ) in the treatment group was 6.69% ± 0.31%, which was significantly higher than that of the BP-PEG-RC48 group without R848 loading (3.89% ± 0.16%) and the PBS group (3.13% ± 0.04%) and other groups. This difference confirmed that photothermal activation could synergistically enhance the recruitment ability of the loaded R848 adjuvant to spleen helper T cells, suggesting that the locally increased temperature mediated by laser might amplify the anti-tumor immune response by promoting drug targeted release or regulating the immune microenvironment.

[0097] To further systematically evaluate the immune activation efficacy of the nanodrug, the present invention quantitatively analyzed the maturity of dendritic cells (DC) in mouse lymph nodes by flow cytometry ( Figure 13 a). The experimental data showed ( Figure 13 b): The proportion of mature DCs (CD80⁺CD86⁺ double positive marker) in the BP-PEG-RC48-R848 combined with near-infrared laser irradiation group (BP-PEG-RC48-R848+L) reached 46.13% ± 2.41%, which was higher than that of the BP-PEG-R848+L group (44.4% ± 1.5%) (***p<0.001), significantly higher than that of the BP-PEG-RC48 group without R848 loading (28.47%±2.74%), while the PBS+L control group only showed 27.57% ±0.85%. This result confirmed that the introduction of the R848 adjuvant significantly enhanced the immunogenicity of the drug through the Toll-like receptor 7 / 8 signaling pathway. More importantly, photothermal stimulation could synergistically promote the activation of antigen-presenting cells, and the proportion of mature DCs was increased by nearly 1.7 times compared with the non-irradiated group, fully reflecting the multiple synergistic mechanisms of photocontrolled drug release and immune activation.

[0098] Effect Example 5

[0099] Taking the sample prepared in Example 1 as an example, to systematically evaluate the in vivo targeting performance of the nano-drug, this study established a HER2-positive tumor-bearing (Balb / c female mice) model and injected a nano-suspension (100 μL / mouse) loaded with the near-infrared fluorescent probe indocyanine green (ICG) via the tail vein. The spatio-temporal distribution characteristics of the fluorescence intensity in the tumor region at 6, 12, 24, 48, and 72 hours after administration were dynamically monitored using a small animal in vivo imaging system ( Figure 14 a). Quantitative analysis showed that the fluorescence signal in the tumor reached its peak at 24 h ( Figure 14 b), indicating that this time window was the optimal treatment intervention node. After euthanasia at the end of the experiment, the tumor tissue and major organs (heart, liver, spleen, lung, kidney) were completely excised, and the tumor targeting efficiency and biodistribution pattern of the nanoparticles were quantitatively revealed by ex vivo fluorescence imaging.

[0100] It should be noted that the fluorescence intensity in the tumor region of the HER2-positive model group was significantly higher than that of the HER2-negative control group 72 hours after administration ( Figure 14 c). Further quantitative analysis of the excised tumors confirmed that the fluorescence accumulation in the positive group was 1.6 times that of the negative group (***p < 0.001, Figure 14 d). Sequential in vivo imaging ( Figure 14 e) and multi-organ biodistribution studies ( Figure 14 f) jointly verified that the nano-drug showed specific targeted accumulation in the HER2-positive model, indicating that the active targeting mechanism mediated by the RC48 antibody effectively enhanced tumor-selective delivery.

[0101] Effect Example 6

[0102] Taking the sample prepared in Example 1 as an example, to systematically evaluate the in vivo anti-tumor performance of the nano-drug, this patent selected 6-8-week-old female Balb / c mice to establish a HER2-positive breast cancer xenograft model. A suspension of EMT6-HER2+ breast cancer cells (1 - 5×10 6cells / mL, 100-200 μL / mouse), and after a 14-day tumor growth cycle, the experimental stage was entered when the tumor volume reached 150 mm³ (calculation formula: V=0.5×long diameter×short diameter²). The tumor-bearing mice were randomly divided into groups (n=6) and given corresponding treatments (100-120 μL / mouse) via tail vein injection. The black phosphorus concentration in the nanomedicine group was uniformly 1 mg / mL. 24 hours after the intervention, the tumor area of ​​the photothermal therapy group was irradiated with 808 nm near-infrared laser (1.0 W / cm², 10 min / mouse), and the temperature changes were monitored in real time using an infrared thermal imaging system (thermal images were recorded at intervals of 1 min). The tumor volume was measured with an electronic vernier caliper every 48 hours during the treatment cycle, and the weight changes of the mice were monitored to evaluate systemic toxicity. The mice were killed at the end of the experiment on the 21st day, and the tumor tissue was completely removed for wet weight measurement and volume calculation. The example figure is shown in the figure below. Figure 15 As shown in a.

[0103] The results showed that the tumor sites of mice in the BP group treated with laser irradiation showed a significant increase in temperature ( Figure 15 b) Since the content of BP in the drugs is the same, the temperature rise curves of the three are very small, and all of them can increase the surface temperature of the mouse tumor to about 56 degrees (n = 3), achieving the effect of photothermal heating, which also indirectly shows that the quantitative determination of BP in the drugs is accurate. Figure 15 As shown in Figure c, after different treatments were performed on tumor-bearing mice, it was found that the BP-PEG-RC48-R848+L (L represents light irradiation during treatment) treatment group (Group 8) had the smallest tumor, indicating that the targeted photothermal chemotherapy mediated by RC48 and R848 simultaneously has a good tumor inhibition effect. Figure 15 d As can be seen, by comparing BP-PEG+L (the fourth group) and BP-PEG-R848+L (the sixth group), it can be found that the drug BP-PEG-R848+L group after loading R848 is significantly smaller than BP-PEG+L; by comparing BP-PEG-RC48 (the fifth group) and BP-PEG-RC48-R848 (the seventh group), it can also be judged that the successful introduction of R848 has significantly enhanced the anti-tumor ability of the drug (the average tumor size of the fifth group is 2.12 times that of the seventh group, p<0.001). Anatomical observation further confirmed ( Figure 15 e) The tumor tissue in the treatment group combined with R848 showed typical necrosis characteristics, and the average size of the tumor was smaller. These differences prove that the drug loaded with R848 has stronger anti-tumor ability in vivo and can play a more effective role after combined with photothermal therapy.

[0104] The above description is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A two-dimensional black phosphorus hybrid nanomedicine modified with RC48 protein, characterized in that, It includes a carrier and an active ingredient loaded on the carrier; the carrier includes two-dimensional black phosphorus nanosheets modified with mPEG-NH2; the active ingredient includes a specific targeting antibody RC48 and an immune adjuvant R848; It includes the following preparation steps: A. Dissolve the bulk black phosphorus crystal powder in an amide organic solvent, ultrasonically vibrate and break it, then centrifuge and wash it with water to obtain an aqueous solution of black phosphorus nanosheets, and store it for later use at low temperature in the dark; B. Mix the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then ultrasonically centrifuge and wash it with water to obtain black phosphorus nanosheets modified with mPEG-NH2; C. Mix the black phosphorus nanosheets modified with mPEG-NH2 with the aqueous solution of the specific targeting antibody, then stir, centrifuge and wash it with water to obtain an intermediate product loaded with the specific targeting antibody; D. Mix the intermediate product loaded with the specific targeting antibody with the alcoholic solution of the immune adjuvant, then stir, centrifuge and wash it with water to obtain the two-dimensional black phosphorus hybrid nanomedicine modified based on the RC48 protein.

2. A preparation method of the two-dimensional black phosphorus hybrid nanomedicine modified based on the RC48 protein according to claim 1, characterized in that, It includes: A. Dissolve the bulk black phosphorus crystal powder in an amide organic solvent, ultrasonically vibrate and break it, then centrifuge and wash it with water to obtain an aqueous solution of black phosphorus nanosheets, and store it for later use at low temperature in the dark; B. Mix the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then ultrasonically centrifuge and wash it with water to obtain black phosphorus nanosheets modified with mPEG-NH2; C. Mix the black phosphorus nanosheets modified with mPEG-NH2 with the aqueous solution of the specific targeting antibody, then stir, centrifuge and wash it with water to obtain an intermediate product loaded with the specific targeting antibody; D. Mix the intermediate product loaded with the specific targeting antibody with the alcoholic solution of the immune adjuvant, then stir, centrifuge and wash it with water to obtain the two-dimensional black phosphorus hybrid nanomedicine modified based on the RC48 protein.

3. The preparation method of the two-dimensional black phosphorus hybrid nanomedicine modified based on the RC48 protein according to claim 2, wherein, The mass-volume ratio of the bulk black phosphorus crystal powder to the amide organic solvent is 50-100 mg: 20-30 mL; the mass ratio of the black phosphorus nanosheets, mPEG-NH2, the specific targeting antibody, and the immune adjuvant is 1: 8-10: 1.5-2.5: 1-1.5; the concentration of the aqueous solution of the black phosphorus nanosheets is 4-6 mg / mL; the concentration of the aqueous solution of mPEG-NH2 is 10-20 mg / mL; the concentration of the aqueous solution of the specific targeting antibody is 5-10 mg / mL; the concentration of the alcoholic solution of the immune adjuvant is 5-10 mg / mL.

4. The preparation method of the two-dimensional black phosphorus hybrid nanomedicine based on the RC48 protein modification according to claim 3, characterized in that, The black phosphorus nanosheets include two-dimensional black phosphorus nanosheets of 100-200 nm; the amide organic solvent includes NMP; mPEG-NH2 includes mPEG-NH2 with a molecular weight of 5000; the alcoholic solution of the immune adjuvant includes the methanol solution of the immune adjuvant.

5. The preparation method of the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 3, characterized in that, It includes: A. Dissolve the bulk black phosphorus crystal powder in the amide organic solvent, first use a probe ultrasonic oscillator to break it up, then perform ultrasonic oscillation cleaning in a low-temperature water bath. After that, centrifuge at 2500 - 3500 rpm for 10 - 15 min to remove the large black phosphorus precipitate. Then centrifuge at 12000 - 13000 rpm and wash with deionized water to obtain an aqueous solution of the black phosphorus nanosheets with uniform dispersion, and store it in the dark at 4 - 8 °C for later use; B. Mix the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then perform ultrasonic bath in the dark at 10 - 15 °C for 2 - 4 h, centrifuge at 12000 - 13000 rpm for 10 - 15 min and wash with deionized water, and collect all the precipitates to obtain the black phosphorus nanosheets modified with mPEG-NH2; C. Mix the black phosphorus nanosheets modified with mPEG-NH2 with the aqueous solution of the specific targeting antibody, stir in the dark for 10 - 14 h, centrifuge at 12000 - 13000 rpm for 10 - 15 min and wash with deionized water, and collect all the precipitates to obtain the intermediate product loaded with the specific targeting antibody; D. Mix the intermediate product loaded with the specific targeting antibody and the alcoholic solution of the immune adjuvant, then stir with an open mouth in the dark for 12 - 16 h, centrifuge at 12000 - 13000 rpm for 10 - 15 min and wash with deionized water, and collect all the precipitates to obtain the two-dimensional black phosphorus hybrid nano-drug modified based on the RC48 protein.

6. The preparation method of the two-dimensional black phosphorus hybrid nanomedicine based on the RC48 protein modification according to claim 5, wherein, In step A, the operation of first using a probe ultrasonic oscillator to break it up and then performing ultrasonic oscillation cleaning in a low-temperature water bath includes: first use a cell crusher probe ultrasonic oscillator to break it up. Under the condition of a power of 810 - 900 W and an ultrasonic frequency of a cycle of turning on for 2.0 s and turning off for 1.0 s, use a No. 6 horn ultrasonic cell crusher to perform ice bath ultrasonic crushing for 60 - 80 min, repeat 3 times, and then perform ultrasonic cleaning in a water bath at 10 - 15 °C for 1 - 1.5 h.

7. Use of the two-dimensional black phosphorus hybrid nano-drug modified based on the RC48 protein according to claim 1 in the preparation of a cancer photothermal immunocomposite therapy targeting drug.

8. Use of the two-dimensional black phosphorus hybrid nanomedicine modified based on the RC48 protein according to claim 7, characterized in that, The cancer is HER2-positive breast cancer.

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