Two-dimensional black phosphorus hybrid nano-drug based on RC48 protein modification as well as preparation method and application of two-dimensional black phosphorus hybrid nano-drug

Abstract

The invention discloses a two-dimensional black phosphorus hybrid nano-drug based on RC48 protein modification as well as a preparation method and application of the two-dimensional black phosphorus hybrid nano-drug. According to the medicine, a metabolizable two-dimensional black phosphorus quantum sheet inorganic carrier is modified by amino polyethylene glycol monomethyl ether, so that the colloidal stability and the biocompatibility are improved, and the antigen capturing capability of antigen presenting cells is enhanced. Furthermore, active targeting of HER2 positive tumors is realized through coupling with dicetumab, and meanwhile, an adjuvant, namely leximod, is loaded, so that immune cells are activated, and macrophages are induced to secrete M1 type cytokines so as to reverse tumor immunosuppression. According to the system, precise treatment is achieved through a targeted recognition-photo-thermal ablation-immune activation synergistic mechanism, the innovation is that RC48 and R848 are integrated on a photosensitive black phosphorus platform for the first time, the tumor microenvironment regulation and control capacity is remarkably enhanced while the mild reaction condition is guaranteed, and a new strategy is provided for targeted immune combined treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of nanomedicine, and in particular relates to a two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification, and a preparation method and application thereof. Background Art

[0002] Breast cancer is one of the most common and deadly malignant tumors in women worldwide, and its incidence rate ranks first among all female malignant tumors. With the advancement 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 treatments include surgical resection, radiotherapy, chemotherapy, targeted therapy, etc. However, these treatments often face problems such as limited effectiveness, high recurrence rate and drug resistance in some cases of advanced breast cancer.

[0003] Disadvantages of existing technologies: 1. Inaccurate positioning: The positioning accuracy of targeted drugs in tumor tissues is poor, resulting in reduced treatment efficiency and possible damage to normal tissues. 2. Single treatment method: Photothermal therapy or immunotherapy alone is difficult to solve the complexity of tumors, especially for HER2-positive breast cancer. Traditional treatment methods are difficult to overcome the problems of immune escape and recurrence. 3. Limitations of immunotherapy: The existence of immunosuppressive mechanisms in the tumor microenvironment makes it difficult for immunotherapy to completely eliminate tumors, and the immune response rate is slow. 4. Material problems: Existing drug carrier materials are mostly non-degradable materials, which may cause biological toxicity or long-term residual problems in the body.

[0004] Specific reasons for the shortcomings: 1. Insufficient targeting: Traditional nanomedicine design fails to fully combine the targeting function for HER2-positive breast cancer. 2. Treatment methods are not combined: 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 of tumor treatment.

[0006] However, although immunotherapy has achieved initial results in some breast cancer patients, there are often some limitations when used alone. First, it is often difficult to completely eradicate local solid tumors when used alone, and there are problems such as low immune response rate and strong immune escape mechanism. Tumor cells often escape recognition of the immune system through an immunosuppressive microenvironment, which makes single immunotherapy less effective in treating advanced breast cancer. Therefore, there is an urgent need for a comprehensive treatment strategy that combines immunotherapy with other treatments to overcome the limitations of existing treatments.

[0007] The ideal treatment plan should quickly eliminate local solid tumors through combined treatment 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 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 quickly heat up under specific lighting 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, the small size effect and easy functional modification of nanomaterials have provided new possibilities 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 immune responses. Therefore, the design of nanomedicines has become a promising treatment method that can achieve targeted drug delivery, immunomodulation, and synergistic effects of photothermal effects during treatment.

[0009] The results of searching domestic and foreign literature and patents on hybrid nanomedicines show that: At present, there are no reports on the application of two-dimensional black hybrid nanomedicines and preparation methods based on RC48 protein modification targeting HER2-positive breast cancer. Summary of the invention

[0010] The purpose of the present invention is to solve the problems of inaccurate in vivo positioning 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 new type of nanomedicine to organically combine photothermal therapy, targeted therapy and immunotherapy to improve the effect of treating HER2-positive breast cancer. The specific goals are as follows: 1. Enhance targeting: Utilize the targeting function of RC48 protein to enable nanomedicine to accurately identify and act on HER2-positive breast cancer cells, thereby reducing damage to normal tissues and improving the accuracy of treatment. 2. Improve the therapeutic effect: Combine photothermal therapy with immunotherapy, quickly destroy tumor tissue through photothermal effect, release tumor antigens, and further stimulate the immune system response, thereby overcoming the limitations of immunotherapy alone and achieving efficient anti-tumor effects. 3. Overcome the immunosuppressive microenvironment: By combining the immune adjuvant R848, immune cells (such as dendritic cells and macrophages) are activated, the immune response ability is enhanced, and the immunosuppressive state of the tumor microenvironment is improved. 4. Optimize the performance of nanocarriers: Use two-dimensional black phosphorus as a carrier to give the drug high photothermal conversion efficiency and complete metabolism, and at the same time improve its colloidal stability and biocompatibility through mPEG-NH2 modification to ensure in vivo safety and degradability. 5. Achieve synergistic treatment: Innovatively integrate photothermal therapy, targeted therapy and immunotherapy into a nanodrug platform, significantly improve the treatment effect through synergistic effects, and reduce recurrence and drug resistance. Through the above design, the present invention provides a new, accurate, efficient and safe HER2-positive breast cancer treatment strategy, which provides a new solution to the problem of advanced breast cancer treatment.

[0011] In order to solve the above technical problems, the present invention adopts the following technical solutions: A two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification comprises a carrier and an active ingredient loaded on the carrier; the carrier comprises a two-dimensional black phosphorus nanosheet modified with mPEG-NH2; the active ingredient comprises a specific targeting antibody and an immune adjuvant.

[0012] Design of novel two-dimensional black phosphorus hybrid nanomedicine: 1. Material selection: Two-dimensional black phosphorus (BP) is used as a nanodrug carrier, which has the following innovative features: high photothermal conversion efficiency, able to heat up rapidly under near-infrared light irradiation; biodegradable, metabolized into non-toxic phosphates, reducing potential biological toxicity. 2. Surface modification: mPEG-NH2 is used to improve the colloidal stability and biocompatibility of the nanosheets, ensure the stability of the drug in the body, and reduce the aggregation tendency of the drug in body fluids.

[0013] Preferably, the specific targeting antibody comprises a monoclonal antibody that specifically targets HER2.

[0014] The nanomedicine platform designed in the present invention is not only applicable to HER2-positive breast cancer, but also has scalability and can be applied to different types of cancer by modifying other targeting molecules.

[0015] Preferably, the specific targeting antibody includes RC48; and the immune adjuvant includes R848.

[0016] Preferably, the active component consists of RC48 antibody targeting HER2 and immune adjuvant R848.

[0017] RC48 is a combination of a monoclonal antibody and a cytotoxic drug (e.g., 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, while the cytotoxic drug acts directly on tumor cells through the guidance of the antibody, thereby achieving a highly effective 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 the drug. Once RC48 binds to HER2-positive tumor cells, enzymes in the cells cause the drug to be released. The drug inhibits the division and proliferation of cancer cells by inhibiting microtubule polymerization, ultimately leading to the death of cancer cells. RC48 is mainly used to treat HER2-positive cancers that are insensitive to or resistant to traditional chemotherapy. In clinical trials, it has shown strong anti-tumor activity, especially for HER2-positive gastric cancer and breast cancer. In some trials, RC48 has shown superior efficacy to traditional monoclonal antibody treatments (such as Herceptin). By modifying the RC48 protein (antibody-drug conjugate targeting HER2-positive breast cancer), the nanomedicine of the present invention can efficiently identify HER2-positive breast cancer cells and achieve precise targeting. The high specificity of RC48 significantly reduces toxicity to normal tissues and reduces side effects; it overcomes the problems of uneven distribution and inaccurate positioning of existing targeted drugs in the body.

[0018] Two-dimensional black phosphorus nanosheets are a new type of two-dimensional inorganic nanomaterial with high specific surface area and photothermal conversion efficiency, and can be completely metabolized into non-toxic phosphates and phosphonates in the body. In this patent, two-dimensional black phosphorus is modified with amino polyethylene glycol monomethyl ether (mPEG-NH2) and loaded with vedicituzumab RC48, and combined with 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, has 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, which plays a role in tumor immunosuppression, realizing targeted photothermal immunotherapy for human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Two-dimensional black phosphorus (BP) is used as a carrier, which has 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 the performance remains stable. Although traditional photothermal therapy materials such as gold nanorods and carbon-based materials have certain effects, they often have problems of bioincompatibility or non-degradability. The BP carrier of the present invention is completely metabolizable, avoiding the biological toxicity caused by residues.

[0019] The coordination principle of the components of the present invention: 1. The synergistic effect of BP-PEG-RC48-R848: BP provides photothermal effect; RC48 is responsible for targeting HER2-positive tumor cells; R848 activates the immune system to achieve tumor ablation and recurrence prevention. 2. Structural characteristics of nanomedicine: Through functional modification, each component works synergistically to enhance the therapeutic effect.

[0020] Most 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 multifunctional synergistic treatment. The present invention integrates photothermal therapy, targeted therapy and immunotherapy into a nano-drug platform for the first time. The three functions work 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.

[0021] A method for preparing the above-mentioned two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification comprises: A. dissolving blocky black phosphorus crystal powder in an amide organic solvent, crushing it by ultrasonic vibration, centrifuging it and washing it with water to obtain an aqueous solution of black phosphorus nanosheets, and storing it at low temperature and away from light for later use; B. mixing the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then ultrasonically centrifuging and washing with water to obtain black phosphorus nanosheets modified with mPEG-NH2; C. Mixing the black phosphorus nanosheet modified with mPEG-NH2 with an aqueous solution of a specific targeting antibody, and then stirring, centrifuging and washing with water to obtain an intermediate product loaded with the specific targeting antibody; D. Mixing the intermediate product loaded with specific targeting antibodies and the alcohol solution of immune adjuvant, and then stirring, centrifuging and washing with water to obtain the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification.

[0022] Precision preparation method of multifunctional drug platform: 1. Liquid phase exfoliation method: NMP dissolution and ultrasonic centrifugation process are used to prepare two-dimensional black phosphorus nanosheets with uniform particle size (100-200 nm). 2. Multi-step modification process: mPEG-NH2, RC48 and R848 are gradually modified by ultrasound, stirring and other methods to ensure the successful combination and functional synergy of each component.

[0023] The preparation process of multifunctional nano drugs in the prior art is complicated, the reaction conditions are harsh, and it is difficult to achieve industrial application. The present invention adopts liquid phase exfoliation method in the preparation of nano materials, which has a simple preparation process and mild conditions, and can achieve large-scale production. The process flow is scientifically designed to ensure the functionality and stability of each component, laying the foundation for clinical transformation. While ensuring the performance, the present invention optimizes the preparation process and has higher operability and promotion value.

[0024] Preferably, the mass volume ratio of the blocky black phosphorus crystal powder to the amide organic solvent includes 50-100 mg: 20-30 mL; the mass feed ratio of the black phosphorus nanosheets, the mPEG-NH2, the specific targeting antibody, and the immune adjuvant includes 1:8-10:1.5-2.5:1-1.5; the concentration of the aqueous solution of the black phosphorus nanosheets includes 4-6 mg / mL; the concentration of the aqueous solution of the mPEG-NH2 includes 10-20 mg / mL; the concentration of the aqueous solution of the specific targeting antibody includes 5-10 mg / mL; the concentration of the alcohol solution of the immune adjuvant includes 5-10 mg / mL.

[0025] Preferably, the black phosphorus nanosheets include two-dimensional black phosphorus nanosheets of 100-200 nm; the amide organic solvent includes NMP; the mPEG-NH2 includes mPEG-NH2 with a molecular weight of 5000; and the alcohol solution of the immune adjuvant includes a methanol solution of the immune adjuvant.

[0026] Preferably, including: A. dissolving the blocky black phosphorus crystal powder in the amide organic solvent, firstly crushing it with ultrasonic vibration of the probe and then cleaning it with ultrasonic vibration of low-temperature water bath, then centrifuging at 2500-3500 rpm for 10-15min to remove the large black phosphorus precipitate, then centrifuging at 12000-13000 rpm and washing with deionized water to obtain an aqueous solution of the black phosphorus nanosheets dispersed evenly, and storing it at 4-8°C away from light for later use; B. Mixing the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then ultrasonicating in a light-proof water bath at 10-15°C for 2-4 hours, centrifuging at 12000-13000 rpm for 10-15 minutes and washing with deionized water, collecting all precipitates, and obtaining the black phosphorus nanosheets modified with mPEG-NH2; C. Mixing the black phosphorus nanosheet modified with mPEG-NH2 with the aqueous solution of the specific targeting antibody, stirring for 10-14 hours in the dark, centrifuging at 12000-13000 rpm for 10-15 minutes and washing with deionized water, collecting all precipitates, and obtaining the intermediate product loaded with the specific targeting antibody; D. Mix the intermediate product loaded with specific targeting antibodies and the alcohol solution of the immune adjuvant, then stir in an open and dark environment for 12-16 hours, centrifuge at 12000-13000 rpm for 10-15 minutes and wash with deionized water, collect all precipitates, and obtain the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification.

[0027] Preferably, in step A, the operation of first using a probe for ultrasonic oscillation and then using a low-temperature water bath for ultrasonic oscillation cleaning includes: first using a cell crusher probe for ultrasonic oscillation and crushing, using an ultrasonic cell crusher with a No. 6 amplitude rod for ultrasonic crushing in an ice bath for 60-80 min under the conditions of a power of 810-900 W and an ultrasonic frequency of a cycle of on 2.0 s and off 1.0 s, repeating 3 times, and then ultrasonic cleaning in a 10-15°C water bath for 1-1.5 h.

[0028] An application of the above-mentioned two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification in the preparation of targeted drugs for photothermal and immune combined treatment of cancer.

[0029] Preferably, the cancer is HER2-positive breast cancer.

[0030] The present invention can be directly used for the photothermal-immunotherapy of cancer, or for preparing related drugs, especially for the photothermal-immunotherapy of in situ breast cancer.

[0031] The implementation of the present invention has the following beneficial effects: 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 of uniform size. The surface covalent modification of amino polyethylene glycol monomethyl ether (mPEG-NH2) significantly improves the colloidal stability and biocompatibility, and enhances the antigen capture ability of antigen-presenting cells. The anti-HER2 antibody RC48 is further modified by directional coupling technology to achieve tumor-specific targeting, and the immune adjuvant R848 is loaded to activate the TLR7 / 8 signaling pathway to form a "photothermal-targeting-immunity" triple synergistic treatment system. The entire process is completed in a 25°C aqueous environment, the reaction conditions are mild and controllable, and it has the potential for industrial production.

[0032] 2. The present invention innovatively integrates RC48 antibody and R848 adjuvant into the photosensitive black phosphorus platform, breaking through the limitations of traditional therapies. Among them, RC48 achieves accurate recognition and drug delivery of HER2-positive tumors through antigen-antibody specific binding, and directly kills tumor cells in combination with the high photothermal conversion efficiency of black phosphorus; R848 activates dendritic cells (DC) and induces macrophages to secrete M1 cytokines, induces the secretion of TNF-α and IL-6, and reverses the tumor immunosuppressive microenvironment. Experiments have shown that the system has a high drug loading rate, and the synergistic effect of photothermal ablation and immune activation is significantly better than single therapy.

[0033] 3. The present invention uses metabolizable two-dimensional black phosphorus as the core carrier, which has both high biosafety 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 temperature rise of tumor tissue is induced by near-infrared light irradiation, which triggers thermal damage and antigen release of tumor cells. R848 activates the anti-tumor ability of the immune system, eliminates residual tumor cells, and prevents recurrence. Compared with the existing technology, its breakthrough lies in the modular integration of antibody targeted therapy, photothermal ablation and immune regulation, which provides a new strategy for the precise combined treatment of HER2-positive breast cancer, and has broad prospects for clinical application.

[0034] 4. The present invention provides a two-dimensional black phosphorus hybrid nanomedicine targeting HER2-positive breast cancer based on RC48 protein modification, which realizes photothermal, targeted and immune combined therapy through the following technical steps: 1. Nanocarrier selection: Two-dimensional black phosphorus (BP) is used as a drug carrier, which has high photothermal conversion efficiency and biodegradability. 2. Multifunctional modification: mPEG-NH2 (amino polyethylene glycol monomethyl ether) is used to improve the colloidal stability and biocompatibility of BP; RC48 (vedicizumab, HER2-targeted antibody) is combined to achieve precise targeting; R848 (immune adjuvant resimod) is loaded to activate the immune system. 3. Comprehensive treatment: Photothermal therapy (PTT) and immunotherapy are integrated to achieve synergistic treatment of HER2-positive breast cancer. Through innovative nanomaterial design and multifunctional modification, the shortcomings of insufficient targeting and single therapeutic effect in the existing technology are overcome, and accurate and efficient treatment of HER2-positive breast cancer is achieved, which significantly improves the efficacy and reduces side effects. This solution provides strong technical support and application prospects for photothermal and immune combined therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The material analysis diagrams prepared at different stages of Example 1 of the present invention, wherein: (a) is the TEM image of BP-PEG-RC48-R848; (b) is the AFM image of BP-PEG-RC48-R848; (c) is the element mapping analysis diagram of BP-PEG-RC48-R848.

[0036] Figure 2 This is a surface potential bar graph of BP-PEG-RC48-R848, its intermediates and other components of Effect Example 1 of the present invention.

[0037] Figure 3 The ultraviolet absorption spectra of Example 1 of the present invention are as follows: (a) ultraviolet absorption spectra of BP-PEG-RC48-R848 solutions with different concentrations of 100, 50, 25, 12.5, and 6.25 μg / mL in the range of 220-280 nm; (b) ultraviolet absorption spectra of R848, BP-PEG-RC48, and BP-PEG-RC48-R848 solutions in the range of 220-280 nm.

[0038] Figure 4 Two temperature curves of Example 1 of the present invention: (a) BP-PEG-RC48-R848 with different concentrations at 1.0 W / cm 2 Temperature rise diagram after 808 nm laser irradiation for 10 min; (b) 100 μg / mL BP-PEG-RC48-R848 at 1.0 W / cm 2Temperature rise-fall curve under 808nm laser irradiation.

[0039] Figure 5 This is the ultraviolet absorption spectrum of the R848 immune adjuvant of Example 1 of the present invention: (a) ultraviolet absorption spectrum of R848 solution with different concentrations in the range of 220-300nm; (b) ultraviolet absorption standard curve of R848 solution at 247nm.

[0040] Figure 6 This is a schematic diagram of the dark cytotoxicity evaluation of Example 2 of the present invention, wherein: the cell survival rate (a) and cell morphology (c) of 4T1 cells after co-culture with different concentrations of RC48 and BP-PEG-RC48-R848 for 24 hours; the cell survival rate (b) and cell morphology (d) of SKBR3 cells after co-culture with different concentrations of RC48 and BP-PEG-RC48-R848 for 24 hours.

[0041] Figure 7 Schematic diagram of cell phototoxicity evaluation of Example 2 of the present invention, wherein: (a) cell survival rate of 4T1 cells after incubation with different concentrations of BP-PEG-RC48-R848 for 4 hours with or without laser irradiation; (b) cell survival rate of SKBR3 cells after incubation with different concentrations of BP-PEG-RC48-R848 for 4 hours with or without laser irradiation (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. +L).

[0042] Figure 8 The phototoxic live-dead double staining images of cells in Example 2 of the present invention, where: (a) 4T1 cells were co-cultured with drugs of different concentrations for 4 h and the power was 1.0 W / cm 2 Calcein-AM / PI double-stained cells after irradiation with 808 nm laser (L) for 10 min; (b) SKBR3 cells were co-cultured with drugs at different concentrations for 4 h and irradiated with 1.0 W / cm 2 Calcein-AM / PI double-stained cells after irradiation with 808 nm laser (L) for 10 min (scale bar is 100 μm).

[0043] Fig. 9 Schematic diagram of cell targeting in Example 2 of the present invention: (a) microscopy and DAPI staining images of 4T1 cells after co-culture with drugs of different concentrations; (b) microscopy and DAPI staining images of SKBR3 cells after co-culture with drugs of different concentrations (scale bar is 50 μm).

[0044] Fig.10The contents of TNF-α (a) and IL-6 (b) cytokines secreted by BMDC after being treated with different samples in Example 3 of the present invention (n = 3, ***P<0.001 vs. BP-PEG-RC48-R848).

[0045] Fig.11 In Example 3 of the present invention, BMDCs were treated with different samples and then matured into DCs (CD80 + CD86 + Representative flow cytometry plots (a) and percentages (b) of BP-PEG-RC48-R848 (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. +BP-PEG-RC48-R848).

[0046] Fig.12 In Example 4 of the present invention, spleen CD3 + CD4 + T cells (a), CD3 + CD8 + Representative flow cytometry plots of T cells (b) and percentage of activated T cells (c, d) (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. +BP-PEG-RC48-R848+L).

[0047] Fig.13 In Example 4 of the present invention, DC maturation (CD80 + CD86 + Representative flow cytometry plots (a) and percentages (b) of BP-PEG-RC48-R848+L (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. +BP-PEG-RC48-R848+L).

[0048] Fig.14 In Example 5 of the present invention, (a) is a comparison graph of fluorescence intensity monitoring of the tumor area at multiple time points after the addition of the drug in the HER2-positive mouse model and a fluorescence intensity quantitative analysis graph (b); (c) is a comparison graph of drug fluorescence intensity in the body of HER2-negative and positive mice 3 days after drug injection; (d) is a tumor fluorescence intensity analysis graph of HER2-negative and positive mice 3 days after drug injection; (e) is a comparison graph of drug fluorescence intensity in the body of HER2-negative and positive mice 24 hours after drug injection; (f) is a comparison graph of the fluorescence intensity accumulation of various organs in the body of HER2-negative and positive mice 3 days after drug injection (n = 3, *P<0.05, **P<0.01, ***P<0.001 vs. HER2+).

[0049] Fig.15 In Example 6 of the present invention, (a) is a schematic diagram of the anti-tumor experiment process in mice; (b) 24 hours after the tail vein injection of the drug, the mouse was irradiated with laser (1.0 W / cm 2 ) of the tumor site; (c) the curve of the change of tumor volume with the number of days after the mice were treated with different methods; after the mice were killed on day 21, the statistical analysis of the tumor weight of the mice treated with different methods (d) and the comparison of the anatomical morphology of tumor tissues in different treatment groups (e). DETAILED DESCRIPTION

[0050] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0051] Example 1 The preparation of BP-PEG-RC48-R848 drug includes the following process: (1) Preparation of black phosphorus nanosheets The powdered black phosphorus crystals were dissolved in NMP by liquid phase exfoliation method, and then ultrasonically oscillated and crushed with a cell crusher probe. The ultrasonic cell crusher with a No. 6 amplitude was ultrasonically crushed in an ice bath for 60 min at a power of 810 W and an ultrasonic frequency of 2.0 s on and 1.0 s off. This was repeated 3 times, and then ultrasonically cleaned in a 10°C water bath for 1 h. After that, the solution was 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 then washed with deionized water to obtain a uniformly dispersed aqueous solution of the black phosphorus nanosheets. Finally, the black phosphorus concentration was quantified with a UV-visible spectrophotometer and stored at 4°C in the dark for later use.

[0052] (2) The black phosphorus nanosheet aqueous solution and mPEG-NH2 aqueous solution (20 mg / mL) prepared above were mixed and sonicated in a light-proof water bath at 10°C for 4 h. The unbound excess mPEG-NH2 was removed by centrifugation at 13,000 rpm for 12 min, and the mixture was washed with deionized water to collect all the precipitates to obtain the BP-PEG aqueous solution.

[0053] (3) The BP-PEG aqueous solution and RC48 aqueous solution (10 mg / mL) prepared above were mixed and stirred in the dark for 12 h. The unbound excess RC48 was removed by centrifugation at 13,000 rpm for 12 min, and the mixture was washed with deionized water. All precipitates were collected to obtain a BP-PEG-RC48 aqueous solution.

[0054] (4) The BP-PEG-RC48 aqueous solution prepared above and the R848 methanol solution (10 mg / mL) were mixed, stirred in an open and dark environment for 12 h, centrifuged at 13,000 rpm for 12 min to remove the unbound excess R848, and washed with deionized water to collect all the precipitates to obtain the BP-PEG-RC48-R848 aqueous solution.

[0055] (5) The BP-PEG-RC48-R848 aqueous solution prepared in (4) of Example 1 and the ICG-NHS alcohol solution (5 mg / mL) were mixed, stirred for 12 h in an open and dark environment, centrifuged at 13,000 rpm for 12 min to remove the unbound excess ICG-NHS, and washed with deionized water to collect all the precipitates to obtain the BP-PEG-RC48-R848-ICG aqueous solution for in vivo targeting evaluation experiments in mice. ICG (indocyanine green) is a near-infrared fluorescent probe, and staining with ICG can be used for in vivo imaging for easy observation.

[0056] Example 2 The preparation of BP-PEG-RC48-R848 drug includes the following process: (1) Preparation of black phosphorus nanosheets The powdered black phosphorus crystals were dissolved in NMP by liquid phase exfoliation method, and then ultrasonically oscillated and crushed with a cell crusher probe. The ultrasonic cell crusher with a No. 6 horn was ultrasonically crushed in an ice bath for 60 min at a power of 900 W and an ultrasonic frequency of 2.0 s on and 1.0 s off. This was repeated 3 times, and then ultrasonically cleaned in a 15°C water bath for 1 h. After that, the black phosphorus was centrifuged at 2500 rpm for 15 min to remove the large black phosphorus precipitate, and then centrifuged at 12000 rpm for 15 min to remove the residual NMP, and then washed with deionized water to obtain a uniformly dispersed aqueous solution of the black phosphorus nanosheets. Finally, the black phosphorus concentration was quantified by a UV-visible spectrophotometer and stored at 8°C in the dark for later use.

[0057] (2) The black phosphorus nanosheet aqueous solution and mPEG-NH2 aqueous solution (15 mg / mL) prepared above were mixed and sonicated in a light-proof water bath at 15°C for 2 h. The unbound excess mPEG-NH2 was removed by centrifugation at 12000 rpm for 15 min, and the mixture was washed with deionized water to collect all the precipitates to obtain the BP-PEG aqueous solution.

[0058] (3) The BP-PEG aqueous solution and RC48 aqueous solution (7 mg / mL) prepared above were mixed and stirred in the dark for 14 h, and the unbound excess RC48 was removed by centrifugation at 12000 rpm for 15 min. The mixture was washed with deionized water and all precipitates were collected to obtain a BP-PEG-RC48 aqueous solution.

[0059] (4) The BP-PEG-RC48 aqueous solution prepared above and the R848 methanol solution (7 mg / mL) were mixed, stirred in an open and dark environment for 16 h, centrifuged at 12,000 rpm for 15 min to remove the unbound excess R848, and washed with deionized water to collect all the precipitates to obtain the BP-PEG-RC48-R848 aqueous solution.

[0060] Example 3 The preparation of BP-PEG-RC48-R848 drug includes the following process: (1) Preparation of black phosphorus nanosheets The powdered black phosphorus crystals were dissolved in NMP by liquid phase exfoliation method, and then ultrasonically oscillated and crushed with a cell crusher probe. The ultrasonic cell crusher with a No. 6 amplitude was ultrasonically crushed in an ice bath for 80 min at a power of 810 W and an ultrasonic frequency of 2.0 s on and 1.0 s off. This was repeated 3 times, and then ultrasonically cleaned in a 12°C water bath for 1.5 h. After that, the black phosphorus was centrifuged at 3500 rpm for 10 min to remove the large black phosphorus precipitate, and then centrifuged at 12500 rpm for 10 min to remove the residual NMP, and then washed with deionized water to obtain a uniformly dispersed aqueous solution of the black phosphorus nanosheets. Finally, the black phosphorus concentration was quantified by a UV-visible spectrophotometer and stored at 6°C in the dark for later use.

[0061] (2) The black phosphorus nanosheet aqueous solution and mPEG-NH2 aqueous solution (10 mg / mL) prepared above were mixed and sonicated in a light-proof water bath at 12°C for 3 h. The unbound excess mPEG-NH2 was removed by centrifugation at 12500 rpm for 10 min, and the mixture was washed with deionized water to collect all the precipitates to obtain the BP-PEG aqueous solution.

[0062] (3) The BP-PEG aqueous solution and RC48 aqueous solution (5 mg / mL) prepared above were mixed and stirred in the dark for 12 h. The unbound excess RC48 was removed by centrifugation at 12500 rpm for 10 min, and the mixture was washed with deionized water to collect all the precipitates to obtain the BP-PEG-RC48 aqueous solution.

[0063] (4) The BP-PEG-RC48 aqueous solution prepared above and the R848 methanol solution (5 mg / mL) were mixed, stirred in an open and dark environment for 14 h, centrifuged at 12500 rpm for 10 min to remove the unbound excess R848, and washed with deionized water to collect all the precipitates to obtain the BP-PEG-RC48-R848 aqueous solution.

[0064] Effect Example 1 Taking the sample prepared in Example 1 as an example, transmission electron microscopy characterization shows that ( Figure 1a), BP-PEG-RC48-R848 nanocomposite presents a typical layered topology, with jagged edges and an average particle size of about 100-200 nm. RC48 is attached to the surface of BPNSs and wraps BPNSs, proving the successful loading of RC48. Atomic force microscopy three-dimensional morphology analysis ( Figure 1 b) Further verification shows that the cross-sectional height distribution is unimodal, with the thickest reaching 23.48 nm and an average thickness of 20.37 ± 3.17 nm, which is significantly lower than that of bare BP nanosheets. The energy dispersion spectrum (Figure 1c) shows that BP-PEG-RC48-R848 is mainly composed of P elements, as well as a large number of other elements such as carbon (C), nitrogen (N) and oxygen (O).

[0065] Zeta potential analysis revealed the hierarchical assembly process of the hybrid nanosystem ( Figure 2 ), PO4 generated by surface oxidation of pristine black phosphorus nanosheets (BPNSs) 3- The group showed significant negative charge (-30.87 ± 0.25 mV). BPNSs and mPEG-NH2 were prepared by electrostatic interaction. After the negatively charged BPNSs were modified by mPEG-NH2 (1.70 ± 0.29mV), the potential of the product BP-PEG was significantly increased to (-14.39 ± 0.18mV). This change was attributed to the electrostatic neutralization effect between the protonated amino group at the end of mPEG-NH2 and the negative charge on the surface of BP. RC48 is a large molecular antibody protein and is therefore neutral (-0.62 ± 0.13mV), so the potential of BP-PEG-RC48 (-14.12 ± 0.21mV) was only slightly adjusted. R848 is electronegative (-17.06 ± 1.37 mV). After BP-PEG-RC48 combined with R848, the potential of the final product BP-PEG-RC48-R848 was significantly reduced to -18.38 ± 0.42 mV, which also proved the successful loading of R848.

[0066] UV-visible absorption spectroscopy analysis showed that ( Figure 3 ), BP, R848, BP-PEG-RC48 and BP-PEG-RC48-R848 all have spectral absorption in the range of 220-280 nm. Figure 3 a shows the UV absorption characteristics of BP-PEG-RC48-R848 at a concentration gradient of 100, 50, 25, 12.5, and 6.25 μg / mL. Figure 3 As shown in b, BP-PEG-RC48-R848 loaded with R848 showed a characteristic absorption peak at 245-248 nm that was completely consistent with that of free R848 ( Figure 3b), further verifying the successful loading of R848.

[0067] To evaluate the effects of 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. Figure 4 As shown in a, the laser light at 808 nm (1.0 W / cm 2 ) After irradiation for 10 minutes, the temperature of the 100 μg / mL BP-PEG-RC48-R848 solution increased significantly to 56.2 ± 0.5℃, and even when the concentration dropped to 50 μg / mL, it still reached 51.1 ± 0.3℃, which fully confirmed that the composite system has concentration-dependent photothermal conversion ability. More importantly, after four temperature rise / fall cycles ( Figure 4 b) The maximum temperature of the material only dropped by about 2%, demonstrating highly stable photothermal performance, which provides a key guarantee for repeated treatment in clinical transformation.

[0068] To ensure the accuracy of drug quantitative analysis, this patent established a standard curve of R848 through UV-visible absorption spectroscopy ( Figure 5 a). The standard sample was prepared by gradient dilution method (15~0.625 μg / mL, 9 concentration points in total). Spectral analysis showed that R848 had a significant characteristic absorption peak at 247nm. The standard curve calculated by the characteristic peak value ( Figure 5 b) shows excellent linear relationship (R 2 = 0.9997), based on which the loading rate of R848 in BP-PEG-RC48-R848 was calculated to be 23.04%, which provided an accurate basis for drug quantification in subsequent efficacy experiments.

[0069] Effect Example 2 Taking the samples prepared in Example 1 as an example, the cytotoxicity of RC48 and BP-PEG-RC48-R848 was evaluated by CCK-8 assay and cell morphology observation. Figure 6 As shown in a, in HER2-low-expressing 4T1 cells, both drugs showed no significant cytotoxicity in the concentration range of 0-50 μg / mL, and the cell survival rate was always maintained above 85%. However, in HER2-high-expressing SKBR3 breast cancer cells ( Figure 6b) Both RC48 and BP-PEG-RC48-R848 showed a dose-dependent killing effect: when the concentration reached 50 μg / mL, the cell survival rate of the free RC48 group was 67.37% ± 6.26%, while the survival rate 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 may enhance the cytotoxicity by enhancing the targeted delivery of drugs. 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 and no obvious membrane rupture or fragmentation. The survival rate was 89.56% ± 6.45%, which was highly consistent with the CCK-8 data. Bright field microscopy images ( Figure 6 d) Further, it was shown that SKBR3 cells showed typical apoptotic morphological characteristics in the BP-PEG-RC48-R848 treatment group, including cell shrinkage, shedding, and cytoplasmic vacuolation, while 4T1 cells maintained normal morphology, which once again confirmed the reliability of the CCK-8 experiment. The above results show that BP-PEG-RC48-R848 has good biocompatibility in HER2-negative cells and exhibits a specific killing effect on HER2-positive tumor cells, providing an experimental basis for the safety and effectiveness of subsequent targeted therapy.

[0070] To verify the synergistic enhancement of the photothermal conversion performance of black phosphorus on the drug killing effect, this study further evaluated the in vitro phototoxic effect of BP-PEG-RC48-R848 on 4T1 (HER2-negative) and SKBR3 (HER2-positive) cells. In the experimental design, cells were co-cultured with different concentrations of nanodrugs for 4 hours, and then 808 nm near-infrared laser (1.0 W / cm 2 ) for 10 min, and the cell viability was quantified by CCK-8 method. Figure 7 As shown in Figure 2, the photothermal synergistic effect significantly improved the cell killing efficiency of the nanomedicine. Figure 7 a) Under laser irradiation, the cytotoxicity of BP-PEG-RC48-R848 showed a concentration-dependent enhancement. When the black phosphorus concentration reached 50 μg / mL, the cell survival rate dropped to 49.93% ± 4.55%, which was about 35% lower than that of the no-light group (survival rate>85%) (p<0.01), indicating that the photothermal effect of black phosphorus can break through the non-targeted 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 about 50% lower than that of the 4T1 group (p < 0.01), confirming the synergistic mechanism of targeted delivery and photothermal effect. This indicates that the local high heat (ΔT ≈ 25°C) generated by black phosphorus nanosheets under near-infrared laser excitation can induce increased tumor cell membrane permeability and promote the intracellular penetration of RC48; at the same time, HER2 targeting allows the drug to accumulate at a higher concentration in SKBR3 cells. It is worth noting that after the drug is loaded with R848, the killing effect on HER2-positive cells is significantly stronger than that of RC48 alone, 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.

[0071] To directly evaluate the photothermal synergistic killing effect of BP-PEG-RC48-R848, this study used live-dead cell double staining (Calcein-AM / PI) to detect HER2-negative 4T1 cells ( Figure 8 a) and HER2-positive SKBR3 cells ( Figure 8 b) Quantitative analysis. In the experimental design, cells were co-cultured with different concentrations of nanodrugs (0-50 μg / mL, based on black phosphorus concentration) for 4 hours, and then irradiated with 808 nm near-infrared laser (power density 1.0 W / cm²) for 10 minutes, and the cell survival status was observed and quantified by fluorescence microscopy. As the concentration of BP-PEG-RC48-R848 increased (0-50μg / mL), the PI red fluorescence signal (marking dead cells) of the two cell lines was significantly enhanced, while the Calcein-AM green fluorescence signal (marking live cells) was synchronously weakened. The SKBR3 cell death rate was higher than that of 4T1 cells, which was consistent with the phototoxic survival rate analysis. It is worth noting that in the control group with PBS plus laser irradiation, a large number of living cells survived, confirming that simple photothermal stimulation had no significant toxicity, while the cell killing of BP-PEG-RC48-R848 depended on the synergistic effect of drug and photothermal. BP-PEG-RC48-R848 significantly enhances the selective killing of HER2-positive tumors by integrating targeted delivery and photothermal conversion properties, providing a key experimental basis for the development of photothermal-chemotherapy synergistic treatment schemes.

[0072] To verify the targeting specificity of BP-PEG-RC48-R848, this study used fluorescence microscopy to observe the aggregation behavior of nanodrugs around HER2-negative (4T1) and positive (SKBR3) breast cancer cells. Fig. 9 As shown in a, the drug is sparsely distributed around 4T1 cells, indicating that its aggregation mainly depends on nonspecific adsorption or passive permeation (such as the EPR effect). Fig. 9In b, the drug forms dense clusters on the surface and pericellular area of ​​SKBR3 cells, and the aggregation effect is far greater than that of 4T1, confirming that RC48-mediated HER2 targeted binding significantly enhances drug enrichment. As a HER2 monoclonal antibody fragment, RC48 drives the nanodrug to anchor on the surface of SKBR3 cell membranes 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 endocytosis.

[0073] Effect Example 3 Taking the sample prepared in Example 1 as an example, in order to evaluate the immune activation effect of BP-PEG-RC48-R848 nanocomplex on bone marrow-derived dendritic cells (BMDC), this study quantitatively detected the secretion levels of proinflammatory cytokines TNF-α and IL-6 by ELISA ( Fig.10 ). Experimental data showed that the nanomedicine loaded with R848 exhibited significant immune activation ability: in terms of TNF-α secretion level ( Fig.10 a), the concentration of BP-PEG-RC48-R848 group reached 277.52 ± 12.38 pg / mL, which was 14.4 times higher than that of PBS control group (19.26 ± 1.47 pg / mL) (P<0.001), and significantly higher than that of BP-PEG-RC48 group without R848 loading (96.52 ± 1.33 pg / mL, P<0.001); in terms of IL-6 secretion level ( Fig.10 b), the concentration of BP-PEG-RC48-R848 group was as high as 181.75 ± 6.35 pg / mL, which was about 7 times higher than that of PBS group (17.54 ± 4.11 pg / mL) and BP-PEG-RC48 group without R848 adjuvant (25.97 ± 2.76 pg / mL) (all P<0.001). Mechanism studies showed that the loaded adjuvant R848 activated the MyD88 / NF-κB signaling pathway mediated by TLR7 / 8 receptors, drove BMDC to differentiate into a pro-inflammatory phenotype, and synergized with nanocarriers to enhance antigen presentation ability. 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.

[0074] 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, and the expression levels of mature DCs markers (CD80+CD86+) were analyzed by flow cytometry. Representative flow cytometry plots ( Fig.11a) The results showed that the proportion of mature DCs in the BP-PEG-RC48-R848 group was significantly higher than that in the other treatment groups. Quantitative analysis showed that ( Fig.11 b), compared with the PBS group (40.63±2.12%), RC48 group (25.83±1.84%) and 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 indicates that BP-PEG-RC48-R848 loaded with adjuvant R848 can activate BMDC cells and increase the proportion of mature BMDC cells. By synergizing the targeted delivery of RC48 and the immune stimulation of R848, the antigen presentation function of DCs is further enhanced.

[0075] Effect Example 4 Taking the sample prepared in Example 1 as an example, a HER2-positive breast cancer transplant tumor model was constructed using 6-8 week old female Balb / c mice. The HER2-positive breast cancer cell suspension of EMT6 (100-200 μL, concentration 1-5×10 6 cells / mL), and when the tumor volume reached 150 mm³, the mice were randomly divided into groups and treated with tail vein injection (100-120 μL / mouse). The experimental group was injected with black phosphorus composite nanodrugs (BP concentration 1 mg / mL), and the control group was given RC48 monotherapy with equivalent loading amounts. The first administration was recorded as day 0, and 24 hours later, the tumor area of ​​the mice in the illumination group was irradiated with 808 nm near-infrared laser (power density 1 W / cm², lasting 10 min). On the third day after treatment, the mice were killed, and the tumor-draining lymph nodes and spleen tissues were aseptically isolated. The tissue samples were placed in 3 mL PBS and mechanically ground to prepare a single cell suspension. After centrifugation at 1500 rpm for 5 min to remove the supernatant, the pre-cooled red blood cell lysis solution was added and incubated in an ice bath for 3 min, and the lysis products were centrifuged again to remove the lysis products. The obtained cell pellet was resuspended in PBS and filtered through a 30 μm cell filter membrane to finally obtain a high-purity single cell suspension. 100 μL of spleen cell suspension was added with CD3, CD4 and CD8 fluorescent antibodies respectively; 100 μL of 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⁺, CD3⁺CD8⁺ in the spleen cell suspension and the expression ratios of CD80⁺CD86⁺ in lymphocytes.

[0076] To evaluate the regulatory effect of BP-PEG-RC48-R848 combined with photothermal therapy on the immune system, flow cytometry was first used to detect the changes in spleen T lymphocyte subsets in mice in different treatment groups ( Fig.12 a and 12b are CD3 +CD4 + and CD3 + CD8 + Flow cytometry representative diagram). Fig.12 c shows that under 808 nm laser irradiation, helper T cells (CD3 + CD4 + ) ratio significantly increased to 11.27%±0.42%, which was about 2.7 times higher than the PBS control group (4.21%±0.19%) (***p<0.001) and 1.6 times higher than the BP-PEG-RC48 group without R848 loading (7%±0.07%). It is worth noting that this ratio is higher than the non-light-treated group (BP-PEG-RC48-R848: 7.84%±0.07%). Fig.12 As shown in d, cytotoxic T lymphocytes (CD3 + CD8 + ) accounted for 6.69%±0.31% in the treatment group, which was significantly higher than other groups such as the BP-PEG-RC48 group (3.89%±0.16%) and PBS group (3.13%±0.04%) without R848 loading. This difference confirmed that photothermal activation can synergistically enhance the recruitment ability of the loaded R848 adjuvant on splenic helper T cells, suggesting that laser-mediated local temperature increase may amplify the anti-tumor immune response by promoting targeted drug release or regulating the immune microenvironment.

[0077] In order to further systematically evaluate the immune activation efficacy of nanomedicines, the present invention quantitatively analyzed the maturity of dendritic cells (DCs) in mouse lymph nodes by flow cytometry ( Fig.13 a). Experimental data show that ( Fig.13 b): The proportion of mature DCs (CD80⁺CD86⁺ double positive markers) 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 44.4% ± 1.5% in the BP-PEG-R848+L group (***p<0.001), and significantly higher than 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 R848 adjuvant significantly enhanced the immunogenicity of the drug through the Toll-like receptor 7 / 8 signaling pathway. More importantly, photothermal stimulation can synergistically promote the activation of antigen-presenting cells, increasing the proportion of mature DCs by nearly 1.7 times compared with the non-irradiated group, which fully reflects the multiple synergistic mechanisms of light-controlled release drugs and immune activation.

[0078] Effect Example 5 Taking the sample prepared in Example 1 as an example, in order to systematically evaluate the in vivo targeting performance of nanomedicine, this study constructed a HER2-positive tumor-bearing (Balb / c female mouse) model, and injected a nanosuspension (100 μL / mouse) loaded with near-infrared fluorescent probe indocyanine green (ICG) through the tail vein. The spatiotemporal distribution characteristics of the fluorescence intensity in the tumor area at 6, 12, 24, 48 and 72 hours after administration were dynamically monitored using a small animal in vivo imaging system ( Fig.14 a), Quantitative analysis showed that the fluorescence signal at the tumor site reached a peak at 24 h ( Fig.14 b), indicating that this time window is the optimal treatment intervention node. After euthanasia at the end of the experiment, tumor tissues and major organs (heart, liver, spleen, lungs, and kidneys) were completely removed, and the tumor targeting efficiency and biodistribution pattern of the nanoparticles were quantitatively revealed by ex vivo fluorescence imaging.

[0079] It is worth noting that the fluorescence intensity of the tumor area in the HER2-positive model group was significantly higher than that in the HER2-negative control group 72 hours after administration ( Fig.14 c), Quantitative analysis of in vitro tumors further confirmed that the fluorescence accumulation in the positive group was 1.6 times that in the negative group (***p<0.001, Fig.14 d). Time-lapse in vivo imaging ( Fig.14 e) and multi-organ biodistribution studies ( Fig.14 f) Joint verification: Nanomedicine showed specific targeted accumulation in HER2-positive models, indicating that the active targeting mechanism mediated by RC48 antibody effectively enhanced tumor-selective delivery.

[0080] Effect Example 6 Taking the sample prepared in Example 1 as an example, in order to systematically evaluate the in vivo anti-tumor performance of the nanomedicine, this patent selected 6-8 week old female Balb / c mice to construct a HER2 positive breast cancer xenograft model. The EMT6-HER2+ breast cancer cell suspension (1-5×10 6 cells / 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. Fig.15 As shown in a.

[0081] The results showed that the tumor sites of mice in the BP group treated with laser irradiation showed a significant increase in temperature ( Fig.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. Fig.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. Fig.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 ( Fig.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.

[0082] 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 nanodrug modified with RC48 protein, characterized in that: It comprises a carrier and an active ingredient loaded on the carrier; the carrier comprises a two-dimensional black phosphorus nanosheet modified by mPEG-NH2; and the active ingredient comprises a specific targeting antibody and an immune adjuvant.

2. The two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 1, characterized in that: The specific targeting antibody includes a monoclonal antibody that specifically targets HER2.

3. The two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 1, characterized in that: The specific targeting antibody includes RC48; the immune adjuvant includes R848.

4. A method for preparing a two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 1, characterized in that: include: A. dissolving blocky black phosphorus crystal powder in an amide organic solvent, crushing it by ultrasonic vibration, centrifuging it and washing it with water to obtain an aqueous solution of black phosphorus nanosheets, and storing it at low temperature and away from light for later use; B. mixing the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then ultrasonically centrifuging and washing with water to obtain black phosphorus nanosheets modified with mPEG-NH2; C. Mixing the black phosphorus nanosheet modified with mPEG-NH2 with an aqueous solution of a specific targeting antibody, and then stirring, centrifuging and washing with water to obtain an intermediate product loaded with the specific targeting antibody; D. Mixing the intermediate product loaded with the specific targeting antibody and the alcohol solution of the immune adjuvant, and then stirring, centrifuging and washing with water to obtain the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification.

5. The method for preparing the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 4, characterized in that: The mass volume ratio of the blocky black phosphorus crystal powder to the amide organic solvent includes 50-100 mg: 20-30 mL; the mass ratio of the black phosphorus nanosheets, the mPEG-NH2, the specific targeting antibody, and the immune adjuvant includes 1:8-10:1.5-2.5:1-1.5; the concentration of the aqueous solution of the black phosphorus nanosheets includes 4-6 mg / mL; the concentration of the aqueous solution of the mPEG-NH2 includes 10-20 mg / mL; the concentration of the aqueous solution of the specific targeting antibody includes 5-10 mg / mL; the concentration of the alcohol solution of the immune adjuvant includes 5-10 mg / mL.

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

7. The method for preparing the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 4, characterized in that: include: A. dissolving the blocky black phosphorus crystal powder in the amide organic solvent, firstly crushing it with ultrasonic vibration of the probe and then cleaning it with ultrasonic vibration of low-temperature water bath, then centrifuging at 2500-3500 rpm for 10-15min to remove the large black phosphorus precipitate, then centrifuging at 12000-13000 rpm and washing with deionized water to obtain an aqueous solution of the black phosphorus nanosheets dispersed evenly, and storing it at 4-8°C away from light for later use; B. Mixing the aqueous solution of the black phosphorus nanosheets with the aqueous solution of mPEG-NH2, then ultrasonicating in a light-proof water bath at 10-15°C for 2-4 hours, centrifuging at 12000-13000 rpm for 10-15 minutes and washing with deionized water, collecting all precipitates, and obtaining the black phosphorus nanosheets modified with mPEG-NH2; C. Mixing the black phosphorus nanosheet modified with mPEG-NH2 with the aqueous solution of the specific targeting antibody, stirring for 10-14 hours in the dark, centrifuging at 12000-13000 rpm for 10-15 minutes and washing with deionized water, collecting all precipitates, and obtaining the intermediate product loaded with the specific targeting antibody; D. Mix the intermediate product loaded with specific targeting antibodies and the alcohol solution of the immune adjuvant, then stir in an open and dark environment for 12-16 hours, centrifuge at 12000-13000 rpm for 10-15 minutes and wash with deionized water, collect all precipitates, and obtain the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification.

8. The method for preparing the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 7, characterized in that: In step A, the operation of first using the probe for ultrasonic oscillation and then using low-temperature water bath ultrasonic oscillation to clean the cells includes: first using the cell crusher probe for ultrasonic oscillation to crush the cells, using an ultrasonic cell crusher with a No. 6 amplitude transformer to ultrasonically crush the cells in an ice bath for 60-80 minutes at a power of 810-900 W and an ultrasonic frequency of 2.0 s on and 1.0 s off, repeating this three times, and then ultrasonically cleaning the cells in a 10-15°C water bath for 1-1.5 hours.

9. An application of the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 1, characterized in that: Application in the preparation of targeted drugs for photothermal and immunotherapy of cancer.

10. The use of the two-dimensional black phosphorus hybrid nanomedicine based on RC48 protein modification according to claim 9, characterized in that: The cancer is HER2-positive breast cancer.

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