Preparation method and application of bimetal composite nanomaterial for targeting salivary gland adenoid cystic carcinoma tumor

Abstract

The invention discloses a preparation method and application of a bimetallic composite nanomaterial for targeting a salivary gland adenoid cystic carcinoma tumor. The prepared bimetallic composite nanomaterial has SACC homologous targeting ability, and is used for systematically activating anti-tumor immune response of cold tumors. Under 808 nm laser irradiation, the nanometer material promotes active oxygen generation and glutathione depletion in tumor cells and aggravates oxidative stress by means of multi-enzyme activity and through photo-thermal therapy / photodynamic therapy / chemical dynamic therapy, generates oxygen to maintain PDT / CDT curative effect, effectively induces tumor cell immunogen cell death, and has a good application prospect. The adaptive anti-tumor immune response and cytotoxic T lymphocyte infiltration are obviously enhanced, so that the growth of primary lesion and metastatic lesion is inhibited. The invention provides an effective NIR-triggered space-time regulation and control system for reversing SACC cold tumor phenotype and inhibiting systemic metastasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanotechnology and biomedical materials, and particularly relates to a preparation method and application of a bimetallic composite nanomaterial particle for targeting salivary adenoid cystic carcinoma tumor. BACKGROUND

[0002] Salivary adenoid cystic carcinoma (SACC) is a highly malignant tumor derived from salivary gland epithelial tissue, and is mainly characterized by strong local infiltration, early perineural invasion (PNI) and late distant metastasis (mainly in the lung). The current standard treatment strategy for SACC includes surgical resection combined with radiotherapy or chemotherapy, but due to its insensitivity to radiotherapy and chemotherapy, combined with the complex tumor location and difficulty in monitoring metastasis, the local recurrence and distant metastasis rates after treatment are still high. More importantly, SACC is a typical "immune cold tumor", with low immune cell infiltration, poor antigen presentation efficiency and weak anti-tumor immune response in the tumor microenvironment, making it difficult for existing immunotherapy strategies to achieve satisfactory results in SACC. Therefore, developing a new treatment platform that combines tumor-targeted imaging, synergistic therapy and immune activation has become an important direction of current clinical research.

[0003] In recent years, nanomedicine has made significant progress in the field of precise tumor treatment, especially multifunctional nanoplatforms, which have been widely used in the treatment of solid tumors due to their excellent targeted delivery capacity, imaging navigation performance and multi-modal treatment potential. However, existing multifunctional nanomaterials still have many shortcomings when dealing with immunosuppressive tumors (such as SACC): on the one hand, most nanoplatforms lack sufficient immune stimulation signal release after promoting tumor cell death, making it difficult to effectively induce immunogenic cell death (ICD); on the other hand, although some nanomaterials have certain immune regulation functions, their imaging accuracy is limited, the treatment is single or the efficacy is unstable, and they cannot meet the integrated needs of diagnosis and treatment. Therefore, there is an urgent need to design a high-performance nanosystem that can efficiently mediate ICD effect, activate immune pathways, and has imaging and multi-modal treatment capabilities, to improve the comprehensive treatment effect on immune "cold tumors".

[0004] Therefore, there is an urgent need to develop a method that can effectively overcome the problem of traditional treatment failing to remove micro-metastases, for precise diagnosis and treatment of SACC. SUMMARY

[0005] The present application aims to solve the problems of poor targeting, limited treatment effect and low immune activation efficiency in the treatment of immunosuppressive tumors (SACC). To this end, a cell membrane coated magnetic bimetallic mesoporous organosilicon nanoparticle (MEM@Fe3O4@MnO2@IR780@MON) is provided, which has imaging, targeting, multi-mode treatment and immune regulation functions, can synergistically realize PTT / PDT / CDT treatment under near-infrared laser irradiation, induce ICD and activate the cGAS-STING pathway, significantly enhance the anti-tumor immune response, and is suitable for precise diagnosis and treatment of SACC and other "cold" tumors.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: A bimetallic composite nanomaterial targeting salivary gland adenoid cystic carcinoma tumor, the nanomaterial comprising Fe3O4@MnO2@MON loaded with IR780, and SACC-LM cell membrane coated on the surface thereof; the Fe3O4@MnO2@MON comprising a Fe3O4@MnO2 bimetallic oxide core and a mesoporous organosilica shell material; The preparation method of the bimetallic composite nanomaterial comprises the following steps: (1) Synthesizing sodium citrate modified Fe3O4 nanoparticles by a solvothermal method; (2) Dispersing the obtained Fe3O4 nanoparticles in deionized water, adding sodium citrate, then adding potassium permanganate solution dropwise, and after heating reaction, centrifuging, washing and freeze-drying, Fe3O4@MnO2 nanoparticles are obtained; (3) Dispersing the Fe3O4@MnO2 nanoparticles in deionized water, using cetyltrimethylammonium bromide as a template agent, tetraethyl orthosilicate as a silicon source, and bis-(gamma-triethoxysilylpropyl) tetrasulfide as an organosilicon source to obtain Fe3O4@MnO2@MON; (4) Mixing Fe3O4@MnO2@MON with IR780 solution, stirring overnight at room temperature, and after centrifuging, washing and freeze-drying, Fe3O4@MnO2@IR780@MON loaded with IR780 is obtained; (5) Mixing Fe3O4@MnO2@IR780@MON with SACC-LM cell membrane, and using ultrasonic combined with lipid extrusion method to coat SACC cell membrane on the surface of Fe3O4@MnO2@IR780@MON, and after centrifuging, washing and freeze-drying, the bimetallic composite nanomaterial is obtained.

[0007] Further, the specific steps of the solvothermal method in step (1) are as follows: sodium citrate, sodium acetate are dissolved in deionized water, then iron chloride hexahydrate is added, and then the mixture is transferred to a reaction kettle for hydrothermal reaction; the mass ratio of sodium citrate, sodium acetate and iron chloride hexahydrate is 1: (4.7-4.8): (2.5-2.6); the hydrothermal reaction temperature is 200 DEG C; and the reaction time is 8 hours.

[0008] Further, the concentration ratio of Fe3O4 nanoparticles, sodium citrate and potassium permanganate in step (2) is 10:1.2:1.58; the heating reaction temperature is 80 DEG C; and the reaction time is 1.5 hours.

[0009] Further, the dosage ratio of Fe3O4@MnO2 nanoparticles, cetyltrimethylammonium bromide, tetraethyl orthosilicate and bis- (gamma-triethoxysilylpropyl) tetrasulfide in step (3) is 150 mg:2.4 g:150 μL: (400-500 μL).

[0010] Further, the reaction temperature in step (3) is 40 DEG C; and the hydrothermal method needs a reaction time of 6 hours.

[0011] Further, in step (4), IR780 and 3-aminopropyltriethoxysilane in the IR780 solution are dissolved in anhydrous ethanol at a molar ratio of 1:27; and the solid-liquid ratio of Fe3O4@MnO2@MON and the IR780 solution is 10 mg:1 mL.

[0012] Further, in step (5), the mass ratio of Fe3O4@MnO2@IR780@MON and SACC-LM cell membrane is 1:2.

[0013] Further, in step (5), the ultrasonic power of the ultrasonic combined lipid extrusion method is 100 W, and the time is 10 min.

[0014] Further, in step (5), the ultrasonic combined lipid extrusion method needs to use polycarbonate membranes, and the diameters are 800 nm and 400 nm, respectively.

[0015] The application also provides application of the bimetallic composite nanomaterial in preparation of a targeted anti-salivary gland adenoid cystic carcinoma tumor drug.

[0016] The application has the following advantages: (1) The present invention provides a cell membrane coated magnetic bimetallic mesoporous organosilicon nanoparticle (MEM@Fe3O4@MnO2@IR780@MON). The nanoparticle can efficiently induce tumor cell immunogenic death, activate the cGAS–STING pathway, and enhance the systemic anti-tumor immune response through the synergistic effect of PTT / PDT / CDT. It has a good therapeutic effect on “cold tumor” (SACC) and has almost no toxic side effects on normal tissue cells.

[0017] (2) Based on the simple and efficient hydrothermal method, sol-gel method and cell membrane encapsulation technology, this invention constructs a nanoplatform with multiple therapeutic and imaging functions. The process is mature, easy to scale up production, and suitable for precision treatment research and clinical translation.

[0018] The MEM@Fe3O4@MnO2@IR780@MON in this invention operates under near-infrared laser conditions (808nm, 2W / cm²). 2 Under irradiation (5 min), it exhibits excellent photothermal and photosensitive response properties, effectively initiating the PTT / PDT / CDT synergistic treatment mechanism, generating a large amount of reactive oxygen species and achieving thermal damage, while simultaneously activating the immune response of tumor cells, overcoming the problem of incomplete removal of occult metastases and perineural invasive lesions in traditional treatments. Specifically, the Fe3O4@MnO2 bimetallic oxide core structure used in this invention possesses synergistic catalytic properties. Fe3O4 exhibits excellent magnetic responsiveness and can generate hydroxyl radicals (·OH) via the Fenton reaction, enhancing the efficacy of chemokinetic therapy (CDT); MnO2 can reduce and release MnO2 in the tumor microenvironment of high H2O2 / acidity / high GSH. 2+ It alleviates hypoxia and improves T1-MRI imaging sensitivity, while accelerating the continuous generation of reactive oxygen species (ROS). The synergistic effect of the components of this nuclear structure enhances treatment depth and efficiency.

[0019] Furthermore, the outer mesoporous organic silica (MON), serving as the shell material, exhibits excellent biocompatibility and degradability. Its abundant surface organic functional groups not only facilitate the covalent modification and stable loading of various functional molecules but also enable responsive structural disintegration in tumor-specific microenvironments (such as acidic or high GSH concentrations), promoting precise release of drugs or active ingredients. Simultaneously, the open mesoporous network structure of MON promotes rapid contact and reaction between active substances (such as H2O2, GSH, and O2) inside and outside tumor cells and catalytic components such as Fe3O4 and MnO2, thereby accelerating the Fenton reaction and ROS generation, enhancing CDT efficacy, and synergistically stimulating the PTT / PDT effect, achieving highly efficient tumor cell killing and providing crucial support for the overall treatment mechanism.

[0020] The outermost layer of the nano platform is coated with a homologous cell membrane derived from SACC-LM tumor cells, which retains its surface specific proteins, sugars and lipids and other biomarkers, and endows the material with good immune escape and targeted delivery capacity. The membrane structure can effectively evade immune system clearance through the "self-recognition" mechanism, prolong the circulation time in vivo, and rely on the chemotaxis and recognition function of the tumor cell membrane to achieve active targeting of homologous tumor tissues, significantly enhancing the accumulation efficiency of therapeutic agents at the tumor site. In addition, the membrane wrapping also acts as a physical barrier to prevent protein adsorption and mononuclear phagocyte system recognition, improving the stability and bioavailability of the material, and maintaining the targeting ability in dynamic blood flow and complex tumor microenvironment, providing a guarantee for precise, safe and efficient anti-tumor therapy.

[0021] (3) The material of the present application is verified by in vitro and in vivo experiments, and has good biocompatibility and hemocompatibility. Even if the concentration reaches 400 μg / mL, no obvious hemolysis or organ toxicity is observed, and it has good clinical safety basis. Through the multifunctional synergistic mechanism and cell membrane targeting modification technology, the present application first realizes the precise treatment strategy of activating the cGAS-STING pathway in SACC, an immunosuppressive tumor, which has significant innovation and application prospect, and is especially suitable for the treatment of "cold tumor" which lacks effective means at present, providing a new technical solution and theoretical basis for the field of tumor immunotherapy.

[0022] In summary, the MEM@Fe3O4@MnO2@IR780@MON nano platform provided by the present application is based on mesoporous organosilica structure, loaded with bimetallic oxide (Fe / Mn) and photosensitizer IR780, and has T1 / T2 dual-mode MRI and NIRF functions. At the same time, the outer coating of SACC cell membrane can endow the nanoparticle with the characteristics of active targeting of tumor lesions. Under the irradiation of 808 nm near-infrared laser, the nano platform can synergistically stimulate PTT / PDT / CDT and other effects, achieving efficient killing of SACC tumor cells. More importantly, while mediating cell immunogenic death, the platform can activate the cGAS-STING signaling pathway and enhance the antigen presentation function of dendritic cells, further stimulating T cell-mediated systemic anti-tumor immune response, thereby effectively overcoming the difficulty that traditional treatment cannot remove micrometastases, providing a theoretical basis and transformation prospect for the precise diagnosis and treatment of SACC. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Transmission electron microscopy image of the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1.

[0024] Figure 2X-ray photoelectron spectrum of the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1; (A) full spectrum; (B) Fe 2p; (C) Mn 2p; (D) Si 2p; (E) S 2p; (F) magnetization curve.

[0025] Figure 3 Catalytic production of dissolved oxygen by the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1 at different concentrations.

[0026] Figure 4 Verification of the depletion performance of the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1 on different concentrations of GSH.

[0027] Figure 5 Verification of the ·OH production capacity of the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1 at different pH (A-B) and different concentrations (C-D).

[0028] Figure 6 Inverted fluorescence microscopic pictures of the intracellular reactive oxygen species produced by the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1.

[0029] Figure 7 Effect of the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1 on the intracellular glutathione consumption.

[0030] Figure 8 Photothermal effect diagram of the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1; wherein (A, D) photothermal conversion performance of the composite nanomaterial at different concentrations; (B, E) photothermal conversion performance of the composite nanomaterial at different power concentrations; (C) 5-cycle heating-cooling curve.

[0031] Figure 9 Biocompatibility (A) and hemocompatibility (B) diagrams of the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1.

[0032] Figure 10 Diagram of the promotion of tumor cell death by the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1 combined with 808 nm laser irradiation.

[0033] Figure 11Figure 1. The MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1 combined with 808 nm laser irradiation promotes immunogenic cell death of tumor cells.

[0034] Figure 12 Figure 2. The MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1 combined with 808 nm laser irradiation activates the cGAS-STING signaling pathway.

[0035] Figure 13 Figure 3. The MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial prepared in Example 1 combined with 808 nm laser irradiation improves the presentation ability of antigen-presenting cells (dendritic cells). DETAILED DESCRIPTION

[0036] The present application is further illustrated by the following examples, which do not limit the scope of the application. It should be noted that, if the following processes are not specifically described in detail, they can be implemented or understood by those skilled in the art according to the prior art. If the reagents or instruments used are not specified by the manufacturer, they are considered to be conventional products that can be purchased on the market.

[0037] In the following, the nanoparticles refer to MEM@Fe3O4@MnO2@IR780@MON unless otherwise specified.

[0038] Example 1 The synthesis of the MEM@Fe3O4@MnO2@IR780@MON composite nanomaterial is as follows: Step 1: Dissolve 0.318 g of sodium citrate in 20 mL of ethylene glycol and stir at 60°C. After complete dissolution, add 1.5 g of sodium acetate, then add 1.8 mL of deionized water to obtain solution A. Dissolve 0.819 g of iron chloride hexahydrate in 10 mL of ethylene glycol and stir at room temperature to form solution B. Mix solutions A and B and stir at 80°C for 20 min. Then, transfer the mixture to a polytetrafluoroethylene-lined high-pressure reaction kettle, seal it, and place it in a 200°C air-drying oven for 8 hours. After the reaction is completed, collect the product by centrifugation and wash it with anhydrous ethanol and deionized water alternately three times. Each time, after ultrasonic dispersion, centrifuge at 12000 rpm for 10 min. Finally, obtain sodium citrate surface-modified Fe3O4 nanoparticles (Fe3O4 NPs), which are freeze-dried and stored at 4°C in a sealed container.

[0039] Step 2: 100 mg of the freeze-dried Fe3O4NPs were ultrasonically dispersed in 10 mL of deionized water for 30 min, 24 mg of sodium citrate was dissolved in 20 mL of deionized water, and then 2.7 mL of the above dispersion was added. After ultrasonic dispersion for another 30 min, the mixture was transferred into a 150 mL three-necked flask and magnetically stirred (600 rpm) for standby. Separately, 31.6 mg of potassium permanganate (KMnO4) was dissolved in 20 mL of deionized water, which was stirred at room temperature until homogeneous, and then slowly added dropwise into the reaction system in the three-necked flask. After the dropwise addition was completed, the mixture was continuously stirred for 1 h, and then heated to 80 °C and stirred for 30 min. After the reaction was completed, the product was repeatedly washed with deionized water until the supernatant was clear, to obtain the Fe3O4@MnO2NPs. After freeze-drying, the product was sealed and stored at 4 °C for standby.

[0040] Step 3: 150 mg of Fe3O4@MnO2NPs were dispersed in 20 mL of deionized water, and ultrasonically dispersed in an ice bath for 20 min to obtain a Fe3O4@MnO2 dispersion. Separately, 1.8 g of CTAB was dissolved in 140 mL of anhydrous ethanol (ultrasonically dispersed in an ice bath), and then added to the above Fe3O4@MnO2 dispersion and ultrasonically dispersed for another 20 min. Subsequently, the mixture was transferred into a 40 °C water bath, and 1.5 mL of concentrated ammonia water (28%) and 150 μL of TEOS were sequentially added, and the mixture was stirred at 600 rpm for 40 min. After the reaction was completed, the product was alternately washed with anhydrous ethanol and deionized water for three times. The precipitate was dispersed in a mixture of 20 mL of anhydrous ethanol and 40 mL of deionized water, and 0.6 g of CTAB was added, and ultrasonically dispersed for 20 min. Then, 2 mL of concentrated ammonia water (28%) and 400 μL of BTES were sequentially added at 30 °C and 150 rpm, and the mixture was continuously stirred for 6 h. After repeated washing for three times, the precipitate was dispersed in a 8 mg / mL sodium chloride methanol solution, and refluxed at 60 °C for 8 h to remove the CTAB, and the operation was repeated for three times. The final product Fe3O4@MnO2@MON was obtained, which was washed, freeze-dried and sealed at 4 °C for storage.

[0041] Step 4: 100 mg of Fe3O4@MnO2@MON was added into 10 mL of an IR780 stock solution (IR780 and 3-aminopropyltriethoxysilane were dissolved in 9 mL of anhydrous ethanol at a molar ratio of 1:27) with a concentration of 1 mg / mL, and stirred at room temperature overnight in the dark. The product was centrifuged at 12000 rpm for 30 min, and the precipitate was collected and alternately washed with anhydrous ethanol and deionized water for three times to obtain Fe3O4@MnO2@IR780@MON. After freeze-drying, the product was sealed and stored at 4 °C.

[0042] Step 5: Cell membrane protein extraction kit was used to extract cell membrane from SACC-LM cells, and the extracted membrane was mixed with Fe3O4@MnO2@IR780@MON at a mass ratio of 2:1, and then ultrasonicated in ice bath for 10 min (power 100 W), followed by extrusion through 800 nm and 400 nm polycarbonate membrane in turn to obtain uniform membrane coating. After centrifugation at 12000 rpm for 20 min, the final product was washed with ice-cold PBS (pH=7.2) for three times, and then freeze-dried and sealed at 4°C for storage to obtain the cell membrane coated MEM@Fe3O4@MnO2@IR780@MON nanoparticles.

[0043] The transmission electron microscopy of the composite material obtained in Example 1 is shown in Figure 1 It can be seen that the core of the synthesized MEM@Fe3O4@MnO2@IR780@MON nanoparticles is a spherical structure composed of manganese dioxide wrapped around magnetite, the outer shell is a mesoporous organosilica layer, and the outermost layer is a thin layer of cell membrane structure, and the size of the composite material is about 200 nm. The X-ray photoelectron detection result of the composite material obtained in Example 1 is shown in Figure 2 It can be seen that the synthesized MEM@Fe3O4@MnO2@IR780@MON nanoparticles have characteristic peaks of Fe3O4, MnO2 and MON.

[0044] Example 2 Synthesis of MEM@Fe3O4@MnO2@IR780@MON composite nanomaterials, the specific steps are as follows: Step 1: Dissolve 0.318 g of sodium citrate in 20 mL of ethylene glycol, and stir at 60°C. After complete dissolution, add 1.5 g of sodium acetate, and then add 1.8 mL of deionized water to obtain solution A. Dissolve 0.819 g of iron chloride hexahydrate in 10 mL of ethylene glycol, and stir at room temperature to form solution B. Mix solutions A and B, and stir at 80°C for 20 min. Then, transfer the mixed solution to a polytetrafluoroethylene lined high-pressure reaction kettle, seal it, and place it in a 200°C air drying oven for 8 hours. After the reaction is completed, the product is collected by centrifugation, and washed with anhydrous ethanol and deionized water alternately for three times. Each time, ultrasonic dispersion is followed by centrifugation at 12000 rpm for 10 min. Finally, sodium citrate surface modified Fe3O4 nanoparticles (Fe3O4 NPs) are obtained, which are freeze-dried and sealed for storage at 4°C.

[0045] Step 2: 100 mg of the freeze-dried Fe3O4NPs were dispersed in 10 mL of deionized water by ultrasonication for 30 min, 24 mg of sodium citrate was dissolved in 20 mL of deionized water, and then 2.7 mL of the above dispersion was added. After ultrasonication for another 30 min, the mixture was transferred into a 150 mL three-necked flask and magnetically stirred (600 rpm) for standby. Separately, 31.6 mg of potassium permanganate (KMnO4) was dissolved in 20 mL of deionized water, which was stirred at room temperature until homogeneous, and then slowly added dropwise into the reaction system in the three-necked flask. After the dropwise addition was completed, the reaction was continued to stir for 1 h, and then the temperature was increased to 80 °C and stirred for 30 min. After the reaction was completed, the product was repeatedly washed with deionized water until the supernatant was clear, to obtain the Fe3O4@MnO2NPs. After freeze-drying, the product was sealed and stored at 4 °C for standby.

[0046] Step 3: 150 mg of Fe3O4@MnO2NPs were dispersed in 20 mL of deionized water by ultrasonication for 20 min in an ice bath to obtain a Fe3O4@MnO2dispersion. Separately, 1.8 g of CTAB was dissolved in 140 mL of anhydrous ethanol (ultrasonication in an ice bath), and then the above Fe3O4@MnO2dispersion was added and ultrasonicated for another 20 min. Subsequently, the mixture was transferred into a 40 °C water bath, and 1.5 mL of concentrated ammonia water (28%) and 150 μL of TEOS were sequentially added, and the mixture was stirred at 600 rpm for 40 min. After the reaction was completed, the product was washed with anhydrous ethanol and deionized water alternately for three times. The precipitate was dispersed in a mixture of 20 mL of anhydrous ethanol and 40 mL of deionized water, and then 0.6 g of CTAB was added and ultrasonicated for 20 min. Subsequently, 2 mL of concentrated ammonia water (28%) and 300 μL of BTES were sequentially added at 30 °C and 150 rpm, and the mixture was stirred for another 6 h. After repeated washing for three times, the precipitate was dispersed in a 8 mg / mL sodium chloride methanol solution, and then refluxed at 60 °C for 8 h to remove the CTAB, which was repeated for three times. The final product Fe3O4@MnO2@MON was obtained, washed, freeze-dried, and then sealed and stored at 4 °C.

[0047] Step 4: 100 mg of Fe3O4@MnO2@MON was added into 10 mL of an IR780 stock solution, and then stirred at room temperature overnight in the dark. The product was centrifuged at 12000 rpm for 30 min, and the precipitate was collected and washed with anhydrous ethanol and deionized water alternately for three times to obtain Fe3O4@MnO2@IR780@MON. After freeze-drying, the product was sealed and stored at 4 °C.

[0048] Step 5: Cell membrane was extracted from SACC-LM cells using cell membrane protein extraction kit, and the extracted membrane was mixed with Fe3O4@MnO2@IR780@MON at a mass ratio of 2:1, and then ultrasonicated in ice bath for 10 min (power 100 W), followed by extrusion through 800 nm and 400 nm polycarbonate membrane in turn to obtain uniform membrane coating. After centrifugation at 12,000 rpm for 20 min, the final product was washed with ice-cold PBS (pH=7.2) for three times, and then freeze-dried and sealed at 4°C for storage to obtain the cell membrane coated MEM@Fe3O4@MnO2@IR780@MON nanoparticles.

[0049] Example 3 The synthesis of MEM@Fe3O4@MnO2@IR780@MON composite nanomaterials is as follows: Step 1: 0.318 g of sodium citrate was dissolved in 20 mL of ethylene glycol, and stirred at 60°C. After complete dissolution, 1.5 g of sodium acetate was added, followed by the addition of 1.8 mL of deionized water to obtain solution A. 0.819 g of iron chloride hexahydrate was dissolved in 10 mL of ethylene glycol, and stirred at room temperature to form solution B. Solution A and B were mixed, and stirred at 80°C for 20 min, and then the mixture was transferred to a polytetrafluoroethylene lined high-pressure reaction kettle, which was sealed and placed in a 200°C air-drying oven for 8 hours. After the reaction was completed, the product was collected by centrifugation, and washed with anhydrous ethanol and deionized water alternately for three times. Each time, the product was ultrasonicated and then centrifuged at 12,000 rpm for 10 min. Finally, the sodium citrate surface modified Fe3O4 nanoparticles (Fe3O4NPs) were obtained, which were freeze-dried and sealed for storage at 4°C.

[0050] Step 2: 100 mg of freeze-dried Fe3O4NPs were ultrasonicated in 10 mL of deionized water for 30 min, and 24 mg of sodium citrate was dissolved in 20 mL of deionized water, followed by the addition of 2.7 mL of the above dispersion liquid. After ultrasonication for 30 min, the mixture was transferred into a 150 mL three-necked flask, and magnetically stirred (600 rpm) for standby. Separately, 31.6 mg of potassium permanganate (KMnO4) was dissolved in 20 mL of deionized water, and stirred uniformly at room temperature. The reaction system in the three-necked flask was added slowly dropwise. After the addition was completed, the stirring was continued for 1 hour, and then the temperature was increased to 80°C for constant temperature stirring for 30 min. After the reaction was completed, the product was repeatedly washed with deionized water until the supernatant was clear, to obtain the manganese dioxide coated magnetite nanoparticles (Fe3O4@MnO2NPs), which were freeze-dried and sealed for storage at 4°C.

[0051] Step 3: 150 mg Fe3O4@MnO2NPs were dispersed in 20 mL deionized water and sonicated in an ice bath for 20 min to obtain a Fe3O4@MnO2 dispersion. Separately, 1.8 g CTAB was dissolved in 140 mL anhydrous ethanol (sonicated in an ice bath), and the above Fe3O4@MnO2 dispersion was added and sonicated for another 20 min. Then the mixture was transferred into a 40 °C water bath, and 1.5 mL concentrated ammonia (28%) and 150 μL TEOS were added in sequence, and the reaction was stirred at 600 rpm for 40 min. After the reaction was completed, the product was washed with anhydrous ethanol and deionized water alternately three times. The precipitate was dispersed in a mixture of 20 mL anhydrous ethanol and 40 mL deionized water, 0.6 g CTAB was added, and the mixture was sonicated for 20 min. Then 2 mL concentrated ammonia and 500 μL BTES were added in sequence at 30 °C and 150 rpm, and the stirring was continued for 6 h. After repeated washing three times, the precipitate was dispersed in a 8 mg / mL sodium chloride methanol solution, and the CTAB was removed by refluxing at 60 °C for 8 h, which was repeated three times. The final product Fe3O4@MnO2@MON was obtained, washed, freeze-dried, and stored at 4 °C.

[0052] Step 4: 100 mg Fe3O4@MnO2@MON was added to 10 mL IR780 stock solution, and the mixture was stirred at room temperature overnight in the dark. The product was collected by centrifugation at 12000 rpm for 30 min, and washed with anhydrous ethanol and deionized water alternately three times to obtain IR780 modified Fe3O4@MnO2@IR780@MON. The product was freeze-dried and stored at 4 °C.

[0053] Step 5: The cell membrane was extracted from SACC-LM cells using a cell membrane protein extraction kit, and the extracted membrane was mixed with Fe3O4@MnO2@IR780@MON at a mass ratio of 2:1, and then sonicated in an ice bath for 10 min (power 100 W). Then the mixture was extruded through 800 nm and 400 nm polycarbonate membranes in sequence to obtain a uniform membrane coating. The final product was centrifuged at 12000 rpm for 20 min, washed with ice-cold PBS (pH=7.2) three times, freeze-dried, and stored at 4 °C to obtain the cell membrane coated MEM@Fe3O4@MnO2@IR780@MON nanoparticles.

[0054] Example 4: Verification of the oxygen dissolving performance of MEM@Fe3O4@MnO2@IR780@MON Step 1: The oxygen production capacity of the nanoparticles was detected using a portable dissolved oxygen meter. Before use, the meter was activated for 1 h, and then calibrated with ultrapure water and 5% sodium sulfite (oxygen-free liquid).

[0055] Step 2: The freeze-dried MEM@Fe3O4@MnO2@IR780@MON (prepared in Example 1, which was used in the following examples) was prepared into a solution with a concentration of 0, 6.25, 12.5, 25, 50, 100, 200, 400 μg / mL using deionized water. 5 mL of the MEM@Fe3O4@MnO2@IR780@MON NPs solution was added to a 15 mL centrifuge tube. The detection parameters were set to 5 s / time, the electrode probe was immersed below the liquid surface, and after standing for 10 s, 40 μL of H2O2 (10 mM) was quickly injected. The change in dissolved oxygen concentration in the solution was recorded within 150 s.

[0056] As shown in Figure 3 , with the increase of the concentration of MEM@Fe3O4@MnO2@IR780@MON, the amount of dissolved oxygen also increased.

[0057] Example 5: Performance verification of MEM@Fe3O4@MnO2@IR780@MON in depleting GSH Step 1: The freeze-dried MEM@Fe3O4@MnO2@IR780@MON was prepared into a solution with a concentration of 0.5 mM using deionized water. An appropriate amount of GSH was added to make the GSH concentration in the mixed solution 0, 0.1, 0.2, 0.4, 0.8, 1.6 mM, respectively, and vortex mixed uniformly. Step 2: The amount of GSH consumed by the nanomaterial was detected using a reduced GSH detection kit according to the manufacturer's instructions.

[0058] As shown in Figure 4 , after different concentrations of GSH reacted with 0.5 mM of MEM@Fe3O4@MnO2@IR780@MON, the remaining GSH content in the supernatant was detected using a GSH detection kit. The results showed that with the increase of the GSH concentration, the total amount of GSH consumed by MEM@Fe3O4@MnO2@IR780@MON also increased, which indirectly proved the degradation of the mesoporous organosilica layer of the material shell by GSH.

[0059] Example 6: Performance verification of MEM@Fe3O4@MnO2@IR780@MON in generating ·OH Effect of different pH buffers on ·OH generation performance: Step 1: Preparation of 3,3',5,5'-tetramethylbenzidine (TMB) solution: TMB was dissolved in 40 mL of ethanol to prepare a solution with a concentration of 5 mM. The prepared solution should be stored in the dark and used only on the same day.

[0060] Step 2: Prepare 500 mL of 0.2 M aqueous sodium phosphate dibasic solution, and then prepare 0.1 M aqueous citric acid solution. Mix the two solutions in different proportions to obtain buffer solutions with different pH values in the range of 3.0-7.4 (specific pH values: 3, 3.4, 4, 4.4, 5, 5.4, 6, 6.4, 6.8, 7.4), and calibrate using a pH meter. The obtained buffer solutions can be stored at 4°C for 3 months.

[0061] Step 3: Weigh the freeze-dried MEM@Fe3O4@MnO2@IR780@MON and prepare a solution with a concentration of 0.5 mg / mL using deionized water. Take 0.4 mL and inject it into a PE tube containing 5 mL of sodium phosphate dibasic-citric acid buffer with different pH values. Add 1 mL of TMB solution (5 mM) and vortex to mix. Then add 1 mL of H2O2 (10 mM) and react for 30 min at room temperature in the dark. Step 4: Remove the unreacted MEM@Fe3O4@MnO2@IR780@MON under the assistance of a magnet, terminate the reaction, and measure the absorbance of the supernatant at 625 nm using a UV spectrophotometer.

[0062] Effect of different concentrations of MEM@Fe3O4@MnO2@IR780@MON on ·OH production performance: Step 1: Weigh the freeze-dried MEM@Fe3O4@MnO2@IR780@MON and prepare solutions with different concentrations (0, 0.6125, 1.25, 2.5, 5.0 mg / mL) using deionized water for standby; Step 2: Add 0.4 mL of MEM@Fe3O4@MnO2@IR780@MON solution with different concentrations, 5 mL of sodium phosphate dibasic-citric acid buffer (pH = 3.4), 1 mL of TMB solution (5 mM), and 1 mL of H2O2 (10 mM) solution to a PE tube in sequence. React for 30 min at room temperature in the dark. Step 3: Remove the unreacted MEM@Fe3O4@MnO2@IR780@MON under the assistance of a magnet, terminate the reaction, and measure the absorbance of the supernatant at 625 nm using a UV spectrophotometer.

[0063] ·OH produced by MEM@Fe3O4@MnO2@IR780@MON at different pH values and different concentrations Figure 5As shown, the MEM@Fe3O4@MnO2@IR780@MON-H2O2-TMB system was reacted at different pH values, and it was found that the catalytic activity of the material gradually increased with the decrease of pH concentration, and reached the highest catalytic activity when pH = 3.4, indicating that the enzyme catalytic performance of the material had acid specificity. Further, the catalytic activity of MEM@Fe3O4@MnO2@IR780@MON with different concentrations was verified under the optimum pH condition, and the results showed that the catalytic activity of •OH generation increased with the increase of the concentration of the material, indicating that the catalysis of the material had concentration dependence.

[0064] Example 7 Performance of intracellular reactive oxygen species Step 1: SACC-LM cells were selected as model cells, and the following experimental groups were set: PBS group, PBS + Laser group, MFMIHMON (MEM@Fe3O4@MnO2@IR780@MON) group, MFMIHMON + Laser group. The cells were inoculated in confocal culture dishes at a density of 1×10 4 / mL and incubated for 24 h to adhere.

[0065] Step 2: Nanoparticles (MEM@Fe3O4@MnO2@IR780@MON, final concentration 400 μg / mL) and PBS were added to the cells in step 1 respectively, and co-cultured for 4 h.

[0066] Step 3: The PBS + Laser group and the MEM@Fe3O4@MnO2@IR780@MON + Laser group were irradiated with an 808 nm near-infrared laser instrument at 2 W / cm 2 for 5 min, and the well plate was placed back in a constant temperature cell incubator at 37°C, 5% CO2 for 20 h.

[0067] Step 4: According to the manufacturer's instructions, the DCFH-DA probe was diluted with serum-free medium at a ratio of 1:1000, and then the old medium was discarded. 1 mL of diluted probe solution was added to each well, and incubated at 37°C, 5% CO2 in a constant temperature cell incubator for 20 min.

[0068] Step 5: The probe-containing medium was aspirated and washed 3 times with serum-free medium, and then observed and photographed under a laser confocal microscope for the intracellular ROS generated.

[0069] The experimental results are shown in Figure 6 DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to generate DCFH, and the intracellular reactive oxygen species can oxidize the non-fluorescent DCFH to generate fluorescent DCF. As Figure 6The green fluorescence in the cells indicates the presence of active oxygen, and the group with MEM@Fe3O4@MnO2@IR780@MON and near-infrared laser irradiation has more green fluorescence, indicating that the synergy of photodynamics and chemical dynamics can produce more active oxygen.

[0070] Example 8: Verification of intracellular glutathione depletion performance Step 1: Select SACC-LM cells as model cells, set the following experimental groups: PBS group, different concentrations of MEM@Fe3O4@MnO2@IR780@MON group (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL). Cells were seeded in 24-well plates at a density of 1 × 10 4 / mL and incubated for 24 h.

[0071] Step 2: Add nanoparticles and PBS to the cells in step 1 respectively, and co-culture for 24 h.

[0072] Step 3: Aspirate the drug-containing culture medium, and wash 3 times with serum-free medium, then detect GSH in cells according to the manufacturer's instructions.

[0073] The depletion level of intracellular GSH is shown in Figure 7 As can be seen, with the increase of material concentration, the intracellular GSH level gradually decreases, and compared with the blank group, the intracellular GSH level of the cells treated with MEM@Fe3O4@MnO2@IR780@MON significantly decreases.

[0074] Example 9: Photothermal effect of MEM@Fe3O4@MnO2@IR780@MON Photothermal conversion performance of different concentrations of MEM@Fe3O4@MnO2@IR780@MON: Step 1: Take the freeze-dried MEM@Fe3O4@MnO2@IR780@MON, prepare solutions with concentrations of 0, 6.25, 12.5, 25, 50, 100, 200, 400 μg / mL using deionized water, and then take 500 μL into EP tubes; Step 2: Use an 808 nm near-infrared laser instrument to irradiate the MEM@Fe3O4@MnO2@IR780@MON aqueous solution with different concentrations in the EP tube at a power of 2 W / cm 2 Every 30 s, record the temperature change using Applent AT4204 multi-channel temperature instrument, and draw the temperature change curve.

[0075] Photothermal conversion performance of MEM@Fe3O4@MnO2@IR780@MON under different powers: Step 1: Take the freeze-dried MEM@Fe3O4@MnO2@IR780@MON, prepare a solution with a concentration of 200 μg / mL using deionized water, and then take 500 μL and add it to an EP tube; Step 2: Use an 808 nm near-infrared laser instrument to irradiate the MEM@Fe3O4@MnO2@IR780@MON aqueous solution in the EP tube at powers of 0.5, 1.0, 1.5, and 2.0 W / cm 2 for 5 min. Use an Applent AT204 multi-channel temperature instrument to record temperature changes every 30 s, and plot the temperature change curve.

[0076] Photothermal stability of MEM@Fe3O4@MnO2@IR780@MON: Step 1: Take the freeze-dried MEM@Fe3O4@MnO2@IR780@MON, prepare a solution with a concentration of 400 μg / mL using deionized water, and then take 500 μL and add it to an EP tube; Step 2: Use an 808 nm near-infrared laser instrument to irradiate the MEM@Fe3O4@MnO2@IR780@MON aqueous solution in the EP tube at a power of 2 W / cm 2 for 5 min, and then naturally cool to room temperature. Repeat the operation continuously for 5 times, use an Applent AT4204 multi-channel temperature instrument to record temperature changes every 30 s, and plot the temperature change curve.

[0077] The photothermal effect diagram is shown in Figure 8 As the material concentration and laser power increase, more heat is generated at the same time, and the temperature rises more obviously, with the highest temperature exceeding 45℃, indicating that the material has the ability to exert a mild photothermal effect against tumor cells. The photothermal stability of the material was evaluated, and the heating-cooling curve remained basically unchanged in 5 cycles of intermittent irradiation experiments, which was reproducible, indicating that the material had good photothermal stability.

[0078] Example 10 Verification of the biocompatibility and blood compatibility of MEM@Fe3O4@MnO2@IR780@MON Verification of biocompatibility: Step 1: Select SACC-LM cells, HN6 cells and HOK cells as model cells, and co-culture with different concentrations of MEM@Fe3O4@MnO2@IR780@MON (final concentration 0 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). SACC-LM cells, HN6 cells and HOK cells were respectively seeded in 96-well plates at a density of 1 × 10 4 / mL and incubated for 24 h.

[0079] Step 2: Add PBS and different concentrations of nanoparticle-containing serum-free pure DMEM medium to the cells in step 1 respectively, and co-culture for 24 h.

[0080] Step 3: Aspirate the culture medium, add freshly prepared CCK-8 solution under light-proof conditions, and co-culture for 1 h. Measure the absorbance at 450 nm under a multifunctional enzyme labeler, and calculate the cell survival rate.

[0081] Blood compatibility verification: Step 1: Collect fresh blood from the heart of a healthy SD rat, and add it to a 15 mL centrifuge tube containing an anticoagulant. After repeated washing at room temperature, add pre-cooled PBS to prepare a solution containing 10% red blood cells.

[0082] Step 2: Mix 50 μL of the solution in step 1 with different concentrations of MEM@Fe3O4@MnO2@IR780@MON, and incubate at 37°C for 1.5 h. Mix 500 μL of a PBS solution containing 10% red blood cells with 500 μL of a PBS solution as a negative control. Mix 50 μL of a PBS solution containing 10% red blood cells with 950 μL of distilled water as a positive control.

[0083] Step 3: After incubation, centrifuge at 2500 rpm for 5 min. Take the upper solution and add it to a 96-well plate to detect the absorbance at 540 nm in an enzyme labeler.

[0084] Biological blood compatibility and blood compatibility are as shown in Figure 9 The results show that when the material concentration is 400 μg / mL, the blood compatibility rate is less than 5%, and the cell survival rate is more than 80%, indicating that the material has good biocompatibility and blood compatibility.

[0085] Example 11 CCK8 characterization of high-efficiency anticancer activity of MEM@Fe3O4@MnO2@IR780@MON Step 1: SACC-LM cells were selected as model cells, and the following experimental groups were set: different concentrations of MEM@Fe3O4@MnO2@IR780@MON combined with near-infrared laser irradiation group. Cells were inoculated in 96-well plates at a density of 1x10 4 / mL and incubated for 24 h to adhere.

[0086] Step 2: After the cells in step 1 were incubated and adhered, the culture medium was removed, and fresh culture medium containing different concentrations of MEM@Fe3O4@MnO2@IR780@MON (final concentration 0, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) was added for co-culture. After 4 hours, 808 nm laser irradiation (2 W / cm 2 , 5 min) was applied, and then incubation was continued for 20 h. The group with a final concentration of 0 was added with an equal amount of PBS.

[0087] Step 3: The culture medium in step 2 was removed, and freshly prepared CCK-8 solution was added under light-free conditions for 1 h. The absorbance value at 450 nm was measured on a multifunctional enzyme label instrument, and the cell survival rate was calculated.

[0088] The tumor killing effect is shown in Figure 10 As can be seen from the figure, with the increase of nanoparticle concentration, the cell survival rate decreases, indicating that the material combined with 808 nm laser irradiation can enhance the killing ability of cancer cells.

[0089] Example 12 MEM@Fe3O4@MnO2@IR780@MON promotes immunogenic death of tumor cells Step 1: SACC-LM cells were selected as model cells, and the following experimental groups were set: Control (PBS) group, MFMIMON (MEM@Fe3O4@MnO2@IR780@MON) group, MFMIMON+Laser (MEM@Fe3O4@MnO2@IR780@MON+808 nm) group. Cells were inoculated in confocal dishes at a density of 1x10 4 / mL and incubated for 24 h to adhere.

[0090] Step 2: Nanoparticles (400 μg / mL) and PBS were added to the cells in step 1, respectively, and co-cultured in serum-free pure DMEM medium for 4 h.

[0091] Step 3: The 808 nm near-infrared laser instrument was used for 2 W / cm 2 irradiation for 5 min, and the plate was placed back in a constant temperature cell incubator at 37°C, 5% CO2 for 30 min.

[0092] Step 4: After aspirating the medium containing the probe and washing 3 times with serum-free medium, the cells were routinely fixed, permeabilized, and serum blocked.

[0093] Step 5: According to the manufacturer's instructions, add CRT, HMGB1 primary antibody working solution, and incubate overnight at 4°C.

[0094] Step 6: Discard the primary antibody culture solution, wash 3 times with serum-free medium, and then add Alexa Fluor 488 conjugated goat anti-rabbit IgG H&L, incubate at room temperature for 1 h.

[0095] Step 7: Take out the confocal slide in the dark environment, add a drop of DAPI-containing anti-fluorescence quenching mounting medium on the coverslip, and then place the confocal slide with cells upside down on the coverslip, and observe under the laser confocal microscope.

[0096] The immunogenic death of tumor cells is manifested as Figure 11 As shown in the figure, in the MEM@Fe3O4@MnO2@IR780@MON group, when the SACC-LM cells were not irradiated with 808 nm laser, the green fluorescence emitted by HMGB1 was mainly concentrated in the nucleus, which was consistent with the localization of HMGB1 observed in the Control group, indicating that the expression of HMGB1 did not increase and did not trigger nuclear-cytoplasmic translocation. At the same time, the same result was observed from the laser confocal image of CRT, that is, the green fluorescence of CRT was limited in the endoplasmic reticulum. When combined with near-infrared laser irradiation, the expression of HMGB1 in the nucleus increased, and the green fluorescence in the cytoplasm also became obvious. At the same time, the content of CRT in the cytoplasm increased (the green fluorescence intensity became larger).

[0097] Example 13 Activation of cGAS-STING signaling pathway Step 1: Select SACC-LM cells as model cells, and set the following experimental groups: PBS group, Laser group (irradiate infrared laser under PBS conditions), MFMIMON (MEM@Fe3O4@MnO2@IR780@MON group), MFMIMON+Laser (MEM@Fe3O4@MnO2@IR780@MON+808nm) group. Cells were seeded in 6-well plates at a density of 3x10 5 / mL and incubated for 24 h.

[0098] Step 2: Add nanoparticles (400 μg / mL) and PBS-free pure DMEM medium to the cells in step 1, respectively, and co-culture for 4 h.

[0099] Step 3: The 808 nm near-infrared laser instrument was used for the combined near-infrared laser irradiation group at 2 W / cm 2 for 5 min, and the hole plate was returned to the constant temperature cell incubator at 37°C, 5% CO2 for 20 h incubation.

[0100] Step 4: The SACC-LM cell protein sample was prepared according to the WB scheme, electrophoresis was performed at 120 V for 75 min using a precast gel of 4-20%, and the gel was subjected to electrotransfer at 200 mA for 2 h to transfer the protein to the PVDF membrane. The blocking, primary antibody incubation, secondary antibody incubation, and Western blot development operations were completed according to the previous steps to verify the changes in the expression levels of cGAS, p -STING, and IFN-β proteins in the cells of different treatment groups.

[0101] The changes in the expression levels of key proteins (cGAS, p -STING, and IFN-β) in the cGAS-STING signaling pathway caused by different treatments of tumor cells are shown in Figure 12 Compared with the PBS group, the PBS + 808 nm group, or the MEM@Fe3O4@MnO2@IR780@MON group, the expression levels of cGAS, p -STING, and IFN-β proteins in the tumor cells of the MEM@Fe3O4@MnO2@IR780@MON + 808 nm group were significantly increased, indicating that the material combined with near-infrared laser irradiation can effectively activate the cGAS-STING signaling pathway and promote the expression level of key proteins.

[0102] Example 14 Enhancement of the presentation ability of antigen-presenting cells by MEM@Fe3O4@MnO2@IR780@MON combined with 808 nm laser irradiation Step 1: SACC-LM cells were selected as model cells, and the following experimental groups were set: PBS group, PBS + Laser group, MEM@Fe3O4@MnO2@IR780@MON group, and MEM@Fe3O4@MnO2@IR780@MON + Laser group. The cells were inoculated in a 6-well plate at a density of 3 × 10 5 / mL for 24 h incubation.

[0103] Step 2: Nanoparticles (400 μg / mL) and PBS were added to the cells in step 1, respectively, and co-cultured in serum-free pure DMEM medium for 4 h.

[0104] Step 3: The 808 nm near-infrared laser instrument was used for the combined near-infrared laser irradiation group at 2 W / cm 2 for 5 min, and the hole plate was returned to the constant temperature cell incubator at 37°C, 5% CO2 for 20 h incubation.

[0105] Step 4: After the probe-containing medium was sucked out and washed with serum-free medium for 3 times, the SACC-LM cells were collected and inoculated into the lower chamber of the Transwell chamber. Meanwhile, the mouse-derived bone marrow cells were induced to differentiate into immature dendritic cells, and the immature dendritic cells were inoculated into the upper chamber of the Transwell chamber at a density of 1x10 6 The immature dendritic cells were inoculated into the upper chamber of the Transwell chamber at a density of 1x10 Step 5: After the incubation, the dendritic cells in the Transwell chamber were collected, and the antibody staining solution (anti-CD86, anti-I-A / I-E) was prepared at a ratio of 1:200 according to the manufacturer's instructions, and flow cytometry detection was performed.

[0106] After the addition of MEM@Fe3O4@MnO2@IR780@MON and combined with 808nm laser irradiation, the tumor cells were co-cultured with immature dendritic cells for 24h, and the ratio of dendritic cell maturation was Figure 13 indicated. After the MEM@Fe3O4@MnO2@IR780@MON combined with 808nm irradiation, the proportion of mature dendritic cells increased significantly, indicating that the material combined with laser irradiation of tumor cells has the ability to promote the maturation of antigen-presenting cells.

[0107] The above examples are only the preferred embodiments of the present application, and are used to explain the present application, but not to limit the present application. The changes, replacements, modifications and the like made by those skilled in the art without departing from the spirit and essence of the present application shall all belong to the protection scope of the present application.

Claims

1. A bimetallic composite nanomaterial for targeting adenoid cystic carcinoma of the salivary gland, characterized in that: The nanomaterials include Fe3O4@MnO2@MON loaded with IR780, and SACC-LM cell membranes coated on its surface; the Fe3O4@MnO2@MON includes Fe3O4@MnO2 bimetallic oxide core and shell material mesoporous organic silica. The preparation method of the bimetallic composite nanomaterial includes the following steps: (1) Sodium citrate-modified Fe3O4 nanoparticles were synthesized by a solvothermal method; (2) The obtained Fe3O4 nanoparticles were dispersed in deionized water, mixed with sodium citrate, and then potassium permanganate solution was added dropwise. After heating and reaction, Fe3O4@MnO2 nanoparticles were obtained by centrifugation, washing and freeze-drying. (3) Fe3O4@MnO2 nanoparticles were dispersed in deionized water, and Fe3O4@MnO2@MON was obtained by reacting with hexadecyltrimethylammonium bromide as a template agent, tetraethyl orthosilicate as a silicon source and bis-(γ-triethoxysilylpropyl)tetrasulfide as an organosilicon source. (4) Fe3O4@MnO2@MON was mixed with IR780 solution and stirred overnight at room temperature. After centrifugation, washing and freeze-drying, Fe3O4@MnO2@IR780@MON loaded with IR780 was obtained. (5) Fe3O4@MnO2@IR780@MON was mixed with SACC-LM cell membrane, and the SACC cell membrane was coated on the surface of Fe3O4@MnO2@IR780@MON by ultrasonic combined lipid extrusion method. After centrifugation, washing and freeze-drying, bimetallic composite nanomaterials were obtained.

2. The bimetallic composite nanomaterial according to claim 1, characterized in that: The specific steps of the solvothermal method in step (1) are as follows: dissolve sodium citrate and sodium acetate in deionized water, then add ferric chloride hexahydrate, and then transfer it to a reaction vessel for hydrothermal reaction; the mass ratio of sodium citrate, sodium acetate and ferric chloride hexahydrate is 1:(4.7~4.8):(2.5~2.6); the temperature of the hydrothermal reaction is 200℃; and the time is 8 hours.

3. The bimetallic composite nanomaterial according to claim 1, characterized in that: In step (2), the concentration ratio of Fe3O4 nanoparticles, sodium citrate and potassium permanganate is 10:1.2:1.58; the heating reaction temperature is 80℃ and the reaction time is 1.5h.

4. The bimetallic composite nanomaterial according to claim 1, characterized in that: In step (3), the ratio of Fe3O4@MnO2 nanoparticles, hexadecyltrimethylammonium bromide, tetraethyl orthosilicate, and bis-(γ-triethoxysilylpropyl)tetrasulfide is 150mg:2.4g:150μL:(400-500μL).

5. The bimetallic composite nanomaterial according to claim 1, characterized in that: The reaction temperature in step (3) is 40°C; the reaction time required by the hydrothermal method is 6 hours.

6. The bimetallic composite nanomaterial according to claim 1, characterized in that: In step (4), IR780 and 3-aminopropyltriethoxysilane are dissolved in anhydrous ethanol at a molar ratio of 1:27 in the IR780 solution, and the solid-liquid ratio of Fe3O4@MnO2@MON to the IR780 solution is 10 mg: 1 mL.

7. The bimetallic composite nanomaterial according to claim 1, characterized in that: In step (5), the mass ratio of Fe3O4@MnO2@IR780@MON to SACC-LM cell membrane is 1:

2.

8. The bimetallic composite nanomaterial according to claim 1, characterized in that: In step (5), the ultrasonic power of the combined ultrasonic and lipid extrusion method is 100W and the time is 10min.

9. The bimetallic composite nanomaterial according to claim 1, characterized in that: The ultrasonic combined lipid extrusion method described in step (5) requires the use of polycarbonate membranes with diameters of 800 nm and 400 nm, respectively.

10. The use of the bimetallic composite nanomaterial as described in any one of claims 1 to 9 in the preparation of a targeted anti-salivary gland adenoid cystic carcinoma tumor drug.

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