Nano immunomodulator as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a nano immunomodulator, which comprises the following steps: mixing 3-amino-1, 2, 4-triazole and oxalic acid, and sequentially carrying out calcination, grinding, acid treatment, ultrasonic stripping and step-by-step centrifugation to obtain a double-defect nitrogen-rich carbon nitride CNO nanosheet; adding the copper chloride solution into the metal-phase molybdenum disulfide MS nanosheet solution, and stirring for reaction to obtain a surface-atomized copper-chelated metal-phase molybdenum disulfide nanosheet CuMS; and dropwise adding the CuMS solution into the double-defect nitrogen-rich carbon nitride CNO nanosheet solution, and reacting to obtain the nano immunomodulator CNO coated CuMS. The invention also discloses the nano immunomodulator prepared by the preparation method and application of the nano immunomodulator in preparation of medicines for treating bone tumors. According to the nano immunomodulator prepared by the preparation method disclosed by the invention, the tumor immune microenvironment is improved through multi-mode catalysis and cell copper death induction effects of piezoelectric catalysis, sonodynamic catalysis and Fenton-like catalysis, so that efficient treatment of malignant bone tumors is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of bone tumors, and particularly relates to a nano-immunomodulator and a preparation method and application thereof. Background Art

[0002] Osteosarcoma (OS) is the most common primary malignant bone tumor, accounting for approximately 20% of primary malignant bone tumors worldwide, primarily affecting children, adolescents, and individuals over 65 years of age. Although advances in surgery, chemoradiotherapy, targeted therapy, and multidisciplinary treatment have significantly improved the five-year survival rate of osteosarcoma patients, the local suppressive immune microenvironment of the tumor significantly impairs response to clinical treatment, can induce metastasis, and can lead to death, making it a critical challenge that needs to be overcome in the clinical treatment of osteosarcoma.

[0003] Catalytic therapy is an emerging treatment method in which bioactive catalytic drugs utilize the unique microenvironmental conditions of tumor tissue or respond to external stimuli such as light, heat, force, sound, and electricity to produce a large number of cytotoxic molecules, such as reactive oxygen species (ROS), through catalytic reactions, thereby inducing tumor cell death. Studies have shown that the large amount of ROS produced by catalytic reactions can not only effectively induce immunogenic death of tumor cells, but also increase the expression and release of damage-associated molecular patterns (DAMPs) such as CALP, ATP, and HMGB1 by tumor cells, thereby promoting the maturation of dendritic cells and the transformation of T lymphocytes to CD8 + T cell differentiation activates the body's anti-tumor immunity and can also regulate the polarization of macrophages in the tumor immune microenvironment from the pro-tumor M2 type to the anti-tumor M1 type, improving the tumor-suppressive immune microenvironment. For example, Chinese Patent Publication No. CN117427184A discloses a nanocatalytic drug T-HCN@CuMS, which achieves efficient treatment of in situ osteosarcoma through the combination of near-infrared photocatalysis, chemical catalysis, and copper apoptosis and surface targeted modification. For example, Chinese Patent Publication No. CN118845654A discloses a thermosensitive nano-drug delivery system co-loaded with disulfiram and copper sulfide, as well as its preparation and application. The thermosensitive nano-drug delivery system co-loaded with disulfiram and copper sulfide is composed of an upper critical solution temperature micelle carrier material poly(acrylonitrile-acrylamide)-polyethylene glycol polymer, disulfiram, and copper sulfide; it achieves copper-enhanced photothermal, chemical, chemokinetic, and copper apoptosis combined therapy, showing great potential in tumor treatment.

[0004] Therefore, developing nanoimmunomodulators with high catalytic activity to improve the immune microenvironment of bone tumors is of great value in increasing the efficacy of clinical treatment for osteosarcoma, improving patient survival rates, and improving patient prognosis. However, the limited penetration of near-infrared light in tissue (<1 cm) and light scattering, the mismatch between the pH of the tumor microenvironment and the optimal pH for Fenton-like catalytic reactions, and the hardened extracellular matrix of tumors hindering the penetration of nanocatalysts into tumor tissues, mean that the activity of nanoimmunomodulators based on photocatalysis and Fenton-like catalytic reactions still needs to be improved. Ultrasound, as a conventional medical imaging technique, has the advantages of being non-invasive and having high tissue penetration (~10 cm).

[0005] Therefore, developing an ultrasound-responsive nanoimmunomodulator with excellent performance through rational design and material preparation is of great significance for improving the tumor immune microenvironment and enhancing the clinical treatment efficacy of osteosarcoma. Summary of the Invention

[0006] The purpose of the present invention is to provide a nano-immunomodulator and its preparation method and application. The prepared nano-immunomodulator improves the tumor immune microenvironment through multimodal catalysis of piezoelectric catalysis, sonodynamic catalysis and Fenton-like catalysis and cell copper death induction, thereby achieving efficient treatment of malignant bone tumors.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] A method for preparing a nano-immunomodulator, comprising:

[0009] (1) 3-amino-1,2,4-triazole and oxalic acid are mixed and then calcined, ground, acid-treated, ultrasonically exfoliated, and centrifuged in steps to obtain double-defect nitrogen-rich carbon nitride (CNO) nanosheets;

[0010] (2) adding a copper chloride solution to a metallic phase molybdenum disulfide MS nanosheet solution and stirring the reaction to obtain metallic phase molybdenum disulfide nanosheets CuMS with surface atomized copper chelated;

[0011] (3) The CuMS solution was added dropwise into the double-defect nitrogen-rich carbon nitride CNO nanosheet solution to react and obtain the nanoimmunomodulator CNO@CuMS.

[0012] The present invention uses double-defect nitrogen-rich carbon nitride nanosheets (CNO) as the base material, and utilizes surface atomized copper chelated metallic phase molybdenum disulfide nanosheets (CuMS) and double-defect nitrogen-rich carbon nitride nanosheets to construct a heterojunction, successfully preparing a nano-immunomodulator CNO@CuMS with multimodal catalysis (piezoelectric catalysis, sonodynamic catalysis, Fenton-like catalysis) and cell copper death induction effects.

[0013] In step (1), the preparation method of the CNO nanosheets is as follows: 3-amino-1,2,4-triazole powder and oxalic acid powder are mixed, calcined in an argon atmosphere, and the calcined product is placed in an air atmosphere for secondary calcination to obtain bulk CNO; the obtained bulk CNO is ground and then subjected to acidification and ultrasonic exfoliation, and CNO nanosheets within a specific range are obtained by step-by-step centrifugation.

[0014] The amount of oxalic acid is 1-5 mmol, the mass of the 3-amino-1,2,4-triazole powder is 1.0 g, the calcination temperature in argon atmosphere is 500-600° C., and the calcination time is 2-4 hours; the secondary calcination temperature in air atmosphere is 500-600° C., and the secondary calcination time is 2-4 hours.

[0015] After ultrasonic exfoliation of the acidified CNO, CNO nanosheets were obtained by stepwise centrifugation in the range of 6000-9000 rpm.

[0016] In step (2), the preparation method of the surface atomized copper chelated metal phase molybdenum disulfide nanosheets CuMS is:

[0017] (2-1) adding ammonium heptamolybdate tetrahydrate and thiourea in a molar mass ratio of (1-3):30 to 30-50 mL of water and hydrothermally reacting them at a reaction temperature of 180-220° C. for 18-24 hours to obtain bulk metallic phase MoS2, followed by ultrasonic exfoliation and stepwise centrifugation to obtain metallic phase MoS2 MS nanosheets in the range of 9000-12000 rpm;

[0018] (2-2) The feeding ratio of MS nanosheets to copper chloride solution is as follows: 0.1-2 mL of 5-10 mM copper chloride solution is added to a solution containing 10 mg of MS nanosheets, and the stirring time is 12-24 hours to allow the copper ions to be fully atomized on the surface of the MS nanosheets.

[0019] In step (3), the added mass of the CuMS is 5-30% of the CNO nanosheets.

[0020] The preparation method further comprises:

[0021] (4) Disperse CNO@CuMS and add PEG 2k -DSPE modified as a nanoimmunomodulator.

[0022] The present invention also provides a nano-immunomodulator obtained by the above preparation method.

[0023] The present invention also provides the use of the nano-immunomodulator in preparing drugs for treating bone tumors.

[0024] The present invention improves the piezoelectric catalytic activity of nitrogen-rich carbon nitride by introducing two defect sites, nitrogen vacancies and oxygen doping, and gives it a new function of sonodynamic catalysis; the use of CuMS with excellent conductive properties and CNO to construct a heterostructure can not only increase the electron migration rate inside the nanoimmunomodulator, increase the electron migration path, prolong the reaction time of active electrons, and further enhance the piezoelectric catalytic activity of CNO, but also introduce the properties of CuMS catalyzing Fenton-like reactions and pH-responsive release of copper ions to induce cell copper death; under ultrasonic stimulation, CNO@CuMS can produce a large amount of cytotoxic reactive oxygen species such as hydrogen peroxide, singlet oxygen and hydroxyl free radicals through piezoelectric-sound dynamic-Fenton-like three-mode catalysis; the combination of three-mode catalysis and induction of copper death overcomes the shortcomings of insufficient activity of nanoimmunomodulators caused by factors such as the limited penetration and light scattering of external light stimulation in tissues, and the mismatch between the tumor microenvironment and the optimal pH conditions of Fenton / Fenton-like reactions.

[0025] The present invention also provides the use of the above-mentioned nano-immunomodulator in combination with the immune checkpoint inhibitor αPD-1 in the preparation of drugs for treating bone tumors.

[0026] The nano-immunomodulator CNO@CuMS provided by the present invention is used to improve the therapeutic efficacy of in situ osteosarcoma immune checkpoint inhibitor therapy. The nano-immunomodulator CNO@CuMS, through the combination of trimodal catalysis and copper-induced death, can induce immunogenic death of bone tumor cells and promote cytotoxic CD8 + It can infiltrate tumors with T cells and NK cells, and induce tumor-associated macrophages to repolarize from the pro-tumor M2 type to the anti-tumor M1 type, thereby improving the immune microenvironment of in situ bone tumors and significantly improving the efficacy of immune checkpoint inhibitors. It is an excellent nano-immunomodulator for malignant bone tumors.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) By regulating chemical composition, introducing defects, adjusting physical phases, atomic doping, and constructing heterostructures, a nanoimmunomodulator with piezoelectric catalysis, sonodynamic catalysis, Fenton-like catalysis, and cell copper death induction was prepared to improve the immune microenvironment of in situ bone tumors and significantly improve the efficacy of immune checkpoint inhibitors.

[0029] (2) The copper ions coordinated and bound to the surface of metallic phase MoS2 nanosheets are reduced to copper atoms in situ, which not only introduces Fenton-like catalytic activity and pH-responsive copper death induction, but also improves the stability and safety of the nanoimmunomodulator. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1Schematic diagram of the preparation and application of nanoimmunomodulator CNO@CuMS.

[0031] Figure 2 Transmission electron microscopy characterization of metallic phase MoS2 nanosheets MS and metallic phase MoS2 nanosheets CuMS with surface atomic copper chelation.

[0032] Figure 3 High-angle annular dark-field microscopy and elemental mapping of CuMS.

[0033] Figure 4 X-ray photoelectron spectroscopy characterization of MS.

[0034] Figure 5 X-ray photoelectron spectroscopy characterization of CuMS.

[0035] Figure 6 is the nonlinear least squares fitting result of the Auger spectrum of Cu element in CuMS.

[0036] Figure 7 Related characterization of different carbon nitride nanosheets.

[0037] Figure 8 Characterization of CNO@CuMS materials.

[0038] Figure 9 Characterization of the catalytic activity of CNO@CuMS.

[0039] Figure 10 Colorimetric determination of hydrogen peroxide production by CNO@CuMS in response to ultrasound stimulation.

[0040] Figure 11 This study investigates the performance of CNO@CuMS in inducing immunogenic death of osteosarcoma K7M2 cells through trimodal catalysis and copper death induction.

[0041] Figure 12 Flow cytometric characterization of the generation of reactive oxygen species by CNO@CuMS in K7M2 osteosarcoma cells via trimodal catalysis.

[0042] Figure 13 To investigate the performance of CNO@CuMS in inducing copper-induced death and immunogenic death of bone tumor cells.

[0043] Figure 14 This is to verify the property of CNO@CuMS in inducing immunogenic death of bone tumors in vivo.

[0044] Figure 15 In vivo experimental verification of CNO@CuMS enhancing the anti-tumor efficacy of the immune checkpoint inhibitor αPD-1.

[0045] Figure 16 Actual pictures of tumors in each treatment group and tumor weight analysis of each group after combined treatment of CNO@CuMS and αPD-1.

[0046] Figure 17 Figure 3 shows the flow cytometric analysis of cell suspensions of tumor tissues in each group after combined treatment with CNO@CuMS and αPD-1 and the detection results of pro-inflammatory factors in serum.

[0047] Figure 18 These are the H&E section staining results of the main organs of mice in each group after combined treatment with CNO@CuMS and αPD-1.

[0048] Figure 19 These are the liver and kidney function test results of mice in each group after combined treatment with CNO@CuMS and αPD-1. DETAILED DESCRIPTION

[0049] Example 1

[0050] The preparation and application of the nano-immunomodulator CNO@CuMS provided by the present invention are as follows Figure 1 Specifically, the preparation method of the nano-immunomodulator CNO@CuMS provided in this embodiment includes:

[0051] After thoroughly mixing 1g of 3-amino-1,2,4-triazole and 4.4mmol of oxalic acid, the mixture was heated to 500°C at a rate of 5°C / min in an Ar atmosphere and held for 3h. Subsequently, 0.5g of the product was calcined again in air to obtain a bulk solid of double-defect C3N5 (CNO) containing N vacancies and O doping. The solid powder was collected and thoroughly ground in an agate mortar. An appropriate amount of the powder was added to a nitric acid solution (5M), incubated in an oil bath at 130°C for 24h, washed with water until neutral, and then redispersed in water. Ultrasonic exfoliation was performed, and CNO nanosheets were obtained by stepwise centrifugation in the range of 6000-9000rpm for use.

[0052] Dissolve 1.236g of ammonium heptamolybdate tetrahydrate and 2.284g of thiourea in 35mL of water, stir thoroughly, and transfer to a hydrothermal reactor. React at 200°C for 24 hours, cool naturally to room temperature, centrifuge, and rinse with pure water to obtain a bulk metallic MoS2 material. Ultrasonic exfoliation of the bulk metallic MoS2 was performed, and MS nanosheets were obtained by stepwise centrifugation at 9000-12000rpm for later use.

[0053] Take 10 mg of the above-mentioned MS nanosheets and evenly disperse them in water. Add 1 mL of 8 mM copper chloride solution, disperse them evenly with ultrasound and stir at room temperature for 24 hours. The product is metal phase molybdenum disulfide nanosheets (CuMS) with surface atomized copper chelate. Centrifuge and wash with water for later use.

[0054] 10 mg of double-defect nitrogen-rich carbon nitride (CNO) nanosheets were evenly dispersed in water, and surface atomized copper-chelated metallic phase molybdenum disulfide nanosheets (CuMS) were added according to the mass fraction of carbon nitride nanosheets of 0-30%. The mixture was stirred at room temperature overnight, centrifuged and resuspended in water to obtain the nanoimmunomodulator CNO@CuMS.

[0055] The nanoimmunomodulator CNO@CuMS was evenly dispersed in 10 mL of water and 20 mg of mPEG was added. 2k -COOH, stirred at 4 °C overnight, centrifuged and resuspended in water to obtain the surface PEGylated nanoimmunomodulator CNO@CuMS, which was used for subsequent in vitro and in vivo experimental studies.

[0056] like Figure 2 As shown, Figure 2 Figures a and b are transmission electron microscope characterization images of the metal phase molybdenum disulfide nanosheets MS and the metal phase molybdenum disulfide nanosheets CuMS with surface atomic copper chelation prepared in this example, respectively.

[0057] Figure 3 a in the figure is a high-angle annular dark field microscope (HAADF) of the CuMS prepared in this example; Figure 3 bd in the figure are the element mapping patterns of the CuMS prepared in this example: wherein b is the Mo element, c is the S element, and d is the Cu element.

[0058] Figure 4 X-ray photoelectron spectroscopy (XPS) characterization of the MS prepared in this example: wherein a is the total element spectrum, b is the XPS characterization of the Mo element, and c is the XPS characterization of the S element.

[0059] Figure 5 X-ray photoelectron spectroscopy (XPS) characterization of the CuMS prepared in this example: wherein a is the total element spectrum, b is the Mo element XPS characterization, c is the S element XPS characterization, and d is the Cu element XPS characterization.

[0060] Figure 6 This is the nonlinear least squares fitting result of the Auger spectrum of the Cu element in the CuMS prepared in this example.

[0061] Preparation of graphite phase carbon nitride (C3N4) and nitrogen-rich carbon nitride (C3N5) for comparison:

[0062] For graphite carbon nitride (C3N4), 10 g of urea was placed in a porcelain crucible with a lid and heated at 5 °C min in air. -1 The temperature was increased to 550 °C and maintained for 2 h.

[0063] For nitrogen-rich carbon nitride (C3N5), 2 g of 3-amino-1,2,4-triazole powder was polymerized in a N2 atmosphere for 3 h (500 °C, heating rate 5 °C min -1 After cooling naturally to room temperature, the final product was collected and ground.

[0064] Take 1g of ground C3N4 or C3N5 powder, add it to 100mL of 5M HNO3 solution, oil bath at 130℃ for 24h, wash with water until neutral, and then ultrasonically treat for 16h. The material in the range of 6000-9000rpm is obtained by step-by-step centrifugation, namely graphite phase carbon nitride (C3N4) and nitrogen-rich carbon nitride (C3N5) nanosheets.

[0065] Figure 7 Related characterization of graphite phase carbon nitride (C3N4), nitrogen-rich carbon nitride (C3N5), and double-defect nitrogen-rich carbon nitride (CNO) nanosheets: Figure 7 ac in the figure are TEM images of three nanosheets, Figure 7 The df in the figure are the butterfly diagram and phase hysteresis curve of the three nanosheets characterized by piezoelectric force microscopy (PFM). Figure 7 The gi in the figure are the amplitude diagrams of the three nanosheets characterized by piezoelectric force microscopy (PFM), Figure 7 jl in the figure represent phase images of the three nanosheets characterized by piezoelectric force microscopy (PFM). Comparison shows that double-defect nitrogen-rich carbon nitride (CNO) exhibits superior piezoelectric properties compared to graphitic carbon nitride (C3N4) and nitrogen-rich carbon nitride (C3N5), making it a promising candidate for the preparation of high-performance ultrasound-responsive nanoimmunomodulators.

[0066] Figure 8 Characterization of materials related to nanoimmunomodulator CNO@CuMS: Figure 8 a in the figure is the UV-visible-near-infrared absorption spectrum of three carbon nitride nanosheets. Figure 8 b in the figure is the electron spin resonance (EPR) characterization of three carbon nitride nanosheets. Figure 8 c in the figure is the Fourier transform infrared spectrum (FT-IR) of three carbon nitride nanosheets. Figure 8 d in the figure is the TEM characterization image of CNO@MS. Figure 8 e in the figure is the TEM characterization image of CNO@CuMS. Figure 8 f is the amplitude butterfly diagram and phase hysteresis curve representing the piezoelectric properties of CNO@MS. Figure 8 g in the figure is the UV-visible-near-infrared absorption spectrum of CNO@CuMS. Figure 8 h in the figure is the UV-visible diffuse reflectance characterization of CNO@CuMS. Figure 8 The i in the figure is the amplitude butterfly diagram and phase hysteresis curve of the piezoelectric properties of CNO@CuMS. Figure 8 The j in the figure is the Mott-Schottky curve test of CNO@CuMS. Figure 8 k in the equation is the electrochemical impedance spectroscopy (EIS) of CNO@CuMS. Figure 8 The l in the figure represents the photocurrent response of CNO@CuMS. The results demonstrate that CNO@CuMS exhibits more ideal piezoelectric and superior electrical properties than CNO@MS (prepared by directly adding the aforementioned MS nanosheets to a solution of double-defect nitrogen-rich carbon nitride (CNO) nanosheets). This paves the way for the rapid migration of active electrons within the nanoimmunomodulator and the realization of high-performance catalysis.

[0067] For 1 mg mL -1 The CNO@CuMS solution was ultrasonically treated (1.0 W cm -2 , 1 MHz, 20 min), and then the H2O2 concentration produced in the supernatant was tested using a H2O2 detection kit (titanium sulfate method). Figure 10 The titanium sulfate colorimetric method was used to determine the hydrogen peroxide produced by CNO@CuMS in response to ultrasonic stimulation.

[0068] Example 1

[0069] Characterization of catalytic activity of CNO@CuMS:

[0070] Aqueous solutions of CNO, CNO@MS, and CNO@CuMS were prepared respectively, and singlet oxygen and superoxide anion scavengers were added. The response intensity of the active oxygen capture probe was measured by electron spin resonance spectroscopy (ESR) at 0 min. The response intensity of the capture probe was measured again after ultrasonic treatment for 10 min to characterize the generation of H2O2 by CNO@CuMS through piezoelectric catalysis and sonodynamic catalysis. 1 The properties of O2. CNO, CNO@MS, and CNO@CuMS were mixed with 100μM H2O2, and then a hydroxyl radical capture probe was added. The response intensity of the hydroxyl radical capture probe was measured at 0min and 10min, respectively, to characterize the properties of CNO@CuMS catalyzing the Fenton-like reaction to produce hydroxyl radicals. The results are shown in Figure 2. Figure 9 As shown in ai.

[0071] Preparation of CNO@CuMS aqueous solution (1.0 mg mL -1 ). PBS with different pH values (7.4 and 5.5) was mixed with CNO@CuMS solution in a volume ratio of 1:1. The mixture was incubated in a shaker at 37°C. 1 mL of the solution was centrifuged at different time points (0, 1, 2, 4, 6, 12, 24 h), and the copper concentration in the supernatant was determined by ICP-MS. The responsive release of copper in CNO@CuMS was calculated. The results are shown in Figure 2. Figure 9 As shown in j.

[0072] Experimental study on the degradation of methylene blue by generating hydroxyl radicals by CNO@CuMS catalyzed Fenton-like reaction. -1 of methylene blue, 10 mM H2O2, and 200 μg mL -1 The mixed solution of CNO@CuMS was prepared by using the principle that hydroxyl radicals generated by copper atoms oxidize the discoloration of methylene blue. After 4 hours, the UV-visible absorption curve of the solution was measured to calculate the oxidation percentage of methylene blue. CNO and CNO@MS were used as control experimental groups. Figure 9 As shown in k.

[0073] Experimental study on the degradation of Rhodamine B by catalyzing the generation of reactive oxygen species in response to ultrasonic stimulation. -1 CNO@CuMS with 10 μg mL -1 Rhodamine B (RhB) mixed solution, ultrasonic treatment conditions are 1.0 MHz, 1.0 W cm -2 , after 20 minutes, the absorbance of the solution at 553 nm was measured and the degradation rate of RhB was calculated. The experimental results are as follows Figure 9 As shown in l.

[0074] Experimental study on the degradation of methylene blue by CNO@CuMS through piezoelectric-sound dynamic=Fenton-like triple mode catalysis to produce a large amount of reactive oxygen species. -1 of methylene blue, 10 mM H2O2, and 200 μg mL -1 The mixed solution of CNO@CuMS was ultrasonicated at 1.0 MHz and 1.0 W cm -2 After 20 minutes, the UV-visible absorption curve of the solution was measured to calculate the oxidation percentage of methylene blue. CNO and CNO@MS were used as control experimental groups. Figure 9 As shown in m.

[0075] Experimental study on the production of a large amount of reactive oxygen species by CNO@CuMS through piezoelectric-sound dynamic=Fenton-like three-mode catalysis to consume GSH. -1 The mixed solution of CNO@CuMS and 1 mM GSH was ultrasonicated for 20 min (1.0 W cm -2 , 1.0MHz), and the residual GSH content was determined using a GSSG / GSH detection kit. Figure 9 As shown in n.

[0076] Figure 9 Characterization of the catalytic activity of CNO@CuMS: Figure 9The ac in the figure is the electron spin resonance spectrum of CNO@CuMS in response to ultrasonic stimulation (1.0 W cm -2 , 1.0MHz) produces the property of singlet oxygen, Figure 9 The df in the figure is the electron spin resonance spectrum of CNO@CuMS in response to ultrasonic stimulation (1.0 W cm -2 , 1.0MHz) produces hydrogen peroxide, Figure 9 gi in the equation is the property of producing hydroxyl radicals by the Fenton-like reaction catalyzed by CNO@CuMS determined by electron spin resonance spectroscopy. Figure 9 j in the figure represents the property of CNO@CuMS releasing copper ions in response to a slightly acidic environment. Figure 9 k in the equation is the property of CNO@CuMS catalyzing the Fenton-like reaction to degrade methylene blue. Figure 9 The l in the figure represents the property of CNO@CuMS to generate reactive oxygen species in response to ultrasonic stimulation to degrade Rhodamine B. Figure 9 m in the equation is the property of CNO@CuMS in degrading methylene blue via piezoelectric-sound-dynamic-Fenton-like three-mode catalysis. Figure 9 n is the property of CNO@CuMS to generate reactive oxygen species and consume glutathione in response to ultrasound stimulation. Figure 9 The o in the graph represents the surface charge property of CNO@CuMS.

[0077] Example 2

[0078] Cell experiments show that CNO@CuMS induces immunogenic death of bone tumor cells through piezoelectric-sound dynamics = Fenton-like triple-mode catalysis and copper death induction.

[0079] K7M2 cells were seeded in 96-well plates and cultured at 37°C overnight. 100 μg mL -1 After incubation for 4-6 h, the CNO@CuMS was ultrasonicated (1.0 MHz, 0.5 W cm -2 , 50% cycle, 3 minutes). CCK-8 detection kit, Live / Dead staining kit, intracellular reactive oxygen species detection kit, mitochondrial membrane potential detection kit, Anexin-V-FITC / PI apoptosis detection kit were used to detect tumor cell survival, intracellular reactive oxygen species production, mitochondrial membrane potential changes, cell membrane phosphatidylserine externalization and other apoptotic phenotypes. Immunofluorescence was used to analyze the accumulation of the copper death marker protein DLAT in the cells. The results are as follows. Figure 11-13 shown.

[0080] To study the endocytosis of nano-immunomodulator CNO@CuMS by tumor cells, K7M2 cells were firstly cultured at a density of 1×10 5The cells were seeded at a density of 1000 / dish in a confocal microscopy dish and cultured at 37°C for 24 hours. CNO@CuMS was labeled with FITC and co-cultured with K7M2 cells for 12 hours. The cells were washed with PBS three times and the cell nuclei were stained with Hoechst 33258 (blue). The endocytosis of CNO@CuMS by tumor cells was observed under a confocal microscope. Figure 11 As shown in b.

[0081] Figure 11 The performance of CNO@CuMS in inducing immunogenic death of osteosarcoma K7M2 cells through trimodal catalysis and copper death induction was investigated: Figure 11 a in the figure is a schematic diagram of the principle. Figure 11 b is the confocal microscopy observation of FITC-labeled CNO@CuMS being internalized by K7M2 osteosarcoma cells. Figure 11 c in the figure shows that CNO@CuMS induces K7M2 cell death without ultrasound stimulation. Figure 11 Figures d and e show that CNO@CuMS induced K7M2 cell death under ultrasound stimulation. Figure 11 Figure f is the live-death fluorescence staining of K7M2 cells induced by CNO@CuMS under ultrasound stimulation. Figure 11 g in the figure is the flow cytometric analysis of K7M2 cell apoptosis induced by CNO@CuMS under ultrasound stimulation. Figure 11 h in the figure is the fluorescent probe detection of reactive oxygen species produced by CNO@CuMS in K7M2 cells through trimodal catalysis.

[0082] The properties of CNO@CuMS in killing bone tumor K7M2 cells under conditions without and with ultrasound are as follows: Figure 11 As shown in the results, under ultrasound-free conditions, CNO@CuMS can produce hydroxyl radicals through Fenton-like catalysis, and release copper ions in response to the slightly acidic environment inside the tumor to induce cell copper death.

[0083] Figure 12 Flow cytometric characterization of the generation of reactive oxygen species by CNO@CuMS in K7M2 osteosarcoma cells via trimodal catalysis: Figure 12 a and d in the equation are hydrogen peroxide. Figure 11 b and e in are singlet oxygen, Figure 11 The c and f in are hydroxyl radicals.

[0084] Figure 12 It shows that under ultrasound stimulation, CNO@CuMS further produces H2O2 and singlet oxygen through piezoelectric catalysis and sonodynamic catalysis, increasing the level of oxidative stress in tumor cells; the three-mode catalysis and copper death-inducing effect can significantly kill K7M2 cells, and tumor cells show apoptotic phenotypes such as mitochondrial depolarization and cell membrane phosphatidylserine externalization.

[0085] Figure 13 Investigation of the performance of CNO@CuMS in inducing copper-induced cell death and immunogenic death of bone tumor cells: Figure 13 a in the figure is a schematic diagram of the principle of inducing cell copper death. Figure 13 b is the confocal microscopy observation of the copper death marker DLAT protein aggregation. Figure 13 Figures c and d are the fluorescence staining and kit determination of cell immunogenic death markers CALR, HMGB1, and ATP. Figure 13 Figure e is a schematic diagram of the principle by which cell immunogenic death promotes dendritic cell maturation.

[0086] Figure 13 The results showed that the copper death marker protein DLAT had already significantly aggregated in the CNO@CuMS-only treatment group, and the aggregation was even more pronounced after CNO@CuMS+US treatment. Furthermore, CNO@CuMS+US treatment induced immunogenic cell death (ICD) in K7M2 osteosarcoma cells in mice, with significantly increased expression of calreticulin CRT on the cell membrane, nuclear high-mobility group box 1 (HMGB1) excretion, and increased extracellular ATP, laying the foundation for the activation of anti-tumor immunity.

[0087] Example 3

[0088] Animal experiments have shown that the nanoimmunomodulator CNO@CuMS induces immunogenic tumor cell death through trimodal catalysis and copper death induction. A mouse orthotopic osteosarcoma model was established using luciferase-labeled K7M2 cells. Six groups were established, including PBS, PBS+US, CNO, CNO+US, CNO@CuMS, and CNO@CuMS+US. The nanoimmunomodulator dosage was 10 mg kg -1 The ultrasonic treatment conditions were 1.0 MHz, 0.5 W cm -2 , 50% cycle, 10 minutes. On the 0th, 7th and 14th days of treatment, in situ fluorescence imaging of tumor tissues in tumor-bearing mice was performed using a small animal light imager to observe the size of the tumor tissue and measure the fluorescence intensity, and the fluorescence intensity was compared and analyzed. The changes in body weight and tumor volume of nude mice in different treatment groups were recorded. After 14 days of treatment, the tumor tissue was separated, the size and weight of the tumor tissue were measured and analyzed, and the relevant markers were stained and observed. After the treatment, H&E staining, immunohistochemical staining of the tumor cell proliferation marker Ki67, immunofluorescence staining of the copper death marker, and immunofluorescence staining of the tumor cell immunogenic death marker were performed on the tumor tissues of the different treatment groups to systematically evaluate the efficacy of the nanoimmunomodulator in inducing immunogenic death of tumors through tri-mode catalysis and copper death induction. The results are as follows Figure 14 shown.

[0089] Figure 14 Verification of the immunogenic death of bone tumors induced by CNO@CuMS in vivo: Figure 14 a in the figure is the CNO@CuMS treatment flow chart. Figure 14 b in the figure is the tumor growth curve of each group of mice during the treatment cycle. Figure 14 c in the figure is the weight change trend of mice during the treatment period. Figure 14 The d in the figure is the tumor growth curve of different individuals during the treatment cycle. Figure 14 The e in the figure is the in vivo fluorescence imaging of tumors in each group of mice on days 0, 7, and 14. Figure 14 Figure f shows H&E staining, cell proliferation immunohistochemical staining, copper death, and immunogenic death marker immunofluorescence staining of tumor tissue sections of mice in each group.

[0090] Figure 14 It was shown that CNO@CuMS has the property of inducing immunogenic cell death in tumor tissue cells in response to ultrasound stimulation through trimodal catalysis and copper death induction.

[0091] Example 4

[0092] Animal experimental study on the effect of nanoimmunomodulator CNO@CuMS on reshaping the immune microenvironment of bone tumors and enhancing the anti-tumor efficacy of immune checkpoint inhibitor αPD-1. Luciferase-labeled K7M2 cells were used to establish an orthotopic osteosarcoma model in mice. When the orthotopic tumor size was 60 mm, the 3 The tumor-bearing mice were randomly divided into 4 groups, with no less than 5 mice in each group. They were treated with different treatments: G1: PBS, G2: αPD-1, G3: CNO@CuMS+US, G4: CNO@CuMS+US+αPD-1. The ultrasonic treatment conditions were 1.0 MHz, 0.5 W cm -2 ,50% cycle,10 minutes,Nano-immunomodulator dose:10 mg kg -1 , αPD-1 dose 20μg / mouse / time.

[0093] On the 0th, 7th and 14th days of treatment, in situ fluorescence imaging of tumor tissues in tumor-bearing mice was performed using a small animal light imager. The size of the tumor tissue was observed and the fluorescence intensity was measured. The fluorescence intensity was compared and analyzed. After 14 days of treatment, the tumor tissue was isolated, the size and weight of the tumor tissue were measured and analyzed, and the relevant markers were stained and observed. After the treatment, H&E staining and immunohistochemical staining of the tumor cell proliferation marker Ki67 were performed on the tumor tissues of the different treatment groups. The cell suspensions of the tumor tissues of each group after combined treatment were subjected to flow cytometry analysis of immune cells and detection of pro-inflammatory factor kits in serum. H&E staining was performed on the main tissues and organs of nude mice in the different treatment groups, and blood routine and liver and kidney function biochemical indicators were measured. Systematic evaluation of the immune microenvironment regulation and toxic side effects of nano-immunomodulators. The results are as follows Figures 15-19 shown.

[0094] Figure 15 In vivo experimental validation of CNO@CuMS enhancing the anti-tumor efficacy of the immune checkpoint inhibitor αPD-1: Figure 15 a in the figure is the flow chart of combined treatment of CNO@CuMS and αPD-1. Figure 15 b in the figure is the tumor growth curve of different individuals during the treatment cycle. Figure 15 c in the figure is the tumor growth curve of each group of mice during the treatment cycle. Figure 15 The d in the figure is the in vivo fluorescence imaging of the tumors in each group of mice on days 0, 7, and 14. Figure 15 The e in the figure is the weight change trend of mice during the treatment period. Figure 15 Figure f shows H&E staining and cell proliferation immunohistochemical staining of tumor tissue sections of mice in each group. Figure 15 g in the figure shows the statistical analysis of the immunohistochemical staining results of the cell proliferation marker Ki67. Figure 16 Actual pictures of tumors in each group after combined treatment with CNO@CuMS and αPD-1 ( Figure 16 Analysis of tumor weight in each group Figure 16 b) in the above example. Figure 17 Flow cytometry analysis of cell suspensions of tumor tissues in each group after combined treatment with CNO@CuMS and αPD-1 and detection of pro-inflammatory factors in serum by kit: Figure 17 a and b in the equation are CD3 + T cells, Figure 17 The cd in is CD3 + CD8 + T cells, Figure 17 ef in the code is CD3 - CD49b + NK cells, Figure 17 The gi in the cells are M1 and M2 macrophages. Figure 17 The j in is the ratio of M1 / M2, Figure 17k in is IL-6, Figure 17 The l in is TNF-α, Figure 17 The m in the table is the IFN-γ level test. Figure 17 n is the number of CD8 + Immunofluorescence staining of T cells. Figure 18 These are the H&E section staining results of the main organs of mice in each group after combined treatment with CNO@CuMS and αPD-1. Figure 19 The al in the figure are the liver and kidney function test results of each group of mice after combined treatment of CNO@CuMS and αPD-1.

[0095] Figures 15-19 This indicates that the use of nanoimmunomodulator CNO@CuMS to regulate the immune microenvironment of tumor tissue can significantly enhance the efficacy of immune checkpoint inhibitor αPD-1. After 14 days of combined treatment, tumor growth was significantly inhibited. Flow cytometric analysis of tumor tissue showed that nanoimmunomodulator can increase CD8 + The infiltration of T cells and NK cells into tumor tissues modulates macrophage polarization toward the anti-tumor M1 phenotype and increases levels of pro-inflammatory cytokines such as IL-6, TNF-α, and IFN-γ. No significant toxic side effects were observed in major tissue and organ sections, blood routine, and liver and kidney function biochemical tests, further demonstrating the ideal biosafety of the nanoimmunomodulator CNO@CuMS.

Claims

1. A method for preparing a nano-immunomodulator, characterized in that: The preparation method comprises: (1) 3-amino-1,2,4-triazole and oxalic acid are mixed and then calcined, ground, acid-treated, ultrasonically exfoliated, and centrifuged in steps to obtain double-defect nitrogen-rich carbon nitride (CNO) nanosheets; (2) adding a copper chloride solution to a metallic phase molybdenum disulfide MS nanosheet solution and stirring the reaction to obtain metallic phase molybdenum disulfide nanosheets CuMS with surface atomized copper chelated; (3) The CuMS solution was added dropwise into the double-defect nitrogen-rich carbon nitride CNO nanosheet solution to react and obtain the nanoimmunomodulator CNO@CuMS.

2. The method for preparing the nano-immunomodulator according to claim 1, wherein In step (1), the preparation method of the CNO nanosheets is as follows: 3-amino-1,2,4-triazole powder and oxalic acid powder are mixed, calcined in an argon atmosphere, and the calcined product is placed in an air atmosphere for secondary calcination to obtain bulk CNO; the bulk CNO is ground and then subjected to acidification and ultrasonic exfoliation, and CNO nanosheets within a specific range are obtained by step-by-step centrifugation.

3. The method for preparing the nano-immunomodulator according to claim 2, characterized in that: The amount of oxalic acid is 1-5 mmol, the mass of the 3-amino-1,2,4-triazole powder is 1.0 g, the calcination temperature in argon atmosphere is 500-600° C., and the calcination time is 2-4 hours; the secondary calcination temperature in air atmosphere is 500-600° C., and the secondary calcination time is 2-4 hours.

4. The method for preparing the nano-immunomodulator according to claim 2, wherein: CNO nanosheets were obtained by stepwise centrifugation in the range of 6000-9000 rpm.

5. The method for preparing the nano-immunomodulator according to claim 1, characterized in that: In step (2), the preparation method of the surface atomized copper chelated metal phase molybdenum disulfide nanosheets CuMS is: (2-1) adding ammonium heptamolybdate tetrahydrate and thiourea in a molar mass ratio of (1-3):30 to 30-50 mL of water and hydrothermally reacting them at a reaction temperature of 180-220° C. for 18-24 hours to obtain bulk metallic phase MoS2, followed by ultrasonic exfoliation and stepwise centrifugation to obtain metallic phase MoS2 MS nanosheets in the range of 9000-12000 rpm; (2-2) The feeding ratio of MS nanosheets to copper chloride solution is as follows: 0.1-2 mL of 5-10 mM copper chloride solution is added to a solution containing 10 mg of MS nanosheets, and the stirring time is 12-24 hours to allow the copper ions to be fully atomized on the surface of the MS nanosheets.

6. The method for preparing the nano-immunomodulator according to claim 1, characterized in that: In step (3), the added mass of the CuMS is 5-30% of the CNO nanosheets.

7. The method for preparing the nano-immunomodulator according to claim 1, characterized in that: The preparation method comprises: (4) Disperse CNO@CuMS and add PEG 2k -DSPE modified as a nanoimmunomodulator.

8. A nano-immunomodulator obtained according to the preparation method according to any one of claims 1 to 7.

9. Use of the nano-immunomodulator according to claim 8 in the preparation of drugs for treating bone tumors.

10. Use of the nano-immunomodulator according to claim 8 in combination with the immune checkpoint inhibitor αPD-1 in the preparation of a drug for treating bone tumors.

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