Application of hydrophilic black phosphorus nanoparticles in preparation of anti-tumor immunologic adjuvant

Abstract

The invention belongs to the field of anti-tumor, and provides application of hydrophilic black phosphorus nanoparticles in preparation of an anti-tumor immunologic adjuvant, the hydrophilic black phosphorus nanoparticles improve the sensitive degree of tumor cells to tumor immunotherapy drugs by improving the oxidative phosphorylation level of the tumor cells and inhibiting expression of tumor cell PD-L1 antibodies, and the anti-tumor immunologic adjuvant is prepared. Synergistic inhibition of the hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs on tumors is achieved, and the hydrophilic black phosphorus nanoparticles are obtained by modifying black phosphorus nanosheets through a hydrophilic high polymer material. The tumor immunotherapy drug is a PD-1 / PD-L1 immune checkpoint inhibitor, and the tumor cells comprise melanoma cells or breast cancer cells. The sensitive degree of tumor cells to tumor immunotherapy drugs can be improved, and the tumor immunotherapy effect is enhanced.

Description

Technical Field

[0001] The invention belongs to the field of anti-tumor, and relates to the application of hydrophilic black phosphorus nanoparticles in the preparation of anti-tumor immune adjuvants. Background Art

[0002] Cancer is one of the most serious diseases threatening human health, second only to cardiovascular disease in terms of mortality. Currently, the main method for treating cancer clinically is systemic chemotherapy, but chemotherapy drugs themselves are highly toxic and have indiscriminate toxicity to tumor cells and normal tissue cells, which can cause serious damage to the patient's health. Therefore, researchers have been actively seeking different methods for tumor treatment.

[0003] Under normal physiological conditions, mitochondrial oxidative phosphorylation (OXPHOS) contributes more than 95% of the energy required by cells through mitochondrial respiration, producing adenosine triphosphate (ATP). The Warburg effect indicates that tumor cells prefer glycolysis, and the uptake and consumption of glucose in the glycolysis process is greater than that in the oxidative phosphorylation process, resulting in the rapid production of ATP (although the efficiency is low), which is conducive to the rapid consumption of energy for tumor cells to proliferate. At the same time, abnormal glycolysis metabolism leads to a surge in lactate, pyruvate and ROS content, acidifies the microenvironment, and promotes tumor progression, invasion and immune escape. In addition, glycolysis also inhibits the activation of the anti-tumor immune microenvironment. The inhibition of glycolysis can enhance the efficacy of CTLA-4 inhibitors and is expected to overcome the resistance to immunotherapy. Therefore, the regulation of tumor energy metabolism from glycolysis to OXPHOS may be an advanced therapeutic strategy.

[0004] Studies have found that some tumors have strong immunogenicity. For these tumors with strong immunogenicity, immunotherapy is currently the most promising treatment method. Common treatment methods include the use of cytokines, vaccines, adoptive T cells, and immune checkpoint inhibitors. PD-1 / PD-L1 antibodies can cut off tumor immune escape and prompt T cells to attack tumor cells by blocking activated T cells from recognizing PD-L1 on the surface of tumor cells. That is, PD-1 / PD-L1 antibodies can restore T cell activity and enhance anti-tumor immune response by specifically blocking the interaction between PD-1 and PD-L1. Therefore, PD-1 monoclonal antibodies are currently the most promising immunotherapy drugs for metastatic tumors. Currently, nivolumab (MDX-1106) and pembrolizumab are more commonly used in clinical practice. They can significantly improve the response rate of some patients with advanced melanoma and prolong survival time. However, in the treatment of solid tumors, the objective response rate of PD1 / PD-L1 blockade therapy rarely reaches 40%, and most patients do not respond to PD1 / PD-L1 treatment or will relapse after treatment.

[0005] Although some researchers have proposed to improve the PD1 / PD-L1 blocking effect of solid tumors through the synergistic treatment of chemotherapy and immunotherapy, the chemotherapy drugs currently used in combination with PD-1 / PD-L1 immune checkpoint inhibitors, such as paclitaxel, gemcitabine, oxaliplatin, pyrrole and doxorubicin, have weak immunogenicity, indiscriminate toxicity and side effects on tumor cells, immune cells and normal tissue cells, large side effects, high cost, instability, easy to produce drug resistance and unclear mechanism of action. Therefore, there is still an urgent need for chemotherapy drugs with low toxicity and side effects, synergistic stimulation of immunity and clear mechanism of action in clinical practice, which can be used in combination with immunosuppressive drugs to produce effective synergistic treatment effects, so as to improve the problems of poor selectivity and multidrug resistance of chemotherapy drugs currently used in synergistic treatment with immunosuppressive drugs. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of hydrophilic black phosphorus nanoparticles in the preparation of anti-tumor immune adjuvants to increase the sensitivity of tumor cells to tumor immunotherapy drugs and enhance the tumor immunotherapy effect.

[0007] The present invention discovered through experiments that hydrophilic black phosphorus nanoparticles have the ability to continuously degrade and metabolize into phosphates inside tumor cells. After treatment with a hydrogel containing hydrophilic black phosphorus nanoparticles and PD-1 / PD-L1 immunosuppressants, the mitochondrial oxidative phosphorylation level of tumor cells was significantly upregulated, accompanied by a decrease in the expression level of PD-L1 protein in tumor cells. In addition, the hydrophilic black phosphorus nanoparticles and PD-1 / PD-L1 immunosuppressants have a synergistic anti-tumor effect. The hydrogel containing hydrophilic black phosphorus nanoparticles and PD-1 / PD-L1 immunosuppressants can effectively inhibit the growth of tumors in melanoma-bearing mice and breast cancer-bearing mice.

[0008] Based on the above experimental results, the present invention provides the following technical solutions:

[0009] The application of hydrophilic black phosphorus nanoparticles in the preparation of anti-tumor immune adjuvants. The hydrophilic black phosphorus nanoparticles increase the oxidative phosphorylation level of tumor cells and inhibit the expression of PD-L1 antibodies in tumor cells, thereby increasing the sensitivity of tumor cells to tumor immunotherapy drugs, thereby achieving synergistic inhibition of tumors by hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs. The hydrophilic black phosphorus nanoparticles are obtained by modifying black phosphorus nanosheets with hydrophilic polymer materials.

[0010] In the above-mentioned technical solution, the tumor immunotherapy drug is a PD-1 / PD-L1 immune checkpoint inhibitor. Further, the PD-1 / PD-L1 immune checkpoint inhibitor includes any one of BMS-1, BMS-202, BMS-1166, nivolumab, pembrolizumab, atezolizumab, ipilimumab and blinatumomab.

[0011] In the technical solution of the above application, it is preferred that the hydrophilic black phosphorus nanoparticles and the tumor immunotherapy drug are combined in a mass ratio of (5 to 10):1 to act on tumor cells.

[0012] In the technical scheme of the above application, hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs are loaded on a pharmaceutically acceptable carrier to act on tumor cells. The pharmaceutically acceptable carrier material includes hydrogels, micelles, liposomes, nanoparticles, nanocapsules, microspheres or microcapsules. Further, when the pharmaceutically acceptable carrier material is a hydrogel, the materials constituting the hydrogel polymer network include but are not limited to poloxamer, polylactic acid, polyethylene glycol, polylactic acid-polyethylene glycol-polylactic acid, hyaluronic acid, collagen, gelatin, dextran, chitosan, and one or more of the derivatives of these polymer compounds. For example, the material constituting the hydrogel polymer network can be a thiolated hyaluronic acid with a structure as shown in formula (I), and the grafting rate of the thiol group in the thiolated hyaluronic acid can be 10% to 30%. The thiolated hyaluronic acid is modified based on hyaluronic acid, and the molecular weight range of the hyaluronic acid used as the modification basis can be 30 to 50 kDa.

[0013]

[0014] In the technical solution of the above application, hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs are prepared into an injection to act on tumor cells. Preferably, the concentration of hydrophilic black phosphorus nanoparticles in the injection is at least 5 mg / mL. For example, the concentration of hydrophilic black phosphorus nanoparticles in the injection can be 5-10 mg / mL.

[0015] In the technical solution of the above application, the hydrophilic black phosphorus nanoparticles are obtained by bonding a hydrophilic polymer material to the surface of a black phosphorus nanosheet or by combining the hydrophilic polymer material through electrostatic action. Further, the hydrophilic polymer material preferably coats the black phosphorus nanosheet. Furthermore, the hydrophilic polymer material includes any one of amino polyethylene glycol, polylysine, polyethyleneimine, and polyamino acid.

[0016] In the technical solution of the above-mentioned drug-loaded complex, the tumor cells include melanoma cells or breast cancer cells.

[0017] In the above-mentioned technical solution, the size of the hydrophilic black phosphorus nanoparticles is between 10 and 300 nm.

[0018] The present invention has confirmed through cell experiments that hydrophilic black phosphorus nanoparticles are metabolized into phosphate after being taken up by B16F10 cells, thereby regulating the metabolic process of B16F10 cells, and improving the oxidative phosphorylation level of B16F10 cells while inhibiting glycolysis of B16F10 cells, which is specifically manifested as: significantly improving the activity of mitochondrial respiratory chain complexes I, II, IV and V of B16F10 cells, increasing ATP content and reducing intracellular lactic acid content. In addition, hydrophilic black phosphorus nanoparticles can significantly downregulate the expression of PD-L1 antibodies on the surface of B16F10 cells. Hydrophilic black phosphorus nanoparticles can selectively inhibit the proliferation and migration of B16F10 cells, but have no cytotoxicity to normal L929 cells.

[0019] The present invention has been confirmed through animal experiments: by injecting a hydrogel containing hydrophilic black phosphorus nanoparticles and a tumor immunotherapy drug BMS-1 ​​into the tumor of a B16F10 tumor-bearing mouse (combined medication), compared with injecting a hydrogel containing only hydrophilic black phosphorus nanoparticles or a hydrogel containing only the tumor immunotherapy drug BMS-1, the combined medication can effectively downregulate the expression level of PD-L1 antibodies in tumor cells of B16F10 tumor-bearing mice, which is beneficial to increase the sensitivity of tumor cells to the tumor immunotherapy drug BMS-1, thereby enhancing the tumor immunotherapy effect. At the same time, compared with the injection of a hydrogel containing only hydrophilic black phosphorus nanoparticles or a hydrogel containing only the tumor immunotherapy drug BMS-1, the combined use of hydrophilic black phosphorus nanoparticles and the tumor immunotherapy drug BMS-1 ​​can more effectively inhibit tumor growth and significantly increase the survival rate of B16F10 tumor-bearing mice and 4T1 tumor-bearing mice.

[0020] Compared with the prior art, the technical solution provided by the present invention produces the following beneficial technical effects:

[0021] 1. The present invention provides an application of hydrophilic black phosphorus nanoparticles in the preparation of anti-tumor immune adjuvants, wherein the hydrophilic black phosphorus nanoparticles inhibit the expression of PD-L1 antibodies in tumor cells by increasing the oxidative phosphorylation level of tumor cells, thereby increasing the sensitivity of tumor cells to tumor immunotherapy drugs, and realizing the synergistic inhibition of tumors by hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs, wherein the hydrophilic black phosphorus nanoparticles are obtained by modifying black phosphorus nanosheets with hydrophilic polymer materials. The hydrophilic black phosphorus nanoparticles of the present invention have obvious cytotoxicity to B16F10 cells, but do not produce cytotoxicity to normal cells such as L929 cells, that is, when the present invention uses hydrophilic black phosphorus nanoparticles to enhance the anti-tumor effect of tumor immunotherapy drugs, it will not have an adverse effect on normal cells, and has the characteristics of low toxicity and side effects, which can improve the problem of large toxicity and side effects of chemotherapy drugs currently used in synergistic treatment with immunosuppressive drugs.

[0022] 2. The present invention has been confirmed through animal experiments: by injecting a hydrogel containing hydrophilic black phosphorus nanoparticles and the tumor immunotherapy drug BMS-1 ​​next to the tumor of B16F10 tumor-bearing mice (combined medication), compared with injecting a hydrogel containing only hydrophilic black phosphorus nanoparticles or a hydrogel containing only the tumor immunotherapy drug BMS-1, the combined medication can effectively downregulate the expression level of PD-L1 antibodies in tumor cells of B16F10 tumor-bearing mice, thereby increasing the sensitivity of tumor cells to the tumor immunotherapy drug BMS-1, thereby enhancing the tumor immunotherapy effect. Compared with the injection of a hydrogel containing only hydrophilic black phosphorus nanoparticles or a hydrogel containing only the tumor immunotherapy drug BMS-1, the combined use of hydrophilic black phosphorus nanoparticles and the tumor immunotherapy drug BMS-1 ​​can more effectively inhibit tumor growth and significantly increase the survival rate of B16F10 tumor-bearing mice and 4T1 tumor-bearing mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are transmission electron micrographs of BPNS and BPP, where (a) and (c) are transmission electron micrographs of BPNS, and (b) and (d) are transmission electron micrographs of BPP.

[0024] Figure 2 This is a high-magnification transmission electron microscopy image of BPP.

[0025] Figure 3 Figure 1 is the physical and chemical characterization results of BPNS, BPP and NH2-PEG. Figure A is the surface potential test results of BPNS, BPP and NH2-PEG, and Figure B is the size distribution diagram of BPNS and BPP.

[0026] Figure 4These are the test results of the cytotoxicity of different concentrations of BPP on L929 cells and B16F10 cells in Example 3.

[0027] Figure 5 The figure shows the inhibition of B16F10 cell migration at different time points by different concentrations of BPP in Example 4, wherein (a) is the scratch healing rate test result, and (b) is an inverted microscope photograph.

[0028] Figure 6 The results of testing the metabolites of BPP in B16F10 cells in Example 5, wherein (a) is a schematic diagram of testing the metabolites of BPP in B16F10 cells, (b) is a graph showing the change of the phosphate content in the cells obtained by testing in step (1) of Example 6 with the culture time, and (c) is a graph showing the change of the phosphate content in the cells obtained by testing in step (2) of Example 6 with the culture time.

[0029] Figure 7 These are the test results of metabolic changes in B16F10 cells after treatment with BPP for different time periods in Example 6, wherein (a) to (e) are changes in the contents of mitochondrial respiratory chain complexes I, II, III, IV and V with culture time, (f) is changes in ATP content with culture time, and (g) and (h) are changes in intracellular and extracellular lactic acid content with culture time.

[0030] Figure 8 These are the test results of the effect of BPP on the expression level of PD-L1 antibody on the surface of B16F10 cells in Example 7, wherein (a) is a flow cytometric graph of the fluorescence expression of PD-L1 in B16F10 cells, and (b) is a quantitative test result of the expression level of PD-L1 antibody.

[0031] Fig. 9 Figure 11 is the tumor growth curve and tumor inhibition rate test results of each experimental group on B16F10 tumor-bearing mice, wherein (a) is the average tumor volume change curve of mice in each experimental group, (b) is the average tumor volume change curve of different mice in each experimental group, and (c) is the calculated result of the tumor inhibition rate of each experimental group.

[0032] Fig.10 This is the survival curve of B16F10 tumor-bearing mice in each experimental group of Example 11.

[0033] Fig.11 The PD-L1 expression of tumor cells in tumor tissues of B16F10 tumor-bearing mice in different experimental groups in Example 12, wherein (a) is a flow cytometer graph of the fluorescence expression of PD-L1 in tumor cells, and (b) is a quantitative test result of the PD-L1 antibody expression level in tumor cells.

[0034] Fig.12 The CD8 + The ratio of T cells and regulatory T cells, where (a) is the CD8 + Flow cytometry of T cells, (b) shows the CD8 + Statistical graph of T cell flow cytometry results, (c) is a flow cytometric graph of regulatory T (Treg) cells in tumor tissue, and (d) is a statistical graph of regulatory T (Treg) cell flow cytometric results in each experimental group.

[0035] Fig.13 Figure 14 is the test results of the tumor growth curve, tumor inhibition rate and survival curve of 4T1 tumor-bearing mice in each experimental group of Example 14, wherein Figure (a) is the average tumor volume change curve of mice in each experimental group, Figure (b) is the calculation result of the tumor inhibition rate of each experimental group, and Figure (c) is the survival curve of 4T1 tumor-bearing mice in each experimental group of this embodiment. DETAILED DESCRIPTION

[0036] The following examples further illustrate the application of the hydrophilic black phosphorus nanoparticles provided by the present invention in the preparation of anti-tumor immune adjuvants. In the following examples, the effect of the combined use of hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs is described by taking the case where the pharmaceutically acceptable carrier material is a hydrogel as an example, but the application of the present invention is not limited to the preparation of hydrophilic black phosphorus nanoparticles, tumor immunotherapy drugs and pharmaceutically acceptable carriers into the form of hydrogels for application. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. The technical personnel in the relevant field make some non-essential improvements and adjustments to the present invention according to the above invention content for specific implementation, which still belongs to the scope of protection of the present invention.

[0037] Example 1

[0038] In this embodiment, black phosphorus nanosheets (BPNS) are prepared by liquid phase exfoliation method, and the steps are as follows:

[0039] 10 mg of black phosphorus (BP) powder was added to 20 mL of N-methyl-2-pyrrolidone (NMP), and the large volume of BP powder was physically broken by ultrasonic treatment with a probe at 0-10°C for 3 h (the conditions of the probe ultrasound were: ultrasonic power 800 W, probe vibration amplitude 20%, and pulse parameters 2s on 1s off), and then the large volume of BP powder was physically broken, and then the water bath ultrasound was performed at 200 W for 30 min at room temperature (about 20°C), and then the obtained suspension was allowed to stand, the supernatant was taken and centrifuged at 7000 rpm for 10 min, and the supernatant was taken and centrifuged at 10000 rpm for 15 min, the supernatant was discarded, the precipitate was collected, and freeze-dried to obtain BPNS, which was then stored at -20°C in vacuum or N2 sealed. This embodiment was carried out under N2 conditions throughout the preparation process to isolate oxygen.

[0040] Example 2

[0041] In this example, hydrophilic black phosphorus nanoparticles (BPP) were prepared. Specifically, the surface of the BPNS described in Example 1 was hydrophilically modified to prepare BPP.

[0042] BPNS was fully dispersed in deionized water to form a 1 mg / mL BPNS dispersion, NH2-PEG was added to the BPNS dispersion at a mass ratio of 4:1, and the mixture was fully mixed and ultrasonicated in an ice bath at a power of 100 W for 10 minutes, and stirred at room temperature for 1 hour, so that NH2-PEG was bound to the surface of BPNS by electrostatic adsorption, and then centrifuged at 12000 rpm for 30 minutes, and the precipitate was collected to obtain BPP, which was freeze-dried and stored at -20°C. The NH2-PEG has an amino group (-NH2) connected to one end of the PEG molecular chain, and the molecular weight of NH2-PEG is 2000 g / mol.

[0043] Figure 1 Figures (a) and (c) are transmission electron micrographs of the BPNS prepared in Example 1. Figure 1 Figures (b) and (d) are transmission electron micrographs of the BPP prepared in Example 2. Figure 1 It can be seen that both BPNS and BPP are in the form of nanosheets, and after being modified by NH2-PEG, a coating layer is formed on the surface of BPNS. Figure 2 It is a high-magnification transmission electron micrograph of the BPP prepared in Example 1, wherein Figures (b) and (c) are partial enlarged views of different parts of Figure (a). Figure 3 Figure A is the surface potential test results of BPNS prepared in Example 1, BPP prepared in Example 2, and NH2-PEG used in the examples. Figure 3 Figure B is a size distribution diagram of BPNS prepared in Example 1 and BPP prepared in Example 2. Figure 3 The Zeta potential in the figure shows that the hydrophilicity of BPNS increases after modification with NH2-PEG. Figure 3 From the size distribution diagram, we can see that after NH2-PEG modification, the size of BPNS increased slightly. The size distribution of BPNS was 50-250 nm, with an average size of 177.27±2.06 nm, and the size distribution of BPP was 60-300 nm, with an average size of 178.13±2.95 nm.

[0044] Example 3

[0045] In this example, the cytotoxicity of the BPP prepared in Example 2 to B16F10 cells (mouse skin melanoma cells) and L929 cells (mouse fibroblasts) was tested.

[0046] B16F10 cells and L929 cells were cultured in a culture dish until they adhered to the wall and proliferated to more than 80% to 90%, and the cells were digested with trypsin and prepared into a single cell suspension, and the number of cells was counted using a cell counting plate. The cells were added to a 96-well plate at 3000 cells / well (100 μL culture medium / well) overnight. After the cells were fully spread, the culture medium supernatant was discarded, and 100 μL of 1640 culture medium containing different concentrations (0, 5, 50, 250, 1000 μg / mL) of BPP was added to each well. Three parallel samples were set for each concentration. After 48 hours of culture, the culture medium supernatant in the well was aspirated, and 100 μL of serum-free fresh culture medium containing 10% CCK8 test solution was added to each well, and 100 μL of pure culture medium was added to the outermost circle of the culture plate to avoid volatilization. Incubate at 37°C in the dark for 2 hours, then shake the 96-well plate to fully dissolve the formazan, and detect the absorbance (OD value) at a wavelength of 450nm on an ELISA reader. Among them, the wells with 100μL of blank culture medium are used as blank wells, the wells with a BPP concentration of 0μg / mL are used as control wells, and the wells with other concentrations of BPP are used as experimental wells. Calculate the cell survival rate according to the following formula:

[0047] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0048] In the above formula, Ab is the OD value of the blank well, Ac is the OD value of the control well, and As is the OD value of the experimental well.

[0049] In this example, the test results of different concentrations of BPP on the cytotoxicity of L929 cells and B16F10 cells are as follows: Figure 4 As shown. Figure 4It can be seen that BPP has no adverse effect on the survival rate of L929 cells, that is, BPP has no cytotoxicity to normal cells; with the increase of BPP concentration, the survival rate of B16F10 cells gradually decreased, especially when the concentration of BPP reached 500μg / mL, the survival rate of B16F10 cells began to decrease significantly.

[0050] Example 4

[0051] In this example, the effect of BPP on the migration of B16F10 cells was tested.

[0052] The ability of BPP to inhibit lateral cell migration was evaluated by scratch test. B16F10 cells were plated at 1.5×10 4 1640 medium / well) was added to a 24-well plate. When the cells adhered to the wall and proliferated to about 80%, a scratch was made vertically from top to bottom in the center of the well plate with a 10 μL pipette tip, and then washed 2 to 3 times with PBS buffer until the cells suspended in the culture medium were removed. Then 0.5 mL of 1640 medium containing different concentrations (0, 5, 50, 250, 500, 1000 μg / mL) of BPP was added to the well plate and cultured for 12 hours. Three parallel samples were set for each concentration. When the culture time was 0, 3, 6 and 9 hours, the lateral migration of cells on both sides of different wells to the blank area in the middle of the scratch was observed under an inverted fluorescence microscope and photographed and recorded. The wells with a BPP concentration of 0 μg / mL were used as control wells. The width of the blank scratches in the wells with different BPP concentrations at different time points was measured using Image J software, and the original scratch width was compared. The scratch healing rate [Width (%)] was calculated according to the following formula:

[0053] Scratch healing rate (%) = (W0-W t ) / W0×100%

[0054] In the above formula, W t represents the width of the scratch at time t, and W0 represents the width of the scratch at time 0 (the original scratch width).

[0055] Figure 5 The figure shows the inhibition of B16F10 cell migration at different time points by different concentrations of BPP in this example, wherein (a) is the scratch healing rate test result, and (b) is an inverted microscope photo. Figure 5 It can be seen that with the increase of BPP concentration, the ability of BPP to inhibit the migration of B16F10 cells gradually increased, especially when the concentration of BPP reached 250 μg / mL, it could significantly inhibit the migration of B16F10 cells.

[0056] Example 5

[0057] In this example, the metabolites of BPP in B16F10 cells were investigated.

[0058] (1) Figure 6 As shown in route d1 in Figure (a), cells were cultured at a rate of 1.5×10 4 Cells were added into a 24-well plate at 1000 μg / mL per well (0.5 mL 1640 medium / well) and cultured overnight to ensure that the cells were fully attached to the wall. The cells were rinsed twice with PBS buffer to remove floating cells, and then the medium was replaced with fresh 1640 medium containing 1000 μg / mL BPP for culture. Eighteen parallel samples were set for each concentration. Three sample wells were randomly selected at culture times of 0, 4, 16, 28, 40 and 52 h, respectively. The cells were washed repeatedly with ultrapure water twice, and the cells were carefully collected in a 1.5 mL enzyme-free centrifuge tube with a cell scraper. The centrifuge was precooled to 4°C, and the cells were centrifuged at 300g for 5 min. The supernatant was removed as much as possible, and the cell pellet was collected. Subsequently, 500 μL of lysis buffer was added to the cell pellet and vortexed for 15 s to fully mix. The cells were incubated in an ice bath for 30 min, and the fully lysed cell fragment suspension was centrifuged at 16000g for 5 min in a centrifuge precooled at 4°C to precipitate excess cell fragments. 500 μL of supernatant was transferred to a new 1.5 mL enzyme-free centrifuge tube, and the intracellular phosphate content of each sample was detected according to the operating instructions of the cell inorganic phosphorus / free phosphate ion colorimetric quantitative detection kit.

[0059] (2) Figure 6 As shown in the d2 route in Figure (a). In order to further confirm the metabolism of BPP in cells, it is excluded that BPP is degraded during the free process outside the cells, which increases the phosphate concentration in the culture medium, thereby affecting the intracellular phosphate concentration and creating the illusion of increased intracellular phosphate concentration. As described in step (1) above, first replace the cell culture medium after overnight attachment with fresh 1640 culture medium containing 1000μg / mL BPP. After culturing for 12 hours, rinse twice with PBS buffer, then remove the culture medium in the supernatant and replace it with normal 1640 culture medium without BPP to continue culturing cells. Set 18 parallel samples for each concentration. Start timing after adding fresh 1640 culture medium with 1000μg / mL BPP. Randomly select 3 sample wells at 0, 12, 16, 28, 40 and 52 hours of culture time, and detect the intracellular phosphate concentration by referring to the operation of step (1).

[0060] Figure 6 Figure (a) is a schematic diagram of testing the metabolites of BPP in B16F10 cells. Figure 6 Figure (b) shows the change of intracellular phosphate content with culture time obtained in step (1). Figure 6Figure (c) shows the change of intracellular phosphate content with culture time obtained in step (2). Figure 6 It can be seen that BPP is taken up by B16F10 cells and metabolized into phosphate.

[0061] Example 6

[0062] In this example, the effect of BPP on glucose metabolism in B16F10 cells was investigated.

[0063] The cells were divided into 9×10 4 1640 cells / well were added to a 6-well plate, and the volume of 1640 culture medium was supplemented to 3 mL. The cells were cultured overnight to ensure that the cells were fully attached to the wall. The culture medium was then replaced with fresh 1640 culture medium containing 1000 μg / mL BPP, and cultured for 0, 16, and 52 hours, respectively. Nine parallel samples were set up at each time point. After the culture was completed, 1.5 mL of the cell culture medium supernatant was collected in a 15 mL centrifuge tube and centrifuged at 1200 rpm for 5 minutes to remove impurities such as dead cell residues and suspended materials in the cell supernatant. Then 1 mL of supernatant was taken from each tube and placed in a new centrifuge tube for later use. At the same time, the cells at the bottom of each group of wells were rinsed 3 times with PBS buffer to remove residual materials, collected by trypsin digestion, and centrifuged at 800 rpm / min for 3 minutes. 6×10 cells were quantitatively collected from each group using a cell counter. 6 The cells were centrifuged twice with PBS buffer, the supernatant was removed, and 1 mL of PBS buffer was added to resuspend the cells and stored.

[0064] The intracellular mitochondrial complex activity, lactate content and ATP content of each sample were detected using mitochondrial respiratory chain complex I, II, III, IV and V activity detection kits, lactate (LA) content detection kits and ATP content detection kits. For specific operations, please refer to the instructions of the above kits.

[0065] Figure 7 The results of the test of metabolic changes in B16F10 cells after BPP treatment for different time periods in this embodiment are shown in Figures (a) to (e) showing changes in the contents of mitochondrial respiratory chain complexes I, II, III, IV and V with culture time, Figure (f) showing changes in ATP content with culture time, and Figures (g) and (h) showing changes in intracellular and extracellular lactic acid content with culture time. Figures 6-7 It can be seen that BPP is metabolized into phosphate after being taken up by B16F10 cells, which can regulate the metabolic process of B16F10 cells, while inhibiting their glycolysis and increasing their oxidative phosphorylation level. Specifically, BPP treatment significantly increases the activity of mitochondrial respiratory chain complexes I, II, IV and V in B16F10 cells, increases ATP content and reduces intracellular lactate content.

[0066] Example 7

[0067] In this example, the effect of BPP on the expression level of PD-L1 antibody on the surface of B16F10 cells was tested.

[0068] Experimental group (BPP group): B16F10 cells were cultured at 2×10 5 The cells were seeded at a density of 100 cells / well (2 mL 1640 medium / well) in a 6-well culture plate and cultured overnight to ensure that the cells were fully attached to the wall. The medium was then replaced with 2 mL 1640 medium containing 1000 μg / mL BPP and cultured for 48 hours.

[0069] Control group: B16F10 cells were cultured at 2×10 5 The cells were seeded at a density of 100 cells / well (2 mL 1640 medium / well) in a 6-well culture plate and cultured overnight to ensure that the cells fully adhered to the wall. The medium was then replaced with 2 mL 1640 medium and cultured for 48 hours.

[0070] The cells were digested with trypsin, washed twice with PBS buffer, centrifuged, and collected with a flow cytometer. The anti-cd274-PE antibody was diluted to 0.2 μg / mL with PBS buffer. The supernatant was removed from each tube of cells, 100 μL of the diluted anti-cd274-PE antibody was added, dispersed and incubated in the dark for 30 minutes. The cells were then washed with PBS buffer and fixed to 300 mL, and then analyzed by flow cytometry. Data were collected using a Gallios flow cytometer and analyzed using FlowJo (Threestar) software.

[0071] Figure 8 is the test result of the effect of BPP on the expression level of PD-L1 antibody on the surface of B16F10 cells in this example, Figure 8 Figure (a) is a flow cytometry graph of the fluorescence expression of PD-L1 in B16F10 cells. Figure 8 Figure (b) is the quantitative test result of PD-L1 antibody expression level. Figure 8 It can be seen that the expression of PD-L1 antibody on the surface of B16F10 cells in the experimental group was significantly reduced, indicating that BPP can significantly downregulate the expression level of PD-L1 antibody on the surface of B16F10 cells, which means that the ability of tumor cells to evade anti-tumor immunity is reduced, which is conducive to the activation of anti-tumor immunity.

[0072] Example 8

[0073] In this embodiment, thiolated hyaluronic acid (HA-SH) is prepared based on sodium hyaluronate (HA), and the steps are as follows:

[0074] HA with a molecular weight of 40 kDa was dissolved in deionized water to form a 5 mg / mL HA solution, and then N-hydroxysuccinimide (NHS) was added to fully dissolve it, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) was added to fully dissolve it, and the pH value of the resulting mixed solution was adjusted to 4.75, and the mixture was reacted at room temperature for 2 hours. Then, an aqueous solution of cysteine ​​hydrochloride (CSH·HCl) was added to control the molar ratio of HA, NHS, EDC·HCl and CSH·HCl to be 1:2:3:3, and the mixture was reacted at room temperature for 24 hours. Subsequently, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8000 to 14000 Da, and dialyzed in deionized water with a pH of 3.5 for 72 hours, during which the deionized water was replaced 5 times a day. After the dialysis is completed, the liquid in the dialysis bag is freeze-dried to obtain sponge-like HA-SH and stored at -20°C. The structure of HA-SH is shown in formula (I). Tests show that the grafting rate of thiol groups in HA-SH is about 16.04%.

[0075]

[0076] Example 9

[0077] In this embodiment, the injectable hydrogel containing BPP is prepared in the following steps:

[0078] (1) The BPP prepared in Example 2 was fully dispersed in ultrapure water to obtain a 12 mg / mL BPP dispersion.

[0079] (2) The HA-SH prepared in Example 8 was dissolved in ultrapure water to obtain a 20 mg / mL HA-SH solution.

[0080] (3) The BPP dispersion and the HA-SH solution were fully mixed at a volume ratio of 1:1, and the concentrations of BPP and HA-SH in the obtained mixed solution were 6 mg / mL and 10 mg / mL, respectively. Under ice bath conditions, the pH value of the obtained mixed solution was adjusted to 7.4 with a filtered and sterilized NaOH solution or / and HCl solution to obtain a gel precursor solution, and the gel precursor solution was allowed to stand until it was transformed into a gel state to obtain an injectable hydrogel containing BPP.

[0081] Example 10

[0082] In this example, an injectable hydrogel containing BMS-1 ​​and BPP was prepared by the following steps:

[0083] (1) The BPP prepared in Example 2 was fully dispersed in ultrapure water to obtain a 24 mg / mL BPP dispersion.

[0084] (2) Dissolve the PD-1 / PD-L1 small molecule inhibitor BMS-1 ​​in ultrapure water to obtain a 4 mg / mL BMS-1 ​​solution.

[0085] (3) The HA-SH prepared in Example 8 was dissolved in ultrapure water to obtain a 20 mg / mL HA-SH solution.

[0086] (4) The BPP dispersion and the BMS-1 ​​solution were mixed at a volume ratio of 1:1 to obtain a mixed solution A, and the mixed solution A was mixed with the HA-SH solution at a volume ratio of 1:1 to obtain a mixed solution B. In the mixed solution B, the concentrations of BMS-1, BPP and HA-SH were 1 mg / mL, 6 mg / mL and 10 mg / mL, respectively. Under ice bath conditions, the pH value of the mixed solution B was adjusted to 7.4 with a filtered and sterilized NaOH solution or / and HCl solution to obtain a gel precursor solution, which was allowed to stand until it turned into a gel state to obtain an injectable hydrogel containing BMS-1 ​​and BPP.

[0087] Comparative Example 1

[0088] In this comparative example, an injectable hydrogel containing the PD-1 / PD-L1 small molecule inhibitor BMS-1 ​​was prepared in the following steps:

[0089] (1) Dissolve the PD-1 / PD-L1 small molecule inhibitor BMS-1 ​​in ultrapure water to obtain a 2 mg / mL BMS-1 ​​solution.

[0090] (2) The HA-SH prepared in Example 8 was dissolved in ultrapure water to obtain a 20 mg / mL HA-SH solution.

[0091] (3) The BMS-1 ​​solution and the HA-SH solution were fully mixed in a volume ratio of 1:1, and the concentrations of BMS-1 ​​and HA-SH in the obtained mixed solution were 1 mg / mL and 10 mg / mL, respectively. Under ice bath conditions, the pH value of the obtained mixed solution was adjusted to 7.4 with a filtered and sterilized NaOH solution or / and HCl solution to obtain a gel precursor solution, which was allowed to stand until it turned into a gel state to obtain an injectable hydrogel containing BMS-1.

[0092] Comparative Example 2

[0093] In this comparative example, a blank injectable hydrogel was prepared in the following steps:

[0094] (1) The HA-SH prepared in Example 8 was dissolved in ultrapure water to obtain a 10 mg / mL HA-SH solution.

[0095] (2) Under ice bath conditions, the pH value of the HA-SH solution is adjusted to 7.4 using a filtered and sterilized NaOH solution or / and HCl solution to obtain a gel precursor solution, which is then allowed to stand until it is converted into a gel state, thereby obtaining a blank injectable hydrogel.

[0096] Embodiment 11

[0097] In this example, the inhibitory effect of BPNS in combination with the immunosuppressant BMS-1 ​​on melanoma was investigated.

[0098] (1) Construct a mouse skin melanoma cell B16F10 tumor-bearing mouse model. When the tumor volume of the mouse reaches 50 mm 3 At about 1:10 pm, B16F10 tumor-bearing mice were randomly divided into 4 groups (n=15), and the following 4 experimental groups were set up:

[0099] PBS group, injected with PBS buffer;

[0100] The PDH group was injected with the gel precursor solution used to prepare the injectable hydrogel containing BMS-1 ​​in Comparative Example 1;

[0101] BPPH group, injected with the gel precursor solution used to prepare the injectable hydrogel containing BPP in Example 7;

[0102] The PD-BPPH group was injected with the gel precursor solution used to prepare the injectable hydrogel containing BMS-1 ​​and BPP in Example 11.

[0103] The B16F10 tumor-bearing mice in the four experimental groups were administered by subcutaneous in situ injection, with a dosage of 50 μL per mouse, that is, 50 μL of PBS buffer or corresponding gel precursor solution was subcutaneously injected into each mouse, specifically, after the PBS buffer or gel precursor solution was prepared under ice bath conditions, it was quickly injected into the tumor. According to the drug release experiment of the drug-loaded hydrogel, 3 days was selected as the dosing interval. During the entire experiment, a total of 3 doses were administered, each with an interval of 3 days. On the 3rd day after the 3rd dose, the mice were dislocated and killed. The tumor volume of each mouse was recorded before each dose and on the 3rd day after the last dose, and the tumor volume change curve and mouse weight change curve of the mice in each experimental group were drawn, and the tumor inhibition rate was calculated.

[0104] (2) In order to further evaluate the prognosis of each hydrogel intervention treatment on mice, a B16F10 tumor-bearing mouse model was constructed according to the operation of step (1) and an experimental group was set up. Then the drug was administered according to the administration method of step (1). During the entire experiment, the drug was administered 4 times, with an interval of 3 days each time. The drug was stopped after the 4th administration, and the mice were raised normally. Starting from the first administration, the survival status of mice in each group was recorded until all the mice in the PBS group were sacrificed. The survival curve of mice in each experimental group was drawn with time as the horizontal axis and the survival rate of mice as the vertical axis.

[0105] Fig. 9Figure 1 is the tumor growth curve and tumor inhibition rate test results of each experimental group in this embodiment on B16F10 tumor-bearing mice, wherein Figure (a) is the average tumor volume change curve of mice in each experimental group, wherein Figure (b) is the average tumor volume change curve of different mice in each experimental group, wherein Figure (c) is the tumor inhibition rate calculation result of each experimental group. Fig.10 : is the survival curve of B16F10 tumor-bearing mice in each experimental group of this example. Figures 9-10 It can be seen that compared with BMS-1 ​​and BPP alone, the PD-BPPH group had a smaller tumor volume and a higher tumor inhibition rate after the combined administration of BPP and BMS-1; on the 24th day after the first administration, the survival rate of mice in the PBS group was 0, the survival rate of mice in the PDH group was 60%, the survival rate of mice in the BPPH group was 80%, and the survival rate of mice in the PD-BPPH group was 100%. This shows that BPP synergizes with the immunosuppressant BMS-1 ​​to produce a significantly better inhibitory effect on melanoma B16F10.

[0106] Example 12

[0107] In this example, the effect of BPP co-immunosuppressant BMS-1 ​​on the expression of PD-L1 antibody in tumor tissue was investigated.

[0108] In order to explore the expression of PD-L1 antibody on the surface of tumor cells in tumor tissue after each hydrogel intervention treatment on mice, a B16F10 tumor-bearing mouse model was constructed according to the operation in step (1) of Example 12, and an experimental group was set for drug treatment. On the third day after the third administration, the mice were killed and soaked in a 75% volume concentration alcohol solution for 10 minutes, kept at room temperature, and the tumor tissue of the mice was removed under a sterile environment. The fascia tissue on the tumor was carefully removed, and after repeated washing with PBS buffer added with double antibodies, a cell sieve (100 μm) was placed in a plate, and the tumor tissue was placed on the sieve and the tumor was cut as much as possible, and then repeatedly ground with a syringe piston until there was no obvious tissue residue in the cell sieve. The sieve was then repeatedly rinsed with whole blood and tissue diluent, and the tissue suspension obtained by grinding was collected, and then filtered with a 70 μm cell sieve to collect a single cell suspension. The single cell suspension was collected in a 15 mL centrifuge tube, and the collected single cell suspension was repeatedly washed twice in a 50 mL centrifuge tube with 100 mL of PBS buffer, and centrifuged repeatedly at 100 rpm to remove cell debris in the supernatant, and further tumor cells were collected. Qualitative and quantitative analysis was performed using a flow cytometer, and the collected tumor cells were dispersed and resuspended in PBS buffer, PE anti-mouse CD274 was added, and incubated at 4 ° C in the dark for 30 minutes, and then centrifuged at 800 rpm for 3 minutes with PBS buffer, and the incubated tumor cells were resuspended with 300 μL PBS buffer, and then tested on the machine to compare and analyze the expression results of PD-L1 antibodies in tumor cells in each experimental group.

[0109] Fig.11 The PD-L1 expression of tumor cells in tumor tissues of B16F10 tumor-bearing mice in different experimental groups of this example, wherein (a) is a flow cytometric graph of the fluorescence expression of PD-L1 in tumor cells, and (b) is a quantitative test result of the PD-L1 antibody expression level in tumor cells. Fig.11 It can be seen that compared with BMS-1 ​​alone and BPP alone, the combination of BPP and BMS-1 ​​can further downregulate the PD-L1 antibody expression level of tumor cells in the tumor tissue of B16F10 tumor-bearing mice.

[0110] Embodiment 13

[0111] In this example, the enhancement of the anti-tumor immune response of the immunosuppressant BMS-1 ​​by BPP was investigated.

[0112] In order to explore the cell quantity and differentiation of infiltrating T lymphocytes in tumor tissue after each hydrogel intervention treatment on mice, single cell suspension was collected according to the operation of Example 12. Tumor lymphocytes were separated using tumor infiltrating tissue lymphocyte separation reagent. The specific operation was as follows: add an appropriate amount of tumor infiltrating tissue lymphocyte separation solution to a 15mL centrifuge tube, add an equal amount of diluted single cell suspension, centrifuge at room temperature, carefully collect lymphocytes in the middle layer of the separation solution, and then repeatedly centrifuge and wash with sterile PBS buffer and resuspend the lymphocytes for use. In the 5 experimental groups (n=3), about 6×10 5 Cells were centrifuged and washed twice with sterile PBS buffer, and each group of cells was collected in a flow cytometer. Qualitative and quantitative analysis was performed using a flow cytometer. Fluorescently labeled antibodies APC anti-mouse CD3, PE anti-mouse CD4, and FITC anti-mouse CD8a were added in sequence according to the recommended dosage in the instructions. Sterile PBS buffer was added to each tube of cells at 4°C, the dye solution was diluted to 100 μL and gently shaken to mix, incubated in the dark for 30 minutes, centrifuged with PBS buffer and collected the cells, and then resuspended with 300 μL PBS buffer for detection.

[0113] Fig.12 The CD8 + The ratio of T cells and regulatory T cells, where (a) is the CD8 + Flow cytometry of T cells, (b) shows the CD8 + Figure (c) is a statistical diagram of the results of T cell flow cytometry, Figure (d) is a statistical diagram of the results of regulatory T (Treg) cell flow cytometry in each experimental group. Fig.12 It can be seen that compared with BMS-1 ​​and BPP alone, the combined administration of BPP and BMS-1 ​​can further increase the expression of CD8 + T cell infiltration and reduced the proportion of regulatory T (Treg) cells. + T cells are killer T cells that can inhibit tumor growth, while T (Treg) cells are regulatory T cells that promote tumor growth. + The changes in T cells and T (Treg) cells indicated that T (Treg) cells activated the anti-tumor immune effect in tumor tissues.

[0114] Embodiment 14

[0115] In this example, the inhibitory effect of BPP synergistically with the immunosuppressant BMS-1 ​​on breast cancer was investigated.

[0116] (1) Construct a mouse model bearing 4T1 breast cancer cells. When the tumor volume of the mouse reaches 100 mm 3 At about 1:10 pm, 4T1 tumor-bearing mice were randomly divided into 4 groups (n=15), and the following 4 experimental groups were set up:

[0117] HASH, the gel precursor solution used to prepare the blank injectable hydrogel in injection comparative example 2;

[0118] The PDH group was injected with the gel precursor solution used to prepare the injectable hydrogel containing BMS-1 ​​in Comparative Example 1;

[0119] BPPH group, injected with the gel precursor solution used to prepare the injectable hydrogel containing BPP in Example 7;

[0120] The PD-BPPH group was injected with the gel precursor solution used to prepare the injectable hydrogel containing BMS-1 ​​and BPP in Example 11.

[0121] The 4T1 tumor-bearing mice in the four experimental groups were administered subcutaneously in situ, with a dosage of 50 μL per mouse, that is, 50 μL of gel precursor solution was injected subcutaneously per mouse. Specifically, after the gel precursor solution was prepared under ice bath conditions, the gel precursor solution was quickly injected next to the tumor. According to the drug release experiment of the drug-loaded hydrogel, 3 days was selected as the dosing interval. During the entire experiment, a total of 5 doses were administered, each with an interval of 3 days. On the 3rd day after the 5th dose, the mice were dislocated and killed. The tumor volume of each mouse was recorded before each dose and on the 3rd day after the last dose, and the tumor volume change curve and mouse weight change curve of the mice in each experimental group were drawn, and the tumor inhibition rate was calculated.

[0122] (2) In order to further evaluate the prognosis of each hydrogel intervention treatment on mice, a 4T1 tumor-bearing mouse model was constructed according to the operation of step (1) and an experimental group was set up. Then the drug was administered according to the administration method of step (1). A total of 5 administrations were performed during the entire experiment, with an interval of 3 days between each administration. The administration was stopped after the 5th administration, and the mice were raised normally. Starting from the first administration, the survival status of mice in each group was recorded until all the mice in the HASH group were sacrificed. The survival curve of mice in each experimental group was drawn with time as the horizontal axis and the survival rate of mice as the vertical axis.

[0123] Fig.13The figure is the test results of tumor growth curve and tumor inhibition rate of 4T1 tumor-bearing mice in each experimental group of this embodiment, wherein (a) is the average tumor volume change curve of mice in each experimental group, wherein (b) is the calculation result of tumor inhibition rate of each experimental group, wherein (c) is the survival curve of 4T1 tumor-bearing mice in each experimental group of this embodiment. Fig.13 It can be seen that compared with BMS-1 ​​and BPP alone, the PD-BPPH group had a smaller tumor volume and a higher tumor inhibition rate after the combination of BPP and BMS-1; on the 34th day after the first administration, the survival rate of mice in the HASH group was 0, the survival rate of mice in the PDH group was 20%, the survival rate of mice in the BPPH group was 70%, and the survival rate of mice in the PD-BPPH group was 100%. This shows that BPP synergizes with the immunosuppressant BMS-1 ​​to produce a significantly better inhibitory effect on mouse breast cancer 4T1.

Claims

1. Application of hydrophilic black phosphorus nanoparticles in the preparation of anti-tumor immune adjuvants. Hydrophilic black phosphorus nanoparticles can increase the oxidative phosphorylation level of tumor cells and inhibit the expression of PD-L1 antibodies in tumor cells, thereby enhancing the sensitivity of tumor cells to tumor immunotherapy drugs, thereby achieving synergistic inhibition of tumors by hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs. The hydrophilic black phosphorus nanoparticles are obtained by modifying black phosphorus nanosheets with hydrophilic polymer materials.

2. The use of the hydrophilic black phosphorus nanoparticles according to claim 1 in the preparation of an anti-tumor immune adjuvant, characterized in that: The tumor immunotherapy drug is a PD-1 / PD-L1 immune checkpoint inhibitor.

3. The use of the hydrophilic black phosphorus nanoparticles in the preparation of an anti-tumor immune adjuvant according to claim 2, characterized in that: The PD-1 / PD-L1 immune checkpoint inhibitor includes any one of BMS-1, BMS-202, BMS-1166, nivolumab, pembrolizumab, atezolizumab, ipilimumab and blinatumomab.

4. The use of the hydrophilic black phosphorus nanoparticles according to any one of claims 1 to 3 in the preparation of an anti-tumor immune adjuvant, characterized in that: The hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs are combined in a mass ratio of (5-10):1 to act on tumor cells.

5. The use of the hydrophilic black phosphorus nanoparticles according to claim 4 in the preparation of an anti-tumor immune adjuvant, characterized in that: The hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs are loaded on a pharmaceutically acceptable carrier to act on tumor cells.

6. The use of the hydrophilic black phosphorus nanoparticles according to claim 4 in the preparation of an anti-tumor immune adjuvant, characterized in that: Hydrophilic black phosphorus nanoparticles and tumor immunotherapy drugs are prepared into an injection to act on tumor cells, and the concentration of the hydrophilic black phosphorus nanoparticles in the injection is at least 5 mg / mL.

7. Use of the hydrophilic black phosphorus nanoparticles according to any one of claims 1 to 3 in the preparation of an anti-tumor immune adjuvant, characterized in that: The tumor cells include melanoma cells or breast cancer cells.

8. Use of the hydrophilic black phosphorus nanoparticles according to any one of claims 1 to 3 in the preparation of an anti-tumor immune adjuvant, characterized in that: The hydrophilic black phosphorus nanoparticles are obtained by bonding a hydrophilic polymer material to the surface of a black phosphorus nanosheet or by combining the hydrophilic polymer material through electrostatic interaction.

9. The use of the hydrophilic black phosphorus nanoparticles according to claim 8 in preparing an anti-tumor immune adjuvant, characterized in that: The hydrophilic polymer material includes any one of amino polyethylene glycol, polylysine, polyethyleneimine and polyamino acid.

10. Use of the hydrophilic black phosphorus nanoparticles according to any one of claims 1 to 3 in the preparation of an anti-tumor immune adjuvant, characterized in that: The size of the hydrophilic black phosphorus nanoparticles is between 10 and 300 nm.

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