Cordyceps polysaccharide composition as well as preparation method and application thereof

Abstract

The invention provides a cordyceps polysaccharide composition as well as a preparation method and application thereof, and belongs to the field of medicines. The cordyceps polysaccharide composition comprises cordyceps polysaccharide, black phosphorus nanosheets, a wrapping material and a functional ligand. The cordyceps polysaccharide composition has a good anti-tumor effect, is low in toxicity and has an excellent effect.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and in particular relates to a cordyceps polysaccharide composition and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy is a treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. Tumor associated macrophages (TAMs) are important immune cells in the tumor microenvironment. They mediate tumor progression by regulating the tumor microenvironment. In vivo, TAMs are mainly divided into two subtypes: tumor-suppressing macrophages (M1) and tumor-promoting macrophages (M2). Generally speaking, M1 macrophages can produce molecules that have cytotoxic effects on tumor cells, such as TNF-α, IL-12, and nitric oxide (NO), and also express immunostimulatory cytokines for the proliferation of T cells and NK cells and anti-tumor effects. In the tumor microenvironment, M2 macrophages release tumor-promoting cytokines (such as IL-4, IL-10, etc.) to promote tumor progression and metastasis. Therefore, inhibiting the M2 phenotype of TAMs cells and promoting their transformation to M1 macrophages is an effective tumor immunotherapy strategy. At the same time, enhancing the ability of phagocytes and promoting the maturation of T cells and DCs cells are also methods of tumor immunotherapy. Studies have shown that cordyceps polysaccharides can achieve anti-tumor effects by stimulating the body's immune function. The results of Sheng et al. showed that cordyceps polysaccharides can promote the expression of cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ in mice and increase protein levels to improve anti-tumor effects. Chen et al. confirmed that the acidic polysaccharides in Cordyceps sinensis activated the NF-κB signaling pathway to promote the transformation of mouse macrophages from M2 phenotype to M1 phenotype, thereby improving the efficiency of tumor treatment. Lee et al. confirmed that the polysaccharides isolated from Cordyceps sinensis enhanced the phagocytic ability of mouse phagocytes by activating NF-κB and three MAPKs signaling pathways. Cordyceps sinensis polysaccharides also inhibit STAT3 phosphorylation and promote the maturation and activation of dendritic cells (DC), thereby exerting anti-tumor effects.

[0003] In recent years, the discovery of black phosphorus (BP), a 2D nanomaterial with special physical and chemical properties, has created opportunities for designing new Cordyceps sinensis polysaccharide nanodrug delivery systems to solve the problems existing in the use of single polysaccharides. BP-based nanodrugs have attracted widespread attention for their sustained release, targeting, high bioavailability, reduced toxicity and other excellent properties. Compared with other two-dimensional materials such as graphene, the folded lattice structure of BP has a higher drug loading capacity. At the same time, BP is a highly efficient photosensitizer with photodynamic therapy (PDT) and photothermal therapy (PTT) effects on tumors. In addition, BP nanosheets can be rapidly degraded into phosphates and phosphonates that are non-toxic to the human body in aqueous media.

[0004] So far, BP nano drug delivery system is mainly combined with small molecule drugs to conduct anti-tumor experimental research, and there is almost no research on loading effective components of Chinese medicine macromolecules. Therefore, it will be a new research direction to form a new nano drug delivery system for BP loading Chinese medicine Cordyceps polysaccharide macromolecules to solve the clinical use of Cordyceps polysaccharide and carry out efficient anti-tumor. Summary of the invention

[0005] The present invention aims to provide a composition and a preparation method and application thereof. The composition has a good anti-tumor effect, and cordyceps polysaccharide and black phosphorus have a synergistic technical effect.

[0006] In a first aspect, the present invention provides a composition.

[0007] In some embodiments, the composition includes Cordyceps polysaccharide, black phosphorus nanosheets, a packaging material, and a functional ligand.

[0008] In some embodiments, the encapsulating material comprises polydopamine or a salt thereof.

[0009] In some embodiments, the functional ligand comprises aminopolyethylene glycol folate.

[0010] In some embodiments, the weight average molecular weight (Mw) of the aminopolyethylene glycol folic acid is 500-4000. In some embodiments, the weight average molecular weight (Mw) of the aminopolyethylene glycol folic acid is 1000-3000. In some embodiments, the weight average molecular weight (Mw) of the aminopolyethylene glycol folic acid is 1500-2500. In some embodiments, the weight average molecular weight (Mw) of the aminopolyethylene glycol folic acid is 500, 1000, 1500, 2000, 2500 or 3000. In some embodiments, the weight average molecular weight (Mw) of the aminopolyethylene glycol folic acid is 2000.

[0011] In some embodiments, the black phosphorus nanosheets in the composition are loaded with Cordyceps polysaccharide.

[0012] In some embodiments, the black phosphorus nanosheets in the composition are loaded with the Cordyceps polysaccharide and then wrapped by the wrapping material to obtain the wrapped composition A.

[0013] In some embodiments, the composition A is linked to the functional ligand.

[0014] In some embodiments, based on the total mass of the composition, the content of the cordyceps polysaccharide is 5.00wt%-40.00wt%. In some embodiments, based on the total mass of the composition, the content of the cordyceps polysaccharide is 10.00wt%-30.00wt%. In some embodiments, based on the total mass of the composition, the content of the cordyceps polysaccharide is 15.00wt%-25.00wt%. In some embodiments, based on the total mass of the composition, the content of the cordyceps polysaccharide is 20.00wt%-22.00wt%. In some embodiments, based on the total mass of the composition, the content of the cordyceps polysaccharide is 21.64wt%. In some embodiments, based on the total mass of the composition, the content of the cordyceps polysaccharide is 5.00wt%, 10wt%, 15wt%, 20wt%, 25wt%, 30wt%, 35wt% or 40.00wt%.

[0015] In some embodiments, based on the total mass of the composition, the content of black phosphorus in the composition is 50.00wt%-85.00wt%. In some embodiments, based on the total mass of the composition, the content of black phosphorus in the composition is 60.00wt%-80.00wt%. In some embodiments, based on the total mass of the composition, the content of black phosphorus in the composition is 50.00wt%, 55.00wt%, 60.00wt%, 65.00wt%, 70.00wt%, 75.00wt%, 80.00wt% or 85.00wt%.

[0016] In some embodiments, based on the total mass of the composition, the content of the encapsulating material is 5.00 wt%-30.00 wt%. In some embodiments, based on the total mass of the composition, the content of the encapsulating material is 5.00 wt%-10.00 wt%. In some embodiments, based on the total mass of the composition, the content of the encapsulating material is 5.00wt%, 6.00wt%, 7.00wt%, 8.00wt%, 9.00wt%, 10.00wt%, 11.00wt%, 12.00wt%, 13.00wt%, 14.00wt%, 15.00wt%, 16.00wt%, 17.00wt%, 18.00wt%, 19.00wt%, 20.00wt%, 21.00wt%, 22.00wt%, 23.00wt%, 24.00wt%, 25.00wt%, 26.00wt%, 27.00wt%, 28.00wt%, 29.00wt% or 30.00wt%.

[0017] In some embodiments, based on the total mass of the composition, the content of the functional ligand is 5.00 wt%-30.00 wt%. In some embodiments, based on the total mass of the composition, the content of the functional ligand is 5.00 wt%-10.00 wt%. In some embodiments, based on the total mass of the composition, the content of the functional ligand is 5.00wt%, 6.00wt%, 7.00wt%, 8.00wt%, 9.00wt%, 10.00wt%, 11.00wt%, 12.00wt%, 13.00wt%, 14.00wt%, 15.00wt%, 16.00wt%, 17.00wt%, 18.00wt%, 19.00wt%, 20.00wt%, 21.00wt%, 22.00wt%, 23.00wt%, 24.00wt%, 25.00wt%, 26.00wt%, 27.00wt%, 28.00wt%, 29.00wt% or 30.00wt%.

[0018] In some embodiments, the method for preparing the Cordyceps polysaccharide comprises:

[0019] (1) Supercritical treatment: cutting or crushing the Cordyceps sinensis, and then subjecting the Cordyceps sinensis to supercritical treatment to obtain the Cordyceps sinensis after supercritical treatment;

[0020] (2) Extraction and concentration: extracting the supercritically treated Cordyceps sinensis with a solvent to obtain an extract, and concentrating the extract to obtain a concentrate;

[0021] (3) alcohol precipitation: taking the concentrated solution and subjecting it to alcohol precipitation, centrifuging to obtain a precipitate, and drying the precipitate to obtain crude Cordyceps polysaccharide;

[0022] (4) protein removal: removing protein from crude Cordyceps polysaccharide by Sevage method to obtain a protein-free Cordyceps polysaccharide solution;

[0023] (5) Dialysis: The deproteinized Cordyceps polysaccharide solution is then dialyzed to obtain a retained solution, which is the dialyzed Cordyceps polysaccharide solution;

[0024] (6) Filtration, alcohol precipitation and freeze-drying: The cordyceps polysaccharide solution after the dialysis is filtered and precipitated with alcohol, centrifuged to obtain a polysaccharide precipitate, and the polysaccharide precipitate is dried or the polysaccharide precipitate is redissolved and then freeze-dried to obtain the cordyceps polysaccharide.

[0025] In some embodiments, the supercritical treatment includes performing CO2 supercritical treatment at a temperature of 50°C-150°C and a pressure of 10MPa-30MPa.

[0026] In some embodiments, the temperature of the supercritical treatment is 50° C. to 150° C. In some embodiments, the temperature of the supercritical treatment is 80° C. to 150° C. In some embodiments, the temperature of the supercritical treatment is 50° C., 60° C., 70° C., 80° C., 90° C., 95° C., 100° C., 105° C., 110° C., 120° C., 130° C., 140° C., or 150° C.

[0027] In some embodiments, the supercritical treatment pressure is 10 MPa-30 MPa. In some embodiments, the supercritical treatment pressure is 10 MPa, 15 MPa, 16 MPa, 17 MPa, 18 MPa, 19 MPa, 20 MPa, 21 MPa, 22 MPa, 23 MPa, 24 MPa, 25 MPa or 30 MPa.

[0028] In some embodiments, the extraction includes mixing the supercritically treated Cordyceps sinensis with a solvent, heating for extraction, filtering, taking the filtrate, and continuing to extract the filter cake with the solvent for 0-4 times (i.e., 0 times, 1 time, 2 times, 3 times or 4 times), combining the filtrate, and obtaining an extract.

[0029] In some embodiments, the concentrating comprises concentrating by reduced pressure concentration.

[0030] In some embodiments, the step (3) of taking the concentrated solution for alcohol precipitation includes mixing the concentrated solution with an ethanol solution of 4 times the volume of the concentrated solution, and placing it at 4°C-10°C (for example: 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C) for 12 hours to 24 hours (for example, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours). In some embodiments, the step (3) of taking the concentrated solution for alcohol precipitation includes mixing the concentrated solution with an ethanol solution of 4 times the volume of the concentrated solution, and placing it at 4°C-8°C for 20 hours to 24 hours.

[0031] In some embodiments, the ethanol solution in step (3) is composed of ethanol and water in a volume ratio of 95:5-100:0. In some embodiments, the ethanol solution in step (3) is composed of ethanol and water in a volume ratio of 95:5, 96:4, 97:3, 98:2, 99:1 or 100:0.

[0032] In some embodiments, the Sevage method includes re-dissolving the crude Cordyceps polysaccharide with water to obtain a re-dissolved solution, mixing the re-dissolved solution with a sevage reagent, centrifuging to remove insoluble matter, obtaining a supernatant, and replacing the re-dissolved solution with the supernatant to repeat the above "mixing with a sevage reagent, centrifuging to remove insoluble matter" operation until no insoluble matter is produced.

[0033] In some embodiments, the re-dissolving the Cordyceps crude polysaccharide with water comprises dissolving the Cordyceps crude polysaccharide in water at a mass ratio of 1:8-1:20 to the Cordyceps crude polysaccharide. In some embodiments, the re-dissolving the Cordyceps crude polysaccharide with water comprises dissolving the Cordyceps crude polysaccharide in water at a mass ratio of 1:8-1:15 to the Cordyceps crude polysaccharide. In some embodiments, the re-dissolving the Cordyceps crude polysaccharide with water comprises dissolving the Cordyceps crude polysaccharide in water at a mass ratio of 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20 to the Cordyceps crude polysaccharide.

[0034] In some embodiments, the volume ratio of the reconstituted solution or supernatant to the sevage reagent is 5: 1-4: 1. In some embodiments, the volume ratio of the reconstituted solution or supernatant to the sevage reagent is 4: 1.

[0035] In some embodiments, the sevage reagent is a mixture of chloroform and n-butanol in a volume ratio of 5:1-4:1. In some embodiments, the sevage reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1.

[0036] In some embodiments, the molecular weight cutoff of the dialysis is 2.0 kDa-5.0 kDa. In some embodiments, the molecular weight cutoff of the dialysis is 2.0 kDa, 3.0 kDa, 3.5 kDa, 4.0 kDa, 4.5 kDa or 5.0 kDa. In some embodiments, the molecular weight cutoff of the dialysis is 3.5 kDa.

[0037] In some embodiments, the dialysate of the dialysis is water.

[0038] In some embodiments, the dialysate is replaced every 1 h-3 h. In some embodiments, the dialysate is replaced every 1 h, 1.5 h, 2 h, 2.5 h or 3 h.

[0039] In some embodiments, the dialysis time is 24 hours to 52 hours. In some embodiments, the dialysis time is 24 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours or 52 hours.

[0040] In some embodiments, the dialyzed Cordyceps polysaccharide solution in step (6) is then filtered and precipitated with alcohol, including filtering the dialyzed Cordyceps polysaccharide solution to obtain a filtrate, adding 4 times the volume of the filtrate to the filtrate and mixing, and placing at 4°C-10°C (for example: 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C) for 12 hours to 24 hours (for example, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours).

[0041] In some embodiments, the ethanol solution in step (3) is composed of ethanol and water in a volume ratio of 95:5-100:0. In some embodiments, the ethanol solution in step (3) is composed of ethanol and water in a volume ratio of 95:5, 96:4, 97:3, 98:2, 99:1 or 100:0.

[0042] In a second aspect, the present invention provides a method for preparing the composition of the first aspect.

[0043] In some embodiments, a method for preparing the composition of the first aspect comprises: loading Cordyceps polysaccharide on black phosphorus nanosheets, then wrapping them with a wrapping material, and finally connecting the wrapping material with a functional ligand, and drying to obtain the composition.

[0044] In some embodiments, a method for preparing the composition of the first aspect comprises:

[0045] (1) Preparation of cordyceps polysaccharide-black phosphorus intermediate: Under the condition of avoiding light and protecting with nitrogen or inert gas, mixing the aqueous solution containing cordyceps polysaccharide with black phosphorus nanosheets, stirring, centrifuging to obtain a precipitate 1, and washing the precipitate 1 with water to obtain a cordyceps polysaccharide-black phosphorus intermediate;

[0046] (2) Preparation of cordyceps polysaccharide-black phosphorus-polydopamine intermediate: under the conditions of avoiding light and protecting with nitrogen or inert gas, mixing the cordyceps polysaccharide-black phosphorus intermediate with a dopamine hydrochloride solution, stirring, centrifuging to obtain a precipitate 2, washing the precipitate 2 with water, and drying the washed precipitate 2 to obtain a cordyceps polysaccharide-black phosphorus-polydopamine intermediate;

[0047] (3) Preparation of Cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition: The functional ligand is combined with the surface of the Cordyceps polysaccharide-black phosphorus-polydopamine intermediate product by Michael addition reaction, and the Cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition is obtained after post-treatment, that is, the composition.

[0048] In some embodiments, the Michael addition reaction comprises: mixing the cordyceps polysaccharide-black phosphorus-polydopamine intermediate with an amino polyethylene glycol folic acid solution, stirring, and reacting under conditions of light protection and nitrogen or inert gas protection.

[0049] In some embodiments, the post-treatment includes: centrifugation to obtain a precipitate 3, washing the precipitate 3 with water, and drying the washed precipitate 3.

[0050] In some embodiments, the concentration of cordyceps polysaccharide in the aqueous solution containing cordyceps polysaccharide in step (1) is 10 mg / mL-50 mg / mL. In some embodiments, the concentration of cordyceps polysaccharide in the aqueous solution containing cordyceps polysaccharide in step (1) is 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL or 50 mg / mL.

[0051] In some embodiments, the mass ratio of cordyceps polysaccharide to black phosphorus nanosheets in the aqueous solution containing cordyceps polysaccharide in the step (1) is 1:1-10:1. In some embodiments, the mass ratio of cordyceps polysaccharide to black phosphorus nanosheets in the aqueous solution containing cordyceps polysaccharide in the step (1) is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. In some embodiments, the mass ratio of cordyceps polysaccharide to black phosphorus nanosheets in the aqueous solution containing cordyceps polysaccharide in the step (1) is 1:1-3:1. In some embodiments, the mass ratio of cordyceps polysaccharide to black phosphorus nanosheets in the aqueous solution containing cordyceps polysaccharide in the step (1) is 2:1.

[0052] In some embodiments, the stirring in step (1) is stirring for 12 hours to 36 hours. In some embodiments, the stirring in step (1) is stirring for 12 hours, 15 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 35 hours or 36 hours.

[0053] In some embodiments, the dopamine hydrochloride solution in step (2) is a solution of dopamine hydrochloride dissolved in Tris buffer.

[0054] In some embodiments, the pH of the dopamine hydrochloride solution in step (2) is 8.0-9.0. In some embodiments, the pH of the dopamine hydrochloride solution in step (2) is 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0055] In some embodiments, the concentration of dopamine hydrochloride in the dopamine hydrochloride solution in step (2) is 8mM-12mM or 10mM. In some embodiments, the concentration of dopamine hydrochloride in the dopamine hydrochloride solution in step (2) is 8mM, 9mM, 10mM, 11mM or 12mM.

[0056] In some embodiments, the mass ratio of the cordyceps polysaccharide-black phosphorus intermediate product to the dopamine hydrochloride in the dopamine hydrochloride solution in the step (2) is 1:2-2:1. In some embodiments, the mass ratio of the cordyceps polysaccharide-black phosphorus intermediate product to the dopamine hydrochloride in the dopamine hydrochloride solution in the step (2) is 1:1.

[0057] In some embodiments, the stirring in step (2) is stirring for 8 hours to 24 hours. In some embodiments, the stirring in step (2) is stirring for 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours or 24 hours.

[0058] In some embodiments, the aminopolyethylene glycol folic acid solution in step (3) is a solution of aminopolyethylene glycol folic acid dissolved in Tris buffer.

[0059] In some embodiments, the pH of the amino polyethylene glycol folic acid solution in step (3) is 8.0-9.0. In some embodiments, the pH of the amino polyethylene glycol folic acid solution in step (3) is 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0060] In some embodiments, the concentration of aminopolyethylene glycol folic acid in the aminopolyethylene glycol folic acid solution in step (3) is 8mM-12mM. In some embodiments, the concentration of aminopolyethylene glycol folic acid in the aminopolyethylene glycol folic acid solution in step (3) is 8mM, 9mM, 10mM, 11mM or 12mM.

[0061] In some embodiments, the mass ratio of the cordyceps polysaccharide-black phosphorus-polydopamine intermediate product in step (3) to the amino-polyethylene glycol folic acid in the amino-polyethylene glycol folic acid solution is 1:2-2:1 or 1:1.

[0062] In some embodiments, the stirring in step (3) is for 6 hours to 12 hours.

[0063] In some embodiments, the drying in step (2) is freeze-drying.

[0064] In some embodiments, the drying in step (3) is freeze-drying.

[0065] In a third aspect, the present invention provides an application of the aforementioned composition or the composition obtained by the aforementioned preparation method.

[0066] A use of the composition of the first aspect or the composition obtained by the preparation method of the second aspect in preparing a product for treating tumors.

[0067] In some embodiments, the tumor comprises a malignant solid tumor such as breast cancer or thyroid cancer.

[0068] Beneficial Effects

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The composition (cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition) provided by the present invention has good anti-tumor effect and low toxicity, and has good application prospects in phototherapy and immunotherapy.

[0071] Terminology

[0072] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0073] The terms "cordyceps" and "cordyceps" in the present invention are interchangeable.

[0074] The term "% vol" means volume percentage.

[0075] The term "wt%" means percent by mass.

[0076] The term "M" means molar concentration, mol / L.

[0077] The term "retained solution" refers to the sample solution that is retained in the dialysis bag or dialysis membrane and is not dialyzed during the dialysis process.

[0078] The term "permeate" refers to the solution outside the dialysis bag (membrane) during dialysis.

[0079] The term “black phosphorus nanosheet” refers to a two-dimensional nanomaterial consisting of a single or several layers of sheet-like black phosphorus.

[0080] In the following, whether or not the word "about" or "approximately" is used, it means within 1%, within 2%, within 5%, within 7%, within 8%, within 10%, within 15%, within 20%, etc. of a given value or range. Alternatively, for those of ordinary skill in the art, the term "about" or "approximately" means within the acceptable standard error range of the mean. Whenever a number with a value of N is disclosed, any number with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15% or N+ / -20% will be explicitly disclosed, where "+ / -" means plus or minus. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1A This is a perspective electron microscope image of BP (black phosphorus nanosheets) in Example 2; wherein the scale is 100 nm.

[0082] Figure 1B This is a perspective electron micrograph of BP / CSP@PDA-PEG-FA in Example 2; wherein the scale bar is 100 nm.

[0083] Figure 1C This is a statistical diagram of the zeta potential of BP (black phosphorus nanosheets) and BP / CSP@PDA-PEG-FA in Example 2.

[0084] Figure 1D FT-IR spectra of BP (black phosphorus nanosheets), BP / CSP and BP / CSP@PDA-PEG-FA in Example 2.

[0085] Figure 1E This is the XPS result diagram of BP (black phosphorus nanosheets), CSP, BP / CSP, BP / CSP@PDA and BP / CSP@PDA-PEG-FA in Example 2.

[0086] Figure 2A This is a perspective electron microscope image of BP / CSP in Example 2.

[0087] Figure 2B This is the size detection result diagram of BP / CSP@PDA in Example 2.

[0088] Figure 2C This is a statistical diagram of the zeta potential of BP / CSP and BP / CSP@PDA in Example 2.

[0089] Figure 3 This is a graph showing the H&E staining results of tumor tissues in different groups after 20 days, starting from the first administration as day 0 in the in vivo anti-cancer evaluation test in Example 4.

[0090] Figure 4 This is the H&E staining result of each major tissue and organ in different groups after 20 days, calculated from the first administration as day 0 in the in vivo anti-cancer evaluation test in Example 4 (Heart: heart; Liver: liver; Spleen: spleen; Lung: lung; Kidney: kidney). DETAILED DESCRIPTION

[0091] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further describe the present invention in detail.

[0092] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0093] Black phosphorus nanosheets were purchased from Nanjing Muke Nanotechnology Co., Ltd.

[0094] Mouse IL-4 (mouse interleukin 4) was purchased from Gibco, and Mice CD86 / BV421 (mouse CD86 / BV421 antibody, i.e., mouse CD86 antibody conjugated with BV421 fluorescent dye) and CD206 / PE (CD206 / PE antibody, i.e., CD206 antibody conjugated with PE fluorescent dye) were from BD Horizon.

[0095] M1 macrophages: M1 macrophages are macrophages that can produce pro-inflammatory cytokines and are also called classical activated macrophages (M1).

[0096] M2 macrophages: M2 macrophages, also known as alternatively activated macrophages (M2), are mainly activated by the inflammatory factor IL-4 and mainly inhibit M1 macrophages by secreting anti-inflammatory cytokines such as IL10.

[0097] Size and zeta potential detection method: The size and zeta potential were measured by Malvern Mastersizer 2000. Before the measurement, the sample was appropriately diluted with deionized water and ultrasonically treated. The data were averaged for 3 times.

[0098] Transmission electron microscopy detection method: Use transmission electron microscopy to observe the size, shape and surface morphology of the product.

[0099] FT-IR (Fourier transform infrared spectroscopy) detection method: Fourier transform infrared spectroscopy is used to detect FT-IR.

[0100] XPS (X-ray photoelectron spectroscopy) detection method: used to analyze the surface chemistry of the sample to be tested, the analyzer uses a monochromatic Alkα X-ray source (1486.6ev photons, 150w).

[0101] Example 1: Preparation of Cordyceps polysaccharide

[0102] 1. Supercritical treatment of Cordyceps sinensis samples: Cut the Cordyceps sinensis into rice-sized pieces, and treat the Cordyceps sinensis with CO2 supercritical fluid for 2 hours at a temperature of 100°C and a pressure of 20MPa to obtain the supercritically treated Cordyceps sinensis;

[0103] 2. Extraction and concentration: Take the Cordyceps sinensis after the supercritical treatment, add water at a ratio of 30 mL of water per 1 g of Cordyceps sinensis, decoct the medicine at a temperature of 100° C. for 2.0 h, filter to obtain a filtrate and a filter cake, and extract the filter cake with water according to the above extraction operation for 2 times, combine all the filtrates, and concentrate under reduced pressure to obtain a concentrate;

[0104] 3. Alcohol precipitation: add 4 times the volume of 95% vol ethanol aqueous solution to the concentrated solution obtained above for alcohol precipitation, store at 4°C for 24 hours, centrifuge at 5000 rpm for 5 minutes, recover the precipitate, and vacuum dry for 24 hours to obtain Cordyceps crude polysaccharide;

[0105] 4. Sevage method to remove protein:

[0106] 4.1 Redissolution: Heat and re-dissolve the crude Cordyceps polysaccharide in water, with the mass ratio of the crude Cordyceps polysaccharide to water being 1:10, to obtain a re-dissolved solution;

[0107] 4.2 Protein removal: add 1 / 4 volume of chloroform and n-butanol mixed reagent (the volume ratio of chloroform and n-butanol is 5:1) to the above-obtained reconstituted solution, shake for 5 minutes, centrifuge to obtain a supernatant and a protein precipitate, remove the protein precipitate, and repeat the protein removal operation of the supernatant according to the above protein removal operation (i.e., replace the reconstituted solution with the supernatant and then perform the protein removal operation) until substantially no protein precipitate appears, thereby obtaining a protein-free Cordyceps polysaccharide solution;

[0108] 5. Dialysis: The cordyceps polysaccharide solution after protein removal is placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed with water, with the water changed every 2 hours, and the dialyzation is performed for 48 hours to obtain a retained solution, which is the cordyceps polysaccharide solution after dialysis;

[0109] 6. Filtration and alcohol precipitation: The dialyzed Cordyceps polysaccharide solution was filtered with a 0.45 μm aqueous phase filter membrane, and the filtrate was added with 4 times the volume of 95% vol ethanol aqueous solution for alcohol precipitation. After being placed at 4°C for 12 hours, it was centrifuged to obtain polysaccharide precipitation;

[0110] 7. Freeze-drying: The polysaccharide precipitate obtained in step 6 is heated and re-dissolved with water, and freeze-dried for 8 hours to obtain Cordyceps polysaccharide (alias: CSP).

[0111] Example 2: Preparation of the composition of the present invention

[0112] Note: The following preparation operations of cordyceps polysaccharide-black phosphorus intermediate, cordyceps polysaccharide-black phosphorus-polydopamine intermediate and cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition are all carried out at least under light-proof conditions.

[0113] 1 Preparation and characterization of Cordyceps polysaccharide-black phosphorus intermediate

[0114] (1) Preparation: Preparation of cordyceps polysaccharide-black phosphorus intermediate (hereinafter referred to as BP / CSP): In the dark and under the protection of nitrogen or inert gas, the cordyceps polysaccharide (CSP) obtained in Example 1 was dissolved in water to a concentration of 25 mg / mL, and then mixed with black phosphorus nanosheets (BP), the mass ratio of cordyceps polysaccharide (CSP) to black phosphorus nanosheets (BP) being 2:1, stirred for 24 hours in the dark, and centrifuged at 14000 rpm for 30 min to obtain a precipitate 1, and the precipitate 1 was washed with water to obtain a cordyceps polysaccharide-black phosphorus intermediate (hereinafter referred to as BP / CSP);

[0115] (2) Characterization: FT-IR spectrum identification, at about 1160cm -1 and about 1080cm -1 There is a vibration peak (see Figure 1D ), which are typical characteristic absorption peaks of α-type glycosidic bonds in CSP, indicating that BP is successfully loaded on CSP.

[0116] 2. Preparation of Cordyceps polysaccharide-black phosphorus-polydopamine intermediate

[0117] Preparation of cordyceps polysaccharide-black phosphorus-polydopamine intermediate (code name: BP / CSP@PDA): Under the condition of avoiding light and protection of nitrogen or inert gas, the cordyceps polysaccharide-black phosphorus intermediate is mixed with Tris buffer containing 10mM dopamine hydrochloride at pH 8.5, the mass ratio of the cordyceps polysaccharide-black phosphorus intermediate to the dopamine hydrochloride in the Tris buffer containing dopamine hydrochloride is 1:1, stirred under the condition of avoiding light, centrifuged to obtain precipitate 2, washed precipitate 2 with water, and dried the washed precipitate 2 to obtain cordyceps polysaccharide-black phosphorus-polydopamine intermediate (code name: BP / CSP@PDA).

[0118] 3. Cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition

[0119] (1) Preparation: Preparation of cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition (alias: BP / CSP@PDA-PEG-FA): The functional ligand is bound to the surface of BP / CSP@PDA by Michael addition reaction (polydopamine and amino polyethylene glycol folic acid are connected by chemical bonds), and the specific operation is as follows: Under the condition of avoiding light and protecting with nitrogen or inert gas, the cordyceps polysaccharide-black phosphorus-polydopamine intermediate product is mixed with Tr containing 10 mM amino polyethylene glycol folic acid. is buffer solution, the cordyceps polysaccharide-black phosphorus-polydopamine intermediate product and the amino polyethylene glycol folic acid in the Tris buffer solution containing 10mM amino polyethylene glycol folic acid at pH 8.5 are fed in a mass ratio of 1:1, stirred for 12 hours under light-proof conditions for reaction, centrifuged to obtain a precipitate 3, washed with water, and dried to obtain a cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition (BP / CSP@PDA-PEG-FA, i.e., the composition of the present invention). The drug loading (LC) (polysaccharide loading rate) of the composition is calculated by the total mass of all polysaccharides added, the mass of polysaccharides remaining after loading, and the mass of BP / CSP@PDA-PEG-FA obtained, which can be calculated according to the following formula:

[0120] LC (%) = (total mass of CSP - mass of CSP in supernatant) ÷ mass of BP / CSP@PDA-PEG-FA × 100%

[0121] The drug loading amount of cordyceps polysaccharide in the composition of the present invention (BP / CSP@PDA-PEG-FA) was detected by the phenol-sulfuric acid method, and the drug loading amount of cordyceps polysaccharide in the composition of the present invention (BP / CSP@PDA-PEG-FA) was calculated to be 21.64wt%.

[0122] (2) Characterization:

[0123] 1) The zeta potentials of BP, BP / CSP, BP / CSP@PDA and BP / CSP@PDA-PEG-FA were measured by Malvern Mastersizer 2000. The results were as follows: the zeta potential of BP was -34.60 mV, the zeta potential of BP / CSP was -5.95 mV, the zeta potential of BP / CSP@PDA was -2.64 mV, and the zeta potential of BP / CSP@PDA-PEG-FA was 6.98 mV, which proved that BP / CSP@PDA-PEG-FA was successfully prepared.

[0124] 2) The particle sizes of BP, BP / CSP, BP / CSP@PDA and BP / CSP@PDA-PEG-FA were measured by Malvern Mastersizer 2000. The results showed that the average particle size of BP was 146 nm, the average particle size of BP / CSP was 202 nm, the average particle size of BP / CSP@PDA was 226 nm, and the average particle size of BP / CSP@PDA-PEG-FA was 245 nm, indicating that the increase in the average particle size indicated the successful loading of CSP, successful encapsulation of PDA, and successful connection of functional ligands.

[0125] 3) BP, BP / CSP, BP / CSP@PDA and BP / CSP@PDA-PEG-FA were detected by transmission electron microscopy. The results are shown in Figure 1A , Figure 1B , Figure 2A and Figure 2B ,From the results, it can be seen that the size of the particles increases ,with the loading of CSP, the encapsulation of PDA and the connection of ,functional ligands, the size of the particles increases continuously, indicating that the loading of CSP is successful, the encapsulation of PDA is successful, and the

[0126] 4) The chemical compositions of BP, CSP, BP / CSP, BP / CSP@PDA and BP / CSP@PDA-PEG-FA were determined by XPS. Figure 1E ), compared with BP, the C peak in the XPS spectra of BP / CSP, BP / CSP@PDA and BP / CSP@PDA-PEG-FA was significantly increased, indicating that the loading of CSP was successful.

[0127] Taking into account the zeta potential, particle size, loading and transmission electron microscopy results of BP, BP / CSP, BP / CSP@PDA and BP / CSP@PDA-PEG-FA, it was shown that BP / CSP@PDA-PEG-FA was successfully prepared.

[0128] Example 3: Preparation of black phosphorus nanoformulation (without cordyceps polysaccharide)

[0129] Based on the preparation method of Example 2, the aqueous solution of cordyceps polysaccharide (CSP) was replaced with water of the same mass, and the remaining operations were the same as in Example 2 to obtain a black phosphorus nanoformulation (also known as BP@PDA-PEG-FA).

[0130] Example 4: Pharmacological Experiment

[0131] 1. In vitro tumor-associated macrophage polarization and TNF-α and NO release experiments

[0132] Experimental operation:

[0133] The following experiments were performed in parallel three times.

[0134] Detection of M1 / M2 content of RAW264.7 cells (RAW264.7 group): RAW264.7 cells were cultured in 1640 complete medium at 37°C and 5% carbon dioxide incubator. The cells were observed once a day and passaged according to the cell growth. The old culture medium was discarded, and the cells were repeatedly rinsed with PBS twice. New culture medium was added, and the cells were scraped off with a cell scraper, counted, and passaged according to different cell passage densities. RAW264.7 cells were incubated in the culture medium for 24 hours, and then the RAW264.7 cells incubated in the above culture medium for 24 hours were double stained with Mice CD86 / BV421 and CD206 / PE for 30 minutes, and then washed twice with PBS. The cells were collected, and the M1 macrophage content and M2 macrophage content were detected by flow cytometry (FACSCalibur, BD), and the macrophage M1 / M2 ratio was calculated.

[0135] M1 / M2 conversion rate detection of RAW264.7 cells without the addition of the test sample (IL-4 group): RAW264.7 cells were cultured in 1640 complete medium at 37°C and 5% carbon dioxide incubator. The cells were observed once a day and passaged according to the cell growth. The old culture medium was discarded, PBS was repeatedly rinsed twice, new culture medium was added, cells were scraped off with a cell scraper, counted, and passaged according to different cell passage densities. RAW264.7 cells were incubated in 20ng / mL Mouse IL-4 culture medium for 24h to generate M2 macrophages. Then, the RAW264.7 cells incubated in 20ng / mL Mouse IL-4 culture medium for 24h were double stained with Mice CD86 / BV421 and CD206 / PE for 30 minutes, and then washed twice with PBS. The cells were collected, and the content of M1 macrophages and M2 macrophages was detected by flow cytometry (FACSCalibur, BD), and the macrophage M1 / M2 ratio was calculated.

[0136] M1 / M2 conversion rate detection of RAW264.7 cells with test samples added (CSP group, BP@PDA-PEG-FA group, BP / CSP@PDA-PEG-FA group): In order to investigate whether Cordyceps polysaccharides, various intermediates or the composition of the present invention can transform M2 macrophages into M1 macrophages, the following grouping experiments were performed: the groups were CSP group, BP@PDA-PEG-FA group, and BP / CSP@PDA-PEG-FA group. According to the above-mentioned "RAW264.7 cell M1 / M2 conversion rate detection without adding the sample to be tested", RAW264.7 cells incubated in 20ng / mL Mouse IL-4 culture medium for 24h were obtained, and then CSP, BP@PDA-PEG-FA, and BP / CSP@PDA-PEG-FA were taken and incubated in 20ng / mL Mouse IL-4 culture medium for 24h. RAW264.7 cells were incubated for 3h, respectively. Among them, the final concentration of CSP in the CSP group (i.e., the concentration after adding the sample to be tested and incubated) was 4.328μg / mL, the final concentration of BP@PDA-PEG-FA in the BP@PDA-PEG-FA group was 15.672μg / mL, and the final concentration of BP / CSP@PDA-PEG-FA in the BP / CSP@PDA-PEG-FA group was 20.000μg / mL. After washing with PBS, each group was double-stained with CD86 / BV421 and CD206 / PE for 30 min, and the content of M1 macrophages and M2 macrophages was detected by flow cytometry, and the macrophage M1 / M2 ratio was calculated.

[0137] Detection of TNF-α cytokine and NO content: After the cells washed with PBS were centrifuged at 1000 rpm for 5 min, the supernatant was taken and the TNF-α detection kit (Thermo Fisher Scientific) and the total NO detection kit (Biyuntian) were used to detect the TNF-α cytokine and NO content, respectively. The results of the tumor-associated macrophage polarization and TNF-α and NO release experiments are shown in Tables 1 and 2.

[0138] Table 1: Effects of different drugs on the ratio of two macrophage phenotypes in RAW264.7 cells

[0139]

[0140] The experimental results in Table 1 show that CSP and BP@PDA-PEG-FA cannot effectively convert M2 into M1, while the composition of the present invention can effectively convert M2 into M1, especially BP / CSP@PDA-PEG-FA has the most obvious effect in converting M2 into M1.

[0141] Table 2: Effects of different drugs on the release of TNF-α and NO from RAW264.7 cells

[0142] Group RAW264.7 group IL-4 group CSP Group BP@PDA-PEG-FA group BP / CSP@PDA-PEG-FA group TNF-α(pg / mL) 3148±101 1651±60 2877±17 1625±30 4109±125 Group RAW264.7 group IL-4 group CSP Group BP@PDA-PEG-FA group BP / CSP@PDA-PEG-FA group NO(μM) 24.73±0.27 20.60±1.99 21.84±0.69 18.95±0.97 30.72±0.61

[0143] The experimental results in Table 2 show that the composition of the present invention is beneficial to promoting the release of TNF-α and NO in RAW264.7 cells, especially BP / CSP@PDA-PEG-FA is more beneficial to promoting the release of TNF-α and NO in RAW264.7 cells.

[0144] 2. In vivo anticancer evaluation

[0145] On the basis of the in vitro experimental results, in vivo experiments were further conducted to study the tumor phototherapy and immunotherapy effects of BP / CSP@PDA-PEG-FA on 4T1 tumor mice.

[0146] Female mice (BALB / c) implanted subcutaneously with 4T1 breast tumor xenografts were used as animal models. 4T1 cells were revived, cultured and collected in the logarithmic growth phase, the culture medium was removed and washed twice with PBS, and the cell survival before and after inoculation was counted. The cell inoculation amount was 5×10 5 Cells were inoculated subcutaneously at 100 μL / mouse. Three times the number of animals expected to be enrolled were inoculated into mice. When the tumor volume could be measured, the tumors were measured and weighed twice a week. Mice with uniform tumor growth and moderate growth rate were selected and grouped according to tumor volume and given medication.

[0147] Tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 .

[0148] When the tumor volume reaches 100 mm 3 When selecting mice with uniform tumor growth and moderate growth rate, group them according to tumor volume. The mice were randomly divided into 6 groups, 5 mice in each group:

[0149] Group 1: CSP;

[0150] Group 2: BP@PDA-PEG-FA;

[0151] Group 3: BP / CSP@PDA-PEG-FA;

[0152] Group 4: BP@PDA-PEG-FA+808nm laser (also known as BP@PDA-PEG-FA+L group);

[0153] Group 5: BP / CSP@PDA-PEG-FA+808nm laser (also known as BP / CSP@PDA-PEG-FA+L group);

[0154] Group 6: PBS (phosphate buffered saline).

[0155] The CSP in the above group is the Cordyceps polysaccharide obtained in Example 1, BP@PDA-PEG-FA is the black phosphorus nanoformulation obtained in Example 3, and BP / CSP@PDA-PEG-FA is the Cordyceps polysaccharide-black phosphorus-polydopamine-aminopolyethylene glycol folic acid composition obtained in Example 2.

[0156] The above groups of mice were intravenously injected with drugs (each group used PBS to dissolve the drug). The first group received a dose of 4.3 mg / kg (i.e., 4.3 mg CSP per kg mouse per dose), the second and fourth groups received a dose of 15.7 mg / kg (i.e., 15.7 mg BP@PDA-PEG-FA per kg mouse per dose); the third and fifth groups received a dose of 20.0 mg / kg (i.e., 20.0 mg BP / CSP@PDA-PEG-FA per kg mouse per dose), and the sixth group received the same volume of PBS as the other groups; the frequency of administration for each group was once every 2 days, for a total of 3 times. Among them, the fourth and fifth groups were treated with 808 nm (1.0 W / cm 2 The tumor site of mice was irradiated with laser for 10 min. The tumor size and body weight were measured every two days.

[0157] Starting from the first administration as day 0, after 20 days, each group performed tumor tissue dissection, soaked in 4% formalin, and embedded in paraffin. TUNEL staining was performed on the tumor tissue to observe the apoptosis of cells in the tumor site. In addition, in order to evaluate the effect of BP / CSP@PDA-PEG-FA on TAMs polarization, the tumor was stained with CD86 / Cy3 antibody and CD206 / FITC antibody (source: Wuhan Sevier Biotechnology Co., Ltd.) to determine the distribution of M1 macrophages and M2 macrophages in the tumor site. At the same time, the tumor was stained with CD4 / FITC antibody and CD8 / Cy3 antibody (source: Wuhan Sevier Biotechnology Co., Ltd.) to evaluate the effect of immunotherapy. Other major organ tissues (including heart, liver, spleen, lung, and kidney) were also stained with hematoxylin and eosin (H&E) to evaluate the toxicity of the preparation.

[0158] Results: See Figure 3 , Figure 4 , Table 3 and Table 4.

[0159] in, Figure 3The experimental results showed that the cells in the PBS group (Group 6) and the BP@PDA-PEG-FA group (Group 2) were closely fused and arranged, and there was no tumor fragmentation. The CSP group (Group 1) and the BP / CSP@PDA-PEG-FA group (Group 3) showed a certain degree of nuclear dissolution and fragmentation, but the effect was general, indicating that the immune anti-tumor effect caused by CSP alone was limited. In the BP@PDA-PEG-FA+808nm laser group (Group 3) and the BP / CSP@PDA-PEG-FA+808nm laser group (Group 5), a large number of tumor cells showed nuclear dissolution, fragmentary necrosis and fragmentation. Among them, the BP / CSP@PDA-PEG-FA+808nm laser group (Group 5) had the most obvious symptoms, indicating that the photothermal synergistic immunotherapy effect was significant.

[0160] Figure 4 The experimental results showed that the H&E staining results of the main tissues and organs of mice in the BP / CSP@PDA-PEG-FA+808nm laser group (Group 5) at the end of the experiment were as follows: Figure 4 As shown, no obvious abnormality was observed, indicating that the composition provided by the present invention is relatively safe to use and has no obvious toxic side effects on tissues and organs.

[0161] Table 3: Effects of different drugs on the changes in mouse tumor volume

[0162]

[0163] Table 4: Effects of different drugs on the endpoint tumor weight of mice

[0164]

[0165] The experimental results in Table 3 and Table 4 show that compared with the PBS group, BP@PDA-PEG-FA group (without laser treatment), CSP group, BP / CSP@PDA-PEG-FA group (without laser treatment) and BP@PDA-PEG-FA+L group, the BP / CSP@PDA-PEG-FA+L group is more conducive to inhibiting tumor growth, and is even better than the combined effects of the CSP group alone and the BP@PDA-PEG-FA+L group alone, with an unexpected synergistic technical effect.

[0166] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A composition, characterized in that It includes Cordyceps polysaccharides, black phosphorus nanosheets, encapsulation materials and functional ligands.

2. The composition according to claim 1, wherein the packaging material comprises polydopamine or a salt thereof; and / or The functional ligand comprises amino polyethylene glycol folic acid; and / or The weight average molecular weight of the aminopolyethylene glycol folic acid is 500-4000 or 2000.

3. The composition according to any one of claims 1 to 2, wherein the black phosphorus nanosheets are loaded with the Cordyceps polysaccharide; Optionally, the cordyceps polysaccharide loaded by the black phosphorus nanosheets is wrapped by the wrapping material to obtain a wrapped composition A; Optionally, the composition A is linked to the functional ligand.

4. The composition according to any one of claims 1 to 3, wherein the content of the cordyceps polysaccharide is 5.00wt%-40.00wt% or 10.00wt%-30.00wt% or 21.64wt% based on the total mass of the composition; and / or Based on the total mass of the composition, the content of black phosphorus in the composition is 50.00wt%-85.00wt% or 60.00wt%-80.00wt%; and / or Based on the total mass of the composition, the content of the encapsulating material is 5.00wt%-30.00wt% or 5.00wt%-10.00wt%; and / or Based on the total mass of the composition, the content of the functional ligand is 5.00 wt%-30.00 wt% or 5.00 wt%-10.00 wt%.

5. The composition according to any one of claims 1 to 4, further comprising a method for preparing Cordyceps polysaccharide, the method for preparing Cordyceps polysaccharide comprising: (1) Supercritical treatment: Cut or crush the Cordyceps sinensis into pieces and then perform supercritical treatment; Obtaining Cordyceps sinensis after supercritical treatment; (2) Extraction and concentration: extracting the supercritically treated Cordyceps sinensis with a solvent to obtain an extract, and concentrating the extract to obtain a concentrate; (3) alcohol precipitation: taking the concentrated solution and subjecting it to alcohol precipitation, centrifuging to obtain a precipitate, and drying the precipitate to obtain crude Cordyceps polysaccharide; (4) protein removal: removing protein from crude Cordyceps polysaccharide by Sevage method to obtain a protein-free Cordyceps polysaccharide solution; (5) Dialysis: The deproteinized Cordyceps polysaccharide solution is then dialyzed to obtain a Cordyceps polysaccharide solution; (6) Filtration, alcohol precipitation and freeze-drying: The cordyceps polysaccharide solution is filtered and precipitated with alcohol, centrifuged to obtain a polysaccharide precipitate, and the polysaccharide precipitate is dried or the polysaccharide precipitate is redissolved and then freeze-dried to obtain the cordyceps polysaccharide.

6. The composition according to claim 5, wherein the supercritical treatment comprises a CO2 supercritical treatment at a temperature of 50°C to 150°C and a pressure of 10 MPa to 30 MPa; and / or The extraction comprises mixing the supercritically treated Cordyceps sinensis with a solvent, heating for extraction, filtering, taking the filtrate, extracting the filter cake with the solvent for 0 to 4 times, combining the filtrate to obtain an extract; and / or The concentration includes concentration by reduced pressure concentration method; and / or The step (3) of taking the concentrated solution for alcohol precipitation comprises mixing the concentrated solution with an ethanol solution of 4 times the volume of the concentrated solution, and placing the mixture at 4° C. to 10° C. for 12 to 24 hours; and / or The ethanol solution in step (3) is composed of ethanol and water in a volume ratio of 95:5-100:0; and / or The Sevage method comprises re-dissolving the crude Cordyceps polysaccharide with water to obtain a re-dissolved solution, mixing the re-dissolved solution with a sevage reagent, removing insoluble matter by centrifugation to obtain a supernatant, replacing the re-dissolved solution with the supernatant, and repeating the above "mixing with a sevage reagent, removing insoluble matter by centrifugation" operation until no insoluble matter is produced; and / or The re-dissolving the crude cordyceps polysaccharide with water comprises dissolving the crude cordyceps polysaccharide with water at a mass ratio of the crude cordyceps polysaccharide to water of 1:8-1:20; and / or The volume ratio of the reconstituted solution or supernatant to the sevage reagent is 5:1-4:1 or 4:1; and / or The sevage reagent is a mixture of chloroform and n-butanol in a volume ratio of 5:1-4:1 or 4:1; and / or The molecular weight cut-off of the dialysis is 2.0 kDa-5.0 kDa or 3.5 kDa; and / or The dialysate of the dialysis is water; and / or The dialysate is replaced every 1-3 hours or 2 hours; and / or The dialysis time is 24 hours to 52 hours or 48 hours; and / or The dialyzed Cordyceps polysaccharide solution in step (6) is then filtered and precipitated with alcohol, including filtering the dialyzed Cordyceps polysaccharide solution to obtain a filtrate, adding an ethanol solution 4 times the volume of the filtrate to the filtrate, and placing the filtrate at 4°C-10°C for 12 hours-24 hours; and / or The ethanol solution in step (3) is composed of ethanol and water in a volume ratio of 95:5-100:

0.

7. A method for preparing the composition according to any one of claims 1 to 6, comprising: The cordyceps polysaccharide is loaded on the black phosphorus nanosheet, which is then wrapped with a wrapping material, and finally connected with the wrapping material with a functional ligand, and dried to obtain the composition.

8. The preparation method according to claim 7, comprising: (1) Preparation of cordyceps polysaccharide-black phosphorus intermediate: Under the condition of avoiding light and protecting with nitrogen or inert gas, mixing the aqueous solution containing cordyceps polysaccharide with black phosphorus nanosheets, stirring, centrifuging to obtain a precipitate 1, and washing the precipitate 1 with water to obtain a cordyceps polysaccharide-black phosphorus intermediate; (2) Preparation of cordyceps polysaccharide-black phosphorus-polydopamine intermediate: under the conditions of avoiding light and protecting with nitrogen or inert gas, the cordyceps polysaccharide-black phosphorus intermediate is mixed with a dopamine hydrochloride solution, stirred, and centrifuged to obtain a precipitate 2, and the precipitate 2 is washed with water and dried to obtain a cordyceps polysaccharide-black phosphorus-polydopamine intermediate; (3) Preparation of Cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition: The functional ligand is combined with the surface of the Cordyceps polysaccharide-black phosphorus-polydopamine intermediate product by Michael addition reaction, and the Cordyceps polysaccharide-black phosphorus-polydopamine-amino polyethylene glycol folic acid composition is obtained after post-treatment, that is, the composition; Optionally, the Michael addition reaction comprises: mixing the cordyceps polysaccharide-black phosphorus-polydopamine intermediate with an aminopolyethylene glycol folic acid solution under conditions of light protection and nitrogen or inert gas protection, stirring, and reacting; Optionally, the post-treatment includes: centrifugation to obtain a precipitate 3, washing the precipitate 3 with water, and drying the washed precipitate 3.

9. The preparation method according to claim 8, The concentration of cordyceps polysaccharide in the aqueous solution containing cordyceps polysaccharide in step (1) is 10 mg / mL-50 mg / mL or 25 mg / mL; and / or The mass ratio of cordyceps polysaccharide to black phosphorus nanosheets in the aqueous solution containing cordyceps polysaccharide in step (1) is 1:1-10:1 or 1:1-3:1 or 2:1; and / or The stirring in step (1) is stirring for 12 hours to 36 hours or 24 hours; and / or The dopamine hydrochloride solution in step (2) is a solution of dopamine hydrochloride dissolved in Tris buffer; and / or The pH of the dopamine hydrochloride solution in step (2) is 8.0-9.0 or 8.5; and / or The concentration of dopamine hydrochloride in the dopamine hydrochloride solution in step (2) is 8mM-12mM or 10mM; and / or The mass ratio of the cordyceps polysaccharide-black phosphorus intermediate product to the dopamine hydrochloride in the dopamine hydrochloride solution in the step (2) is 1:2-2:1 or 1:1; and / or The stirring in step (2) is for 8 hours to 24 hours or 12 hours; and / or The aminopolyethylene glycol folic acid solution in step (3) is a solution of aminopolyethylene glycol folic acid dissolved in Tris buffer; and / or The pH of the amino polyethylene glycol folic acid solution in step (3) is 8.0-9.0 or 8.5; and / or The concentration of amino polyethylene glycol folic acid in the amino polyethylene glycol folic acid solution in step (3) is 8mM-12mM or 10mM; and / or The mass ratio of the cordyceps polysaccharide-black phosphorus-polydopamine intermediate product to the amino-polyethylene glycol folic acid in the amino-polyethylene glycol folic acid solution in the step (3) is 1:2-2:1 or 1:1; and / or The stirring in step (3) is for 6 hours to 12 hours; and / or The drying in step (2) is freeze-drying; and / or The drying in step (3) is freeze-drying.

10. Use of the composition according to any one of claims 1 to 6 or the composition obtained by the preparation method according to any one of claims 7 to 9 in preparing a product for treating tumors; Optionally, the tumor comprises breast cancer or thyroid cancer.

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