CN114469953B

CN114469953B - Antitumor pharmaceutical composition with synergistic effect, nano preparation, and preparation method and application thereof

Info

Publication number: CN114469953B
Application number: CN202210132859.2A
Authority: CN
Inventors: 邓意辉; 范垂众; 李聪; 路双; 刘欣荣; 宋艳志
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2022-02-14
Filing date: 2022-02-14
Publication date: 2023-07-14
Anticipated expiration: 2042-02-14
Also published as: CN114469953A

Abstract

An antitumor pharmaceutical composition with synergistic effect, a nano-preparation and a preparation method and application thereof belong to the technical field of biological medicines, and in particular relate to a pharmaceutical composition formed by a cationic drug of arbeli and mitoxantrone, a nano-preparation and a preparation method and application thereof. The nano preparation is formed by self-assembling Abeli, mitoxantrone and a polyanion material according to a proportion. The pharmaceutical composition reduces the dosage of single medicine through the synergistic effect between medicines, and achieves the effect of 1+1> 2. The nanometer preparation can be remarkably accumulated at tumor sites, reverse immunosuppression microenvironment, reduce toxic and side effects and improve the curative effect of the medicine. In addition, the medicine in the nano preparation has the function of synergic anti-metastasis, and provides a new strategy for the treatment of clinical tumor patients.

Description

Antitumor pharmaceutical composition with synergistic effect, nano preparation, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an antitumor pharmaceutical composition with a synergistic effect, a nano preparation containing the pharmaceutical composition, and a preparation method and application thereof.

Background

Tumor (Tumor), a disease caused by the disorder of the control of the cell division proliferation mechanism. In addition to uncontrolled division, cancer cells can also invade surrounding normal tissues locally and metastasize to other parts of the body even via the circulatory system or lymphatic system in the body. According to 2020 global cancer statistics report (Global Cancer Statistics 2020), about 1930 ten thousand cancer cases are newly diagnosed worldwide, and the number of cancer deaths can reach 1000 ten thousand.

Tumor metastasis refers to the process by which tumor cells spread from the primary tumor site, colonize and grow in organs other than the primary tumor. In tumor patients, about 90% of deaths are associated with metastasis. Tumor cells first break away from the primary tumor, migrate, invade, reach different sites through blood and lymphatic vessels, and subsequently adhere and grow. Compared with primary tumor, the tumor metastasis has wide distribution, small volume, high non-uniformity and difficult surgical excision. Therefore, there is a need to find an effective and safe transfer-targeted therapeutic strategy to increase patient survival and reduce side effects of chemotherapeutic agents.

In recent years, with the continuous development of biomedical technology, emerging therapies such as molecular targeted therapy and immunotherapy are also continuously entering clinic to become new strategies for tumor treatment. However, due to the complex pathological mechanism of tumors, whether traditional or novel biological drugs, single drug therapy relying on a single anti-tumor mechanism is difficult to obtain a substantial clinical benefit. Based on the pathological complexity of tumors, the combined use of antitumor drugs has gradually become a new clinical treatment trend.

Combination administration, i.e., simultaneous administration of two or more drugs, becomes critical to achieving long-term prognosis and reducing adverse side effects. The combined treatment can regulate different signal paths in tumor cells, cause synergistic effect, additive effect and reinforcing effect, and greatly enhance the treatment effect. By the year 2020, there are nearly 6000 tumor combinations registered in clinical laboratory, and it is seen that antitumor combinations have been gradually valued and developed into mainstream therapeutic regimens in clinic.

With the rapid development of nanomedicine, a nanomedicine delivery system is expected to improve the shortcomings of the existing anti-tumor multi-drug combination delivery strategy and enhance the treatment effect. The penetration of related research has provided more new possibilities for co-delivery of multiple drugs, and tumor multi-drug combination strategies based on nano-drug delivery systems have more varied functionalities than traditional "cocktail" therapies and have shown better therapeutic effects.

Neutrophils are the most abundant circulating leukocytes in humans and are important immune cells against infection. However, recent studies have found that neutrophils are one of the main driving forces for the formation of cancer metastasis, and are involved in the overall process of cancer metastasis, and exert various pro-metastatic effects during metastasis. As tumor metastasis progresses, the number of neutrophils in the circulation increases and can be recruited preferentially and in large numbers to the site of metastasis. Given the spontaneous, massive recruitment of neutrophils to the site of metastasis and their diverse functions, it is appreciated that neutrophils can serve as a vehicle for drug delivery to enhance therapeutic effects.

Abetaciclib (ABE), an inhibitor of Cyclin Dependent Kinases (CDKs) 4 and 6, has become an effective drug for the treatment of breast cancer. Several studies have shown that ABE may affect a variety of immune cells in the tumor microenvironment, including inhibiting proliferation of Treg cells and enhancing activation of tumor-infiltrating T cells (TILs). However, ABE is less effective against certain tumors, possibly associated with tumor microenvironments lacking TILs infiltration and "immune coldness", and thus strategies for combination therapy may increase the anti-tumor effects of ABE. Mitoxantrone can induce immunogenic cell death (immunogenic cell death, ICD), increase immune responses, and overcome the "immune-cold" tumor microenvironment by recruiting TILs. It is synergistic with ABE and may be used in raising the curative effect.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art, providing an anti-tumor pharmaceutical composition with a synergistic effect, and preparing a nano preparation thereof. The pharmaceutical composition and the nano preparation can utilize the synergistic effect, the additive effect and the enhancement effect caused by combined administration to effectively improve the immunosuppression microenvironment and the systemic immune macro environment of tumors and tumor metastasis positions and improve the curative effect of the medicine.

It is still another object of the present invention to provide the application of the pharmaceutical composition and the nano-preparation in preparing medicines for resisting solid tumors and treating tumor metastasis.

The solid tumor comprises oral cancer, gastrointestinal cancer, colon cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, bladder cancer, pancreatic cancer, bone cancer, liver cancer, gall bladder cancer, kidney cancer, skin cancer and testicular cancer, melanoma and sarcoma. The tumor metastasis organ is lung, other side lung, liver, lymph, bone, brain, adrenal gland.

In order to achieve the above purpose, the following technical scheme is adopted:

an antitumor pharmaceutical composition with synergistic effect comprises Abeli and mitoxantrone. The mass ratio of the abbe-cilia to the mitoxantrone is (8:1) - (1:6), preferably (4:1) - (1:2).

The concentration of the arbeli in the pharmaceutical composition is 0.1 mg.mL ^-1 -50mg·mL ^-1 Preferably 1 mg.mL ^-1 -40mg·mL ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Mitoxantrone concentration of 0.1 mg/mL ^-1 -20mg·mL ^-1 Preferably 0.5 mg/mL ^-1 -15mg·mL ^-1 . The pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials and carriers.

The pharmaceutical composition is used for synergistically inhibiting the growth of tumor cells and improving the immunosuppressive microenvironment and the immune large environment.

The invention also provides a dosage form of the pharmaceutical composition, which comprises any one of a solid preparation, a liquid preparation and a semisolid preparation. Further the dosage form is a nano-formulation comprising a polyion complex, a phospholipid complex, a liposome, a nanoparticle, a nanocapsule and a polymer micelle.

Under certain conditions, oppositely charged polyelectrolyte interactions are capable of forming polyelectrolyte complexes, also known as polyionic complexes (Polyionic complex, PIC). Polyelectrolytes capable of reacting include acids, bases, salts and the like in polymeric form, even including certain biological macromolecules and ionic surfactants.

A significant feature of polyion complexes in this application of drug delivery is that their core can act as a microreservoir for charged compounds (e.g., genes, enzymes, drugs, etc.), thereby modulating their inherent properties such as stability, solubility, and reactivity.

The polyion compound disclosed by the invention refers to a compound with the average particle size of nanometer level, wherein the average particle size of the compound is the nanometer level, and the compound is the nanometer level.

Wherein the mass ratio of the polyanionic material to the pharmaceutical composition is (1:2) to (30:1), preferably (1:1) to (10:1). The polyanion material is any one of polysialic acid, hyaluronic acid, alginic acid, polyglutamic acid, heparin, chondroitin sulfate, dextran sulfate and succinyl gelatin. The polymerization degree of the polysialic acid is 2-1000, preferably 20-400; the polymerization degree of the hyaluronic acid is 10-5000, preferably 50-500; the polymerization degree of the alginic acid is 300-1000, preferably 600-800; the polyglutamic acid has a polymerization degree of 1000 to 15000, preferably 2000 to 5000.

Sialic Acid (SA), also known as sugar acid, is a class of nine-carbon monosaccharides linked mainly in short chain residues to the ends of glycoproteins, glycolipids and oligosaccharides via alpha-glycosidic bonds, and is ubiquitous on mammalian cell membrane surfaces. In addition, many pathogens utilize the SA to "dress" themselves to mask autoantigen epitopes, inhibit alternative activation pathways of complement, reduce immunogenicity and thus successfully escape attack by the host immune system. Polysialic acid (PSA) is a homopolymer of multiple SA monomers linked at a-2, 8 and/or a-2, 9. PSA has a structure similar to that of a sugar chain signal molecule at the end of lipopolysaccharide, lipoprotein, etc. on the surface of human cells, and is non-immunogenic in humans. PSA exists mainly in the form of complex with other molecules such as neural cell adhesion molecules, lactose, proteins or lipids in higher organisms, and the unique properties and biological functions of polysialic acid make the PSA increasingly wide application in the fields of food health care, medicine, cosmetics and the like, and as a high value-added material, the research and application values of the PSA are also increasingly prominent.

The invention also provides a preparation method of the polyion compound, which comprises the following steps:

(1) Dissolving the medicinal composition with pure water to obtain solution A with concentration of 0.1 mg.mL ^-1 ～10mg·mL ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The mass ratio of the abbe-cilia to the mitoxantrone in the pharmaceutical composition is (8:1) - (1:6), preferably (4:1) - (1:2);

(2) Dissolving polyanion material in pure water to obtain solution B with concentration of 0.1 mg.mL ^-1 ～100mg·mL ^-1 ；

(3) Mixing the solution A and the solution B to obtain the polyion compound. Wherein the mass ratio of the polyanionic material to the pharmaceutical composition is (1:2) to (30:1), preferably (1:1) to (10:1), and more preferably 2:1.

According to specific conditions, glucose, trehalose, sucrose, lactose, mannitol, sorbitol, xylitol and glycerol can be adopted to adjust osmotic pressure so as to meet the injection requirements. Or lyophilizing or spray drying, wherein the lyoprotectant can be mannitol, trehalose, sorbitol, sucrose, lactose, maltose, and dextran to obtain solid product.

Other adjuvants such as EDTA (disodium salt, calcium sodium salt), human serum albumin, poloxamer, dextran, polyvinylpyrrolidone, polysaccharides (such as Ginseng radix polysaccharide, radix astragali polysaccharide, and lentinan) and pH regulator can also be added into the preparation.

In the preparation prepared from the pharmaceutical composition of the abbe-xili and the mitoxantrone, the auxiliary materials used for the preparation are on the premise of not reacting with the pharmaceutical composition or not affecting the curative effect of the medicament according to the invention according to different preparation forms and preparation specifications.

The invention combines the Abeli and the mitoxantrone for the first time, and the Abeli and the mitoxantrone are compounded in the pharmaceutical composition of the invention to have a synergistic effect, obtain higher anti-tumor activity at low concentration and have the advantages of high efficiency and low toxicity. Mitoxantrone is used to increase immune response, overcoming the "immune cold" tumor microenvironment by recruiting TILs, and producing a synergistic effect with the inhibition of Treg cell proliferation and the enhancement of tumor infiltrating T cell (TILs) activation by arbelide. Meanwhile, the compound with the average particle size of nano-scale and co-carried by the Abeli and the mitoxantrone is prepared, the particle size is about 100nm, and the tumor targeting nano injection is further prepared, and the 4T1 breast cancer lung metastasis mice show excellent anti-tumor effect after injection. The nanoscale composite is also characterized by the following: 1. the preparation process does not need specific synthesis conditions or additional reagents, and has good biocompatibility, biodegradability, no toxicity and no immunogenicity. The preparation method is simple and convenient, and is suitable for large-scale production; 2. targeting lung metastases with self neutrophil migration, converting disadvantages to advantages; 3. reverse the immunosuppression microenvironment, influence the systemic tumor large environment and completely eliminate the metastasis. The drug effect of the nano-scale compound of the invention is far better than that of a drug composition solution, a single drug preparation and simultaneous or sequential administration.

Most importantly, the polyanion material modified arbelide and mitoxantrone co-supported polyion complex expands the tumor treatment spectrum from solid tumors such as in-situ lung cancer, in-situ breast cancer and the like to tumors and metastatic tumors of organs such as lung, liver, lymph, bone, brain, adrenal gland and the like.

The dosage, route of administration or form of preparation of the pharmaceutical composition of the present invention may be varied appropriately, but it is premised on ensuring that the pharmaceutical composition can achieve an effective efficacy in vivo.

The invention has the beneficial effects that:

the pharmaceutical composition has a synergistic effect, and can be applied to preparation of the pharmaceutical composition for preventing and/or treating solid tumors and tumor metastasis through targeting of nano preparations and neutrophils. The medicine composition and the nano preparation provided by the invention can also be applied to the aspects of inflammatory diseases and autoimmune diseases, and provide technical guidance for the combination of medicines and the development of a nano medicine delivery system.

Drawings

FIG. 1 is a photograph of a nanocomposite and a Transmission Electron Microscope (TEM) image; a: abeli-polysialic acid nanocomposite B: mitoxantrone-polysialic acid nanocomposite C: an abbe-mitoxantrone-polysialic acid nanocomposite;

FIG. 2 is an infrared spectrum of different substances and nanocomposites;

FIG. 3 is an in vitro release process of a nanocomposite;

FIG. 4 is a flow cytometry to detect neutrophil purity;

FIG. 5 shows the expression level of L-selectin on neutrophil membrane;

FIG. 6 is an examination of the uptake of nanocomposites by neutrophils; a: confocal microscopy imaging technique B: flow cytometry;

FIG. 7 is a graph showing the effect of CCK8 assay on neutrophil viability of nanocomposites;

FIG. 8 is a graph showing the effect of MTT assay on 4T1 tumor cell viability of nanocomposites;

FIG. 9 is a graph showing the change of neutrophil count in the lung over time after tumor lung metastasis modeling;

FIG. 10 is in vivo fluorescence imaging of nanocomposites in 4T1 breast cancer lung metastasis BALB/c mice; a: pulmonary transfer mice in vivo fluorescence presentation B: fluorescence imaging of isolated viscera;

FIG. 11 shows the lung tissue distribution of the nanocomposite in a 4T1 breast cancer lung metastasis BALB/c mouse;

FIG. 12 is a photograph of lung of a 4T1 breast cancer lung metastasis BALB/c mouse;

FIG. 13 is a section of 4T1 breast cancer lung metastasis BALB/c murine lung tissue;

FIG. 14 is a graph showing the number of lung metastasis nodules in a 4T1 breast cancer lung metastasis BALB/c mouse;

FIG. 15 shows survival curves of 4T1 breast cancer lung metastasis BALB/c mice;

FIG. 16 shows weight change in 4T1 breast cancer lung metastasis BALB/c mice;

FIG. 17 is lung weight of 4T1 breast cancer lung metastasis BALB/c mice;

FIG. 18 shows the index of inhibition of lung metastasis BALB/c murine metastasis from 4T1 breast cancer;

FIG. 19 is a significant tissue section of a 4T1 breast cancer lung metastasis BALB/c mouse;

FIG. 20 is a graph of 4T1 breast cancer lung metastasis BALB/c murine lung CD4 ⁺ T，CD8 ⁺ T cell change; a: flow cytometry results B: CD4 ⁺ T cell C: CD8 ⁺ T cells;

FIG. 21 shows changes in lung Treg cells of 4T1 breast cancer lung metastasis BALB/c mice; a: flow cytometry results B: regulatory T cells;

FIG. 22 shows changes in lung cytokine in mice with 4T1 breast cancer lung metastases BALB/c.

Detailed Description

The main reagents, instruments and animals adopted in the invention are as follows:

instrument: BS124s electronic analytical balance (saidolis company, germany); DF-101S heat collection type constant temperature heating magnetic stirrer (consolidating Yingyu to Hua instrument factory); PB-10 pH meter (Sidoris Corp., germany); TDL-80-2S centrifuge (Shanghai Anting scientific instruments Co.); UV 1801-type ultraviolet-visible spectrophotometer (beijing rayleigh analytical instruments limited); thermo Scientific Forma CO ₂ Carbon dioxide incubator (Shanghai Seiemerle Feier technologies Co., ltd.); TM-4 dynamic sterilizer (Shenyang Tianmeida scientific instruments Co., ltd.); SCIENTZ-30F freeze dryer (Ningbo Xinzhi Biotechnology Co., ltd.); BALB/c mice (18-22 g, male, university of Shenyang pharmacy laboratory animal center); 0.45 μm polyethylene microporous filter membrane (Shanghai Mohs science equipments Co., ltd.).

Reagent: in the present invention, the polymerization degree of the PSA used is 2, 3, 4, 5, 6, 10, 16, 32, 100, 130 to 170, 200, 270, respectively, the corresponding molecular weights are 644.50, 957.73, 1270.97, 1584.21, 1897.45, respectively, and the average molecular weights are 3000, 5000, 16000, 30000,4 to 5 ten thousand and 6 to 8 ten thousand daltons, respectively. PSA with degrees of polymerization of 2, 3, 4, 5, 6 was purchased from Nacalai company, usa; PSA having a degree of polymerization of 7 to 10 is purchased from Nacalaitesque, japan; PSA (average molecular weight 11.0kDa, polydispersity (p.d.) 1.17; average molecular weight 22.7kDa, polydispersity index (p.d.) 1.34; average molecular weight 39.0kDa, polydispersity (p.d.) 1.40), from Camida, irish; the average molecular weight of 30kDa was obtained from Carbosynth Inc. (cabosson chemical technology (Suzhou)) of England; the PSA of the remaining molecular weight is self-made. Abeli bulk drug (ABE, beijing Huavone Libo Co., ltd., purity is not less than 99%); mitoxantrone bulk drug (MIT, beijing Huafeng Libo Co., ltd., purity > 99%); absolute ethanol (analytically pure, tianjin dense euler reagent development center); 5% glucose injection (5% Glu, chenxin pharmaceutical Co., ltd.); 50% glucose injection (50% Glu, tianjin pharmaceutical Co., ltd.); sterilized water for injection (Shijia four-medicine Co., ltd.).

Example 1

Cell Activity assay

The study used breast cancer cell line 4T1, cells in 5% CO ₂ Culturing in corresponding culture solution under the conditions of 95% air, saturated humidity and 37 ℃, and adding 10% new born calf serum. In each experiment, the seeding density of cells was 5×10 ⁴ /mL。

1. mu.L (about 5X 10) of 4T1 cell suspension ⁴ Per mL of cells) was added to a 96-well plate (edge wells were filled with sterile water or PBS). Blank wells (with medium, no cells) and control wells (medium without drug, with cells) were set, and 3 duplicate wells were set for each group.

2. Placing at 37deg.C, 5% CO ₂ Incubate for 0.5h and observe under inverted microscope.

3. mu.L of the drug dilution medium to be tested at different concentrations was added to each well and incubated at 37 ℃.

4. 10. Mu.LCCK-8 solution was added to each well and incubated for 4h at 37 ℃.

5. The absorbance value (SoftMax Pro 7.1) was determined for each well at 450 nm.

6. Analysis of results: the OD value of each test well was subtracted by either the zeroed well OD value or the control well OD value. OD values for each replicate well were averaged. Cell viability% = (dosing cell OD-blank OD)/(control cell OD-blank OD) ×100%

The combination of abbe-cili and mitoxantrone was analyzed for synergy, additive effects, or antagonism by the combination index method set forth in Zhou Teshi.

The experimental results show that the ABE and MIT solutions used alone have inhibition effect on proliferation of 4T1 tumor cells, and the inhibition effect on cell growth is gradually enhanced along with the increase of the drug concentration, and the dose dependency exists. After the two drugs are combined, the inhibition rate is obviously enhanced compared with that of the single drug at the same dosage. For 4T1 cells, the synergistic ratio of the two ranges from (4:1) to (1:2).

TABLE 1 inhibition of 4T1 cells by two medicinal fluids acting alone and in unequal ratio

Based on the medium-effect Principle (or Chou-Talalay combined index method), the CompuSyn statistical software is used for drawing a combination index curve under different effects, and whether a synergistic effect exists between two medicines is quantitatively evaluated from the relationship between the effect of combining the two medicines and the combination index. Combination index ci=d ₁ /D _X1 +D ₂ /D _X2 +αD ₁ D ₂ /D _X1 D _X2 Wherein D is ₁ 、D ₂ For generating X effect when two medicines are singly used, the respective concentration of the two medicines is D _X1 、D _X2 The concentration of each of the two drugs when combined produces an X effect. Since ABE and MIT act differently, α=0 was taken in this experiment. When CI is less than 1, the combined effect of the two medicines is synergistic, and the smaller the numerical value is, the stronger the synergistic effect is indicated; when ci=1, the combined effect of the two drugs is additive; when CI is more than 1, the combined effect of the two medicines is antagonism. Through calculation, the combination index CI values of the ABE and the MIT are smaller than 1 in different mass ratios, and CI=0.82 when the mass ratio of the ABE to the MIT is 4:1; ci=0.53 when the mass ratio of ABE to MIT is 2:1; when the mass ratio of ABE to MIT is 1:2, ci=0.78, indicating that ABE and MIT have a better synergistic effect, and the synergistic effect is strongest when the mass ratio is 2:1.

Example 2

Preparation of Abeli-mitoxantrone-polysialic acid (average molecular weight 30000 Dalton, degree of polymerization 100) polyion Complex (Abeli-mitoxantrone-polysialic acid nanocomposite)

In order to enhance the combined application effect of the drug of the abbe-cilia and the mitoxantrone in the mass ratio of (4:1) - (1:2), a polyion compound, taking polysialic acid as an example, is prepared by utilizing the acting force of charges and a polyanion material.

Precisely weighing 20mg of Abeli hydrochloride (ABE) and dissolving in 10mL of sterilized water for injection to obtain 2 mg.mL ^-1 Abbe-S Li Rongye (ABE-S) hydrochloride; precisely weighing 10mg mitoxantrone hydrochloride (MIT) and dissolving in 10mL sterilized water for injection to obtain 1 mg.mL ^-1 Mitoxantrone hydrochloride solution (MIT-S); precisely weighing 60mg of PSA, and dissolving in 20mL of sterilized water for injection to obtain 3mg.mL ^-1 Polysialic acid solution (PSA-S) of (C). Placing the drug mixed solution of ABE-S and MIT-S on a magnetic stirrer, keeping the stirring rotation speed at 100rpm, then dropwise adding PSA-S into the drug mixed solution, and incubating for 30min to obtain the Abeli-mitoxantrone-polysialic acid polyion compound. Finally, 50% glucose injection is adopted to adjust to isotonicity, wherein the final concentration of the Abeli is 1 mg.mL ^-1 Mitoxantrone final concentration of 0.5 mg.mL ^-1 . The prescription screening and optimizing results are shown in table 1, the electron microscope image is shown in figure 1, and the infrared spectrogram is shown in figure 2.

Experimental results show that when the mass ratio of polysialic acid to cationic drug (the drug composition of Abeli-mitoxantrone) is 1/2, a large amount of free cationic drug cannot be wrapped, and the administration dosage cannot be satisfied after the free drug is removed. When the mass ratio of polysialic acid to cationic medicine is increased to 2/1, the nano preparation has higher loading efficiency, and the average particle size and the polydispersity coefficient are better. With further increase of the amount of polysialic acid, the average particle size becomes larger, and the nano-preparation shows obvious opalescence although the compounding efficiency becomes higher. Based on the above results, we prefer a mass ratio of polysialic acid/cationic drug of 2/1 to ensure that the average particle size of the nano-formulation is suitable, no significant opalescence occurs and the particle size is evenly distributed.

TABLE 2 polyion Complex prescription screening and optimization

When the mass ratio of polysialic acid to cationic medicine is 2/1, the Abeli-mitoxantrone-polysialic acid polyion compound is prepared by adopting PSA with average molecular weight of 644.50, 957.73, 1270, 1585, 1900, 3000, 5000, 10000, 20000, 30000,4-5 ten thousand and 6-8 ten thousand daltons, and the result shows that when the average molecular weight is less than 3000, the stability of the preparation is less than 1 month (the average particle size is more than 230nm and filtration sterilization is difficult); when the average molecular weight is 3000-10000, the stability of the preparation is less than 5 months (the average grain diameter is more than 200 nm); when the average molecular weight is 10000-80000 daltons, the average grain diameter is 60-200nm, and the stability of the preparation is more than 6 months; when the average molecular weight is 20000-50000 dalton, the average particle diameter is 80-160nm, and the stability of the preparation is more than 6 months. Thus, the PSA molecular weight is selected to be 10000 to 80000 daltons, preferably 20000 to 50000 daltons.

Example 3

In vitro release investigation of polysialic acid nanocomposites

Drug release from polysialic acid nanocomposites was analyzed by dialysis bag method. The polysialic acid nano-complex is transferred into a treated dialysis bag, placed in phosphate buffer solution and dialyzed at a constant temperature of 37 ℃ +/-2 ℃ in a dark place. At a specific time point 2mL of dialysate was collected and an equal volume of blank release medium was replenished. The sample was diluted with the mobile phase and the released amounts of the drugs were analyzed at 296 and 658nm, respectively, using an ultraviolet spectrophotometer, and the cumulative release amount R of the drugs was calculated according to the following formula _n ％。

Wherein V is ₀ To release the medium volume, C _n For the n-th sampling concentration, V is the sampling volume, M _t Is the whole medicine concentration, i is an integer more than or equal to 1.

From the results, it was found that ABE-S and MIT-S were rapidly released at pH7.4 (37 ℃ C.), which is probably caused by rapid diffusion of free drug molecules through the dialysis membrane. The drug release rate of the nanocomposite set was significantly slower than the free drug, indicating that the nanocomposite can effectively delay the release of the drug in the in vivo circulation. (FIG. 3).

Example 4

And (5) enriching peripheral blood neutrophils by density gradient centrifugation and detecting purity.

The neutrophils are separated and purified by using a mouse neutrophil separation liquid kit (LZS 1100) of the company of Sijin company of biological products technology, and the whole process sample, reagent and experimental environment are all carried out at 20+/-2 ℃.

1. Preparing anticoagulated blood with heparin content of 10IU.mL ^-1 Whole blood.

2. A separate tube was taken, 3mL of separation solution I (outsourced) was added, and then 80% concentration separation solution II (separation solution I: sample dilution=4:1 mixture) was added: 1.5mL, forming a gradient interface.

3. Preparing a blood sample: after the anticoagulated blood and erythrocyte sedimentation liquid are uniformly mixed according to the proportion of 1:1, the mixture is carefully added on the gradient interface of the separation liquid, and the mixture is centrifuged for 20min at 800 g.

4. After centrifugation, two white cyclic cell layers appear under the plasma layer, the lower white cyclic neutrophil layer (mixed with a small amount of red blood cells) is taken out, 3-5 times of volume of cleaning solution is added for uniform mixing, and 400g is centrifuged for 10min.

5. Centrifuging and discarding the supernatant to obtain the neutral granulocyte.

To further quantitatively confirm the purity of neutrophils, we labeled neutrophils specifically with FITC-conjugated monoclonal anti-mouse Ly-6G/Ly-6C antibodies from bioleged Inc. The isolated and purified mouse neutrophils were washed once with PBS, gently blown into cells in RPMI-1640 medium containing 10% fetal bovine serum to form a cell suspension (1X 10) ⁶ personal/mL), transferred to a cell culture flask, placed at 37 ℃,5% co ₂ Culturing in an incubator for 0.5h. After taking out the flask, FITC-conjugated monoclonal anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (250 ng. Multidot.mL) was added ^-1 ) At 5% CO ₂ Culturing in an incubator at 37 ℃ for 0.5h, collecting cells, centrifuging at 5000rpm for 3min, and removing supernatant. Washing cells with PBS, centrifuging at 5000rpm for 3min, and discarding supernatantCells were resuspended by adding 200. Mu.LPBS, and the fluorescence intensity of the samples were measured on a flow cytometer, 1X 10 samples were collected for each sample ⁴ Cells were analyzed for fluorescence intensity using FITC channel and data were analyzed using FlowJo 10.4 (macOSMontrey 12.2) software to give a neutrophil purity of 95.3%. (see FIG. 4)

Example 5

Westernblot method for detecting expression quantity of L-selectin on neutrophil membrane

PBNs were isolated from normal mice and transfer mice and then cultured in 6-well plates (2 x 10 per well ⁵ Cells) for 1h, cells were lysed on ice using RIPA lysis buffer with protease inhibitors for 5min. After centrifugation of the lysate at 12,000rpm for 10min, the supernatant was used to quantify total protein using the enhanced BCA protein assay kit. Proteins were loaded onto SDS-PAGE of the same concentration and then transferred onto polyvinylidene fluoride (PVDF) membranes, blocked with skimmed milk. The primary antibody to CD62L and beta-actin was incubated overnight at 4℃for 1h at room temperature with HRP-conjugated secondary antibody and finally visualized on film.

During inflammation, L-selectin is highly expressed on neutrophil membranes, whereas neutrophil selective phagocytosis of polysialic acid nanocomposites is mediated by L-selectin. Compared with normal mice, the tumor lung metastasis mice highly express L-selectin on the neutrophil membrane. The results are shown in FIG. 5.

Example 6

Examination of the uptake of polysialic acid nanocomposites by neutrophils

The fluorescent probe DiR-labeled nanocomposite sets were prepared at a defined weight ratio (DiR: psa=1:2). DiR was dissolved in ethanol and then evaporated to near dryness. The aqueous PSA solution was added to the ethanol solution of DiR with stirring and stirred for 20min. After the preparation was completed, the ethanol was further removed in vacuo at room temperature.

Gently beating the isolated and purified neutrophils with RPMI-1640 medium without fetal bovine serum to form a cell suspension, and preparing into a concentration of 5×10 ⁵ Cell suspension at 1X 10 per mL ⁶ Density of wells/well inoculated into 6 well plates, 2mL of culture was added to each wellRadical at 37℃in 5% CO ₂ Culturing in an incubator for 0.5h. After the plate was removed, 100. Mu.L of the culture medium containing the sterile fluorescent probe DiR solution and the PSA-DiR nanocomposite was replaced with 5% CO ₂ Culturing in an incubator at 37 ℃ for 0.5h, collecting cells, centrifuging at 5000rpm for 3min, and removing supernatant. The cells were resuspended in PBS and washed, centrifuged at 5000rpm for 3min, and the supernatant was discarded. The fluorescence intensity of the samples was measured on a flow cytometer and the data was analyzed using FlowJo 10.4 (macos monitor 12.2) software. At the same time, another group of neutrophils was treated with FITC-Ly6G/Ly6C antibody (250 ng. ML ^-1 ) Cell membranes were labeled and nuclear counterstaining was performed using the nuclear dye DAPI. After the staining was completed, the cells were washed three times with cold PBS. And (3) lightly spin-drying the climbing slices, dripping the anti-fluorescence quenching sealing slice on a glass slide, taking out the cover glass from the culture plate, buckling the surface with cells attached on the glass slide downwards, removing bubbles, sucking up the overflowed sealing slice, sealing edges, and finally placing the climbing slices under a laser confocal microscope for observation and photographing.

Competitive inhibition experiments prove that the polysialic acid modified nano-composite can enhance the uptake of the neutrophils to the medicine through receptor-ligand specific recognition. Excess polysialic acid solution (10.0 mg. ML) was first used ^-1 ) Competing with neutrophils, inhibiting receptor recognition function on the cell surface, and then removing the supernatant by centrifugation and collecting the cells. Mouse neutrophils were incubated with polysialic acid modified nanocomposites for 2h at 37 ℃ (referred to as competitive inhibition group). The supernatant was removed by centrifugation and cells were collected, and after fixing the cells by adding 4% paraformaldehyde, the results were examined using a flow cytometer and a laser confocal microscope, as shown in fig. 6.

Laser confocal microscopy results showed that polysialic acid nanocomposites significantly enhanced DiR fluorescence signal in neutrophils compared to the solution set. It is shown that polysialic acid can effectively promote the uptake of the nano-complex by neutrophils, which is important for the drug to exert an in vivo immunotherapeutic effect.

The flow cytometry results further showed that the polysialic acid nanocomposite showed a stronger fluorescent signal in neutrophils compared to the solution group, suggesting a significant increase in the uptake of the formulation by the cells. In competitive inhibition experiments, the pre-incubation of polysialic acid solution significantly inhibited neutrophil uptake of polysialic acid nanocomposites. This result is consistent with laser confocal microscopy, indicating that free polysialic acid can bind to polysialic acid receptors on the cell surface as a competitive inhibitor, blocking the ability of polysialic acid nanocomposites to bind to neutrophil receptors, resulting in lower cellular uptake.

Example 7

CCK8 method for detecting influence of nano-composite on neutrophil viability

In order to examine the influence of the nano-composite on the viability of the neutrophils, considering that the survival time of the neutrophils is 6-8h, the MTT method (12 h in the color development process) cannot be used for measurement, so that the CCK8 method which can develop color only for 4h is adopted for measurement.

1. 100. Mu.L (about 5X 10) of neutrophil suspension ⁴ Per mL of cells) was added to a 96-well plate (edge wells were filled with sterile water or PBS). Blank wells (with medium, no cells) and control wells (medium without drug, with cells) were set, and 3 duplicate wells were set for each group.

3. mu.L of polysialic acid nano-complexes with different concentrations to be detected are added to each well, and incubated at 37 ℃.

CCK8 experimental results show that the concentration of the abbe-cilia and the mitoxantrone is 0.01-10 mug.mL ^-1 Within the scope, the cell viability of the solution group was significantly lower than that of the polysialic acid nanocomposite group. At 5. Mu.g.mL ^-1 Abeli and 2.5. Mu.g.mL ^-1 Is incubated at mitoxantrone concentrationThe neutrophil viability of the abbe-mitoxantrone-polysialic acid nanocomposite set was still over 75% during cell culture, and we therefore considered that we performed subsequent experiments (abbe-cele concentration of 5 μg mL ^-1 Mitoxantrone concentration of 2.5 μg mL ^-1 ) Neutrophil viability was not affected. The results are shown in FIG. 7.

Example 8

Investigation of Release behavior of neutrophils after uptake of nanocomposites

The MTT method is used for measuring cytotoxicity of the medicine released by the neutrophils on the tumor cells of the 4T1 mice, and the experimental method is as follows

(1) Neutrophils (1X 10) ⁶ Individual cells/ml) were incubated with different concentrations of drug or polysialic acid nanocomposites for 30 min, then separated by centrifugation and resuspended in DMEM medium containing 10% fetal bovine serum.

(2) 4T1 tumor cell suspensions were added to 96-well plates (edge wells filled with sterile water or PBS) at a cell density of 1X 10 cells per well ⁵ And each. Blank wells (with medium, no cells) and control wells (medium without drug, with cells) were set, and 3 duplicate wells were set for each group.

(3) The neutrophils obtained above were cultured for another 8 hours and then centrifuged. The medium of 4T1 cells was replaced with the supernatant released from neutrophils and incubated for a further 48h and observed under an inverted microscope.

(4) mu.L of the drug to be detected and the polysialic acid nano-complex with different concentrations are added into each hole, and the mixture is incubated for 48 hours at 37 ℃.

(5) mu.L of MTT solution was added to each well and incubated at 37℃for 4h.

(6) Formazan produced by living cells was lysed by adding triplex solution overnight to each well.

(7) The absorbance of each well was measured at a wavelength of 570 nm.

Analysis of results: the OD value of each test well was subtracted by either the zeroed well OD value or the control well OD value. OD values for each replicate well were averaged. Cell viability% = (dosing cell OD-blank OD)/(control cell OD-blank OD) ×100% and half Inhibition Concentration (IC) of cells was calculated ₅₀ )。

From the cytotoxicity analysis results, the viability of 4T1 tumor cells decreased with increasing drug concentration in the neutrophil medium, indicating that the polysialic acid nanocomposites released by neutrophils still retain their biological activity. The drug solution has only weak growth inhibition effect on 4T1 tumor cells, probably because the drug solution influences the activity of neutrophils to a certain extent, and the uptake efficiency of the polysialic acid nano-composite by the neutrophils is higher. The results indicate that neutrophils can phagocytose polysialic acid nanocomposites and deliver the formulation to sites of tumor, inflammation, etc. The results are shown in FIG. 8.

Example 9

Changes over time in the amount of neutrophils in the lung after tumor lung metastasis model establishment

The stored mouse breast cancer 4T1 cell cryopreservation tube is taken out of the liquid nitrogen and quickly placed into water at 37 ℃ for resuscitation. The resuscitated 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and when the activity of the tumor cells is more than 95%, physiological saline is added to dilute the cell suspension, and the dilution factor is adjusted. The 4T1 cell suspension was tail vein injected into mice using 75% alcohol for sterilization. After intravenous injection of

4T1 tumor cells

0, 3, 6, 9, 12 and 15 days in the tail of the mice, the neck skin of the anesthetized mice was incised after iodine disinfection. The salivary glands and muscles were blunt-separated with forceps to expose the trachea. Suture under the trachea, make a V-shaped incision in the cartilage ring in the middle of the trachea, insert the lavage needle into the trachea for about 0.5 cm, ensure that the lungs are not injured, ligate and fix. 1mL of PBS was slowly injected into the lungs of the mice, and repeated 3 times. Approximately 1.5mL of lung lavage fluid was collected and centrifuged for 3min. The cell pellet was washed 3 times with PBS and resuspended in paraformaldehyde. Finally, adding a neutrophil marker FITC-labeled anti-Ly6G/Ly6C antibody, and incubating for 20min in a dark environment at 4 ℃ to label neutrophils. After fixing the cells with 4% paraformaldehyde, the fluorescence intensity of the samples was measured on a flow cytometer via FITC channel and the experimental data was analyzed with FlowJo 10.4 (macos monterey 12.2) software.

Previous studies have shown that, in comparison with other immune cells,neutrophils are not present in large numbers in the primary tumor microenvironment, but are the primary immune cells that increase in the metastatic lung. To study the changes in the number of neutrophils recruited to the lung during metastasis, we analyzed lung lavage fluid at various time points after establishment of breast cancer lung metastases in mice using flow cytometry. Transfer Ly6G in the lungs of mice compared to the lungs of blank mice ⁺ Ly6C ⁺ The neutrophil count increased dramatically, reached a peak at day 12, and remained stable. Whereas neutrophils were only viable in mice for 18h, this suggests that tumor metastasis can lead to continued migration of neutrophils to the lungs. The results are shown in FIG. 9.

Example 10

Nanocomposite in vivo fluorescence imaging in 4T1 breast cancer lung metastasis BALB/c mice

The stored mouse breast cancer 4T1 cell cryopreservation tube is taken out of the liquid nitrogen and quickly placed into water at 37 ℃ for resuscitation. The resuscitated 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and when the activity of the tumor cells is more than 95%, physiological saline is added to dilute the tumor cells into the cell suspension, and the dilution factor is adjusted. The mice were then sterilized with 75% alcohol and the 4T1 cell suspension was tail-injected intravenously. The mice were randomly divided into 2 groups, i.e. a fluorescent probe DiR solution group and a fluorescent probe DiR polysialic acid nanocomposite group, 8 per group, on day 7 post-inoculation. 1 mg/kg ^-1 The dose tail vein is injected with fluorescent probe DiR solution and fluorescent probe DiR polysialic acid nano compound, living body fluorescent imaging is carried out at 1, 4, 8 and 24 hours respectively, and the fluorescence intensity of the in vivo tumor tissue of each group of mice is measured. Mice were sacrificed after 24h, tumors were isolated for ex vivo organ imaging with other major organs, and the average fluorescence intensity of each organ was measured.

The experimental result shows that the fluorescence of the nano probe is gradually detected by the lung of the mouse along with the extension of time, and the fluorescence intensities of the two groups reach peak values at 24 hours. In contrast, the fluorescent signal of the fluorescent probe DiR solution group is mainly distributed in the liver, while the signal of the lung is weak. These results indicate that the polysialic acid modified nano-composite can increase the uptake of neutrophils in the blood circulation, and the circulating neutrophils phagocytosing the nano-preparation carry the drug to cross the vascular wall and enter the lung under the drive of tumor chronic inflammation, thereby causing more drug to stay in the lung. Tumors and other major organs were collected 24h after injection of the formulation and the biodistribution of the formulation in mice was examined. In breast cancer lung metastasis mouse tissues, polysialic acid nanocomposites exhibit a stronger lung metastasis targeting ability. The results are shown in FIG. 10.

Example 11

Nanocomposite distribution in pulmonary tissue of 4T1 breast cancer pulmonary metastasis BALB/c mice

To further demonstrate that polysialic acid can effectively enhance drug phagocytosis by neutrophils in mice, lung-metastatic mice were divided into four groups, first into the blocking neutrophil group and the unblocking neutrophil group. Then, respectively injecting a fluorescent probe DiR solution or a fluorescent probe DiR polysialic acid nano-composite, marking neutrophils in lung metastasis tissues by using a neutrophil marker Ly6G/Ly6C, and observing the uptake of tumor-related neutrophils to the medicine.

Frozen section making

(1) Tissue fixation:

fresh tumor tissue is fixed by the fixing liquid for more than 24 hours, and the tissue of the target part is leveled by a scalpel after being taken out.

(2) Dehydrating:

placing the trimmed tissue in a 15% sucrose solution, dehydrating and sinking in a refrigerator at 4 ℃, and transferring the trimmed tissue into a 30% sucrose solution, dehydrating and sinking in a refrigerator at 4 ℃.

(3) OCT embedding:

taking out the dehydrated tissue, slightly sucking the surface water, then placing the surface water on an embedding table upwards, dripping an OCT embedding agent around the tissue, placing the embedding table on a frozen microtome for quick freezing embedding, and slicing after OCT becomes white and hard. The fresh tissue is frozen directly, the tissue of the target part is leveled by a scalpel directly without fixed dehydration, and then the tissue is embedded by an OCT embedding agent.

(4) Slicing:

the embedding table is fixed on a slicing machine, the tissue surface is roughly cut and flattened, slicing can be started, the slicing thickness is 8-10 mu m, a clean glass slide is placed above the cut tissue piece, and the tissue can be attached to the glass slide. The label is stored at-20 ℃ for standby after the label is written on the sheet.

(5) Immunofluorescent staining:

tumor sections were incubated with primary antibody for 8h at 4 ℃ and then with fluorescein-conjugated secondary antibody for 1h at room temperature.

(6) Nuclear dyeing:

the slide was washed 3 times with PBS (pH 7.4) on a decolorizing shaker in the absence of light for 5min each. And (3) dripping DAPI dye liquor after spin-drying the slices, and dying the nuclei at room temperature for 10min in a dark place.

(7) Sealing piece:

the slide was washed 3 times with PBS (pH 7.4) on a decolorizing shaker in the absence of light for 5min each. And (5) after the slices are dried, sealing the tablets by using anti-fluorescence quenching.

(8) And (5) microscopic examination and photographing:

sections were observed under a fluorescence microscope and images were acquired.

From immunofluorescence experimental results of lung sections, it was found that the fluorescent co-localization signal was stronger in the polysialic acid nanocomposite group Ly6G/Ly 6C-labeled neutrophils than in the fluorescent probe DiR solution group. These results indicate that polysialic acid is effective in increasing the uptake of the nanocomposite by neutrophils, which is consistent with the in vitro cellular uptake results. To further verify that the accumulation of polysialic acid nanocomposites in the lung was mediated by neutrophils, we injected anti-Ly6G antibodies intravenously into mice with tumor lung metastases, and set up a model of neutrophil depletion. After depletion of neutrophils, the targeting of polysialic acid nanocomposites was almost completely lost and the fluorescent signal was similar to that of the fluorescent probe DiR solution set. The results are shown in FIG. 11.

Example 12

Anti-tumor study of polysialic acid nano-composite on 4T1 tumor-bearing mice

The stored mouse breast cancer 4T1 cell cryopreservation tube is taken out of the liquid nitrogen and quickly placed into water at 37 ℃ for resuscitation. In vitro culturing of resuscitated 4T1 cell suspension in an inverted microscopeCount down, dilute the cell suspension with normal saline and adjust dilution factor when the activity of tumor cells is greater than 95%. The 4T1 cell suspension was inoculated onto a third pair of breast pads of mice, each of which was inoculated with 0.1mL, and 56 mice were inoculated, and the mice were randomly divided into 7 groups, i.e., a control group, a arbeli solution group, a mitoxantrone solution group, a pharmaceutical composition solution group of abbe-mitoxantrone, an abbe-polysialic acid nanocomposite group, a mitoxantrone-polysialic acid nanocomposite group, and an abbe-mitoxantrone-polysialic acid nanocomposite group, each group being 8. Each group of mice was dosed at 5 mg.kg of arbelide 3 times (3, 5, 7 days after inoculation) starting on day 3 after inoculation 1 time every 2 days ^-1 Mitoxantrone dosage is 2.5 mg.kg ^-1 The control group was given 5% dextrose injection. Data on body mass, mortality events, etc. were recorded throughout the pharmacodynamic trial.

The anti-tumor experimental result shows that the tumor inhibition rate is as follows in sequence: the abbe-mitoxantrone-polysialic acid nanocomposite group > abbe-weli-polysialic acid nanocomposite group > abbe-mitoxantrone pharmaceutical composition solution group > mitoxantrone solution group > abbe-weli solution group > control group. Tumor volumes increased continuously during the experiments in the arbelii solution group and in the mitoxantrone solution group. The drug composition solution group of the abbe-mitoxantrone has better tumor inhibition effect, which can be explained by lower bioavailability of the drug solution, but the abbe-west and the mitoxantrone play a synergistic effect, so that immune response of the whole body and tumor microenvironment is stimulated, and the anti-tumor effect is enhanced. The nanocomposite group can remarkably inhibit the tumor growth of tumor-bearing mice and prolong the survival time of the mice. Notably, the tumor volume of the mice of the abbe-mitoxantrone-polysialic acid nanocomposite group was significantly inhibited, with an average tumor inhibition of 72.45% at 26 days, significantly higher than the abbe-polysialic acid nanocomposite group and mitoxantrone-polysialic acid nanocomposite group. In addition, for the in situ triple negative breast cancer 4T1 model, the EPR effect is weaker due to the tumor located in the mouse breast, resulting in poor clinical treatment. The experimental results before the subject group show that the tumor inhibition rate of the PEG doxorubicin liposome on the 4T1 tumor-bearing mice is lower than 40 percent, which is far less than that of the Abeli-mitoxantrone-polysialic acid nano-composite group. Meanwhile, it can be observed that the lung weight of the mice in the Abeli-mitoxantrone-polysialic acid nanocomposite group is far smaller than that of the mice in the control group, and is close to that of the mice in the blank group after dissecting the mice.

Example 13

Pharmacodynamics evaluation of polysialic acid nano-composite on anti-tumor metastasis of 4T1 breast cancer lung metastasis BALB/c mice

The stored mouse breast cancer 4T1 cell cryopreservation tube is taken out of the liquid nitrogen and quickly placed into water at 37 ℃ for resuscitation. The resuscitated 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and when the activity of the tumor cells is more than 95%, physiological saline is added to dilute the tumor cells into the cell suspension, and the dilution factor is adjusted. The 4T1 cell suspension was tail vein injected into mice using 75% alcohol for sterilization. On day 3 after tumor bearing, mice were randomly divided into 7 groups, namely, a control group, an abbe-siroli solution group, a mitoxantrone solution group, a pharmaceutical composition solution group of abbe-siroli-mitoxantrone, an abbe-polysialic acid nanocomposite group, a mitoxantrone-polysialic acid nanocomposite group, and an abbe-siroli-mitoxantrone-polysialic acid nanocomposite group, each group of 8. Each group of mice was dosed at 5 mg.kg of arbelide 3 times (3, 5, 7 days after inoculation) starting on day 3 after inoculation 1 time every 2 days ^-1 Mitoxantrone dosage is 2.5 mg.kg ^-1 The control group was given 5% dextrose injection. Data on body mass, mortality events, etc. were recorded throughout the pharmacodynamic trial. On day 18 of tumor cell inoculation, all groups of lung specimens were resected and photographed while examining lung weight and number of metastases to assess inhibition of lung metastases. Hematoxylin and eosin (H) for pulmonary specimens &E) Staining and observation with a reverse microscope. Mice used for survival studies were treated in the same manner.

We analyzed the lung photographs of mice and the lung HE-stained metastatic nodules (dark-colored nuclei). Except for the abbe-mitoxantrone-polysialic acid nanocomposite, all groups clearly found metastases, indicating that the polysialic acid nanocomposite has a pronounced anti-metastatic capacity. The results are shown in FIGS. 12 and 13.

The solution group slightly inhibited lung metastasis of breast cancer compared with the control group, and the abbe-polysialic acid nano-composite group and the mitoxantrone-polysialic acid nano-composite group exert moderate anti-metastasis effect. The average number of transfer knots per lung of the abbe-mitoxantrone-polysialic acid nanocomposite group was 11, which is 10.3%, 12.4%, 13.7%, 15.1%, 29.3% and 28.8% of the control group, the abbe-celecoxib solution group, the mitoxantrone solution group, the abbe-mitoxantrone pharmaceutical composition solution group, the abbe-polysialic acid nanocomposite group and the mitoxantrone-polysialic nanocomposite group, respectively. The results are shown in FIG. 14.

The long-term survival of breast cancer lung metastasis mice was assessed by experiments for up to 40 days. The survival rate of the abbe-mitoxantrone-polysialic acid nanocomposite group was highest compared to all other groups and metastasis could be almost completely eliminated. The results are shown in FIG. 15.

The weight change of mice was analyzed during the test, and the safety of the nanocomposite was studied. No major changes in body weight occurred in the group injected with the nanocomposite. In particular, mice in the abbe-mitoxantrone-polysialic acid nanocomposite group had steadily increased body weight, while the control group and mitoxantrone solution group had decreased body weight, which may be related to the effect of lung metastases on the quality of life of mice or toxicity of mitoxantrone. The results are shown in FIG. 16.

The lung weight of adult mice is a constant value, related to the weight of the metastases. The average lung weight of mice treated with the solution (Abeli solution group: 0.62.+ -. 0.06g; mitoxantrone solution group: 0.35.+ -. 0.06 g) or single drug formulation (Abeli-polysialic acid nanocomposite group: 0.29.+ -. 0.02g; mitoxantrone-polysialic acid nanocomposite group: 0.28.+ -. 0.04 g) was significantly lower than that of mice of the control group (0.91.+ -. 0.02 g). Among all treatment groups, mice treated with the abbe-mitoxantrone-polysialic acid nanocomposite had the lowest lung weights (0.18±0.01 g), closest to the lung weights of the mice in the blank group (0.18±0.04 g). The results are shown in FIG. 17.

The Spleen index (Spleen index) is the ratio of Spleen mass (mg) to body mass (g) and reflects the immune function of the organism to a certain extent. The spleen is a peripheral immune organ, is a place where T, B lymphocytes settle, is also a place where immune cells execute immune response functions, and can secrete bioactive mediators (such as cytokines and the like). The spleen index of mice is commonly used in the literature to evaluate the effect of drugs on the immune system. However, the spleen index formula is observed to find that the unit of spleen mass/body mass is mg/g, the unit is not uniform, the spleen and body weight data cannot be intuitively obtained from the data, and the spleen and body weight data are simply compared with a control group as a measurement standard.

The lung weight of adult mice is a constant value, and when lesions occur in the lung (e.g., lung cancer, tumor lung metastasis, chronic obstructive pulmonary disease (Chronic obstructive pulmonary disease, COPD), pulmonary fibrosis, pulmonary edema, acute lung injury, tuberculosis), the lung becomes heavier. These lesions can lead to tumor growth, destruction of the alveolar walls and bronchioles, exudation of lung tissue, and other proliferative responses. Taking the spleen index as an example, we propose related index concepts of lung cancer inhibition index, lung metastasis inhibition index, chronic obstructive pulmonary disease treatment index, pulmonary fibrosis inhibition index, pulmonary edema inhibition index, lung injury treatment index, pulmonary tuberculosis inhibition index, and the like. In addition, by studying the change in lung weight in a population, quality of life conditions, such as pneumoconiosis, in a certain area or in a certain occupational population can be obtained.

To distinguish the indices associated with pulmonary diseases, we named the pulmonary index of particular utility as "pulmonary variability index (Lung Diseaseindex, LD) _index ) "(the formula is: LD (laser diode) _index =body weight (g)/lung weight (g)) and "lung weight index (lungwight index, LW _index ) "(the formula is: LW (LW) _index = Lung weight in the control and treatment groups (g)/Lung weight in the blank groups (g)). The above index of lung lesions can be replaced by both indices.

By comparing the lung weights after tumor metastasis with the lung photographs, it was found that the more and more severe tumor metastasis, the greater the lung mass of the mice. We can verify this finding by comparing the number of nodules to the lung redo ratio. The number of nodules is positively correlated with the weight of the lung, so we can think that measuring lung weight can infer the number of nodules, and more accurate data is obtained.

While lung weight and body weight have been considered classical parameters for anti-metastatic experiments, they are mutually independent indicators when evaluating anti-metastatic effects and toxicity. Here we propose "metastasis inhibition index (Metastasis inhibition index, MI _index ) "as a comprehensive parameter for evaluating anti-metastatic effect and toxicity".

The formula for the metastasis inhibition index is as follows:

MI _index ＝body weight(g)/lung weight(g).

the metastasis inhibition index may also reflect small and intrapulmonary metastases that cannot be directly observed to obtain more accurate values. The mice treated with the abbe-mitoxantrone-polysialic acid nanocomposite had significantly greater metastasis inhibition index than the other groups, indicating that the abbe-mitoxantrone-polysialic acid nanocomposite was the least toxic and the best anti-metastasis effect. However, the transfer inhibition index was the smallest in the abbe-cili solution group and the mitoxantrone solution group among all the treatment groups, almost the same as the control group. These data indicate that toxicity of the arbekiln solution group and mitoxantrone solution group results in the lowest Quality of life (QOL). Overall, the metastasis inhibition index provides a new evaluation index that more significantly reflects the differences in efficacy between treatment groups to improve the accuracy of anti-metastasis evaluation and promote clinical transformations of the underlying study. The results are shown in FIG. 18 and Table 3

TABLE 3 results of anti-4T 1 tumor metastasis experiments with different polysialic acid nanocomposites

Group of	Index of metastasis inhibition
		Control group	16.7±0.38
Abeli solution group	24.1±0.46
		Mitoxantrone solution sets	48.0±7.38
Abeli-mitoxantrone pharmaceutical composition solution set	57.2±2.84
		Abeli-polysialic acid nanocomposite set	71.8±13.9
Mitoxantrone-polysialic acid nanocomposite sets	73.4±8.35
		Abeli-mitoxantrone-polysialic acid nanocomposite set	108.6±7.48
Blank group	125.1±5.68

The receptors for PSA are highly expressed on the surface of a variety of leukocytes, including peripheral blood neutrophils (Peripheral blood neutrophils, PBN) that are greatly increased in tumor and tumor metastasis mediated chronic inflammatory responses. By combining with Selectin, siglec on the surfaces of neutrophils and other cells, the neutrophils penetrate through the vascular wall function by unique deformation, and release the medicine in tissues, so that the purpose of targeted delivery of the medicine is realized, and the curative effect is improved.

Mouse respiration experiment

In general, mice with tumor lung metastases have a smaller lung volume, resulting in poor breathing and increased respiratory rate. Thus, the following is adopted: the "mouse breath experiment" study related tumor metastasis indices.

In the experimental group, the respiration times of the mice in 1min are recorded, and the result shows that the average respiration times of the mice in the control group in 1min are 276 times which are far greater than 163 times of the mice in the blank group, thus indicating that the respiration times of the mice are influenced by tumor lung metastasis. In contrast, the average 1 minute breath times of mice of the abbe-mitoxantrone-polysialic acid nanocomposite group were 180 times, indicating that the polysialic acid nanocomposite can effectively inhibit metastasis with little impact on the lung capacity of the mice.

Example 14

Important tissue section of 4T1 breast cancer lung metastasis mice

To examine the effect of each nanocomposite on the major organs of mice, after the end of the anti-tumor metastasis experiment in 4T1 breast cancer lung metastasis mice, the heart, liver, spleen, kidney of each group of tumor-bearing mice were collected for HE pathological sections.

Paraffin section production

(1) Tissue fixation

Freshly stripped heart, liver, spleen, lung, kidney tissue was washed with PBS (ph 7.4), fixed with 4% paraformaldehyde for more than 24h, after fixation was completed, the tissue was removed from the fixation solution and trimmed with a surgical knife in a fume hood.

(2) Flushing

Placing the trimmed tissue blocks into an embedding box, placing the embedding box under distilled water, and flushing the tissues with lower running water speed for 12-24 h.

(3) Dewatering

Placing the embedding box into a hanging basket, placing into a dehydrator, dehydrating with ethanol with different gradients, sequentially reacting with 75% ethanol for 4h,85% ethanol and 90% ethanol for 2h, reacting with 95% ethanol for 1h, and dehydrating with anhydrous ethanol for 2 times.

(4) Transparent and wax-impregnated

Transfer each tissue into a closed vessel, xylene: ethanol (1:1, v/v) for 5-10 min, xylene I for 5-10 min, and xylene II for 5-10 min. After the transparency is finished, each tissue block is immersed in the completely melted paraffin (an incubator at 60 ℃) for 1h, and then the tissue block is transferred into new paraffin liquid for secondary paraffin dipping for 1h.

(5) Embedding

The tissue immersed with the wax is placed in an embedding machine for embedding. Firstly, putting melted wax into an embedding frame, taking out tissues from a dehydration box before the wax is solidified, putting the tissues into the embedding frame according to the requirement of an embedding surface, and attaching corresponding labels. Cooling at-20deg.C, taking out the wax block from the embedding frame after the wax is solidified, and trimming the wax block.

(6) Slicing

Placing the wax block on a paraffin slicer for continuous slicing, wherein the thickness of the slice is 5 mu m, floating the slice on the slice spreading machine, flattening the tissue at 40 ℃ with warm water, taking out the tissue with an anti-drop glass slide, and baking the slice in a 60 ℃ oven. And after the water is baked and the wax is baked, taking out the glass slide, and preserving at normal temperature for standby.

(7) HE staining

(8) Dewaxing paraffin section to water

The tissue slice is soaked in xylene I and xylene II solution for 20min,100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol for 5min, and finally washed with distilled water for 10min.

(9) Nuclear dyeing

The slices are placed into hematoxylin dye liquor for dyeing for 8min, distilled water is used for washing to remove floating color, then 1% hydrochloric acid alcohol is used for differentiating for 20s, and finally distilled water is used for washing to turn blue.

Chromatin

The sections were stained in eosin dye solution for 3min and rinsed with distilled water for 20s.

De-water sealing sheet

Soaking the slices sequentially with 95% alcohol I and 95% alcohol II for 5min, then with absolute alcohol I and absolute alcohol II for 5min, then with xylene I and xylene II for 5min, dehydrating and transparentizing, finally taking out the slices from xylene, air drying, and sealing with neutral resin.

Microscopic examination photographing

After the sealing sheet is dried, the glass slide is placed under an inverted microscope for observation.

From the observation of each organ section, each nanocomposite group was complete in mouse cardiomyocytes and no myocardial rupture was observed. For pathological sections of liver, the integrity of liver cell nucleus is not damaged, the morphology of liver cells and liver sinus epithelial cells is normal, and liver sinus has no congestion phenomenon. In addition, glomeruli and tubular epithelial cells were intact, kidney vesicles were normal, and no bleeding or inflammatory infiltrate was observed in all mouse kidney sections. No histopathological abnormalities, lesions, or degenerations were shown in the individual formulation groups in the spleen sections of the mice. The above results indicate that after 3 drug treatments, the mouse organs including heart, liver, spleen and kidney do not show histopathological abnormalities, lesions or degenerations, demonstrating that each of the nanocomposites can be safely used for mouse tumor treatment. The results are shown in FIG. 19.

Example 15

Pulmonary immune cell changes in 4T1 breast cancer lung metastasis mice

To investigate the triggering of the anti-tumor immune response by each treatment regimen, T cell infiltration in lung tissue of mice in each treatment group was determined using flow cytometry. The stored mouse breast cancer 4T1 cell cryopreservation tube is taken out of the liquid nitrogen and quickly placed into water at 37 ℃ for resuscitation. The resuscitated 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and when the activity of the tumor cells is more than 95%, physiological saline is added to dilute the tumor cells into the cell suspension, and the dilution factor is adjusted. Tail vein injection of 4T1 cell suspension into mice using 75% alcohol sterilizationIn vivo. On day 3 after tumor bearing, mice were randomly divided into 7 groups, namely, a control group, an abbe-siroli solution group, a mitoxantrone solution group, a pharmaceutical composition solution group of abbe-siroli-mitoxantrone, an abbe-polysialic acid nanocomposite group, a mitoxantrone-polysialic acid nanocomposite group, and an abbe-siroli-mitoxantrone-polysialic acid nanocomposite group, each group of 8. Each group of mice was dosed at 5 mg.kg of arbelide 3 times (3, 5, 7 days after inoculation) starting on day 3 after inoculation 1 time every 2 days ^-1 Mitoxantrone dosage is 2.5 mg.kg ^-1 The control group was given 5% dextrose injection. Data on body mass, mortality events, etc. were recorded throughout the pharmacodynamic trial. On day 18 of tumor cell inoculation, mice were sacrificed and tumor tissues were collected. Cutting tumor tissue to about 2X 2mm ³ Is placed in a newly prepared digestive juice (containing 0.5 mg.mL) ^-1 Collagenase IV and 0.2 mg.mL ^-1 DNase I) was incubated at 37℃for 1h. Undigested tissue pieces were filtered using a 70- μm nylon screen and the single cell suspension was collected by centrifugation. The resulting tumor cells were centrifuged at 1200rpm for 5min and resuspended in erythrocyte lysate to eliminate erythrocytes. After washing the cells with fresh PBS, anti-CD 3-FITC, anti-CD 8-APC, anti-CD 4-PerCP, anti-CD 25-APC and anti-Foxp 3-PE antibodies were added and stained at 4℃for 30min to label the T cells and detected by flow cytometry.

The results show that the immunosuppressive microenvironment of the lung impedes the improvement of the effector T cell function by abbe's, whereas mitoxantrone promotes the immunogenic effect at the site of metastasis, releasing the signal that elicits the immune response, thus providing the opportunity to heat the immune "cold" tumor. Abeli and mitoxantrone cooperate to achieve the best immune response for cancer immunotherapy. Flow cytometry results showed that the abbe-mitoxantrone-polysialic acid nanocomposite would be CD8 ⁺ The ratio of T cells was increased to 12.6% ± 1.0%. CD4 of Abeli-mitoxantrone-polysialic acid nanocomposite set ⁺ And CD8 ⁺ The ratio of T cells was 7.9-fold and 4.6-fold, respectively, compared to the control group. Regulatory T cells are suppressive immune cells that suppress the whole body by inducing and maintaining tolerance to self-antigensA sexual immune response. The regulatory T cell ratio of the abbe-mitoxantrone-polysialic acid nanocomposite group was reduced to 7.2% ± 1.0% compared to the control group. The regulatory T cell ratio was the lowest for the abbe-mitoxantrone-polysialic acid nanocomposite group compared to the other groups. The results are shown in FIGS. 20 and 21.

Example 16

Changes in lung cytokines in 4T1 breast cancer lung metastasis mice

To examine the changes in lung cytokines in mice with lung metastasis in each treatment regimen, the changes in cytokines in lung tissue of mice in each treatment group were measured by ELISA. The stored mouse breast cancer 4T1 cell cryopreservation tube is taken out of the liquid nitrogen and quickly placed into water at 37 ℃ for resuscitation. The resuscitated 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and when the activity of the tumor cells is more than 95%, physiological saline is added to dilute the tumor cells into the cell suspension, and the dilution factor is adjusted. The 4T1 cell suspension was tail vein injected into mice using 75% alcohol sterilization. On day 3 after tumor bearing, mice were randomly divided into 7 groups, namely, a control group, an abbe-siroli solution group, a mitoxantrone solution group, a pharmaceutical composition solution group of abbe-siroli-mitoxantrone, an abbe-polysialic acid nanocomposite group, a mitoxantrone-polysialic acid nanocomposite group, and an abbe-siroli-mitoxantrone-polysialic acid nanocomposite group, each group of 8. Each group of mice was dosed at 5 mg.kg of arbelide 3 times (3, 5, 7 days after inoculation) starting on day 3 after inoculation 1 time every 2 days ^-1 Mitoxantrone dosage is 2.5 mg.kg ^-1 The control group was given 5% dextrose injection. Data on body mass, mortality events, etc. were recorded throughout the pharmacodynamic trial. On day 18 of tumor cell inoculation, mice were sacrificed and tumor tissues were collected. After sufficiently grinding the lung tissue with a tissue refiner, it was centrifuged at 10000rpm for 10min. The concentrations of cytokines IL-10, TGF-beta, IFN-gamma and TNF-alpha in the supernatants were determined according to the instructions of each ELISA kit.

Regulatory T cells (Treg cells) are antigen-specific suppressor cells that exert immunosuppressive effects, primarily by releasing IL-10 or TGF- β. The lung immunocytokine levels measured by ELISA are shown below,among the groups, the lowest levels of IL-10 and TGF- β in the abbe-mitoxantrone-polysialic acid nanocomposite group indicate that the abbe-mitoxantrone-polysialic nanocomposite can significantly suppress adaptive T cells, improving the immunosuppressive environment. Inflammatory mediators TNF-alpha and IFN-gamma play an important role in anti-tumor immunity and promote CD8 ⁺ Proliferation and differentiation of T cells and enhancement of anti-tumor activity of the cells. Compared with other groups, the Abeli-mitoxantrone-polysialic acid nano-composite group obviously enhances the release of inflammatory mediators, reverses the immunosuppressive microenvironment and realizes the optimal immune response of cancer immunotherapy. The results are shown in FIG. 22.

Example 17

Preparation of other polyion complexes

Accurately weighing 20mg of Abeli hydrochloride, and dissolving in 10mL of sterilized water for injection to obtain 2mg.mL ^-1 Is a solution of arbeli hydrochloride; precisely weighing 10mg mitoxantrone hydrochloride and dissolving in 10mL sterilized water for injection to obtain 1 mg.mL ^-1 Mitoxantrone hydrochloride solution; precisely weighing 60mg of polyanion material (polysialic acid, hyaluronic acid, polyacrylic acid, alginic acid, carboxymethyl cellulose, carrageenan, polyglutamic acid, polyitaconic acid, heparin, gelatin, polybetaine, chondroitin sulfate, dextran sulfate, polymethyl methacrylate, polystyrene sulfonic acid, succinic acid and human serum albumin) and dissolving in 20mL of sterilized injection water to obtain 3 mg.mL ^-1 Is a polyanion solution of (a). Placing the drug composition solution of the abbe-sitagliptin and the mitoxantrone on a magnetic stirrer, keeping the stirring rotation speed at 100rpm, then dropwise adding the polyanion solution into the drug solution, and incubating for 30min to obtain the abbe-sitagliptin-polyion compound.

The result shows that the carboxymethyl cellulose can not be compounded with cationic drugs to obtain a polyion compound; polyion compound prepared by compounding polyacrylic acid, carrageenan, polyitaconic acid, gelatin, polybetaine, polymethyl methacrylate and cationic medicine is unstable; the pre-experimental effect of the drug effect of the nano-composite prepared by using the human blood albumin is poor, and the possible reason is presumably that the blood of the mice contains a large amount of protein, which can interfere with the targeting effect of the nano-composite and can not cause the nano-particles to accumulate at the tumor and the tumor metastasis sites; the nanocomposite prepared with succinic acid has good stability in vitro, but may be dissociated after injection into the body, and its drug effect is similar to that of the solution group. After the carrageenan compound is injected, the mice have inflammation, which indicates that a certain amount of carrageenan can induce inflammation and is not suitable for being used as a drug carrier;

Example 18

Pharmacodynamic evaluation of other polyion complexes against 4T1 breast cancer and pulmonary metastasis

The polyanion material is selected from hyaluronic acid (polymerization degree is 200), alginic acid (polymerization degree is 600), polyglutamic acid (polymerization degree is 3000), heparin, chondroitin sulfate, dextran sulfate and succinyl gelatin in the mass ratio of Abelide and mitoxantrone (4:1) - (1:2) for compatibility administration. Abeli is administered at a dose of 5 mg.kg ^-1 Mitoxantrone is administered at a dose of 2.5 mg.kg ^-1 . Accurately weighing 20mg of Abeli hydrochloride, and dissolving in 10mL of sterilized water for injection to obtain 2mg.mL ^-1 Is a solution of arbeli hydrochloride; precisely weighing 10mg mitoxantrone hydrochloride and dissolving in 10mL sterilized water for injection to obtain 1 mg.mL ^-1 Mitoxantrone hydrochloride solution; precisely weighing 60mg of polyanion compound, and dissolving in 20mL of sterilized water for injection to obtain 3mg.mL ^-1 Is a polyanion solution of (a). Placing the medicinal composition solution on a magnetic stirrer, keeping the stirring rotation speed at 100rpm, then dropwise adding the polyanion solution into the medicinal solution, and incubating for 30min to obtain the Abeli-mitoxantrone-polyion compound. Finally, 50% glucose injection is adopted to adjust to isotonicity, wherein the final concentration of the Abeli is 1 mg.mL ^-1 Mitoxantrone final concentration of 0.5 mg.mL ^-1 。

The stored mouse breast cancer 4T1 cell cryopreservation tube is taken out of the liquid nitrogen and quickly placed into water at 37 ℃ for resuscitation. The resuscitated 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and when the activity of the tumor cells is more than 95%, physiological saline is added to dilute the tumor cells into the cell suspension, and the dilution factor is adjusted. Sterilizing with 75% alcohol, and collecting 4T1 cell suspensionInjected into mice. On day 3 post-tumor bearing, mice were randomly divided into 24 groups of 8 mice each. Each group of mice was dosed at 5 mg.kg of arbelide 3 times (3, 5, 7 days after inoculation) starting on day 3 after inoculation 1 time every 2 days ^-1 Mitoxantrone dosage is 2.5 mg.kg ^-1 The control group was given 5% dextrose injection. Data on body mass, mortality events, etc. were recorded throughout the pharmacodynamic trial. The results are shown in Table 4.

TABLE 4 results of anti-4T 1 experiments with different polyanionic Polymer modified complexes

Group of	Index of metastasis inhibition
		Solution set	16.7±0.38
Hyaluronic acid	102.1±8.21
		Alginic acid	85.2±4.72
Polyglutamic acid	93.6±2.83
		Heparin	22.9±7.10
Chondroitin sulfate	88±8.42
		Dextran sulfate	73±9.42
Succinylated gelatin	86±10.41

After injection of heparin complex, mice showed bleeding and death, which were higher than the control group. The study proves that heparin can cause bleeding of mice due to the anticoagulation effect of heparin, and is not suitable for being compounded with cationic drugs to be used as a drug carrier.

Claims

1. The nanometer preparation of the anti-tumor pharmaceutical composition with the synergistic effect is characterized by comprising abbe seli and mitoxantrone, wherein the nanometer preparation is a polyion compound which is composed of a polyanion material and the anti-tumor pharmaceutical composition with the synergistic effect;

wherein the mass ratio of the polyanion material to the antitumor drug composition with synergistic effect is (1:2) - (30:1); the polyanion material is any one of polysialic acid, hyaluronic acid, alginic acid, polyglutamic acid, heparin, chondroitin sulfate, dextran sulfate and succinyl gelatin; the polymerization degree of the polysialic acid is 2-1000; the polymerization degree of the hyaluronic acid is 10-5000; the polymerization degree of the alginic acid is 300-1000; the polymerization degree of the polyglutamic acid is 1000-15000.

2. A method for preparing a nano-preparation of the anti-tumor pharmaceutical composition with synergistic effect as claimed in claim 1, comprising the following steps:

(1) Dissolving the medicinal composition with pure water to obtain solution A with concentration of 0.1 mg.mL ^-1 ～10mg·mL ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The mass ratio of the abbe-cilia to the mitoxantrone in the pharmaceutical composition is (8:1) - (1:6);

(2) Dissolving polyanionic material in pure waterObtaining solution B with the concentration of 0.1 mg.mL ^-1 ～100mg·mL ^-1 ；

(3) Mixing the solution A and the solution B to obtain a polyion compound; wherein the mass ratio of the polyanionic material to the pharmaceutical composition is (1:2) - (30:1).

3. Use of a nano-preparation of an antitumor pharmaceutical composition with a synergistic effect as claimed in claim 1 for the preparation of a medicament for treating breast cancer lung metastasis.