CN114469953A

CN114469953A - Antitumor drug composition with synergistic effect, nano preparation, preparation method and application thereof

Info

Publication number: CN114469953A
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: 2022-05-13
Anticipated expiration: 2042-02-14
Also published as: CN114469953B

Abstract

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

Description

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

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-tumor medicine composition with a synergistic effect, a nano preparation containing the medicine composition, and a preparation method and application of the nano preparation.

Background

Tumor (Tumor), a disease caused by disorders that control cell division and proliferation mechanisms. In addition to uncontrolled division, cancer cells can locally invade surrounding normal tissues and even migrate to other parts of the body via the systemic circulation or lymphatic system. According to 2020 Global Cancer Statistics reports (Global Cancer Statistics 2020), about 1930 million new Cancer cases are diagnosed globally, and the number of Cancer deaths is estimated to be 1000 million.

Tumor metastasis is 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 detach from the primary tumor, migrate, invade, reach different sites through blood and lymph vessels, then adhere and grow. Compared with the primary tumor, the tumor metastasis is widely distributed, small in size, high in nonuniformity and difficult to excise by operation. Therefore, there is a need to find an effective and safe metastatic targeted therapy strategy to improve the survival rate of patients and reduce the side effects of chemotherapeutic drugs.

In recent years, with the continuous development of biomedical technology, new therapies such as molecular targeted therapy and immunotherapy are continuously entering the clinic to become new strategies for tumor therapy. However, due to the complex pathological mechanism of tumor, no matter traditional chemical drugs or novel biological drugs, the single-drug therapy only relying on a single anti-tumor mechanism is difficult to obtain a substantial clinical benefit. Based on the complexity of tumor pathology, the combined use of antitumor drugs has gradually become a new clinical treatment trend.

Combination, i.e. the simultaneous administration of two or more drugs, becomes critical to achieve long-term prognosis and to reduce adverse side effects. Combination therapy can modulate different signaling pathways in tumor cells, leading to synergistic, additive, and potentiating effects, greatly enhancing therapeutic efficacy. As far as 2020, nearly 6000 tumor combination tests were registered in clinicaltralals. gov, it can be seen that the anti-tumor combination drugs have gradually gained attention in clinic and gradually developed into the mainstream treatment scheme.

With the rapid development of nano medicine, a nano drug delivery system is expected to improve the defects of the existing anti-tumor multi-drug combined delivery strategy and improve the treatment effect. The related research provides more new possibilities for the co-delivery of multiple drugs, and the tumor multi-drug combination strategy based on the nano-drug delivery system has more diversified functionalities compared with the traditional 'cocktail' therapy and has shown better treatment effect.

Neutrophils, the most abundant circulating leukocytes in the human body, are important immune cells for protection against infection. However, recent studies have found that neutrophils are one of the main driving forces for cancer metastasis formation, participate in the whole process of cancer metastasis, and play multiple metastasis-promoting roles in the metastasis process. As tumor metastasis progresses, the number of circulating neutrophils increases and can be recruited preferentially and in large numbers to the site of metastasis. In view of the spontaneous, massive recruitment of neutrophils to the metastatic site and their multiple functions, it is recognized that neutrophils can serve as a vehicle for drug delivery to enhance therapeutic efficacy.

Abelib (Abemaciclib, ABE), an inhibitor of Cyclin Dependent Kinases (CDKs) 4 and 6, has become an effective drug for treating breast cancer. Several studies have shown that ABE may affect a variety of immune cells in the tumor microenvironment, including inhibiting Treg cell proliferation and enhancing tumor infiltrating T cell (TILs) activation. However, ABE is less effective in certain tumors, possibly associated with a tumor microenvironment lacking TILs infiltration and "immune cold", and therefore combination therapy strategies may enhance the anti-tumor effect of ABE. Mitoxantrone can induce Immunogenic Cell Death (ICD), increase immune response, and overcome the "immune cold" tumor microenvironment by recruiting TILs. It has synergistic effect with ABE and is expected to raise the curative effect.

Disclosure of Invention

The invention aims to provide an anti-tumor drug composition with synergistic effect and prepare a nano preparation thereof aiming at the defects of the prior art. The pharmaceutical composition and the nano preparation can effectively improve the immunosuppressive microenvironment and the general immune environment of the tumor and the tumor metastasis part by utilizing the synergistic effect, the accumulation effect and the enhancement effect caused by the combined medication, and improve the curative effect of the medicine.

The invention further aims to provide application of the pharmaceutical composition and the nano preparation in preparation of anti-solid tumor and tumor metastasis treatment drugs.

The solid tumors comprise oral cancer, gastrointestinal cancer, colon cancer, gastric cancer, pulmonary tract cancer, lung cancer, breast cancer, ovarian cancer, prostatic cancer, uterine cancer, endometrial cancer, cervical cancer, bladder cancer, pancreatic cancer, bone cancer, liver cancer, gall bladder cancer, kidney cancer, skin cancer, testicular cancer, melanoma and sarcoma. The tumor metastasis organs are lung, lung on the other side, liver, lymph, bone, brain and adrenal gland.

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

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

The concentration of Abelide in the pharmaceutical composition is 0.1 mg/mL^-1-50mg·mL^-1Preferably 1 mg/mL^-1-40mg·mL^-1(ii) a The mitoxantrone concentration was 0.1 mg/mL^-1-20mg·mL^-1Preferably 0.5 mg/mL^-1-15mg·mL^-1. The pharmaceutical composition further comprises a pharmaceutically acceptable carrierAdjuvant and carrier.

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

The invention also provides a dosage form of the pharmaceutical composition, which comprises any one of a solid preparation, a liquid preparation and a semi-solid preparation. The dosage form is a nano preparation and comprises a polyion compound, a phospholipid compound, a liposome, nanoparticles, a nanocapsule and a polymer micelle.

Under certain conditions, oppositely charged polyelectrolytic interactions can form polyelectrolyte complexes, also known as Polyionic complexes (PICs). The polyelectrolytes capable of reacting include acids, bases, salts and the like in the form of polymers, and even include certain biological macromolecules and ionic surfactants.

The application of polyion complex in drug delivery has the remarkable characteristic that the core of the polyion complex can be used as a trace reservoir of charged compounds (such as genes, enzymes, drugs and the like) so as to adjust the inherent characteristics of the polyion complex, such as stability, solubility and reactivity.

The polyion complex is a complex with the average particle size of the common carrier (namely the pharmaceutical composition) of Abeli and mitoxantrone modified by polyanion materials at a nanometer level.

Wherein the mass ratio of the polyanionic material to the pharmaceutical composition is (1:2) - (30:1), preferably (1:1) - (10: 1). The polyanionic 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, and 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 polymerization degree of the polyglutamic acid is 1000-15000, preferably 2000-5000.

Sialic Acid (SA), also known as sugar acids, is a class of nine-carbon monosaccharides that are predominantly short-chain residues linked by α -glycosidic linkages to the termini of glycoproteins, glycolipids and oligosaccharides, and are ubiquitous on the surface of mammalian cell membranes. In addition, many pathogens "dress" themselves with SA to mask self epitopes, inhibit alternative complement activation pathways, reduce immunogenicity and thus successfully escape the attack of the host immune system. Polysialic acid (PSA) is a homopolymer of a plurality of SA monomers linked in α -2,8 and/or α -2, 9. PSA has a structure similar to the structure of a signal molecule of a sugar chain at the end of lipopolysaccharide, lipoprotein, etc. on the surface of a human cell, and is non-immunogenic in the human body. In higher organisms, PSA exists mainly in the form of complexes with other molecules such as nerve cell adhesion molecules, lactose, proteins or lipids, and the unique property and biological function of polysialic acid make the application of the polysialic acid in the fields of food health care, medicine, cosmetics and the like increasingly wider, so that the research and application value of the polysialic acid is increasingly highlighted as a high value-added material.

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

(1) dissolving the pharmaceutical composition in pure water to obtain solution A with concentration of 0.1 mg/mL^-1～10mg·mL^-1(ii) a The mass ratio of Abelide to mitoxantrone in the pharmaceutical composition is (8:1) - (1:6), preferably (4:1) - (1: 2);

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

(3) And mixing the solution A and the solution B to obtain the polyion compound. The mass ratio of the polyanionic material to the pharmaceutical composition is (1:2) - (30:1), preferably (1:1) - (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 requirement. Or freeze drying or spray drying, wherein the freeze-drying protective agent can be mannitol, trehalose, sorbitol, sucrose, lactose, maltose, or dextran, to obtain solid product.

Other adjuvants such as EDTA (disodium salt, calcium sodium salt), human serum albumin, poloxamer, dextran, polyvinylpyrrolidone, polysaccharides (such as panaxan, Astragalus polysaccharides, lentinan) and pH regulator can also be added into the formulation.

In the preparation prepared from the pharmaceutical composition of Abelide and mitoxantrone, the auxiliary materials used in the preparation are on the premise of not reacting with the pharmaceutical composition or influencing the curative effect of the medicament according to different preparation forms and preparation specifications.

According to the invention, the Abelix and the mitoxantrone are combined for the first time, and the combination of the Abelix and the mitoxantrone in the pharmaceutical composition has a synergistic effect, and the pharmaceutical composition has the advantages of high antitumor activity at low concentration, high efficiency and low toxicity. Mitoxantrone is used for increasing immune response, tumor microenvironment of 'immune cold' is overcome by recruiting TILs, and synergistic effect is generated by inhibiting Treg cell proliferation and enhancing activation of tumor infiltrating T cells (TILs) of Abeli. And simultaneously, the compound with the average particle size of about 100nm, which is carried by Abelide and mitoxantrone together, is prepared, so that the tumor-targeted nano injection is further prepared, and the excellent anti-tumor effect is shown after the injection of 4T1 breast cancer lung metastasis mice. The nano-scale compound also has the following characteristics: 1. the preparation process does not need specific synthesis conditions or additional reagents, and has good biocompatibility, biodegradability, non-toxicity and no immunogenicity. The preparation method is simple and convenient and is suitable for large-scale production; 2. the self neutrophil migration is utilized to target a lung metastasis, so that the disadvantage is converted into the advantage; 3. reversing immunosuppressive microenvironment, affecting the large tumor environment of the whole body, and completely eliminating metastasis. The efficacy of the nano-scale compound of the invention is far superior to that of a pharmaceutical composition solution, a single drug preparation, and simultaneous or sequential administration.

Most importantly, the polyanion material modified Abetiril and mitoxantrone co-carried polyion compound 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 formulation of the pharmaceutical composition of the present invention may be varied as appropriate, but it is essential to ensure that the pharmaceutical composition achieves effective therapeutic effect in vivo.

The invention has the beneficial effects that:

the pharmaceutical composition has a synergistic effect, and can be applied to preparation of drugs for preventing and/or treating solid tumors and tumor metastasis through targeting of the nano preparation and the neutrophils. The pharmaceutical 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 development of drug combination and nano drug delivery systems.

Drawings

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

FIG. 2 is a graph of infrared spectra of various substances and nanocomposites;

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

FIG. 4 shows the purity of neutrophils as detected by flow cytometry;

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

FIG. 6 is a study of the uptake of the nanocomposite by neutrophils; a: confocal microscopy imaging technique B: flow cytometry;

FIG. 7 is a CCK8 method for detecting the effect of the nano-composite on the activity of neutrophils;

FIG. 8 is a MTT method for detecting the effect of the nano-composite on the viability of 4T1 tumor cells;

FIG. 9 is a graph of the amount of neutrophils in the lung as a function of time after the tumor lung metastasis model was established;

FIG. 10 shows fluorescence imaging of the nanocomposites in 4T1 mice with lung metastasis of breast cancer BALB/c; a: lung metastasis mice in vivo fluorescence imaging B: in vitro organ fluorescence imaging;

FIG. 11 shows the distribution of the nano-complexes in lung tissues of BALB/c mice with 4T1 mammary cancer lung metastasis;

FIG. 12 is a photograph of the lungs of BALB/c mouse with 4T1 mammary cancer lung metastasis;

FIG. 13 is a section of 4T1 lung tissue from BALB/c mice with metastatic breast cancer;

FIG. 14 shows the number of BALB/c mouse lung metastasis nodules in 4T1 mammary cancer lung metastasis;

FIG. 15 is a BALB/c mouse survival curve for lung metastasis of 4T1 breast cancer;

FIG. 16 shows the change in body weight of BALB/c mice with 4T1 mammary cancer lung metastasis;

FIG. 17 shows the lung weights of BALB/c mice with 4T1 mammary cancer lung metastasis;

FIG. 18 is the BALB/c mouse metastasis suppression index for 4T1 breast cancer lung metastasis;

FIG. 19 is a section of important tissues of BALB/c mouse with 4T1 mammary cancer lung metastasis;

FIG. 20 shows the lung metastasis of 4T1 breast cancer BALB/c mouse lung CD4⁺T，CD8⁺(ii) a T cell change; a: flow cytometry results B: CD4⁺T cell C: CD8⁺A T cell;

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

FIG. 22 shows the change of BALB/c mouse lung cytokine in lung metastasis of 4T1 breast cancer.

Detailed Description

The main reagents, instruments and equipment used in the invention and animals:

the instrument comprises the following steps: BS124s electronic analytical balance (sydoris, germany); DF-101S heat collection type constant temperature heating magnetic stirrer (Guyi City Yingyu Yuhua apparatus factory); a PB-10 type pH meter (Siderelis, Germany); TDL-80-2S centrifuge (Shanghai' an pavilion scientific instruments factory); UV1801 type ultraviolet-visible spectrophotometer (beijing rayleigh analytical instruments ltd); thermo Scientific Forma CO₂Carbon dioxide incubators (shanghai semer feishel technologies ltd); TM-4 dynamic sterilizer (Shenyang Meida scientific instruments Co., Ltd.); SCIENTZ-30F lyophilizer (Ningbo Xinzhi Biotech Co., Ltd.); BALB/c mice (18-22 g, female, Shenyang university of pharmacy animal center); 0.45 μm polyvinylidene microporous membrane (Shanghai Movix scientific instruments Co., Ltd.).

Reagent testing: in the invention, the polymerization degrees of the PSAs are respectively 2, 3, 4, 5, 6, 10, 16, 32, 100, 130-170, 200 and 270, the corresponding molecular weights are respectively 644.50, 957.73, 1270.97, 1584.21 and 1897.45, and the average molecular weights are respectively 3000, 5000, 16000, 30000, 4-5 ten thousand and 6-8 ten thousand daltons. PSAs with a degree of polymerization of 2, 3, 4, 5, 6 were purchased from Nacalai, USA; PSAs with a degree of polymerization of 7 to 10 were purchased from Nacalaitesque, japan; PSA (average molecular weight 11.0kDa, polydispersity (p.d.). sub.1.17; average molecular weight 22.7kDa, polydispersity index (p.d.). sub.1.34; average molecular weight 39.0kDa, polydispersity (p.d.). sub.1.40) from Camida, Ireland; average molecular weight 30kDa was obtained from Carbosynth, UK (Calboses chemical technology, Suzhou, Ltd.); PSA of the remaining molecular weight is self-made. Abeli bulk drugs (ABE, Beijing Feng Libokozhi Co., Ltd., purity not less than 99%); mitoxantrone bulk drug (MIT, Beijing Huafeng Bibock technologies, Inc., purity is more than or equal to 99%); absolute ethanol (analytically pure, department of Tianjin, Mimi European chemical reagent development center); 5% glucose injection (5% Glu, Chenxin pharmaceutical Co., Ltd.); 50% glucose injection (50% Glu, Xinzheng GmbH, Tianjin pharmaceutical group); sterilized water for injection (Shijiazhuang Siyao Co., Ltd.).

Example 1

Cell viability assay

The research applies a breast cancer cell line 4T1, and cells are cultured in 5% CO₂95% air, saturated humidity and 37 ℃, cultured in the corresponding culture solution, and 10% newborn calf serum was added. In each experiment, the cells were seeded at a density of 5X 10⁴/mL。

1. 100. mu.L (about 5X 10) of 4T1 cell suspension was added⁴mL cells) were added to a 96-well plate (marginal wells filled with sterile water or PBS). Blank wells (medium, no cells) and control wells (medium without drug, cells) were set, and 3 more wells were set for each group.

2. Standing at 37 deg.C for 5% CO₂Incubate for 0.5h and observe under an inverted microscope.

3. Add 10. mu.L of the drug dilution medium to be tested at different concentrations to each well and incubate at 37 ℃.

4. Add 10. mu.L of LCCK-8 solution to each well and incubate at 37 ℃ for 4 h.

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

6. And (4) analyzing results: the OD value of the zeroing well or the control well was subtracted from the OD value of each test well. The OD values of each replicate well were averaged. Cell viability% ((dosed cells OD-blank OD)/(control cells OD-blank OD) × 100%)

Whether the combination of Abelix and mitoxantrone has synergistic effect, additive effect or antagonistic effect is analyzed by a combination index method created by Wetts.

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

TABLE 1 the individual action and the combined non-equal ratio of the two solutions in the inhibition of 4T1 cells

Taking the Median-effect Principle (middle effect Principle or Chou-Talalay combined index method) as an evaluation basis, drawing a combination index curve under different effects by using CompuSyn statistical software, and quantitatively evaluating whether a synergistic effect exists between the two medicines according to the relationship between the effect of the combination of the two medicines and the combination index. Common usage index CI ═ D₁/D_X1+D₂/D_X2+αD₁D₂/D_X1D_X2Wherein D is₁、D₂The concentrations of the two drugs when used singly, D_X1、D_X2Is produced when two medicines are used togetherThe concentration of each of the two drugs is responsible for the X effect. Because the mechanism of action of ABE is different from that of MIT, α is 0 in this experiment. When CI is less than 1, the effect of the two medicines is synergistic, and the smaller the numerical value is, the stronger the synergistic effect is; when CI is 1, the effect of combining the two medicines is addition; when CI is more than 1, the effect of the two drugs is antagonism. Through calculation, the CI values of the combined indexes of the ABE and the MIT are both less than 1 when the mass ratio of the ABE to the MIT is different, and when the mass ratio of the ABE to the MIT is 4:1, the CI is 0.82; when the mass ratio of ABE to MIT is 2:1, CI is 0.53; when the mass ratio of ABE to MIT is 1:2, CI is 0.78, which shows that ABE and MIT have 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 daltons, degree of polymerization 100) polyion complex (Abeli-mitoxantrone-polysialic acid nanocomposite)

In order to enhance the drug combination effect of Abelide and mitoxantrone in the mass ratio of (4:1) - (1:2), a polyion complex is prepared by utilizing charge acting force and polyanion materials, taking polysialic acid as an example.

Precisely weighing 20mg Abelide hydrochloride (ABE) and dissolving in 10mL sterilized water for injection to obtain 2 mg/mL^-1Abelian hydrochloride solution (ABE-S); precisely weighing 10mg mitoxantrone hydrochloride (MIT) and dissolving in 10mL sterilized water for injection to obtain 1 mg/mL^-1Mitoxantrone hydrochloride solution (MIT-S); precisely weighing 60mg PSA, and dissolving in 20mL sterilized water for injection to obtain 3 mg/mL^-1The polysialic acid solution (PSA-S). And (3) placing the drug mixed solution of ABE-S and MIT-S on a magnetic stirrer, keeping the stirring 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 complex. Finally, the mixture is adjusted to be isotonic by adopting 50 percent glucose injection, wherein the final concentration of Abeli is 1 mg.mL^-1Final concentration of mitoxantrone 0.5 mg/mL^-1. The prescription screening and optimizing results are shown in table 1, the electron microscope picture is shown in attached figure 1, and the infrared spectrogram is shown in attached figure 2.

The experimental result shows that when the mass ratio of the polysialic acid to the cationic drug (the pharmaceutical composition of Abelide-mitoxantrone) is 1/2, a large amount of free cationic drug cannot be wrapped, and the dosage cannot be satisfied after the free drug is removed. When the mass ratio of the polysialic acid to the cationic drug is improved to 2/1, the group of nano preparations has higher loading efficiency and better average particle size and polydispersity. With further increase in the amount of polysialic acid used, the average particle diameter became larger although the complexing efficiency became higher, and the nano-formulation appeared noticeably opalescent. Based on the above results, we prefer the ratio of polysialic acid/cationic drug mass to be 2/1 to ensure that the average particle size of the nano-preparation is appropriate, no significant opalescence occurs and the particle size is uniformly distributed.

TABLE 2 polyion Complex formulation screening and optimization

When the mass ratio of the polysialic acid to the cationic drug is 2/1, PSA with the 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 is adopted to prepare the Abeli-mitoxantrone-polysialic acid polyion complex, 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 the filtering sterilization is difficult to adopt); when the average molecular weight is 3000-10000, the preparation stability is less than 5 months (the average particle size is more than 200 nm); when the average molecular weight is 10000-80000 daltons, the average particle size is 60-200nm, and the preparation stability is more than 6 months; when the average molecular weight is 20000-50000 daltons, the average particle size is 80-160nm, and the preparation stability is more than 6 months. Therefore, the molecular weight of PSA is selected to be 10000-80000 daltons, preferably 20000-50000 daltons.

Example 3

In vitro release study of polysialic acid nanocomplexes

The drug release of the polysialic acid nanocomplexes was analyzed by the dialysis bag method. Transferring the polysialic acid nanocomplex into a treated dialysis bag, placing the dialysis bag in a phosphate buffer solution, and keeping the dialysis bag at 37 ℃ +/-2 ℃And (5) performing light constant temperature dialysis. At a particular time point, 2mL of dialysate was collected and supplemented with an equal volume of blank release medium. Diluting the sample with mobile phase, analyzing the amount of released drug at 296 nm and 658nm respectively by using ultraviolet spectrophotometer, and calculating the cumulative release amount R of drug according to the following formula_n％。

Wherein, V₀To release the volume of medium, C_nConcentration at the nth sampling, V is the sampling volume, M_tI is an integer greater than or equal to 1 for the total drug concentration.

From the results, it was found that ABE-S and MIT-S were rapidly released at pH7.4(37 ℃), which is probably caused by rapid diffusion of free drug molecules through the dialysis membrane. The drug release rate of the nanocomposite group was significantly slower than that of 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) performing density gradient centrifugation to enrich peripheral blood neutrophils and detecting the purity.

The neutrophils are separated and purified by adopting a mouse neutrophil separating medium kit (LZS1100) of a tertiary ocean biological product science and technology Limited company in Tianjin, and the whole process of samples, reagents and experimental environment is carried out at the temperature of 20 +/-2 ℃.

1. Preparing anticoagulant blood, the adding amount of heparin is 10 IU.mL^-1Whole blood.

2. 3mL of a separation solution I (purchased from outsourcing) was added to one centrifuge tube, and then an 80% concentration separation solution II (a mixture of the separation solution I: a sample diluent: 4:1) was added: 1.5mL, forming a gradient interface.

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

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

5. Centrifuging and then removing the supernatant to obtain the neutrophil.

To further quantify the purity of neutrophils, we specifically labeled neutrophils with a FITC-conjugated monoclonal antibody to mouse Ly-6G/Ly-6C from Biolegend. Washing the separated and purified mouse neutrophilic granulocyte with PBS, adding RPMI-1640 culture medium containing 10% fetal calf serum, and gently blowing to obtain cell suspension (1 × 10)⁶one/mL), transferred to a cell culture flask, placed at 37 ℃ in 5% CO₂Culturing in an incubator for 0.5 h. After the culture flask was taken out, an FITC-conjugated monoclonal antibody, Ly-6G/Ly-6C (Gr-1) antibody (250 ng. mL), was added^-1) At 5% CO₂Culturing at 37 deg.C for 0.5h, collecting cells, centrifuging at 5000rpm for 3min, and removing supernatant. Adding PBS to wash the cells, centrifuging at 5000rpm for 3min, discarding the supernatant, adding 200 μ L PBS to resuspend the cells, detecting the fluorescence intensity of the samples on a flow cytometer, collecting 1 × 10 samples⁴The cells were analyzed for fluorescence intensity using FITC channel and FlowJo 10.4(macOSMonterey 12.2) software, and the purity of neutrophils was 95.3%. (see FIG. 4)

Example 5

Westernblot method for detecting expression quantity of L-selectin on neutrophile granulocyte cell membrane

PBNs were isolated from normal and transfer mice and cultured in 6-well plates (2X 10 per well)⁵Individual cells) for 1h, cells were lysed using RIPA lysis buffer with protease inhibitors on ice for 5 min. After centrifugation of the lysates at 12,000rpm for 10min, the supernatants were assayed for total protein using the enhanced BCA protein assay kit. The proteins were loaded on SDS-PAGE at the same concentration and then transferred to polyvinylidene fluoride (PVDF) membrane for blocking with skim milk. CD62L and β -actin primary antibodies were incubated overnight at 4 ℃ with HRP conjugated secondary antibodies for 1h at room temperature and finally developed on film.

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

Example 6

Examination of uptake of polysialic acid nanocomposite by neutrophil

The fluorescent probe DiR-labeled set of nanocomposites was prepared at a determined 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 DiR ethanol solution with stirring and stirred for 20 min. After preparation was complete, the ethanol was further removed in vacuo at room temperature.

Gently beating the separated and purified neutrophils with RPMI-1640 culture medium without fetal calf serum to obtain cell suspension, and preparing into 5 × 10⁵Cell suspension at 1X 10/mL⁶The density of each well was inoculated into 6-well plates, 2mL of the medium was added to each well, and the plates were incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 0.5 h. After the plate was removed, 100. mu.L of the culture medium containing the sterile fluorescent probe DiR solution and PSA-DiR nanocomposite was replaced with 5% CO₂Culturing at 37 deg.C for 0.5h, collecting cells, centrifuging at 5000rpm for 3min, and removing supernatant. Adding PBS to resuspend the cells, washing, centrifuging at 5000rpm for 3min, and discarding the supernatant. The fluorescence intensity of the samples was measured on a flow cytometer and the data analyzed using FlowJo 10.4(macOSMonterey 12.2) software. Meanwhile, another group of neutrophils was treated with FITC-Ly6G/Ly6C antibody (250ng mL)^-1) The cell membrane was labeled and nuclear counterstaining was performed using the nuclear dye DAPI. After staining was completed, the cells were washed three times with cold PBS. And (3) spin-drying the slide gently, dripping the anti-fluorescence quenching sealing piece on the glass slide, taking out the cover glass from the culture plate, downwards buckling the surface attached with the cells on the glass slide, removing bubbles, sucking the overflowing sealing piece, sealing the edge, and finally placing the slide under a laser confocal microscope for observation and photographing.

Competitive inhibition experiments are adopted to prove that the nano-composite modified by the polysialic acid can enhance the drug uptake of the neutrophil granulocytes through receptor-ligand specific recognition. An excess of polysialic acid solution (10.0 mg. multidot.mL) was first used^-1) With neutral particle sizeCells, competitively inhibit receptor recognition function on the cell surface, then supernatant is removed by centrifugation and cells are collected. The mouse neutrophils were further co-incubated with the polysialic acid modified nanocomplexes at 37 ℃ for 2h (referred to as competitive inhibition group). The supernatant was removed by centrifugation and the cells were collected, and after fixing the cells by adding 4% paraformaldehyde, they were examined by flow cytometry and laser confocal microscopy, and the results are shown in FIG. 6.

Laser confocal microscopy results show that the polysialic acid nanocomplexes significantly enhanced DiR fluorescence signals in neutrophils compared to the solution group. The polysialic acid is shown to effectively promote the uptake of the nano-composite by the neutrophils, which is important for the medicine to play the role of in vivo immunotherapy.

Flow cytometry results further showed that the polysialic acid nanocomplexes exhibited stronger fluorescence signals 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 uptake of polysialic acid nanocomplexes by neutrophils was significantly inhibited after pre-incubation with a polysialic acid solution. This result is consistent with confocal laser 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 nanocomplexes to bind to neutrophil receptors, resulting in lower cellular uptake.

Example 7

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

In order to examine the influence of the nano-composite on the activity of the neutrophils, the survival time of the neutrophils is 6-8h, and the measurement cannot be carried out by using an MTT method (the developing process is 12h), so that the CCK8 method which can develop the color only in 4h is adopted for measurement.

1. A suspension of neutrophils (about 5X 10) in an amount of 100. mu.L was prepared⁴mL cells) were added to a 96-well plate (marginal wells filled with sterile water or PBS). Blank wells (medium, no cells) and control wells (medium without drug, cells) were set, and 3 more wells were set for each group.

2. Standing at 37 ℃ for 5%CO₂Incubate for 0.5h and observe under an inverted microscope.

3. mu.L of the polysialic acid nanocomplexes to be detected at different concentrations were added to each well and incubated at 37 ℃.

4. Add 10. mu.L of LCCK-8 solution to each well and incubate at 37 ℃ for 4 h.

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

The CCK8 experiment result shows that the concentration of Abelide and mitoxantrone is 0.01-10 mu g.mL^-1Within the range, the cell viability of the solution group was significantly lower than that of the polysialic acid nanocomposite group. At 5. mu.g.mL^-1Abelide and 2.5. mu.g.mL^-1The neutrophil activity of the Abeli-mitoxantrone-polysialic acid nanocomposite group was still more than 75% when the cells were incubated at the concentration of mitoxantrone (Abeli concentration: 5. mu.g.mL), and therefore, it is considered that we performed the subsequent experiments (Abeli concentration: 5. mu.g.mL)^-1Mitoxantrone concentration 2.5. mu.g.mL^-1) The neutrophil viability was not affected. The results are shown in FIG. 7.

Example 8

Examination of release behavior of neutrophils after uptake of nanocomplexes

The MTT method is adopted to measure the cytotoxicity of the drug released by the neutrophils on the tumor cells of the 4T1 mouse

(1X 10) neutrophilic granulocytes⁶Individual cells/ml) were co-cultured with various concentrations of the drug or polysialic acid nanocomplex for 30 minutes, and then separated by centrifugation and resuspended in DMEM medium containing 10% fetal bovine serum.

② adding 4T1 tumor cell suspension into 96-well plate (the edge hole is filled with sterile water or PBS), the cell density is 1X 10 per hole⁵And (4) respectively. Blank wells (medium, no cells) and control wells (medium without drug, cells) were set, and 3 more wells were set for each group.

③ culturing the obtained neutrophilic granulocyte for 8h, and then centrifuging. The medium of 4T1 cells was replaced with the supernatant released from neutrophils, cultured for another 48h, and observed under an inverted microscope.

And fourthly, adding 10 mu L of the drugs to be detected with different concentrations and the polysialic acid nano-composite into each hole, and incubating for 48 hours at 37 ℃.

Add 10. mu.L MTT solution into each well, incubate 4h at 37 ℃.

Sixthly, adding a triple solution into each hole to dissolve formazan generated by living cells overnight.

And measuring the light absorption value of each hole at the wavelength of 570 nm.

And (4) analyzing results: the OD value of the zeroing well or the control well was subtracted from the OD value of each test well. The OD values of each replicate well were averaged. Cell viability% ((dosed cells OD-blank OD)/(control cells OD-blank OD) × 100%), and half Inhibitory Concentration (IC) of cells was calculated₅₀)。

From the results of the cytotoxicity analysis, the survival rate of 4T1 tumor cells decreased with the increase of the drug concentration in the neutrophil medium, indicating that the polysialic acid nanocomplexes released from 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 the 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 the polysialic acid nano-complex and deliver the preparation to the tumor, inflammation and other parts. The results are shown in FIG. 8.

Example 9

The change of the quantity of the neutrophils in the lung along with the time after the establishment of the tumor lung metastasis model

And (3) taking the preserved mouse breast cancer 4T1 cells out of the liquid nitrogen, freezing and storing the tubes, and rapidly putting the tubes into water at 37 ℃ for resuscitation. The recovered 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, diluted into cell suspension by adding physiological saline when the activity of the tumor cells is more than 95 percent, and the dilution factor is adjusted. The 4T1 cell suspension was injected into mice tail vein after disinfection with 75% alcohol. The neck skin of anesthetized mice was incised after iodine sterilization after injection of 4T1 tumor cells in the tail vein of the mice for 0, 3, 6, 9, 12, and 15 days. Salivary glands and muscles were bluntly separated with forceps to expose trachea. Suture under trachea, make a V-shaped incision on cartilage ring in trachea middle, insert lavage needle about 0.5 cm into trachea to ensure no damage to lung, 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 3 min. The cell pellet was washed 3 times with PBS and resuspended in paraformaldehyde. Finally, the anti-Ly6G/Ly6C antibody labeled with a neutrophil marker FITC is added, and neutrophils are labeled by incubating for 20min in a dark environment at 4 ℃. After the cells were fixed with 4% paraformaldehyde, the fluorescence intensity of the samples was measured by FITC channel on a flow cytometer and the experimental data was analyzed using FlowJo 10.4(macOSMonterey 12.2) software.

Previous studies have shown that neutrophils are not abundant in the primary tumor microenvironment compared to other immune cells, 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 lung metastases of breast cancer in mice using flow cytometry. Ly6G in lungs of metastatic mice compared to lungs of naive mice⁺Ly6C⁺The neutrophil number increased dramatically, peaking at day 12 and then remaining stable. Given that neutrophils survive only 18h in mice, this suggests that tumor metastasis can lead to the continued migration of neutrophils into the lung. The results are shown in FIG. 9.

Example 10

Fluorescence imaging of nano-composite in 4T1 breast cancer lung metastasis BALB/c mouse in vivo

The preserved mouse breast cancer 4T1 cell cryopreservation tube is taken out from liquid nitrogen and is quickly placed in water at 37 ℃ for resuscitation. The recovered 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and diluted into cell suspension by adding physiological saline when the activity of the tumor cells is more than 95 percent and the dilution ratio is adjusted. The 4T1 cell suspension was injected tail vein into mice after being sterilized with 75% alcohol. On day 7 after inoculationThe mice were randomly divided into 2 groups, i.e., a fluorescent probe DiR solution group and a fluorescent probe DiR polysialic acid nanocomplex group, with 8 mice each. At a dose of 1 mg/kg^-1And injecting a fluorescent probe DiR solution and a fluorescent probe DiR polysialic acid nano-composite into the tail vein of the dose, performing in-vivo fluorescence imaging for 1 hour, 4 hours, 8 hours and 24 hours respectively, and measuring the fluorescence intensity of in-vivo tumor tissues of each group of mice. Mice were sacrificed by decapitation after 24h, tumors were isolated from other major organs for ex vivo organ imaging, and the mean fluorescence intensity for each organ was measured.

The experimental result shows that the fluorescence of the nano probe is gradually detected in the lung of the mouse along with the prolonging of the time, and the fluorescence intensities of the two groups reach the peak value within 24 hours. In contrast, the fluorescence signals of the fluorescent probe DiR solution group are mainly distributed in the liver, while the signals of the lung are weak. These results indicate that polysialic acid modified nanocomplexes can increase the uptake of neutrophils in the blood circulation, and circulating neutrophils phagocytosing the nano-preparations carry the drugs across the blood vessel wall into the lung under the drive of chronic inflammation of the tumor, thereby causing more drugs to be retained 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, the polysialic acid nanocomplexes exhibit stronger lung metastasis targeting ability. The results are shown in FIG. 10.

Example 11

Distribution condition of nano-composite in lung tissue of BALB/c mouse with 4T1 breast cancer lung metastasis

To further demonstrate that polysialic acid can effectively improve the phagocytosis of drugs by neutrophils in mice, the mice with lung metastasis are divided into four groups, namely a blocked neutrophil group and an unblocked neutrophil group. Then, a fluorescent probe DiR solution or a fluorescent probe DiR polysialic acid nano-complex is injected respectively, the neutrophil in the lung metastasis tissue is labeled by using a neutrophil marker Ly6G/Ly6C, and the drug uptake of tumor-associated neutrophils is observed.

Frozen section preparation

Tissue fixation:

fresh tumor tissue is fixed by fixing liquid for more than 24h, taken out and flattened by a scalpel.

And (2) dehydrating:

placing the trimmed tissue in 15% sucrose solution, dehydrating and precipitating at 4 deg.C in refrigerator, transferring into 30% sucrose solution, dehydrating and precipitating at 4 deg.C in refrigerator.

③ OCT embedding:

and taking out the dehydrated tissue, slightly sucking surface water, then upwards placing the dehydrated tissue on an embedding platform with the section facing upwards, dripping OCT embedding agent on the periphery of the tissue, placing the embedding platform on a freezing microtome for quick-freezing embedding, and slicing after the OCT becomes white and hard. The direct frozen section of the fresh tissue does not need to be fixed and dehydrated, and the section is embedded by OCT embedding medium after the tissue of the target part is flattened by a scalpel directly.

Cutting into slices:

fixing the embedding table on a slicer, roughly cutting, trimming and flattening the tissue surface, then starting slicing, wherein the slicing thickness is 8-10 μm, and flatly placing a clean glass slide on the cut tissue piece to stick the tissue on the glass slide. The film is stored at-20 ℃ for later use after the label is written.

Immunofluorescence staining:

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

Sixthly, dyeing the core:

the slides were washed in PBS (pH7.4) with shaking in the dark on a destaining shaker for 3 times, 5min each time. After the slices are dried, DAPI dye liquor is dripped to dye the kernels for 10min in a dark place at room temperature.

Seventh, sealing sheet:

the slides were washed in PBS (pH7.4) with shaking in the dark on a destaining shaker for 3 times, 5min each time. The slices are dried and then sealed by an anti-fluorescence quenching sealing agent.

Eighthly, microscopic examination and photographing:

the sections were observed under a fluorescent microscope and images were collected.

According to the immunofluorescence experiment result of the lung section, compared with the fluorescent probe DiR solution group, the fluorescence co-localization signal is stronger in the neutral granulocyte labeled by the polysialic acid nano-complex group Ly6G/Ly 6C. These results indicate that polysialic acid is effective in increasing the uptake of the nanocomplexes by neutrophils, which is consistent with the in vitro cellular uptake results. To further verify that the accumulation of polysialic acid nanocomplexes in the lung is mediated by neutrophils, we injected the anti-Ly6G antibody intravenously into mice with tumor lung metastasis to establish a neutrophil depletion model. After the neutrophilic granulocyte is exhausted, the targeting property of the polysialic acid nano-composite almost completely disappears, and the fluorescence signal is similar to that of a fluorescent probe DiR solution group. The results are shown in FIG. 11.

Example 12

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

The preserved mouse breast cancer 4T1 cell cryopreservation tube is taken out from liquid nitrogen and is quickly placed in water at 37 ℃ for resuscitation. The recovered 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and diluted into cell suspension by adding physiological saline when the activity of the tumor cells is more than 95 percent and the dilution ratio is adjusted. Disinfecting by using 75% alcohol, inoculating 4T1 cell suspension on a third pair of breast pads of mice, inoculating 0.1mL of each mouse, inoculating 56 mice, and randomly dividing the mice into 7 groups, namely a control group, an Abeli solution group, a mitoxantrone solution group, an Abeli-mitoxantrone pharmaceutical composition solution group, an Abeli-polysialic acid nano-complex group, a mitoxantrone-polysialic acid nano-complex group and an Abeli-mitoxantrone-polysialic acid nano-complex group, wherein each group comprises 8 mice. Each group of mice was administered 1 time every 2 days on day 3 after inoculation and 3 times in total (3, 5, 7 days after inoculation), and the Abeli dose was 5 mg/kg^-1The mitoxantrone dose was 2.5mg kg^-1The control group was given 5% glucose injection. Throughout the duration of the pharmacodynamic test, data were recorded for body mass, death events, etc.

The result of an anti-tumor experiment shows that the tumor inhibition rate is as follows in sequence: the Abeli-mitoxantrone-polysialic acid nano-composite group is the Abeli-polysialic acid nano-composite group, and the Abeli-polysialic acid nano-composite group is the Abeli-polysialic acid nano-composite group. Tumor volumes continued to increase during the experiments in the abelian solution group and mitoxantrone solution group. The better tumor inhibition effect of the Abeli-mitoxantrone medicinal composition solution group can be explained as that the bioavailability of the medicinal solution is lower, but the Abeli and the mitoxantrone play a synergistic effect to stimulate the immune reaction of the whole body and a tumor microenvironment, thereby enhancing the anti-tumor effect. The nano-composite group can obviously inhibit the tumor growth of tumor-bearing mice and prolong the survival time of the mice. It is worth noting that the tumor volume of the mice in the Abeli-mitoxantrone-polysialic acid nano-complex group is obviously inhibited, and the average tumor inhibition rate of the mice in the Abeli-mitoxantrone-polysialic acid nano-complex group is 72.45 percent at 26 days, which is obviously higher than that of the Abeli-polysialic acid nano-complex group and the mitoxantrone-polysialic acid nano-complex group. In addition, for the in situ triple negative breast cancer 4T1 model, EPR effect is weak because the tumor is located in the mouse mammary gland, resulting in poor clinical treatment effect. The experimental results before the subject group show that the tumor inhibition rate of the PEG adriamycin liposome to 4T1 tumor-bearing mice is lower than 40 percent, which is far inferior to the Abeli-mitoxantrone-polysialic acid nano-composite group. Meanwhile, after the mice are dissected, the lung weight of the Abeli-mitoxantrone-polysialic acid nano-composite mice is observed to be far less than that of the control mice and close to that of the blank mice.

Example 13

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

The preserved mouse breast cancer 4T1 cell cryopreservation tube is taken out from liquid nitrogen and is quickly placed in water at 37 ℃ for resuscitation. The recovered 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and diluted into cell suspension by adding physiological saline when the activity of the tumor cells is more than 95 percent and the dilution ratio is adjusted. The 4T1 cell suspension was injected into mice tail vein after being sterilized with 75% alcohol. On day 3 after tumor loading, the mice were randomly divided into 7 groups, i.e., control group, Abeli solution group, mitoxantrone solution group, Abeli-mitoxantrone pharmaceutical composition solution group, Abeli-polysialic acidAcid nanocomposite group, mitoxantrone-polysialic acid nanocomposite group and Abeli-mitoxantrone-polysialic acid nanocomposite group, each group containing 8. Each group of mice was administered 1 time every 2 days on day 3 after inoculation and 3 times in total (3, 5, 7 days after inoculation), and the Abeli dose was 5 mg/kg^-1The mitoxantrone dose was 2.5mg kg^-1The control group was given 5% glucose injection. Throughout the duration of the pharmacodynamic test, data were recorded for body mass, death events, etc. On day 18 of tumor cell inoculation, lung specimens of all groups were excised and photographed, while examining lung weight and number of metastases, to assess inhibition of lung metastases. Hematoxylin and eosin (H) for lung specimens&E) Staining and observation with an inverted microscope. Mice for survival studies were treated in the same manner.

We analyzed photographs of the mouse lung and HE-stained metastatic nodules (dark-stained nuclei) in the lung. Except for the Abeli-mitoxantrone-polysialic acid nano-composite, metastasis can be clearly found in all groups, and the polysialic acid nano-composite has obvious anti-metastasis capability. The results are shown in FIGS. 12 and 13.

Compared with the control group, the solution group can slightly inhibit the lung metastasis of the breast cancer, and the Abeli-polysialic acid nano-complex group and the mitoxantrone-polysialic acid nano-complex group play a moderate anti-metastatic role. The average number of transferred nodules per lung of the group of the Abeli-mitoxantrone-polysialic acid nanocomposites was 11, which was 10.3%, 12.4%, 13.7%, 15.1%, 29.3% and 28.8% of the group of the control group, the group of the Abeli solution, the group of the mitoxantrone solution, the group of the Abeli-mitoxantrone pharmaceutical composition solution, the group of the Abeli-polysialic acid nanocomposites and the group of the mitoxantrone-polysialic acid nanocomposites. The results are shown in FIG. 14.

Long-term survival of breast cancer lung metastasis mice was assessed by up to 40 day experiments. Compared with all other groups, the Abeli-mitoxantrone-polysialic acid nanocomposite group had the highest survival rate and almost completely eliminated metastasis. The results are shown in FIG. 15.

The change in body weight of the mice was analyzed during the experiment and the safety of the nanocomposite was investigated. In the group injected with the nanocomplexes, the body weight did not change much. In particular, the body weight of the mice in the Abeli-mitoxantrone-polysialic acid nanocomposite group steadily increased, while the body weight of the control group and the mitoxantrone solution group decreased, which may be related to the effect of lung metastases on the quality of life of the mice or toxicity of mitoxantrone. The results are shown in FIG. 16.

The lung weight of adult mice is a constant value, correlated with the weight of metastases. The mean lung weight of mice treated with the solution (Abelix solution group: 0.62. + -. 0.06 g; mitoxantrone solution group: 0.35. + -. 0.06g) or the single agent (Abelix-polysialic acid nanocomposite group: 0.29. + -. 0.02 g; mitoxantrone-polysialic acid nanocomposite group: 0.28. + -. 0.04g) was significantly lower than that of mice of the control group (0.91. + -. 0.02 g). In all treatment groups, the lung weight of mice treated with Abeli-mitoxantrone-polysialic acid nanocomplexes was lowest (0.18. + -. 0.01g), closest to that of 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 can reflect the immune function of the organism to a certain extent. The spleen is a peripheral immune organ, is a place where T, B lymphocytes are resident, is also a place where immune cells perform an immune response function, and can secrete bioactive media (such as cytokines and the like). The spleen index of mice is commonly used in the literature to assess the effect of drugs on the immune system. However, the observation of the spleen index formula shows that the unit of spleen mass/body mass is mg/g, the unit is not uniform, the data of spleen and body weight cannot be intuitively obtained from the data, and the data is only used as a measurement standard to be simply compared with a control group.

The lung weight of adult mice is a constant value, and when the lung is diseased (such as lung cancer, tumor lung metastasis, Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis, pulmonary edema, acute lung injury, tuberculosis), the lung becomes heavy. These lesions can lead to tumor growth, destruction of the alveolar walls and bronchioles, exudation of lung tissue, and other proliferative responses. Taking spleen index as an example, the concepts of related indexes such as 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 are provided. Furthermore, by studying the changes in lung weight in a population, a quality of life situation, such as pneumoconiosis, can be obtained for a certain region or for a certain professional population.

To distinguish indices associated with Lung disease, we named the Lung index with particular utility as "Lung disease index (LD)_index) "(the formula is: LD_indexBody weight (g)/lung weight (g)) and lung weight index (LW)_index) "(the formula is: LW_indexLung 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 can be seen that the more tumor metastasis, the more severe the mouse, the greater the lung mass. We can validate 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 the lung weight can deduce the number of nodules, and obtain more accurate data.

Although lung weight and body weight have been considered as classical parameters for anti-metastatic experiments, they are independent indicators when evaluating anti-metastatic effects and toxicity. Here, we propose a "Metastasis inhibition index (MI)_index) "as a comprehensive parameter for the evaluation of the efficacy and toxicity of the anti-metastatic agent.

The formula for the metastasis inhibition index is as follows:

MI_index＝body weight(g)/lung weight(g).

the metastasis suppression index may also reflect small and intra-pulmonary metastases that cannot be directly observed to obtain more accurate values. The transfer inhibition index of mice treated by the Abeli-mitoxantrone-polysialic acid nano-composite is obviously greater than that of other groups, which shows that the Abeli-mitoxantrone-polysialic acid nano-composite has the lowest toxicity and the best anti-transfer effect. However, the metastasis inhibition index was the smallest in the Abeli solution group and the mitoxantrone solution group among all treatment groups, and was almost the same as that in the control group. These data indicate that toxicity of the Abelix solution group and the mitoxantrone solution group resulted in the lowest Quality of life (QOL). In general, the metastasis inhibition index provides a new evaluation index, which can more obviously reflect the curative effect difference among treatment groups, so as to improve the accuracy of anti-metastasis evaluation and promote the clinical transformation of basic research. 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
Abelide solution group	24.1±0.46
		Mitoxantrone solution group	48.0±7.38
Abelide-mitoxantrone pharmaceutical composition solution set	57.2±2.84
		Abeli-polysialic acid nano-composite group	71.8±13.9
Mitoxantrone-polysialic acid nanocomposite sets	73.4±8.35
		Abeli-mitoxantrone-polysialic acid nano-composite group	108.6±7.48
Blank group	125.1±5.68

The receptor for PSA is highly expressed on the surface of a variety of leukocytes, including Peripheral Blood Neutrophils (PBN) which are highly increased in chronic inflammatory responses mediated by tumors and tumor metastases. Through combination with selectins and siglecs on the surfaces of cells such as neutrophils, the drug is released in tissues by utilizing the unique function of the neutrophils in deforming and penetrating through the blood vessel wall, the aim of targeted drug delivery is fulfilled, and the curative effect is improved.

Mouse breath test

Generally, mice with tumor lung metastases will have a smaller lung volume, resulting in poor breathing and increased respiratory frequency. Therefore, the following steps are adopted: the mouse breath experiment researches related tumor metastasis indexes.

In the experimental group, the breathing frequency of the mice within 1min is recorded, and the result shows that the average breathing frequency of the control group of mice within 1min is 276 times which is far greater than 163 times of the blank group, which indicates that the lung metastasis of the tumor affects the breathing frequency of the mice. Compared with the Abeli-mitoxantrone-polysialic acid nano-composite group, the average 1-minute breathing frequency of the mice is 180 times, which shows that the polysialic acid nano-composite can effectively inhibit metastasis and has little influence on the lung capacity of the mice.

Example 14

4T1 section of important tissue of mouse with lung metastasis of breast cancer

In order to examine the influence of each nano-composite on the main organs of the mice, after the anti-tumor metastasis experiment of 4T1 breast cancer lung metastasis mice is finished, the heart, the liver, the spleen and the kidney of each group of tumor-bearing mice are collected and used for HE pathological sections.

Paraffin section preparation

Tissue fixation

The freshly stripped heart, liver, spleen, lung and kidney tissues are washed with PBS (pH7.4), fixed with 4% paraformaldehyde for more than 24h, taken out from the fixing solution after fixation is finished, and flattened by using a surgical knife in a fume hood.

② washing

And (3) placing the trimmed tissue block into an embedding box, placing the embedding box under distilled water, flushing the tissue at a lower flowing speed for 12-24 hours.

(iii) dehydration

Putting the embedding box into a hanging basket, putting into a dehydrating machine, dehydrating with ethanol with different gradients, sequentially reacting with 75% ethanol for 4h, 85% ethanol and 90% ethanol for 2h respectively, reacting with 95% ethanol for 1h, and then reacting with anhydrous ethanol for 2 times to dehydrate.

Fourthly, transparent and wax dipping

Each tissue was transferred into a closed container, xylene: the ethanol (1:1, v/v) acts for 5-10 min, the xylene I acts for 5-10 min, and the xylene II acts for 5-10 min. After the transparence is finished, each tissue block is immersed in completely melted paraffin (a thermostat at 60 ℃) for 1h, and then the tissue blocks are transferred into new paraffin liquid for secondary paraffin immersion for 1 h.

Fifthly, embedding

The tissues soaked with wax are placed in an embedding machine for embedding. Firstly, molten wax is put into an embedding frame, before the wax is solidified, the tissue is taken out from a dehydration box, put into the embedding frame according to the requirements of an embedding surface and pasted with a corresponding label. And (4) freezing and cooling at the temperature of minus 20 ℃, taking the wax block out of the embedding frame after the wax is solidified, and trimming the wax block.

Sixthly, slicing

Placing the wax block on a paraffin slicer to continuously slice, wherein the slice thickness is 5 μm, floating the slice on a spreading machine, flattening the tissue with warm water at 40 deg.C, taking out the tissue with an anti-drop glass slide, and baking the slice in an oven at 60 deg.C. And after the water and the wax are baked, taking out the glass slide, and storing at normal temperature for later use.

Seventhly, HE dyeing

Dewaxing the paraffin wax section to water

Soaking the tissue slices in xylene I and xylene II solution for 20min, respectively, soaking in 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5min, and washing with distilled water for 10 min.

Ninthly dye the nucleus

The slices are placed into hematoxylin staining solution for staining for 8min, washed by distilled water to remove loose color, then differentiated by 1% hydrochloric acid alcohol for 20s, and finally washed by distilled water to turn blue.

Property of infection of red

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

Dehydration seal

Soaking the slices in 95% ethanol I and 95% ethanol II for 5min, respectively, soaking in anhydrous ethanol I and anhydrous ethanol II for 5min, respectively, soaking in xylene I and xylene II for 5min, dehydrating, air drying, and sealing with neutral gum.

Microscopic examination photograph

After the mounting is dried, the slide is placed under an inverted microscope for observation.

As a result of observation of each organ section, the cardiomyocytes of each nanocomposite group mouse were intact and no myocardial rupture phenomenon was observed. For pathological liver sections, the liver cell nucleus is intact, the shapes of the epithelial cells of the liver cells and the liver sinuses are normal, and the liver sinuses have no hyperemia. In addition, glomerular and tubular epithelial cells were intact, the renal capsule was normal, and no bleeding or inflammatory infiltration was observed in all mouse kidney sections. No histopathological abnormalities, lesions or degeneration were shown in each preparation group in mouse spleen sections. The above results indicate that after 3 drug treatments, mouse organs including heart, liver, spleen and kidney showed no histopathological abnormalities, lesions or degeneration, demonstrating that each nanocomposite can be safely used for mouse tumor treatment. The results are shown in FIG. 19.

Example 15

4T1 pulmonary immune cell changes in mice with pulmonary metastasis of breast cancer

To examine the triggering of the anti-tumor immune response for each treatment regimen, flow cytometry was used to determine the T cell infiltration in lung tissue of mice in each treatment group. The preserved mouse breast cancer 4T1 cell cryopreservation tube is taken out from liquid nitrogen and is quickly placed in water at 37 ℃ for resuscitation. The recovered 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and diluted into cell suspension by adding physiological saline when the activity of the tumor cells is more than 95 percent and the dilution ratio is adjusted. The 4T1 cell suspension was injected into mice tail vein after being sterilized with 75% alcohol. On day 3 after tumor loading, the mice were randomly divided into 7 groups, i.e., a control group, an abbeli solution group, a mitoxantrone solution group, an abbeli-mitoxantrone pharmaceutical composition solution group, an abbeli-polysialic acid nanocomposite group, a mitoxantrone-polysialic acid nanocomposite group, and an abbeli-mitoxantrone-polysialic acid nanocomposite group, with 8 mice per group. Each group of mice was administered 1 time every 2 days on day 3 after inoculation and 3 times in total (3, 5, 7 days after inoculation), and the Abeli dose was 5 mg/kg^-1The mitoxantrone dose was 2.5mg kg^-1The control group was given 5% glucose injection. Data on body mass, death events, etc. were recorded throughout the duration of the pharmacodynamic test. On day 18 of tumor cell inoculation, mice were sacrificed and tumor tissue was collected. The tumor tissue is cut to about 2X 2mm³The small pieces are placed in newly prepared digestive juice (containing 0.5 mg. mL)^-1Collagenase IV and 0.2 mg/mL^-1DNase I), incubation was carried out for 1h at 37 ℃. The undigested tissue mass was filtered using a 70- μm nylon mesh 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 addedStaining was performed at 4 ℃ for 30min to label T cells and detected by flow cytometry.

The results show that the immunosuppressive microenvironment formed in the lungs prevents abbeli from improving effector T cell function, whereas mitoxantrone promotes immunogenic effects at the metastatic sites, releasing signals that elicit an immune response, thus providing the opportunity to "warm" the immune "cold" tumor. Abelide and mitoxantrone act synergistically to achieve optimal immune response for cancer immunotherapy. Flow cytometry results showed that Abeli-mitoxantrone-polysialic acid nanocomplexes conjugated CD8⁺The ratio of T cells increased to 12.6% ± 1.0%. CD4 of Abeli-mitoxantrone-polysialic acid nano-complex group⁺And CD8⁺The ratio of T cells was 7.9-fold and 4.6-fold that of the control group, respectively. Regulatory T cells are suppressive immune cells that suppress the systemic immune response by inducing and maintaining tolerance to self-antigens. The regulatory T cell ratio of the abelian-mitoxantrone-polysialic acid nanocomposite group decreased to 7.2% ± 1.0% compared to the control group. The regulatory T cell fraction was lowest for the abelmoscil-mitoxantrone-polysialic acid nanocomplex group compared to the other groups. The results are shown in FIGS. 20 and 21.

Example 16

Changes in lung cytokines in 4T1 mammary cancer pulmonary metastasis mice

To examine the changes in lung cytokines in the lung metastatic mice in each treatment protocol, the changes in cytokines in lung tissue of mice in each treatment group were measured by ELISA. The preserved mouse breast cancer 4T1 cell cryopreservation tube is taken out from liquid nitrogen and is quickly placed in water at 37 ℃ for resuscitation. The recovered 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and diluted into cell suspension by adding physiological saline when the activity of the tumor cells is more than 95 percent and the dilution ratio is adjusted. 4T1 cell suspension was injected tail vein into mice using 75% alcohol for sterilization. On day 3 after tumor loading, the mice were randomly divided into 7 groups, i.e., control group, Abeli solution group, mitoxantrone solution group, Abeli-mitoxantrone pharmaceutical composition solution group, Abeli-polysialic acid nanocomposite group, mitoxantrone-polysialic acidA nanocomposite group and an Abeli-mitoxantrone-polysialic acid nanocomposite group, each group comprising 8. Each group of mice was administered 1 time every 2 days on day 3 after inoculation and 3 times in total (3, 5, 7 days after inoculation), and the Abeli dose was 5 mg/kg^-1The mitoxantrone dose was 2.5mg kg^-1The control group was given 5% glucose injection. Throughout the duration of the pharmacodynamic test, data were recorded for body mass, death events, etc. On day 18 of tumor cell inoculation, mice were sacrificed and tumor tissue was collected. After fully grinding lung tissue with a tissue homogenizer, centrifugation was carried out at 10000rpm for 10 min. The concentrations of the cytokines IL-10, TGF- β, IFN- γ and TNF- α in the supernatants were determined according to the instructions of the respective ELISA kits.

Regulatory T cells (Treg cells) are antigen-specific suppressor cells that exert immunosuppressive effects, primarily through the release of IL-10 or TGF- β. The lung immunocytokine level measured by ELISA was shown as follows, and in each group, the IL-10 and TGF-beta levels in the Abeli-mitoxantrone-polysialic acid nanocomposite group were the lowest, indicating that the Abeli-mitoxantrone-polysialic acid nanocomposite could significantly inhibit adaptive regulatory T cells and improve the immunosuppressive environment. The inflammatory mediators TNF-alpha and IFN-gamma play an important role in anti-tumor immunity and can promote CD8⁺Proliferation and differentiation of T cells, and enhancement of anti-tumor activity of cells. Compared with other groups, the Abeli-mitoxantrone-polysialic acid nano-composite group obviously enhances the release of inflammation mediators, reverses 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

Precisely weighing 20mg Abelide hydrochloride, and dissolving in 10mL sterile water for injection to obtain 2 mg/mL^-1Abelian hydrochloride solution; precisely weighing 10mg mitoxantrone hydrochloride, and dissolving in 10mL sterile water for injection to obtain 1 mg/mL^-1The mitoxantrone hydrochloride solution; accurately weighing polyanionic material (polysialic acid, hyaluronic acid, polyacrylic acid, alginic acid, carboxymethyl cellulose, carrageenan, polyglutamic acid, polyitaconic acid, heparin, etc.) 60mg,Gelatin, polybetaine, chondroitin sulfate, dextran sulfate, polymethyl methacrylate, polystyrene sulfonic acid, succinic acid and human serum albumin) in 20mL sterile water for injection to obtain 3 mg/mL^-1A polyanion solution of (1). And (3) placing the pharmaceutical composition solution of Abelide and mitoxantrone on a magnetic stirrer, keeping the stirring speed at 100rpm, then dropwise adding the polyanion solution into the pharmaceutical solution, and incubating for 30min to obtain the Abelide-mitoxantrone-polyion compound.

The results show that carboxymethyl cellulose cannot be complexed with cationic drugs to obtain polyion complexes; a polyion compound prepared by compounding polyacrylic acid, carrageenan, polyitaconic acid, gelatin, polybetaine, polymethyl methacrylate and a cationic drug is unstable; the efficacy of the nano-composite prepared by using human serum albumin is not good in experimental effect, and presumably, the reason is that the blood of a mouse contains a large amount of protein, the targeting effect of the nano-composite is interfered, and the nano-particles cannot be accumulated in tumors and tumor metastasis parts; the nano-composite prepared by using succinic acid has good stability in vitro, but can be dissociated after being injected into the body, and the drug effect of the nano-composite is similar to that of a solution group. Wherein, after injecting the carrageenan compound, the mouse has inflammation, which indicates that a certain amount of carrageenan can induce the 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 lung metastasis

Within the mass ratio (4:1) - (1:2) of Abeli and mitoxantrone, the polyanion materials including hyaluronic acid (polymerization degree of 200), alginic acid (polymerization degree of 600), polyglutamic acid (polymerization degree of 3000), heparin, chondroitin sulfate, dextran sulfate and succinyl gelatin are selected for compatibility administration. Abelide is administered in an amount of 5 mg/kg^-1The dose of mitoxantrone administered is 2.5mg kg^-1. Precisely weighing 20mg Abelide hydrochloride, and dissolving in 10mL sterile water for injection to obtain 2 mg/mL^-1Abelian hydrochloride solution; precisely weighing 10mg mitoxantrone hydrochloride, and dissolving in 10mL sterile water for injection to obtain 1 mg/mL^-1The mitoxantrone hydrochloride solution; precisely weighing 60mg polyanion compound, and dissolving in 20mL sterilized water for injection to obtain 3 mg/mL polyanion compound^-1A polyanion solution of (1). And (3) placing the medicinal composition solution on a magnetic stirrer, keeping the stirring 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, the mixture is adjusted to be isotonic by adopting 50 percent glucose injection, wherein the final concentration of Abeli is 1 mg.mL^-1Final mitoxantrone concentration of 0.5 mg/mL^-1。

The preserved mouse breast cancer 4T1 cell cryopreservation tube is taken out from liquid nitrogen and is quickly placed in water at 37 ℃ for resuscitation. The recovered 4T1 cell suspension is cultured in vitro, counted under an inverted microscope, and diluted into cell suspension by adding physiological saline when the activity of the tumor cells is more than 95 percent and the dilution ratio is adjusted. 4T1 cell suspension was injected tail vein into mice using 75% alcohol for sterilization. On day 3 post-tumor loading, mice were randomized into 24 groups of 8 mice each. Each group of mice was administered 1 time every 2 days on day 3 after inoculation and 3 times in total (3, 5, 7 days after inoculation), and the Abeli dose was 5 mg/kg^-1The mitoxantrone dose was 2.5mg kg^-1The control group was given 5% glucose injection. Throughout the duration of the pharmacodynamic test, data were recorded for body mass, death events, etc. The results are shown in Table 4.

Table 4 results of the anti-4T 1 experiment for complexes modified with different polyanionic polymers

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 the injection of heparin complex, mice developed bleeding and death phenomena, with a higher mortality rate than the control group. The research proves that the heparin can cause the bleeding of mice due to the anticoagulant effect, and is not suitable to be compounded with cationic drugs to be used as a drug carrier.

Claims

1. An antitumor drug composition with a synergistic effect is characterized by comprising Abelide and mitoxantrone, wherein the mass ratio of Abelide to mitoxantrone is (8:1) - (1: 6).

2. The synergistic antitumor pharmaceutical composition as claimed in claim 1, wherein the mass ratio of Abelide to mitoxantrone is (4:1) - (1: 2).

3. The synergistic antitumor pharmaceutical composition as claimed in claim 1, wherein the concentration of Abelix in said pharmaceutical composition is 0.1 mg-mL^-1-50mg·mL^-1The concentration of mitoxantrone was 0.1 mg/mL^-1-20mg·mL^-1。

4. The synergistic antitumor pharmaceutical composition as claimed in claim 1, further comprising pharmaceutically acceptable adjuvants and carriers.

5. A dosage form of the synergistic antitumor pharmaceutical composition as claimed in any one of claims 1 to 4, comprising any one of a solid preparation, a liquid preparation and a semi-solid preparation.

6. The synergistic antitumor pharmaceutical composition dosage form as claimed in claim 5, wherein the dosage form is a nano preparation comprising polyion complex, phospholipid complex, liposome, nanoparticle, nanocapsule and polymeric micelle.

7. The dosage form of synergistic antitumor pharmaceutical composition as claimed in claim 6, wherein said polyion complex is composed of polyanionic material and synergistic antitumor pharmaceutical composition;

wherein the mass ratio of the polyanionic material to the antitumor drug composition with the synergistic effect is (1:2) - (30: 1); the polyanionic 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.

8. A method for preparing a dosage form of the synergistic antitumor pharmaceutical composition as claimed in claim 6, wherein the preparation of the polyion complex comprises the steps of:

(1) dissolving the pharmaceutical composition in pure water to obtain solution A with concentration of 0.1 mg/mL^-1～10mg·mL^-1(ii) a The mass ratio of Abelide to mitoxantrone in the pharmaceutical composition is (8:1) - (1: 6);

(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).

9. The application of the nanometer preparation of the antitumor drug composition with the synergistic effect of any one of claims 1 to 4 and the antitumor drug composition with the synergistic effect of any one of claims 6 to 7 in the preparation of drugs for resisting solid tumors and treating tumor metastasis.

10. The use of claim 9, wherein the solid tumors comprise oral cancer, gastrointestinal cancer, colon cancer, gastric cancer, cancer of the pulmonary tract, lung cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, bladder cancer, pancreatic cancer, bone cancer, liver cancer, gallbladder cancer, kidney cancer, skin cancer and testicular cancer, melanoma and sarcoma; the tumor metastasis organs are lung, lung on the other side, liver, lymph, bone, brain and adrenal gland.