A polymer for treating cancer metastasis and its preparation method and application

Abstract

The present invention discloses a polymer for treating cancer metastasis and its preparation method and application, and relates to the field of biomedicine. The polymer is a cationic polymer and / or a cationic polymer nanoparticle, and the cationic polymer nanoparticle is formed by self-assembly of a block copolymer in an aqueous phase, and the block copolymer is composed of a chain segment of an electrically neutral polymer and a chain segment of a cationic polymer. The present application uses a cationic polymer and a cationic polymer nanoparticle having nucleic acid binding affinity and capable of accumulating in the liver to remove NETs that can induce cancer metastasis and accumulate in large quantities in organs. Both the soluble cationic polymer material and the cationic polymer nanoparticle material can compete for binding to NET-DNA from the NET-DNA and CCDC25 complex, thereby blocking the migration of cancer cells mediated by it.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polymer for treating cancer metastasis and a preparation method and application thereof. Background Art

[0002] Nearly 90% of cancer patient deaths are caused by distant cancer metastasis. Unfortunately, because most cancers are biologically heterogeneous, with rapid regeneration of heterogeneous cell subpopulations, the outcome of cancer metastasis depends on multiple interactions with homeostatic mechanisms, which greatly increases the difficulty of inhibiting metastasis, so traditional treatments are resistant to cancer metastasis. Although traditional treatments such as chemotherapy can shrink the lesions of the primary tumor to an undetectable level, they are less effective in metastasis and cause extensive side effects, which in turn deteriorate the quality of life of patients.

[0003] Recent studies have shown that the DNA component of neutrophil extracellular traps (NETs) is associated with cancer metastasis. Neutrophil extracellular traps (NETs) consist of DNA nets and associated proteolytic enzymes, released by neutrophils, the most abundant white blood cells in human blood. NETs are secreted into extracellular tissues, where they capture microorganisms to resist invading pathogens. Although NETs can kill microorganisms during infection, excessive NET formation can induce proinflammatory behavior that damages cells and the host. Studies have shown that NETs play a key role in cancer metastasis. NETs can trap and attract cancer cells to spread to distant organs, while metastatic cancer cells further promote neutrophil formation and support NETs to metastasize in a mutually reinforcing manner. At the same time, NETs DNA has also been identified as a receptor for the cancer cell transmembrane protein CCDC25. Excessive accumulation of NETs has been found in the liver and lungs of patients with breast cancer or colon cancer metastasis. Therefore, it is urgent to study the interaction between blocking NET-DNA and CCDC25 to effectively limit cancer metastasis and reduce mortality. Summary of the invention

[0004] The present invention provides a polymer for treating cancer metastasis, a preparation method and an application thereof, and provides a polymer material that can competitively bind to NET-DNA to block the interaction between NET-DNA and CCDC25 to effectively inhibit cancer metastasis.

[0005] In order to solve the above technical problems, one of the purposes of the present invention is to provide a polymer for treating cancer metastasis, wherein the polymer is a cationic polymer and / or cationic polymer nanoparticles, wherein the cationic polymer nanoparticles are formed by self-assembly of a block copolymer in an aqueous phase, wherein the block copolymer is composed of segments of an electrically neutral polymer and segments of a cationic polymer, wherein the cationic polymer comprises one or more of positively charged poly(methyl)acrylate 2-(dimethylamino)ethyl ester, poly-α-aminovalerolactone, poly(2-dimethylaminoethylthio)caprolactone, polylysine, polyarginine and polyaspartic acid amino derivatives, or copolymers thereof; wherein the electrically neutral polymer comprises one or more of polylactic acid-glycolic acid random copolymer, hydrophobic polyphosphate, hydrophobic polycarbonate, polyethylene glycol, polycaprolactone and hydrophobic polyamino acid, wherein the hydrophobic polyamino acid is one or more of polytyrosine, polyalanine and polyphenylalanine.

[0006] As a preferred embodiment, the number average molecular weight of the polymer chain segment is 3k-500k, the weight average molecular weight is 3k-500k, the polymer dispersity coefficient is 1-1.5, the particle size of the cationic polymer nanoparticles is 30nm-200nm, and the polydispersity index is 0.1-0.3.

[0007] As a preferred embodiment, the block copolymer is polytyrosine-poly(N-(N'N-dimethylethylenediamine)asparagine) with a BLA amination rate of 100%.

[0008] As a preferred embodiment, the chemical structural formula of the block copolymer is:

[0009] As a preferred embodiment, the cationic polymer is poly-N-(N,N-dimethylethylenediamine)asparagine with an amination rate of (BLA) benzyl aspartate of 10% to 100%.

[0010] By adopting the above scheme, the purpose of grafting N'N-dimethylethylenediamine in this application is to graft cations onto the polyaspartic acid segment, thereby achieving charged properties. The higher the amination rate, the more positive charges it carries, and the stronger its ability to compete for binding to NET-DNA.

[0011] As a preferred embodiment, the chemical structural formula of the cationic polymer is:

[0012] In order to solve the above technical problems, a second object of the present invention provides an application of a polymer for treating cancer metastasis in preparing a kit for treating cancer metastasis.

[0013] As a preferred embodiment, the reagent of the kit for inhibiting cancer metastasis is an intravenous injection.

[0014] In order to solve the above technical problems, the third object of the present invention provides a method for preparing cationic polymer nanoparticles, comprising the following steps:

[0015] (1) dissolving (S)-4-(4-hydroxybenzyl)oxazolidine-2,5-dione in N'N-dimethylformamide, adding n-butylamine to carry out a ring-opening polymerization reaction, precipitating in cold ether, filtering, and drying to obtain a first intermediate product;

[0016] (2) mixing the first intermediate product, (4S)-4-[(4-hydroxyphenyl)methyl]-2,5-oxazolidinedione, N'N-dimethylformamide and dichloromethane for polymerization reaction, precipitating in cold ether, filtering and drying to obtain a second intermediate product;

[0017] (3) subjecting the second intermediate product to an aminolysis reaction with dimethylethylenediamine, dialyzing the product in an organic solvent to remove dimethylethylenediamine, distilling off the organic solvent, and drying to obtain a block copolymer;

[0018] (4) dissolving the block copolymer in a dimethyl sulfoxide aqueous solution at a concentration of 5-15 mg / mL, then adding the block copolymer dropwise into water until the polymer concentration reaches 1-3 mg / mL, dialyzing to remove the dimethyl sulfoxide, and making the solution into PBS with a concentration of 10 mmol / L, thereby preparing a PBS solution containing cationic polymer nanoparticles.

[0019] As a preferred embodiment, in steps (1), (2) and (3), the reaction temperature is 40°C-70°C and the reaction time is more than 40 hours.

[0020] As a preferred embodiment, in step (1), the polymerization reaction synthesizes a first intermediate product with a degree of polymerization of 10-40; in step (2), the polymerization reaction synthesizes a second intermediate product with a degree of polymerization of 50-400.

[0021] By adopting the above scheme, the first intermediate product is polymerized to form a hydrophobic segment, and the second intermediate product is polymerized to produce a hydrophilic segment. The degree of polymerization of the hydrophilic and hydrophobic segments will affect the particle size of the nanoparticles after self-assembly. The increase in the degree of polymerization and the excessive length of the hydrophobic segment of the first intermediate product will cause the morphology of the nanoparticles finally formed by self-assembly to change, and the hydrophilicity of the entire polymer will decrease, and it may even be insoluble in water. If the hydrophobic segment is reduced too much, the polymer cannot be assembled into nanoparticles. If the degree of polymerization of the hydrophilic segment and the hydrophobic segment is increased or decreased in proportion at the same time, it will lead to an increase or decrease in the particle size of the nanoparticles. There is no effect within a certain range, but if the change is too large, the particle size is too small, the effect will be poor, and if the particle size is too large, it will affect safety.

[0022] As a preferred embodiment, in step (1), the molar ratio of (S)-4-(4-hydroxybenzyl)oxazolidine-2,5-dione to n-butylamine is 15.7:0.7, and the degree of polymerization of the first intermediate product is 20.

[0023] As a preferred embodiment, in step (2), the molar ratio of the first intermediate product to (4S)-4-[(4-hydroxyphenyl)methyl]-2,5-oxazolidinedione is 0.083:13.3, and the degree of polymerization of the second intermediate product is 160.

[0024] As a preferred embodiment, in step (3), the molar ratio of the second intermediate product to dimethylethylenediamine is 1:(160-480).

[0025] In order to solve the above technical problems, the fourth object of the present invention provides a method for preparing a cationic polymer, comprising the following steps:

[0026] (1) (4S)-4-[(4-hydroxyphenyl)methyl]-2,5-oxazolidinedione, n-butylamine, N'N-dimethylformamide, and dichloromethane are mixed, and the polymerization reaction is carried out under nitrogen protection. The mixture obtained after the reaction is precipitated in cold ether and vacuum dried to obtain PBLA;

[0027] (2) PBLA is dissolved in anhydrous DMSO, dimethylethylenediamine is added, and after the polymerization reaction, the mixture solution is dialyzed to remove excess dimethylethylenediamine, and then the solvent is removed by rotary evaporation and vacuum dried to obtain a cationic polymer.

[0028] As a preferred embodiment, in steps (1) and (2), the reaction temperature is 40°C-70°C and the reaction time is more than 40 hours.

[0029] As a preferred embodiment, in step (1), the molar ratio of (4S)-4-[(4-hydroxyphenyl)methyl]-2,5-oxazolidinedione to n-butylamine is 1205:8.

[0030] As a preferred embodiment, in step (2), the molar ratio of PBLA to dimethylethylenediamine is 1:(16-360).

[0031] As a preferred embodiment, the amination rate of the cationic polymer is 100%, and in step (2), the molar ratio of PBLA to dimethylethylenediamine is 1:(144-360).

[0032] By adopting the above scheme, the present application uses aspartic acid benzyl ester endocyclic anhydride monomer (BLA-NCA) to polymerize to obtain polyaspartic acid substituted by benzyl ester, and then replaces the benzyl group with dimethylethylenediamine. The ultimate goal is to obtain polyaspartic acid with the side chain replaced by dimethylethylenediamine. This is to introduce tertiary amines to make the polymer have a positive charge.

[0033] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0034] The present application uses cationic polymers and cationic polymer nanoparticles with high nucleic acid binding affinity and capable of accumulating in the liver to clear NETs that have accumulated in large quantities in the liver before the levels in plasma increase. Both soluble cationic polymer materials and cationic polymer nanoparticle materials can greatly compete for NET-DNA from the NET-DNA and CCDC25 complex, thereby blocking its-mediated chemotaxis and migration of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : A schematic diagram of the synthesis of the cationic polymer nanoparticles of the present invention and a schematic diagram of the competitive binding of the cationic polymer nanoparticles to NET-DNA;

[0036] Figure 2 : The synthesis route of the cationic polymer and cationic polymer nanoparticles in Examples 1-2 of the present invention (Note: A-synthesis route of the cationic polymer in Example 1; B-synthesis route of the cationic polymer nanoparticles in Example 2);

[0037] Figure 3 : is the hydrogen nuclear magnetic resonance spectrum of the cationic polymer in Example 1 of the present invention ( 1 HNMR) spectrum;

[0038] Figure 4 : is the hydrogen nuclear magnetic resonance spectrum of the cationic polymer nanoparticles in Example 2 of the present invention ( 1 HNMR) spectrum;

[0039] Figure 5 : PBLA in Example 1 of the present invention and PTyr in Example 2 20 -b-PBLA 160 Gel permeation chromatography elution time curve;

[0040] Figure 6 : DLS particle size and TEM results of cationic polymer nanoparticles in Example 2 of the present invention (Note: left - DLS; right - TEM);

[0041] Figure 7 : The observation results of cytoskeleton remodeling in different groups in Example 1 of the present invention;

[0042] Figure 8 : The results of cell chemotaxis tests of different groups in Example 2 of the present invention;

[0043] Fig. 9 : The results of the in vivo anti-cancer cell metastasis experiment in the 4T1 mouse model in Example 3 of the present invention (Note: A-experimental progress diagram; B-digital photo of mouse liver, white arrows represent cancer metastasis; C-luminescence intensity of mouse liver, the stronger the luminescence, the more metastatic cancer cells; D-40× low-power microphotograph of mouse liver H&E stained section; E-200× high-power microphotograph of mouse liver H&E stained section, the dotted circled part represents tumor infiltration, and the black arrow represents immune cell infiltration; F-luminescence intensity of mouse lung, the stronger the luminescence, the more metastatic cancer cells; G-40× low-power microphotograph of mouse lung H&E stained section; H-200× high-power microphotograph of mouse lung H&E stained section, the dotted circled part represents tumor infiltration, and the black arrow represents immune cell infiltration; I-survival curve of mice from 13 days to 28 days);

[0044] Fig.10 :The results of the in vivo anti-cancer cell metastasis experiment in the MDA-MB-231 human orthotopic breast cancer mouse model in the effect verification example 4 of the present invention (Note: A-experimental progress diagram; B-digital photo of mouse liver, white arrows represent cancer metastasis; C-luminescence intensity of mouse liver, the stronger the luminescence, the more metastatic cancer cells; D-200× high-power microscopic photo of mouse liver H&E stained section, the dotted circled part represents tumor infiltration, and the black arrow represents immune cell infiltration; E-luminescence intensity of mouse lung, the stronger the luminescence, the more metastatic cancer cells; F-200× high-power microscopic photo of mouse lung H&E stained section, the dotted circled part represents tumor infiltration, and the black arrow represents immune cell infiltration; G-statistical results of mouse weight change; H-survival curve of mouse modeling from 50 days to 137 days; I-NET expression in mouse liver immunofluorescence staining experiment; J-NET expression in mouse lung immunofluorescence staining experiment, white arrows represent NET expression);

[0045] Fig.11 : The results of the in vivo anti-cancer cell metastasis experiment in the HCT116 human colon cancer mouse model in Example 5 of the present invention (Note: A-schematic diagram of the experimental progress; B-representative morphology of mouse liver metastatic nodules; C-quantitative analysis of mouse liver metastatic nodules; D-luminescence intensity of mouse liver; E-quantitative analysis of luminescence intensity of mouse liver; F-40× low-power micrograph of H&E stained sections of mouse liver; G-200× high-power micrograph of H&E stained sections of mouse liver, the dotted line circled part represents tumor infiltration, and the black arrow represents immune cell infiltration; H-quantitative analysis of tumor infiltration area of ​​mouse liver). DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] This application uses cationic polymer materials to electrostatically bind NET-DNA as a new anti-metastasis strategy, such as Figure 1 As shown, the strong electrostatic interaction between cationic polymer nanoparticles cANP and DNA can compete with CCDC25 for binding to NET-DNA, thereby reducing the chemotactic ability of NET-DNA during cancer metastasis, and blocking the interaction between NET-DNA and CCDC25 can inhibit cancer metastasis. The use of soluble polymer materials or their nanoparticle materials with high nucleic acid binding affinity and accumulation in the liver can greatly compete with NET-DNA from the NET-DNA and CCDC25 complex, thereby blocking its mediated cancer cell chemotaxis and migration, and is used to clear NETs that accumulate in large quantities in the liver before the level in plasma increases. This treatment effectively inhibits cancer metastasis in the 4T1 orthotopic breast cancer tumor mouse model, and the same therapeutic effect was also observed in the human breast cancer MDA-MB-231 and colon cancer HCT116 metastasis models, indicating that cationic polymer materials have significant therapeutic effects in blocking cancer metastasis.

[0048] Embodiment 1

[0049] A method for preparing a cationic polymer, the synthetic route is as follows Figure 2 As shown in A, the following steps are included:

[0050] (1) Synthesis of PBLA: In a 100 mL Schlenk flask, 3 g (BLA-NCA, purity 99.5%, source leaf biotechnology, 12.05 mmol) (4S)-4-[(4-hydroxyphenyl)methyl]-2,5-oxazolidinedione was added, followed by 10 mL of a solution of 5.9 mg n-butylamine (purity 98%, Aladdin, 0.08 mmol) in N'N-dimethylformamide (DMF) and 48 mL of dichloromethane. The polymerization reaction was carried out under nitrogen protection at 40°C for 48 h. The mixture obtained after the reaction was precipitated in a large amount of cold ether and the reaction was repeated three times. It was then dried in vacuum at room temperature for 24 h to obtain PBLA.

[0051] According to 1H NMR (Bruker AVANCE III 400MHz) spectrum analysis, the monomer conversion rate of the polymer is 96% and the degree of polymerization is 144; Figure 5 As shown, the polymer polydispersity Mw / Mn was analyzed by gel permeation chromatography (GPC) with DMF as eluent, and the result was 1.07.

[0052] (2) Synthesis of PAsp: First, 0.3 g of PBLA (0.01 mmol) was dissolved in 6 mL of anhydrous DMSO and placed in three Schlenk flasks. Then, 320 mg (3.6 mmol, 2.5 equivalents of benzyl), 77.9 mg (0.88 mmol, about 0.61 equivalents of benzyl) and 14.2 mg (0.16 mmol, about 0.11 equivalents of benzyl) of dimethylethylenediamine (dmeen, purity 98%, Aladd in), after aminolysis reaction at 40°C for 48 hours, the mixture solution was dialyzed in methanol (molecular weight cutoff: 3500Da) for 48 hours to remove excess dimethylethylenediamine, and then the solvent was removed by rotary evaporation and vacuum drying to obtain PAsp homopolymers with BLA amination rates of 100%, 50% and 10%, which were called poly (N-(N,N-dimethylethylenediamine)asparagine)PAsp-100%, PAsp-50% and PAsp-10%, respectively.

[0053] Embodiment 2

[0054] A method for preparing cationic polymer nanoparticles, the synthetic route is as follows Figure 2 As shown in B, the following steps are included:

[0055] (1) Synthesis of PTyr-NH2: In a glove box, 3 g (15.7 mmol, Tyr-NCA, purity 97%, Mack Lin) (S)-4-(4-hydroxybenzyl)oxazolidine-2,5-dione was dissolved in 50 mL DMF and placed in a 100 mL Schlenk flask. Then, 54.7 mg (0.7 mmol) of n-butylamine was added. The polymerization reaction was carried out at 40°C in a nitrogen atmosphere for 48 h. The resulting mixture was then precipitated in a large amount of cold ether. This was repeated three times. After filtration and vacuum drying for 24 hours, the degree of polymerization was 20 according to 1H NMR spectroscopy analysis, i.e., the product was PTyr 20 -NH2.

[0056] (2)PTyr 20 -b-PBLA 160 Synthesis: 0.25 g (0.083 mmol) PTyr 20-NH2, 3.32g (13.3mmol) BLA-NCA, 10mL DMF and 50mL dichloromethane were charged into a 100mL Schlenk flask, and then polymerized at 40°C for 48h. The mixture obtained after the reaction was precipitated in a large amount of cold ether, and the reaction was repeated three times, and then vacuum dried at room temperature for 24h. According to 1H NMR spectrum analysis, the PBLA monomer conversion rate was 99%, the degree of polymerization was 160, and the diblock copolymer PTyr 20 -b-PBLA 160 , using DMF as the eluent, such as Figure 5 As shown, the polymer polydispersity Mw / Mn was 1.10 as measured by GPC.

[0057] (3)PTyr 20 -b-PAsp 160 -100% synthesis: Dissolve 0.3 g PTyr in 6 mL DMSO. 20 -b-PBLA 160 (0.0075 mmol), then 249.2 mg of dimethylethylenediamine (2.82 mmol, about 2.4 times the equivalent of benzyl) was added, and after aminolysis at 40°C for 48 hours, the mixture solution was dialyzed in methanol for 48 hours (molecular weight cutoff: 3500 Da) to remove excess dimethylethylenediamine, and then the solvent was removed by rotary evaporation and vacuum dried to obtain PTyr 20 -b-PAsp 160 - 100% block copolymer.

[0058] (4) Self-assembly preparation of cANP-100% nanoparticles: PTyr 20 -b-PAsp 160 -100% was first dissolved in dimethyl sulfoxide (DMSO) and the polymer concentration in the mixture was controlled to be 10 mg / mL. 20 -b-PAsp 160 -100% DMSO solution was slowly dripped into deionized water until the polymer concentration reached 2 mg / mL. The mixture solution was dialyzed against deionized water (molecular weight cutoff: 3500Da) to remove DMSO. A mixture of Na2HPO4 and KH2PO4 was added to the cANP-100% solution to change the aqueous solution into PBS buffer in which the phosphate concentration was 10 mM.

[0059] Effect Verification Example 1

[0060] Cationic polymers and nanoparticles were used to inhibit cancer cell skeleton remodeling:

[0061] To evaluate the inhibitory effect of PAsp in Example 1 and cANP in Example 2 on NET-DNA-induced tumor cell cytoskeleton remodeling, 2×10 3 MDA-MB-231 cells (human breast cancer cells) were placed in 8-well plates. After overnight incubation, the old medium was replaced with a new serum-free medium containing 5 g / mL NET-DNA and 50 g / mL PAsp-100% or cANP-100%. PBS was used as the blank group. After incubation at 37°C for 3 hours, the supernatant in the wells was taken and stained with Alexa Fluor TM The filamentous actin (F-actin) of MDA-MB-231 cells was stained with 555Phal oid in, and the cell nuclei were stained with DAPI, and then observed under a confocal microscope. For quantitative analysis, the cell filamentous processes were counted from 10 areas in each well, and 3 independent experiments were performed simultaneously.

[0062] The experimental results show that Figure 7 As shown, after the addition of cationic polymers and cationic polymer nanoparticle materials, the behavior of NET-DNA in changing the cytoskeleton was greatly blocked, among which the cationic polymer nanoparticle sample was the most significant, and cytoskeleton remodeling is necessary for cancer cell migration.

[0063] Effect Verification Example 2

[0064] Cationic polymers and nanoparticles inhibit cancer cell migration (cell chemotaxis assay):

[0065] In order to evaluate the chemotactic activity of PAsp in Example 1 and cANP in Example 2 on tumor cells, 3×10 6 MDA-MB-231 cells with a concentration of 10 μg / mL were moved to the seeding chamber of the μ-S l ide chemotaxis chamber. After the cancer cells adhered to the seeding chamber, the left reservoir was filled with 5μg / mL NET-DNA, 5μg / mL NET-DNA+50μg / mL PAsp-100%, or 5μg / mL NET-DNA+50μg / mL cANP-100% PBS solution, and the right reservoir was filled with only PBS solution. Then, a LionheartTMFX automatic live cell imager was used to capture cancer cell images every 8 minutes in the chemotaxis chamber for 7-12 hours, and then the cancer cell migration trajectory was analyzed.

[0066] The results show that if Figure 8 As shown, compared with the NET-DNA control group, the cationic polymers and cationic polymer nanoparticles in the experimental group can significantly inhibit the migration of cancer cells caused by NET-DNA.

[0067] Effect Verification Example 3

[0068] In vivo anticancer cell metastasis experiment of cationic polymers and cationic nanoparticles in inhibiting 4T1 orthotopic breast cancer tumor mice:

[0069] (1) Grouping: Balb / c mice weighing 20 g were divided into a model group, three PAsp-100% administration groups in Example 1 (different concentrations, 5 mg / kg, 10 mg / kg, and 20 mg / kg), and three cANP-100% administration groups in Example 2 (different concentrations, 5 mg / kg, 10 mg / kg, and 20 mg / kg), for a total of 7 groups, with 8 mice in each group;

[0070] (2) Modeling: Mice were anesthetized with isoflurane gas, the skin on the surface of the mammary gland was incised, and 5 × 10 5 4T1-luc cells were resuspended in 100 μL PBS and injected into the fourth mammary fat pad on the right side of BALB / c mice using a syringe, and then the incision was sutured;

[0071] (3) Administration: Starting from the 8th day after model establishment, 5 mg / kg, 10 mg / kg, and 20 mg / kg of PAsp or cANP in PBS solution were injected into the tail vein of the corresponding groups of mice every two days, and the model group mice were injected with an equal volume of PBS solution. The administration lasted for 20 days.

[0072] (4) Recording the changes in tumor volume and body weight of mice: During the administration period, the length and width of the in situ tumor were measured and recorded using a vernier caliper every two days, and the in situ tumor volume was calculated; the weight of the mice was weighed and recorded using an electronic balance;

[0073] (5) Monitoring tumor metastasis: During the administration period, the IVIS Lumina X5 (PerkinElmer) luminescence imager was used to detect tumor metastasis in mice. After the mice were sacrificed, the luminescence intensity of the in situ tumors and different organs was analyzed using the luminescence imager;

[0074] (6) Blood collection and tissue sections: On the 28th day of modeling, i.e., 20 days after drug administration, blood and serum were collected and the mice were euthanized. The heart, liver, spleen, lung, and kidney were taken for tissue section research. Photos of organs such as the liver and lungs where cancer metastasis occurred were taken with a camera. Fig. 9 As shown in BH.

[0075] The results show that if Fig. 9As shown, both PAsp and cANP have an inhibitory effect on the liver and lung metastasis of breast cancer. Compared with the model group, the tumor infiltration area and immune cell recruitment in the liver and lungs of the treated mice have decreased significantly, and the visible cancer metastases have also decreased significantly. The survival curve also shows that the lifespan of the treated mice is extended compared with the untreated model group. At the same time, as the therapeutic concentration of PAsp or cANP increases, the inhibitory effect on tumors becomes more obvious, and the therapeutic effect of cANP on tumors is better than that of PAsp. It is speculated that this is because the nanoparticles stay in the liver for a long time, have a longer onset time, and will not be metabolized immediately.

[0076] Effect Verification Example 4

[0077] In vivo anticancer cell metastasis experiment of cationic polymers and cationic nanoparticles in inhibiting MDA-MB-231 orthotopic breast cancer tumors in mice:

[0078] (1) Grouping: NOD / SCI D mice weighing 20 g were divided into two groups, each of which included three subgroups, one model group, one PAsp-100% administration group of Example 1 (concentration of 10 mg / kg), and one cANP-100% administration group of Example 2 (concentration of 10 mg / kg). The cancer cells injected into the first group were MDA-MB-231-l uc, and the cancer cells injected into the second group were MDA-MB-231. There were 6 groups in total, with 4 mice in each group. The first group could monitor the metastasis of cancer in real time due to the access of l uc groups, and the second group was used to evaluate the effect of cationic materials on extending the life span of mice.

[0079] (2) Modeling: Mice were anesthetized with isoflurane gas, the skin on the surface of the mammary gland was incised, and 2 × 10 6 MDA-MB-231-l uc cells were resuspended in 100 μL PBS and injected into the fourth mammary fat pad on the right side of NOD / SCI D mice using a syringe, and then the incision was sutured;

[0080] (3) Administration: Starting from the 8th day after model establishment, 10 mg / kg PAsp or cANP in PBS solution was injected into the tail vein of the corresponding groups of mice every two days, and the model group mice were injected with an equal volume of PBS solution. The administration lasted for 46 days.

[0081] (4) Recording the changes in tumor volume and body weight of mice: During the administration period, the length and width of the in situ tumor were measured and recorded using a vernier caliper every two days, and the in situ tumor volume was calculated; the weight of the mice was weighed and recorded using an electronic balance;

[0082] (5) Monitoring tumor metastasis: During the administration period, the IVIS Lumina X5 (PerkinElmer) luminescence imager was used to detect tumor metastasis in mice. After the mice were sacrificed, the luminescence intensity of the in situ tumors and different organs was analyzed using the luminescence imager;

[0083] (6) Blood collection and tissue sectioning: On the 54th day of modeling, i.e., 46 days after drug administration, blood and serum were collected and the mice in the first group were euthanized. The hearts, livers, spleens, lungs, and kidneys were taken for tissue section research, and photos of organs such as the liver and lungs where cancer metastasis occurred were taken with a camera.

[0084] The results show that if Fig.10 As shown, both PAsp and cANP have an inhibitory effect on the liver and lung metastasis of MDA-MB-231 human breast cancer. Compared with the model group, the tumor infiltration area and immune cell recruitment in the liver and lungs of treated mice were significantly reduced, and the visible cancer metastases were also significantly reduced. The expression of NET in the liver and lungs was also significantly reduced. The survival curve also showed that the lifespan of treated mice was extended compared with the untreated model group, and the therapeutic effect of cANP on tumors was better than that of PAsp.

[0085] Effect Verification Example 5

[0086] In vivo anticancer cell metastasis experiment of cationic polymers and cationic nanoparticles in inhibiting HCT116 human colon cancer in mice:

[0087] (1) Grouping: NOD / SCI D mice weighing 20 g were divided into a model group (PBS), a PAsp-100% administration group in Example 1 (concentration of 10 mg / kg), and a cANP-100% administration group in Example 2 (concentration of 10 mg / kg), for a total of 3 groups, with 6 mice in each group;

[0088] (2) Modeling: Mice were anesthetized with isoflurane gas, and the abdominal cavity was opened. 1×10 6 HCT116-l uc cells were resuspended in 50 μL PBS and injected into the NOD / SC ID spleen using a syringe, and then the incision was sutured;

[0089] (3) Administration: Starting from the 8th day after model establishment, 10 mg / kg PAsp or cANP in PBS solution was injected into the tail vein of the corresponding group of mice every two days, and the model group mice were injected with an equal volume of PBS solution. The administration lasted for 36 days.

[0090] (4) Monitoring tumor metastasis: During the administration period, the IVIS Lumina X5 (PerkinElmer) luminescence imager was used to detect tumor metastasis in mice. After the mice were sacrificed, the luminescence intensity of the in situ tumors and different organs was analyzed using the luminescence imager;

[0091] (5) Blood collection and tissue sections: On the 54th day of modeling, i.e., 46 days after drug administration, blood and serum were collected and the mice in the first group were euthanized. The hearts, livers, spleens, lungs, and kidneys were taken for tissue section research, and photos of organs such as the liver and lungs where cancer metastasis occurred were taken with a camera;

[0092] The results show that if Fig.11 As shown, both PAsp and cANP have an inhibitory effect on the liver and lung metastasis of human colon cancer. Compared with the model group, the tumor infiltration area and immune cell recruitment in the liver and lungs of the treated mice were significantly reduced, and the cancer metastases visible to the naked eye were also significantly reduced. In addition, the therapeutic effect of cANP on tumors is better than that of PAsp.

[0093] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. It is particularly pointed out that for those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cationic polymer nanoparticle for treating cancer metastasis, characterized in that: The cationic polymer nanoparticles are formed by self-assembly of a block copolymer in an aqueous phase, and the chemical structural formula of the block copolymer is: , the preparation method of the block copolymer comprises the following steps: (1) dissolving (S)-4-(4-hydroxybenzyl)oxazolidine-2,5-dione in N,N-dimethylformamide, adding n-butylamine to carry out a ring-opening polymerization reaction, filtering and drying after precipitation in cold ether to obtain a first intermediate product, and synthesizing a first intermediate product with a degree of polymerization of 10-40 by polymerization reaction; (2) mixing the first intermediate product, (4S)-4-[(4-hydroxyphenyl)methyl]-2,5-oxazolidinedione, N,N-dimethylformamide and dichloromethane for polymerization reaction, filtering and drying after precipitation in cold ether to obtain a second intermediate product, and synthesizing a second intermediate product with a degree of polymerization of 50-400 by polymerization reaction; (3) The second intermediate product is subjected to an aminolysis reaction with dimethylethylenediamine, the product is dialyzed in an organic solvent to remove dimethylethylenediamine, the organic solvent is distilled off, and the block copolymer is obtained after drying.

2. The cationic polymer nanoparticles for treating cancer metastasis according to claim 1, characterized in that The number average molecular weight of the block copolymer is 3k-500k, the weight average molecular weight is 3k-500k, the polymer dispersity coefficient is 1-1.5, the particle size of the cationic polymer nanoparticles is 30nm-200nm, and the polydispersity index is 0.1-0.

3.

3. A cationic polymer for treating cancer metastasis, characterized in that: The chemical structural formula of the cationic polymer is: , the preparation method of the cationic polymer comprises the following steps: (1) (4S)-4-[(4-Hydroxyphenyl)methyl]-2,5-oxazolidinedione was mixed with n-butylamine, N,N-dimethylformamide and dichloromethane, and the polymerization reaction was carried out under nitrogen protection. The mixture obtained after the reaction was precipitated in cold ether and vacuum dried to obtain PBLA. The degree of polymerization of PBLA was 144. (2) PBLA is dissolved in anhydrous DMSO, dimethylethylenediamine is added, and after the aminolysis reaction, the mixture solution is dialyzed to remove excess dimethylethylenediamine, and then the solvent is removed by rotary evaporation and vacuum dried to obtain a cationic polymer.

4. Use of the cationic polymer nanoparticles for treating cancer metastasis according to claim 1 or 2 in preparing a kit for treating cancer metastasis.

5. Use of the cationic polymer for treating cancer metastasis according to claim 3 in preparing a kit for treating cancer metastasis.

6. A method for preparing cationic polymer nanoparticles for treating cancer metastasis as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) dissolving (S)-4-(4-hydroxybenzyl)oxazolidine-2,5-dione in N,N-dimethylformamide, adding n-butylamine to carry out a ring-opening polymerization reaction, precipitating in cold ether, filtering and drying to obtain a first intermediate product; (2) mixing the first intermediate product, (4S)-4-[(4-hydroxyphenyl)methyl]-2,5-oxazolidinedione, N,N-dimethylformamide and dichloromethane for polymerization reaction, precipitating in cold ether, filtering and drying to obtain a second intermediate product; (3) subjecting the second intermediate product to an aminolysis reaction with dimethylethylenediamine, dialyzing the product in an organic solvent to remove dimethylethylenediamine, distilling off the organic solvent, and drying to obtain a block copolymer; (4) The block copolymer is dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5-15 mg / mL, and then added dropwise into water until the polymer concentration reaches 1-3 mg / mL. The DMSO is dialyzed to remove the DMSO, and a mixture of Na2HPO4 and KH2PO4 is added to convert the aqueous solution into PBS buffer solution, and the PBS concentration in the solution is adjusted to 10 mmol / L, thereby obtaining a PBS solution containing cationic polymer nanoparticles.

7. The method for preparing cationic polymer nanoparticles for treating cancer metastasis according to claim 6, characterized in that: In steps (1), (2) and (3), the reaction temperature is 40°C-70°C and the reaction time is more than 40 hours.

8. A method for preparing a cationic polymer for treating cancer metastasis according to claim 3, characterized in that: The following steps are involved: (1) (4S)-4-[(4-hydroxyphenyl)methyl]-2,5-oxazolidinedione is mixed with n-butylamine, N,N-dimethylformamide and dichloromethane, and the polymerization reaction is carried out under nitrogen protection. The mixture obtained after the reaction is precipitated in cold ether and vacuum dried to obtain PBLA; (2) PBLA is dissolved in anhydrous DMSO, dimethylethylenediamine is added, and after the aminolysis reaction, the mixture solution is dialyzed to remove excess dimethylethylenediamine, and then the solvent is removed by rotary evaporation and vacuum dried to obtain a cationic polymer.

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