阅读说明：本技术 一种药物共担载纳米颗粒及其制备方法和应用 (Drug co-supported nano-particles and preparation method and application thereof ) 是由 田华雨 胡莹莹 郭兆培 林琳 陈杰 于海洋 陈学思 于 2021-07-29 设计创作，主要内容包括：本发明属于医疗领域,尤其涉及一种药物共担载纳米颗粒及其制备方法和应用。本发明提供的药物共担载纳米颗粒制备方法包括以下步骤：a)将高分子载体、至少两种小分子药物和有机溶剂混合,得到混合溶液；所述高分子载体为甲氧基聚乙二醇改性聚谷氨酸；所述小分子药物为小分子疏水药物和/或小分子亲水药物；b)将所述混合溶液滴加到水中,搅拌,透析,得到药物共担载纳米颗粒。本发明提供的制备方法工艺简单,条件温和,易于操作,适合大规模生产应用；采用该方法制备的药物共担载纳米颗粒具有十分优异的治疗效果,在有效抑制肿瘤生长的同时能极大地降低药物对机体的副作用,具有十分广阔的市场前景。(The invention belongs to the field of medical treatment, and particularly relates to a drug co-supported nanoparticle and a preparation method and application thereof. The preparation method of the drug co-supported nanoparticles provided by the invention comprises the following steps: a) mixing a macromolecular carrier, at least two micromolecular medicines and an organic solvent to obtain a mixed solution; the polymer carrier is methoxy polyethylene glycol modified polyglutamic acid; the small molecular drug is a small molecular hydrophobic drug and/or a small molecular hydrophilic drug; b) and dripping the mixed solution into water, stirring and dialyzing to obtain the drug co-supported nano particles. The preparation method provided by the invention has the advantages of simple process, mild condition, easy operation and suitability for large-scale production and application; the drug co-supported nanoparticles prepared by the method have excellent treatment effect, can greatly reduce the side effect of the drug on organisms while effectively inhibiting the growth of tumors, and have wide market prospect.)

1. A preparation method of drug co-supported nanoparticles comprises the following steps:

a) mixing a macromolecular carrier, at least two micromolecular medicines and an organic solvent to obtain a mixed solution;

the polymer carrier is methoxy polyethylene glycol modified polyglutamic acid; the small molecular drug is a small molecular hydrophobic drug and/or a small molecular hydrophilic drug;

b) and dripping the mixed solution into water, stirring and dialyzing to obtain the drug co-supported nano particles.

2. The preparation method according to claim 1, wherein the number average molecular weight of the methoxypolyethylene glycol segment in the methoxypolyethylene glycol-modified polyglutamic acid is 500 to 10000Da, and the number average molecular weight of the polyglutamic acid segment is 10000 to 70000 Da;

the molar ratio of the repeating unit corresponding to the methoxypolyethylene glycol to the repeating unit corresponding to the polyglutamic acid in the methoxypolyethylene glycol modified polyglutamic acid is (1-20): 1.

3. the method of claim 1, wherein the small molecule drug is selected from at least two of mitoxantrone, doxorubicin, JQ1, camptothecin, gambogic acid, indocyanine green, IR783, ABT263, and NLG 919.

4. The preparation method according to claim 1, wherein the mass ratio of the polymeric carrier to the small-molecule drug is (0.01-100): 1.

5. the preparation method according to claim 1, wherein the total concentration of the polymeric carrier and the small molecule drug in the mixed solution is not less than 0.01 mg/mL.

6. The method according to claim 1, wherein the volume of the water is 5 to 20 times the volume of the mixed solution.

7. The preparation method according to claim 1, wherein the dropping speed is 0.1 to 5 mL/min; the stirring time is more than or equal to 0.5 min; the dialysis time is 0.5-3 days.

8. A drug co-supported nanoparticle comprises a polymer carrier and at least two small molecule drugs supported on the polymer carrier;

the polymer carrier is methoxy polyethylene glycol modified polyglutamic acid;

the small molecule drug is a small molecule hydrophobic drug and/or a small molecule hydrophilic drug.

9. The drug-co-supported nanoparticle according to claim 8, wherein the particle size of the drug-co-supported nanoparticle is 50 to 200 nm.

10. The use of the drug co-supported nanoparticles prepared by the preparation method of any one of claims 1 to 7 or the drug co-supported nanoparticles of any one of claims 8 to 9 in the preparation of a medicament for treating tumors.

Technical Field

The invention belongs to the field of medical treatment, and particularly relates to a drug co-supported nanoparticle and a preparation method and application thereof.

Background

Tumor refers to a new organism formed by local tissue cell proliferation under the action of various tumorigenic factors, because the new organism mostly presents space-occupying block-shaped protrusions, which is also called neoplasm. According to the cellular characteristics of the new organism and the degree of harm to the organism, the tumors are divided into two categories of benign tumors and malignant tumors, wherein the malignant tumors can be divided into carcinoma and sarcoma, the carcinoma refers to the malignant tumor derived from epithelial tissue, and the sarcoma refers to mesenchymal tissue including fibrous connective tissue, fat, muscle, vessel, bone, cartilage tissue and the like.

At present, tumors still are the biggest killers harmful to human life health, and the traditional means for treating tumors include operations, chemotherapy and radiotherapy. Wherein, because the operation and the radiotherapy can only be used for the local treatment of the tumor, but no strategy is provided for the tumor metastasis, the chemotherapy can lead the medicine to be spread over most organs and tissues of the whole body along with the blood circulation, and becomes an effective means for treating the tumor, particularly the middle and late stage tumor. However, how to effectively deliver therapeutic drugs to tumor tissues is the biggest challenge facing current tumor chemotherapy.

Disclosure of Invention

In view of the above, the invention aims to provide a drug-co-supported nanoparticle, and a preparation method and an application thereof.

The invention provides a preparation method of drug co-supported nanoparticles, which comprises the following steps:

a) mixing a macromolecular carrier, at least two micromolecular medicines and an organic solvent to obtain a mixed solution;

the polymer carrier is methoxy polyethylene glycol modified polyglutamic acid; the small molecular drug is a small molecular hydrophobic drug and/or a small molecular hydrophilic drug;

b) and dripping the mixed solution into water, stirring and dialyzing to obtain the drug co-supported nano particles.

Preferably, the number average molecular weight of a methoxy polyethylene glycol chain segment in the methoxy polyethylene glycol modified polyglutamic acid is 500-10000 Da, and the number average molecular weight of a polyglutamic acid chain segment is 10000-70000 Da;

the molar ratio of the repeating unit corresponding to the methoxypolyethylene glycol to the repeating unit corresponding to the polyglutamic acid in the methoxypolyethylene glycol modified polyglutamic acid is (1-20): 1.

preferably, the small molecule drug is selected from at least two of mitoxantrone, doxorubicin, JQ1, camptothecin, gambogic acid, indocyanine green, IR783, ABT263, and NLG 919.

Preferably, the mass ratio of the high molecular carrier to the small molecular drug is (0.01-100): 1.

preferably, the total concentration of the macromolecular carrier and the small molecular drug in the mixed solution is more than or equal to 0.01 mg/mL.

Preferably, the volume of the water is 5-20 times of the volume of the mixed solution.

Preferably, the dropping speed is 0.1-5 mL/min; the stirring time is more than or equal to 0.5 min; the dialysis time is 0.5-3 days.

The invention provides a drug co-supported nanoparticle, which comprises a high molecular carrier and at least two small molecular drugs supported on the high molecular carrier;

the polymer carrier is methoxy polyethylene glycol modified polyglutamic acid;

the small molecule drug is a small molecule hydrophobic drug and/or a small molecule hydrophilic drug.

Preferably, the particle size of the drug co-supported nanoparticles is 50-200 nm.

The invention also provides the drug co-supported nanoparticles prepared by the preparation method of the technical scheme or the application of the drug co-supported nanoparticles in the preparation of the tumor treatment drug.

Compared with the prior art, the invention provides a drug co-supported nanoparticle and a preparation method and application thereof. The preparation method of the drug co-supported nanoparticles provided by the invention comprises the following steps: a) mixing a macromolecular carrier, at least two micromolecular medicines and an organic solvent to obtain a mixed solution; the polymer carrier is methoxy polyethylene glycol modified polyglutamic acid; the small molecular drug is a small molecular hydrophobic drug and/or a small molecular hydrophilic drug; b) and dripping the mixed solution into water, stirring and dialyzing to obtain the drug co-supported nano particles. The method provided by the invention uses methoxy polyethylene glycol modified polyglutamic acid (PLG-g-mPEG) as a drug carrier, and carries various hydrophilic/hydrophobic micromolecular drugs efficiently through electrostatic interaction and hydrophobic interaction. Compared with single loading, the co-loading mode of the multiple medicines can obviously improve the loading efficiency of the medicines, particularly the hydrophobic medicines; meanwhile, the particle size of the drug co-supported nanoparticles prepared by the method is stable and controllable, the particle size range is 50-200 nm, the drug co-supported nanoparticles have a good high-permeability long-retention effect (EPR effect) in vivo, the effective transportation and enrichment of the drug at a tumor part can be realized, the treatment effect is good, and the side effect is low; moreover, the drug co-supported nanoparticles prepared by the method also have certain pH response release performance, and can realize the release of tumor site specific drugs, thereby further reducing the side effects of the drugs; in addition, the drug co-supported nanoparticles prepared by the method can be used for different combination treatments according to the selected drugs, and have certain universality. The preparation method provided by the invention has the advantages of simple process, mild condition, easy operation and suitability for large-scale production and application; the drug co-supported nanoparticles prepared by the method have excellent treatment effect, can greatly reduce the side effect of the drug on organisms while effectively inhibiting the growth of tumors, and have wide market prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph of MTO release rate of co-supported nanoparticles provided in example 4 of the present invention in solutions of different pH values;

FIG. 2 is a bar graph of cell viability of 4T1 cells provided in example 5 of the present invention after treatment with various drugs;

FIG. 3 is a bar graph of cell viability of 4T1 cells provided in example 7 of the present invention after treatment with various drugs.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a preparation method of drug co-supported nanoparticles, which comprises the following steps:

a) mixing a macromolecular carrier, at least two micromolecular medicines and an organic solvent to obtain a mixed solution;

b) and dripping the mixed solution into water, stirring and dialyzing to obtain the drug co-supported nano particles.

In the preparation method provided by the invention, in the step a), the macromolecule carrier is methoxy polyethylene glycol modified polyglutamic acid (PLG-g-mPEG), which consists of a polyglutamic acid (PLG) main chain and methoxy polyethylene glycol (mPEG) branched chains grafted on the main chain, and the specific structure of the macromolecule carrier can be shown as the formula (I):

in the preparation method provided by the invention, in the step a), the number average molecular weight of a methoxypolyethylene glycol chain segment in the methoxypolyethylene glycol modified polyglutamic acid is preferably 500-10000 Da, and specifically can be 500Da, 1000Da, 2000Da, 3000Da, 4000Da, 5000Da, 6000Da, 7000Da, 8000Da, 9000Da or 10000 Da; the number average molecular weight of a polyglutamic acid chain segment in the methoxypolyethylene glycol modified polyglutamic acid is preferably 10000-70000 Da, and specifically can be 10000Da, 15000Da, 20000Da, 21600Da, 25000Da, 30000Da, 35000Da, 40000Da, 45000Da, 50000Da, 55000Da, 60000Da, 65000Da or 70000 Da; the molar ratio of the repeating unit corresponding to the methoxypolyethylene glycol to the repeating unit corresponding to the polyglutamic acid in the methoxypolyethylene glycol modified polyglutamic acid is preferably (1-20): 1, more preferably (1 to 15): 1, most preferably (1-10): 1, specifically 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10: 1.

In the preparation method provided by the invention, in the step a), the methoxypolyethylene glycol modified polyglutamic acid can be commercially available, and the solution can be prepared according to the following steps:

i) mixing methoxy polyethylene glycol (mPEG) and polyglutamic acid (PLG) in an organic solvent for reaction to obtain a reaction product;

ii) mixing the reaction product with a condensing agent for reaction, and performing post-treatment to obtain the methoxy polyethylene glycol modified polyglutamic acid.

In the preparation step of the methoxypolyethylene glycol modified polyglutamic acid, in step i), the number average molecular weight of the methoxypolyethylene glycol is preferably 500-10000 Da, and specifically can be 500Da, 1000Da, 2000Da, 3000Da, 4000Da, 5000Da, 6000Da, 7000Da, 8000Da, 9000Da or 10000 Da; the number average molecular weight of the polyglutamic acid is preferably 10000-70000 Da, and specifically can be 10000Da, 15000Da, 20000Da, 21600Da, 25000Da, 30000Da, 35000Da, 40000Da, 45000Da, 50000Da, 55000Da, 60000Da, 65000Da or 70000 Da; the mole ratio of the repeating units of the methoxypolyethylene glycol and the polyglutamic acid is preferably (1-20): 1, more preferably (1 to 15): 1, most preferably (1-10): 1, specifically 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10: 1.

In the preparation step of the methoxypolyethylene glycol modified polyglutamic acid, in step i), the temperature of the mixing reaction is preferably 30-50 ℃, and more preferably 40 ℃; the mixing reaction time is preferably 1-5 h, and more preferably 2 h.

In the preparation step of the methoxypolyethylene glycol-modified polyglutamic acid provided by the invention, in the step ii), the condensing agent comprises 4-Dimethylaminopyridine (DMAP) and/or diisopropylcarbodiimide; the ratio of the amount of the 4-dimethylaminopyridine to the amount of the polyglutamic acid which is a raw material for preparing the reaction product is preferably 0.48 mmol: (1-5) g, more preferably 0.48 mmol: 2g of the total weight of the mixture; the ratio of the amount of the diisopropylcarbodiimide to the amount of polyglutamic acid which is a raw material for preparing the reaction product is preferably 4.8 mmol: (1-5) g, more preferably 4.8 mmol: 2g of the total weight.

In the step of preparing the methoxypolyethylene glycol-modified polyglutamic acid provided by the invention, in the step ii), the mixing reaction is carried out under stirring conditions; the temperature of the mixing reaction is preferably 15-35 ℃, and more preferably 25 ℃ (room temperature); the mixing reaction time is preferably 1 to 5 days, and more preferably 3 days.

In the step of preparing the methoxypolyethylene glycol-modified polyglutamic acid provided by the present invention, in step ii), the post-treatment is preferably performed in the following manner: sequentially carrying out diethyl ether precipitation, washing, vacuum drying, distilled water dialysis and freeze-drying.

In the preparation method provided by the invention, in the step a), the small molecule drug is a small molecule hydrophobic drug and/or a small molecule hydrophilic drug, and the relative molecular mass of the small molecule drug is usually less than 1000; the small molecule hydrophobic drugs comprise but are not limited to anthracyclines including antitumor chemotherapeutic drugs mitoxantrone and adriamycin, and small molecule hydrophobic drugs with tumor immune microenvironment regulation effects, and the small molecule hydrophilic drugs comprise but are not limited to small molecule imaging reagents containing sulfonic acid groups. In the present invention, the small molecule drug preferably includes at least two of Mitoxantrone (MTO), Doxorubicin (DOX), JQ1, camptothecin, Gambogic Acid (GA), indocyanine green, IR783, ABT263 and NLG919, wherein the chemical structures of JQ1, IR783, ABT263 and NLG919 are as follows:

in one embodiment provided by the invention, in step a), the small molecule drug is selected from mitoxantrone and JQ1, and the mass ratio of mitoxantrone to JQ1 is preferably 1: (5-15), specifically 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15, and most preferably 1: 9.

In another embodiment provided by the present invention, in step a), the small molecule drug is selected from mitoxantrone and gambogic acid, and the mass ratio of mitoxantrone to gambogic acid is preferably 1: (0.5-5), specifically 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, most preferably 1:2.

In other embodiments provided herein, in step a), the small molecule drug is selected from mitoxantrone and ABT263, and the mass ratio of mitoxantrone to ABT263 is preferably 1: (5-15), specifically 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15, and most preferably 1: 10.

In other embodiments provided by the present invention, in step a), the small molecule drug is mitoxantrone and doxorubicin, and the mass ratio of mitoxantrone to doxorubicin is preferably 1: (0.2 to 3), specifically 1:0.2, 1:0.5, 1:0.7, 1:1, 1:1.2, 1:1.5, 1:1.7, 1:2, 1:2.3, 1:2.5, 1:2.7 or 1:3, and most preferably 1:1.

In other embodiments provided by the present invention, in step a), the small molecule drug is mitoxantrone and NLG919, and the mass ratio of mitoxantrone to NLG919 is preferably 1: (5-15), specifically 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15, and most preferably 1: 10.

In other embodiments provided by the present invention, in step a), the small molecule drug is selected from doxorubicin and JQ1, and the mass ratio of doxorubicin to JQ1 is preferably 1: (5-15), specifically 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15, and most preferably 1: 10.

In other embodiments provided by the present invention, in step a), the small molecule drug is selected from doxorubicin and gambogic acid, and the mass ratio of doxorubicin to gambogic acid is preferably 1: (0.5-5), specifically 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, most preferably 1:2.

In other embodiments provided by the present invention, in step a), the small molecule drug is selected from doxorubicin and ABT263, and the mass ratio of doxorubicin to ABT263 is preferably 1: (5-15), specifically 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15, and most preferably 1: 10.

In other embodiments provided by the present invention, in step a), the small molecule drug is selected from doxorubicin and NLG919, and the mass ratio of doxorubicin to NLG919 is preferably 1: (5-15), specifically 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15, and most preferably 1: 10.

In other embodiments provided herein, in step a), the small molecule drug is selected from JQ1 and ABT263, and the mass ratio of JQ1 to ABT263 is preferably 5: (10-50), specifically 5:10, 5:15, 5:20, 5:25, 5:30, 5:35, 5:40, 5:45 or 5:50, most preferably 5: 25.

In other embodiments provided by the present invention, in step a), the small molecule drug is selected from camptothecin and JQ1, and the mass ratio of camptothecin to JQ1 is preferably 1: (0.5-5), specifically 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, most preferably 1:2.

In other embodiments provided by the present invention, in step a), the small molecule drug is selected from camptothecin and ABT263, and the mass ratio of camptothecin to ABT263 is preferably 1: (0.5-5), specifically 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, most preferably 1:2.

In other embodiments provided by the present invention, in step a), the small molecule drug is selected from gambogic acid and JQ1, and the mass ratio of gambogic acid to JQ1 is preferably 2: (5-15), specifically 2:5, 2:6, 2:7, 2:8, 2:9, 2:10, 2:11, 2:12, 2:13, 2:14 or 2:15, and most preferably 2: 10.

In other embodiments provided by the present invention, in step a), the small molecule drug is selected from indocyanine green and gambogic acid, and the mass ratio of the indocyanine green to the gambogic acid is preferably 1: (2-10), specifically 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, and most preferably 1: 5.

In other embodiments provided herein, in step a), the small molecule drug is selected from IR783 and gambogic acid, and the mass ratio of IR783 to gambogic acid is preferably 1: (2-10), specifically 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, and most preferably 1: 5.

In the preparation method provided by the invention, in the step a), the mass ratio of the macromolecular carrier to the micromolecular drug is preferably (0.01-100): 1, more preferably (0.05 to 10): 1, most preferably (0.1 to 5):1, specifically 0.1:1, 0.12:1, 0.15:1, 0.16:1, 0.18:1, 0.2:1, 0.23:1, 0.25:1, 0.27:1, 0.3:1, 0.32:1, 0.34:1, 0.37:1, 0.4:1, 0.42:1, 0.45:1, 0.47:1, 0.5:1, 0.52:1, 0.55:1, 0.57:1, 0.6:1, 0.62:1, 0.65:1, 0.67:1, 0.7:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, 1.2:1, 1.5:1, 4:1, or 4: 1.

In the preparation method provided by the invention, in the step a), the organic solvent can be mutually soluble with water and can dissolve the carrier and the small molecule drug, preferably methanol and/or dimethyl sulfoxide, and more preferably dimethyl sulfoxide.

In the preparation method provided by the invention, in the step a), the total concentration of the macromolecular carrier and the micromolecular drug in the mixed solution is preferably not less than 0.01mg/mL, more preferably 0.1-50 mg/mL, most preferably 1-20 mg/mL, and most preferably 10-20 mg/mL.

In the preparation method provided by the invention, in the step a), preferably, the polymer carrier and the small molecule drug are respectively mixed with the organic solvent, and then the obtained polymer carrier solution is mixed with the small molecule drug solution. Wherein the concentration of the macromolecular carrier solution is preferably not less than 0.01mg/mL, more preferably 0.1-50 mg/mL, most preferably 1-20 mg/mL, and most preferably 20 mg/mL; the concentration of the small molecular drug solution is preferably not less than 0.01mg/mL, more preferably 0.1-50 mg/mL, most preferably 1-20 mg/mL, and most preferably 10 mg/mL.

In the preparation method provided by the invention, in the step a), the mixing temperature is preferably 15-35 ℃, and more preferably 25 ℃ (room temperature); the mixing time is not particularly limited, and the components may be sufficiently dissolved and uniformly mixed.

In the preparation method provided by the invention, in the step b), the water is preferably ultrapure water; the volume of the water is preferably 5 to 20 times, more preferably 5 to 15 times, and most preferably 10 times of the volume of the mixed solution.

In the preparation method provided by the invention, in the step b), the dripping temperature is preferably 15-35 ℃, and more preferably 25 ℃ (room temperature); the dropping speed is preferably 0.1-5 mL/min, more preferably 0.1-3 mL/min, and most preferably 1-2 mL/min.

In the preparation method provided by the invention, in the step b), the stirring is preferably rapid stirring; the stirring temperature is preferably 15-35 ℃, and more preferably 25 ℃ (room temperature); the stirring time is preferably not less than 0.5min, more preferably 3-20 min, and most preferably 5 min.

In the preparation method provided by the invention, in the step b), the cut-off molecular weight of a dialysis bag adopted by dialysis is preferably 2000-5000, and more preferably 3500; the dialysis temperature is preferably 15-35 ℃, and more preferably 25 ℃ (room temperature); the dialysis time is preferably 0.5 to 3 days, more preferably 1 to 3 days, and most preferably 2 days.

The invention also provides the drug co-supported nanoparticles prepared by the method of the technical scheme. The drug co-supported nanoparticles provided by the invention comprise a high molecular carrier and at least two small molecular drugs supported on the high molecular carrier; wherein, the polymer carrier and the small molecule drug are introduced above and are not described herein again; the particle size of the drug co-supported nanoparticles is preferably 50-200 nm, specifically 50nm, 53.8nm, 55nm, 60.8nm, 65nm, 66.1nm, 70nm, 75nm, 78.2nm, 80nm, 85nm, 90nm, 90.2nm, 95nm, 100nm, 105nm, 110nm, 113nm, 115nm, 117.8nm, 118.6nm, 120nm, 123.8nm, 125nm, 130nm, 135nm, 136.9nm, 137.9nm, 140nm, 145nm, 150nm, 155nm, 155.5nm, 160nm, 165nm, 169nm, 170nm, 171.6nm, 175nm, 180nm, 181.7nm, 185nm, 190nm, 195nm or 200nm, and the nanoparticles in the above particle size range have a good EPR effect in vivo.

The invention also provides an application of the drug co-supported nanoparticles in the technical scheme or the drug co-supported nanoparticles prepared by the preparation method in the technical scheme in the preparation of tumor treatment drugs. In addition, although the invention emphasizes the application of the drug in the preparation of tumor treatment drugs, the application range of the drug co-loaded nanoparticles is not limited to the tumor treatment field, and the drug co-loaded nanoparticles can be applied to different fields according to the curative effect of the selectively loaded drug.

The technical scheme provided by the invention utilizes methoxy polyethylene glycol modified polyglutamic acid as a drug carrier, and various hydrophilic/hydrophobic micromolecule drugs are efficiently carried through electrostatic interaction and hydrophobic interaction. Compared with single loading, the co-loading mode of the multiple medicines can obviously improve the loading efficiency of the medicines, particularly the hydrophobic medicines; meanwhile, the particle size of the drug co-supported nanoparticles prepared by the technical scheme of the invention is stable and controllable, the particle size range is 50-200 nm, the drug co-supported nanoparticles have a good enhanced permeability and retentivity effect (EPR effect) in vivo, the effective transportation and enrichment of the drug at a tumor part can be realized, the treatment effect is good, and the side effect is low; moreover, the drug co-supported nanoparticles prepared by the technical scheme of the invention also have certain pH response release performance, and can realize the release of tumor site specific drugs, thereby further reducing the side effects of the drugs; in addition, the drug co-supported nanoparticles prepared by the technical scheme of the invention can be used for different combination treatments according to the selected drugs, and have certain universality. The technical scheme provided by the invention has the advantages of simple process, mild condition, easy operation and suitability for large-scale production and application; the drug co-supported nanoparticles prepared by the technical scheme have excellent treatment effect, can greatly reduce the side effect of the drug on organisms while effectively inhibiting the growth of tumors, and have wide market prospect.

For the sake of clarity, the following examples are given in detail.

Example 1

1) Preparation of Polymer Carrier PLG-g-mPEG:

dissolving 2g of polyglutamic acid (number average molecular weight 21600Da) and a required amount of methoxypolyethylene glycol (number average molecular weight 5000Da) in 80 ml of anhydrous DMF according to a repeating unit molar ratio of 5:1 of methoxypolyethylene glycol and polyglutamic acid, and then reacting at 40 ℃ for 2 hours; after the reaction, the temperature of the mixture was reduced to room temperature, and then 0.48mmol of DMAP and 4.8mmol of diisopropylcarbodiimide were added to the mixture, followed by stirring at room temperature for 3 days; after stirring was completed, the mixture was precipitated with ether and washed twice, dried under vacuum, dialyzed against distilled water for 2 days, and finally lyophilized to obtain PLG-g-mPEG.

2) Preparation of drug co-supported nanoparticles:

dissolving the PLG-g-mPEG high molecular carrier prepared in the step 1) in dimethyl sulfoxide, wherein the dissolving concentration is 20 mg/mL; then, respectively dissolving the small molecular drugs (mitoxantrone, JQ1) in dimethyl sulfoxide, wherein the dissolving concentration is 10 mg/mL; mixing the carrier solution and the medicine solution at room temperature according to the corresponding dosage proportion, and then slowly dripping (2.0mL/min) the mixed solution into ultrapure water, wherein the volume of the ultrapure water is ten times that of the dimethyl sulfoxide; after the dropwise addition, stirring at room temperature for 5min, then placing the stirred mixed solution into a dialysis bag with the molecular weight cutoff of 3500, and dialyzing at room temperature for 2 days to obtain the drug co-supported nanoparticles.

3) Determination of drug loading rate of drug co-supported nanoparticles:

dispersing the drug co-supported nanoparticles prepared in the step 2) in an aqueous solution with the concentration of 1mg/mL, diluting 200 mu L of the nanoparticle dispersed aqueous solution with a dimethyl sulfoxide solvent, carrying out vortex oscillation for 15s, detecting an absorption peak of a drug Mitoxantrone (MTO) at 621nm by using an ultraviolet spectrophotometer, and measuring the drug loading of the mitoxantrone; the same method was used to dissolve the free drug and the peak absorbance of the free drug was measured at different concentrations and used for standard curve plotting. For the drug loading rate of the drug JQ1, the prepared drug co-supported nanoparticles are dispersed in an aqueous solution, then the dispersion liquid is freeze-dried, 10mg of the dispersion liquid is used for element analysis and detection, and the content of JQ1 in the nanoparticles is quantified by detecting S element.

The results of the drug loading rate measurement of the drug co-loaded nanoparticles of different PLG-g-mPEG and drug dosage ratios are detailed in Table 1:

table 1 example 1 drug loading rate of drug co-supported nanoparticles

In table 1, DLE represents the drug loading rate.

As can be seen from the data in Table 1, in the vector PLG-g-mPEG: MTO: the JQ1 ratio was 2:1:9 for optimum drug loading. In addition, the carrier with the same proportion is used for independently loading the mitoxantrone and JQ1, the drug loading of the mitoxantrone is 31.1 percent, and the drug loading is not obviously reduced; however, the drug loading rate of JQ1 is reduced to 15.3%, and obvious precipitation occurs in the drug loading process, which shows that the drug loading rate of the hydrophobic drug can be obviously improved by the drug co-loading system.

Example 2

1) Preparation of drug co-supported nanoparticles:

dissolving the PLG-g-mPEG polymer carrier prepared in the step 1) of the example 1 in dimethyl sulfoxide to obtain a dissolved concentration of 20 mg/mL; then, respectively dissolving the micromolecule drugs (mitoxantrone and gambogic acid) in dimethyl sulfoxide, wherein the dissolving concentration is 10 mg/mL; mixing the carrier solution and the medicine solution at room temperature according to the corresponding dosage proportion, and then slowly dripping (2.0mL/min) the mixed solution into ultrapure water, wherein the volume of the ultrapure water is ten times that of the dimethyl sulfoxide; after the dropwise addition, stirring at room temperature for 5min, then placing the stirred mixed solution into a dialysis bag with the molecular weight cutoff of 3500, and dialyzing at room temperature for 2 days to obtain the drug co-supported nanoparticles.

3) Determination of drug loading rate of drug co-supported nanoparticles:

dispersing the drug co-supported nanoparticles prepared in the step 2) in an aqueous solution with the concentration of 1mg/mL, diluting 200 mu L of the nanoparticle dispersed aqueous solution with a dimethyl sulfoxide solvent, carrying out vortex oscillation for 15s, detecting an absorption peak of a drug Mitoxantrone (MTO) at 621nm by using an ultraviolet spectrophotometer, and measuring the drug loading of the mitoxantrone; the same method was used to dissolve the free drug and the peak absorbance of the free drug was measured at different concentrations and used for standard curve plotting. For the drug loading rate of the drug Gambogic Acid (GA), the absorption peak of the drug GA was detected at 363nm, which was also measured by ultraviolet spectrophotometry.

The results of the drug loading rate measurement of the drug co-loaded nanoparticles of different PLG-g-mPEG and drug dosage ratios are shown in Table 2:

table 2 example 2 drug loading rate of drug co-supported nanoparticles

In table 2, DLE represents the drug loading rate.

As can be seen from the data in Table 2, in the vector PLG-g-mPEG: MTO: the GA ratio is 1.5:1:0.5, and the drug loading of the two drugs is the highest. In addition, GA and mitoxantrone are independently supported by using carriers with the same proportion, the drug loading of the mitoxantrone is 36.4 percent, and the drug loading is not obviously reduced; however, the drug loading rate of the gambogic acid is reduced to 5.3%, and obvious precipitation appears in the drug loading process, which shows that the drug co-loading system can obviously improve the drug loading rate of the hydrophobic drug.

Example 3

Preparation of drug co-supported nanoparticles:

dissolving the PLG-g-mPEG polymer carrier prepared in the step 1) of the example 1 in dimethyl sulfoxide to obtain a dissolved concentration of 20 mg/mL; then dissolving different small molecular drugs in dimethyl sulfoxide respectively, wherein the dissolving concentration is 10 mg/mL; mixing the carrier solution and the medicine solution at room temperature according to the corresponding dosage proportion, and then slowly dripping (2.0mL/min) the mixed solution into ultrapure water, wherein the volume of the ultrapure water is ten times that of the dimethyl sulfoxide; after the dropwise addition, stirring at room temperature for 5min, then placing the stirred mixed solution into a dialysis bag with the molecular weight cutoff of 3500, and dialyzing at room temperature for 2 days to obtain the drug co-supported nanoparticles. The dosage ratios of the different supported drugs and the PLG-g-mPEG to the drugs are shown in Table 3:

table 3 table of raw material information of example 3

Numbering	Medicine 1	Medicine 2	The mass ratio of the PLG-g-mPEG to the medicament 1 to the medicament 2
				1	Mitoxantrone	JQ1	2:1:9
2	Mitoxantrone	Gambogic acid	2:1:2
				3	Mitoxantrone	ABT263	2:1:10
4	Mitoxantrone	Adriamycin	2:1:1
				5	Mitoxantrone	NLG919	2:1:10
6	Adriamycin	JQ1	2:1:10
				7	Adriamycin	Gambogic acid	2:1:2
8	Adriamycin	ABT263	2:1:10
				9	Adriamycin	NLG919	2:1:10
10	JQ1	ABT263	3:5:25
				11	Camptothecin	JQ1	2:1:2
12	Camptothecin	ABT263	2:1:2
				13	Gambogic acid	JQ1	2:2:10
14	Indocyanine green	Gambogic acid	2:1:5
				15	IR783	Gambogic acid	2:1:5

The prepared drug-loaded nanoparticles were subjected to particle size detection using a potentiometric particle sizer, and the results are shown in table 4:

table 4 particle size of drug co-loaded nanoparticles prepared in example 3

Numbering	Co-carried nanoparticle size (nm)
		1	113
2	169
		3	60.8
4	78.2
		5	118.6
6	137.9
		7	123.8
8	53.8
		9	136.9
10	66.1
		11	171.6
12	281.7
		13	117.8
14	90.2
		15	155.5

As can be seen from Table 4, the particle size of the drug-loaded nanoparticles prepared in the above examples is stabilized at 50-200 nm, so that the drug-loaded nanoparticles can have a good EPR effect in vivo.

Example 4

The co-supported nanoparticles prepared in the number 1 of example 3 were dispersed in an aqueous solution at a concentration of 0.5mg/mL and a volume of 3mL, and then the aqueous solution of nanoparticles was placed in a dialysis bag with a cut-off molecular weight of 3500, respectively placed in buffers with pH values of 7.2, 6.5, and 5.0, shaken in a 37 ℃ incubator, and 2mL of supernatant buffer was taken for drug detection at 0h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, and 72h, and 2mL of fresh buffer was added at the same time of each dot taking. And detecting the 621nm wavelength absorption value of the supernatant buffer solution by an ultraviolet spectrophotometer to further calculate the drug release amount of the Mitoxantrone (MTO) at different times.

The experimental results are shown in fig. 1, and fig. 1 is a graph of the release rate of MTO of the co-supported nanoparticles provided in example 4 of the present invention in solutions with different pH values. As can be seen from fig. 1, mitoxantrone drug release was slowest in pH 7.2 buffer; under the condition of pH value of 6.5, the release amount is not obviously different from that of pH value of 7.2; however, under more acidic conditions, mitoxantrone was released rapidly at pH 5.0 and at 48h 2 times the release in pH 7.2. The experimental phenomenon shows that the nano-drug prepared by the method has certain pH response release performance for the anthracycline, and the performance is favorable for realizing the specific release of the drug at the tumor part and reducing the side effect of the drug in vivo.

Example 5

The drug co-loaded nanoparticles prepared in example 3, No. 1, were used for in vitro anti-tumor therapy. 4T1 cells were selected for in vitro tumor suppression experiments. 4T1 cells were plated at 1X 10 per well⁴Was grown in 96-well plates and cultured overnight. Then adding drugs with different concentrations into the cells, culturing for 4h, removing the culture medium supernatant, replacing the fresh culture medium, continuing culturing for 44h, adding 20 mu L of thiazole blue solution (5mg/mL) into each hole of a 96-hole plate, continuing culturing for 4h at 37 ℃, adding dimethyl sulfoxide for dissolving, and measuring the absorbance value of each hole at 490 nm. Cell viability was calculated using the following formula:

cell viability (%) ═ a sample/a blank × 100.

The experimental results are shown in fig. 2, and fig. 2 is a bar graph of cell survival rates of 4T1 cells treated with different drugs according to example 5 of the present invention; in FIG. 2, MTO is the free chemotherapeutic mitoxantrone; JQ1 is a free small molecule inhibitor JQ 1; the MTO + JQ1 is a free compound of the two medicines, and the mass ratio of the two medicines is MTO: JQ1 ═ 1: 9; MJ @ NPs are drug co-loaded nanoparticles prepared as in example 3, No. 1; the concentrations of each drug in the column diagram correspond from left to right to 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL, 0.0625. mu.g/mL, and 0.032. mu.g/mL, respectively.

Two conclusions can be drawn from the above experiments: 1) the experimental result shows that the chemotherapeutic drug mitoxantrone and the small molecular inhibitor JQ1 have very good drug synergistic effect, the drug synergistic index is 0.48, and the two drugs have stronger killing capability on tumor cells when being carried together; 2) compared with free drugs, the formed co-loaded nano-drug has relatively weak tumor cell killing capacity, which may be related to slow release of the drug, and is beneficial to reducing the side effect of the nano-drug on normal tissues.

Example 6

The drug co-loaded nanoparticles prepared in example 3, No. 1, were used for in vivo anti-tumor therapy. The in vivo anti-tumor experiment adopts 4T1 tumor model, about 20g Balb/C mice, and tumor inoculationFirst, 4T1 cells in logarithmic growth phase were taken, digested with trypsin, and then mixed with trypsin, 1X 10 cells in cell culture³Centrifuge at rpm for 5min, wash twice with PBS, and suspend the cells with PBS. 2X 10 for each mouse⁶Cells were inoculated in the mouse axilla. After 7 days, the average tumor size is as large as 80mm³The in vivo anti-tumor treatment is carried out. The nano-drug prepared in the number 1 of example 3 or equivalent free drug was injected into mice via tail vein, and the dosage was 5mg/kg mitoxantrone and JQ135mg/kg, and only one administration was performed. The change in tumor volume and mouse body weight was followed after dosing for a total of 20 days throughout the experiment. Tumor growth after treatment is shown in table 5:

TABLE 5 tumor weights after different treatments

Administration of drugs	Tumor weight after treatment (g)
		PBS	1.92
Free MTO	0.5
		Free JQ1	0.53
Free MTO + JQ1	0.19
		Example 3 number 1 nanoparticles	0.07

The results of in vivo tumor inhibition experiments show that 1) free mitoxantrone single drug (MTO) and JQ1 single drug have obvious inhibition effect on tumor growth; 2) after the free two medicines are combined for treatment, the weight of the tumor is 0.4 times of that of the two single medicines, which shows that the two medicines have good medicine synergistic treatment effect; 3) after the nanoparticles in the number 1 of the example 3 are treated, the tumor growth is obviously inhibited, the tumor inhibition effect is the best in all treatments, the tumor weight is 0.37 times of that of the free medicine group, and the comparison of the free medicine group and the EPR effect shows that the nanoparticles can be better accumulated in the tumor part, effectively carry the nanoparticles to the tumor part and kill the tumor.

Example 7

The drug co-loaded nanoparticles prepared in example 3, No. 2, were used for in vitro anti-tumor therapy. 4T1 cells were selected for in vitro tumor suppression experiments. 4T1 cells were plated at 1X 10 per well⁴Was grown in 96-well plates and cultured overnight. Then adding drugs with different concentrations into the cells, culturing for 4h, removing the culture medium supernatant, replacing the fresh culture medium, continuing culturing for 44h, adding 20 mu L of thiazole blue solution (5mg/mL) into each hole of a 96-hole plate, continuing culturing for 4h at 37 ℃, adding dimethyl sulfoxide for dissolving, and measuring the absorbance value of each hole at 490 nm. Cell viability was calculated using the following formula:

cell viability (%) ═ a sample/a blank × 100.

The experimental results are shown in fig. 3, and fig. 3 is a bar graph of cell survival rates of 4T1 cells treated with different drugs according to example 7 of the present invention; in fig. 3, MTO/GA is a free Mitoxantrone (MTO) and Gambogic Acid (GA) complex formulation, the mass ratio of MTO: GA 1: 2; MG @ NPs were drug co-loaded nanoparticles prepared as in example 3, No. 2.

As can be seen from fig. 3, compared with the free drug, the formed co-loaded nano-drug has relatively weak tumor cell killing capacity, which may be related to the slow release of the drug, which is beneficial to reduce the side effect of the nano-drug on normal tissues.

Example 8

Dispersing the drug co-loading nanoparticles prepared in the number 6 of the example 3 in an aqueous solution with the concentration of 1mg/mL, diluting 200 mu L of the nanoparticle dispersed aqueous solution by using a dimethyl sulfoxide solvent, performing vortex oscillation for 15s, and detecting the drug loading rate of adriamycin (DOX) by using an ultraviolet spectrophotometer; the same method was used to dissolve the free drug and the peak absorbance of the free drug was measured at different concentrations and used for standard curve plotting. For the drug loading rate of the drug JQ1, the prepared drug co-supported nanoparticles are dispersed in an aqueous solution, then the dispersion liquid is freeze-dried, 10mg of the dispersion liquid is used for element analysis and detection, and the content of JQ1 in the nanoparticles is quantified by detecting S element. The drug loading rate of the drug co-loaded nanoparticles is detailed in the following table:

table 6 drug loading ratio of drug co-supported nanoparticles of example 3 No. 6

Medicine	Drug Loading Rate (%)
		DOX	30.4
JQ1	66.7

In addition, under the same drug proportion, two drugs are independently loaded, the drug loading rates of DOX and JQ1 are respectively 29.7% and 12.6%, and obvious precipitation occurs in the drug loading process, which indicates that the co-loading can effectively improve the drug loading rate of the hydrophobic drug.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.