CN112516329A

CN112516329A - Self-assembled combined drug carrier based on high-molecular prodrug and application thereof

Info

Publication number: CN112516329A
Application number: CN202011579337.4A
Authority: CN
Inventors: 莫然; 沈诗洋; 章颖; 刘华宇
Original assignee: China Pharmaceutical University
Current assignee: Nanjing Ningdan New Drug Technology Co ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2021-03-19
Anticipated expiration: 2040-12-28
Also published as: CN112516329B

Abstract

The invention discloses a self-assembled combined drug carrier constructed based on polysaccharide macromolecule prodrugs and application thereof in drug-resistant tumor combined drug therapy, belonging to the technical field of pharmaceutical preparations. The invention designs and synthesizes a polysaccharide macromolecule prodrug, which is formed by connecting a chemotherapeutic drug and a low-oxygen responsive hydrophobic group on a polysaccharide macromolecule polymer; the polysaccharide polymer prodrug is used for constructing a combined drug delivery system for co-loading a drug resistance reducing inducer and a chemotherapeutic drug, and the delivery system can gradually and sequentially release two drugs in drug-resistant tumor cells or/and tumor stem cells so as to solve the technical problem of poor combined effect of the two drugs.

Description

Self-assembled combined drug carrier based on high-molecular prodrug and application thereof

Technical Field

The invention belongs to the technical field of medicinal preparations, and particularly relates to a self-assembled combined medicament carrier constructed based on a polysaccharide macromolecule prodrug and application thereof in drug-resistant tumor combined medicament treatment.

Background

Chemotherapy (chemotherapy) is one of the main therapeutic modalities for malignant tumors in the clinic. However, chemotherapy drugs have short half-life in vivo and poor tumor targeting, so that many drugs cannot reach tumor tissues after administration, resulting in limited therapeutic effect, and the drugs are distributed in normal tissues in large quantities, resulting in great toxic and side effects. With the continuous development and maturation of polymer chemistry, the polymer prodrug is used as a novel drug delivery system, is formed by derivatization of chemotherapeutic drugs or covalently coupled to polymer polymers, has the advantages of increasing the tumor targeting property and selectivity of the drugs, improving the drug concentration in tumor tissues, reducing the distribution of the drugs in normal visceral organs, reducing toxic and side effects and the like, and is widely researched at home and abroad. However, tumor resistance remains one of the major causes of failure of tumor therapy. The molecular mechanism of tumor drug resistance is complex, and the molecular mechanism mainly comprises high-expression drug efflux protein on the surface of tumor cells, high-expression anti-apoptosis protein in the tumor cells and the like. For example: drug efflux proteins (P-glycoprotein (P-gp) and ATP-binding transporter 2(ABCG2) and the like) actively transport chemotherapeutic drugs in tumor cells to the outside of cells by means of energy provided by ATP, so that the intracellular drug concentration is always lower than the effective treatment concentration; the anti-apoptosis protein (survivin protein and B Cell Lymphoma (BCL) -2 protein) inhibits apoptosis induced by chemotherapeutic drugs by blocking apoptosis signal channels such as Caspase, NF-kB and the like. In recent years, research has found that a group of cells with stem cell characteristics, called stem cell-like tumor cells (tumor stem-like cells for short), exist in tumor tissues, and the cells have self-renewal, multidirectional differentiation capacity and strong tumorigenicity. The tumor stem-like cells highly express drug efflux protein, and have extremely strong DNA repair and active oxygen scavenging capacity, so that the tumor stem-like cells have high tolerance to radiotherapy and chemotherapy.

In order to overcome the drug resistance of the tumor, a combination drug combination treatment strategy is adopted, the chemosensitizer is combined with the chemotherapeutic drug, the chemosensitizer is utilized to reduce the chemoresistance of the drug-resistant tumor, the drug resistance of the tumor is overcome, the treatment effect of the chemotherapeutic drug on the drug-resistant tumor is enhanced, and the synergistic anti-tumor effect is exerted. However, the chemical structures of the chemotherapy drug and the chemotherapy sensitizer are different, so that the pharmacokinetic behaviors and the tissue distribution rules of the chemotherapy drug and the chemotherapy sensitizer in vivo are greatly different, and the optimal synergistic drug ratio in the whole process that the two drugs are distributed to tumor tissues and then enter tumor cells after being administered is difficult to maintain through the traditional physically mixed 'cocktail' type administration mode, so that the drug combination cannot achieve the expected effect. The micro-nano carrier-based combined drug co-delivery system can solve the problem. The combined medicine is loaded in the same carrier, so that the synergistic proportion of the medicine after administration can be maintained, the action time of the medicine is prolonged, and the targeting property of the medicine is improved. However, the action mechanism of the chemosensitizer and the chemotherapeutic drug determines the action sequence of the two drugs in the process of treating the drug-resistant tumor. If the chemotherapeutic medicament is released from the carrier simultaneously or before the chemotherapeutic sensitizer, the released chemotherapeutic medicament cannot kill unsensitized drug-resistant tumor cells and even can enhance the drug resistance or dryness of the cells. Therefore, based on the biological characteristics of the drug-resistant tumor cells and the tumor stem-like cells, the chemosensitizer and the chemotherapeutic drug are required to be used together for treating the drug-resistant tumor, the synergistic ratio of the two drugs is required to be maintained, and the two drugs are required to play roles in cells at proper time based on the action mechanisms of the chemosensitizer and the chemotherapeutic drug, so that the synergistic antitumor efficacy is enhanced.

Disclosure of Invention

The invention aims to solve the technical problem that the combined effect of a chemotherapeutic sensitizer and a chemotherapeutic drug on drug-resistant tumor is poor, and provides a polysaccharide polymer prodrug coupled with the chemotherapeutic drug and a hypoxia-responsive hydrophobic group, which can be self-assembled and physically embedded into the chemotherapeutic sensitizer to obtain a combined drug carrier, and is finally used for co-delivery of the chemotherapeutic sensitizer and the chemotherapeutic drug, so that the synergistic drug-resistant tumor resistance of the chemotherapeutic sensitizer and the chemotherapeutic drug is improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

a polysaccharide macromolecule prodrug comprises a macromolecule polysaccharide polymer, wherein a chemotherapy drug and a hypoxia-responsive hydrophobic group are modified on the macromolecule polysaccharide polymer, and the chemotherapy drug and the hypoxia-responsive hydrophobic group are respectively connected with the macromolecule polysaccharide polymer through amido bonds or ester bonds;

the high molecular polysaccharide polymer is selected from hyaluronic acid, heparin or carboxyl glucan;

the chemotherapeutic drug is selected from camptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, topotecan, vinblastine, vincristine, vinorelbine, vindesine, cytarabine, gemcitabine, ancitabine, 5-fluorouracil, 6-mercaptopurine, methotrexate, doxorubicin, epirubicin, doxorubicin, daunorubicin, mitoxantrone, paclitaxel, docetaxel, nimustine, teniposide, etoposide, aminoglutethimide or melphalan;

the low-oxygen-responsive hydrophobic group is a 2-nitroimidazole group or an azobenzene group.

Furthermore, a connecting arm is arranged between the high molecular polysaccharide polymer and the chemotherapeutic drug, the connecting arm is an active oxygen responsive connecting arm, one end of the active oxygen responsive connecting arm is connected with the high molecular polysaccharide polymer through an amido bond or an ester bond, and the other end of the active oxygen responsive connecting arm is connected with the chemotherapeutic drug.

Further, the active oxygen-responsive linker arm is oxalate or thioketal.

A polymer micelle for treating drug-resistant tumor is formed by self-assembling the polysaccharide macromolecule prodrug in water;

the polymer micelle is coated with a chemotherapeutic sensitizer, wherein the chemotherapeutic sensitizer is selected from the group consisting of goropamide, quinidine, verapamil, chlorpromazine, cyclosporine A, reserpine, digoxin, amiodarone, progesterone, xyloflavone, tamoxifen, felodipine, nifedipine, erythromycin, flufenamic acid, diltiazem, valcephradine, bilactada, elcridad, oxazolineda, lovastatin, simvastatin, atorvastatin, rosuvastatin, curcumin, ginsenoside, eltrombopag, fumagillotoxin C, lapatinib, neomycin, sulfasalazine, febuxostat, YM-155, LLP-3, vexinetox, navitoclax, obatocleate, ABT-737, BM-1074 or Marinpyrole A.

A polymer hydrogel for treating drug-resistant tumor is formed by self-assembling the polysaccharide macromolecule prodrug and the polymer micelle in water.

A polymeric micelle for treating common tumor or tumor stem-like cell-related drug-resistant tumor is formed by self-assembling the polysaccharide macromolecule prodrug in water;

the polymer micelle is coated with a differentiation inducer, and the differentiation inducer is selected from all-trans retinoic acid, 1, 3-cis retinoic acid, 9-cis retinoic acid, retinol, retinal, arsenic trioxide, arsenic sulfide, bone morphogenetic protein 7, bone morphogenetic protein 2 or vismodegib.

A polymer hydrogel for treating common tumor or tumor stem-like cell-related drug-resistant tumor is formed by self-assembling the polysaccharide high-molecular prodrug and the polymer micelle in water.

The invention provides a polysaccharide macromolecule prodrug with double modification, wherein a chemotherapeutic drug and a hydrophobic group with low oxygen response are modified on a macromolecule polysaccharide polymer, and the polysaccharide macromolecule prodrug can be prepared into a combined drug release system coated with a chemotherapeutic sensitizer or a differentiation inducer. Because the chemosensitizer or differentiation inducer is loaded in the micelle carrier formed by the polysaccharide macromolecule prodrug through physical action, the chemosensitizer or differentiation inducer can be released when entering drug-resistant tumor cells or tumor stem cells to play a role in reducing drug resistance, and the chemotherapeutics covalently connected to the polysaccharide molecules through chemical bonds can be later than the chemotherapeutics which reduce the drug-resistance inducer, thereby further playing a curative effect.

Drawings

FIG. 1 shows the release process of the self-assembled drug carrier based on the high molecular prodrug in drug-resistant tumor cells, tumor stem-like cells and normal tumor cells.

FIG. 2 shows the release results of the drug in the polymer micelle of example 1 in a low oxygen environment.

FIG. 3 is a graph showing the down-regulation effect of the polymer micelle of example 1 on the drug-resistant protein in the drug-resistant tumor cell.

FIG. 4 is a graph showing the inhibition of the survival rate of the drug-resistant tumor cells by the polymer micelle in example 1.

Fig. 5 is a graph showing the change in the blood concentration ratio of the drug loaded on the polymer micelle in example 1 in vivo.

FIG. 6 is the results of the in vivo antitumor activity of the polymer micelle in example 1.

FIG. 7 is a photograph of the polymer hydrogel of example 2.

FIG. 8 shows the release of the drug from the polymer hydrogel of example 2 in a hypoxic environment.

FIG. 9 shows the results of the in vivo antitumor activity of the polymer hydrogel in example 2.

FIG. 10 shows the release results of the drug in the polymeric micelle of example 3 in a low oxygen and active oxygen environment.

FIG. 11 shows the results of the polymer micelle of example 3 for up-regulating the reactive oxygen species level of tumor stem-like cells and the intracellular release of chemotherapeutic agents.

FIG. 12 is the inhibitory effect of the polymer micelle of example 3 on stem-associated protein in tumor stem-like cells.

FIG. 13 shows the results of the inhibition of the survival rate of tumor stem-like cells by the polymer micelle in example 3.

Fig. 14 is an in vivo pharmacokinetic examination and a change in blood concentration ratio of the polymer micelle in example 3.

FIG. 15 shows the results of the inhibition of tumor growth by the polymer micelle of example 3 on tumor stem-like cell-rich tumors.

FIG. 16 shows the release of the drug from the polymer hydrogel of example 4 in a hypoxic and reactive oxygen environment.

FIG. 17 shows the results of the inhibition of tumor growth by the polymer hydrogel of example 4 on tumor-rich stem-like cells.

Detailed Description

The invention designs and synthesizes a polysaccharide polymer prodrug, and utilizes the polysaccharide polymer prodrug to construct and obtain a combined drug delivery system which is commonly loaded with a drug resistance reducing inducer (a chemotherapy sensitizer or a differentiation inducer) and a chemotherapy drug, and the delivery system can gradually and sequentially release the two drugs in drug-resistant tumor cells or/and tumor stem-like cells so as to solve the technical problem of poor combined effect of the two drugs.

In the present invention, the tumor stem-like cell-related drug-resistant tumor refers to a tumor tissue rich in undifferentiated/poorly differentiated tumor stem-like cells, thereby causing the tumor tissue to have high drug resistance.

The polysaccharide macromolecule prodrug comprises a macromolecule polysaccharide polymer, wherein a chemotherapeutic drug and a hypoxia-responsive hydrophobic group are modified on the macromolecule polysaccharide polymer, and the chemotherapeutic drug and the hypoxia-responsive hydrophobic group are respectively connected with the macromolecule polysaccharide polymer through amido bonds or ester bonds; the high molecular polysaccharide polymer is selected from hyaluronic acid, heparin or carboxyl glucan; the chemotherapeutic drug is selected from camptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, topotecan, vinblastine, vincristine, vinorelbine, vindesine, cytarabine, gemcitabine, ancitabine, 5-fluorouracil, 6-mercaptopurine, methotrexate, doxorubicin, epirubicin, doxorubicin, daunorubicin, mitoxantrone, paclitaxel, docetaxel, nimustine, teniposide, etoposide, aminoglutethimide or melphalan; the low-oxygen-responsive hydrophobic group is a 2-nitroimidazole group or an azobenzene group.

As shown in A in figure 1, the polysaccharide macromolecule prodrug has amphipathy, can self-assemble in water to form a polymer micelle, and embeds a chemosensitizer to form a combined drug delivery carrier for treating drug-resistant tumors. The combined drug delivery carrier can maintain the synergistic drug ratio of the chemotherapeutic sensitizer and the chemotherapeutic drug in the tumor tissue. In a tumor tissue hypoxia microenvironment, a hypoxia-responsive hydrophobic group in a polysaccharide polymer prodrug can generate structural change, and an original hydrophobic structure is converted into a hydrophilic structure, so that the hydrophobic acting force in micelles is reduced, the micelles are dissociated, and the embedded chemosensitizer is released quickly. Because the chemotherapeutic drug is covalently linked to the high molecular weight polysaccharide polymer through ester or amide bonds, its release is dependent on hydrolysis of the covalent bond by esterases or amidases in the resistant cells. Thus, the rate of release of the chemosensitizer physically embedded in the micelle at low oxygen is much higher than the rate of release of the covalently coupled chemotherapeutic drug. In drug-resistant tumor cells, the chemosensitizer released first can reduce the drug resistance of the cells, and the chemotherapeutic drug released later can kill the tumor cells after the drug resistance is obviously reduced, so that the synergistic efficiency of the two drugs is greatly improved.

In the present invention, the preparation method of the polymer micelle comprises: dissolving a chemosensitizer in an organic solvent to obtain an organic phase, dissolving a polysaccharide macromolecule prodrug in water to obtain an aqueous phase, then blending the two phases, performing ultrasonic emulsification, removing the organic solvent by adopting vacuum decompression after the two phases are finished, and then passing through a water system filter membrane to obtain a polymer micelle, namely the combined drug delivery carrier for treating the drug-resistant tumor.

Further, the chemosensitizer is selected from the group consisting of gallopamil, quinidine, verapamil, chlorpromazine, cyclosporin A, reserpine, digoxin, amiodarone, progesterone, xyloflavone, tamoxifen, felodipine, nifedipine, erythromycin, flufenamic acid, diltiazem, valcephrade, bilactad, eticridade, azolquinda, lovastatin, simvastatin, atorvastatin, rosuvastatin, curcumin, ginsenoside, eltrombopag, fumonisin C, lapatinib, neomycin, azasulfapyridine, febuxostat, YM-155, LLP-3, veocoltax, navitoclax, Obatoclax Mesylate, ABT-737, BM-1074, Marinpyrole A.

In addition, in order to further improve the distribution of the drug in the high drug resistance tumor tissue and realize the in-situ administration of the tumor, the combined drug delivery carrier and the high-concentration polysaccharide macromolecule prodrug can be further assembled in water to form a combined drug hydrogel carrier. The preparation method comprises the following steps: and adding the combined drug delivery carrier into a high-concentration polysaccharide polymer prodrug water solution, and standing after vortex to obtain the polysaccharide polymer prodrug.

On the other hand, aiming at tumor cells with strong drug resistance, namely tumor stem-like cells, the invention further performs structural derivatization on the polysaccharide polymer prodrug, and specifically, the chemotherapeutic drug is covalently connected with a polymer polysaccharide polymer through an active oxygen responsive connecting arm to obtain the polysaccharide polymer prodrug. The active oxygen-responsive linker arm is selected from oxalate or thioketal.

As shown in B in FIG. 1, the polysaccharide polymer prodrug can also self-assemble to form micelles, and load a differentiation inducer to obtain a combined drug delivery carrier. Particularly, the combined drug delivery carrier can rapidly release a physically-embedded differentiation inducer in response to hypoxia in tumor stem-like cells, and chemotherapy drugs are stably modified on polysaccharide high molecular polymers and are not released in the tumor stem-like cells due to the strong active oxygen scavenging capacity of the tumor stem-like cells, wherein the active oxygen level is low. The differentiation inducer released firstly induces the differentiation of the tumor stem-like cells, reduces the drug resistance of the tumor stem-like cells, enhances the sensitivity of the tumor stem-like cells to chemotherapeutic drugs, and simultaneously, in the differentiation process, because the biosynthesis is increased, the intracellular active oxygen level is greatly increased, the active oxygen responsive connecting arms in the polysaccharide macromolecule prodrug are degraded, the chemotherapeutic drugs are separated from the polysaccharide macromolecule polymer and released in the differentiated tumor cells. The method regulates and controls timely and sequential release of a differentiation inducer and a chemotherapeutic drug based on physiological signal changes in the undifferentiated and differentiated processes of the tumor stem-like cells, and greatly improves the killing power of the chemotherapeutic drug on the tumor stem-like cells. Meanwhile, in non-dry common tumor cells, because the intracellular active oxygen level is higher, the polysaccharide macromolecule prodrug can be rapidly degraded to release chemotherapeutic drugs and kill the tumor cells. The combined drug delivery carrier can be assimilated and can simultaneously remove tumor stem-like cells and common tumor cells, and overcome tumor heterogeneity and dry-related tumor drug resistance.

In the present invention, the preparation method of the polymer micelle comprises: dissolving a differentiation inducer in an organic solvent to obtain an organic phase, dissolving a polysaccharide macromolecule prodrug in water to obtain an aqueous phase, blending the two phases, performing ultrasonic emulsification, removing the organic solvent by adopting vacuum decompression after the two phases are finished, and passing through a water system filter membrane to obtain a polymer micelle, namely the combined drug delivery carrier.

Further, the differentiation inducer is selected from the group consisting of all-trans retinoic acid, 1, 3-cis retinoic acid, 9-cis retinoic acid, retinol, retinal, arsenic trioxide, arsenic sulfide, bone morphogenetic protein 7, bone morphogenetic protein 2, and vismodegib.

In addition, in order to further improve the distribution of the drug in tumor tissues rich in tumor stem-like cells and realize tumor in-situ administration, the combined drug delivery carrier and the high-concentration polysaccharide macromolecule prodrug can be further assembled in water to form a combined drug hydrogel carrier. The preparation method comprises the following steps: and adding the combined drug delivery carrier into a high-concentration polysaccharide polymer prodrug water solution, and standing after vortex to obtain the polysaccharide polymer prodrug.

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

Example 1

Polymeric micelles for drug-resistant tumor therapy

Synthesis of polysaccharide polymer prodrug

Cholesterol chloroformate (502mg) and triethylamine (120mg) are added into 20mL of dichloromethane to be stirred, p-aminoazobenzene (368mg) is continuously added to react for 12 hours, reaction liquid is collected after the reaction is finished, vacuum spin-drying is carried out, and a product is purified and separated by silica gel column chromatography to obtain the cholesterol formyl p-aminoazobenzene. 2-nitroimidazole (302mg) and potassium carbonate (557mg) were added to 10mL of N, N-dimethylformamide and stirred, 6- (Boc-amino) bromohexane (782mg) was then added thereto, the reaction was heated to 80 ℃ and refluxed for 4 hours, and after the reaction was completed, spin-dried at 70 ℃ to obtain a brown solid. After extraction and drying, the product 1- (6-Bo-aminohexyl) -2-nitroimidazole is obtained. 1- (6-Boc-aminohexyl) -2-nitroimidazole (493mg) was dissolved in 10mL of 1.25M hydrochloric acid-methanol solution and the reaction was stirred overnight at room temperature to remove the Boc protecting group. After the reaction is finished, the reaction solution is dried by spinning at 40 ℃ to obtain the 6- (2-nitroimidazolyl) hexylamine.

Hyaluronic acid (HA,90KDa,196mg), heparin sodium (HP,15KDa,150mg), and carboxydextran (CDT,15KDa,137mg) were dissolved in 10mL of water, followed by addition of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDCI) (190mg) and N-hydroxysuccinimide (NHS) (143mg), stirring at room temperature for 0.5h, addition of cholesteryl paraaminoazobenzene (197mg) dissolved in 5mL of Dimethylsulfoxide (DMSO), and reaction was continued with stirring at room temperature for 24 h. Then putting the reaction solution into a dialysis bag (3.5kDa) and dialyzing in water for 24h, collecting the liquid in the dialysis bag and freeze-drying to obtain the cholesterol azobenzene group modified polysaccharide derivatives c-HA, c-HP and c-CDT.

HA (90kDa,205mg), HP (15kDa,144mg) and CDT (15kDa,125mg) were dissolved in 25mL of water, EDCI (571mg) and NHS (571mg) were added thereto, and after stirring at room temperature for 0.5h, 6- (2-nitroimidazolyl) hexylamine (155mg) was added thereto, and the reaction was continued with stirring at room temperature for 24 h. Then putting the reaction solution into a dialysis bag (14KDa) to be dialyzed in water for 24h, collecting the liquid in the dialysis bag and freeze-drying to obtain the polysaccharide derivatives n-HA, n-HP and n-CDT modified by the 2-nitroimidazole group.

Respectively dissolving appropriate amounts of c-HA, c-HP, c-CDT, n-HA, n-HP and n-CDT in 25mL of water, adding EDCI and NHS, stirring at room temperature for 0.5h, then respectively adding Camptothecin (CPT), 10-Hydroxycamptothecin (HCPT), 7-ethyl-10-hydroxycamptothecin (SN-38), Vinblastine (VLB), Doxorubicin (DOX), Epirubicin (EPI), Mitoxantrone (MIT), Paclitaxel (PTX) and Docetaxel (DTX), and continuously stirring at room temperature for 24 h. Then putting the reaction solution into a dialysis bag, dialyzing in water for 24h, collecting the liquid in the dialysis bag and freeze-drying to obtain polysaccharide polymer prodrugs c-HA-CPT, c-HA-HCPT, c-HA-SN-38, n-HA-VLB, c-HP-DOX, n-HP-EPI, c-CDT-MIT, n-CDT-PTX and n-CDT-DTX.

Secondly, preparation of polymer micelle loaded with combined drug

The polymer micelle loaded with the combined medicament is prepared by adopting a single emulsification method. Dissolving cyclosporine A (CSA), Quinidine (QD), Reserpine (RSP), wood flavone (GST), Tamoxifen (TAM), Nifedipine (NDP), Diltiazem (DTZ), Curcumin (CUR), fumonisin C (FTC), YM-155(YM) and ABT-737(ABT) in 1mL of chloroform to obtain an organic phase. Dissolving polysaccharide polymer prodrug c-HA-CPT, c-HA-HCPT, c-HA-SN-38, n-HA-VLB, c-HP-DOX, n-HP-EPI, c-CDT-MIT, n-CDT-PTX and n-CDT-DTX in 5mL of water to obtain an aqueous phase. Placing the water phase in ice water bath, dropwise adding the organic phase into the water phase under probe ultrasound, and continuing ultrasound for 20min after the dropwise addition. And (3) vacuumizing the obtained emulsion at 40 ℃, performing rotary evaporation to remove the organic solvent, and filtering the emulsion through a 220nm filter membrane to obtain the polymer micelle loaded with the combined drug. The particle size and polydispersity of each micelle group were measured using a particle sizer, and the results are shown in table 1.

TABLE 1 particle size and polydispersity index of drug combination loaded polymeric micelles

Third, drug release

The chemotherapy drug (CPT or MIT) and the chemotherapy sensitizer (CSA or FTC) are loaded into the polysaccharide high-molecular micelle modified by the hypoxia response group through a single emulsification method, so as to obtain the polymer micelle (CSA/CPT/HA and FTC/MIT/CDT) of the physical co-loaded combined drug. 2mL of the combined drug-loaded polymeric micelles (CSA/CPT-HA, FTC/MIT-CDT, YM/PTX-CDT and ABT/DTX-CDT) and the polymeric micelles (CSA/CPT/HA and FTC/MIT/CDT) physically co-loaded with the combined drug were loaded into dialysis bags, and the dialysis bags were placed in 60mL of dialysis medium (PBS containing 2% Tween 80). NADPH was added to a final concentration of 100. mu.M and liver microsomes at 10mg/mL, and nitrogen was introduced to displace air to simulate a hypoxic microenvironment. Under stirring, 200 μ L of dialysis medium was taken at different time points, and the drug concentration was measured by high performance liquid chromatography, and the drug release amount was calculated to draw a drug release curve. The results are shown in fig. 2, and the release rate of the chemosensitizer physically embedded in the polymer micelle loaded with the combined drug is significantly higher than that of the chemoconjugate chemotherapeutic drug under the low oxygen environment. The polymer micelle is proved to be capable of responding to a hypoxic microenvironment to rapidly release the chemotherapy sensitizer, so that the loaded combined drug chemotherapy sensitizer and the chemotherapy drug are released in a differential speed (in sequence).

Fourth, evaluation of drug resistance related protein down-regulated by polymer micelle loaded with combined drug

Human breast cancer drug-resistant cells (MCF-7/MDR) cells at 1 × 10⁵The density of each well was inoculated in a 6-well plate, and the drug-loaded polymeric micelles (CSA/CPT-HA, FTC/MIT-CDT, YM/PTX-CDT and ABT/DTX-CDT) and the physical co-loading combination were addedPolymer micelles of the drug (CSA/CPT/HA, FTC/MIT/CDT, YM/PTX/CDT and ABT/DTX/CDT) were cultured for 48 h. And carrying out fluorescent labeling on the related drug-resistant protein of the treated drug-resistant cells by adopting a fluorescent antibody immunostaining method, and detecting the protein level of the fluorescent labeling by adopting a flow cytometry. The results are shown in figure 3, compared with the untreated control group, the drug resistance related proteins (P-gp, ABCG2, survivin and BCL-2 protein) in the drug resistant cells treated by the polymer micelle loaded with the chemosensitizer are all significantly reduced. The polymer micelle loaded with the combined drug is proved to be capable of obviously reducing the drug resistance of the drug-resistant tumor cells.

Fifthly, evaluating in vitro cytotoxicity of polymer micelle loaded with combined drug on drug-resistant tumor cells

Golopami (GAL), Digoxin (DIG), amiodarone (BTL), Felodipine (FDP), Erythromycin (EM), LLP-3(LLP), BM-1074(BM) are used as chemosensitizers to prepare polymer micelles (GAL/CPT-HA, DIG/CPT-HA, BTL/CPT-HA, FDP/CPT-HA, EM/CPT-HA, LLP/CPT-HA and BM/CPT-HA) loaded with the combination drug with the polysaccharide polymer prodrug c-HA-CPT according to the method described in the second part. MCF-7/MDR cells at 5X 10³The density of each well is inoculated in a 96-well culture plate, and the polymer micelle loaded with the combined drug and the free combined drug (chemosensitizer and chemotherAN _ SNy drug) or the polymer micelle loaded with the combined drug (CSA/CPT-HA and FTC/MIT-CDT) and the polymer micelle loaded with the combined drug (CSA/CPT/HA and FTC/MIT/CDT) at different concentrations are added. After culturing for 96h in a hypoxic incubator, cell viability was determined and cell viability was calculated using the CCK8 assay. The results are shown in fig. 4, compared to the polymer micelle of free combination drug and physical co-loading combination drug, the polymer micelle of loaded combination drug has stronger cytotoxicity to drug-resistant tumor cells. The polymer micelle loaded with the combined drug is proved to be capable of maintaining the synergistic ratio of the two drugs and simultaneously gradually releasing the two drugs in the drug-resistant tumor cells, thereby enhancing the synergistic killing efficiency of the two drugs on the drug-resistant tumor cells.

Six, pharmacokinetic investigation of polymer micelle loaded with combined drug

The polymer micelle (CSA/CPT-HA and FTC/MIT-CDT) loaded with the combined medicament and the free combined medicament (CSA + CPT and FTC + MIT) are injected into the body of an experimental rat in the tail vein, blood is respectively taken at different time points, blood plasma is taken by centrifugation, and the blood concentration is determined after the extraction by an organic solvent. The results are shown in fig. 5, where the polymeric micelles can maintain the dose ratio of the combined drugs for a long period of time, compared to the free drug mixture.

Seven, evaluation of in vivo antitumor activity of polymer micelle loaded with combined drug

The MCF-7/MDR cells were resuspended in a mixed solution of PBS and Matrigel (1:1) at 1X 10⁷One mouse (nu/nu, female, 6 weeks old, 20g) was inoculated with one/one of the number of mice to construct a drug-resistant tumor mouse model. Tumor-bearing mice were grouped, and the drug-loaded polymeric micelles (CSA/CPT/HA and FTC/MIT/CDT) and polymeric micelles (CSA/CPT-HA and FTC/MIT-CDT) were administered once every two days, four times, respectively, by tail vein injection of physiological Saline (salt), free combination drugs (CSA + CPT and FTC + MIT), physical co-loaded combination drugs. The tumor volume was monitored by measuring the major and minor diameters of the tumor, calculating the tumor volume (volume ═ major diameter × minor diameter/2). The results are shown in fig. 6, compared with the free combination drug, the combination drug micelle has stronger effect of inhibiting the growth of the drug-resistant tumor, which proves that the micelle carrying the combination drug chemosensitizer and the chemo-therapeutic drug maintains the drug ratio of the two drugs and improves the effect of the drug synergistic anti-drug-resistant tumor. The anti-tumor effect of the combined drug micelle with the timely gradual drug release function is stronger than that of the physical co-loading combined drug micelle without the gradual drug release characteristic, so that the micelle with the gradual drug release characteristic firstly releases the chemotherapeutic sensitizer, reduces the resistance of the drug-resistant tumor cells to the chemotherapeutic drug, then releases the chemotherapeutic drug and obviously enhances the effect of the two drugs on the synergistic inhibition of the drug-resistant tumor growth under the condition of maintaining the drug synergistic ratio.

Example 2

Polymer hydrogel for drug-resistant tumor therapy

Synthesis of polysaccharide polymer prodrug

c-HA, c-CDT, n-HA, n-HP, n-CDT were dissolved in 25mL of water, EDCI and NHS were added, and after stirring at room temperature for 0.5h, cytarabine (CC), topotecan (TPT), Methotrexate (MTX), nimustine (ANU), and etoposide (VP) were added, and the reaction was continued with stirring at room temperature for 24 h. Then putting the reaction solution into a dialysis bag, putting the dialysis bag into water for dialysis for 24h, collecting the liquid in the dialysis bag and freeze-drying to obtain the polysaccharide macromolecule prodrug c-HA-CC, c-CDT-TPT, n-HA-MTX, n-HP-ANU and n-CDT-VP.

Preparation of polymer hydrogel loaded with combined drug

Verapamil (VER), Chlorpromazine (CPZ), ginsenoside Rg1(GSG), and ginsenoside Rb1(GSB) were mixed with polysaccharide polymer prodrugs c-HA-CC, c-CDT-TPT, n-HA-MTX, n-HP-ANU, and n-CDT-VP to prepare a drug-loaded polymer micelle as described in example 1. And (3) blending 1mL of micelle with a corresponding polysaccharide macromolecule prodrug solution, uniformly mixing by vortex, and standing at normal temperature to form polymer hydrogel VEP/CC-HA, VEP/TPT-CDT, CPZ/MTX-HA, GSG/ANU-HP or GSB/VP-CDT for loading the combined drug. The gel forming effect is shown in figure 7, water, VEP/CC-HA, VEP/TPT-CDT, CPZ/MTX-HA, GSG/ANU-HP and GSB/VP-CDT are respectively arranged from left to right, and the assembled system is found to form hydrogel.

Third, drug release

1mL of the drug-loaded polymer hydrogel (VEP/CC-HA and CPZ/MTX-HA) and the polymer hydrogel physically co-loaded with the drug (VEP/CC/HA and CPZ/MTX/HA) were placed in flasks, 20mL of dialysis medium (PBS containing 2% Tween 80) was added, and NADPH and 10mg/mL of liver microsomes were added to a final concentration of 100. mu.M, and air was replaced with nitrogen to simulate a hypoxic microenvironment. Under stirring, 200 μ L of dialysis medium was taken at different time points, and the drug concentration was measured by high performance liquid chromatography, and the drug release amount was calculated to draw a drug release curve. The results are shown in fig. 8, and the release rate of the chemosensitizer physically embedded in the polymer hydrogel loaded with the combined drug is significantly higher than that of the chemoconjugate chemotherapeutic drug under the low oxygen environment. The polymer hydrogel is proved to be capable of responding to a hypoxic microenvironment to rapidly release the chemotherapy sensitizer, and differential (sequential) release of the loaded combination drug chemotherapy sensitizer and the chemotherapy drug is realized.

Fourth, evaluation of in vivo antitumor Activity of Polymer hydrogel loaded with combination drug

Drug-resistant breast cancer (MCF-7/MDR) tumor mice are grouped, physiological Saline (Saline), free combined drugs (VEP + CC and CPZ + MTX), polymer hydrogel of physical co-loaded combined drugs (VEP/CC/HA and CPZ/MTX/HA) and polymer hydrogel of loaded combined drugs (VEP/CC-HA and CPZ/MTX-HA) are respectively injected in situ to tumors, and the tumor volume change is monitored after administration once. The results are shown in fig. 9, the anti-tumor effect of the combined drug hydrogel with timely gradual drug release function is stronger than that of the physical co-loading combined drug hydrogel and free combined drug without gradual drug release property, which proves that under the condition of maintaining the drug synergistic ratio, the hydrogel with gradual drug release property firstly releases the chemotherapy sensitizer, reduces the resistance of the drug-resistant tumor cells to the chemotherapy drugs, then releases the chemotherapy drugs, and can obviously enhance the effect of the two drugs for synergistically inhibiting the growth of the drug-resistant tumor.

Example 3

Polymer micelle for treating drug-resistant tumor related to tumor stem-like cells

Synthesis of polysaccharide polymer prodrug

Oxalyl chloride (632mg) was dissolved in 5mL of anhydrous dichloromethane and placed on an ice-water bath with stirring, and 2-azidoethanol (87.2mg) was dissolved in 5mL of anhydrous dichloromethane and slowly added dropwise to the reaction solution, and the reaction was continued on the ice-water bath with stirring for 4 hours. After completion of the reaction, spin-dried to give a light brown oil. The resulting oil was redissolved in 20mL of anhydrous dichloromethane and stirred in an ice-water bath, followed by dissolving Camptothecin (CPT), triethylamine (110mg) and 4-dimethylaminopyridine (25.6mg) in 10mL of anhydrous dichloromethane, slowly dropwise adding the solution to the reaction mixture, and continuing the reaction for 2h in an ice-water bath. Extracting the reaction solution, and spin-drying to obtain a crude product. Finally, the active oxygen responding oxalate coupled CPT prodrug molecule is obtained through silica gel column chromatography purification. An appropriate amount of n-HA was dissolved in 25mL of water, EDCI and NHS were added thereto, and after stirring at room temperature for 0.5h, amino-dibenzocyclooctyne (125mg) was added thereto, and the reaction was continued with stirring at room temperature for 24 h. Then the reaction solution is filled into a dialysis bag (14kDa) and dialyzed in water for 48 hours, and the liquid in the dialysis bag is collected and freeze-dried to obtain the product. Mixing the product with grassThe acid ester-coupled CPT prodrug molecule is dissolved in 50mL of a mixed solution of DMSO and water (DMSO: H)₂O ═ 4:1, v: v), the reaction was stirred at room temperature for 24 h. Then filling the reaction solution into a dialysis bag to dialyze in water for 48h, collecting the liquid in the dialysis bag and freeze-drying to obtain the polysaccharide macromolecule prodrug n-HA-oxa-CPT.

Anhydrous acetone and anhydrous 3-mercaptopropionic acid are taken, stirred for 4 hours at room temperature after dry hydrogen chloride is introduced, and then the reaction is stopped in an ice salt mixture to realize crystallization. Washing the product, filtering the product by using normal hexane and cold water, and drying the product in vacuum to obtain the product, namely the thioketal dipropionic acid. The ketothiodipropionic acid was dissolved in anhydrous DMF and NHS and EDCI were added to the mixture under nitrogen. After stirring for 2h, CPT and Doxorubicin (DOX) were added dropwise and reacted for a further 24h to give the crude product. Purifying by silica gel column chromatography to obtain active oxygen-responsive ketothiol prodrug molecules. Dissolving the active oxygen response ketodithiol prodrug molecule in 25mL of water, adding EDCI and NHS, stirring at room temperature for 0.5h, dissolving appropriate amounts of n-HA and n-CDT in 25mL of water respectively, and continuing to stir at room temperature for 24 h. Then putting the reaction solution into a dialysis bag (14kDa) and dialyzing in water for 48h, collecting the liquid in the dialysis bag and freeze-drying to obtain the polysaccharide macromolecule prodrug n-HA-tkt-DOX and n-CDT-tkt-CPT.

Secondly, preparation of polymer micelle loaded with combined drug

The polymer micelle loaded with the combined medicament is prepared by adopting a single emulsification method. All-trans retinoic acid (ATRA), 1, 3-Cis Retinoic Acid (CRA), 9-cis retinoic acid (TRA), Retinol (RTO), Retinal (RTE), Vismodegib (VMG), and polysaccharide polymer prodrug. 3mg ATRA, CRA, TRA, RTO, RTE, VMG were dissolved in 1mL chloroform to obtain an organic phase, and the polysaccharide prodrug molecules n-HA-oxa-CPT, n-HA-tkt-DOX, and n-CDT-tkt-CPT were dissolved in 5mL water to obtain an aqueous phase. Placing the water phase in ice water bath, ultrasonically dropwise adding the organic phase into the water phase under probe ultrasound, and continuing ultrasound for 20min after dropwise adding. And (3) vacuumizing the obtained emulsion at 40 ℃, performing rotary evaporation to remove the organic solvent, and filtering with a 220nm filter membrane to obtain the polymer micelle loaded with the combined drug. The particle size and polydispersity of each micelle group were measured using a particle sizer, and the results are shown in table 2.

TABLE 2 particle size and polydispersity index of drug combination loaded polymer micelles

Third, drug release

2mL of the drug combination-loaded polymeric micelles (ATRA/CPT-HA, CRA/CPT-HA, RTO/DOX-HA and RTE/CPT-CDT) were loaded into a dialysis bag and placed in 60mL of dialysis medium (PBS with 2% Tween 80). NADPH was added to a final concentration of 100. mu.M and liver microsomes at 10mg/mL, and air was replaced with nitrogen to simulate a hypoxic microenvironment. After 48h, hydrogen peroxide was added to the system at a final concentration of 100 μ M to simulate an environment of high reactive oxygen levels for a further 48 h. Under stirring, 200 μ L of dialysis medium was taken at different time points, and the drug concentration was measured by high performance liquid chromatography, and the drug release amount was calculated to draw a drug release curve. The results are shown in fig. 10, where the release rate of the differentiation-inducing agent physically entrapped in the polymer micelles loaded with the combination drug is significantly higher than the release rate of the chemo-conjugated chemotherapeutic drug under hypoxic environment. And under the subsequent hydrogen peroxide environment, the micelle can generate a responsive rapid release chemotherapeutic drug. The release of the two drugs is respectively associated with two different stimuli, and obvious gradual release behaviors are presented. The polymer micelle is proved to be capable of responding to a hypoxia microenvironment to rapidly release a differentiation inducer and responsively release chemotherapeutic drugs in an active oxygen microenvironment, so that differential (gradual) release of the loaded combined drug differentiation inducer and the chemotherapeutic drugs is realized.

Fourthly, the polymer micelle loaded with the combined medicine can up-regulate the active oxygen level in the tumor stem-like cells and evaluate the intracellular release of the chemotherapeutic medicine

Adopting lateral group cell flow sorting technology to obtain MCF-7 tumor stem-like cells, and dividing the MCF-7 tumor stem-like cells into 1 × 10 cells⁵The density of each hole is inoculated in a 6-hole low-adhesion culture plate, and polymer micelles (ATRA/CPT-HA) loaded with the combination drug and polymer micelles (CPT-HA) loaded with the single chemotherapeutic drug are respectively added. After incubating for 48h under the condition of hypoxia, the intracellular active oxygen of the cells is stained by a DCFH-DA staining methodFluorescence labeling is carried out, and the active oxygen concentration is measured by adopting a flow cytometer. And (3) quantitatively detecting the intracellular CPT by adopting high performance liquid chromatography, and inspecting the intracellular release of the CPT. The results are shown in fig. 11, and the reactive oxygen species level in the tumor stem-like cells is significantly increased after the treatment of the polymer micelle loaded with the combined drug. The polymer micelle loaded with the combined drug is proved to be capable of releasing a differentiation inducer ATRA in the tumor stem-like cells under low oxygen, the released ATRA induces the differentiation of the tumor stem-like cells, and the intracellular active oxygen concentration of the differentiated cells is greatly increased. The elevation of intracellular reactive oxygen species triggers the responsive release of the chemotherapeutic agent CPT.

Fifthly, the polymer micelle loaded with the combined medicine can down regulate the dryness-related protein in the tumor stem-like cells

Human triple negative breast cancer HCC70, HCC1937 and SUM-159PT tumor stem-like cells are obtained by adopting a serum-free suspension culture enrichment tumor stem-like cell method. MCF-7, HCC70, HCC1937 and SUM-159PT tumor stem-like cells at 1 × 10⁵The density of each well is inoculated in a 6-well low-adhesion culture plate, and different polymer micelles (ATRA/CPT-HA and RTO/DOX-HA) loaded with the combination drugs, corresponding polymer micelles (ATRA/HA and CPT-HA) loaded with the differentiation inducer and the chemotherapeutic drugs singly and polymer micelles (ATRA/CPT/HA) loaded with the combination drugs physically are added respectively. And after incubation for 48 hours under a hypoxia condition, immunofluorescence labeling is carried out on the dryness-related protein in the cells by adopting an immunofluorescence technology, and the expression quantity of the dryness-related protein is measured by adopting a flow cytometer. The result is shown in fig. 12, compared with the control group, the polymeric micelle loaded with the combined drug can significantly reduce the expression of the sternness related proteins Oct-4, Sox-2, Nanog and ABCG2 in the tumor stem-like cells, and the polymeric micelle is proved to responsively release a differentiation inducer under hypoxia, induce the differentiation of the tumor stem-like cells, significantly reduce the expression of the intracellular stem-related proteins and reduce the sternness.

Sixthly, evaluation of in vitro cytotoxicity of polymer micelle loaded with combined drug on tumor stem-like cells

Human breast cancer MCF-7, HCC70, HCC1937 and SUM-159PT tumor stem-like cells at a ratio of 5 × 10³The density of each well is inoculated in a 96-well low-adhesion culture plate, and the polymerization of the loaded combined medicament is respectively addedThe micelle (ATRA/CPT-HA), the corresponding polymeric micelles (ATRA/HA and CPT-HA) loaded with differentiation inducer and chemotherapeutic drug singly and the polymeric micelles (ATRA/CPT/HA) loaded with combined drug physically. After incubation for 96h under hypoxic conditions, cell viability was determined and cell viability was calculated using the CCK8 assay. The results are shown in fig. 13, compared with the free combination drug and other control micelles, the combination drug loaded polymer micelle with timely gradual release property has the highest cytotoxicity to the tumor stem-like cells. The polymer micelle loaded with the combined medicament can maintain the synergistic ratio of the two medicaments, and gradually releases the two medicaments in the tumor stem-like cells, thereby enhancing the synergistic killing efficiency of the two medicaments on the tumor stem-like cells.

Pharmacokinetic study of polymer micelle loaded with combined drug

Injecting polymer micelle (ATRA/CPT-HA) loaded with the combined drug and tail vein of free combined drug (ATRA + CPT) into experimental rat, taking blood at different time points, centrifuging to take blood plasma, and determining blood concentration after organic solvent extraction. The results are shown in fig. 14, and the polymeric micelles can significantly prolong the plasma half-life of ATRA and CPT compared to the free drug mixture, and can maintain the ratio of the two drug doses for a long time.

Nine, evaluation of in vivo antitumor Activity of drug-loaded Polymer micelle

MCF-7, HCC70 and SUM-159PT tumor dry-like cells were resuspended in a mixed solution of PBS and Matrigel (1:1) at 1X 10⁶One/one number was inoculated into mice (NOD/SCID, female, 6 weeks old, 20g) mammary fat pad, and a mouse model enriched for tumor stem-like cells was constructed. Tumor-bearing mice were grouped, and tail vein injection of physiological Saline (Saline), single-loading differentiation-inducing agent and chemotherapeutic drug-loaded polymeric micelles (ATRA/HA and CPT-HA), physical co-loading drug-loaded polymeric micelles (ATRA/CPT/HA) and drug-loaded polymeric micelles (ATRA/CPT-HA) was performed once every two days, four times of administration. Measuring the long diameter and the short diameter of the tumor, calculating the tumor volume, and monitoring the change of the tumor volume. The results are shown in FIG. 15, and the anti-tumor effect of the combined drug micelle with timely gradual drug release function is shown compared with the control polymer micelleThe enhancement proves that under the condition of maintaining the synergistic ratio of the medicaments, the micelle with the gradual medicament release characteristic firstly releases the differentiation inducer, reduces the resistance of the tumor stem-like cells to the chemotherapeutic medicament, then releases the chemotherapeutic medicament, and obviously enhances the effect of the two medicaments for synergistically inhibiting the growth of the tumor rich in the stem-like cells.

Example 4

Polymer hydrogel for treating drug-resistant tumor related to tumor stem-like cells

Preparation of polymer hydrogel loaded with combined drug

The polymer hydrogel loaded with the combined drug is prepared by an assembly method. Arsenic trioxide (ASO), arsenic sulfide (ASS), bone morphogenetic protein 7(BMP7) and bone morphogenetic protein 2(BMP2) were combined with polysaccharide polymer prodrugs n-HA-oxa-CPT and n-CDT-tkt-DOX to prepare drug-loaded polymeric micelles in accordance with the methods described in example 3. 1mL of micelle is taken to be mixed with the corresponding polysaccharide macromolecule prodrug solution, after vortex mixing, the mixture is stood at normal temperature to form polymer hydrogels ASO/CPT-HA, ASS/DOX-CDT, BMP7/CPT-HA and BMP2/DOX-CDT which are loaded with the combined drug of the differentiation inducer, and each group of hydrogels have good and stable properties.

Second, drug release

1mL of the drug combination loaded polymer hydrogel (ASO/CPT-HA, ASS/DOX-CDT, BMP7/CPT-HA and BMP2/DOX-CDT) was placed in a flask and added to 60mL of dialysis medium (PBS with 2% Tween 80). Adding NADPH with final concentration of 100 μ M and liver microsomes with final concentration of 10mg/mL, introducing nitrogen to replace air, simulating hypoxic microenvironment, and after 48h, adding hydrogen peroxide with final concentration of 100 μ M to simulate high reactive oxygen environment for 48 h. Under stirring, 200 μ L of dialysis medium was taken at different time points, and the drug concentration was measured by high performance liquid chromatography, and the drug release amount was calculated to draw a drug release curve. The results are shown in fig. 16, where the release rate of the differentiation-inducing agent physically entrapped in the polymer hydrogel loaded with the combined drug is significantly higher than the release rate of the chemically conjugated chemotherapeutic drug in the hypoxic environment. Under the subsequent hydrogen peroxide environment, the hydrogel can quickly release chemotherapeutic drugs in response. The release of the two drugs is respectively associated with two different stimuli, and the obvious gradual release phenomenon is presented. The polymer hydrogel is proved to be capable of responding to a hypoxia microenvironment to rapidly release a differentiation inducer and rapidly release chemotherapeutic drugs in an active oxygen microenvironment, so that differential (gradual) release of the loaded combined drug differentiation inducer and the chemotherapeutic drugs is realized.

Thirdly, evaluation of in vivo antitumor activity of polymer hydrogel loaded with combined drug

The MCF-7 tumor stem-like cell-loaded mice are grouped, and physiological Saline (Saline), free combined drugs (ASO + CPT and ASS + DOX), polymer hydrogel for physically loading the combined drugs (ASO/CPT/HA and ASS/DOX/CDT) and polymer hydrogel for loading the combined drugs (ASO/CPT-HA and ASS/DOX-CDT) are respectively injected into tumors in situ, and the change of the tumor volume is monitored after once administration. The results are shown in fig. 17, the anti-tumor effect of the combined drug hydrogel with timely gradual drug release function is stronger than that of the physical co-loading combined drug hydrogel without gradual drug release property, which proves that under the condition of maintaining the synergistic ratio of the drugs, the hydrogel with gradual drug release property firstly releases the differentiation inducer, reduces the resistance of tumor stem-like cells to chemotherapeutic drugs, releases the chemotherapeutic drugs, and can obviously enhance the effect of the two drugs on synergistically inhibiting the tumor growth of the stem-like cells.

Claims

1. A polysaccharide polymer prodrug, comprising: the chemotherapy drug and hypoxia responsive hydrophobic group are respectively connected with the high molecular polysaccharide polymer through amido bonds or ester bonds;

2. The polysaccharide macromolecule prodrug of claim 1, wherein: a connecting arm is arranged between the high molecular polysaccharide polymer and the chemotherapeutic drug, the connecting arm is an active oxygen responsive connecting arm, one end of the active oxygen responsive connecting arm is connected with the high molecular polysaccharide polymer through an amido bond or an ester bond, and the other end of the active oxygen responsive connecting arm is connected with the chemotherapeutic drug;

the active oxygen responsive connecting arm is oxalate or thioketal.

3. A polymeric micelle for use in the treatment of a drug-resistant tumor, comprising: formed by self-assembly of the polysaccharide polymer prodrug of claim 1 in water;

the polymer micelle is coated with a chemotherapy sensitizer, and the chemotherapy sensitizer is selected from the group consisting of goropamide, quinidine, verapamil, chlorpromazine, cyclosporine A, reserpine, digoxin, amiodarone, progesterone, xyloflavone, tamoxifen, felodipine, nifedipine, erythromycin, flufenamic acid, diltiazem, valcephradine, bilactad, elcridad, oxazolindac, lovastatin, simvastatin, atorvastatin, rosuvastatin, curcumin, ginsenoside, eltrombopag, fumatotoxin C, lapatinib, neomycin, sulfasalazine, febuxostat, YM-155, LLP-3, venetocox, navitoclax, obatoclaxax, ABTACOCOCILAX, ABT-737, BM-1074 or Marinopyrorol A.

4. A polymeric hydrogel for the treatment of a drug-resistant tumor, characterized by: formed by self-assembly in water of the polysaccharide polymer prodrug of claim 1 and the polymer micelle of claim 3.

5. A polymeric micelle for use in the treatment of a tumor, comprising: formed by self-assembly of the polysaccharide polymer prodrug of claim 2 in water;

6. The polymeric micelle of claim 5, wherein: the tumor is a common tumor or a tumor stem-like cell related drug-resistant tumor.

7. A polymer hydrogel for treating a tumor, comprising: formed by self-assembly in water of the polysaccharide polymer prodrug of claim 2 and the polymer micelle of claim 5.

8. The polymer hydrogel of claim 7, wherein: the tumor is a common tumor or a tumor stem-like cell related drug-resistant tumor.