CN112516329B - Self-assembled combined drug carrier based on macromolecule prodrug and application thereof - Google Patents

Self-assembled combined drug carrier based on macromolecule prodrug and application thereof Download PDF

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CN112516329B
CN112516329B CN202011579337.4A CN202011579337A CN112516329B CN 112516329 B CN112516329 B CN 112516329B CN 202011579337 A CN202011579337 A CN 202011579337A CN 112516329 B CN112516329 B CN 112516329B
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drug
tumor
polymer
polysaccharide
prodrug
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CN112516329A (en
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莫然
沈诗洋
章颖
刘华宇
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Nanjing Ningdan New Drug Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention discloses a self-assembled combination drug carrier constructed based on polysaccharide polymer prodrug and application thereof in drug-resistant tumor combined drug treatment, belonging to the technical field of pharmaceutical preparations. The invention designs and synthesizes a polysaccharide high molecular prodrug, wherein the polysaccharide high molecular prodrug is formed by connecting a chemotherapeutic drug and a hypoxia-responsive hydrophobic group on a polysaccharide high molecular polymer; the polysaccharide macromolecule prodrug is utilized to construct a combined drug delivery system for co-carrying the inducer for reducing drug resistance and the chemotherapeutic drugs, and the delivery system can release the two drugs step by step and sequentially in drug-resistant tumor cells or/and tumor stem cells so as to solve the technical problem of poor combined action effect of the two drugs.

Description

Self-assembled combined drug carrier based on macromolecule prodrug and application thereof
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a self-assembled combined pharmaceutical carrier constructed based on polysaccharide polymer prodrugs and application thereof in drug-resistant tumor combined drug treatment.
Background
Chemotherapy (chemotherapy) is one of the main modes of treatment for clinical malignancies. However, the in vivo half-life of the chemotherapeutic drug is short, and the tumor targeting is poor, so that a plurality of drugs cannot reach tumor tissues after administration, the treatment effect is limited, and a large amount of drugs are distributed in normal tissues, so that the toxic and side effects are large. With the continuous development and maturation of polymer chemistry, the polymer prodrug is used as a novel drug delivery system, is formed by derivatizing or covalently coupling a chemotherapeutic drug to a polymer, has the advantages of increasing the tumor targeting and selectivity of the drug, improving the drug concentration in tumor tissues, reducing the distribution of the drug in normal organs, reducing toxic and side effects and the like, and is widely studied at home and abroad. However, tumor resistance remains one of the main causes of failure in tumor treatment. The molecular mechanism of tumor drug resistance is complex, and mainly comprises high expression of drug efflux proteins on the surface of tumor cells, high expression of anti-apoptosis proteins in the tumor cells, and the like. For example: drug efflux proteins (P-glycoprotein (P-gp) and ATP-binding transporter 2 (ABCG 2), etc.) actively transport the chemotherapeutic drugs in tumor cells to the outside by means of energy supplied by ATP, resulting in intracellular drug concentrations consistently below the effective therapeutic concentration; anti-apoptotic proteins (survivin protein and B Cell Lymphoma (BCL) -2 protein) inhibit chemotherapy-induced apoptosis by blocking apoptosis signaling pathways such as Caspase and NF- κb. In recent years, it has been found that a group of cells having stem cell characteristics, called stem cell-like tumor cells (abbreviated as tumor stem-like cells), exist in tumor tissue, and such cells have self-renewal, multipotency and strong tumorigenicity. The tumor stem-like cells highly express the 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 combined drug combined treatment strategy is adopted, and the chemosensitizer and the chemotherapeutic drug are combined, so that the chemoresistance of the drug-resistant tumor is reduced by using the chemosensitizer, 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, due to different chemical structures, the chemical structures of the chemotherapeutic drugs and the chemosensitizer are greatly different in the pharmacokinetic behavior and the tissue distribution rule of the chemotherapeutic drugs and the chemosensitizer, and the optimal synergistic drug ratio of the two drugs in the whole process of being distributed to tumor tissues and then entering the tumor cells after being dosed is difficult to maintain by the traditional physical mixed 'cocktail' type dosing mode, so that the drug combination often cannot reach the expected effect. A combined drug co-delivery system based on micro-nano carriers can solve this problem. The combined medicines are co-carried in the same carrier, so that the synergistic ratio of the medicines after administration can be maintained, the acting time of the medicines is prolonged, and the targeting property of the medicines is improved. However, the mechanism of action of chemosensitizers and chemotherapeutic drugs determines the order of action of the two drugs during treatment of resistant tumors. If the chemotherapeutic agent is released from the carrier simultaneously or prior to the chemosensitizer, the released chemotherapeutic agent cannot kill the unsensitized drug-resistant tumor cells and even enhances the drug resistance or dryness thereof. Therefore, based on the biological characteristics of drug-resistant tumor cells and tumor stem-like cells, not only is the combination of a chemosensitizer and a chemotherapeutic drug required to treat drug-resistant tumors, but also the synergistic proportion of the two drugs is required to be maintained, and meanwhile, based on the action mechanism of the chemosensitizer and the chemotherapeutic drug, the two drugs are required to timely act in cells, so that the synergistic anti-tumor efficacy is enhanced.
Disclosure of Invention
The invention aims to solve the technical problem that the combination of a chemosensitizer and a chemotherapeutic drug has poor effect on the treatment of drug-resistant tumors, and provides a polysaccharide macromolecule prodrug coupled with the chemotherapeutic drug and a hydrophobic group with low oxygen responsiveness, which can self-assemble and physically embed the chemosensitizer to obtain a combined drug carrier, and is finally used for co-delivery of the chemosensitizer and the chemotherapeutic drug to improve the synergistic drug-resistant tumor resistance of the two drugs.
In order to achieve the above object, the present invention adopts the following technical scheme:
a 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 an amide bond or an ester bond;
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, decitabine, 5-fluorouracil, 6-mercaptopurine, methotrexate, doxorubicin, epirubicin, doxorubicin, daunorubicin, mitoxantrone, paclitaxel, docetaxel, nimustine, teniposide, etoposide, aminoglutethimide or melphalan;
The hypoxia-responsive hydrophobic group is a 2-nitroimidazole group or an azo phenyl 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 amide bond or an ester bond, and the other end of the active oxygen responsive connecting arm is connected with the chemotherapeutic drug.
Further, the reactive oxygen species-responsive linking arm is an oxalate or a ketal.
A polymer micelle for treating drug-resistant tumors is formed by self-assembling the polysaccharide macromolecule prodrug in water;
the polymeric micelles are entrapped with a chemosensitizer selected from the group consisting of gossypol, quinidine, verapamil, chlorpromazine, cyclosporin a, reserpine, digoxin, amiodarone, progesterone, lignan, tamoxifen, felodipine, nifedipine, erythromycin, flufenamic acid, diltiazem, valpuidamba, brinodalidade, zoolada, lovastatin, simvastatin, atorvastatin, rosuvastatin, curcumin, ginsenoside, eltrombopag, fumagillin C, lapatinib, novobiocin, sulfasalazine, febuxostat, YM-155, LLP-3, venetoclax, navitoclax, obatoclax Mesylate, ABT-737, BM-1074 or Marinopyrrole a.
A polymer hydrogel for treating drug-resistant tumor is prepared from the high-molecular polyose precursor and the polymer micelle through self-assembling in water.
The polymer micelle for treating common tumor or tumor stem-like cell related drug resistant tumor is formed by self-assembling the polysaccharide polymer 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, retinaldehyde, 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 macromolecule prodrug and the polymer micelle in water.
The invention provides a polysaccharide macromolecule prodrug with double modification, which is prepared by modifying a chemotherapeutic drug and a hydrophobic group with low oxygen response on a macromolecule polysaccharide polymer. The chemosensitizer or differentiation inducer is loaded in the micelle carrier formed by the polysaccharide macromolecule prodrug through physical action, and can be released when entering drug-resistant tumor cells or tumor stem cells, so that the effect of reducing drug resistance is exerted, and the release of the drug-resistance inducer is further exerted after the chemosensitizer which is covalently connected to the polysaccharide molecule through chemical bonds.
Drawings
FIG. 1 shows the intracellular drug release process of the self-assembled composite drug carrier based on the macromolecule prodrug in the invention in drug-resistant tumor cells, tumor stem-like cells and common 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 shows the down-regulating effect of the polymer micelle on drug-resistant proteins in drug-resistant tumor cells in example 1.
FIG. 4 shows inhibition of drug-resistant tumor cell viability by the polymer micelles of example 1.
FIG. 5 shows the change in the ratio of the blood concentration of the polymer micelle-supported drug in the body in example 1.
FIG. 6 shows the in vivo antitumor activity of the polymer micelle of example 1.
FIG. 7 is a photograph of the polymer hydrogel of example 2.
FIG. 8 shows the release of drug from the polymer hydrogel of example 2 in a low oxygen environment.
FIG. 9 shows the in vivo antitumor activity of the polymer hydrogel of example 2.
FIG. 10 shows the release results of the drug in the polymer micelle of example 3 in a low oxygen and active oxygen environment.
FIG. 11 shows the results of the polymer micelle in example 3 for up-regulating active oxygen levels in tumor stem-like cells and intracellular release of chemotherapeutic agents.
FIG. 12 shows the inhibition of stem-related proteins in tumor stem-like cells by polymer micelles of example 3.
FIG. 13 shows the result of inhibition of tumor stem-like cell viability by the polymer micelle of example 3.
FIG. 14 shows in vivo pharmacokinetic studies of the polymer micelles of example 3, and changes in the blood concentration ratio.
FIG. 15 shows the result of inhibition of tumor growth by the polymer micelle of example 3 on tumor-rich stem-like cells.
FIG. 16 shows the results of drug release in low oxygen and active oxygen environments from the polymer hydrogels of example 4.
FIG. 17 shows the result of the inhibition of tumor growth by the polymer hydrogel of example 4 on tumor-enriched stem-like cells.
Detailed Description
The invention designs and synthesizes a polysaccharide polymer prodrug, and utilizes the polysaccharide polymer prodrug to construct a combined drug delivery system of a drug resistance reducing inducer (chemosensitizer or differentiation inducer) and a chemotherapeutic drug, and the delivery system can release the two drugs step by step and sequentially in drug-resistant tumor cells or/and tumor stem-like cells so as to solve the technical problem of poor combined action effect of the two drugs.
In the present invention, a tumor stem-like cell-related drug resistant tumor refers to a tumor tissue enriched with undifferentiated/poorly differentiated tumor stem-like cells, thereby resulting in a tumor tissue having high drug resistance.
The polysaccharide high molecular prodrug comprises a high molecular polysaccharide polymer, wherein a chemotherapeutic drug and a hypoxia-responsive hydrophobic group are modified on the high molecular polysaccharide polymer, and the chemotherapeutic drug and the hypoxia-responsive hydrophobic group are respectively connected with the high molecular polysaccharide polymer through an amide bond or an ester bond; 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, decitabine, 5-fluorouracil, 6-mercaptopurine, methotrexate, doxorubicin, epirubicin, doxorubicin, daunorubicin, mitoxantrone, paclitaxel, docetaxel, nimustine, teniposide, etoposide, aminoglutethimide or melphalan; the hypoxia-responsive hydrophobic group is a 2-nitroimidazole group or an azo phenyl group.
As shown in fig. 1 a, since the polysaccharide polymer prodrug has amphipathy, the polysaccharide polymer prodrug can self-assemble in water to form polymer micelles, and the chemotherapy sensitizer is embedded to form a combined drug delivery carrier for treating drug-resistant tumors. The combined drug delivery vehicle can maintain the synergistic drug ratio of the chemosensitizer and the chemotherapeutic drug in the tumor tissue. In the tumor tissue hypoxia microenvironment, the hypoxia-responsive hydrophobic group in the polysaccharide macromolecule prodrug can change in structure, and the original hydrophobic structure is converted into a hydrophilic structure, so that the hydrophobic acting force in the micelle is reduced, the micelle is dissociated, and the embedded chemical sensitizer is rapidly released. Since the chemotherapeutic agent is covalently linked to the polymer via an ester or amide bond, its release is dependent on hydrolysis of the covalent bond by esterases or amidases in the drug resistant cells. Thus, the release rate of the chemosensitizer physically entrapped in the micelle is much higher at hypoxia than the release rate of the covalently coupled chemotherapeutic drug. In drug-resistant tumor cells, the chemical sensitizer released first can reduce the drug resistance of the cells, and the chemotherapeutic drug released later kills the tumor cells after the drug resistance is obviously reduced, so that the synergistic efficiency of the two drugs is greatly improved.
In the invention, the preparation method of the polymer micelle comprises the following steps: dissolving a chemosensitizer in an organic solvent to obtain an organic phase, dissolving a polysaccharide polymer prodrug in water to obtain a water phase, blending the two phases, performing ultrasonic emulsification, removing the organic solvent by vacuum decompression after the completion, and then passing through a water-based filter membrane to obtain a polymer micelle, thus the polymer micelle is a combined drug delivery carrier for treating drug-resistant tumors.
Further, the chemosensitizer is selected from the group consisting of gossypol, quinidine, verapamil, chlorpromazine, cyclosporin A, reserpine, digoxin, amiodarone, progesterone, lignan, tamoxifen, felodipine, nifedipine, erythromycin, flufenamic acid, diltiazem, valproad, brikodada, ricnidazole, zooladine, lovastatin, simvastatin, atorvastatin, rosuvastatin, curcumin, ginsenoside, eltrombopa, fumagillin C, lapatinib, novobiocin, sulfasalazine, febuxostat, YM-155, LLP-3, venetoclax, navitoclax, obatoclax Mesylate, ABT-737, BM-1074, marinopyrole A.
In addition, in order to further improve the distribution of the drug in the high-drug-resistance tumor tissue and realize in-situ tumor administration, the combined drug delivery carrier and the high-concentration polysaccharide polymer prodrug can be further assembled in water to form a combined drug hydrogel carrier. The preparation method comprises the following steps: adding the combined drug delivery carrier into high-concentration polysaccharide macromolecule pre-aqueous solution, and standing after vortex.
On the other hand, aiming at tumor cells-tumor stem-like cells with strong drug resistance, the invention further carries out structural derivatization on the polysaccharide high polymer prodrug, specifically, the polysaccharide high polymer prodrug is obtained by covalently connecting a chemotherapeutic drug with a high polymer polysaccharide polymer through an active oxygen responsive connecting arm. The reactive oxygen species-responsive linker arm is selected from oxalate or ketal.
As shown in fig. 1B, the polysaccharide polymer prodrug can also self-assemble to form micelles and load differentiation inducers to give a combination drug delivery vehicle. In particular, in hypoxic tumor stem-like cells, the combination drug delivery vehicle can respond to rapid release of physically entrapped differentiation inducer, whereas due to the strong active oxygen scavenging capacity of tumor stem-like cells, wherein the active oxygen levels are low, chemotherapeutic drugs are stably modified on the polysaccharide high molecular polymer and are not released in tumor stem-like cells. The released differentiation inducer 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 the chemotherapeutic drugs, greatly increases the intracellular active oxygen level due to the increase of biosynthesis in the differentiation process, degrades the active oxygen responsive connecting arm in the polysaccharide high polymer prodrug, and releases the chemotherapeutic drugs from the polysaccharide high polymer to the differentiated tumor cells. Based on the undifferentiated tumor stem-like cells and the physiological signal change in the differentiation process, the timely and sequential release of the differentiation inducer and the chemotherapeutic drugs is regulated, so that the killing power of the chemotherapeutic drugs on the tumor stem-like cells is greatly improved. Meanwhile, in common non-stem-like tumor cells, the polysaccharide polymer prodrug can be rapidly degraded due to higher intracellular active oxygen level, so that the chemotherapeutic drug is released, and the tumor cells are killed. The combined drug delivery carrier can be used for removing tumor stem-like cells and common tumor cells while being assimilated, and overcomes tumor heterogeneity and stem-related tumor drug resistance.
In the invention, the preparation method of the polymer micelle comprises the following steps: dissolving a differentiation inducer in an organic solvent to obtain an organic phase, dissolving a polysaccharide polymer prodrug in water to obtain a water phase, then blending the two phases, performing ultrasonic emulsification, removing the organic solvent by vacuum decompression after the completion, and then passing through a water-based filter membrane to obtain a polymer micelle, thus obtaining the combined drug delivery carrier.
Further, the differentiation inducer is selected from all-trans retinoic acid, 1, 3-cis retinoic acid, 9-cis retinoic acid, retinol, retinaldehyde, arsenic trioxide, arsenic sulfide, bone morphogenetic protein 7, bone morphogenetic protein 2, vismodegib.
In addition, in order to further improve the distribution of the drug in the tumor tissue rich in the tumor stem-like cells and realize the in-situ administration of the tumor, the combined drug delivery carrier and the high-concentration polysaccharide polymer prodrug can be further assembled in water to form a combined drug hydrogel carrier. The preparation method comprises the following steps: adding the combined drug delivery carrier into high-concentration polysaccharide macromolecule pre-aqueous solution, and standing after vortex.
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
Example 1
Polymeric micelles for drug-resistant tumor treatment
1. Synthesis of polysaccharide polymer prodrugs
Cholesterol chloroformate (502 mg) and triethylamine (120 mg) were added to 20mL of methylene chloride and stirred, p-aminoazobenzene (368 mg) was further added to react for 12 hours, the reaction solution was collected after the completion of the reaction, dried by vacuum, and the product was purified and separated by silica gel column chromatography to obtain cholesterol formyl p-aminoazobenzene. 2-nitroimidazole (302 mg) and potassium carbonate (557 mg) were added to 10mL of N, N-dimethylformamide and stirred, 6- (Boc-amino) bromohexane (782 mg) was further added, the reaction was heated to 80℃and refluxed for 4 hours, and after completion of the reaction, dried by spinning at 70℃to obtain a brown solid. After drying by extraction, the product 1- (6-Bo-aminohexyl) -2-nitroimidazole is obtained. 1- (6-Boc-Aminohexyl) -2-nitroimidazole (493 mg) was dissolved in 10mL of 1.25M hydrochloric acid-methanol solution, and the reaction was stirred at room temperature overnight to remove the Boc protecting group. After the reaction, the reaction solution was spin-dried at 40℃to give 6- (2-nitroimidazolyl) hexylamine.
Hyaluronic acid (HA, 90kDa,196 mg), heparin sodium (HP, 15kDa,150 mg) and carboxydextran (CDT, 15KDa,137mg) were dissolved in 10mL of water, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (190 mg) and N-hydroxysuccinimide (NHS) (143 mg) were added thereto, and after stirring at room temperature for 0.5h, cholesterol formyl-p-aminoazobenzene (197 mg) previously dissolved in 5mL of dimethyl sulfoxide (DMSO) was added thereto, and stirring reaction was continued at room temperature for 24h. Then, the reaction solution is put into a dialysis bag (3.5 kDa) and dialyzed in water for 24 hours, and the liquid in the dialysis bag is collected and freeze-dried to obtain polysaccharide derivatives c-HA, c-HP and c-CDT modified by cholesterol azo phenyl groups.
HA (90 kDa,205 mg), HP (15 kDa,144 mg), CDT (15 kDa,125 mg) were dissolved in 25mL of water, EDCI (571 mg) and NHS (571 mg) were added thereto, and after stirring at room temperature for 0.5h, 6- (2-nitroimidazolyl) hexylamine (155 mg) was added thereto, and the reaction was continued at room temperature for 24h with stirring. Then, the reaction solution is put into a dialysis bag (14 kDa) and dialyzed for 24 hours in water, and the liquid in the dialysis bag is collected and freeze-dried to obtain the polysaccharide derivatives n-HA, n-HP and n-CDT modified by the 2-nitroimidazole groups.
Dissolving appropriate amounts of c-HA, c-HP, c-CDT, n-HA, n-HP and n-CDT in 25mL of water respectively, adding EDCI and NHS, stirring at room temperature for 0.5h, 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) respectively, and continuing stirring at room temperature for 24h. Then, the reaction solution is put into a dialysis bag to be dialyzed for 24 hours in water, and the liquid in the dialysis bag is collected and freeze-dried to obtain polysaccharide macromolecule prodrugs of 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.
2. Preparation of polymer micelle loaded with combined medicines
And preparing the polymer micelle loaded with the combined medicine by adopting a single emulsification method. Cyclosporin A (CSA), quinidine (QD), reserpine (RSP), lignan (GST), tamoxifen (TAM), nifedipine (NDP), diltiazem (DTZ), curcumin (CUR), fumagillin C (FTC), YM-155 (YM), ABT-737 (ABT) were dissolved in 1mL chloroform to obtain an organic phase. Polysaccharide high molecular 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, n-CDT-DTX were dissolved in 5mL of water to obtain an aqueous phase. The aqueous phase is placed in an ice water bath, the organic phase is dropwise added into the aqueous phase under the ultrasonic action of a probe, and the ultrasonic action is continued for 20min after the completion of the dropwise addition. And (3) vacuumizing the obtained emulsion at 40 ℃ to remove the organic solvent by rotary evaporation, and then passing through a 220nm filter membrane to obtain the polymer micelle loaded with the combined drug. The particle size and polydispersity of each group of micelles were measured using a particle size meter, and the results are shown in table 1.
TABLE 1 particle size and polydispersity of polymeric micelles loaded with drug combinations
3. Drug release
And co-loading a chemotherapy drug (CPT or MIT) and a chemotherapy sensitizer (CSA or FTC) into the polysaccharide macromolecule micelle modified by the hypoxia response group through a single emulsification method to obtain the polymer micelle (CSA/CPT/HA and FTC/MIT/CDT) of the physical co-carried combined drug. 2mL of polymer micelles loaded with the combination drug (CSA/CPT-HA, FTC/MIT-CDT, YM/PTX-CDT and ABT/DTX-CDT) were loaded into dialysis bags, and the dialysis bags were placed in 60mL of dialysis medium (PBS containing 2% Tween 80). NADPH at a final concentration of 100. Mu.M and 10mg/mL of liver microsomes were added and the air was replaced with nitrogen to simulate a hypoxic microenvironment. 200 mu L of dialysis medium is taken at different time points under stirring, the drug concentration is measured by adopting a high performance liquid chromatography, the drug release amount is calculated, and a drug release curve is drawn. As shown in fig. 2, the release rate of the chemotherapeutic sensitizer physically entrapped in the polymeric micelles loaded with the combination drug was significantly higher than that of the chemically coupled chemotherapeutic drug in a hypoxic environment. The polymer micelle can respond to the low-oxygen microenvironment to quickly release the chemotherapeutic sensitizer, so that the loaded combined drug chemotherapeutic sensitizer and the chemotherapeutic drug are released in a differential (sequential) manner.
4. Evaluation of drug-resistance-related proteins downregulated by drug-loaded polymeric micelles
Human breast cancer resistant cells (MCF-7/MDR) were cultured at 1X 10 5 Density of individual/well was inoculated into 6-well culture plates, and polymer micelles loaded with the combination drugs (CSA/CPT-HA, FTC/MIT-CDT, YM/PTX-CDT and ABT/DTX-CDT) and polymer micelles physically co-loaded with the combination drugs (CSA/CPT/HA, FTC/MIT/CDT, YM/PTX/CDT and ABT/DTX/CDT) were added, respectively, and cultured for 48 hours. And (3) carrying out fluorescent marking on related drug-resistant proteins of the treated drug-resistant cells by adopting a fluorescent antibody immunostaining method, and detecting the protein level of the fluorescent marking by adopting a flow cytometry. The results are shown in FIG. 3, and compared with the untreated control group, the drug-resistance related proteins (P-gp, ABCG2, survivin, and BCL-2 proteins) in the drug-resistant cells treated by the polymer micelle loaded with the chemosensitizer are significantly down-regulated. Proved that the polymer micelle loaded with the combined medicine can obviously reduce the toleranceDrug resistance of tumor cells.
5. In vitro cytotoxicity evaluation of drug-resistant tumor cells by polymer micelle loaded with combined drug
The polymer micelle loaded with the combination drug (GAL/CPT-HA, DIG/CPT-HA, BTL/CPT-HA, FDP/CPT-HA, EM/CPT-HA, LLP/CPT-HA and BM/CPT-HA) is prepared by taking Goropamide (GAL), digoxin (DIG), amiodarone (BTL), felodipine (FDP), erythromycin (EM), LLP-3 (LLP), BM-1074 (BM) as a chemosensitizer and polysaccharide macromolecular prodrug c-HA-CPT according to the method described in the second part. MCF-7/MDR cells were grown at 5X 10 3 The density of individual/wells was inoculated into 96-well plates, and polymer micelles loaded with the combination drug and free combination drug (chemosensitizer+chemodrug) were added, or polymer micelles loaded with the combination drug (CSA/CPT-HA and FTC/MIT-CDT) and polymer micelles physically co-loaded with the combination drug (CSA/CPT/HA and FTC/MIT/CDT) at different concentrations. After 96h of culture in a hypoxia incubator, cell viability was determined using CCK8 assay and cell viability was calculated. The results are shown in fig. 4, and the polymer micelle loaded with the combination drug has stronger cytotoxicity to drug-resistant tumor cells than the polymer micelle loaded with the free combination drug and the physical co-carried combination drug. The polymer micelle loaded with the combined medicine can maintain the cooperative proportion of the two medicines, simultaneously release the two medicines in the medicine-resistant tumor cells step by step, and enhance the cooperative killing efficiency of the two medicines on the medicine-resistant tumor cells.
6. Pharmacokinetic investigation of polymeric micelles loaded with combination drugs
The polymer micelle loaded with the combined drug (CSA/CPT-HA and FTC/MIT-CDT) and the free combined drug (CSA+CPT and FTC+MIT) are injected into the experimental rat in tail vein, blood is taken at different time points respectively, plasma is taken by centrifugation, and the blood concentration is measured after extraction by an organic solvent. The results are shown in fig. 5, where the polymer micelles maintain the dose ratio of the combination drug for a longer period of time than the free drug mixture.
7. Evaluation of in vivo antitumor Activity of Polymer micelles loaded with Combined drug
MCF-7/MDR cells were resuspended in a mixed solution of PBS and Matrigel (1:1) at 1X 10 7 Number of individuals/individuals were inoculated into mammary fat pads of mice (nu/nu, female, 6 weeks old, 20 g) to construct drug-resistant tumor mouse models. Tumor-bearing mice were grouped and administered once every two days, four times, with physiological Saline (Saline), free combination drug (csa+cpt and ftc+mit), polymer micelle of physical co-carried combination drug (CSA/CPT/HA and FTC/MIT/CDT), and polymer micelle of loaded combination drug (CSA/CPT-HA and FTC/MIT-CDT), respectively. Tumor length and diameter were measured, tumor volume was calculated (volume=length diameter×short diameter/2), and tumor volume change was monitored. The results are shown in figure 6, compared with the free combination drug, the combination drug micelle has stronger effect of inhibiting the growth of drug-resistant tumors, and the fact that the micelle carrying the combination drug chemosensitizer and the chemotherapy drug together maintains the drug ratio of the two drugs, improves the effect of drug synergy on drug-resistant tumors. The anti-tumor effect of the combined drug micelle with the timely gradual drug release function is stronger than that of the physical co-carried combined drug micelle without the gradual drug release characteristic, and the fact that the micelle with the gradual drug release characteristic releases the chemosensitizer at first, reduces the resistance of drug-resistant tumor cells to the chemo-therapeutic drugs, releases the chemo-therapeutic drugs again, and remarkably enhances the effect of the synergistic inhibition of the two drugs on the growth of the drug-resistant tumor is proved.
Example 2
Polymer hydrogels for drug-resistant tumor treatment
1. Synthesis of polysaccharide polymer prodrugs
c-HA, c-CDT, n-HA, n-HP and n-CDT are respectively dissolved in 25mL of water, EDCI and NHS are added, and after stirring for 0.5h at room temperature, cytarabine (CC), topotecan (TPT), methotrexate (MTX), nimustine (ANU) and etoposide (VP) are respectively added, and stirring reaction is continued for 24h at room temperature. And then the reaction solution is filled into a dialysis bag and placed into water for dialysis for 24 hours, and the liquid in the dialysis bag is collected and freeze-dried to obtain polysaccharide macromolecule prodrugs of c-HA-CC, c-CDT-TPT, n-HA-MTX, n-HP-ANU and n-CDT-VP.
2. Preparation of polymer hydrogel loaded with combined medicines
Verapamil (VER), chlorpromazine (CPZ), ginsenoside Rg1 (GSG) and ginsenoside Rb1 (GSB) are prepared into polymer micelles loaded with combined drugs together with polysaccharide high polymer prodrugs c-HA-CC, c-CDT-TPT, n-HA-MTX, n-HP-ANU and n-CDT-VP according to the method in the example 1. Mixing 1mL of micelle with the corresponding polysaccharide macromolecule prodrug solution, vortex mixing uniformly, 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 loaded with the combined drug. The gel forming effect is shown in figure 7, and from left to right, the gel forming effect is respectively water, VEP/CC-HA, VEP/TPT-CDT, CPZ/MTX-HA, GSG/ANU-HP and GSB/VP-CDT, and the assembly system can form hydrogel.
3. Drug release
1mL of the drug-loaded polymer hydrogel (VEP/CC-HA and CPZ/MTX-HA) and the physical drug-loaded polymer hydrogel (VEP/CC/HA and CPZ/MTX/HA) were placed in a flask, 20mL of dialysis medium (PBS containing 2% Tween 80) was added, and NADPH at a final concentration of 100. Mu.M and 10mg/mL of liver microsomes were added, and air was replaced with nitrogen to simulate a hypoxic microenvironment. 200 mu L of dialysis medium is taken at different time points under stirring, the drug concentration is measured by adopting a high performance liquid chromatography, the drug release amount is calculated, and a drug release curve is drawn. As a result, as shown in fig. 8, the release rate of the chemotherapeutic sensitizer physically entrapped in the polymer hydrogel loaded with the combination drug was significantly higher than that of the chemically coupled chemotherapeutic drug in a hypoxic environment. The polymer hydrogel can respond to the low-oxygen microenvironment to quickly release the chemotherapeutic sensitizer, so that the loaded combined drug chemotherapeutic sensitizer and the chemotherapeutic drug are released in a differential (sequential) manner.
4. Evaluation of in vivo antitumor Activity of Polymer hydrogels loaded with combination drugs
Tumor volume changes were monitored by grouping drug-resistant breast cancer (MCF-7/MDR) tumor mice, injecting normal Saline (Saline), free drug combinations (VEP+CC and CPZ+MTX), polymer hydrogels loaded with the drug combinations physically together (VEP/CC/HA and CPZ/MTX/HA), and polymer hydrogels loaded with the drug combinations (VEP/CC-HA and CPZ/MTX-HA) in situ in the tumor, respectively, once. As shown in figure 9, the anti-tumor effect of the combined medicine hydrogel with the timely gradual release function is stronger than that of the physical co-carried combined medicine hydrogel without the gradual release characteristic and the free combined medicine, and the results prove that the hydrogel with the gradual release characteristic firstly releases the chemosensitizer, reduces the resistance of the drug-resistant tumor cells to the chemo-therapeutic medicine and then releases the chemo-therapeutic medicine under the condition of maintaining the drug synergistic proportion, and can obviously enhance the effect of the two medicines on the synergistic inhibition of the growth of the drug-resistant tumor.
Example 3
Polymer micelle for tumor stem-like cell related drug resistant tumor treatment
1. Synthesis of polysaccharide polymer prodrugs
Oxalyl chloride (632 mg) was dissolved in 5mL of anhydrous dichloromethane and stirred on an ice-water bath, 2-azidoethanol (87.2 mg) was dissolved in 5mL of anhydrous dichloromethane and slowly added dropwise to the reaction solution, followed by stirring on an ice-water bath for 4 hours. After completion of the reaction, spin-drying gave a light brown oil. The resulting oil was redissolved in 20mL of anhydrous dichloromethane and placed on an ice-water bath with stirring, then Camptothecin (CPT) and triethylamine (110 mg) and 4-dimethylaminopyridine (25.6 mg) were dissolved in 10mL of anhydrous dichloromethane, slowly added dropwise to the reaction solution, and the reaction was continued on the ice-water bath for 2h. The reaction liquid is extracted and then dried by spin to obtain a crude product. And finally, purifying by silica gel column chromatography to obtain the active oxygen response oxalate coupled CPT prodrug molecule. 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 (125 mg) was added thereto, and the reaction was continued at room temperature for 24h with stirring. Then the reaction solution is put into a dialysis bag (14 kDa) and dialyzed in water for 48 hours, and the liquid in the dialysis bag is collected and lyophilized to obtain the product. The product and oxalate-coupled CPT prodrug molecule were then dissolved in 50mL of a mixed solution of DMSO and water (DMSO: H) 2 O=4:1, v:v), the reaction was stirred at room temperature for 24h. And then the reaction solution is put into a dialysis bag to be dialyzed for 48 hours in water, and the liquid in the dialysis bag is collected and freeze-dried to obtain the polysaccharide macromolecule prodrug n-HA-oxa-CPT.
Taking anhydrous acetone and anhydrous 3-mercaptopropionic acid, stirring the mixture for 4 hours at room temperature by introducing dry hydrogen chloride, and stopping the reaction in an ice salt mixture to realize crystallization. The product is washed, filtered by normal hexane and cold water, and dried in vacuum to obtain the product, thioketal dipropionic acid. The ketal dipropionic acid was dissolved in anhydrous DMF and NHS and EDCI were added to the above mixture under nitrogen. After stirring for 2h, CPT and Doxorubicin (DOX) were added dropwise and reacted further for 24h to give a crude product which was dried by spin. Purifying by silica gel column chromatography to obtain active oxygen response ketal prodrug molecule. The active oxygen response ketal prodrug molecules are dissolved in 25mL of water, EDCI and NHS are added, after stirring for 0.5h at room temperature, a proper amount of n-HA and n-CDT are respectively dissolved in 25mL of water, and stirring reaction is continued for 24h at room temperature. And then, the reaction solution is filled into a dialysis bag (14 kDa) and dialyzed in water for 48 hours, and the liquid in the dialysis bag is collected and freeze-dried to obtain polysaccharide macromolecule prodrugs n-HA-tkt-DOX and n-CDT-tkt-CPT.
2. Preparation of polymer micelle loaded with combined medicines
And preparing the polymer micelle loaded with the combined medicine by adopting a single emulsification method. All-trans retinoic acid (ATRA), 1, 3-Cis Retinoic Acid (CRA), 9-cis retinoic acid (TRA), retinol (RTO), retinaldehyde (RTE), vismodegib (VMG), and polysaccharide polymer prodrugs. 3mg of ATRA, CRA, TRA, RTO, RTE, VMG was dissolved in 1mL of chloroform to obtain an organic phase, and polysaccharide prodrug molecules n-HA-oxa-CPT, n-HA-tkt-DOX and n-CDT-tkt-CPT were dissolved in 5mL of water to obtain an aqueous phase. The water phase is placed in an ice water bath, the organic phase is added dropwise to the water phase by ultrasound under the ultrasound of a probe, and ultrasound is continued for 20min after the addition is completed. And (3) vacuumizing the obtained emulsion at 40 ℃ to remove the organic solvent by rotary evaporation, and then passing through a 220nm filter membrane to obtain the polymer micelle loaded with the combined drug. The particle size and polydispersity of each group of micelles were measured using a particle size meter, and the results are shown in table 2.
TABLE 2 particle size and polydispersity of drug-loaded polymeric micelles
3. Drug release
2mL of drug-loaded polymer micelles (ATRA/CPT-HA, CRA/CPT-HA, RTO/DOX-HA and RTE/CPT-CDT) were placed in dialysis bags and placed in 60mL of dialysis medium (PBS containing 2% Tween 80). NADPH at a final concentration of 100. Mu.M and 10mg/mL of liver microsomes were added and the air was replaced by nitrogen to simulate a hypoxic microenvironment. After 48 hours, hydrogen peroxide was added to the system at a final concentration of 100. Mu.M to simulate a high reactive oxygen species environment for 48 hours. 200 mu L of dialysis medium is taken at different time points under stirring, the drug concentration is measured by adopting a high performance liquid chromatography, the drug release amount is calculated, and a drug release curve is drawn. As a result, as shown in fig. 10, the release rate of the differentiation inducer physically entrapped in the polymer micelle loaded with the combination drug was significantly higher than that of the chemically coupled chemotherapeutic drug in the hypoxic environment. And in the subsequent hydrogen peroxide environment, the micelle can release the chemotherapeutic drug rapidly in response. The release of the two drugs was associated with two different stimuli, respectively, exhibiting a distinct progressive release behavior. The polymer micelle can respond to the low-oxygen microenvironment to quickly release the differentiation inducer, and responds to the release of the chemotherapeutic drugs in the active oxygen microenvironment to realize the differential (gradual) release of the loaded combined drug differentiation inducer and the chemotherapeutic drugs.
4. Polymer micelle loaded with combined drug up-regulates active oxygen level in tumor stem-like cells and intracellular release evaluation of chemotherapeutic drugs
MCF-7 tumor stem-like cells are obtained by adopting a side group cell flow separation technology, and are processed by a method of 1 multiplied by 10 5 The density of each well was inoculated into a 6-well low-adhesion culture plate, and polymer micelles loaded with the combination drug (ATRA/CPT-HA) and polymer micelles loaded with the chemotherapeutic drug alone (CPT-HA) were added, respectively. After 48h incubation under hypoxia, intracellular active oxygen of the cells was fluorescently labeled by DCFH-DA staining and the active oxygen concentration was measured by flow cytometry. And (3) quantitatively detecting the intracellular CPT by adopting high performance liquid chromatography, and examining the intracellular release of the CPT. The results are shown in fig. 11, and the active oxygen level in the tumor stem-like cells is significantly increased after the treatment of the polymer micelle loaded with the combination drug. The polymer micelle loaded with the combined medicine can release the differentiation inducer ATRA in the tumor stem-like cells under the condition of hypoxia, and the released ATRA induces the differentiation of the tumor stem-like cells, so that the intracellular active oxygen concentration of the differentiated cells is greatly increased. Elevated triggering of intracellular reactive oxygen speciesResponsive release of therapeutic drug CPT.
5. Polymer micelle loaded with combined drugs down-regulates stem-related proteins in tumor stem-like cells
The serum-free suspension culture enrichment tumor stem-like cell method is adopted to obtain the human triple negative breast cancer HCC70, HCC1937 and SUM-159PT tumor stem-like cells. MCF-7, HCC70, HCC1937 and SUM-159PT tumor stem-like cells were cultured at 1×10 5 The density of each hole is inoculated into a 6-hole low-adhesion culture plate, and polymer micelles (ATRA/CPT-HA and RTO/DOX-HA) loaded with different combined medicines, corresponding polymer micelles (ATRA/HA and CPT-HA) loaded with single differentiation inducer and chemotherapeutic medicines and polymer micelles (ATRA/CPT/HA) loaded with the combined medicines physically are respectively added. After 48h incubation under the hypoxia condition, immunofluorescence technology is adopted to carry out immunofluorescence labeling on the dry related protein in the cells, and a flow cytometer is adopted to measure the dry related protein expression quantity. As shown in fig. 12, compared with the control group, the polymer micelle loaded with the combined drug can obviously reduce the expression of the stem related proteins Oct-4, sox-2, nanog and ABCG2 in the tumor stem-like cells, and proves that the polymer micelle responds to release of the differentiation inducer under hypoxia to induce the differentiation of the tumor stem-like cells, obviously reduce the expression of the intracellular stem related proteins and reduce the dryness.
6. In vitro cytotoxicity evaluation 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 were cultured at 5×10 3 The density of each hole is inoculated into a 96-hole low-adhesion culture plate, and polymer micelles loaded with combined drugs (ATRA/CPT-HA), corresponding polymer micelles loaded with differentiation inducer and chemotherapeutic drugs (ATRA/HA and CPT-HA) and polymer micelles physically co-loaded with the combined drugs (ATRA/CPT/HA) are respectively added. After 96h incubation under hypoxia, cell viability was determined using CCK8 assay and cell viability was calculated. As shown in fig. 13, the polymer micelle loaded with the combination drug, which has the characteristic of timely stepwise drug release, has the strongest cytotoxicity to the tumor stem-like cells compared with the free combination drug and other control group micelles. It has been demonstrated that polymeric micelles loaded with the combination drug are capable of sustainingThe synergistic ratio of the two drugs and the release of the two drugs in the tumor stem-like cells step by step enhances the synergistic killing efficiency of the two drugs on the tumor stem-like cells.
7. Pharmacokinetic investigation of polymeric micelles loaded with combination drugs
The polymer micelle loaded with the combined drug (ATRA/CPT-HA) and the tail vein of the free combined drug (ATRA+CPT) are injected into the body of an experimental rat, blood is taken at different time points respectively, plasma is taken by centrifugation, and the blood concentration is measured after extraction by an organic solvent. The results are shown in figure 14, where the polymeric micelles significantly extend the plasma half-life of ATRA and CPT and maintain the two dose ratio over time, as compared to the free drug mixture.
9. Evaluation of in vivo antitumor Activity of Polymer micelles loaded with Combined drug
MCF-7, HCC70 and SUM-159PT tumor stem-like cells were resuspended in a mixed solution of PBS and Matrigel (1:1) at 1X 10 6 Number of individuals/individuals were inoculated into mammary fat pads of mice (NOD/SCID, female, 6 weeks old, 20 g) to construct a mouse model enriched for tumor stem-like cells. Tumor-bearing mice were grouped and administered with physiological Saline (Saline), polymer micelles of single-carrier differentiation inducer and chemotherapeutic drug (ATRA/HA and CPT-HA), polymer micelles of physical co-carrier combination drug (ATRA/CPT/HA), and polymer micelles of carrier combination drug (ATRA/CPT-HA), respectively, once every two days, four times. And measuring the long diameter and the short diameter of the tumor, calculating the tumor volume, and monitoring the change of the tumor volume. As shown in fig. 15, compared with the control polymer micelle, the anti-tumor effect of the composite drug micelle with timely gradual drug release function is obviously enhanced, and the fact that the micelle with gradual drug release characteristic releases the differentiation inducer first, reduces the resistance of tumor stem-like cells to chemotherapeutic drugs and releases the chemotherapeutic drugs, and the effect of the two drugs on synergistically inhibiting the growth of tumors rich in stem-like cells is obviously enhanced.
Example 4
Polymer hydrogel for tumor stem-like cell related drug resistant tumor treatment
1. Preparation of polymer hydrogel loaded with combined medicines
The polymer hydrogel loaded with the combined medicine is prepared by an assembly method. Arsenic trioxide (ASO), arsenic sulfide (ASS), bone morphogenetic protein 7 (BMP 7) and bone morphogenetic protein 2 (BMP 2) were prepared into polymer micelles loaded with the combination drug with polysaccharide high molecular prodrugs n-HA-oxa-CPT and n-CDT-tkt-DOX according to the related method in example 3. 1mL of micelle is taken to be mixed with the corresponding polysaccharide macromolecule prodrug solution, after vortex mixing is carried out, the mixture is stood at normal temperature, and polymer hydrogels ASO/CPT-HA, ASS/DOX-CDT, BMP7/CPT-HA and BMP2/DOX-CDT of the load combination drugs of the load differentiation inducer can be formed, and each group of hydrogels HAs good and stable properties.
2. Drug release
1mL of the drug-loaded polymer hydrogels (ASO/CPT-HA, ASS/DOX-CDT, BMP7/CPT-HA, and BMP 2/DOX-CDT) were placed in a flask and 60mL of dialysis medium (PBS containing 2% Tween 80) was added. NADPH with a final concentration of 100 mu M and 10mg/mL of liver microsomes were added, nitrogen was introduced to replace air, the microenvironment of low oxygen was simulated, and after 48 hours, hydrogen peroxide with a final concentration of 100 mu M was added to the system to simulate the environment of high active oxygen level for 48 hours. 200 mu L of dialysis medium is taken at different time points under stirring, the drug concentration is measured by adopting a high performance liquid chromatography, the drug release amount is calculated, and a drug release curve is drawn. As a result, as shown in fig. 16, the release rate of the differentiation inducer physically entrapped in the polymer hydrogel loaded with the combination drug was significantly higher than that of the chemically coupled chemotherapeutic drug in the hypoxic environment. In the subsequent hydrogen peroxide environment, the hydrogel can release the chemotherapeutic drugs relatively quickly in response. The release of the two drugs is associated with two different stimuli, respectively, exhibiting a distinct progressive release profile. The polymer hydrogel can respond to the low-oxygen microenvironment to quickly release the differentiation inducer and quickly release the chemotherapeutic drugs in the active oxygen microenvironment, so that the differential (gradual) release of the loaded combined drug differentiation inducer and the chemotherapeutic drugs is realized.
3. Evaluation of in vivo antitumor Activity of Polymer hydrogels loaded with combination drugs
Tumor volume changes were monitored by grouping MCF-7 tumor stem-like cell mice, injecting normal Saline (Saline), free drug combinations (ASO+CPT and ASS+DOX), polymer hydrogels of physical co-loaded drug combinations (ASO/CPT/HA and ASS/DOX/CDT), and polymer hydrogels loaded with drug combinations (ASO/CPT-HA and ASS/DOX-CDT) in situ into the tumor, respectively, once. As shown in figure 17, the anti-tumor effect of the combined medicine hydrogel with the timely gradual release function is stronger than that of the physical co-carried combined medicine hydrogel without the gradual release characteristic, and the results prove that the hydrogel with the gradual release characteristic releases the differentiation inducer first to reduce the resistance of the tumor stem-like cells to the chemotherapeutic medicine and then releases the chemotherapeutic medicine under the condition of maintaining the medicine synergistic ratio, so that the effect of the two medicines on the synergistic inhibition of the growth of the tumor rich in the stem-like cells can be obviously enhanced.

Claims (8)

1. A polysaccharide polymer prodrug, characterized in that: the preparation method comprises a high-molecular polysaccharide polymer, wherein a chemotherapeutic drug and a hypoxia-responsive hydrophobic group are modified on the high-molecular polysaccharide polymer, and the chemotherapeutic drug and the hypoxia-responsive hydrophobic group are respectively connected with the high-molecular polysaccharide polymer through an amide bond or an ester bond;
The high molecular polysaccharide polymer is selected from hyaluronic acid, heparin or carboxyl glucan;
the chemotherapy medicine is selected from camptothecine, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, vinblastine, cytarabine, methotrexate, doxorubicin, epirubicin, mitoxantrone, paclitaxel, docetaxel, nimustine and etoposide;
the hypoxia-responsive hydrophobic group is a 2-nitroimidazole group or an azo phenyl group.
2. The polysaccharide polymer 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 amide 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 oxalic ester or ketal.
3. A polymeric micelle for use in the treatment of a drug resistant tumor, characterized by: formed by self-assembly of the polysaccharide polymer prodrug of claim 1 in water;
the polymer micelle is coated with a chemosensitizer, wherein the chemosensitizer is selected from the group consisting of goropamide, quinidine, verapamil, chlorpromazine, cyclosporine A, reserpine, digoxin, amiodarone, lignan, tamoxifen, felodipine, nifedipine, erythromycin, diltiazem, curcumin, ginsenoside, fumagillin C, YM-155, LLP-3, ABT-737 and BM-1074.
4. A polymer hydrogel for use in the treatment of drug resistant tumors, characterized by: formed by self-assembly of the polysaccharide macromolecule prodrug of claim 1 and the polymeric micelle of claim 3 in water.
5. A polymeric micelle for treating a tumor, characterized by: formed by self-assembly of the polysaccharide polymer prodrug of claim 2 in water;
the polymer micelle is coated with a differentiation inducer, wherein 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.
6. The polymeric micelle of claim 5 in which: the tumor is common tumor or tumor stem cell related drug resistant tumor.
7. A polymer hydrogel for treating a tumor, characterized in that: formed by self-assembly of the polysaccharide macromolecule prodrug of claim 2 and the polymeric micelle of claim 5 in water.
8. The polymer hydrogel of claim 7, wherein: the tumor is common tumor or tumor stem cell related drug resistant tumor.
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