Core-shell structure drug-loaded magnetic-thermal hydrogel microsphere for treating drug-resistant ovarian cancer and preparation method thereof

Abstract

The invention relates to a core-shell structure drug-loaded magnetic-thermal hydrogel microsphere for treating drug-resistant ovarian cancer and a preparation method thereof, belonging to the field of biological and medical micro-nano materials and technologies, and the core-shell structure drug-loaded magnetic-thermal hydrogel microsphere is formed by combining an inner core and a shell layer through covalent crosslinking and electrostatic interaction. An anti-tumor drug cis-platinum and magnetic nanoparticles are loaded on an inner core, and an AZ191 inhibitor is loaded on a chitosan shell, so that preferential release of AZ191 is realized to inhibit a drug-resistant signal channel and reduce the drug resistance of cancer cells, and the cis-platinum is slowly released after the action is completed to kill tumor cells. And the magnetic nanoparticles in the inner core can be heated under an external magnetic field, so that multifunctional synergistic treatment of magnetic hyperthermia, drug resistance inhibition and chemotherapy is realized. Chitosan and sodium alginate are biodegradable materials, so that the utilization rate of the embedded medicine can be increased, and meanwhile, the medicine can be metabolized out of a body. The preparation method does not relate to toxic cross-linking agents and organic solvents, and the prepared hydrogel core-shell microspheres have no biotoxicity. Complicated operation steps are not needed, the technological process is simple, the preparation period is short, and the application range is wide.

Description

Technical Field

[0001] The invention belongs to the field of biological and medical micro-nano materials and technology, and relates to a core-shell structure drug-loaded magnetic thermal water gel microsphere for treating drug-resistant ovarian cancer. Background Art

[0002] Ovarian cancer is a common gynecological malignancy with a poor prognosis. Its high mortality rate is mainly attributed to the hidden nature of the disease and the fact that patients are usually in the advanced stage when diagnosed. In addition, due to the heterogeneity and complexity of ovarian cancer, many patients will gradually develop drug resistance during chemotherapy, which greatly reduces the treatment effect and increases the risk of recurrence. Currently, cisplatin is the main chemotherapy drug for the treatment of ovarian cancer. Its mechanism of action is to induce cross-linking of cancer cell DNA chains, interfere with DNA replication and transcription, and thus lead to cell apoptosis. However, drug-resistant cancer cells reduce the efficacy of cisplatin through a variety of mechanisms, such as activating DNA damage repair pathways, overexpressing drug efflux pumps, and activating specific signaling pathways, thereby enabling cancer cells to acquire tolerance to chemotherapy drugs.

[0003] DYRK1B is a cell cycle regulatory protein that is associated with a variety of physiological and pathological processes such as cell differentiation, metabolic regulation, apoptosis and cancer. It can help cancer cells escape immune surveillance and chemotherapy by regulating anti-apoptotic pathways and inhibiting cell cycle progression. DYRK1B is overexpressed in cancers such as ovarian cancer, thereby promoting tumor cell survival, migration and the formation of drug resistance, and its specific inhibitor AZ191 has been shown to significantly increase the sensitivity of tumor cells to chemotherapeutic drugs by inhibiting DYRK1B activity. Therefore, the combination of AZ191 and cisplatin can significantly improve the efficacy of anti-tumor drugs. The key is to rationally design a drug release system to achieve the sequential release of AZ191 and cisplatin, which can not only effectively overcome tumor resistance, but also improve the effect of anti-tumor treatment.

[0004] The present invention proposes a strategy for treating drug-resistant ovarian cancer using core-shell structured drug-loaded hydrogel microspheres. By loading the AZ191 inhibitor in the shell layer and cisplatin in the core layer, combined with the diffusion mechanism and degradability of the hydrogel material, the rapid release of AZ191 can be achieved to inhibit the drug resistance signaling pathway of cancer cells, thereby reducing their drug resistance. Subsequently, cisplatin continues to act on tumor cells through the slow release of the core layer, further achieving efficient killing. In addition, the self-controlled temperature magnetic nanoparticles doped in the core layer (invention patent "A method for preparing high-heat-generating self-controlled temperature magnetic nanoparticles" (publication number CN108373174B)) are heated to 42-45°C under the action of an alternating magnetic field through hysteresis loss and eddy current effect, giving the microspheres a magnetic hyperthermia function, effectively killing tumor cells. The multifunctional core-shell structured drug-loaded magnetic thermal water gel microspheres combine the multiple synergistic effects of hyperthermia, drug resistance inhibition and chemotherapy, providing an efficient comprehensive treatment plan for the treatment of drug-resistant ovarian cancer.

[0005] The existing core-shell structure drug carriers are not suitable for the requirements of the present invention, and their main disadvantages are as follows:

[0006] (1) The core and shell of the core-shell structure detach due to insufficient binding force, especially in complex biological environments, resulting in reduced functionality.

[0007] (2) The preparation process of core-shell hydrogel microspheres is complicated, including emulsification, multiple cross-linking, drying and other processes, which not only increases the time and economic cost, but also may lead to uneven structure or fluctuations in drug loading; at the same time, the chemical cross-linking agents (such as aldehydes) and organic solvents introduced during the preparation and cleaning process will bring certain biological toxicity.

[0008] (3) Due to the weak mechanical properties of matrix materials such as sodium alginate and chitosan, core-shell hydrogel microspheres may experience structural rupture or loss of function when subjected to external forces (such as extrusion and shear). Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention proposes a core-shell structure drug-loaded hydrogel microsphere for treating drug-resistant ovarian cancer and a preparation method thereof, which has the following advantages:

[0010] (1) Precise design and functional realization of the core-shell;

[0011] The core prepared by the spray method is formed by cross-linking sodium alginate, and the anti-tumor drug cisplatin and self-controlling magnetic nanoparticles are loaded in the core. Subsequently, the chitosan layer is combined with the core through covalent cross-linking and electrostatic interaction. The shell is rich in AZ191 inhibitors, which can be released before cisplatin, inhibit the resistance-related DYRK1B signaling pathway, and increase the sensitivity of tumor cells to cisplatin, thereby achieving the combined and sequential release of drugs. At the same time, the self-controlling magnetic nanoparticles doped in the core layer can be heated to 42-45°C under the action of an alternating magnetic field, killing tumor cells, thereby giving the microspheres a multifunctional synergistic killing effect of ovarian cancer cells with heat-inhibition of resistance-chemotherapy.

[0012] (2) Multiple cross-linking mechanisms enhance stability;

[0013] The core is initially bonded to the shell through the electrostatic interaction between the carboxyl and amino groups; subsequently, the chitosan shell further forms covalent bonds through the crosslinking agent, which increases the crosslinking density and stability of the core-shell structure. This design effectively overcomes the problem of delamination and shelling that may occur in traditional core-shell hydrogels.

[0014] (3) Green preparation process;

[0015] The entire preparation process does not involve toxic cross-linking agents and organic solvents, which avoids the leakage of fat-soluble drugs and ensures the biocompatibility of the microspheres.

[0016] (4) Mechanical properties can be controlled;

[0017] When preparing the shell layer, compounding a small amount of polyvinyl alcohol gel in a certain proportion can effectively improve the mechanical properties of the hydrogel microspheres, thereby achieving the regulation of the mechanical properties of the microspheres during the preparation process.

[0018] In order to achieve the above technical purpose, the technical solution adopted by the present invention is:

[0019] A core-shell structure drug-loaded magnetic hydrogel microsphere for treating drug-resistant ovarian cancer cells. The core-shell structure drug-loaded magnetic hydrogel microsphere is a core-shell structure, comprising a core and a shell, and the two parts are combined by covalent crosslinking and electrostatic action.

[0020] The inner core is a chitosan sodium alginate inner core: formed by sodium alginate hydrogel through ion crosslinking, the inner core is loaded with anti-tumor drug cisplatin and self-controlling temperature magnetic nanoparticles, cisplatin is used for chemotherapy to kill tumor cells, and the self-controlling temperature magnetic nanoparticles can be heated to 42-45°C in an alternating magnetic field through hysteresis loss and eddy current effect to achieve magnetic hyperthermia function, that is, the magnetic nanoparticles in the inner core can be heated under an external magnetic field to achieve magnetic hyperthermia-resistance inhibition-chemotherapy multifunctional synergistic treatment;

[0021] The shell layer is a chitosan shell: the shell layer is loaded with the DYRK1B inhibitor AZ191, and AZ191 can be quickly released in the hydrogel shell layer to inhibit the drug resistance-related DYRK1B signaling pathway and enhance the sensitivity of tumor cells to cisplatin, thereby achieving staged release of the drug and combined treatment.

[0022] The combination between the core and the shell is as follows:

[0023] The electrostatic effect is: electrostatic adsorption between the negatively charged carboxyl groups in sodium alginate and the positively charged amino groups in chitosan;

[0024] The covalent cross-linking: through the cross-linking reaction of genipin, a covalent bond is formed between the surface of the sodium alginate core and the chitosan molecular chain, thereby enhancing the bonding strength between the core and the shell layer and avoiding the shelling phenomenon.

[0025] A method for preparing core-shell structure drug-loaded magnetic hydrogel microspheres for treating drug-resistant ovarian cancer cells, wherein the preparation method firstly prepares a sodium alginate core hydrogel coated with anti-tumor drug cisplatin and self-controlling temperature magnetic nanoparticles by spraying; then, the core hydrogel is reacted with the chitosan shell containing the AZ191 inhibitor by covalent crosslinking and electrostatic action to form core-shell structure drug-loaded magnetic hydrogel microspheres. Specifically, the method comprises the following steps:

[0026] In the first step, sodium alginate powder is dissolved in deionized water to obtain a sodium alginate aqueous solution; cisplatin and magnetic nanoparticles are evenly mixed with the sodium alginate solution to obtain a solution for preparing the inner core.

[0027] Furthermore, in the first step, the concentration of the sodium alginate aqueous solution is 0.5-2% (w / v), and 50-100 mg of cisplatin and 100-200 mg of magnetic nanoparticles are added to every 20-40 ml of the sodium alginate solution.

[0028] The second step is to prepare sodium alginate core hydrogel microspheres, specifically:

[0029] 2.1) The core solution is passed through an air flow atomizer nozzle to prepare atomized droplets, and a calcium chloride solution is placed outside the air flow atomizer nozzle to crosslink the atomized droplets. After the droplets are crosslinked in calcium chloride, the sodium alginate microspheres are separated using a magnet.

[0030] 2.2) The separated microspheres are immersed in anhydrous ethanol for dehydration treatment, and then the dehydrated microspheres are separated and added into a mixed solution of a cross-linking agent and ethanol for immersion and absorption to obtain sodium alginate core hydrogel microspheres.

[0031] Furthermore, in the above 2.1), a calcium chloride solution with a concentration of 5-10% (w / v) is placed 30-50 cm away from the nozzle of the airflow atomizer to crosslink the atomized droplets; the droplet crosslinking time is 30-60 min; and the dehydration treatment time is 30-60 min.

[0032] Furthermore, in the mixed solution of the cross-linking reagent and ethanol in 2.2), the concentration of the cross-linking reagent is 1-6% (w / v), the concentration of ethanol is 75% (v / v), and the rest is water; the cross-linking reagent is genipin.

[0033] Furthermore, in the above 2.2), after immersion and absorption for 8-16 hours, the microspheres are washed with deionized water for 2-3 times to obtain hydrogel microspheres with a sodium alginate core.

[0034] The third step is to prepare the shell solution, specifically:

[0035] 3.1) dissolving chitosan powder in acetic acid solution to obtain chitosan acetic acid solution;

[0036] 3.2) The AZ191 inhibitor was added into ethanol and dissolved to obtain an AZ191 ethanol solution.

[0037] 3.3) Adding the AZ191 ethanol solution to a saturated aqueous solution containing carboxymethyl-β-cyclodextrin, and after ultrasonic treatment, mixing it with the chitosan acetic acid solution to obtain a shell layer solution.

[0038] Furthermore, in the above 3.1), the concentration of chitosan in acetic acid solution is 2-6% (w / v).

[0039] Furthermore, in the above 3.2), 3-8 mg of AZ191 inhibitor is added to every 0.5 ml of ethanol.

[0040] Furthermore, in 3.3), 0.5 ml of AZ191 ethanol solution and 25-80 ml of chitosan acetic acid solution are added to every 1 ml of saturated aqueous solution containing carboxymethyl-β-cyclodextrin. In 3.3), the ultrasonic treatment time is 30-60 min.

[0041] The fourth step is to prepare core-shell structure drug-loaded magnetic hydrogel microspheres, specifically:

[0042] 4.1) adding the sodium alginate core hydrogel microspheres obtained in the second step into the polyvinyl alcohol aqueous solution and mechanically stirring to make them dispersed evenly;

[0043] 4.2) Add the shell solution prepared in the third step to the uniformly dispersed solution in 4.1). After stirring and reacting for 4 hours, wash with deionized water, freeze-dry and store to obtain core-shell structure drug-loaded magnetic hydrothermal water gel microspheres.

[0044] Furthermore, in the above 4.1), the concentration of polyvinyl alcohol in the polyvinyl alcohol aqueous solution is 3-8% (w / v); and 50-100 mg of sodium alginate core hydrogel microspheres are added to every 5-10 ml of polyvinyl alcohol aqueous solution.

[0045] Furthermore, in the above 4.2), the volume of the added shell solution is 25-80 ml.

[0046] A use of a core-shell structure drug-loaded magnetic thermothermal gel microsphere, the microsphere is used for the treatment of drug-resistant ovarian cancer, through a triple synergistic treatment method of magnetic hyperthermia-inhibition of drug resistance-chemotherapy, during the treatment process:

[0047] Rapid release of AZ191 can inhibit drug resistance in cancer cells.

[0048] The slow release of cisplatin can continuously kill cancer cells.

[0049] The magnetothermal effect of the self-controlled temperature magnetic nanoparticles raises the temperature to 42-45°C in an alternating magnetic field, enhancing the chemotherapy effect and accelerating the death of tumor cells.

[0050] In order to construct a drug delivery system with loading and sequential release of AZ191 inhibitor and cisplatin and magnetic thermal function, which is used to kill highly resistant ovarian cancer tumor cells, the present invention designs a core-shell structure hydrogel drug-loaded magnetic thermal microsphere. First, the sodium alginate core loaded with cisplatin and self-controlled temperature magnetic nanoparticles is prepared by air flow atomization technology, and further coated with a chitosan shell loaded with AZ191 inhibitor, so as to achieve a precise core-shell structure and magnetic thermal-resistance inhibition-chemotherapy synergistic treatment function.

[0051] In the process of coating the shell, in order to enhance the bonding strength between the core and the shell, the following technical measures were taken:

[0052] (1) Pre-dehydration treatment: Before coating the chitosan layer, the sodium alginate core is dehydrated with anhydrous ethanol. This process improves the core's absorption capacity for the chitosan crosslinker, thereby significantly increasing the crosslinking density between the core and the shell.

[0053] (2) Multiple cross-linking mechanisms:

[0054] Electrostatic interaction: The core of sodium alginate is rich in negatively charged carboxyl groups, while the shell of chitosan is rich in positively charged amino groups. The two are tightly combined through electrostatic adsorption, providing initial stability.

[0055] Chemical cross-linking: The dehydrated inner core can absorb more chitosan cross-linking agent and achieve chemical cross-linking with the chitosan outer shell through covalent bonds, further enhancing the mechanical strength and stability of the microspheres.

[0056] (3) Adjustable mechanical properties:

[0057] Adding a certain amount of polyvinyl alcohol solution to the shell solution can effectively improve the mechanical properties of the microspheres. This is because the physically cross-linked polyvinyl alcohol molecular chains can form microcrystalline regions, produce a large number of hydrogen bonds and crystallization regions, and significantly improve the mechanical strength of the microspheres.

[0058] This core-shell structure design not only ensures the layered loading and sequential release of the two drugs, but also optimizes the physical and chemical properties of the hydrogel, providing an effective solution for the precise treatment of highly drug-resistant tumors.

[0059] The beneficial effects of the present invention are:

[0060] (1) The core-shell structured drug-loaded magnetic hot water gel microspheres prepared by the present invention have the function of sequentially releasing the core-shell double-layer loaded drugs and simultaneously killing tumor cells by magnetic hyperthermia. The shell layer preferentially releases the AZ191 inhibitor, first reducing the drug resistance of ovarian cancer cells; secondly, it releases the anti-tumor drug cisplatin in the inner core layer, which efficiently kills cancer cells. The microspheres heated to the temperature of magnetic hyperthermia can cooperate with chemotherapy to kill tumor cells and enhance the treatment effect.

[0061] (2) The whole process of the preparation method of the present invention does not involve toxic crosslinking agents and organic solvents, and the prepared hydrogel core-shell microspheres are non-biotoxic and degradable. Moreover, the method can be completed in one step without complicated operation steps, and the process is simple, the preparation cycle is short, and the application range is wide. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of drug release and degradation of core-shell structure drug-loaded hydrogel;

[0063] Figure 2 The figure is a flow chart for the preparation of core-shell structure drug-loaded hydrogel;

[0064] Figure 3 Optical microscope images of core-shell microspheres and inner-shell microspheres under bright field, and fluorescence images under excitation wavelengths of 488 nm and 543 nm, respectively;

[0065] Figure 4 The mass spectra of the standard sample of AZ191 inhibitor and the mass spectra of the drug release medium after the core-shell microspheres released AZ191; Figure 4 (a) is the mass spectrum of the standard sample of AZ191 inhibitor. Figure 4 (b) is the mass spectrum of the medium after drug release of the core-shell structure. DETAILED DESCRIPTION

[0066] The present invention is further described below in conjunction with specific embodiments.

[0067] Example 1

[0068] In the first step, sodium alginate powder is dissolved in deionized water to obtain a sodium alginate aqueous solution with a concentration of 0.5% (w / v); 50 mg of cisplatin, 100 mg of magnetic nanoparticles and 20 ml of the sodium alginate solution are evenly mixed to obtain a solution for preparing the core.

[0069] The second step is to prepare sodium alginate core hydrogel microspheres, specifically:

[0070] 2.1) The core solution was passed through an air flow atomizer nozzle to prepare atomized droplets, and a 5% (w / v) calcium chloride solution was placed 30 cm away from the air flow atomizer nozzle to crosslink the atomized droplets. After the droplets were crosslinked in calcium chloride for 30 minutes, the sodium alginate microspheres were separated using a magnet.

[0071] 2.2) The separated microspheres were immersed in anhydrous ethanol for dehydration for 60 min, the dehydrated microspheres were separated, added into 10 ml of 1% (w / v) genipin ethanol solution for immersion absorption for 8 h, and then washed with deionized water for 3 times to obtain sodium alginate core hydrogel microspheres.

[0072] The third step is to prepare the shell solution, specifically:

[0073] 3.1) dissolving chitosan powder in acetic acid solution to obtain a chitosan acetic acid solution with a concentration of 2% (w / v);

[0074] 3.2) 3 mg of AZ191 inhibitor was dissolved in 0.5 ml of ethanol to obtain AZ191 ethanol solution.

[0075] 3.3) The AZ191 ethanol solution was added to 1 ml of a saturated aqueous solution containing carboxymethyl-β-cyclodextrin, and after ultrasonic treatment for 30 min, it was mixed evenly with 25 ml of chitosan acetic acid solution to obtain a shell layer solution.

[0076] The fourth step is to prepare core-shell structure drug-loaded magnetic hydrogel microspheres, specifically:

[0077] 4.1) Add 50 mg of sodium alginate core hydrogel microspheres obtained in the second step to 5 ml of 3%

[0078] (w / v) aqueous solution of polyvinyl alcohol, and mechanically stirred to make it dispersed evenly;

[0079] 4.2) Add 25 ml of the shell solution prepared in step 3 to the uniformly dispersed solution in step 4.1). After stirring and reacting for 4 hours, wash with deionized water, freeze-dry and store to obtain core-shell structure drug-loaded magnetic hydrothermal water gel microspheres. The preparation flow chart is as follows: Figure 2 shown.

[0080] The prepared core-shell microspheres were imaged by optical microscope under bright field and fluorescence imaging under different excitation lights (488, 543 nm). Figure 3 As shown. It can be seen that compared with the inner core microspheres, the outer layer of the core-shell structure microspheres shows obvious fluorescence. This is because the cross-linked product of chitosan and genipin can produce fluorescence under multi-band excitation light, indicating that the core-shell structure successfully covers the inner core with a shell layer.

[0081] Example 2

[0082] In the first step, sodium alginate powder is dissolved in deionized water to obtain a sodium alginate aqueous solution with a concentration of 1% (w / v); 75 mg of cisplatin, 150 mg of magnetic nanoparticles and 30 ml of the sodium alginate solution are evenly mixed to obtain a solution for preparing the core.

[0083] The second step is to prepare sodium alginate core hydrogel microspheres, specifically:

[0084] 2.1) The core solution was passed through an air flow atomizer nozzle to prepare atomized droplets, and a 7.5% (w / v) calcium chloride solution was placed 40 cm away from the air flow atomizer nozzle to crosslink the atomized droplets. After the droplets were crosslinked in calcium chloride for 40 minutes, the sodium alginate microspheres were separated using a magnet.

[0085] 2.2) The separated microspheres were immersed in anhydrous ethanol for dehydration for 60 minutes, the dehydrated microspheres were separated, added into 10 ml of 4% (w / v) genipin ethanol solution for immersion absorption for 12 hours, and then washed with deionized water for 3 times to obtain sodium alginate core hydrogel microspheres.

[0086] The third step is to prepare the shell solution, specifically:

[0087] 3.1) dissolving chitosan powder in acetic acid solution to obtain a chitosan acetic acid solution with a concentration of 3% (w / v);

[0088] 3.2) 5 mg of AZ191 inhibitor was dissolved in 0.5 ml of ethanol to obtain AZ191 ethanol solution.

[0089] 3.3) The AZ191 ethanol solution was added to 1 ml of a saturated aqueous solution containing carboxymethyl-β-cyclodextrin, and after ultrasonic treatment for 40 min, it was mixed evenly with 50 ml of chitosan acetic acid solution to obtain a shell layer solution.

[0090] The fourth step is to prepare core-shell structure drug-loaded magnetic hydrogel microspheres, specifically:

[0091] 4.1) Add 75 mg of sodium alginate core hydrogel microspheres obtained in the second step to 7 ml of 5%

[0092] (w / v) aqueous solution of polyvinyl alcohol, and mechanically stirred to make it dispersed evenly;

[0093] 4.2) Add 50 ml of the shell solution prepared in step 3 to the uniformly dispersed solution in step 4.1). After stirring and reacting for 4 hours, wash with deionized water, freeze-dry and store to obtain core-shell structure drug-loaded magnetic hydrothermal water gel microspheres. The preparation flow chart is as follows: Figure 2 shown.

[0094] Sequential drug release and degradation behavior of core-shell microspheres Figure 1 As shown. It can first release the AZ191 inhibitor in the shell, and then release the cisplatin in the core, effectively killing tumor cells. Under the action of an external alternating magnetic field, the self-controlled temperature magnetic nanoparticles coated in the core can be heated to 42-45°C, achieving hyperthermia killing of tumors, thereby achieving multiple therapeutic effects of hyperthermia-inhibition of drug resistance-chemotherapy. The core-shell gel material of the microsphere is degradable, and can be degraded and metabolized out of the body after it has played its role.

[0095] Example 3

[0096] In the first step, sodium alginate powder is dissolved in deionized water to obtain a sodium alginate aqueous solution with a concentration of 2% (w / v); 100 mg of cisplatin, 200 mg of magnetic nanoparticles and 40 ml of the sodium alginate solution are evenly mixed to obtain a solution for preparing the core.

[0097] The second step is to prepare sodium alginate core hydrogel microspheres, specifically:

[0098] 2.1) The core solution was passed through an air flow atomizer nozzle to prepare atomized droplets, and a 10% (w / v) calcium chloride solution was placed 50 cm away from the air flow atomizer nozzle to crosslink the atomized droplets. After the droplets were crosslinked in calcium chloride for 60 minutes, the sodium alginate microspheres were separated using a magnet.

[0099] 2.2) The separated microspheres were immersed in anhydrous ethanol for dehydration for 60 minutes, the dehydrated microspheres were separated, added into 10 ml of 6% (w / v) genipin ethanol solution for immersion absorption for 16 hours, and then washed with deionized water for 3 times to obtain sodium alginate core hydrogel microspheres.

[0100] The third step is to prepare the shell solution, specifically:

[0101] 3.1) dissolving chitosan powder in acetic acid solution to obtain a chitosan acetic acid solution with a concentration of 2% (w / v);

[0102] 3.2) 8 mg of AZ191 inhibitor was dissolved in 0.5 ml of ethanol to obtain an AZ191 ethanol solution.

[0103] 3.3) The AZ191 ethanol solution was added to 1 ml of a saturated aqueous solution containing carboxymethyl-β-cyclodextrin, and after ultrasonic treatment for 60 min, it was mixed evenly with 80 ml of chitosan acetic acid solution to obtain a shell layer solution.

[0104] The fourth step is to prepare core-shell structure drug-loaded magnetic hydrogel microspheres, specifically:

[0105] 4.1) Add 50 mg of sodium alginate core hydrogel microspheres obtained in the second step to 10 ml of 8%

[0106] (w / v) aqueous solution of polyvinyl alcohol, and mechanically stirred to make it dispersed evenly;

[0107] 4.2) Add 80 ml of the shell solution prepared in the third step to the uniformly dispersed solution in 4.1). After stirring and reacting for 4 hours, wash with deionized water, freeze-dry and store to obtain core-shell structure drug-loaded magnetic hydrothermal water gel microspheres. The preparation flow chart is as follows: Figure 2 shown.

[0108] The standard sample of AZ191 inhibitor and the medium after drug release of core-shell microspheres were analyzed by mass spectrometry. Figure 4 shown. Figure 4 a is the mass spectrum of the standard sample of AZ191 inhibitor, which shows an obvious molecular ion peak at m / z 430. Figure 4 b is the mass spectrum of the medium after drug release from the core-shell structure, which also shows a strong ion peak at m / z 430, indicating that the AZ191 inhibitor was successfully loaded and released in the core-shell structure.

[0109] The above-described embodiments merely express the implementation methods of the present invention, but they should not be understood as limiting the scope of the present invention. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A core-shell structure drug-loaded magnetic thermal water gel microsphere for treating drug-resistant ovarian cancer, characterized in that: The core-shell structure drug-loaded magnetic hydrogel microsphere is a core-shell structure, comprising a core and a shell, and the two parts are combined by covalent cross-linking and electrostatic action.

2. The core-shell structure drug-loaded magnetic thermal water gel microspheres for treating drug-resistant ovarian cancer according to claim 1, characterized in that: The core is a chitosan sodium alginate core: formed by sodium alginate hydrogel through ion cross-linking, and the core is loaded with anti-tumor drug cisplatin and self-temperature-controlled magnetic nanoparticles; The shell layer is a chitosan shell: the DYRK1B inhibitor AZ191 is loaded in the shell layer.

3. A method for preparing the core-shell structure drug-loaded magnetic hydrogel microspheres for treating drug-resistant ovarian cancer cells according to claim 1 or 2, characterized in that: The preparation method firstly prepares a sodium alginate core hydrogel coated with the anti-tumor drug cisplatin and self-controlling temperature magnetic nanoparticles by a spray method; then, the core hydrogel reacts with the chitosan shell containing the AZ191 inhibitor on the outside through covalent crosslinking and electrostatic action to form a core-shell structure drug-loaded magnetic hydrothermal water gel microsphere.

4. The method for preparing a core-shell structure drug-loaded magnetic hydrogel microsphere according to claim 3, characterized in that: The preparation method specifically comprises the following steps: The first step is to dissolve sodium alginate powder in deionized water to obtain a sodium alginate aqueous solution; cisplatin and magnetic nanoparticles are mixed evenly with the sodium alginate solution to obtain a solution for preparing the core; The second step is to prepare sodium alginate core hydrogel microspheres, specifically: 2.1) The core solution is passed through an air flow atomizer nozzle to prepare atomized droplets, and a calcium chloride solution is placed outside the air flow atomizer nozzle to cross-link the atomized droplets; after the droplets are cross-linked in the calcium chloride, the sodium alginate microspheres are separated using a magnet; 2.2) soaking the separated microspheres in anhydrous ethanol for dehydration, separating the dehydrated microspheres, and adding them into a mixed solution of a cross-linking agent and ethanol for soaking and absorption to obtain sodium alginate core hydrogel microspheres; The third step is to prepare the shell solution, specifically: 3.1) dissolving chitosan powder in acetic acid solution to obtain chitosan acetic acid solution; 3.2) adding the AZ191 inhibitor to ethanol and dissolving it to obtain an AZ191 ethanol solution; 3.3) adding the AZ191 ethanol solution to a saturated aqueous solution containing carboxymethyl-β-cyclodextrin, and mixing it with the chitosan acetic acid solution after ultrasonic treatment to obtain a shell layer solution; The fourth step is to prepare core-shell structure drug-loaded magnetic hydrogel microspheres, specifically: 4.1) adding the sodium alginate core hydrogel microspheres obtained in the second step into the polyvinyl alcohol aqueous solution and mechanically stirring to make them dispersed evenly; 4.2) adding the shell solution prepared in the third step to the uniformly dispersed solution in 4.1); stirring and reacting for 4 hours, washing with deionized water, freeze-drying and storing, to obtain core-shell structured drug-loaded magnetic thermal water gel microspheres.

5. The method for preparing a core-shell structure drug-loaded magnetic hydrogel microsphere according to claim 4, characterized in that: In the first step, the concentration of the sodium alginate aqueous solution is 0.5-2% (w / v), and 50-100 mg of cisplatin and 100-200 mg of magnetic nanoparticles are added to every 20-40 ml of the sodium alginate solution.

6. The method for preparing a core-shell structure drug-loaded magnetic hydrogel microsphere according to claim 4, characterized in that: In the second step: In the above 2.1), a calcium chloride solution with a concentration of 5-10% (w / v) is placed at a distance of 30-50 cm from the nozzle of the airflow atomizer to crosslink the atomized droplets; the droplet crosslinking time is 30-60 min; the dehydration treatment time is 30-60 min; In the above 2.2), in the mixed solution of the cross-linking reagent and ethanol: the concentration of the cross-linking reagent is 1-6% (w / v), the concentration of ethanol is 75% (v / v), and the rest is water; the cross-linking reagent is genipin; and the immersion absorption time is 8-16h.

7. The method for preparing a core-shell structure drug-loaded magnetic hydrogel microsphere according to claim 4, characterized in that: In the third step: In the above 3.1), the concentration of chitosan acetic acid solution is 2-6% (w / v); In the above 3.2), 3-8 mg of AZ191 inhibitor is added to every 0.5 ml of ethanol; In the above 3.3), 0.5 ml of AZ191 ethanol solution and 25-80 ml of chitosan acetic acid solution are added to every 1 ml of saturated aqueous solution containing carboxymethyl-β-cyclodextrin and mixed evenly; the ultrasonic treatment time is 30-60 min.

8. The method for preparing a core-shell structure drug-loaded magnetic hydrogel microsphere according to claim 4, characterized in that: In the fourth step, the concentration of polyvinyl alcohol in the polyvinyl alcohol aqueous solution is 3-8% (w / v); 50-100 mg of sodium alginate core hydrogel microspheres and 25-80 ml of shell solution are added to every 5-10 ml of polyvinyl alcohol aqueous solution.

9. Use of the core-shell structure drug-loaded magnetic hot water gel microspheres according to any one of claims 1-2, characterized in that: The microspheres are used for the treatment of drug-resistant ovarian cancer through a triple synergistic treatment method of magnetic hyperthermia-suppression of drug resistance-chemotherapy.

10. The use of the core-shell structure drug-loaded magnetic hot water gel microspheres according to any one of claims 1 to 2 according to claim 9, characterized in that: When applied, the multifunctional synergistic treatment of magnetic hyperthermia-inhibition of drug resistance-chemotherapy is specifically as follows: the rapid release of AZ191 can inhibit the drug resistance of cancer cells; The slow release of cisplatin can continuously kill cancer cells; The magnetocaloric effect of the self-temperature-controlled magnetic nanoparticles raises the temperature to 42-45°C in an alternating magnetic field, accelerating the death of tumor cells.

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