Hydrogel microspheres for treating drug-resistant ovarian cancer and a method for preparing the same

Abstract

The application discloses a hydrogel microsphere for treating drug-resistant ovarian cancer and a preparation method thereof. The microsphere is composed of a chitosan-modified 3,5-dihydroxybenzyl acetate derivative and a lycorine N-demethylation derivative, a chemotherapeutic drug (paclitaxel / cisplatin), hyaluronic acid, glycerol, glutaraldehyde and a copper death mixture (dithiofrane-copper-carrier and quercetin nanocrystals). The microsphere is formed through a microfluidic technology and has the characteristics of multi-mechanism synergy (inhibition of drug-resistant genes + induction of copper death + chemotherapy), efficient targeting (chitosan-enhanced biocompatibility) and controllable slow release (hyaluronic acid-glutaraldehyde crosslinking). In vitro / in vivo experiments show that the tumor inhibition rate of the microsphere reaches 75% at the highest, the safety is good, the process is mature, and the microsphere is suitable for large-scale production, thereby providing an innovative solution for drug-resistant ovarian cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a hydrogel microsphere for treating drug-resistant ovarian cancer and a preparation method thereof. BACKGROUND

[0002] Ovarian cancer is one of the most deadly malignant tumors in the field of gynecology, which seriously threatens the life and health of women. Because of its insidious onset, more than 70% of patients are in the advanced stage when diagnosed, at which time the tumor cells have often spread throughout the abdominal cavity, greatly increasing the difficulty of treatment. In recent years, although medical technology has been continuously improving, the 5-year survival rate of ovarian cancer patients is still not optimistic. One of the key factors is the development of drug resistance, especially in patients with recurrence, the problem of drug resistance is increasingly prominent, which greatly reduces the treatment effect and seriously affects the prognosis and quality of life of patients.

[0003] At present, the treatment methods for ovarian cancer mainly include surgery, chemotherapy, radiotherapy and targeted therapy, etc. Surgery can remove tumor tissue as much as possible, but for patients with advanced extensive metastasis, it is difficult to completely remove the tumor, and the residual tumor cells after surgery are prone to recurrence. Chemotherapy plays an important role in the treatment of ovarian cancer. However, long-term use of chemotherapeutic drugs can lead to drug resistance of tumor cells, making the effect of chemotherapy gradually decrease. For example, common chemotherapeutic drugs such as paclitaxel, cisplatin and carboplatin, drug-resistant ovarian cancer cells can escape the killing effect of drugs through various mechanisms, such as overexpression of drug efflux pump, abnormal apoptosis pathway, etc., resulting in failure of chemotherapy. Radiotherapy can control local tumors to a certain extent, but it also faces the problem of tumor cell resistance. Targeted therapy has a certain specificity, but it is only effective for patients with specific gene mutations or abnormal target expression, and some patients may also develop drug resistance during treatment, so the scope of application is relatively limited.

[0004] In order to overcome the difficulties in the treatment of drug-resistant ovarian cancer, hydrogel microspheres as a new type of drug carrier have shown great application potential in the field of tumor treatment due to their good biocompatibility, adjustable drug release characteristics and the ability to achieve local drug delivery. However, the existing hydrogel microspheres for the treatment of ovarian cancer still have many shortcomings in composition and performance: for example, some hydrogel microspheres have single drug types, which are difficult to act on multiple drug resistance mechanism targets of tumor cells; the drug encapsulation efficiency and drug loading capacity of some hydrogel microspheres are low, which leads to insufficient effective concentration of drugs in the body and affects the treatment effect; the stability and degradation characteristics of some hydrogel microspheres in the body are not ideal, which cannot achieve sustained and stable release of drugs.

[0005] It is worth noting that in the research of anti-ovarian cancer drug resistance, the role of anti-mutagenic active agent is gradually valued, which can intervene in the molecular mechanism related to tumor cell drug resistance, inhibit the development of drug resistance caused by gene mutation, epigenetic change and other reasons of tumor cells, but in the existing related research, the application of anti-mutagenic active agent has limitations, such as insufficient activity, poor targeting and the like, and it is difficult to fully exert its potential of anti-drug resistance, therefore, it is of urgent practical demand and important clinical significance to develop a kind of hydrogel microspheres taking modified anti-mutagenic active agent as core, which can effectively treat drug-resistant ovarian cancer, has unique composition and excellent performance, and a preparation method thereof. SUMMARY

[0006] In view of the insufficient activity, poor targeting and the like, and the difficulty in fully exerting the anti-drug resistance, the present application provides a kind of hydrogel microspheres for treating drug-resistant ovarian cancer and a preparation method thereof, the modified anti-mutagenic active agent inhibits the expression of drug-resistant genes, the copper death mixture induces non-apoptotic cell death, and the chemotherapeutic drug directly kills, so that the three are combined to break through the single drug resistance mechanism.

[0007] In one aspect, the present application provides a kind of hydrogel microspheres for treating drug-resistant ovarian cancer, which is composed of chitosan modified modified anti-mutagenic active agent, chemotherapeutic drug, hyaluronic acid, glycerol, glutaraldehyde and copper death mixture.

[0008] Preferably, the chitosan modified modified anti-mutagenic active agent is a mixture of 3,5-dihydroxybenzyl acetate derivative and lycorine N-demethylation derivative and is modified on the surface by chitosan, and the copper death mixture is a combination of disulfiram-copper-carrier and quercetin nanocrystals in a mass ratio of 1:1.

[0009] Preferably, the chemotherapeutic drug is any one of paclitaxel and cisplatin.

[0010] Preferably, the mass ratio of the chitosan modified modified anti-mutagenic active agent, the chemotherapeutic drug, the hyaluronic acid, the glycerol, the glutaraldehyde and the copper death mixture is (6-9):(4-7):(18-22):(6-9):(1.5-2.5):(12-14).

[0011] In another aspect, the present application provides a kind of preparation method of hydrogel microspheres for treating drug-resistant ovarian cancer, which comprises the following steps:

[0012] (1) Preparation of the modified anti-mutagenic agent modified by chitosan: 3,5-dihydroxybenzyl acetate derivative and lycorine N-demethylation derivative were mixed uniformly at a mass ratio of 1:1, then chitosan was dissolved in acetic acid solution to prepare a chitosan solution with a mass fraction of 1%-3%, the mixed anti-mutagenic agent was slowly added to the chitosan solution, and the reaction was stirred at 30-40°C for 2-4h. After the reaction was completed, the chitosan-modified modified anti-mutagenic agent was obtained by centrifugation, washing, and freeze-drying (-50°C, 24h).

[0013] (2) Preparation of copper death mixture:

[0014] 1) Preparation of disulfiram-copper complex: disulfiram and CuSO4·5H2O were reacted (40°C, 2h) in an ethanol-water mixed solvent (ethanol: water = 4:1) at a molar ratio of 2:1, the precipitate was collected by centrifugation (10000rpm, 15min), and the disulfiram-copper complex was obtained after washing and drying;

[0015] 2) Disulfiram-copper-carrier: phospholipid, DOTAP and disulfiram-copper complex were dissolved in chloroform-methanol (2:1) at a mass ratio of 6:3:1 to obtain disulfiram-copper-carrier by thin film hydration method;

[0016] 3) Copper death mixture: quercetin nanocrystals and disulfiram-copper-carrier were mixed at a mass ratio of 1:1, and incubated at room temperature for 30min. The particle size change was detected by dynamic light scattering; the unabsorbed quercetin nanocrystals were removed by centrifugation (3000rpm, 5min), and the copper death mixture was collected by centrifugation.

[0017] (3) Preparation of hydrogel microspheres: hyaluronic acid was dissolved in deionized water to prepare a hyaluronic acid solution with a mass fraction of 2%-5%, glycerol and glutaraldehyde were added to the hyaluronic acid solution, and then the modified anti-mutagenic agent modified by chitosan prepared in step (1) and the copper death mixture prepared in step (2) and the chemotherapeutic drug were added and stirred uniformly to obtain a mixed solution; the mixed solution was prepared into hydrogel microspheres by microfluidic technology.

[0018] Preferably, the preparation method of the 3,5-dihydroxybenzyl acetate derivative is as follows: 3,5-dihydroxybenzyl and acetic anhydride are reacted at 60-70℃ under stirring at a speed of 200-300 rpm for 3-5 h in a molar ratio of 1:1.2-1:1.5 and in the presence of pyridine in an amount of 10-15% of the mass of 3,5-dihydroxybenzyl; after the reaction is completed, the reaction solution is poured into ice water to precipitate a solid, which is filtered, washed with deionized water for 3-5 times, and then recrystallized from ethanol, the amount of ethanol being 5-8 times the mass of the solid, the recrystallization temperature being controlled at 40-50℃, and the cooling speed being 0.5-1℃ / min, to obtain the 3,5-dihydroxybenzyl acetate derivative.

[0019] Preferably, the preparation method of the lycorine N-demethylation derivative is as follows: lycorine and boron tribromide are reacted at -10-0℃ under stirring at a speed of 250-350 rpm for 8-12 h in a molar ratio of 1:3-1:4 and in the presence of dichloromethane in an amount of 8-10 times the mass of lycorine; after the reaction is completed, the reaction is quenched by slowly adding ice water, and the pH is adjusted to 8-9 by sodium hydroxide solution, the organic phase is separated, dried with anhydrous sodium sulfate, and then purified by column chromatography, the eluent of the silica gel column being dichloromethane:methanol=10:1, to obtain the lycorine N-demethylation derivative.

[0020] Preferably, in the step (1), the centrifugal speed is 8000-10000 rpm, and the centrifugal time is 10 min-15 min; the washing is performed by using deionized water and anhydrous ethanol alternately for 3-5 times.

[0021] Preferably, in the step (2), the preparation method of the quercetin nanocrystal is as follows: quercetin and PVP (1:2 in mass ratio) are dissolved in ethanol, and then injected into ultrapure water (magnetic stirring, 500 rpm), the ethanol is evaporated, the precipitate is collected by centrifugation (8000 rpm, 10 min), and then resuspended in PBS (pH 7.4) after washing, to obtain the quercetin nanocrystal.

[0022] Preferably, in the step (3), when the microfluidic technology is used to prepare the hydrogel microspheres, the flow rate of the inner phase is 0.1 mL / min-0.3 mL / min, and the flow rate of the outer phase is 1 mL / min-3 mL / min.

[0023] The embodiments of the present application have the following beneficial effects:

[0024] (1)Multi-mechanism synergistic treatment of drug-resistant ovarian cancer: by modifying the anti-mutagenic activity agent (3,5-dihydroxy benzyl acetate derivative and lycorine N-demethylation derivative) to inhibit the expression of drug resistance genes of tumor cells, reduce drug resistance; copper death mixture (dithiol- copper-carrier and quercetin nanocrystals) induces non-apoptotic cell death (copper death) of tumor cells, breaks through the drug resistance limit of traditional apoptosis pathway; chemotherapeutic drugs (paclitaxel, cisplatin) directly kill tumor cells, realize the synergistic treatment of multiple action mechanisms, and significantly improve the curative effect.

[0025] (2)Enhance drug targeting and stability: the surface of the modified anti-mutagenic active agent is modified by chitosan, which improves the biocompatibility and targeting of the drug, and reduces the toxicity to normal cells; the copper death mixture enhances the enrichment and retention of the drug in the tumor tissue through the synergistic effect of the nanocarrier (phospholipid / DOTAP) and quercetin nanocrystals.

[0026] (3)Controllable release and long-acting effect: the hyaluronic acid matrix combined with glutaraldehyde cross-linking forms a stable three-dimensional network structure, realizing the slow release of chemotherapeutic drugs and active ingredients, and prolonging the drug action time; the microspheres prepared by microfluidic technology have uniform particle size and high drug encapsulation efficiency, ensuring the accuracy and repeatability of in vivo release.

[0027] (4)Overcome the defects of the prior art: solve the problems of single drug easy to be drug-resistant, low drug loading capacity, and unstable release, etc., significantly improve the treatment effect through compound formula and optimized process, and have potential breakthrough effect on paclitaxel, cisplatin and other drug-resistant cases.

[0028] (5)The preparation process can be scaled up: the preparation method (derivative synthesis, nanocrystal loading, microsphere forming) of each component has clear steps and optimized parameters, which is suitable for industrial production and has high clinical transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the in vitro release rate diagram of each group of test example 1 of the present application;

[0030] Figure 2 is the drug resistance related protein expression amount diagram of each group of test example 3 of the present application. DETAILED DESCRIPTION

[0031] The technical solutions of the present application will be described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0032] The reagents and equipment used in the embodiments of the present disclosure are all conventional and commercially available.

[0033] Preparation Example 1

[0034] Method for preparing 3,5-dihydroxybenzyl acetate derivative

[0035] Take 10 g of 3,5-dihydroxybenzyl with a purity of ≥99%, 13.2 g of acetic anhydride with a purity of ≥99.8%, and 1 g of pyridine as a catalyst, and add them to a round-bottom flask, connect a spherical condenser, and place it on a 60°C magnetic stirrer at 250 rpm for 4 h to perform the reaction. After the reaction is completed, pour the reaction liquid into ice water, and acetic anhydride and pyridine are soluble in water, while the polarity of the ester derivative is reduced, and the solubility in water is small. The product is precipitated by "solvent polarity mutation", and at the same time, the ice water can quench the unreacted anhydride and reduce the solubility of the product, thereby improving the precipitation efficiency. Collect 9.5 g of solid, wash it with deionized water for 3 times, and add 70 g of ethanol to the solid to recrystallize at a temperature of 45°C. The cooling speed is 0.5-1°C / min, and finally 9 g of 3,5-dihydroxybenzyl acetate derivative is obtained.

[0036] Preparation Example 2

[0037] Preparation of lycoramine N-demethylation derivative

[0038] (1) Take 10 g of lycoramine with a purity of ≥99.5%, add 80 mL of dichloromethane to a 250 mL dry round-bottom flask, and stir until completely dissolved. Place the flask in a -10°C low-temperature cooling bath, connect a condenser, and replace the air in the system with nitrogen. Keep the nitrogen flowing slowly (50 mL / min) and slowly add 43.2 g of boron tribromide with a purity of ≥99.8% through a syringe, ensuring that the reaction system does not exceed 0°C. Turn on the magnetic stirrer and set the stirring speed to 250 rpm. The reaction continues for 8 h. During the reaction, monitor the reaction progress every 2 h using thin layer chromatography (TLC) with dichloromethane:methanol=10:1 as the developing agent, and observe the disappearance of the lycoramine raw material spot. At the same time, high-performance liquid chromatography (HPLC) can be used to assist in monitoring the reaction progress to ensure complete reaction.

[0039] (2) After the reaction is completed, slowly pour the reaction liquid into a beaker containing 200 mL of ice water to quench the reaction, and continuously stir to avoid local overheating. Adjust the pH to 8-9, transfer the neutralized mixture to a separatory funnel, and let it stand to separate the layers. Separate the lower organic phase, transfer it to a conical flask, add about 10 g of anhydrous sodium sulfate, and shake for 10 min to dry. After the solution is clear, filter out the sodium sulfate solid.

[0040] (3) Preparation of silica gel column: 300 g of 100-200 mesh silica gel was packed into a column with dichloromethane by wet method, and no air bubbles were ensured in the column. The column efficiency was detected.

[0041] (4) Sample loading and elution: The dried organic phase was concentrated to about 10 mL, and was carefully added to the top of the silica gel column. Elution was performed with dichloromethane:methanol=10:1 as eluent, and the flow rate was controlled at 1-2 drops per second. During the elution process, the composition of the eluent was monitored in real time to avoid loss of the target product.

[0042] (5) Collection and concentration: The eluent containing the target product was collected by HPLC detection, and after being combined, the solvent was removed by distillation under reduced pressure to obtain 6 g of a solid N-demethylated derivative of lycorine with a purity of 97%.

[0043] Preparation Example 3

[0044] Preparation of quercetin nanocrystals

[0045] (1) In a 50 mL centrifuge tube, 100 mg of quercetin with a purity of ≥98% was accurately weighed, and 200 mg of PVP K30 was added to 10 mL of anhydrous ethanol. The mixture was stirred in a 37°C constant temperature water bath for 30 min with a magnetic stirrer (500 rpm) until the solution was clear and transparent.

[0046] (2) 100 mL of ultrapure water was prepared (pre-cooled to 4°C) and poured into a 250 mL beaker. The magnetic stirring was started (500 rpm, stirring bar diameter 15 mm). The clear solution was slowly injected into the ultrapure water through a syringe (needle diameter 0.8 mm) at a speed of ≤1 mL / min. A white turbid liquid was formed, and the stirring was continued for 30 min. The ethanol diffusion into the water phase was confirmed by gas chromatography detection.

[0047] (3) The turbid liquid was transferred to a rotary evaporator and evaporated at 40°C under a vacuum degree of ≤20 mbar for 30 min. The solution was transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm and 4°C for 10 min in a refrigerated centrifuge. The supernatant was discarded.

[0048] (4) 10 mL of ultrapure water was added to the precipitate, and the precipitate was resuspended by vortexing for 1 min. The precipitate was centrifuged again at 8000 rpm for 5 min, and the washing was repeated for 3 times. The precipitate was resuspended with 100 mg of PBS (pH 7.4) to prepare a quercetin nanocrystal dispersion. The quercetin nanocrystals were obtained by freeze-drying at -40°C for 24 h.

[0049] Example 1

[0050] Preparation of hydrogel microspheres for treating drug-resistant ovarian cancer (paclitaxel as a chemotherapeutic drug)

[0051] Components and mass ratio: chitosan modified modified anti-mutagenic active agent: paclitaxel: hyaluronic acid: glycerol: glutaraldehyde: copper death mixture = 7.5:5:20:7.5:2:12.

[0052] (1) Preparation of chitosan modified modified anti-mutagenic active agent:

[0053] 1) Mix 5g-3,5-dihydroxybenzyl acetic acid ester derivative and 5g lycorine N-demethylation derivative at a mass ratio of 1:1, place them in a 50mL glass beaker, use a magnetic stirrer at 300rpm, 25℃, mix for 30min, ensure uniform dispersion, and mix the anti-mutagenic active agent;

[0054] 2) Dissolve 2g chitosan (degree of deacetylation ≥95%) in 100mL 1% acetic acid solution (v / v), magnetic stirring at 500rpm, 35℃, 2h to complete dissolution, to obtain a 2% chitosan solution, slowly add the above mixed anti-mutagenic active agent to the chitosan solution, keep the 35℃ constant temperature water bath, continue to stir for 3h (300rpm), to obtain the chitosan modified modified anti-mutagenic active agent;

[0055] 3) Purification and drying: transfer the above chitosan modified modified anti-mutagenic active agent to a centrifuge tube, centrifuge at 9000rpm for 12min, discard the supernatant, and wash the precipitate with deionized water (50mL x 2 times) and anhydrous ethanol (50mL x 2 times) alternately, centrifuge (9000rpm, 10min) after each washing, finally the precipitate is placed in a freeze dryer (-50℃, 24h), to obtain white powder of chitosan modified modified anti-mutagenic active agent, with a yield of 90%.

[0056] (2) Preparation of copper death mixture:

[0057] 1) Dissolve disulfiram (2mmol, 0.48g) and CuSO4·5H2O (1mmol, 0.25g) in 50mL ethanol-water mixed solvent (ethanol:water = 4:1, v / v), react in a 40℃ water bath for 2h, the solution gradually changes from blue to dark green precipitate, centrifuge (10000rpm, 15min), collect the precipitate, wash with ethanol 3 times, vacuum dry (25℃, 12h), to obtain a dark green disulfiram-copper complex;

[0058] 2) Dissolve 6g phospholipid, 3g-DOTAP and 1g disulfiram-copper complex in chloroform-methanol (2:1, v / v, 30mL), rotary evaporation (40℃, 100rpm) to form a uniform film, then rehydrate (PBS buffer, pH7.4, 50mL) to obtain disulfiram-copper-carrier;

[0059] 3) Mix 12 g quercetin nanocrystals with 12 g disulfiram-copper-carrier at 1:1 mass ratio, incubate at room temperature for 30 min, detect the particle size distribution (target range: 150 ± 20 nm) by dynamic light scattering (DLS), centrifuge (3000 rpm, 5 min) to remove the unabsorbed quercetin, resuspend the precipitate in PBS to obtain the copper death mixture.

[0060] (3) Preparation of hydrogel microspheres:

[0061] 1) Dissolve 20 g of hyaluronic acid (molecular weight 200 kDa) in 667 mL of deionized water to prepare a 3% solution, magnetically stir (600 rpm, 4 h) until transparent gel is obtained, to obtain a hyaluronic acid solution;

[0062] 2) Add to the hyaluronic acid solution in order: 7.5 g glycerol: as a plasticizer, stir for 10 min, 2 g glutaraldehyde (25% aqueous solution): crosslinking agent, slowly drop to avoid local aggregation, 7.5 g chitosan-modified modified antimutagenic active agent, 12 g copper death mixture, 5 g paclitaxel, continue stirring for 1 h until homogeneous;

[0063] 3) Microfluidic molding: use a microfluidic device (inner diameter 0.5 mm nozzle), set parameters: inner phase (mixed solution) flow rate: 0.2 mL / min outer phase (mineral oil) flow rate: 2 mL / min collect microspheres in 0.1% Tween 80 solution to solidify, sieve (particle size range 100-150 μm), wash with PBS 3 times, store at 4°C.

[0064] Example 2

[0065] Replace paclitaxel with cisplatin (adjust mass ratio)

[0066] Components and mass ratio: chitosan-modified modified antimutagenic active agent: paclitaxel: hyaluronic acid: glycerol: glutaraldehyde: copper death mixture = 7:5:20:7:2:12.

[0067] (1) Preparation of chitosan-modified modified antimutagenic active agent: same as Example 1.

[0068] (2) Preparation of copper death mixture: same as Example 1.

[0069] (3) Preparation of hydrogel microspheres:

[0070] 1) Same as Example 1;

[0071] 2) Add to the hyaluronic acid solution in order: 7 g glycerol: as a plasticizer, stir for 10 min, 2 g glutaraldehyde (25% aqueous solution): crosslinking agent, slowly drop to avoid local aggregation, 7 g chitosan-modified modified antimutagenic active agent, 12 g copper death mixture, 5 g cisplatin, continue stirring for 1 h until homogeneous;

[0072] 3) Microfluidic molding: same as Example 1.

[0073] Example 3

[0074] Low proportion formulation verification (lower limit)

[0075] Components and mass ratio: Chitosan modified modified antimutagenic active agent: paclitaxel: hyaluronic acid: glycerol: glutaraldehyde: copper death mixture = 6:4:18:6:1.5:12.

[0076] (1) Preparation of chitosan modified modified antimutagenic active agent: same as Example 1.

[0077] (2) Preparation of copper death mixture: same as Example 1.

[0078] (3) Preparation of hydrogel microspheres:

[0079] 1) 18 g of hyaluronic acid (molecular weight 200 kDa) was dissolved in 667 mL of deionized water to prepare a 3% solution, and magnetic stirring (600 rpm, 4 h) was performed until a transparent gel was obtained, to obtain a hyaluronic acid solution;

[0080] 2) To the hyaluronic acid solution, 6 g of glycerol was added as a plasticizer, stirred for 10 min, 1.5 g of glutaraldehyde (25% aqueous solution) was added as a crosslinking agent, and the solution was slowly added to avoid local aggregation, 6 g of chitosan modified modified antimutagenic active agent, 12 g of copper death mixture, and 4 g of paclitaxel were added, and stirring was continued for 1 h until homogeneity was achieved;

[0081] 3) Microfluidic molding: same as Example 1.

[0082] Example 4

[0083] High drug loading formulation (upper limit)

[0084] Components and mass ratio: Chitosan modified modified antimutagenic active agent: paclitaxel: hyaluronic acid: glycerol: glutaraldehyde: copper death mixture = 9:7:22:9:2.5:14.

[0085] (1) Preparation of chitosan modified modified antimutagenic active agent: same as Example 1.

[0086] (2) Preparation of copper death mixture: same as Example 1.

[0087] (3) Preparation of hydrogel microspheres:

[0088] 1) 22 g of hyaluronic acid (molecular weight 200 kDa) was dissolved in 667 mL of deionized water to prepare a 3% solution, and magnetic stirring (600 rpm, 4 h) was performed until a transparent gel was obtained, to obtain a hyaluronic acid solution;

[0089] 2) To the hyaluronic acid solution, add sequentially: 9 g glycerol: as plasticizer, stir for 10 min, 2.5 g glutaraldehyde (25% aqueous solution): crosslinking agent, slowly drop to avoid local aggregation, 6 g chitosan-modified modified antimutagenic active agent, 14 g copper death mixture, 7 g paclitaxel, continue stirring for 1 h to homogenize;

[0090] 3) Microfluidic molding: same as Example 1.

[0091] Comparative Example 1

[0092] Preparation of hydrogel microspheres without copper death mixture

[0093] Components and mass ratio: without copper death mixture, the rest of the components are the same as Example 1.

[0094] (1) Preparation of chitosan-modified modified antimutagenic active agent: same as Example 1.

[0095] (2) Preparation of hydrogel microspheres:

[0096] 1) Dissolve 20 g of hyaluronic acid (molecular weight 200 kDa) in 667 mL of deionized water to prepare a 3% solution, magnetically stir (600 rpm, 4 h) to a transparent gel, and obtain a hyaluronic acid solution;

[0097] 2) To the hyaluronic acid solution, add sequentially: 7.5 g glycerol: as plasticizer, stir for 10 min, 2 g glutaraldehyde (25% aqueous solution): crosslinking agent, slowly drop to avoid local aggregation, 7.5 g chitosan-modified modified antimutagenic active agent, 5 g paclitaxel, continue stirring for 1 h to homogenize;

[0098] (3) Microfluidic molding: same as Example 1.

[0099] Comparative Example 2

[0100] Preparation of hydrogel microspheres with chitosan instead of chitosan-modified modified antimutagenic active agent

[0101] Components and mass ratio: chitosan: paclitaxel: hyaluronic acid: glycerol: glutaraldehyde: copper death mixture = 7.5:5:20:7.5:2:12.

[0102] (1) Preparation of chitosan: directly use 7.5 g of chitosan powder (degree of deacetylation ≥95%).

[0103] (2) Preparation of copper death mixture: same as Example 1.

[0104] (3) Preparation of hydrogel microspheres:

[0105] 1) Same as Example 1;

[0106] 2) To the hyaluronic acid solution, add sequentially: 7.5 g glycerol: as plasticizer, stir for 10 min, 2 g glutaraldehyde (25% aqueous solution): crosslinking agent, slowly drop to avoid local aggregation, 7.5 g chitosan, 12 g copper death mixture, 5 g paclitaxel, continue stirring for 1 h to homogenize;

[0107] 3) Microfluidic molding: same as Example 1.

[0108] Comparative Example 3

[0109] Preparation of hydrogel microspheres by traditional emulsification method

[0110] Components and mass ratio: same as Example 1.

[0111] (1) Preparation of modified anti-mutagenic active agent modified by chitosan: same as Example 1.

[0112] (2) Preparation of copper death mixture: same as Example 1.

[0113] (3) Preparation of hydrogel microspheres:

[0114] 1) Same as Example 1;

[0115] 2) Same as Example 1;

[0116] 3) Preparation by emulsification method: drop the mixed solution into 500 mL mineral oil containing 0.1% Tween 80, mechanically stir (600 rpm) for 30 min to form microspheres, and then sieve (particle size range 100-150 μm) and wash with PBS.

[0117] Test Example 1

[0118] In vitro drug release kinetics test

[0119] (1) Experimental method: place the hydrogel microspheres of each group in PBS buffer at 37°C, pH 7.4, and stir magnetically at 100 rpm; take 1 mL sample at 0, 2, 4, 8, 12, 24, 48, and 72 h, respectively, while supplementing with an equal volume of fresh PBS, detect the concentration of paclitaxel or cisplatin by HPLC, calculate the cumulative release rate, and the release rate of each group is shown in Table 1. Figure 1

[0120] (2) The experimental data are shown in Table 1:

[0121] Table 1 Cumulative release rate of each group

[0122] Group 48h cumulative release rate 72h cumulative release rate Release pattern Example 1 68.5% 89.2% Biphasic release: 45% release in the first 24h, followed by slow release Example 2 (cisplatin) 65.3% 85.7% Similar to paclitaxel group, slightly lower initial release Example 3 (low proportion) 62.1% 82.3% Release rate is slightly slower, possibly related to low component concentration Example 4 (high drug loading) 72.8% 92.5% High drug loading group releases faster initially, but converges at 72h Comparative Example 1 (no copper death mixture) 58.9% 78.6% Release rate is significantly reduced, possibly due to differences in matrix structure Comparative Example 2 (chitosan substitution) 55.2% 75.1% Release curve is not smooth, with a burst release phenomenon (52% release at 24h) Comparative Example 3 (traditional emulsion method) 45.6% 68.3% Release rate is the slowest, with uneven particle size leading to differences in release

[0123] (3) Experimental results:

[0124] ​The microspheres prepared by microfluidic technology in Examples 1-4 exhibit controllable sustained-release characteristics; the addition of the copper death mixture (Examples 1-4) can optimize the matrix network structure and promote uniform drug release; the release rate of Comparative Example 1 is reduced by 10%-15% due to the lack of this component; the traditional emulsification method (Comparative Example 3) has a non-uniform particle size, low drug encapsulation rate, and significantly poorer release stability than the microfluidic technology.

[0125] Test Example 2

[0126] Detection of Anti-tumor Activity (MTT method)

[0127] (1) Experimental method: the cisplatin-resistant ovarian cancer cell line (A2780 / DDP, Shanghai Jixiang Biotechnology Co., Ltd.) was selected, and 1×10 4 cells / well were inoculated in a 96-well plate, and after 24 h, each group of hydrogel microspheres (drug concentration equivalent to free paclitaxel 10 μg / mL) was added, and incubated for 48 h. MTT solution (5 mg / mL) was added, incubated at 37°C for 4 h, DMSO was used to dissolve the formazan crystals, and the OD 570 nm was measured by a microplate reader.

[0128] (2) The experimental data are shown in Table 2:

[0129] Table 2 Cumulative release rate of each group

[0130] Group Cell survival rate (%) IC50 (μg / mL) Example 1 23.5±2.1 5.2±0.3 Example 2 (cisplatin) 28.7±1.8 6.1±0.5 Example 3 (low proportion) 35.6±3.2 7.8±0.6 Example 4 (high drug loading) 19.2±1.5 4.8±0.2 Comparative Example 1 (no copper death mixture) 47.9±4.3 12.5±1.1 Comparative Example 2 (chitosan substitution) 52.1±3.8 15.7±1.3 Comparative Example 3 (traditional emulsion method) 61.4±5.2 18.9±1.5 Free paclitaxel 38.2±2.9 9.3±0.8

[0131] (3) Experimental results: the cell survival rates of Examples 1-4 were significantly lower than those of free paclitaxel and the comparative examples, indicating that the synergistic effect of multiple components can enhance the resistance to drug resistance; the addition of the copper death mixture (Examples 1-4) reduces the IC50 by 40%-50%, confirming that it breaks through the traditional apoptosis resistance mechanism by inducing copper death; the cell survival rate of Comparative Example 1 increases by 20%-30% due to the lack of a modified anti-mutagenic agent, indicating that the anti-mutagenic agent modified by chitosan is crucial for inhibiting the expression of drug resistance genes.

[0132] Test Example 3

[0133] Detection of Drug Resistance Related Protein Expression-Western Blot method

[0134] (1) Experimental method:

[0135] 1) Cell treatment: the cisplatin-resistant ovarian cancer cell line (A2780 / DDP) was selected, and 5×10 5 cells / well were inoculated in a 6-well plate, and incubated for 24 h, and each group of hydrogel microspheres (drug concentration equivalent to free paclitaxel 10 μg / mL) was added, and a blank control group (only PBS) and a free paclitaxel group (10 μg / mL) were set up, and incubated for 48 h.

[0136] 2) Protein extraction: discard the culture medium, wash the cells twice with pre-cooled PBS, add RIPA lysis buffer (containing 1% protease inhibitor), lyse on ice for 30 min, centrifuge (12000 rpm, 15 min, 4°C) to collect the supernatant, determine the protein concentration by BCA method, and dilute to a uniform concentration (2 μg / μL);

[0137] 3) Western Blot steps: take 30 μg of protein sample, separate by SDS-PAGE electrophoresis, and transfer to PVDF membrane; after blocking (5% skim milk, room temperature for 1 h), incubate the first antibody (4°C overnight): anti-P-gp (1:1000, Abeam, ab170904); anti-BCL-2 (1:1000, Cell Signaling Technology, #2872); anti-copper death marker protein FDX1 (1:1000, Proteintech, 14950-1-AP) anti-β-actin (1:5000, internal reference, Sigma-Aldrich, A5441); after washing, incubate HRP-labeled secondary antibody anti-rabbit IgG (1:5000, Proteintech, SA00001-2) at room temperature for 1 h (internal reference uses anti-mouse IgG, 1:5000, Proteintech, SA00001-1), ECL development, ImageJ quantitative analysis of band gray value.

[0138] (2) Experimental data: as shown in Table 3, the expression amount is as shown in Figure 2 ;

[0139] Table 3 Expression amount of each group

[0140] Group P-gp expression (%) BCL-2 expression (%) FDX1 expression (%) Blank control group 100±5.2 100±4.8 100±3.5 Free paclitaxel group 85.3±4.1 78.6±3.9 105.2±4.7 Example 1 (paclitaxel microspheres) 42.7±3.5* 35.2±2.8* 215.6±8.3* Example 2 (cisplatin microspheres) 45.1±3.8* 38.7±3.1* 208.4±7.9* Comparative Example 1 (no copper death) 68.9±4.6 62.4±4.2 110.3±5.1 Comparative Example 2 (chitosan substitution) 75.2±4.3 70.8±4.0 108.7±4.8

[0141] (* indicates p<0.01 compared with the blank control group)

[0142] (3) Experimental results: Drug resistance inhibition: In Examples 1-2, the modified anti-mutagenic agent significantly down-regulated the expression of P-gp and BCL-2 (by 50-60%), confirming that it enhances the sensitivity of chemotherapy by inhibiting drug resistance genes and apoptosis escape pathways; Comparative Examples 1-2 significantly reduced the effect of drug resistance protein inhibition due to the lack of key components; Copper death mechanism verification: FDX1 expression in Examples 1-2 was significantly increased (about 2 times), indicating that the copper death mixture successfully activated the non-apoptotic cell death pathway; Comparative Example 1 (without copper death mixture) did not show FDX1 up-regulation, indicating that copper death induction depends on the synergistic effect of disulfiram-copper-carrier and quercetin; Synergistic effect: The multi-component combination of hydrogel microspheres (anti-mutagenic + copper death + chemotherapy) can simultaneously target drug resistance proteins and cell death pathways, breaking through the limitations of traditional treatment; This test confirmed that the hydrogel microspheres achieve multi-mechanism synergistic anti-tumor effect by down-regulating drug resistance proteins (P-gp, BCL-2) and activating the copper death pathway (FDX1), providing a molecular level basis for overcoming ovarian cancer drug resistance.

[0143] Test Example 4

[0144] In vivo anti-tumor efficacy evaluation (drug-resistant ovarian cancer mouse model)

[0145] (1) Experimental animals:

[0146] 1) Animal strain: 64 SPF female BALB / c nude mice (6-8 weeks old, 18-22 g), 8 mice per group, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.;

[0147] 2) Rearing conditions: Environment: constant temperature (22±2°C), constant humidity (50±10%), 12h light / dark cycle, acclimatization for 7 days, feed: SPF sterile mouse feed, free access to water (treated with high pressure sterilization) Experimental protocols were approved by the Institutional Animal Ethics Committee.

[0148] (2) Experimental methods:

[0149] 1) Preparation of cell suspension A2780 / DDP cells: cultured to the logarithmic growth phase, 0.25% trypsin digestion, washed with PBS twice, centrifuged at 1000 rpm for 5 min, resuspended with serum-free RPMI 1640 medium, and adjusted to a cell concentration of 5×10 7 cells / mL (live cell rate ≥95% by trypan blue staining);

[0150] 2) Drug preparation: Example 1, 3-4 hydrogel microspheres: suspended in PBS, ultrasonic dispersion for 30 s, concentration adjusted to contain paclitaxel 5 mg / kg (dose volume calculated according to mouse body weight, such as 20 g mouse 0.2 mL); Comparative Examples 1-3 and free paclitaxel group: prepared in the same way, the drug concentration is equivalent to the example group;

[0151] 3) Establishment of nude mouse tumor xenograft model

[0152] 4) Cell inoculation: subcutaneously inject A2780 / DDP cell suspension (100 μL / each, containing 5 x 10 6 cells) on the right side of the back of the nude mouse, gently press the needle eye after injection to prevent cell overflow, monitor the injection site daily after inoculation, and when the tumor volume reaches 100-150 mm³, include it in the experimental group;

[0153] 5) Group administration as shown in Table 4:

[0154] Table 4 Drug administration of each group

[0155] Group Treatment method Number of animals Administration route Administration frequency Blank control group PBS 0.2 mL / each 8 Intraperitoneal injection Administered once every 3 days for 3 weeks Example 1 Hydrogel microspheres (containing paclitaxel 5 mg / kg) 8 Intraperitoneal injection Administered once every 3 days for 3 weeks Example 3 Low proportion hydrogel microspheres (containing paclitaxel 5 mg / kg) 8 Intraperitoneal injection Administered once every 3 days for 3 weeks Example 4 High drug loading hydrogel microspheres (containing paclitaxel 5 mg / kg) 8 Intraperitoneal injection Administered once every 3 days for 3 weeks Comparative Example 1 No copper death mixture microspheres 8 Intraperitoneal injection Administered once every 3 days for 3 weeks Comparative Example 2 Chitosan substitution microspheres 8 Intraperitoneal injection Administered once every 3 days for 3 weeks Comparative Example 3 Traditional emulsion method microspheres 8 Intraperitoneal injection Administered once every 3 days for 3 weeks Free paclitaxel group Free paclitaxel (5 mg / kg) 8 Intraperitoneal injection Administered once every 3 days for 3 weeks

[0156] (3) Data collection: every 3 days (on the day of administration) measure the longest diameter (L) and the perpendicular short diameter (W) of the tumor with a vernier caliper, calculate the volume according to the formula: tumor volume (mm³) = 0.5 x L x W², record the body weight change (g), evaluate the drug toxicity, sacrifice all mice on the 21st day, peel off the tumor, weigh it on an electronic balance, calculate the tumor inhibition rate: tumor inhibition rate (%) = [1 - average tumor weight of treatment group / average tumor weight of blank group] x 100%, data as shown in Table 5:

[0157] Table 5 Tumor inhibition effect

[0158] Group Tumor volume (21st day, mm³) Tumor inhibition rate (vs blank) Survival time (median, days) Blank control group 1200±150 - 35±3 Free paclitaxel group 800±90 33.3% 45±4 Example 1 group 350±50* 70.8%* >60* Example 3 group (low proportion) 450±60* 62.5%* 55±5* Example 4 group (high drug loading) 300±40* 75.0%* >60* Comparative Example 1 group (no copper death) 600±70 50.0% 50±5 Comparative Example 2 group (chitosan substitution) 700±80 41.7% 48±4 Comparative Example 3 group (traditional emulsion method) 750±85 37.5% 46±4

[0159] *: p < 0.01.

[0160] (4) Histological analysis:

[0161] 1) Sacrifice all mice on the 21st day, fix part of the tumor specimen in 4% paraformaldehyde, H&E staining: evaluate the degree of tumor necrosis (scoring criteria: 0-no necrosis, 1-mild necrosis (<25%), 2-moderate necrosis (25%-50%), 3-significant necrosis (50%-75%), 4-widespread necrosis (>75%)) as shown in Table 6;

[0162] 2) Immunohistochemistry (IHC): Ki-67 (proliferation marker, Abeam ab15580) Cleaved Caspase-3 (apoptosis marker, CST #9664) FDX1 (copper death marker, Proteintech 14950-1-AP);

[0163] 3) Safety evaluation: Blood biochemistry: ALT, AST, BUN, Cr (liver and kidney function), major organ pathology: heart, liver, spleen, lung, kidney H&E staining.

[0164] 4) Experimental results: Example 1 group: FDX1 expression increased (IHC score 3+), confirming copper death activation Ki-67 decreased by 50%, Cleaved Caspase-3 increased by 3 times, suggesting proliferation inhibition + apoptosis synergy; Example 3 group: slightly lower effect than Example 1, related to lower active ingredient ratio; Example 4 group: strongest tumor suppression effect, Toxicity Control 1 group: no significant change in FDX1, tumor suppression effect depends on copper death mixture Control 2 group: weak Ki-67 inhibition, confirming the key role of modified anti-mutagenic active agent Control 3 group: uneven drug release, resulting in the worst efficacy. Safety data: Example 1-4 groups: stable body weight (fluctuation <5%), normal ALT / AST, no pathological damage to organs Free paclitaxel group: 15% body weight loss, mild liver damage (ALT increased by 2 times).

[0165] Table 6 H&E staining tumor necrosis results

[0166] Group H&E score (mean ± SD) Representative pathological features Blank control group 0.2±0.1 Tumor cells are dense, mitotic figures are common, and there is no necrosis Free paclitaxel group 1.8±0.3 Scattered necrotic foci (about 30%), with apoptotic bodies visible around Example 1 group 3.5±0.4* Large necrosis (about 70%), with a small amount of tumor cells remaining at the edge, accompanied by lymphocyte and macrophage infiltration Example 3 group (low proportion) 2.7±0.3* Multiple necrosis (about 50%), with pyknosis visible in the survival area Example 4 group (high drug loading) 3.8±0.2* Extensive necrosis (>80%), with only sporadic tumor cells, and obvious fibrosis Comparative Example 1 group (no copper death) 1.5±0.2 Local necrosis (about 20%), weak inflammatory response Comparative Example 2 (chitosan replacement) 1.2±0.3 Small amount of punctate necrosis (<15%), tumor cells proliferating actively Comparative Example 3 (traditional emulsification method) 1.0±0.2 Few necrotic foci (about 10%), poor effect due to uneven drug distribution

[0167] (5) Experimental results: Optimal efficacy group: Example 4 group (high drug loading) showed the most significant tumor necrosis (score 3.8±0.2), with a necrosis area of >80%, followed by Example 1 group (standard formulation) (score 3.5±0.4), both of which were significantly better than the free paclitaxel group (p<0.01); The absence of copper death mixture (Control 1 group) reduced the efficacy by 57% (score from 3.5→1.5); The replacement of modified anti-mutagenic active agent (Control 2 group) resulted in a 66% reduction in efficacy (score from 3.5→1.2), and the traditional emulsification method (Control 3 group) had the worst efficacy, confirming the key role of microfluidic technology in uniform drug distribution.

[0168] In summary, the application provides a hydrogel microsphere for treating drug-resistant ovarian cancer and a preparation method thereof, which significantly improves the treatment effect through multi-mechanism synergy. The hydrogel microsphere is composed of a modified anti-mutagenic active agent, a chemotherapeutic drug, hyaluronic acid, glycerol, glutaraldehyde and a copper death mixture, and each component realizes synergistic effect through optimized ratio and preparation process. Specifically, the multi-mechanism synergistic treatment: the modified anti-mutagenic active agent (3,5-dihydroxybenzyl acetate derivative and lycorine N-demethylation derivative) inhibits the expression of drug-resistant genes and reduces the drug resistance of tumor cells; the copper death mixture (disulfiram-copper-carrier and quercetin nanocrystal) induces non-apoptotic cell death of tumor cells, breaking through the drug resistance limit of the traditional apoptosis pathway. The chemotherapeutic drug (paclitaxel, cisplatin) directly kills tumor cells, realizing synergistic treatment of multiple action mechanisms. The preparation process advantage: the hydrogel microspheres prepared by microfluidic technology have uniform particle size and high drug encapsulation rate, ensuring the accuracy and stability of drug release. The preparation method of each component is clear, the parameters are optimized, and it is suitable for industrial production, having high clinical transformation potential; the experimental verification effect: in vitro and in vivo experiments show that the hydrogel microspheres can significantly inhibit the proliferation of drug-resistant ovarian cancer cells, down-regulate the expression of drug resistance proteins (P-gp, BCL-2), and activate the copper death pathway (FDX1); in a drug-resistant ovarian cancer mouse model, the hydrogel microspheres show significant tumor inhibition effect (the highest tumor inhibition rate is 75%), and have good safety without obvious toxic side effects; the comparative advantage: compared with the microspheres prepared by the traditional emulsion method, the drug release of the microspheres of the application is more uniform, and the curative effect is more significant. The lack of any key component (such as copper death mixture or modified anti-mutagenic active agent) will cause a significant decrease in curative effect, confirming the necessity of multi-component synergy. The innovation of the application lies in overcoming the problems of single drug such as easy drug resistance, low drug loading and unstable release through complex formula and optimized process, providing a new effective way for the treatment of drug-resistant ovarian cancer, and having important clinical application value.

[0169] Although the embodiments of the application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to the embodiments without departing from the principles and spirit of the application, and any equivalent changes and improvements made within the scope of the application should still belong to the patent coverage of the application.

Claims

1. A hydrogel microsphere for treating drug-resistant ovarian cancer, characterized in that, The hydrogel microspheres are made of a chitosan-modified modified anti-mutagenic active agent, a chemotherapeutic drug, hyaluronic acid, glycerol, glutaraldehyde and a copper death mixture, wherein; The chitosan-modified modified anti-mutagenic active agent is a mixture of 3,5-dihydroxybenzyl acetate derivatives and lycorine N-demethylation derivatives and is modified on the surface by chitosan, and the copper death mixture is a combination of disulfiram-copper-carrier and quercetin nanocrystals in a mass ratio of 1:1; The preparation of the chitosan-modified modified anti-mutagenic active agent: uniformly mix 3,5-dihydroxybenzyl acetate derivatives and lycorine N-demethylation derivatives in a mass ratio of 1:1, then dissolve chitosan in an acetic acid solution to prepare a chitosan solution with a mass fraction of 1%-3%, slowly add the mixed anti-mutagenic active agent to the chitosan solution, stir and react at 30-40℃ for 2-4h, after the reaction is completed, centrifuge, wash, freeze-dry, and store at-50℃ for 24h to obtain the chitosan-modified modified anti-mutagenic active agent; The preparation method of the 3,5-dihydroxybenzyl acetate derivative is as follows: 3,5-dihydroxybenzyl and acetic anhydride are mixed in a molar ratio of 1:1.2-1:1.5 under the catalysis of pyridine with a dosage of 10-15% of the mass of 3,5-dihydroxybenzyl, and the reaction is carried out at 60-70℃ under stirring at a speed of 200-300rpm for 3-5h; after the reaction is completed, the reaction solution is poured into ice water to precipitate a solid, which is filtered and washed with deionized water for 3-5 times, then recrystallized with ethanol, the amount of ethanol is 5-8 times the mass of the solid, the recrystallization temperature is controlled at 40-50℃, and the cooling speed is 0.5-1℃ / min to obtain the 3,5-dihydroxybenzyl acetate derivative; The preparation method of the lycorine N-demethylation derivative is as follows: lycorine and boron tribromide are mixed in a molar ratio of 1:3-1:4 in dichloromethane solvent with a dosage of 8-10 times the mass of lycorine, and the reaction is carried out at-10-0℃ under nitrogen protection and magnetic stirring at a speed of 250-350rpm for 8-12h; after the reaction is completed, the reaction is quenched by slowly adding ice water, the pH is adjusted to 8-9 with sodium hydroxide solution, the organic phase is separated, dried with anhydrous sodium sulfate, and then purified by column chromatography, and the silica gel column eluent is dichloromethane:methanol=10:1 to obtain the lycorine N-demethylation derivative.

2. The hydrogel microspheres for treating drug-resistant ovarian cancer according to claim 1, wherein The chemotherapeutic drug is any one of paclitaxel and cisplatin.

3. The hydrogel microspheres for treating drug-resistant ovarian cancer according to claim 1, wherein The mass ratio of the chitosan-modified modified anti-mutagenic active agent, the chemotherapeutic drug, hyaluronic acid, glycerol, glutaraldehyde and the copper death mixture is (6-9):(4-7):(18-22):(6-9):(1.5-2.5):(12-14).

4. A method of preparing hydrogel microspheres for the treatment of drug resistant ovarian cancer, characterized by, The preparation method comprises the following steps: (1) Preparation of the modified anti-mutagenic agent modified by chitosan: uniformly mix 3,5-dihydroxybenzyl acetic acid ester derivative and lycorine N-demethylation derivative according to a mass ratio of 1:1, then dissolve chitosan in an acetic acid solution to prepare a chitosan solution with a mass fraction of 1%-3%, slowly add the mixed anti-mutagenic agent to the chitosan solution, and stir and react at 30-40°C for 2-4 hours; after the reaction is completed, centrifugal separation, washing, and freeze-drying are performed at-50°C for 24 hours to obtain the modified anti-mutagenic agent modified by chitosan, wherein; the preparation method of the 3,5-dihydroxybenzyl acetic acid ester derivative is as follows: 3,5-dihydroxybenzyl and acetic anhydride are reacted according to a molar ratio of 1:1.2-1:1.5 under the catalysis of pyridine with a dosage of 10-15% of the mass of 3,5-dihydroxybenzyl, at 60-70°C, and at a stirring speed of 200-300 rpm for 3-5 hours; after the reaction is completed, the reaction solution is poured into ice water to precipitate a solid, which is filtered and washed with deionized water for 3-5 times, and then recrystallized with ethanol, the amount of ethanol being 5-8 times the mass of the solid, the recrystallization temperature being controlled at 40-50°C, and the cooling speed being 0.5-1°C / min to obtain the 3,5-dihydroxybenzyl acetic acid ester derivative; the preparation method of the lycorine N-demethylation derivative is as follows: lycorine and boron tribromide are reacted according to a molar ratio of 1:3-1:4 in dichloromethane solvent with a dosage of 8-10 times the mass of lycorine, at-10-0°C, under the protection of nitrogen, and at a magnetic stirring speed of 250-350 rpm for 8-12 hours; after the reaction is completed, the reaction is quenched by slowly adding ice water, the pH is adjusted to 8-9 with sodium hydroxide solution, the organic phase is separated, and the obtained product is dried with anhydrous sodium sulfate and then purified by column chromatography, with dichloromethane:methanol=10:1 as the eluent of the silica gel column to obtain the lycorine N-demethylation derivative; (2) Preparation of the copper death mixture: 1) Preparation of disulfiram-copper complex: disulfiram and CuSO4·5H2O are reacted according to a molar ratio of 2:1 in an ethanol-water mixed solvent at 40°C for 2 hours, centrifugal separation is performed, the precipitate is collected, washed, and dried to obtain the disulfiram-copper complex; 2) Disulfiram-copper-carrier: phospholipid, DOTAP, and the disulfiram-copper complex are dissolved in chloroform-methanol according to a mass ratio of 6:3:1 to obtain the disulfiram-copper-carrier by the thin film hydration method; 3) Copper death mixture: quercetin nanocrystals and the disulfiram-copper-carrier are mixed according to a mass ratio of 1:1, incubated at room temperature for 30 minutes, and the change in particle size is detected by dynamic light scattering; the unabsorbed quercetin nanocrystals are removed by centrifugal separation, and the precipitate is collected to obtain the copper death mixture. (3) Preparation of hydrogel microspheres: hyaluronic acid is dissolved in deionized water to prepare a hyaluronic acid solution with a mass fraction of 2%-5%, glycerol and glutaraldehyde are added to the hyaluronic acid solution, and after stirring uniformly, the modified anti-mutagenic active agent prepared in step (1) and the copper death mixture and the chemotherapeutic drug prepared in step (2) are added, and stirring is continued until the mixture is uniformly stirred to obtain a mixed solution; the mixed solution is prepared into hydrogel microspheres by microfluidic technology.

5. The method of claim 4, wherein the hydrogel microspheres are prepared by the method of claim 1, wherein the drug is selected from the group consisting of paclitaxel, docetaxel, and combinations thereof. In the step (1), the centrifugal speed is 8000-10000 rpm, and the centrifugal time is 10 min-15 min; the washing is performed by using deionized water and anhydrous ethanol alternately for 3-5 times.

6. The method of claim 4, wherein the hydrogel microspheres are prepared by the method of claim 1, wherein the drug is selected from the group consisting of paclitaxel, docetaxel, and combinations thereof. In the step (2), the preparation method of the quercetin nanocrystal is as follows: quercetin and PVP are dissolved in ethanol at a mass ratio of 1:2, injected into ultrapure water, magnetically stirred, evaporated to remove ethanol, centrifuged to collect the precipitate, and resuspended in PBS after washing to obtain the quercetin nanocrystal.

7. The method of claim 4, wherein the hydrogel microspheres are prepared for the treatment of drug resistant ovarian cancer. In the step (3), when the microfluidic technology is used to prepare the hydrogel microspheres, the inner phase flow rate is 0.1 mL / min-0.3 mL / min, and the outer phase flow rate is 1 mL / min-3 mL / min.

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