Paclitaxel-loaded reduction-responsive prodrug micelles, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a paclitaxel-loaded reduction-responsive prodrug micelle as well as a preparation method and application thereof. The paclitaxel-loaded reduction-responsive prodrug micelle prepared by the application has a hydrophilic end and a hydrophobic end, which are respectively polyethylene glycol (mPEG) and a drug indomethacin (IND), has a synergistic anti-tumor effect, reverses the multi-drug resistance of the loaded drug paclitaxel, increases the sensitivity of drug-resistant cells to paclitaxel, introduces a disulfide bond as a reduction-sensitive response linker between the hydrophilic end and the hydrophobic end, and responds to the high concentration of GSH in tumor cells to realize the targeted and accurate release of the drug in the tumor cells; the prodrug micelle realizes the goals of solubilization of a poorly soluble drug, tumor targeting and accurate drug release at a tumor site, and can enhance the drug efficacy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a paclitaxel-loaded reduction-responsive prodrug micelle, its preparation method, and its application. Background Technology

[0002] Cancer is one of the most serious diseases threatening human health worldwide. The development of cancer is a highly complex process, generally involving multiple genes and multi-step regulation. Currently, chemotherapy remains one of the most widely used and effective methods in clinical treatment. However, traditional chemotherapy is a systemic treatment, lacking selectivity in the tissues or cells targeted by the drug. While killing tumor cells, it can also damage normal tissues, leading to severe toxic side effects. Furthermore, conventional chemotherapy techniques typically use single-drug administration, but single-drug therapy often fails to achieve the desired therapeutic effect and easily leads to drug resistance.

[0003] The most common chemotherapy drugs in clinical practice include paclitaxel, doxorubicin, and docetaxel. Among them, paclitaxel (PTX) is a broad-spectrum anticancer drug. However, paclitaxel has poor water solubility, is prone to multidrug resistance, and conventional paclitaxel preparations lack targeted therapy. On the one hand, this leads to serious toxic side effects on normal tissues (such as the cardiovascular system and kidneys) during clinical use; on the other hand, insufficient drug concentration at the tumor site results in poor therapeutic efficacy and low bioavailability, thus limiting its chemotherapy effects and clinical application.

[0004] To address the challenges of clinical application of chemotherapy drugs, the development of polymer-based reduction-responsive prodrugs has received increasing attention, leading to the development and use of various polymer prodrug micelles. Polymer prodrug micelles are block copolymers formed by covalently binding drugs to a polymer backbone and then self-assembling in water. They represent a special type of nanomedicine delivery system where the drug acts as a carrier in the construction of the polymer prodrug micelles. Polymer prodrug micelles exhibit good sustained-release properties and stability, and can also serve as carriers for nanomedicines, enabling combined drug delivery by encapsulating different or identical chemotherapeutic drugs. A key challenge in achieving effective combined drug delivery is how to target, enrich, and activate the drug at the disease site, achieving responsive release and thus reducing systemic toxicity while maintaining antitumor effects. To address this issue, various responsive activation technologies have been developed and utilized.

[0005] However, some micellar carriers have poor biodegradability, making them difficult for the body to metabolize naturally. Therefore, there is an urgent need to provide an effective solution that, while possessing good biocompatibility, can overcome the shortcomings of traditional paclitaxel formulations, such as poor water solubility, easy induction of multidrug resistance, and insufficient targeting. Summary of the Invention

[0006] The purpose of this invention is to provide a paclitaxel-loaded reduction-responsive prodrug micelle, its preparation method, and its application, thereby solving the problems of poor water solubility, easy generation of multidrug resistance, and insufficient targeting of traditional paclitaxel formulations.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles, comprising the following steps:

[0009] (1) Succinic anhydride, polyethylene glycol and anhydrous toluene are mixed and heated to undergo reflux reaction to obtain mPEG-COOH polymer; mPEG-COOH polymer, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide and chloroform are mixed to undergo first activation reaction to obtain activated mPEG-COOH polymer;

[0010] A methanol solution of di-tert-butyl dicarbonate, a methanol solution of cystamine dihydrochloride, and triethylamine were mixed to carry out a first substitution reaction to obtain the Boc-SS-NH2 polymer.

[0011] (2) The activated mPEG-COOH polymer is mixed with the Boc-SS-NH2 polymer to carry out a second substitution reaction to obtain the mPEG-SS-Boc polymer;

[0012] (3) Add HCl solution to the methanol solution of mPEG-SS-Boc polymer to carry out amide condensation reaction, then add activated indomethacin solution to adjust the pH to alkaline, react under a protective atmosphere, dialyze and freeze dry to obtain the paclitaxel carrier, i.e. PSI polymer.

[0013] (4) PSI polymer, paclitaxel and methanol are mixed under light-protected conditions, the methanol is removed after dissolution, and then is hydrated at a constant temperature to obtain paclitaxel-loaded reduction-responsive prodrug micelles.

[0014] Preferably, in the method for preparing a paclitaxel-loaded reduction-responsive prodrug micelle, in step (1), the mass-to-volume ratio of the ditert-butyl dicarbonate, the cystamine dihydrochloride, and the triethylamine is 0.5–0.8 g: 0.9–1.0 g: 2.4–2.7 mL; and the time for the first substitution reaction is 2–4 h.

[0015] Preferably, in the method for preparing a paclitaxel-loaded reduction-responsive prodrug micelle, in step (2), the mass ratio of the activated mPEG-COOH polymer to the Boc-SS-NH2 polymer is 1.5-2.5:0.8-1.2; and the second substitution reaction time is 20-26 h.

[0016] Preferably, in the method for preparing a paclitaxel-loaded reduction-responsive prodrug micelle, in step (3), the activated indomethacin solution is prepared by mixing indomethacin, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide and dimethyl sulfoxide to carry out a second activation reaction to obtain the activated indomethacin solution.

[0017] Preferably, in the method for preparing a paclitaxel-loaded reduction-responsive prodrug micelle, in step (3), the mass ratio of the mPEG-SS-Boc polymer to the indomethacin raw material for preparing the activated indomethacin solution is 4 to 6:1.

[0018] Preferably, in the method for preparing a paclitaxel-loaded reduction-responsive prodrug micelle, in step (3), the reaction temperature under a protective atmosphere is 15-25°C, and the reaction time under a protective atmosphere is 24-52 h.

[0019] Preferably, in the method for preparing a paclitaxel-loaded reduction-responsive prodrug micelle, in step (4), the mass ratio of the PSI polymer to the paclitaxel is 16-32:1.6-3.2.

[0020] Preferably, in the method for preparing a paclitaxel-loaded reduction-responsive prodrug micelle, in step (4), the mass ratio of the PSI polymer to the paclitaxel is 28:2.8.

[0021] The present invention also provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles, resulting in paclitaxel-loaded reduction-responsive prodrug micelles.

[0022] The present invention also provides the application of the aforementioned paclitaxel-loaded reduction-responsive prodrug micelles in the preparation of a medicament for treating breast cancer.

[0023] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) In terms of material selection, the hydrophilic end of this invention uses polyethylene glycol (mPEG), which has advantages such as good biocompatibility, biodegradability, and low toxicity. It is widely used in the construction of polymers and can self-assemble into micelles in aqueous liquids, thereby prolonging the circulation time of drugs in vivo, achieving the purpose of slow drug release, reducing the side effects of polymeric drugs, and improving efficacy. The hydrophobic end uses the drug indomethacin (IND), which is chemically bonded into the carrier to form polymer prodrug micelles. The prodrug micelles formed by IND as a micelle carrier material can encapsulate the chemotherapy drug PTX, increasing the drug loading of PTX. IND can also inhibit the activity of the MRP1 promoter, reduce the multidrug resistance of tumor cells to PTX, and synergistically improve the anti-tumor effect of PTX, realizing the dual function of IND as a carrier and drug.

[0025] (2) Compared with normal tissues, tumor cells have high expression of glutathione (GSH). Based on this characteristic, the present invention designs reduction-responsive prodrug micelles. A disulfide bond is introduced between the hydrophilic and hydrophobic ends as a linker arm for reduction-sensitive response, so that the micelles are structurally intact in normal tissues, preventing premature leakage of anti-tumor drugs and accumulating more in the tumor site. When in the environment of high concentration of GSH in tumor cells, its structure will be destroyed, the micelles will lyse and release the drug, achieving targeted drug release and enhancing drug efficacy.

[0026] (3) The paclitaxel-loaded reduction-responsive prodrug micelles of the present invention have the functions of improving drug loading, increasing drug stability and reducing drug toxicity. The prodrug micelles are a biodegradable system that can be biodegraded after drug release without producing toxic side effects on the human body.

[0027] (4) This invention synthesizes targeted paclitaxel-loaded reduction-responsive mPEG-SS-IND prodrug micelles, which can self-assemble into micelles in water, achieving the goals of solubilizing poorly soluble drugs, targeting tumors, and precisely releasing drugs to tumor sites, thereby enhancing drug efficacy and providing great potential for the transformation of polymer prodrug micelles into clinical applications.

[0028] (5) The present invention uses thin film hydration method to prepare prodrug micelles. The prodrug micelle solution has a light blue opalescence, uniform particle size, and high encapsulation efficiency. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0030] Figure 1The synergistic effect curves of FPX and IND obtained by the Chou-Talalay combined index method are shown in (a) the median effect curve, (b) the dose-response curve, and (c) the Fa-CI curve.

[0031] Figure 2 The mPEG-COOH polymer obtained in step (1) of Example 1 1 H-NMR characterization diagram;

[0032] Figure 3 The Boc-SS-NH2 polymer obtained in step (1) of Example 1 1 H-NMR characterization diagram;

[0033] Figure 4 The mPEG-SS-Boc polymer obtained in step (2) of Example 1 1 H-NMR characterization diagram;

[0034] Figure 5 The PSI polymer obtained in step (3) of Example 1 1 H-NMR characterization diagram;

[0035] Figure 6 For the PI polymer obtained in step (1) of Comparative Example 1 1 H-NMR characterization diagram;

[0036] Figure 7 The infrared spectrum of the PSI polymer obtained in step (3) of Example 1;

[0037] Figure 8 This is the infrared spectrum of the IND.

[0038] Figure 9 The infrared spectrum of the PI polymer obtained in step (1) of Comparative Example 1 is shown.

[0039] Figure 10 The particle size distribution diagram of PSI / PTX obtained in Example 1 is shown.

[0040] Figure 11 The Zeta potential diagram of PSI / PTX obtained in Example 1;

[0041] Figure 12 TEM image of PSI / PTX obtained in Example 1;

[0042] Figure 13 The in vitro release diagrams of PTX in different release media are shown for PSI / PTX prepared in Example 1, PI / PTX prepared in Comparative Example 1, and PTX technical drug.

[0043] Figure 14The in vitro release diagrams of IND in different release media are shown for PSI / PTX prepared in Example 1, PI / PTX prepared in Comparative Example 1, and IND technical grade drug.

[0044] Figure 15 The uptake of different drugs by MCF-7 / PTX cells;

[0045] Figure 16 The effect of different treatment groups on the migration ability of MCF-7 / PTX cells;

[0046] Figure 17 The effects of the PSI polymer prepared in step (3) of Example 1 and the PI polymer prepared in step (1) of Comparative Example 1 on the survival rate of MCF-7 cells;

[0047] Figure 18 The effect of the PSI polymer prepared in step (3) of Example 1 and the PI polymer prepared in step (1) of Comparative Example 1 on the survival rate of MCF-7 / PTX cells;

[0048] Figure 19 The effect of different drug groups on the survival rate of MCF-7 cells;

[0049] Figure 20 The effect of different drug groups on the survival rate of MCF-7 / PTX cells;

[0050] Figure 21 The effects of different drug groups on MRP1 protein expression in MCF-7 / PTX cells;

[0051] Figure 22 The properties of the PSI / PTX prodrug micelles obtained in Examples 1-5 are shown, where (a) is the encapsulation efficiency, (b) is the drug loading, and (c) is the particle size. Detailed Implementation

[0052] This invention provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles, comprising the following steps:

[0053] (1) Succinic anhydride, polyethylene glycol and anhydrous toluene are mixed and heated to undergo reflux reaction to obtain mPEG-COOH polymer; mPEG-COOH polymer, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide and chloroform are mixed to undergo first activation reaction to obtain activated mPEG-COOH polymer;

[0054] A methanol solution of di-tert-butyl dicarbonate, a methanol solution of cystamine dihydrochloride, and triethylamine were mixed to carry out a first substitution reaction to obtain the Boc-SS-NH2 polymer.

[0055] (2) The activated mPEG-COOH polymer is mixed with the Boc-SS-NH2 polymer to carry out a second substitution reaction to obtain the mPEG-SS-Boc polymer;

[0056] (3) Add HCl solution to the methanol solution of mPEG-SS-Boc polymer to carry out amide condensation reaction, then add activated indomethacin solution to adjust the pH to alkaline, react under a protective atmosphere, dialyze and freeze dry to obtain the paclitaxel carrier, i.e. PSI polymer.

[0057] (4) PSI polymer, paclitaxel and methanol are mixed under light-protected conditions, the methanol is removed after dissolution, and then is hydrated at a constant temperature to obtain paclitaxel-loaded reduction-responsive prodrug micelles.

[0058] In this invention, in step (1), the molecular weight of the polyethylene glycol is preferably 550 to 2000, more preferably 1000 to 2000, and even more preferably 2000.

[0059] In this invention, in step (1), the anhydrous toluene is first evaporated to remove water using a rotary evaporator before use.

[0060] In this invention, in step (1), the molar volume ratio of the succinic anhydride, the polyethylene glycol and the anhydrous toluene is preferably 1.5-4.5 mmol: 0.5-2.0 mmol: 100-300 mL, more preferably 2.5-3.3 mmol: 0.8-1.1 mmol: 180-210 mL, and even more preferably 3 mmol: 1 mmol: 200 mL.

[0061] In this invention, in step (1), the temperature of the reflux reaction is preferably 95-120°C, more preferably 105-110°C, and even more preferably 110°C; the time of the reflux reaction is preferably 60-85 min, more preferably 65-75 min, and even more preferably 70 min.

[0062] In this invention, step (1) preferably includes the following steps after the reflux reaction: concentrating the product obtained from the reflux reaction under reduced pressure to remove the organic solvent, precipitating it with excess ice-cold ether after cooling, filtering and drying.

[0063] In this invention, in step (1), the mass-to-volume ratio of the mPEG-COOH polymer, the N-hydroxysuccinimide, the N,N'-dicyclohexylcarbodiimide, and the trichloromethane is preferably 4-6 g: 0.7-1.2 g: 0.8-1.3 g: 180-225 mL, more preferably 4.5-5.5 g: 0.9-1.2 g: 0.8-1.1 g: 195-210 mL, and even more preferably 5 g: 1 g: 1 g: 200 mL.

[0064] In this invention, in step (1), the temperature of the first activation reaction is preferably 15-25°C, more preferably 18-22°C, and even more preferably 20°C; the time of the first activation reaction is preferably 4-6.5h, more preferably 4.5-5.5h, and even more preferably 5h.

[0065] In this invention, in step (1), the purpose of the first activation reaction is to activate the carboxyl groups of the mPEG-COOH polymer.

[0066] In this invention, in step (1), the mass-to-volume ratio of the ditert-butyl dicarbonate, the cystamine dihydrochloride, and the triethylamine is preferably 0.5–0.8 g: 0.9–1.1 g: 2.4–2.7 mL, more preferably 0.6–0.7 g: 1.0–1.1 g: 2.5–2.6 mL, and even more preferably 0.7 g: 1.0 g: 2.5 mL.

[0067] In this invention, in step (1), the concentration of di-tert-butyl dicarbonate in the methanol solution of di-tert-butyl dicarbonate is preferably 0.12-0.16 g / mL, more preferably 0.13-0.15 g / mL, and even more preferably 0.14 g / mL.

[0068] In this invention, in step (1), the concentration of cystamine dihydrochloride in the methanol solution of cystamine dihydrochloride is preferably 0.18-0.22 g / mL, more preferably 0.19-0.21 g / mL, and even more preferably 0.2 g / mL.

[0069] In this invention, in step (1), the process of mixing the methanol solution of di-tert-butyl dicarbonate, the methanol solution of cystamine dihydrochloride, and triethylamine is preferably as follows: after stirring and mixing the methanol solution of cystamine dihydrochloride and triethylamine, the methanol solution of di-tert-butyl dicarbonate is added dropwise; the stirring and mixing temperature is preferably 15-25°C, more preferably 20-24°C, and even more preferably 20°C; the stirring and mixing time is preferably 5-15 min, more preferably 8-12 min, and even more preferably 10 min.

[0070] In this invention, in step (1), the temperature of the first substitution reaction is preferably 15-25°C, more preferably 20-24°C, and even more preferably 20°C; the time of the first substitution reaction is preferably 2-4 hours, more preferably 2.5-3.5 hours, and even more preferably 3 hours.

[0071] In this invention, step (1) further includes the following steps after the first substitution reaction: adding purified water to the product of the first substitution reaction for washing and filtration, and then freeze-drying the filtrate.

[0072] In this invention, in step (2), the mass ratio of the activated mPEG-COOH polymer raw material mPEG-COOH polymer to the Boc-SS-NH2 polymer is preferably 1.5-2.5:0.8-1.2, more preferably 1.8-2.2:0.9-1.1, and even more preferably 2:1.

[0073] In this invention, in step (2), the temperature of the second substitution reaction is preferably 15-25°C, more preferably 15-20°C, and even more preferably 20°C; the time of the second substitution reaction is preferably 20-26h, more preferably 22-25h, and even more preferably 24h.

[0074] In this invention, step (2) preferably includes the following steps after the second substitution reaction: concentrating the product obtained from the second substitution reaction under reduced pressure to remove the organic solvent, precipitating it with excess ice-cold ether after cooling, filtering, and drying.

[0075] In this invention, in step (3), the method for preparing the activated indomethacin solution is as follows: indomethacin, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide and dimethyl sulfoxide are mixed and subjected to a second activation reaction to obtain the activated indomethacin solution.

[0076] In this invention, the preferred mass-to-volume ratio of indomethacin, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide, and dimethyl sulfoxide is 4–6 g: 0.7–1.8 g: 0.5–1.2 g: 80–120 mL, more preferably 4.5–6 g: 0.8–1.1 g: 0.7–1 g: 90–105 mL, and even more preferably 5 g: 1 g: 1 g: 100 mL; the preferred temperature for the second activation reaction is 45–65 °C, more preferably 50–60 °C, and even more preferably 50 °C; the preferred time for the second activation reaction is 0.5–1.75 h, more preferably 0.75–1.25 h, and even more preferably 1 h.

[0077] In this invention, the purpose of the second activation reaction is to activate the carboxyl group of indomethacin.

[0078] In this invention, in step (3), the concentration of the methanol solution of the mPEG-SS-Boc polymer is preferably 0.2 to 1 g / mL, more preferably 0.25 to 0.5 g / mL, and even more preferably 0.25 g / mL.

[0079] In this invention, in step (3), the mass-to-volume ratio of the mPEG-SS-Boc polymer to the HCl solution is preferably 1.5-4g:4-6.5mL, more preferably 2-3.5g:4.5-6mL, and even more preferably 2.5g:5mL.

[0080] In this invention, in step (3), the concentration of the HCl solution is preferably 1 to 3.5 mol·L⁻¹. -1 More preferably, it is 1.5–2.5 mol·L⁻¹ -1 More preferably 2 mol·L -1 .

[0081] In this invention, in step (3), the mass ratio of the mPEG-SS-Boc polymer to the indomethacin raw material used to prepare the activated indomethacin solution is preferably 4 to 6:1, more preferably 5 to 6:1, and even more preferably 5:1.

[0082] In this invention, the pH-adjusting agent in step (3) is preferably triethylamine; this invention does not limit the pH value for adjusting the pH to alkalinity, and any solution known to those skilled in the art can be used.

[0083] In this invention, in step (3), the reaction temperature under a protective atmosphere is preferably 15-25°C, more preferably 17-23°C, and even more preferably 20°C; the reaction time under a protective atmosphere is preferably 24-52h, more preferably 32-48h, and even more preferably 48h; the protective atmosphere is preferably nitrogen.

[0084] In this invention, in step (3), the dialysis is performed using purified water, and the dialysis time is preferably 24 to 48 hours, more preferably 36 to 48 hours, and even more preferably 48 hours.

[0085] In this invention, in step (4), the mass ratio of the PSI polymer to the paclitaxel is preferably 16-32:1.6-3.2, more preferably 20-28:2.0-2.8, and even more preferably 28:2.8.

[0086] In this invention, in step (4), the mass-to-volume ratio of the PSI polymer to the methanol is preferably 10-35 mg: 5-20 mL, more preferably 15-25 mg: 10-15 mL, and even more preferably 20 mg: 15 mL.

[0087] In this invention, in step (4), the temperature for removing methanol is preferably 30-70°C, more preferably 40-60°C, and even more preferably 45°C.

[0088] In this invention, in step (4), the temperature of the constant temperature hydration is preferably 30-70°C, more preferably 30-50°C, and even more preferably 40°C; the time of the constant temperature hydration is preferably 0.5-4h, more preferably 0.5-2h, and even more preferably 1h.

[0089] In this invention, step (4) further includes a filtration step after the constant temperature hydration, wherein the filter membrane used for filtration is preferably 0.45 μm.

[0090] The present invention also provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles, resulting in paclitaxel-loaded reduction-responsive prodrug micelles.

[0091] The present invention also provides the application of the aforementioned paclitaxel-loaded reduction-responsive prodrug micelles in the preparation of a medicament for treating breast cancer.

[0092] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0093] Experimental Example 1

[0094] The culture medium consisted of fetal bovine serum solution (osmolality of 280 mOmsmol / kg, volume of 10 mL) and penicillin-streptomycin solution (concentration of 10000 μg·mL). -1 It is prepared in a volume of 1 mL.

[0095] Weigh out the PTX and IND active pharmaceutical ingredients separately, dissolve them in DMSO, transfer them to a clean bench, dilute with culture medium, and prepare a concentration of 5 mg / mL. -1 PTX drug solutions with concentrations of 1.25, 2.5, 5, 10, and 20 mg / mL were used. -1 The IND drug solutions, with PTX to IND mass ratios of 4:1, 2:1, 1:1, 1:2, and 1:4, are PTX+IND combined drug solutions.

[0096] Paclitaxel-resistant human breast cancer cells (MCF-7 / PTX cells) in the logarithmic growth phase were digested with trypsin at 1000 rpm. -1 Centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant, add culture medium, and mix well. Add MCF-7 / PTX cells at a ratio of 10:1. 4The cells were seeded at the specified density in a 96-well plate. The outermost wells were supplemented with 100 μL of polybutylene succinate (PBS), and the remaining wells were supplemented with 100 μL of cell suspension. The cells were gently shaken to mix and then incubated in a constant temperature incubator. After 24 hours of incubation, the plate was removed and the culture medium was discarded. Each well was then supplemented with 100 μL of culture medium, different concentrations of PTX drug solution, different concentrations of IND drug solution, or five different ratios of PTX+IND combined drug solution. Each ratio of PTX+IND combined drug solution was prepared with five different concentrations: 0.25, 0.5, 1, 2, and 4 mg / mL. -1 Three replicates were set up for each concentration. After incubating the plate in an incubator for 48 hours, the plate was removed, and 10 μL of CCK-8 solution was added to each well. The plate was incubated for another 3 hours, and the OD value of each well at 425 nm was measured using a microplate reader. The cell viability was calculated using the following formula:

[0097]

[0098] OD 实验组 The OD values ​​of the experimental group cells (including cells, culture medium, CCK-8, and the test solution) are OD values. 对照组 The OD value of the control group cells (including cells, culture medium, and CCK-8) is shown. 空白组 The OD value of the blank group cells (including culture medium and CCK-8) is shown.

[0099] The drug effect, or inhibition rate (Fa), is calculated as: Fa = 1 - (A value of experimental group - A value of blank group) / (A value of control group - A value of blank group), where A is the absorbance at 425 nm. According to the intermediate-effect equation Fa / fu = (D / Dm)m, taking the logarithm of both sides, lg(Fa / fu) = mlgD - mlgDm. Let a = -mlgDm, b = m, x = lgD, y = lg(Fa / fu), resulting in the equation y = bx + a; where Fa is the drug effect, fu = 1 - Fa, D is the drug concentration, m is the slope, and Dm is the intermediate-effect concentration, i.e., the drug concentration at 0.5 effect points.

[0100] Based on the above formula, the intermediate-acting concentrations (Dm) of the two drugs, used alone and in combination, were calculated. Then, the corresponding drug concentrations (D) for various effects were calculated for both drugs, used alone or in combination. The interaction between PTX and IND was analyzed using Calcusyn software. The combination index (CI) was calculated using the Chou-Talalay method. The CI value can analyze the nature and degree of synergistic, additive, and antagonistic effects of PTX and IND when used in combination. The formula for the combination index is as follows:

[0101] CI = D1 / D m1 +D2 / D m2

[0102] Among them, D m1 D m2 The CI represents the dosage of each drug required to achieve the same therapeutic effect when used alone; D1 and D2 represent the dosages of drug 1 and drug 2 required when used in combination to achieve a certain therapeutic effect (e.g., a cell survival inhibition rate of 50%). CI>1 indicates that the two drugs are antagonistic, CI=1 indicates a simple additive effect, and CI<1 indicates that the two drugs have a synergistic effect when used in combination.

[0103] The synergistic index of the two drugs at different concentration ratios is shown in the table below. The results indicate that the CI values ​​of the two drugs at different ratios are all less than 1, suggesting a good synergistic effect. Furthermore, the CI values ​​of PTX:IND at drug ratios of 2:1 to 1:2 are all less than 0.3, indicating a strong synergistic effect. The synergistic index is lowest and the synergistic effect is strongest at PTX:IND = 1:1. The synergistic effect curves of FPX and IND obtained by the Chou-Talalay synergistic index method were plotted using Calcusyn software, and the results are shown below. Figure 1 As shown. According to Figure 1 (a) median effect curve and Figure 1 (b) The dose-response curves show that the combined use of PTX and IND is more effective than the use of PTX or IND alone. Figure 1 (c) The Fa-CI curve shows that the CI value is less than 1 at different ratios, which also indicates that the combination of PTX and IND has a good synergistic effect.

[0104] Table 1. IC50 under different PTX and IND combination drug ratios 50 Value and CI value

[0105]

[0106] Example 1

[0107] This embodiment provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles, including the following steps:

[0108] (1) Accurately weigh 0.3 g succinic anhydride (SA, i.e., 0.3 mmol) and 2 g mPEG (molecular weight 2000, 0.1 mmol), dissolve in 20 mL anhydrous toluene (the anhydrous toluene was evaporated to remove water beforehand using a rotary evaporator), heat to 110 °C and reflux for 70 min, concentrate under reduced pressure to remove the organic solvent, cool and precipitate with excess ice-cold diethyl ether, filter and dry to obtain mPEG-COOH polymer. The reaction equation is as follows:

[0109]

[0110] Accurately weigh 0.5g of mPEG-COOH polymer, 0.1g of N-hydroxysuccinimide (NHS) and 0.1g of N,N'-dicyclohexylcarbodiimide (DCC), dissolve them in 20mL of chloroform, and carry out the activation reaction at 20℃ for 5h to activate the carboxyl group of mPEG-COOH to obtain the activated mPEG-COOH polymer;

[0111] Weigh 0.7 g of di-tert-butyl dicarbonate and dissolve it in 5 mL of methanol; weigh 1.0 g of cystamine dihydrochloride and dissolve it in 5 mL of methanol, add 2.5 mL of triethylamine, stir at 20 °C for 10 min until completely dissolved, then slowly add the methanol solution of di-tert-butyl dicarbonate dropwise, react at 20 °C for 3 h, add 20 mL of purified water to wash and filter, freeze-dry the filtrate to obtain the Boc-SS-NH2 polymer. The reaction equation is as follows:

[0112]

[0113] (2) The activated mPEG-COOH polymer obtained in step (1) was mixed with 0.25 g of Boc-SS-NH2 polymer and reacted at 20 °C for 24 h. The organic solvent was removed by concentration under reduced pressure. After cooling, the polymer was precipitated with excess ice-cold ether, filtered and dried to obtain the mPEG-SS-Boc polymer. The reaction equation is as follows:

[0114]

[0115] (3) Accurately weigh 0.5g IND, 0.1g DCC, and 0.1g NHS and dissolve them in 10mL of dimethyl sulfoxide (DMSO). Stir at 50℃ for 1h to activate the carboxyl group of IND and obtain an activated indomethacin solution.

[0116] Weigh 2.5g of mPEG-SS-Boc polymer and dissolve it in 10mL of methanol. Add 5mL of 2mol·L⁻¹ to the solution. -1 The HCl solution was reacted at 20°C under nitrogen protection for 1 hour. After the reaction, the solvent was concentrated under reduced pressure. Then, activated indomethacin solution was added, and triethylamine was added to adjust the pH to 10.72. The reaction was carried out at 20°C under nitrogen protection for 48 hours. After the reaction, the mixture was dialyzed with purified water for 48 hours and then lyophilized to obtain the paclitaxel carrier, i.e., the PSI polymer. The reaction equation is as follows:

[0117]

[0118] (4) 20 mg of PSI polymer and 2.0 mg of paclitaxel were added to a reaction flask, and 10 mL of methanol was added. The mixture was sonicated under light-protected conditions to completely dissolve the polymer. After dissolution, the methanol was evaporated at 45 °C to form a thin film. The film was then placed at 30 °C for 1 h of constant temperature hydration and filtered through a 0.45 μm filter membrane to obtain paclitaxel-loaded reduction-responsive prodrug micelles, denoted as PSI / PTX. At this time, the mass ratio of PTX loaded in the prodrug micelles to IND grafted onto the PSI polymer was approximately 1:1.

[0119] Comparative Example 1

[0120] This comparative example provides a method for preparing paclitaxel-loaded PI prodrug micelles, including the following steps:

[0121] (1) Accurately weigh 0.358 g IND, 0.309 g DCC (1.5 mmol) and 0.122 g 4-diaminopyridine (DMAP, i.e., 1 mmol) and dissolve them in 10 mL DMSO. Stir at 50 °C for 1 h to activate the IND carboxyl group; weigh 4.5 g mPEG and dissolve it in 20 mL DMSO. Add the mPEG solution to the above activated IND solution and react at 20 °C for 24 h. After the reaction is complete, dialyze with purified water for 48 h and freeze dry to obtain the PI polymer. The reaction equation is as follows:

[0122]

[0123] (2) 20 mg of PI polymer and 2 mg of paclitaxel were added to a reaction flask, 10 mL of methanol was added, and the mixture was sonicated under light-protected conditions to completely dissolve it. After dissolution, the methanol was evaporated at 45 °C to form a thin film. The film was placed at 30 °C for 1 h of constant temperature hydration and filtered through a 0.45 μm filter membrane to obtain paclitaxel-loaded PI prodrug micelles, denoted as PI / PTX.

[0124] 1 H-NMR characterization

[0125] Nuclear magnetic resonance (NMR) was used to analyze mPEG-COOH polymers, Boc-SS-NH2 polymers, mPEG-SS-Boc polymers, PSI polymers, and PI polymers using deuterated DMSO as the solvent. 1 H-NMR characterization.

[0126] Example 1: The mPEG-COOH polymer obtained in step (1) 1 The H-NMR characterization results are as follows: Figure 2As shown, the chemical shifts are as follows: 12.16 ppm (-COOH), 3.38~3.63 ppm (-O-CH2-CH2-O-), 3.24 ppm (-OCH3), 2.40~2.48 ppm (-CH2-CH2-COOH). The characteristic peak of the α-carboxyl group (-COOH) appears in the figure, indicating that the synthesis of mPEG-COOH was successful.

[0127] Example 1: The Boc-SS-NH2 polymer obtained in step (1) 1 The H-NMR characterization results are as follows: Figure 3 As shown, the chemical shifts are as follows: 7.03 ppm is the characteristic peak of the amide bond (-CONH-), 2.68~3.50 ppm (-SS-CH2-CH2-), 1.37 ppm (-CH2-NH2), 1.19 ppm ((CH3)3-). The presence of the characteristic peak of the β-amide bond (-CONH-) in the figure indicates that the synthesis of Boc-SS-NH2 was successful.

[0128] Example 1 The mPEG-SS-Boc polymer prepared in step (2) 1 The H-NMR characterization results are as follows: Figure 4 As shown, the chemical shifts are as follows: 8.06 and 7.07 ppm are the characteristic peaks of the two amide bonds (-CONH-) in mPEG-SS-(a) and -SS-Boc-(b), 2.18 to 3.59 ppm (-SS-CH2-CH2-), and the carboxyl group (-COOH) peak at 12.16 ppm disappears, indicating that the synthesis of mPEG-SS-Boc was successful.

[0129] The PSI polymer obtained in step (3) of Example 1 1 The H-NMR characterization results are as follows: Figure 5 As shown, the chemical shifts are as follows: 8.07 ppm is the characteristic peak of the amide bond (-CONH-) at -SS-IND and mPEG-SS-, the characteristic peak of the amide bond (-SS-Boc-) at 7.07 ppm disappears, indicating that there is no -Boc structure in the polymer, 6.71~7.67 ppm is the characteristic peak of hydrogen on the benzene ring of IND, and 2.23~3.59 ppm (-SS-CH2-CH2-, -O-CH2-CH2-O-) indicate that the PSI polymer was successfully synthesized.

[0130] Comparative Example 1: The PI polymer obtained in step (1) 1 The H-NMR characterization results are as follows: Figure 6As shown, the chemical shifts are as follows: 6.70–7.68 ppm are the characteristic peaks of hydrogen on the benzene ring of IND, 3.47–3.79 ppm are the peaks of the methylene group adjacent to the ester bond formed between mPEG and IND (-COO-CH2-), 3.33–3.43 ppm (-O-CH2-CH2-O-), 3.24 ppm (-OCH3), and 2.22 ppm (-CH3), indicating that the PI polymer was successfully synthesized.

[0131] FT-IR characterization

[0132] Fourier transform infrared spectroscopy was used to characterize IND, PSI and PI polymers. IND, PSI and PI samples were mixed with 100 times their volume of spectral grade potassium bromide, slowly ground until homogeneous, compressed into tablets and detected.

[0133] Figure 7 The infrared spectrum of the PSI polymer obtained in step (3) of Example 1 is shown at 3446 cm⁻¹. -1 The peak for the NH stretching vibration of the amide bond is 2885 cm⁻¹. -1 The peak for CH stretching vibration is 1735 cm⁻¹. -1 The peak represents the stretching vibration of the carbonyl group (-C=O) in the mPEG segment, at 1627 cm⁻¹. -1 The peak represents the stretching vibration of amide I with the carbonyl group, at 1487 cm⁻¹. -1 The peak represents the in-plane bending vibration of NH in amide II, at 1281 cm⁻¹. -1 The presence of a CN vibration peak in amide III indicates successful synthesis of the PSI polymer.

[0134] Figure 9 The infrared spectrum of the PI polymer obtained in step (1) of Comparative Example 1 shows the main characteristic peak of PI at 2885 cm⁻¹. -1 This is a CH stretching vibration, 1735cm -1 This is the stretching vibration peak of the carbonyl group (-C=O) in the ester bond formed between mPEG and IND, at 1112 cm⁻¹. -1 The peak at this location corresponds to the stretching vibration of the COC (ether bond). (Through...) Figure 9 The infrared spectrum of the PI polymer shown is as follows: Figure 8 As can be seen from the comparison of the infrared spectra of IND shown, the PI polymer has a wavelength of 2885 cm⁻¹. -1 The PI polymer exhibits strong absorption at this point, while IND shows weaker absorption. This indicates that, compared to IND, the proportion of the CH stretching vibration peak in the PI polymer is significantly increased. Furthermore, compared to IND, the PI polymer structure shows a stronger absorption at 1691 cm⁻¹. -1 The absorption peak shifted towards higher wavenumbers, indicating that the hydroxyl group on the carboxyl group in the IND structure was replaced by other groups, meaning that IND and mPEG are linked by an ester bond, further demonstrating the successful synthesis of the PI polymer.

[0135] Figure 10 The particle size distribution diagram of PSI / PTX prepared in Example 1 shows that the particle size of the prodrug micelles is 170.2 ± 2.56 nm. Figure 11 The Zeta potential diagram of the PSI / PTX prepared in Example 1 shows that the potential of the prodrug micelles is -28.01±0.06mV. Figure 12 The image shows a TEM image of the PSI / PTX prepared in Example 1. The results show that the prodrug micelles have small particle size and are evenly distributed.

[0136] Results of in vitro drug release experiments as follows Figure 13 and Figure 14 As shown, in a glutathione (GSH)-containing medium, the cumulative release rates of PTX and IND in the PSI / PTX prodrug micelles after 48 hours were 92.96% and 75.62%, respectively. In a GSH-free medium, the cumulative release rates after 48 hours were only 17.59% and 13.59%, respectively. This indicates that in the presence of GSH, disulfide bonds can break to generate thiol groups, disrupting the micelle structure and thus rapidly releasing the drug. This verifies that the prodrug micelles prepared in this invention possess reduction-sensitive drug release characteristics, which facilitates rapid drug release triggered by the tumor cell microenvironment after the carrier is taken up by tumor cells, enhancing the anti-tumor effect.

[0137] The ability of prodrug micelles from Example 1 to enter MCF-7 / PTX cells was observed using an inverted fluorescence microscope. The lipid-soluble fluorescent dye coumarin 6 (Cou6) was used instead of PTX in the experiment, and the lipid-soluble fluorescent dye CY5 was used instead of IND. DAPI staining solution was used to stain the cell nuclei. The targeted nanodrugs and non-targeted nanodrugs were incubated with MCF-7 / PTX for 4 hours and then observed under an inverted fluorescence microscope. Results are as follows: Figure 15 As shown, after incubation with targeted prodrug micelles, the intracellular fluorescence intensity of MCF-7 / PTX cells was significantly higher than that of cells in the non-targeted nanodrug group. This indicates that the paclitaxel-loaded reduced prodrug micelles have breast cancer cell targeting properties, are more easily taken up by breast cancer cells, and are beneficial for drug delivery.

[0138] The inhibitory effects of different drug groups on the migration ability of MCF-7 / PTX cells were analyzed using a cell scratch assay. Eight groups were set up: PTX, IND, and PTX / IND (1:1 strong synergistic ratio) active pharmaceutical ingredients, PSI / PTX and PI / PTX prodrug micelles, PSI and PI polymer groups, and a blank control (cells only). Monolayer cells were scratched using sterile 10 μL pipette tips. After washing with PBS, different drug groups were added. Samples were taken and photographed under a microscope at 0 and 48 h. The photographed locations were the same at each time point. The results are shown below. Figure 16As shown, microscopic observation after 48 hours revealed that the PSI / PTX prodrug micelle group exhibited a stronger inhibitory effect on cell migration, indicating that the PSI / PTX prodrug micelles released a greater amount of drug intracellularly. This suggests that the PSI / PTX prodrug micelles are more stable extracellularly, and the high GSH environment intracellularly allows for faster and greater drug release, resulting in a better inhibitory effect on MCF-7 / PTX cell migration.

[0139] The anti-breast cancer efficacy of prodrug micelles was evaluated using the CCK-8 cytotoxicity assay. MCF-7 and MCF-7 / PTX cells were cultured at 1×10⁻⁶ cells / cells. 4 Seeds were seeded at a density of 100 cells / well in 96-well plates and incubated overnight. 100 μL of culture medium containing different polymers (PI and PSI) was added to achieve final polymer concentrations of 1, 10, 50, 100, and 200 μg / mL. -1 After incubation for 48 hours, 10 μL of LCK-8 solution was added to each well, and incubation continued for another 3 hours. The absorbance at 425 nm was then measured using a microplate reader. The results are as follows: Figure 17 , 18 As shown in the figure. The results indicate that after treatment with different concentrations of polymer, the survival rates of MCF-7 and MCF-7 / PTX cells were both above 90%, indicating that PI and PSI polymers did not have significant toxic effects on these two cell types.

[0140] MCF-7 and MCF-7 / PTX cells were loaded at 1×10 4 Seeds were seeded at a density of [number] micelles / well in 96-well plates and incubated overnight. Targeted prodrug micelles and non-targeted prodrug micelles (PSI / PTX prepared in Example 1 and PI / PTX prepared in Comparative Example 1) were added, and incubation was continued for 48 hours. 10 μL of CCK-8 solution was added to each well, and incubation was continued for another 3 hours. The absorbance at 425 nm was then measured using a microplate reader. The results are as follows: Figure 19 , 20 As shown in the figure. The results indicate that the PSI / PTX prodrug micelles exhibit stronger cytotoxicity, which is attributed to the breakage of the -SS- bonds in the carrier material under the stimulation of high intracellular GSH concentrations, resulting in greater drug release and stronger cytotoxicity to MCF-7 and MCF-7 / PTX cells.

[0141] Intracellular MRP1 protein expression was determined using Western blot. VEGFR-2 is an important tyrosine kinase receptor; inhibiting VEGFR-2 activation can suppress tumor cell activity and has a synergistic inhibitory effect on tumor cells. To investigate the effect of different polymeric micelle solutions on protein expression, five groups were set up: PTX, IND, PTX / IND, PSI / PTX, and a blank control (containing only cells). First, protein was extracted from MCF-7 / PTX, then protein quantification was performed using a BCA protein assay kit, and finally, MRP1 protein expression was analyzed by Western blot. The results are shown below. Figure 21 As shown in the results, MRP1 protein expression was high in MCF-7 / PTX cells. PTX alone had no significant effect on expression, while IND, PTX+IND raw materials, and PSI / PTX prodrug micelles significantly downregulated MRP1 protein expression. This indicates that IND can reverse cell resistance to PTX, increase its sensitivity, promote apoptosis in drug-resistant MCF-7 / PTX cells, and improve the therapeutic effect of breast cancer.

[0142] Example 2

[0143] This embodiment provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles. The difference from Example 1 is that the amount of paclitaxel in step (4) is changed from 2.0 mg to 1.6 mg, while the other conditions and steps are the same as in Example 1.

[0144] Example 3

[0145] This embodiment provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles. The difference from Example 1 is that the amount of paclitaxel in step (4) is changed from 2.0 mg to 2.4 mg, while the other conditions and steps are the same as in Example 1.

[0146] Example 4

[0147] This embodiment provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles. The difference from Example 1 is that the amount of paclitaxel in step (4) is changed from 2.0 mg to 2.8 mg, while the other conditions and steps are the same as in Example 1.

[0148] Example 5

[0149] This embodiment provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles. The difference from Example 1 is that the amount of paclitaxel in step (4) is changed from 2.0 mg to 3.2 mg, while the other conditions and steps are the same as in Example 1.

[0150] The encapsulation efficiency, drug loading, and particle size of the PSI / PTX prodrug micelles obtained in Examples 1-5 were determined, and the effect of PTX dosage on the properties of the prodrug micelles was investigated. The results are as follows: Figure 22 As shown. From Figure 22 It can be seen that different dosages have a significant impact on the prodrug micelle size, encapsulation efficiency, and drug loading. The drug loading of the prodrug micelles is directly proportional to the PTX dosage; the more PTX is added, the more drug is encapsulated in the prodrug micelles. The PSI / PTX prodrug micelle encapsulation efficiency initially increases and then decreases with increasing dosage. This may be because the hydrophobic core space of the prodrug micelles is limited, and the encapsulation of PTX in the hydrophobic core has reached saturation, thus the encapsulation efficiency decreases. The particle size decreases with increasing dosage, possibly because PTX is encapsulated within the hydrophobic core of the prodrug micelles. The strong interaction between PTX and the IND in the hydrophobic core of the prodrug micelles reduces the core space, thereby decreasing the overall particle size of the prodrug micelles. Considering the trends in particle size, encapsulation efficiency, and drug loading, a dosage of 2.0–2.8 mg was selected.

[0151] Example 6

[0152] This embodiment provides a method for preparing paclitaxel-loaded reduction-responsive prodrug micelles, including the following steps:

[0153] Steps (1) to (3) are the same as steps (1) to (3) in Example 1;

[0154] (4) 28 mg of PSI polymer and 2.8 mg of paclitaxel were added to a reaction flask, and 15 mL of methanol was added. The mixture was sonicated under light-protected conditions to completely dissolve the polymer. After dissolution, the methanol was evaporated at 45 °C to form a thin film. The film was then placed at 40 °C for 1 h of constant temperature hydration and filtered through a 0.45 μm filter membrane to obtain paclitaxel-loaded reduction-responsive prodrug micelles, denoted as PSI / PTX. At this time, the mass ratio of PTX loaded in the prodrug micelles to IND grafted onto the PSI polymer was approximately 1:1.

[0155] Tests showed that the prodrug micelles prepared in Example 6 had the highest encapsulation efficiency and drug loading, smaller particle size, and better reproducibility. Three repeated tests of Example 6 showed minimal deviations in various indicators, indicating that the preparation process of this invention is stable, and the actual drug ratio of PTX to IND in the dosage form is close to 1:1, ensuring a strong synergistic effect between the two drugs.

[0156] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing paclitaxel-loaded reduction-responsive prodrug micelles for treating breast cancer, characterized in that, Includes the following steps: (1) Succinic anhydride, polyethylene glycol and anhydrous toluene are mixed and heated to undergo reflux reaction to obtain mPEG-COOH polymer; mPEG-COOH polymer, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide and chloroform are mixed to undergo first activation reaction to obtain activated mPEG-COOH polymer; A methanol solution of ditert-butyl dicarbonate, a methanol solution of cystamine dihydrochloride, and triethylamine were mixed to carry out a first substitution reaction to obtain the Boc-SS-NH2 polymer. (2) The activated mPEG-COOH polymer is mixed with the Boc-SS-NH2 polymer to carry out a second substitution reaction to obtain the mPEG-SS-Boc polymer; (3) Add HCl solution to the methanol solution of mPEG-SS-Boc polymer to carry out amide condensation reaction, then add activated indomethacin solution to adjust the pH to alkaline, react under a protective atmosphere, dialyze and freeze dry to obtain the paclitaxel carrier, i.e. PSI polymer. In step (3), the activated indomethacin solution is prepared by mixing indomethacin, N,N'-dicyclohexylcarbodiimide, N-hydroxysuccinimide and dimethyl sulfoxide to carry out a second activation reaction to obtain the activated indomethacin solution. In step (3), the mass ratio of the mPEG-SS-Boc polymer to the indomethacin used to prepare the activated indomethacin solution is 4~6:1; (4) PSI polymer, paclitaxel and methanol were mixed under light-protected conditions, and the methanol was removed after dissolution. Then, constant temperature hydration was performed to obtain paclitaxel-loaded reduction-responsive prodrug micelles for the treatment of breast cancer. In step (4), the mass ratio of the PSI polymer to the paclitaxel is 16~32:1.6~3.

2.

2. The method for preparing paclitaxel-loaded reduction-responsive prodrug micelles for treating breast cancer as described in claim 1, characterized in that, In step (1), the mass-to-volume ratio of the ditert-butyl dicarbonate, the cystamine dihydrochloride, and the triethylamine is 0.5~0.8 g: 0.9~1.1 g: 2.4~2.7 mL; the time for the first substitution reaction is 2~4 h.

3. A method for preparing paclitaxel-loaded reduction-responsive prodrug micelles for treating breast cancer as described in claim 1 or 2, characterized in that, In step (2), the mass ratio of the activated mPEG-COOH polymer to the Boc-SS-NH2 polymer is 1.5~2.5:0.8~1.2; the second substitution reaction takes 20~26 h.

4. A method for preparing paclitaxel-loaded reduction-responsive prodrug micelles for treating breast cancer as described in claim 1 or 2, characterized in that, In step (3), the reaction temperature under a protective atmosphere is 15~25 °C, and the reaction time under a protective atmosphere is 24~52 h.

5. The method for preparing paclitaxel-loaded reduction-responsive prodrug micelles for treating breast cancer as described in claim 1, characterized in that, In step (4), the mass ratio of the PSI polymer to the paclitaxel is 28:2.

8.

6. A method for preparing paclitaxel-loaded reduction-responsive prodrug micelles for treating breast cancer according to any one of claims 1 to 5, resulting in paclitaxel-loaded reduction-responsive prodrug micelles for treating breast cancer.

7. The use of the paclitaxel-loaded reduction-responsive prodrug micelles of claim 6 for treating breast cancer in the preparation of a medicament for treating breast cancer.

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