A dual-sensitivity micelle co-loaded with emodin and chlorambucil, its preparation method and application

Abstract

This invention belongs to the field of nanomedicine delivery systems, specifically relating to a dual-sensitivity co-loaded emodin and chlorambucil micelles, their preparation method, and applications. The polymer structure is as follows: Using polymethacrylate as the linking unit, this invention designs and synthesizes a dendritic polymer p(mPEG-co-HPBE-co-EMD) grafted with polyethylene glycol monomethyl ether, p-hydroxymethylphenylboronic acid pinacol ester, and emodin, and uses it for encapsulating chlorambucil. This co-loaded polymer micelle can be used for the co-delivery of emodin and chlorambucil. This co-loaded micelle improves the water solubility of the drug, has a suitable particle size, and dual ROS / pH sensitivity, enabling targeted release of emodin and chlorambucil at tumor sites, achieving synergistic treatment with chemotherapy and oxidative therapy. In the formula, n = 30-85, x:y:z = 1:2-5:1-3.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery systems, specifically relating to a dual-sensitivity co-loaded emodin and chlorambucil micelles, their preparation method, and their applications. Background Technology

[0002] Polymer micelles have been widely studied in the field of cancer treatment due to their advantages such as suitable particle size, good stability, increased solubility of poorly soluble drugs, targeted and slow release of drugs to tumor sites, reduced adverse reactions, and improved drug bioavailability. However, the therapeutic efficiency of conventional polymer micelles is greatly limited by various drug barriers in vivo, such as adsorption by plasma proteins, phagocytosis by macrophages, and clearance by the kidneys.

[0003] Emodin (EMD) is a hydroxyanthraquinone compound that has therapeutic effects on various tumors and can upregulate ROS levels in tumor cells. However, its extremely low water solubility limits its clinical application. Chlorambucil (CLB) is a broad-spectrum antitumor drug, but its poor water solubility and tendency to bind to GSH and be effluxed, leading to drug resistance, also limit its in vivo antitumor efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide a dual-sensitivity co-loaded emodin and chlorambucil micelle, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A ROS / pH dual-sensitive co-supported polymer has the following polymer structural formula.

[0007]

[0008] In the formula, n = 30 - 85, x:y:z (number of substituents) = 1:2 - 5:1 - 3.

[0009] An application of the ROS / pH dual-sensitive polymer, wherein the polymer is used as a hydrophobic drug carrier.

[0010] A dual-sensitivity co-loaded emodin and chlorambucil micelle, wherein the co-loaded micelles are the polymer and chlorambucil as described in claim 1, wherein the mass ratio of chlorambucil to polymer is 1:5-10.

[0011] The preparation of the polymer is as follows:

[0012] Step (1): Synthesis of mPEG-MA reactant monomers: Polyethylene glycol monomethyl ether, methacryloyl chloride and triethylamine are dissolved in solvent A in a molar ratio of 1:1-3:1-3 and reacted at room temperature for 8-12 hours. The mixture is then precipitated 2-3 times in solvent B. The filter cake is then vacuum dried to obtain the mPEG-MA monomer compound.

[0013] The specific reaction formula is as follows:

[0014]

[0015] Step (2): Synthesis of HPBE-MA reactant monomers: Pinacol ester of p-hydroxymethylphenylboronic acid, methacrylamide chloride, and triethylamine were dissolved in solvent A in a molar ratio of 1:1-3:1-3 and reacted at room temperature for 8-12 hours. The HPBE-MA monomer compound was obtained by washing, drying, and purification. The washing, drying, and purification were carried out by washing with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and deionized water in sequence, drying with anhydrous sodium sulfate, and purification by silica gel column chromatography.

[0016] The specific reaction formula is as follows:

[0017]

[0018] Step (3): Synthesis of EMD-MA reactant monomers: Emodin, methacryloyl chloride, and triethylamine were dissolved in solvent A in a molar ratio of 1:1-3:1-3 and reacted at room temperature for 8-12 hours. The EMD-MA monomer compounds were obtained by washing, drying, and purification. The washing, drying, and purification were carried out by washing with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and deionized water in sequence, drying with anhydrous sodium sulfate, and purification by silica gel column chromatography.

[0019] The specific reaction formula is as follows:

[0020]

[0021] Step (4): Synthesis of p(mPEG-co-HPBE-co-EMD) polymer. The mPEG-MA obtained in step (1), HPBE-MA obtained in step (2), and EMD-MA obtained in step (3) are mixed in toluene at a molar ratio of 1:3-5:2. AIBN is added, and the reaction is carried out at 65°C for 20-24 hours under nitrogen protection. The mixture is precipitated 2-3 times in solvent B, redissolved in acetone, purified by dialysis, and freeze-dried to obtain p(mPEG-co-HPBE-co-EMD) polymer.

[0022] The specific reaction formula is as follows:

[0023]

[0024] The molecular weight of the polyethylene glycol monomethyl ether mentioned in step (1) is 1500-4000; the amount of AIBN used in step (4) is 0.2-4.0% of the total mass of the monomers.

[0025] Solvent A mentioned in steps (1), (2) and (3) is any one of dichloromethane, trichloromethane, and acetone.

[0026] Solvent B mentioned in steps (1) and (4) can be any one of n-hexane, diethyl ether, or petroleum ether.

[0027] A method for preparing dual-sensitivity co-loaded emodin and chlorambucil micelles involves mixing chlorambucil and polymer p(mPEG-co-HPBE-co-EMD) at a mass ratio of 1:5-10 in solvent C, dispersing the mixture uniformly by ultrasonication, removing solvent C by rotary evaporation, adding a hydration medium at 30-50℃ for uniform hydration and dispersion, and filtering to obtain p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles.

[0028] The specific reaction formula is as follows:

[0029]

[0030] The solvent C mentioned in step (5) is any one of methanol-acetone mixed solution and tetrahydrofuran; the hydration medium is any one of deionized water, PBS and physiological saline.

[0031] An application of the aforementioned dual-sensitivity co-loaded emodin and chlorambucil micelles, specifically their application in the preparation of antitumor drugs.

[0032] The beneficial effects of this invention are:

[0033] This invention designs and synthesizes a dendritic polymer p(mPEG-co-HPBE-co-EMD) grafted with polyethylene glycol monomethyl ether, p-hydroxymethylbenzyl borate, and emodin, using poly(methacrylate) as a linker unit. This polymeric micelle is then used to encapsulate chlorambucil. This co-loaded polymer micelle can be used for the co-delivery of emodin and chlorambucil. The co-loaded micelle improves the water solubility of the drug, possesses suitable particle size and dual ROS / pH sensitivity, enabling targeted release of emodin and chlorambucil at the tumor site, achieving synergistic treatment with chemotherapy and oxidative therapy. Attached Figure Description

[0034] Figure 1 This is the 1H NMR spectrum of mPEG-MA.

[0035] Figure 2This is the hydrogen NMR spectrum of EMD-MA.

[0036] Figure 3 This is the 1H NMR spectrum of HPBE-MA.

[0037] Figure 4 The image shows the proton NMR spectrum of p(mPEG-co-HPBE-co-EMD).

[0038] Figure 5 Transmission electron microscopy (TEM) image of p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles.

[0039] Figure 6 This is a graph showing the particle size variation of p(mPEG-co-HPBE-co-EMD) in environments with high levels of H2O2 or low pH.

[0040] Figure 7 This is a graph comparing the changes in tumor volume and tumor inhibition rate after treatment with p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles.

[0041] Figure 8 The images show the changes in body weight and HE staining of major organs in mice treated with p(mPEG-co-HPBE-co-EMD)@CLB co-loaded micelles. Detailed Implementation

[0042] The following description, in conjunction with the accompanying drawings, further illustrates an embodiment of the method for preparing a dual-sensitive co-loaded emodin and chlorambucil micelles according to the present invention.

[0043] This invention utilizes methacrylate as the linking unit, polyethylene glycol monomethyl ether (mPEG) as the hydrophilic end, and EMD and pinacol hydroxymethylphenylboronic acid (HPBE), which exhibits dual ROS / pH sensitivity, as the hydrophobic ends to prepare an amphiphilic dendritic polymer p(mPEG-co-HPBE-co-EMD), which is then used to encapsulate hydrophobic CLBs. This polymer micelle exhibits dual sensitivity to high levels of ROS and low pH in the tumor microenvironment, enabling it to induce oxidative damage by promoting ROS generation and consuming GSH, and to achieve targeted therapy of tumor cells in synergistic with chemotherapy.

[0044] Example 1

[0045] Step (1): Polyethylene glycol monomethyl ether, methacryloyl chloride, and triethylamine were dissolved in dichloromethane at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 hours. The mixture was precipitated three times in anhydrous diethyl ether, and the filter cake was dried under vacuum to obtain the mPEG-MA monomer compound.

[0046] like Figure 1The image shows the 1H NMR spectrum (400MHz, CDCl3) of the mPEG-MA monomer. Characteristic peaks for -CH=CH- are 6.13ppm and 5.58ppm, for -O-CH2- is 4.30ppm, for -O-CH2-CH2-O- is 3.5-3.8ppm, for -OCH3 is 3.38ppm, and for -CH3 is 1.98ppm.

[0047] Step (2): Pinacol ester of p-hydroxymethylphenylboronic acid, methacrylamide chloride, and triethylamine were dissolved in dichloromethane at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 h. After washing with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and deionized water, the mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether, v / v = 1:5) to obtain the HPBE-MA monomer compound.

[0048] like Figure 2 The image shows the 1H NMR spectrum (400MHz, CDCl3) of the HPBE-MA monomer. The peaks at 7.80ppm and 7.37ppm are characteristic peaks of Ph-H, 6.15ppm and 5.58ppm are characteristic peaks of -CH=CH-, 5.20ppm is a characteristic peak of -O-CH2, 1.96ppm is a characteristic peak of -CH3, and 1.34ppm is a characteristic peak of -CH3 in the pinacol ester structure.

[0049] Step (3): Synthesis of EMD-MA reactant monomers. Emodin, methacryloyl chloride, and triethylamine were dissolved in acetone at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 hours. The mixture was washed sequentially with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and deionized water. After washing, the mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (mobile phase: methanol / dichloromethane, v / v = 1:10) to obtain the EMD-MA monomer compound.

[0050] like Figure 3 The image shows the 1H NMR spectrum (400MHz, CDCl3) of the EMD-MA monomer. Characteristic peaks for Ar-OH are 12.20ppm and 11.97ppm, for Ar-H are 7.65ppm, 7.58ppm, and 7.11ppm, for -CH=CH- are 6.40ppm and 5.85ppm, for Ar-CH3 is 2.45ppm, and for -CH3 is 2.08ppm.

[0051] Step (4): Dissolve the mPEG-MA obtained in step (1), HPBE-MA obtained in step (2), and EMD-MA obtained in step (3) in toluene at a molar ratio of 1:5:2, add 2.0% AIBN, react at 65°C for 24 h under nitrogen protection, precipitate three times in anhydrous diethyl ether, redissolve in acetone, and then dialyze in a dialysis bag with a molecular weight cutoff of 3500 for 3 days. Freeze-dry to obtain p(mPEG-co-HPBE-co-EMD) polymer, where n = 36-40.

[0052] like Figure 4 The image shows the 1H NMR spectrum (400MHz, CDCl3) of the p(mPEG-co-HPBE-co-EMD) polymer. The peaks are: 11.65–12.34 ppm for Ar-OH, 6.72–8.25 ppm for Ph-H and Ar-H, 4.02–5.76 ppm for -O-CH2-, 3.25–4.02 ppm for -O-CH2-CH2-O- and -OCH3, and 0.63–2.58 ppm for Ar-CH3 and -CH3. Further analysis shows that x:y:z = 1:4.3:1.7.

[0053] Step (5): Chlorobutachlor and p(mPEG-co-HPBE-co-EMD) obtained in step (4) are mixed at a mass ratio of 1:5 and dissolved in a methanol-acetone mixed solution. The mixture is ultrasonically dispersed, the organic solvent is removed by rotary evaporation, and the mixture is hydrated and dispersed evenly in a hydration medium at 40°C. The mixture is then filtered through a 0.22 μm microporous membrane to obtain p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles. The drug loading of EMD is 10.2%, and the drug loading of CLB is 13.4%.

[0054] like Figure 5 The image shown is a transmission electron microscope (TEM) image of p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles. The particle size of the p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles is approximately 35 nm, with a uniform particle size distribution and a near-spherical shape.

[0055] Example 2

[0056] Step (1): Polyethylene glycol monomethyl ether, methacryloyl chloride, and triethylamine were dissolved in dichloromethane at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 hours. The mixture was precipitated three times in anhydrous diethyl ether, and the filter cake was dried under vacuum to obtain the mPEG-MA monomer compound.

[0057] Step (2): Pinacol ester of p-hydroxymethylphenylboronic acid, methacrylamide chloride, and triethylamine were dissolved in dichloromethane at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 h. After washing with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and deionized water, the mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether, v / v = 1:5) to obtain the HPBE-MA monomer compound.

[0058] Step (3): Synthesis of EMD-MA reactant monomers. Emodin, methacryloyl chloride, and triethylamine were dissolved in acetone at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 hours. The mixture was washed sequentially with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and deionized water. After washing, the mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (mobile phase: methanol / dichloromethane, v / v = 1:10) to obtain the EMD-MA monomer compound.

[0059] Step (4): Dissolve the mPEG-MA obtained in step (1), HPBE-MA obtained in step (2), and EMD-MA obtained in step (3) in toluene at a molar ratio of 1:4:2, add 2.0% AIBN, react at 65°C for 24 h under nitrogen protection, precipitate three times in anhydrous diethyl ether, redissolve in acetone, and then dialyze in a dialysis bag with a molecular weight cutoff of 3500 for 3 days. Freeze-dry to obtain p(mPEG-co-HPBE-co-EMD) polymer, where n = 36-40.

[0060] Step (5): Mix chlorambucil and p(mPEG-co-HPBE-co-EMD) obtained in step (4) at a mass ratio of 1:5 in a methanol-acetone mixed solution, ultrasonically disperse evenly, remove organic solvent by rotary evaporation, add hydration medium at 40℃ to hydrate and disperse evenly, and filter through a 0.22μm microporous membrane to obtain p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles.

[0061] Example 3

[0062] Step (1): Polyethylene glycol monomethyl ether, methacryloyl chloride, and triethylamine were dissolved in dichloromethane at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 hours. The mixture was precipitated three times in anhydrous diethyl ether, and the filter cake was dried under vacuum to obtain the mPEG-MA monomer compound.

[0063] Step (2): Pinacol ester of p-hydroxymethylphenylboronic acid, methacrylamide chloride, and triethylamine were dissolved in dichloromethane at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 h. After washing with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and deionized water, the mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (mobile phase: ethyl acetate / petroleum ether, v / v = 1:5) to obtain the HPBE-MA monomer compound.

[0064] Step (3): Synthesis of EMD-MA reactant monomers. Emodin, methacryloyl chloride, and triethylamine were dissolved in acetone at a molar ratio of 1:1.5:1.5 and reacted at room temperature for 12 hours. The mixture was washed sequentially with dilute hydrochloric acid solution, saturated sodium bicarbonate solution, and deionized water. After washing, the mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (mobile phase: methanol / dichloromethane, v / v = 1:10) to obtain the EMD-MA monomer compound.

[0065] Step (4): Dissolve the mPEG-MA obtained in step (1), HPBE-MA obtained in step (2), and EMD-MA obtained in step (3) in toluene at a molar ratio of 1:3:2, add 2.0% AIBN, react at 65°C for 24 h under nitrogen protection, precipitate three times in anhydrous diethyl ether, redissolve in acetone, and then dialyze in a dialysis bag with a molecular weight cutoff of 3500 for 3 days. Freeze-dry to obtain p(mPEG-co-HPBE-co-EMD) polymer, where n = 36-40.

[0066] Step (5): Mix chlorambucil and p(mPEG-co-HPBE-co-EMD) obtained in step (4) at a mass ratio of 1:5 in a methanol-acetone mixed solution, ultrasonically disperse evenly, remove organic solvent by rotary evaporation, add hydration medium at 40℃ to hydrate and disperse evenly, and filter through a 0.22μm microporous membrane to obtain p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles.

[0067] Application examples

[0068] The following describes the dual sensitivity and pharmacodynamic studies of the dual-sensitivity micelles co-loaded with emodin and chlorambucil, based on specific application examples.

[0069] The environmental sensitivity of the polymer p(mPEG-co-HPBE-co-EMD) prepared in Example 1 was investigated by dynamic light scattering (DLS), and the particle size change of the polymer was measured in environments with 10 mM hydrogen peroxide (H2O2) and pH 5.0.

[0070] like Figure 6 The figure shows the particle size variation of p(mPEG-co-HPBE-co-EMD) in environments with high levels of H2O2 or low pH. As time increases, the micelle size of the polymer p(mPEG-co-HPBE-co-EMD) rapidly exhibits a multi-peak state, indicating that it has dual ROS / pH sensitivity.

[0071] The mPEG-MA and HPBE-MA monomers prepared in Example 1 were dissolved in toluene at a molar ratio of 1:7, and 2.0% AIBN was added. The mixture was reacted at 65°C for 24 hours under nitrogen protection, precipitated three times in anhydrous diethyl ether, and then reconstituted in acetone. The resulting product was then placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed for 3 days. Finally, it was freeze-dried to obtain the p(mPEG-co-HPBE) polymer.

[0072] Chlorobutyric acid mustard and p(mPEG-co-HPB) were mixed at a mass ratio of 1:5 and dissolved in a methanol-acetone mixture. The mixture was ultrasonically dispersed, and the organic solvent was removed by rotary evaporation. The mixture was then hydrated and dispersed uniformly in a hydration medium at 40°C. Finally, the mixture was filtered through a 0.22 μm microporous membrane to obtain p(mPEG-co-HPBE)@CLB single-drug micelles.

[0073] The p(mPEG-co-HPB-co-EMD) prepared in Example 1 was dissolved in a methanol-acetone mixed solution, ultrasonically dispersed, and the organic solvent was removed by rotary evaporation. The mixture was then hydrated and dispersed uniformly in a hydration medium at 40°C, and filtered through a 0.22 μm microporous membrane to obtain p(mPEG-co-HPBE-co-EMD) single drug-loaded micelles.

[0074] Using the p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles prepared in Example 1 as a model, and the p(mPEG-co-HPBE) and p(mPEG-co-HPBE-co-EMD) single-loaded drug micelles obtained above as controls, the pharmacodynamic properties of the dual-sensitive co-loaded emodin and chlorambucil micelles of the present invention were investigated.

[0075] The animal model used in the following experiments was established using the ectopic transplantation method. Specifically, the following steps were followed: Six-week-old Balb / c mice were used, and 4T1 cell suspension (5 × 10⁻⁶ cells) was subcutaneously injected into the right axilla. 6 (Number of tumors / mL, 200μL / tumor). Wait until the tumor grows to 100-150mm. 3 The administration began at a specific time. Mice were randomly divided into 4 groups and administered the drug once every 2 days. Each group received a tail vein injection of saline, p(mPEG-co-HPBE-co-EMD), p(mPEG-co-HPBE)@CLB, or p(mPEG-co-HPBE-co-EMD)@CLB, respectively, at a fixed dose (CLB: 6 mg / kg, EMD: 4.5 mg / kg). The administration was repeated for 5 consecutive times, and tumor volume (V) was recorded. Mouse tumor volume was calculated using the formula V = (a × b) / ... 2 The value is calculated as 1 / 2, where a is the long diameter of the tumor and b is the short diameter of the tumor. After the drug administration cycle ended, mice were sacrificed on day 14 after the start of drug administration. The major organs of each group of mice were isolated, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and then subjected to HE staining analysis.

[0076] like Figure 7 The figure shows a comparison of tumor volume changes and tumor inhibition rates after treatment with p(mPEG-co-HPBE-co-EMD)@CLB co-loaded micelles. Under the above dosing regimens, the tumor inhibition rates of p(mPEG-co-HPBE-co-EMD), p(mPEG-co-HPBE)@CLB, and p(mPEG-co-HPBE-co-EMD)@CLB were 37.4%, 57.6%, and 67.7%, respectively. Among them, the dual-sensitivity micelles co-loaded with emodin and chlorambucil showed the slowest tumor growth rate and the best tumor inhibition effect.

[0077] like Figure 8 The image shows the changes in body weight and HE staining of major organs in mice treated with p(mPEG-co-HPBE-co-EMD)@CLB co-loaded micelles. During the administration period, there were no significant changes in body weight in any group of mice, indicating good safety. No inflammation or necrosis was observed in the major organs of both the drug-treated group and the Saline-injected group via HE staining, suggesting that p(mPEG-co-HPBE-co-EMD), p(mPEG-co-HPBE)@CLB, and p(mPEG-co-HPBE-co-EMD)@CLB have no significant toxic side effects on any organ in mice and exhibit good in vivo safety.

[0078] The above results demonstrate that the dual-sensitivity co-loaded emodin and chlorambucil micelles p(mPEG-co-HPBE-co-EMD)@CLB prepared in this invention exhibit dual ROS / pH sensitivity, enabling targeted drug release in the tumor environment with minimal toxicity and good in vivo safety. Furthermore, they demonstrate stronger antitumor activity compared to single-loaded micelles of emodin or chlorambucil.

Claims

1. A ROS / pH dual-sensitive co-supported polymer, characterized in that: The polymer structural formula is as follows: p(mPEG-co-HPBE-co-EMD) In the formula, n = 30 - 85, x:y:z (number of substituents) = 1:2 - 5:1 - 3.

2. An application of the ROS / pH dual-sensitive co-supported polymer according to claim 1, characterized in that: The polymer is used as a hydrophobic drug carrier.

3. A dual-sensitivity micelle co-loaded with emodin and chlorambucil, characterized in that: The co-loaded drug micelles are the polymer and chlorambucil as described in claim 1, wherein the mass ratio of chlorambucil to polymer is 1:5-10.

4. The dual-sensitivity co-loaded emodin and chlorambucil micelles according to claim 3, characterized in that: The preparation of the polymer is as follows: Step (1): Synthesis of mPEG-MA reactant monomers: dissolve polyethylene glycol monomethyl ether, methacryloyl chloride and triethylamine in solvent A at a molar ratio of 1:1-3:1-3, react at room temperature for 8-12 h, precipitate 2-3 times in solvent B, and vacuum dry the filter cake to obtain the mPEG-MA monomer compound. Step (2): Synthesis of HPBE-MA reactant monomers: Pinacol ester of p-hydroxymethylphenylboronic acid, methacrylamide chloride, and triethylamine were dissolved in solvent A in a molar ratio of 1:1-3:1-3 and reacted at room temperature for 8-12 h. The mixture was then washed, dried, and purified to obtain the HPBE-MA monomer compound. Step (3): Synthesis of EMD-MA reactant monomers: Emodin, methacryloyl chloride and triethylamine were dissolved in solvent A in a molar ratio of 1:1-3:1-3 and reacted at room temperature for 8-12 h. The mixture was washed, dried and purified to obtain the EMD-MA monomer compound. Step (4): Synthesis of p(mPEG-co-HPBE-co-EMD) polymer. The mPEG-MA obtained in step (1), HPBE-MA obtained in step (2), and EMD-MA obtained in step (3) are mixed and dissolved in toluene in a molar ratio of 1:2-5:1-3. AIBN is added, and the reaction is carried out at 65°C for 20-24 h under nitrogen protection. The mixture is precipitated 2-3 times in solvent B, redissolved in acetone, purified by dialysis, and freeze-dried to obtain p(mPEG-co-HPBE-co-EMD) polymer.

5. The dual-sensitivity co-loaded emodin and chlorambucil micelles according to claim 4, characterized in that: The molecular weight of the polyethylene glycol monomethyl ether mentioned in step (1) is 1500-4000; the amount of AIBN used in step (4) is 0.2-4.0% of the total mass of the monomers.

6. The dual-sensitivity co-loaded emodin and chlorambucil micelles according to claim 4, characterized in that: Solvent A mentioned in steps (1), (2) and (3) is any one of dichloromethane, trichloromethane and acetone.

7. The dual-sensitivity co-loaded emodin and chlorambucil micelles according to claim 4, characterized in that: Solvent B mentioned in steps (1) and (4) can be any one of n-hexane, diethyl ether, or petroleum ether.

8. A method for preparing the dual-sensitivity co-loaded emodin and chlorambucil micelles as described in claim 3, characterized in that: Chlorobutyric acid mustard and polymer (p(mPEG-co-HPBE-co-EMD)) were mixed at a mass ratio of 1:5-10 and dissolved in solvent C. The mixture was ultrasonically dispersed, solvent C was removed by rotary evaporation, and the mixture was hydrated and dispersed evenly in a hydration medium at 30-50℃. After filtration, p(mPEG-co-HPBE-co-EMD)@CLB co-loaded drug micelles were obtained.

9. The method for preparing dual-sensitivity co-loaded emodin and chlorambucil micelles according to claim 8, characterized in that: The solvent C is any one of methanol-acetone mixed solution and tetrahydrofuran; the hydration medium is any one of deionized water, PBS, and physiological saline.

10. The application of the dual-sensitivity co-loaded emodin and chlorambucil micelles as described in claim 3, characterized in that: The application of the dual-sensitivity co-loaded emodin and chlorambucil micelles in the preparation of antitumor drugs.

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