Preparation and Drug Loading Performance Study Methods of PEGMA-g-PEGMA-b-PCL Polymer Micelles

Abstract

This invention discloses a method for preparing and studying the drug-loading properties of PEGMA-g-PEGMA-b-PCL polymer micelles, belonging to the field of drug carrier technology. The method first synthesizes a triblock graft copolymer using the ATRP method, then completes micelle self-assembly through solution dropwise addition, rotary evaporation, and membrane filtration. Using a hydrophobic antitumor drug as a model, drug-loaded micelles are prepared, and drug loading, release, stability, and cytotoxicity are simultaneously tested. This invention designs a hydrophilic grafted PEGMA-g-PEGMA structure and optimizes the preparation process parameters. The resulting micelles have a spherical core-shell structure with a particle size of 80-180 nm, exhibiting good self-assembly stability and pH response characteristics. The drug loading is ≥8.5%, the encapsulation efficiency is ≥80%, drug release is slow under physiological conditions, and the cumulative drug release rate in the tumor microenvironment is 75%-90% after 72 hours. Furthermore, it exhibits good storage stability and excellent biocompatibility, enabling efficient drug encapsulation and tumor microenvironment-responsive release. This method has significant application value in the preparation of targeted therapeutic carriers for hydrophobic antitumor drugs.

Description

Technical Field

[0001] This invention relates to the field of drug carrier technology, specifically to a method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles and studying their drug loading performance. Background Technology

[0002] Polymer micelles, as a novel type of nanomedicine carrier, have been widely studied and applied in the delivery of hydrophobic antitumor drugs due to the unique advantages of their core-shell structure. Polycaprolactone (PCL) is a preferred material for the hydrophobic core due to its good biodegradability and biocompatibility, while polyethylene glycol monomethyl ether methacrylate (PEGMA), as a hydrophilic shell, can improve the water solubility and in vivo circulation stability of the micelles. The polymer micelles formed by the block synthesis of these two materials have become an important research direction for hydrophobic drug carriers. The application of controlled polymerization methods such as atom transfer radical polymerization (ATRP) has also provided technical support for the precise synthesis of these polymers, promoting the development of micelle carriers in targeted tumor therapy.

[0003] While existing linear block polymer micelles based on PEGMA and PCL can achieve the encapsulation and delivery of hydrophobic drugs, they have significant structural defects. The hydrophilic segments are mostly linear structures, lacking the steric hindrance effect brought about by graft modification, resulting in insufficient self-assembly stability of the micelles, a wide particle size distribution, and a polydispersity index (PDI) that easily exceeds the reasonable range. At the same time, the drug loading sites are limited, and the drug loading and encapsulation efficiency are difficult to meet the high efficiency requirements of clinical applications. Furthermore, the pH-responsive drug release characteristics are not significant, and non-specific drug leakage is prone to occur in physiological environments. In the tumor microenvironment, rapid drug release cannot be achieved, affecting the therapeutic effect.

[0004] Furthermore, the existing polymer micelle preparation process parameters lack systematic optimization. In some processes, unreasonable settings for parameters such as temperature and rotation speed during rotary evaporation can easily lead to micelle aggregation, further reducing particle size uniformity and drug loading stability. Simultaneously, conventional micelles exhibit poor storage stability; particle size easily changes significantly when stored at room temperature, resulting in precipitation and stratification. Moreover, the biocompatibility of blank micelles needs improvement, as some carrier materials can exhibit toxicity to normal cells, limiting their practical pharmaceutical translation applications. Additionally, the existing mainstream PCL-PEG linear block micelles still have significant room for improvement in terms of the precision of drug release in response to the tumor microenvironment, drug loading efficiency, and tumor cell inhibitory activity, making it difficult to simultaneously meet the multiple requirements of efficient drug loading, precise drug release, and good biocompatibility.

[0005] Therefore, a method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles and studying their drug loading properties was proposed to solve the above problems. Summary of the Invention

[0006] 1. The technical problem to be solved by the present invention

[0007] The purpose of this invention is to propose a method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles and studying their drug loading performance in order to solve the following problems existing in the prior art: (1) To solve the technical problems of poor self-assembly stability, low drug loading efficiency and poor drug release characteristics caused by insufficient structural design of existing polymer micelle carriers. Traditional linear block micelles have no grafting modification of hydrophilic segments and weak steric hindrance effect. Not only are the micelles wide in size distribution and easy to aggregate during storage, but they also have limited drug loading sites, low drug loading and encapsulation efficiency. At the same time, they are prone to non-specific drug leakage under physiological environment and slow drug release in tumor microenvironment, which makes it impossible to achieve precise targeted drug delivery.

[0008] (2) Solve the technical problems of lack of systematic optimization of existing micelle preparation process parameters and insufficient comprehensive application performance. The unreasonable setting of key parameters in the existing preparation process can easily cause micelle aggregation, affecting particle size uniformity and drug loading stability. In addition, conventional micelle carriers generally have problems of poor storage stability and poor biocompatibility. Some carriers are toxic to normal cells. At the same time, the existing mainstream micelles cannot meet the multiple application requirements of efficient drug loading, precise pH-responsive drug release and strong tumor cell inhibition activity, which limits the actual pharmaceutical transformation.

[0009] 2. Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles, comprising the following steps: Synthesis of S1.PEGMA-g-PEGMA-b-PCL triblock graft copolymer: Using ethyl α-bromoisobutyrate as an initiator and CuBr / 2,2'-bipyridine as a catalytic system, atom transfer radical polymerization (ATRP) was employed. First, polyethylene glycol monomethyl ether methacrylate (PEGMA) monomer was polymerized to obtain a PEGMA homopolymer. Then, a second portion of PEGMA monomer was added, controlling the grafting degree to 30%-50%, followed by... Continued polymerization yields the grafted polymer PEGMA-g-PEGMA; finally, ε-caprolactone (ε-CL) monomer is added, and polymerization is carried out at 60-80℃ for 4-8 hours to obtain PEGMA-g-PEGMA-b-PCL triblock graft copolymer, wherein the mass ratio of PEGMA-g-PEGMA segments to PCL segments is 1:0.8-1:1.5, the number average molecular weight of the copolymer is 15000-35000 Da, and the molecular weight distribution index is 1.10-1.30; S2. Preparation of self-assembly of polymer micelles: The PEGMA-g-PEGMA-b-PCL triblock graft copolymer synthesized in S1 was dissolved in dichloromethane to prepare a polymer solution with a concentration of 5-15 mg / mL. This solution was slowly added dropwise to deionized water at 37℃ at a dropping rate of 1-3 mL / min. After the addition was completed, stirring was continued for 1-2 h. Then, dichloromethane in the system was removed by rotary evaporation at a temperature of 35-45℃, a rotation speed of 100-150 r / min, and an evaporation time of 30-60 min to obtain a preliminary micelle solution. The preliminary micelle solution was filtered through a 0.22 μm filter membrane to remove unassembled polymers, resulting in a PEGMA-g-PEGMA-b-PCL polymer micelle solution with a micelle size of 80-180 nm, a polydispersity index (PDI) of 0.15-0.30, and a Zeta potential of -10 to -25 mV.

[0010] Preferably, in S1, the molar ratio of ethyl α-bromoisobutyrate, CuBr, and 2,2'-bipyridine is 1:1.0-1.2:2.0-2.4; the molar ratio of the first part of PEGMA monomer to initiator is 50-80:1, the molar ratio of the second part of PEGMA monomer to initiator is 20-40:1, and the molar ratio of ε-CL monomer to initiator is 80-120:1; the polymerization reaction is carried out under an inert gas protection environment, the inert gas being nitrogen or argon, and the ventilation time is 15-30 minutes to completely remove air; The number average molecular weight of the PEGMA monomer in S1 is 500-1500 Da; the ε-CL monomer is purified by vacuum distillation before use to remove impurities and moisture; after the polymerization reaction is completed, the reaction solution is poured into an excess of cold diethyl ether to precipitate, allowed to stand for 12-24 hours, filtered and the precipitate is collected, and placed in a vacuum drying oven and dried at 40-50℃ for 24-48 hours to obtain the purified PEGMA-g-PEGMA-b-PCL triblock graft copolymer; the temperature of the cold diethyl ether is -10 to 0℃, and the amount used is 3-5 times the volume of the reaction solution.

[0011] Preferably, the volume ratio of dichloromethane to deionized water in S2 is 1:8 to 1:15; the rotary evaporation process is carried out under reduced pressure with a vacuum degree of 0.06-0.08 MPa; the membrane filtration is performed under aseptic conditions, and the filtered micelle solution is stored in a refrigerator at 4°C for no more than 72 hours.

[0012] The method for studying the drug loading performance of PEGMA-g-PEGMA-b-PCL polymer micelles, using a hydrophobic antitumor drug as a model drug, includes the preparation of drug-loaded micelles, drug loading performance testing, and in vitro drug release performance testing. The specific steps are as follows: (1) Preparation of drug-loaded micelles: The model drug is dissolved in dichloromethane to prepare a drug solution with a concentration of 2-8 mg / mL. The drug solution is mixed with the polymer solution in S1 of claim 1 at a drug to polymer mass ratio of 1:5-1:20. The mixture is ultrasonically dispersed for 5-10 min at an ultrasonic power of 100-200 W to obtain a drug-polymer mixed solution. The mixed solution is slowly added dropwise to deionized water at 37°C at a dropping rate of 1-3 mL / min. After the addition is completed, the mixture is stirred for 1-2 h. The dichloromethane is removed by rotary evaporation. After filtration, a drug-loaded PEGMA-g-PEGMA-b-PCL polymer micelle solution is obtained. (2) Drug loading performance test: The drug-loaded micelles and free drug were separated by high-speed centrifugation at a speed of 10,000-15,000 r / min for 20-30 min. The supernatant was collected and the concentration of free drug in the supernatant was determined by ultraviolet-visible spectrophotometry. The drug loading (DL) and encapsulation efficiency (EE) were calculated. Among them, the drug loading (DL) = (mass of drug in drug-loaded micelles / total mass of drug-loaded micelles) × 100%, and the encapsulation efficiency (EE) = (mass of drug in drug-loaded micelles / total mass of drug) × 100%. The drug loading is required to be ≥8.5% and the encapsulation efficiency is required to be ≥80%. (3) In vitro drug release performance test: Take the drug-loaded micelle solution, place it in a dialysis bag, put the dialysis bag into PBS buffer, and conduct in vitro drug release experiment under constant temperature shaking conditions of 37℃ and 100-120r / min; take samples at 1, 2, 4, 8, 12, 24, 48 and 72h respectively, and determine the concentration of drug in the release solution by UV-Vis spectrophotometry and calculate the cumulative drug release rate; at the same time, PBS buffer with different pH values ​​(pH=5.0, pH=6.5, pH=7.4) are set up to investigate the effect of pH value on drug release performance; it is required that the cumulative drug release rate is 30%-50% in the physiological environment of pH=7.4 and 72h in the tumor microenvironment of pH=5.0, and the cumulative drug release rate is 75%-90% in the tumor microenvironment of pH=5.0, showing obvious pH-responsive drug release characteristics.

[0013] Preferably, the model drug is doxorubicin (DOX), paclitaxel (PTX), or curcumin (CUR); the detection wavelength of the ultraviolet-visible spectrophotometry in step (2) is adjusted according to the model drug, with the detection wavelength of doxorubicin being 480-490 nm, the detection wavelength of paclitaxel being 225-235 nm, and the detection wavelength of curcumin being 425-435 nm; before detection, a standard curve for the corresponding drug needs to be plotted, and the correlation coefficient R² of the standard curve is ≥0.999.

[0014] Preferably, the stability test of the drug-loaded micelles is also included. Specifically, the drug-loaded micelle solution is stored at 4℃ and 25℃, and samples are taken at 1, 3, 5 and 7 days respectively. The particle size and polydispersity index (PDI) of the micelles are determined by dynamic light scattering (DLS). It is required that after 7 days of storage, the particle size change rate is ≤15%, the PDI change is ≤0.05, and there is no obvious precipitation or stratification.

[0015] Preferably, the assay also includes cytotoxicity testing of drug-loaded micelles. Specifically, using the MTT assay with tumor cells as the research object, a blank control group, a free drug group, and groups with different concentrations of drug-loaded micelles are set up. After culturing for 24-48 hours, the cell viability of each group is measured, and the half-maximal inhibitory concentration (IC50) is calculated. 50 ); requires IC of drug-loaded micelle assembly 50 ≤IC50 of the free drug group 50 Furthermore, the cell survival rate of the blank micelle group was ≥85%, demonstrating that the micelles have good biocompatibility and antitumor activity.

[0016] A PEGMA-g-PEGMA-b-PCL polymer micelle is disclosed, wherein the micelle has a spherical core-shell structure, with a core of hydrophobic PCL segments and a shell of hydrophilic grafted PEGMA-g-PEGMA segments. The micelle has a particle size of 80-180 nm, a polydispersity index (PDI) of 0.15-0.30, a zeta potential of -10 to -25 mV, and a critical micelle concentration (CMC) of 0.01-0.05 mg / mL. It exhibits good self-assembly stability and pH response characteristics.

[0017] The application of a PEGMA-g-PEGMA-b-PCL polymer micelle in the preparation of a hydrophobic drug carrier, wherein the hydrophobic drug is an antitumor drug, and the micelles can achieve efficient drug loading and tumor microenvironment-responsive release for targeted tumor therapy.

[0018] Compared with existing technologies, the method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles and studying their drug loading performance provided by this invention has the following advantages: (1) Advantages of structural design: The PEGMA-g-PEGMA-b-PCL triblock graft copolymer designed in this scheme introduces PEGMA-g-PEGMA hydrophilic graft segments, which form a stronger steric hindrance effect compared with the traditional linear block structure, greatly improving the self-assembly stability of micelles, making the micelle size more uniform and the PDI lower. At the same time, it increases the drug loading sites, significantly improving the drug loading and encapsulation efficiency, and optimizing the core performance of micelles from the structural level.

[0019] (2) Advantages of process parameters: This scheme systematically optimizes the key process parameters for polymer micelle preparation, especially the precise control of parameters such as temperature, rotation speed, and vacuum degree of rotary evaporation, which effectively avoids micelle aggregation and ensures the uniformity of micelle particle size and drug loading stability; at the same time, it clarifies process details such as solution ratio, dropping acceleration rate, and stirring time, making the preparation process highly repeatable, verifying the rationality and optimization of the process, and making it suitable for large-scale preparation.

[0020] (3) Advantages of drug loading and release: The polymer micelles in this scheme have both high efficiency of drug loading and precise pH response drug release characteristics. The drug loading and encapsulation efficiency are much higher than those of conventional micelle carriers. In addition, the drug is slowly released in the physiological environment of pH=7.4, which effectively reduces non-specific leakage. In the acidic tumor microenvironment of pH=5.0, the drug is released rapidly. The cumulative drug release rate reaches 75%-90% in 72h, and the drug release accuracy is greatly improved. It can significantly increase the drug concentration at the tumor site and enhance the anti-tumor effect.

[0021] (4) Comprehensive performance advantages: The micelles in this scheme have excellent storage stability and biocompatibility. After storage at room temperature (25℃) for 7 days, the particle size change rate is ≤15%, and there is no precipitation or stratification, which meets the storage requirements for actual pharmaceutical applications. The blank micelles have extremely low toxicity to normal cells, with a cell survival rate of ≥85%. Moreover, the drug-loaded micelles have stronger tumor cell inhibitory activity, with a half-maximal inhibitory concentration (IC50) of ≥85%. so It achieves dual optimization in biocompatibility and antitumor activity, with overall performance far exceeding existing technologies, compared to free drugs and traditional micelle carriers. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the process for synthesizing and preparing micelles from the PEGMA-g-PEGMA-b-PCL triblock graft copolymer of the present invention. Figure 2 The following are the particle size and PDI change curves of drug-loaded micelles stored at 4°C and 25°C for 7 days in an embodiment of the present invention. Figure 3 This is a schematic diagram of the doxorubicin standard curve according to an embodiment of the present invention; Figure 4 This is a schematic diagram of the cumulative drug release rate curves of drug-loaded micelles in different pH PBS buffers over 72 hours, according to an embodiment of the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0025] Example 1, please refer to Figures 1 to 4 As shown: To address the problems mentioned in the technical solutions, this application provides a method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles and studying their drug loading performance, including the following steps: Synthesis of S1.PEGMA-g-PEGMA-b-PCL triblock graft copolymer: Using ethyl α-bromoisobutyrate as an initiator and CuBr / 2,2'-bipyridine as a catalytic system, atom transfer radical polymerization (ATRP) was employed. First, polyethylene glycol monomethyl ether methacrylate (PEGMA) monomer was polymerized to obtain a PEGMA homopolymer. Then, a second portion of PEGMA monomer was added, controlling the grafting degree to 30%-50%, followed by... Continued polymerization yields the grafted polymer PEGMA-g-PEGMA; finally, ε-caprolactone (ε-CL) monomer is added, and polymerization is carried out at 60-80℃ for 4-8 hours to obtain PEGMA-g-PEGMA-b-PCL triblock graft copolymer, wherein the mass ratio of PEGMA-g-PEGMA segments to PCL segments is 1:0.8-1:1.5, the number average molecular weight of the copolymer is 15000-35000 Da, and the molecular weight distribution index is 1.10-1.30; S2. Preparation of self-assembly of polymer micelles: The PEGMA-g-PEGMA-b-PCL triblock graft copolymer synthesized in S1 was dissolved in dichloromethane to prepare a polymer solution with a concentration of 5-15 mg / mL. This solution was slowly added dropwise to deionized water at 37℃ at a dropping rate of 1-3 mL / min. After the addition was completed, stirring was continued for 1-2 h. Then, dichloromethane in the system was removed by rotary evaporation at a temperature of 35-45℃, a rotation speed of 100-150 r / min, and an evaporation time of 30-60 min to obtain a preliminary micelle solution. The preliminary micelle solution was filtered through a 0.22 μm filter membrane to remove unassembled polymers, resulting in a PEGMA-g-PEGMA-b-PCL polymer micelle solution with a micelle size of 80-180 nm, a polydispersity index (PDI) of 0.15-0.30, and a Zeta potential of -10 to -25 mV.

[0026] In S1, the molar ratio of ethyl α-bromoisobutyrate, CuBr, and 2,2'-bipyridine is 1:1.0-1.2:2.0-2.4; the molar ratio of PEGMA monomer to initiator in the first part is 50-80:1, the molar ratio of PEGMA monomer to initiator in the second part is 20-40:1, and the molar ratio of ε-CL monomer to initiator is 80-120:1; the polymerization reaction is carried out under an inert gas atmosphere, with nitrogen or argon as the inert gas, and the venting time is 15-30 minutes to completely remove air; The number average molecular weight of PEGMA monomer in S1 is 500-1500 Da; the ε-CL monomer is purified by vacuum distillation before use to remove impurities and moisture; after the polymerization reaction is completed, the reaction solution is poured into an excess of cold diethyl ether to precipitate, allowed to stand for 12-24 hours, filtered and the precipitate is collected, and placed in a vacuum drying oven and dried at 40-50℃ for 24-48 hours to obtain the purified PEGMA-g-PEGMA-b-PCL triblock graft copolymer; the temperature of the cold diethyl ether is -10 to 0℃, and the amount used is 3-5 times the volume of the reaction solution.

[0027] The volume ratio of dichloromethane to deionized water in S2 is 1:8-1:15; the rotary evaporation process is carried out under reduced pressure with a vacuum degree of 0.06-0.08 MPa; the membrane filtration is performed under aseptic conditions, and the filtered micelle solution is stored in a refrigerator at 4°C for no more than 72 hours.

[0028] Furthermore, this protocol proposes a method for studying the drug loading performance of PEGMA-g-PEGMA-b-PCL polymer micelles, using a hydrophobic antitumor drug as a model drug. The method includes the preparation of drug-loaded micelles, detection of drug loading performance, and detection of in vitro drug release performance. The specific steps are as follows: (1) Preparation of drug-loaded micelles: The model drug is dissolved in dichloromethane to prepare a drug solution with a concentration of 2-8 mg / mL. The drug solution is mixed with the polymer solution in S1 of claim 1 at a drug to polymer mass ratio of 1:5-1:20. The mixture is ultrasonically dispersed for 5-10 min at an ultrasonic power of 100-200 W to obtain a drug-polymer mixed solution. The mixed solution is slowly added dropwise to deionized water at 37°C at a dropping rate of 1-3 mL / min. After the addition is completed, the mixture is stirred for 1-2 h. The dichloromethane is removed by rotary evaporation. After filtration, a drug-loaded PEGMA-g-PEGMA-b-PCL polymer micelle solution is obtained. (2) Drug loading performance test: The drug-loaded micelles and free drug were separated by high-speed centrifugation at a speed of 10,000-15,000 r / min for 20-30 min. The supernatant was collected and the concentration of free drug in the supernatant was determined by ultraviolet-visible spectrophotometry. The drug loading (DL) and encapsulation efficiency (EE) were calculated. Among them, the drug loading (DL) = (mass of drug in drug-loaded micelles / total mass of drug-loaded micelles) × 100%, and the encapsulation efficiency (EE) = (mass of drug in drug-loaded micelles / total mass of drug) × 100%. The drug loading is required to be ≥8.5% and the encapsulation efficiency is required to be ≥80%. (3) In vitro drug release performance test: Take the drug-loaded micelle solution, place it in a dialysis bag, put the dialysis bag into PBS buffer, and conduct in vitro drug release experiment under constant temperature shaking conditions of 37℃ and 100-120r / min; take samples at 1, 2, 4, 8, 12, 24, 48 and 72h respectively, and determine the concentration of drug in the release solution by UV-Vis spectrophotometry and calculate the cumulative drug release rate; at the same time, PBS buffer with different pH values ​​(pH=5.0, pH=6.5, pH=7.4) are set up to investigate the effect of pH value on drug release performance; it is required that the cumulative drug release rate is 30%-50% in the physiological environment of pH=7.4 and 72h in the tumor microenvironment of pH=5.0, and the cumulative drug release rate is 75%-90% in the tumor microenvironment of pH=5.0, showing obvious pH-responsive drug release characteristics.

[0029] The model drugs are doxorubicin (DOX), paclitaxel (PTX), or curcumin (CUR); the detection wavelength of the UV-Vis spectrophotometry in step (2) is adjusted according to the model drug. The detection wavelength of doxorubicin is 480-490nm, the detection wavelength of paclitaxel is 225-235nm, and the detection wavelength of curcumin is 425-435nm; before detection, a standard curve of the corresponding drug needs to be plotted, and the correlation coefficient R² of the standard curve is ≥0.999.

[0030] It also includes the stability test of drug-loaded micelles. Specifically, the drug-loaded micelle solution is stored at 4℃ and 25℃, and samples are taken at 1, 3, 5 and 7 days. The particle size and polydispersity index (PDI) of the micelles are determined by dynamic light scattering (DLS). After 7 days of storage, the particle size change rate is required to be ≤15%, the PDI change is required to be ≤0.05, and there should be no obvious precipitation or stratification.

[0031] This also includes the cytotoxicity assay of drug-loaded micelles. Specifically, the MTT assay was used with tumor cells as the research object. A blank control group, a free drug group, and groups with different concentrations of drug-loaded micelles were set up. After culturing for 24-48 hours, the cell viability of each group was measured, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ); requires IC of drug-loaded micelle assembly 50 ≤IC50 of the free drug group 50Furthermore, the cell survival rate of the blank micelle group was ≥85%, demonstrating that the micelles have good biocompatibility and antitumor activity.

[0032] Furthermore, based on the above scheme, this paper proposes a PEGMA-g-PEGMA-b-PCL polymer micelle. The micelle has a spherical core-shell structure, with a core of hydrophobic PCL segments and a shell of hydrophilic grafted PEGMA-g-PEGMA segments. The particle size is 80-180 nm, the polydispersity index (PDI) is 0.15-0.30, the zeta potential is -10 to -25 mV, and the critical micelle concentration (CMC) is 0.01-0.05 mg / mL. It exhibits good self-assembly stability and pH response characteristics.

[0033] The application of a PEGMA-g-PEGMA-b-PCL polymer micelle in the preparation of a hydrophobic drug carrier, wherein the hydrophobic drug is an anti-tumor drug, and the micelles can achieve efficient drug loading and tumor microenvironment-responsive release for targeted tumor therapy.

[0034] Example 2: Based on Example 1, but with some differences, the preparation method and drug loading performance study method of PEGMA-g-PEGMA-b-PCL polymer micelles proposed in this invention will be described below with reference to specific examples and accompanying drawings. The specific content is as follows.

[0035] In this embodiment, doxorubicin (DOX) was used as a hydrophobic antitumor model drug. The preparation of PEGMA-g-PEGMA-b-PCL polymer micelles and the detection of drug loading performance were carried out. Intermediate values ​​were selected to ensure experimental repeatability. The specific implementation process is as follows.

[0036] (a) Synthesis of PEGMA-g-PEGMA-b-PCL triblock graft copolymer; 1. Reagents and proportions: Ethyl α-bromoisobutyrate (initiator), CuBr, and 2,2'-bipyridine in a molar ratio of 1:1.1:2.2; PEGMA monomer with a number average molecular weight of 1000 Da was selected, with the first part of PEGMA and initiator in a molar ratio of 65:1, the second part of PEGMA and initiator in a molar ratio of 30:1, and the ε-CL monomer and initiator in a molar ratio of 100:1; the ε-CL monomer was purified by vacuum distillation before use.

[0037] 2. Polymerization reaction: Nitrogen gas was introduced into the reaction system for 20 min to completely purge the air. The first part of PEGMA was first polymerized using the ATRP method to obtain a homopolymer. The second part of PEGMA was added to continue the polymerization, and the grafting degree was controlled at 40% to obtain PEGMA-g-PEGMA. Finally, ε-CL was added, and the temperature was raised to 70℃ and maintained for 6 h for polymerization. The mass ratio of PEGMA-g-PEGMA to PCL segments was 1:1.2.

[0038] 3. Purification treatment: After the reaction is completed, the reaction solution is poured into 4 times the volume of -5℃ cold diethyl ether to precipitate, and allowed to stand for 18 hours. The precipitate is then filtered and collected. The precipitate is placed in a vacuum drying oven at 45℃ and dried for 36 hours to obtain the purified copolymer. The number average molecular weight is 25000 Da and the molecular weight distribution index is 1.20.

[0039] (ii) Preparation of polymer micelles through self-assembly; 1. Take the purified copolymer and dissolve it in dichloromethane to prepare a polymer solution of 10 mg / mL. The volume ratio of dichloromethane to deionized water is 1:12.

[0040] 2. Slowly add the polymer solution dropwise to deionized water at 37°C at a rate of 2 mL / min, and continue stirring for 1.5 h after the addition is complete.

[0041] 3. Dichloromethane was removed by rotary evaporator under reduced pressure. The parameters were set as follows: temperature 40℃, rotation speed 120r / min, vacuum degree 0.07MPa, and evaporation time 45min, to obtain the initial micelle solution.

[0042] 4. After sterile filtration through a 0.22 μm filter membrane, the final micelle solution was obtained and stored at 4°C. Analysis showed that the micelle size was 130 nm, PDI 0.22, Zeta potential -18 mV, and CMC 0.03 mg / mL. Figure 2 As shown in the figure, this is a quantitative detection curve of the storage stability of drug-loaded micelles. The horizontal axis represents the storage time (days), and the vertical axis represents the particle size change rate (%) and PDI change value. It intuitively presents the stability data under two conditions: 4℃ and 25℃. After 7 days of storage at 4℃, the particle size change rate was 6% and the PDI change was 0.02; after 7 days of storage at 25℃, the particle size change rate was 12% and the PDI change was 0.04. There was no obvious precipitation or stratification, which verifies that the micelles meet the stability index requirements at both temperatures.

[0043] (III) Preparation of drug-loaded micelles; 1. Dissolve doxorubicin in dichloromethane to prepare a drug solution of 5 mg / mL.

[0044] 2. Mix the drug solution with the polymer solution at a drug-polymer mass ratio of 1:12, and ultrasonically disperse the mixture at 150W power for 8 minutes to obtain a drug-polymer mixed solution.

[0045] 3. Add the solution dropwise to deionized water at 37℃ at a rate of 2 mL / min, stir for 1.5 h, remove dichloromethane by rotary evaporation, filter through a 0.22 μm filter membrane to obtain the drug-loaded micelle solution.

[0046] (iv) Drug loading and performance testing; 1. Drug loading capacity: High-speed centrifugation at 12000 r / min for 25 min was used to separate free drug. The supernatant was analyzed by UV-Vis spectrophotometry at 485 nm. The standard curve R... 2 =0.9995, the calculated drug loading is 10.2% and the encapsulation efficiency is 88.5%. Figure 3 As shown in the figure, this is the standard working curve for the detection of doxorubicin by ultraviolet-visible spectrophotometry. The horizontal axis represents the doxorubicin concentration (μg / mL), and the vertical axis represents the absorbance value. The fitted linear equation is y=0.058x+0.002, with a correlation coefficient R²=0.9995 (≥0.999), which meets the standard curve requirements for drug concentration quantification in drug loading performance testing and provides a quantitative basis for subsequent calculation of drug loading and encapsulation efficiency.

[0047] 2. In vitro drug release performance: Drug-loaded micelles were placed in dialysis bags and then in PBS buffer solutions at pH 5.0, pH 6.5, and pH 7.4, respectively. The solutions were incubated at 37°C with constant shaking at 110 rpm. Samples were taken at specified time intervals for analysis. The results showed that the cumulative drug release rate after 72 hours was 42% at pH 7.4 and 83% at pH 5.0. Figure 4 As shown in the figure, this is the detection curve of the pH-responsive drug release characteristics of micelles. The horizontal axis is the drug release time (h), and the vertical axis is the cumulative drug release rate (%) over 72 hours. It shows the drug release pattern of micelles in three PBS buffer solutions with pH=5.0, pH=6.5, and pH=7.4: the cumulative drug release rate over 72 hours is 42% at pH=7.4 and reaches 83% at pH=5.0, clearly demonstrating the rapid drug release characteristics of micelles in the acidic tumor microenvironment and the slow drug release characteristics in the physiological environment.

[0048] 3. Stability test: The drug-loaded micelle solutions were stored at 4℃ and 25℃ for 7 days, respectively. The particle size change rate was 6% and the PDI change was 0.02 at 4℃; the particle size change rate was 12% and the PDI change was 0.04 at 25℃. No precipitation or stratification was observed.

[0049] 4. Cytotoxicity assay: Using HepG2 liver cancer cells as the research object, the cell viability was detected by MTT assay after 48 hours of culture. The cell viability of the blank micelle group was 92%, and the IC50 of the drug-loaded micelle group was [missing data]. 50 =0.85μg / mL, IC50 of free doxorubicin group 50 =1.2μg / mL.

[0050] To highlight the advantages of the PEGMA-g-PEGMA-b-PCL polymer micelles of this invention, three comparative examples were set up to compare the polymer structure, preparation process, and conventional micelle carriers. Doxorubicin was used as the model drug in all comparative examples, and the operations were consistent with the examples except for the variables. The specific design is as follows: Comparative Example 1: PEGMA-b-PCL micelles without grafting structure; Variables: The PEGMA grafting process was omitted, and linear PEGMA-b-PCL diblock copolymers were synthesized directly. All other preparation, drug loading, and detection parameters remained the same as in the previous examples. Design objective: To verify the effect of the hydrophilic grafted PEGMA-g-PEGMA segments on improving micelle self-assembly stability and drug loading performance.

[0051] Comparative Example 2: PEGMA-g-PEGMA-b-PCL micelles with varying rotary evaporation process parameters; Variables: The rotary evaporation temperature was changed to 55℃ and the rotation speed to 80 r / min; all other preparation, drug loading, and detection parameters remained the same as in the previous example. Design objective: To verify the rationality of the rotary evaporation process parameters in this scheme, and the impact of the process on micelle particle size uniformity and drug loading stability.

[0052] Comparative Example 3: Conventional PCL-PEG linear block micelles; Variables: Micelles were prepared using PCL-PEG linear diblock copolymers commonly used in existing technologies. Other preparation, drug loading, and detection parameters were consistent with the examples. Design objective: To compare the core advantages of the micelles in this scheme with conventional micelles in terms of pH-responsive drug release, drug loading performance, and cytotoxicity.

[0053] The above embodiments and comparative data statistics are as follows: The core detection indicators of the embodiment and the three comparative examples were quantitatively compared. The data is shown in the table below, and the advantages of this technical solution are intuitively demonstrated through the data differences: Table 1 is a quantitative comparison and statistical table of core detection indicators.

[0054] The data in the table above shows that this solution has the following advantages over existing technologies; 1. Structural advantages: Compared with Comparative Example 1, the PEGMA-g-PEGMA hydrophilic grafted segments in this scheme result in smaller micelle size and lower PDI, increasing drug loading and encapsulation efficiency by 56.9% and 42%, respectively. Furthermore, the pH-responsive drug release characteristics are more significant, demonstrating that the grafted structure can improve the self-assembly stability and drug loading capacity of micelles, while optimizing drug release behavior.

[0055] 2. Process advantages: Compared with Comparative Example 2, the rotary evaporation process parameters (35-45℃, 100-150r / min) of this scheme can effectively avoid micelle agglomeration and ensure particle size uniformity. Moreover, high temperature and low rotation speed will lead to a decrease in micelle drug loading performance and loss of pH responsiveness, which proves the rationality and optimization of the process parameters of this scheme.

[0056] 3. Performance advantages: Compared with Comparative Example 3 (the mainstream carrier of the existing technology), the drug loading and encapsulation efficiency of the micelles in this scheme are increased by 30.8% and 17.7% respectively, the drug release rate in the tumor microenvironment is increased by 22.1%, and the drug release rate in the physiological environment is reduced by 23.6%. Moreover, it has stronger cytotoxicity and better biocompatibility, which proves that the micelles in this scheme have significant advantages over the existing conventional micelles in terms of efficient drug loading, precise pH-responsive drug release, and tumor cell inhibition.

[0057] 4. Stability Advantage: The micelles in this solution showed a particle size change rate of only 12% after being stored at 25℃ for 7 days, which is much lower than that of Comparative Example 1 (28%), Comparative Example 2 (35%), and Comparative Example 3 (22%). Moreover, there was no precipitation or stratification, which proves that it has good storage stability and is more suitable for practical pharmaceutical applications.

[0058] Please refer to the above work process. Figures 1 to 4 .

[0059] It should be noted that the term "comprising" or any other variation thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0060] Although embodiments of the invention 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 these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles, characterized in that, Includes the following steps: Synthesis of S1.PEGMA-g-PEGMA-b-PCL triblock graft copolymer: Using ethyl α-bromoisobutyrate as an initiator and CuBr / 2,2'-bipyridine as a catalytic system, atom transfer radical polymerization (ATRP) was employed. First, polyethylene glycol monomethyl ether methacrylate (PEGMA) monomer was polymerized to obtain a PEGMA homopolymer. Then, a second portion of PEGMA monomer was added, controlling the grafting degree to 30%-50%, followed by... Continued polymerization yields the grafted polymer PEGMA-g-PEGMA; finally, ε-caprolactone (ε-CL) monomer is added, and polymerization is carried out at 60-80℃ for 4-8 hours to obtain PEGMA-g-PEGMA-b-PCL triblock graft copolymer, wherein the mass ratio of PEGMA-g-PEGMA segments to PCL segments is 1:0.8-1:1.5, the number average molecular weight of the copolymer is 15000-35000 Da, and the molecular weight distribution index is 1.10-1.30; S2. Preparation of self-assembly of polymer micelles: The PEGMA-g-PEGMA-b-PCL triblock graft copolymer synthesized in S1 was dissolved in dichloromethane to prepare a polymer solution with a concentration of 5-15 mg / mL. This solution was slowly added dropwise to deionized water at 37℃ at a dropping rate of 1-3 mL / min. After the addition was completed, stirring was continued for 1-2 h. Then, dichloromethane in the system was removed by rotary evaporation at a temperature of 35-45℃, a rotation speed of 100-150 r / min, and an evaporation time of 30-60 min to obtain a preliminary micelle solution. The preliminary micelle solution was filtered through a 0.22 μm filter membrane to remove unassembled polymers, resulting in a PEGMA-g-PEGMA-b-PCL polymer micelle solution with a micelle size of 80-180 nm, a polydispersity index (PDI) of 0.15-0.30, and a Zeta potential of -10 to -25 mV.

2. The method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles according to claim 1, characterized in that, The molar ratio of ethyl α-bromoisobutyrate, CuBr, and 2,2'-bipyridine in S1 is 1:1.0-1.2:2.0-2.4; the molar ratio of PEGMA monomer to initiator in the first part is 50-80:1, the molar ratio of PEGMA monomer to initiator in the second part is 20-40:1, and the molar ratio of ε-CL monomer to initiator is 80-120:1; the polymerization reaction is carried out under an inert gas atmosphere, which is nitrogen or argon, and the venting time is 15-30 minutes to completely remove air; The number-average molecular weight of the PEGMA monomer in S1 is 500-1500 Da; Before use, the ε-CL monomer is purified by vacuum distillation to remove impurities and moisture. After the polymerization reaction is completed, the reaction solution is poured into an excess of cold diethyl ether to precipitate the precipitate. After standing for 12-24 hours, the precipitate is collected by filtration and placed in a vacuum drying oven at 40-50℃ for 24-48 hours to obtain the purified PEGMA-g-PEGMA-b-PCL triblock graft copolymer. The temperature of the cold diethyl ether is -10 to 0℃, and the amount used is 3-5 times the volume of the reaction solution.

3. The method for preparing PEGMA-g-PEGMA-b-PCL polymer micelles according to claim 1, characterized in that, The volume ratio of dichloromethane to deionized water in S2 is 1:8-1:15; the rotary evaporation process is carried out under reduced pressure with a vacuum degree of 0.06-0.08 MPa; the membrane filtration is performed under aseptic conditions, and the filtered micelle solution is stored in a refrigerator at 4°C for no more than 72 hours.

4. A method for studying the drug loading performance of PEGMA-g-PEGMA-b-PCL polymer micelles obtained by any one of the preparation methods described in claims 1-3, characterized in that, Using hydrophobic antitumor drugs as model drugs, the preparation of drug-loaded micelles, drug loading performance testing, and in vitro drug release performance testing are carried out through the following specific steps: (1) Preparation of drug-loaded micelles: The model drug is dissolved in dichloromethane to prepare a drug solution with a concentration of 2-8 mg / mL. The drug solution is mixed with the polymer solution in S1 of claim 1 at a drug to polymer mass ratio of 1:5-1:

20. The mixture is ultrasonically dispersed for 5-10 min at an ultrasonic power of 100-200 W to obtain a drug-polymer mixed solution. The mixed solution is slowly added dropwise to deionized water at 37°C at a dropping rate of 1-3 mL / min. After the addition is completed, the mixture is stirred for 1-2 h. The dichloromethane is removed by rotary evaporation. After filtration, a drug-loaded PEGMA-g-PEGMA-b-PCL polymer micelle solution is obtained. (2) Drug loading performance test: The drug-loaded micelles and free drug were separated by high-speed centrifugation at a speed of 10,000-15,000 r / min and a centrifugation time of 20-30 min. The supernatant was taken and the concentration of free drug in the supernatant was determined by ultraviolet-visible spectrophotometry. The drug loading (DL) and encapsulation efficiency (EE) were calculated. (3) In vitro drug release performance test: Take the drug-loaded micelle solution, place it in a dialysis bag, put the dialysis bag into PBS buffer, and conduct in vitro drug release experiment under constant temperature shaking conditions of 37℃ and 100-120r / min; take samples at 1, 2, 4, 8, 12, 24, 48 and 72h respectively, and determine the concentration of drug in the release solution by UV-Vis spectrophotometry and calculate the cumulative drug release rate; at the same time, PBS buffer with different pH values ​​are set to examine the effect of pH value on drug release performance; it is required that the cumulative drug release rate is 30%-50% in the physiological environment of pH=7.4 and 75%-90% in the tumor microenvironment of pH=5.0, showing obvious pH-responsive drug release characteristics.

5. The method for studying the drug loading performance of PEGMA-g-PEGMA-b-PCL polymer micelles according to claim 4, characterized in that, The model drug is doxorubicin (DOX), paclitaxel (PTX), or curcumin (CUR); the detection wavelength of the ultraviolet-visible spectrophotometry in step (2) is adjusted according to the model drug. The detection wavelength of doxorubicin is 480-490nm, the detection wavelength of paclitaxel is 225-235nm, and the detection wavelength of curcumin is 425-435nm; before detection, a standard curve of the corresponding drug needs to be plotted, and the correlation coefficient R² of the standard curve is ≥0.

999.

6. The method for studying the drug loading performance of PEGMA-g-PEGMA-b-PCL polymer micelles according to claim 4, characterized in that, It also includes the stability test of drug-loaded micelles. Specifically, the drug-loaded micelle solution is stored at 4℃ and 25℃, and samples are taken at 1, 3, 5 and 7 days. The particle size and polydispersity index (PDI) of the micelles are determined by dynamic light scattering (DLS). After 7 days of storage, the particle size change rate is required to be ≤15%, the PDI change is required to be ≤0.05, and there should be no obvious precipitation or stratification.

7. The method for studying the drug loading performance of PEGMA-g-PEGMA-b-PCL polymer micelles according to claim 4, characterized in that, This also includes the cytotoxicity assay of drug-loaded micelles. Specifically, the MTT assay was used with tumor cells as the research object. A blank control group, a free drug group, and groups with different concentrations of drug-loaded micelles were set up. After culturing for 24-48 hours, the cell viability of each group was measured, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ); requires IC of drug-loaded micelle assembly 50 ≤IC50 of the free drug group 50 Furthermore, the cell survival rate of the blank micelle group was ≥85%, demonstrating that the micelles have good biocompatibility and antitumor activity.

8. A PEGMA-g-PEGMA-b-PCL polymer micelle, characterized in that, The PEGMA-g-PEGMA-b-PCL polymer micelles are prepared by any one of the preparation methods described in claims 1-3. The micelles have a spherical core-shell structure, with a core of hydrophobic PCL segments and a shell of hydrophilic grafted PEGMA-g-PEGMA segments. The particle size is 80-180 nm, the polydispersity index (PDI) is 0.15-0.30, the zeta potential is -10 to -25 mV, and the critical micelle concentration (CMC) is 0.01-0.05 mg / mL. They exhibit good self-assembly stability and pH response characteristics.

9. The application of the PEGMA-g-PEGMA-b-PCL polymer micelles according to claim 8 in the preparation of hydrophobic drug carriers, characterized in that, The hydrophobic drug is an anti-tumor drug, and the micelles can achieve efficient drug loading and tumor microenvironment-responsive release for targeted tumor therapy.

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