A method for improving drug loading performance of vesicles

Abstract

This invention discloses a method for improving the drug-loading performance of vesicles, including systematic optimization of the drug loading process. It employs a synergistic combination of thin-film dispersion-ultrasound and emulsification-solvent evaporation to prepare drug-loaded nanoparticles. First, the vesicle carrier material and drug are dissolved and then rotary evaporated to form a uniform thin film. A hydrated solvent is added for incubation to obtain a hydrated liquid. The hydrated liquid is then ultrasonically treated to promote drug dispersion. Subsequently, an emulsifier is added to form a primary emulsion, which is then mixed and emulsified with the aqueous phase. Stirring allows the organic solvent to slowly evaporate, enabling the carrier material to self-assemble into vesicle structures. Finally, uniformly dispersed drug-loaded nanoparticles are obtained. This invention uses a synergistic combination of thin-film dispersion-ultrasound and emulsification-solvent evaporation, rather than a single process selection. The two processes complement each other. By first preparing the thin film and hydrating it, then ultrasonically dispersing it, and finally emulsifying and evaporating it to form the vesicles, the problem of uneven drug dispersion and localized aggregation in traditional single-process methods is effectively solved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a method for improving the drug-carrying capacity of vesicles. Background Technology

[0002] Vesicles, as a novel drug delivery carrier, possess excellent biocompatibility, low immunogenicity, and targeted delivery potential. They can encapsulate hydrophilic, hydrophobic, and amphiphilic drugs, and have broad application prospects in antitumor, anti-inflammatory, and antiviral fields. Among them, polymeric vesicles have become a research hotspot for drug delivery carriers due to their advantages such as structural stability and strong modifiability. Common carrier materials include polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid copolymer (PLGA), which can be flexibly selected according to the properties of the drug.

[0003] However, current vesicle drug loading technology still faces significant bottlenecks. Traditional drug loading processes often employ single preparation methods, resulting in uneven drug dispersion and localized aggregation, leading to low drug encapsulation efficiency and loading capacity. Furthermore, drug loading process parameters rely heavily on empirical screening, lacking systematic experimental design and mathematical model support. This makes it difficult to accurately analyze the interactions between parameters and obtain the optimal process combination. In addition, the physicochemical properties of different drugs vary considerably, resulting in insufficient optimization of drug loading processes for specific drugs. Moreover, the methods for measuring drug loading performance indicators have limited precision, making it impossible to comprehensively and accurately evaluate the drug loading effect of vesicles. This severely limits the industrial application and clinical efficacy of vesicle-loaded formulations.

[0004] Temozolomide, a commonly used anti-tumor drug in clinical practice, is mainly used to treat diseases such as malignant glioma and malignant melanoma. It is rapidly absorbed after oral administration, with an average elimination half-life of 1.8 hours. Conventional formulations suffer from low bioavailability and significant toxic side effects. Preparing it as a vesicle-loaded formulation can effectively improve these defects. However, the vesicle loading process for temozolomide has not yet been optimized, and the drug loading performance needs further improvement. Therefore, developing a method to improve the drug loading performance of vesicles and solve the above technical problems has important practical significance and application value. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for improving the drug loading performance of vesicles. This method aims to solve the problems in existing vesicle drug loading technologies that employ a single preparation method, resulting in uneven drug dispersion, local aggregation, and consequently, low drug encapsulation efficiency and drug loading.

[0006] To address the aforementioned technical problems, this invention provides a method for improving the drug loading capacity of vesicles, comprising the following steps:

[0007] S1. System optimization of drug loading process: Drug-loaded nanoparticles are prepared by combining thin film dispersion-ultrasound method and emulsification-solvent evaporation method. First, the vesicle carrier material and drug are dissolved and then formed into a uniform thin film by rotary evaporation. Aqueous solvent is added and incubated to obtain hydrated liquid. Then, the hydrated liquid is ultrasonically treated to promote drug dispersion. Subsequently, an emulsifier is added to form a primary emulsion and mixed with the aqueous phase for emulsification. The organic solvent is slowly evaporated by stirring to realize the self-assembly of the carrier material to form vesicle structure, and finally drug-loaded nanoparticles with uniform drug dispersion are obtained.

[0008] S2. Based on the response surface methodology, the experimental design was carried out. Based on the drug-loaded nanoparticles prepared in step S1, the drug-to-carrier ratio, ultrasonic time, and emulsification rate were selected as the factors to be investigated. Combined with the Box-Behnken experimental design, the drug encapsulation efficiency and drug loading were used as the response values. The interaction between the factors was analyzed by establishing a mathematical model, and the optimal combination of drug loading process parameters was selected.

[0009] S3. Temozolomide, a commonly used antitumor drug in clinical practice, was selected as the model drug. High performance liquid chromatography combined with centrifugation and ultrafiltration was used to accurately determine the core performance indicators of the drug-loaded vesicles, including drug encapsulation efficiency and drug loading. At the same time, an in vitro release experiment was conducted using a Franz diffusion cell to determine the in vitro drug release characteristics of the vesicles and to comprehensively evaluate the drug loading effect.

[0010] S4. The drug-loaded vesicle solution prepared by the optimal process selected in step S2 is purified to remove unencapsulated free drug, unassembled carrier material and other impurities from the system. After purification, the vesicle solution is stored under low temperature conditions. The particle size and polydispersity index of the vesicles are measured periodically and the vesicle morphology is observed to verify the long-term stability of the drug-loaded vesicles and ensure that they do not aggregate, rupture and leak drugs during storage.

[0011] Preferably, the specific operation of the combination of film dispersion-ultrasound method and emulsification-solvent evaporation method in step S1 is as follows: First, a suitable vesicle carrier material is selected, and the carrier material and drug are dissolved together in an organic solvent according to a preset ratio. The mixed solution is placed in a rotary evaporator and evaporated under preset temperature and speed conditions to completely remove the organic solvent until a uniform and dense honeycomb film is formed on the inner wall of the container. A preset hydration solvent is added to the container, and it is incubated in a 37°C constant temperature water bath for a preset time to fully hydrate the film and obtain a uniform hydrated liquid. Then, the hydrated liquid is placed in an ultrasonic cell disruptor and ultrasonically treated for a preset time at a preset power. The ultrasonic cavitation effect destroys the membrane structure and promotes the initial dispersion of drug molecules in the hydrated liquid. After uniform dispersion and ultrasonic treatment, an appropriate amount of emulsifier is added to the hydrated liquid and stirred evenly to form a stable primary emulsion. The primary emulsion is then mixed with water in a preset volume ratio and stirred and emulsified at a preset emulsification speed to form a stable oil-in-water (O / W) emulsion system. Finally, the emulsion is placed in a magnetic stirrer and stirred continuously at room temperature and a preset stirring speed for a preset time to allow the residual organic solvent in the emulsion to slowly evaporate. As the solvent evaporates, the carrier material gradually self-assembles to form a vesicle structure, and the drug is uniformly encapsulated inside the vesicles, ultimately obtaining uniformly dispersed and morphologically uniform drug-loaded nanoparticles, i.e., crude drug-loaded vesicles. During the emulsification process, an appropriate amount of stabilizer can be added as needed to improve the stability of the emulsion and prevent vesicle aggregation.

[0012] Preferably, the carrier material is one or more of polylactic acid, polyglycolic acid, or a polylactic acid-glycolic acid copolymer; the organic solvent is anhydrous ethanol; the rotary evaporator is used at a temperature of 30-50°C and a rotation speed of 50-100 r / min, with the evaporation time sufficient to completely remove the organic solvent and form a uniform honeycomb film; the hydration solvent is purified water, PBS buffer, or 0.9% physiological saline; the hydration incubation time is 30-60 min to ensure sufficient hydration of the film and formation of a uniform film. The emulsion should be homogeneous; the ultrasonic cell disruptor should have an ultrasonic power of 200-400W and an ultrasonic time of 5-30min; the volume ratio of the primary emulsion to the aqueous phase should be 1:3-1:8, the emulsification rate should be 1000-5000r / min, and the emulsification time should be based on the formation of a stable emulsion; the evaporation time of the organic solvent should be 1-6h, and the stirring speed should be 100-300r / min; the emulsifier should be one or more of Tween 80 and Span 80; and the stabilizer should be one or more of mannitol and sucrose.

[0013] Preferably, the response surface methodology in step S2 adopts a Box-Behnken experimental design, and the specific range of the factors under investigation is as follows: drug to carrier ratio of 1:2-1:10, ultrasonic time of 5-30 min, emulsification rate of 1000-5000 r / min, organic solvent evaporation time of 1-6 h, and hydration time of 30-60 min.

[0014] Preferably, the specific determination conditions for the high performance liquid chromatography method in step S3 are as follows: the chromatographic column is XB-C18 (250mm×4.6mm, 5μm), the mobile phase is acetonitrile / 0.1% ammonia (volume ratio 80:20), the flow rate is 1.0mL·min-1, the detection wavelength is 220nm, the column temperature is room temperature, and the injection volume is 20μL; the specific conditions for the centrifugal ultrafiltration method are as follows: the centrifugation speed is 8000-10000r / min, the centrifugation time is 10-15min, and the ultrafiltration membrane pore size is 0.22μm.

[0015] Preferably, the encapsulation efficiency and drug loading in step S3 are calculated using the following formula:

[0016] Encapsulation efficiency (EE, %) = (1 − W free / W total) × 100%;

[0017] Drug loading (DL, %) = (W total − W free) / (W total lipids + W total − W free) × 100%;

[0018] Wherein, Wfree represents the content of unencapsulated free drug, calculated by determining the drug concentration in the supernatant using high-performance liquid chromatography (HPLC) and combining it with the solution volume; Wtotal represents the total drug content in the drug-loaded system, calculated by determining the drug concentration in the vesicle fragmentation fluid using HPLC and combining it with the solution volume; and Wtotal represents the total carrier material content in the drug-loaded system, which is the mass of carrier material added during the experiment minus the mass of unassembled carrier material.

[0019] Preferably, the specific steps for conducting the in vitro release experiment using the Franz diffusion cell in step S3 are as follows:

[0020] Isolated rat abdominal skin was fixed onto a diffusion chamber to determine the effective permeation area. Drug-loaded vesicle solution was added to the drug delivery chamber, and a pre-prepared receiving solution was added to the receiving chamber. An in vitro release experiment was conducted under pre-prepared stirring speed and constant temperature conditions. Samples were taken at pre-prepared time points and an equal volume of receiving solution was added. After the samples were filtered through a filter membrane, the cumulative drug release was determined by HPLC. An in vitro release curve was plotted to comprehensively evaluate the drug sustained-release performance of the vesicles.

[0021] Preferably, the specific conditions for the in vitro release experiment in step S3 are: the effective permeation area of ​​the Franz diffusion cell is 3.14 cm². 2The receiving solution was physiological saline containing 30% ethanol. The stirring speed was 250 r·min, the experimental temperature was 32±0.5℃, and the sampling time points were 1h, 2h, 4h, 6h, 8h, 12h, and 24h. The sample volume was 1mL each time. After sampling, an equal volume of fresh receiving solution was added immediately. The sample was filtered through a 0.22μm microporous membrane and then detected by high performance liquid chromatography.

[0022] Preferably, the purification method in step S4 employs either centrifugation or gel filtration chromatography.

[0023] The specific conditions for the centrifugation method are: centrifugation speed of 8000-12000 r / min, centrifugation time of 10-15 min, and multiple centrifugations to ensure that impurities are completely removed;

[0024] The specific conditions for the gel filtration chromatography method are as follows: Sephadex G-50 dextran gel is used as the chromatography medium, PBS buffer is used as the elution buffer, the target elution peak is collected, and the purified drug-loaded vesicles are obtained.

[0025] The low-temperature refrigeration condition is 4°C, and the storage time is not less than 28 days.

[0026] Preferably, the model drug may also be other commonly used clinical antitumor drugs such as paclitaxel, docetaxel, and doxorubicin.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention employs a synergistic combination of thin-film dispersion-ultrasound and emulsification-solvent evaporation methods, rather than a single process selection. The two processes complement each other, effectively solving the problems of uneven drug dispersion and localized aggregation in traditional single-process methods by first preparing and hydrating the thin film, then ultrasonically dispersing it, and finally emulsifying and evaporating it to form the vesicle. This promotes the uniform distribution of drug molecules within the vesicle carrier, successfully preparing well-dispersed drug-loaded nanoparticles and laying a solid foundation for improved drug loading performance. Simultaneously, process details are optimized, such as screening for the optimal organic and hydration solvents, further improving the stability and uniformity of vesicle preparation. Response surface methodology is used for experimental design, establishing mathematical models to quantify the impact of various process parameters and their interactions on drug loading performance. This avoids the subjectivity and blindness of empirical screening, enabling precise selection of the optimal combination of process parameters, significantly improving drug encapsulation efficiency and drug loading. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0030] This invention provides a method for improving the drug loading capacity of vesicles, comprising the following steps:

[0031] S1. System optimization of drug loading process: Drug-loaded nanoparticles (i.e., crude drug-loaded vesicles) are prepared by combining thin-film dispersion-ultrasound method and emulsification-solvent evaporation method. Through the synergistic effect of the two processes, uniform dispersion of drug in vesicle carrier is achieved, thereby obtaining drug-loaded nanoparticles. A suitable vesicle carrier material is selected, which is one or a mixture of polylactic acid (PLA), polyglycolic acid (PGA), or polylactic acid-glycolic acid copolymer (PLGA). The carrier material and drug are dissolved together in an organic solvent at a predetermined ratio. The organic solvent is preferably anhydrous ethanol. To avoid using toxic and expensive solvents such as dichloromethane, place the mixed solution in a rotary evaporator and evaporate it at the preset temperature and speed to completely remove the organic solvent until a uniform and dense honeycomb film forms on the inner wall of the container. The rotary evaporation temperature is 30-50℃, and the speed is 50-100 r / min. The evaporation time is based on completely removing the organic solvent and forming a uniform honeycomb film. Add an hydration solvent to the container; purified water is preferred, but it can be replaced with PBS buffer or 0.9% physiological saline as needed. Incubate the membrane in a constant temperature water bath at 37℃ for 30-60 min to fully hydrate it and obtain a hydrated solution. Place the hydrated solution in an ultrasonic cell disruptor and sonicate it at 200-400W power for 5-30 min. The ultrasonic cavitation effect disrupts the membrane structure and promotes the initial dispersion of drug molecules. Then, add an appropriate amount of emulsifier, such as one or more of Tween 80 and Span 80, to the sonicated hydrated solution and stir until homogeneous to form a primary emulsion. Mix the primary emulsion with an aqueous phase at a predetermined volume ratio of 1:3-1:8 and stir at 1000-5000 rpm. Emulsifying at a stirring speed of 100-300 rpm to form a stable oil-in-water (O / W) emulsion system. The emulsion is then placed in a magnetic stirrer and stirred for 1-6 hours at room temperature and 100-300 rpm to allow the residual organic solvent to slowly evaporate. As the solvent evaporates, the carrier material gradually self-assembles to form vesicle structures, and the drug is uniformly encapsulated inside the vesicles, ultimately obtaining uniformly dispersed drug-loaded nanoparticles (i.e., crude drug-loaded vesicles). Stabilizers can be added during the emulsification process as needed. Stabilizers are one or more of mannitol and sucrose to improve emulsion stability and prevent vesicle aggregation.

[0032] S2. Experimental design based on response surface methodology: Using the drug-loaded nanoparticles prepared in step S1 as a basis, experimental design was conducted using response surface methodology. The Box-Behnken experimental design was adopted to screen the optimal combination of drug loading process parameters, as detailed below:

[0033] Identifying influencing factors: Key parameters that significantly affect the drug loading performance of vesicles were selected as factors for investigation using response surface methodology, including drug-to-carrier ratio (1:2-1:10), sonication time (5-30 min), emulsification rate (1000-5000 r / min), organic solvent evaporation time (1-6 h), and hydration time (30-60 min). Among these, drug-to-carrier ratio, sonication time, and emulsification rate were the core factors for investigation.

[0034] Design of response surface experiments: The Box-Behnken experimental design was adopted, with drug encapsulation efficiency and drug loading as response values. Three levels were set for each factor. Multiple parallel experiments were conducted according to the experimental design scheme. Each experiment was repeated three times, and the average value was taken as the experimental result.

[0035] Mathematical model establishment and parameter selection: The experimental data were analyzed by multiple regression using Design-Expert software to establish a quadratic polynomial mathematical model between the response value (encapsulation efficiency and drug loading) and the factors under investigation. The influence of each factor and its interaction on drug loading performance was analyzed. Through model validation and ridge analysis, the optimal combination of drug loading process parameters was selected to ensure that the encapsulation efficiency and drug loading reach the maximum value, while ensuring that the vesicle morphology is uniform and the stability is good.

[0036] S3. Model drug selection and precise determination of drug loading performance:

[0037] For the selection of model drugs, temozolomide, a commonly used anti-tumor drug in clinical practice, was chosen as the model drug. Temozolomide is suitable for the treatment of newly diagnosed glioblastoma multiforme, glioblastoma multiforme that has relapsed or progressed after conventional treatment, or anaplastic astrocytoma. Its physicochemical properties are compatible with vesicle carriers, and there is an urgent clinical need for it. By optimizing its drug delivery process, it can directly provide technical reference for clinical application.

[0038] Drug loading performance indicators were determined using high-performance liquid chromatography (HPLC) as the core method, combined with centrifugation and ultrafiltration, to accurately measure key indicators of vesicle drug loading performance, including encapsulation efficiency, drug loading capacity, and in vitro release characteristics, as detailed below:

[0039] Encapsulation efficiency and drug loading determination: The optimal process drug-loaded vesicle solution obtained in step S2 was used to separate the free drug from the drug-loaded vesicles by centrifugation and ultrafiltration. The supernatant (free drug) and vesicle lysis solution (total drug) were collected. The centrifugation speed for ultrafiltration was 8000-10000 r / min, the centrifugation time was 10-15 min, and the ultrafiltration membrane pore size was 0.22 μm. The drug concentration was then determined by HPLC. An XB-C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was acetonitrile / 0.1% ammonia (volume ratio 80:20), the flow rate was 1.0 mL·min⁻¹, the detection wavelength was 220 nm, the column temperature was room temperature, and the injection volume was 20 μL. The encapsulation efficiency and drug loading were calculated using the following formula:

[0040] Encapsulation efficiency (EE, %) = (1 − W free / W total) × 100%;

[0041] Drug loading (DL, %) = (W total − W free) / (W total lipids + W total − W free) × 100%;

[0042] Wherein, Wfree represents the content of unencapsulated free drug, calculated by determining the drug concentration in the supernatant using high-performance liquid chromatography (HPLC) and combining it with the solution volume; Wtotal represents the total drug content in the drug-loaded system, calculated by determining the drug concentration in the vesicle fragmentation fluid using HPLC and combining it with the solution volume; Wtotal represents the total carrier material content in the drug-loaded system, which is the mass of carrier material added during the experiment minus the mass of unassembled carrier material.

[0043] In vitro release characteristics determination: Exposed rat abdominal skin was fixed to a diffusion cell to determine the effective permeation area. Drug-loaded vesicle solution was added to the drug delivery cell, and a pre-prepared receiving solution was added to the receiving cell. In vitro release experiments were conducted under pre-prepared stirring speed and constant temperature conditions. Samples were taken at pre-prepared time points, and an equal volume of receiving solution was added. After filtration, the cumulative drug release was determined by HPLC, and in vitro release curves were plotted to comprehensively evaluate the sustained-release performance of the vesicles. The effective permeation area of ​​the Franz diffusion cell was 3.14 cm². 2 The receiving solution was physiological saline containing 30% ethanol. The stirring speed was 250 r·min, the experimental temperature was 32±0.5℃, and the sampling time points were 1h, 2h, 4h, 6h, 8h, 12h, and 24h. The sample volume was 1mL each time, and an equal volume of fresh receiving solution was added immediately after sampling. The samples were filtered through a 0.22μm microporous membrane and then detected by high performance liquid chromatography. The in vitro release curve was plotted with the sampling time as the abscissa and the cumulative drug release amount as the ordinate to evaluate the sustained-release performance of the vesicles. It was required that the cumulative drug release amount after 24h be 70%-80%, showing a good sustained-release effect.

[0044] S4. Purify the drug-loaded vesicle solution prepared by the optimal process selected in step S2. The purification method may be either centrifugation or gel filtration chromatography. For centrifugation, the specific conditions are: centrifugation speed 8000-12000 r / min, centrifugation time 10-15 min, with multiple centrifugations to ensure complete removal of impurities. For gel filtration chromatography, the specific conditions are: using Sephadex G-50 dextran gel as the chromatography medium, PBS buffer as the elution buffer, collecting the target elution peak, obtaining the purified drug-loaded vesicles, and removing unencapsulated free vesicles from the system. Drug, unassembled carrier materials, and other impurities were purified and stored in a refrigerator at 4°C for at least 28 days. During this period, the particle size, polydispersity index (PDI), and morphology of the vesicles were periodically monitored. The vesicle particle size was required to show no significant change within 28 days, the PDI was required to remain below 0.25, the morphology was required to be uniform, and there was no aggregation, rupture, or drug leakage. The particle size and polydispersity index (PDI) of the vesicles were determined by dynamic light scattering (DLS), and the morphology of the vesicles was observed by transmission electron microscopy (TEM) to verify the long-term stability of the vesicles and ensure that no aggregation, rupture, or drug leakage occurred during storage.

[0045] In this embodiment, the model drug can also be other commonly used clinical antitumor drugs such as paclitaxel, docetaxel, and doxorubicin. After replacement, it is only necessary to adapt and adjust the types and amounts of organic solvents, hydration solvents, and emulsifiers in step S1 and the range of process parameters in step S2 according to the physicochemical properties of the drug (hydrophilicity, molecular weight, etc.) to effectively improve the drug vesicle loading performance. The particle size range of the drug-loaded nanoparticles is 100-500 nm, the polydispersity index (PDI) is ≤0.3, and the morphology is spherical or near-spherical, with good dispersibility and stability.

[0046] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention. The experimental instruments, reagents and materials used in this embodiment are all commercially available conventional products. Among them, polylactic acid (PLA, molecular weight 50,000-100,000 Da) and polylactic acid-glycolic acid copolymer (PLGA, lactic acid-glycolic acid molar ratio 50:50, molecular weight 50,000-100,000 Da) were purchased from Sigma-Aldrich; temozolomide (purity ≥99%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; anhydrous ethanol, Tween 80, Span 80, mannitol, sucrose and other reagents were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.; rotary evaporator (RE-52AA) The following instruments were purchased: A JY92-IIN ultrasonic cell disruptor (from Shanghai Yarong Biochemical Instrument Factory); a Nippon Chemi-Conductor Biotechnology Co., Ltd. (from Ningbo Xinzhi Biotechnology Co., Ltd.); an Agilent 1260 high-performance liquid chromatograph (from Agilent Technologies); a Zetasizer Nano ZS90 dynamic light scattering system (from Malvern Instruments Ltd.); a JEM-1230 transmission electron microscope (from NEC Corporation); a Franz RC-6 diffusion cell (from Tianjin Pharmacopoeia Standard Instrument Factory); and ultrafiltration tubes (0.22 μm pore size) from Millipore. All experimental procedures were performed strictly according to aseptic techniques. Experimental data were statistically analyzed using SPSS 26.0 software, with P < 0.05 considered statistically significant.

[0047] Example 1

[0048] A method for improving the drug-loading performance of vesicles, using temozolomide as a model drug and polylactic-glycolic acid copolymer (PLGA) as the vesicle carrier material, employs a synergistic combination of thin-film dispersion-ultrasound method and emulsification-solvent evaporation method to prepare drug-loaded nanoparticles. The process parameters are optimized using response surface methodology. The specific steps are as follows:

[0049] S1. System optimization of drug delivery process:

[0050] Accurately weigh 0.4 g of PLGA and 0.1 g of temozolomide, and place them in a 50 mL round-bottom flask at a mass ratio of 4:1. Add 20 mL of anhydrous ethanol as an organic solvent, place the flask on a magnetic stirrer, and stir at 200 r / min for 30 min at room temperature until PLGA and temozolomide are completely dissolved to form a homogeneous and transparent mixed solution. Fix the round-bottom flask on a rotary evaporator, set the rotation temperature to 40℃, the rotation speed to 80 r / min, and the vacuum degree to 0.08 MPa, and continue rotary evaporation for 30 min to completely remove anhydrous ethanol until a uniform, dense, and undamaged honeycomb film forms on the inner wall of the round-bottom flask. Add 10 mL of purified water to the flask as a hydration solvent, place the flask in a 37℃ constant temperature water bath shaker, set the shaking frequency to 100 r / min, and incubate for 45 min to allow the film to fully hydrate and detach, forming a uniform hydrated solution. During this period, gently shake the flask once every 10 min to ensure complete hydration. Transfer the hydrated solution to 50 mL centrifuge tubes and place them in an ultrasonic cell disruptor, set the ultrasonic power to 300 W, and the ultrasonic mode to pulse (3 s sonication, 3 s interval) for 15 min. The ultrasonic cavitation effect disrupts the membrane structure and promotes the initial uniform dispersion of temozolomide molecules in the hydrated solution. Keep the centrifuge tubes in an ice bath during the ultrasonic process. To prevent drug degradation due to excessive temperature, after ultrasonic treatment, 0.1g Tween 80 was added to the hydrated liquid as an emulsifier. The mixture was placed on a magnetic stirrer and stirred at 300r / min for 10min at room temperature to form a stable primary emulsion. The primary emulsion was then mixed with 50mL of purified water (primary emulsion to water phase volume ratio 1:5) and transferred to a high-speed disperser. The emulsification speed was set to 3000r / min and stirred for 20min to form a stable oil-in-water (O / W) emulsion system. The emulsion was uniformly milky white and did not show any layering. The emulsion was transferred to a 100mL beaker and placed on a magnetic stirrer. The mixture was stirred continuously at 200r / min at room temperature for 4h to allow the residual anhydrous ethanol in the emulsion to slowly evaporate. PLGA gradually self-assembled into vesicle structures during the solvent evaporation process. Temozolomide was uniformly encapsulated inside the vesicles, and finally, uniformly dispersed and morphologically uniform drug-loaded nanoparticles (drug-loaded vesicle crude product) were obtained and stored in a refrigerator at 4℃ for later use.

[0051] S2. Optimization of process parameters based on response surface methodology;

[0052] S21. Identify influencing factors. Based on the results of previous single-factor experiments, select three core factors that significantly affect the drug loading performance (encapsulation efficiency and drug loading) of temozolomide vesicles: drug-to-carrier ratio (A), ultrasonic time (B), and emulsification speed (C). Each factor has three gradient levels, specifically: A1=1:2, A2=1:4, A3=1:6; B1=10min, B2=15min, B3=20min; C1=2000r / min, C2=3000r / min, C3=4000r / min.

[0053] S22. Response surface methodology: The Box-Behnken experimental design method was adopted, with the encapsulation efficiency (Y1, %) and drug loading (Y2, %) of temozolomide as the two core response values. Seventeen parallel experiments were designed, with each experiment repeated three times. The average of the three experimental results was taken as the final experimental data. The experimental design scheme and results are shown in Table 1 below (the data in the table are the average ± standard deviation of the three repeated experiments).

[0054] Table 1

[0055] Experiment number Drug-to-carrier ratio (A) Ultrasound time (B, min) Emulsification rate (C, r / min) Encapsulation efficiency (Y1, %) Drug loading (Y2, %) 1 1:4（A2） 15（B2） 3000（C2） 88.6±1.2 11.2±0.5 2 1:2（A1） 15（B2） 2000（C1） 75.3±1.5 8.9±0.4 3 1:4（A2） 10（B1） 2000（C1） 80.5±1.1 9.8±0.3 4 1:6（A3） 15（B2） 2000（C1） 82.7±1.3 8.5±0.2 5 1:4（A2） 20（B3） 2000（C1） 83.2±1.4 10.1±0.4 6 1:2（A1） 15（B2） 4000（C3） 78.6±1.2 9.2±0.3 7 1:4（A2） 10（B1） 4000（C3） 81.8±1.0 10.3±0.5 8 1:6（A3） 15（B2） 4000（C3） 85.4±1.1 9.0±0.2 9 1:4（A2） 20（B3） 4000（C3） 84.5±1.3 10.5±0.4 10 1:2（A1） 10（B1） 3000（C2） 76.8±1.4 9.0±0.3 11 1:6（A3） 10（B1） 3000（C2） 83.5±1.2 8.7±0.2 12 1:2（A1） 20（B3） 3000（C2） 79.2±1.1 9.3±0.3 13 1:6（A3） 20（B3） 3000（C2） 86.1±1.3 9.2±0.2 14 1:4（A2） 15（B2） 3000（C2） 88.3±1.2 11.0±0.4 15 1:4（A2） 15（B2） 3000（C2） 88.8±1.1 11.3±0.5 16 1:4（A2） 15（B2） 3000（C2） 88.5±1.0 11.1±0.4 17 1:4（A2） 15（B2） 3000（C2） 88.7±1.2 11.2±0.5

[0056] S23. Model Establishment and Parameter Selection: Using Design-Expert 12.0 software, a multiple regression analysis was performed on the above 17 sets of experimental data to establish a quadratic polynomial mathematical model between the response values ​​(encapsulation rate Y1, drug loading Y2) and the investigated factors (A, B, C). The regression equation for encapsulation rate (Y1) is: Y1=88.60+2.35A+1.82B+1.56C-0.85AB-0.72AC-0.68BC-3.25A²-2.86B²-2.53C², and the regression equation for drug loading (Y2) is: Y2=11.20+0.45A+0.62B+0.38C-0.25AB-0.21AC-0.18BC-0.85A²-0.72B²-0.65C².

[0057] Analysis of variance (ANOVA) was performed on the regression models. The results showed that the encapsulation efficiency model had an R² of 0.9682 and an adjusted R² of 0.9364, with P < 0.0001, indicating a good model fit and statistical significance. The drug loading model also showed a good fit with an R² of 0.9578 and an adjusted R² of 0.9156, with P < 0.0001. The model analysis revealed that the influence of each factor on encapsulation efficiency and drug loading was in the following order: drug-to-carrier ratio (A) > sonication time (B) > emulsification rate (C), and there were significant interactions among these factors. Ridge analysis and... Model optimization was performed, and the optimal combination of drug loading process parameters was selected as follows: drug-to-carrier ratio 1:4 (A2), ultrasonic time 15 min (B2), and emulsification speed 3000 r / min (C2). At this time, the model predicted an encapsulation efficiency of 88.7% and a drug loading of 11.2%. To verify the reliability of the model, three verification experiments were conducted according to this optimal parameter combination. The encapsulation efficiency was measured to be 88.4 ± 1.1%, and the drug loading was 11.1 ± 0.4%. The relative errors between the predicted and actual values ​​were 0.34% and 0.89%, respectively, both ≤ 5%, indicating that the optimal combination of process parameters has good reliability and repeatability.

[0058] S3. Precise determination of drug loading capacity:

[0059] S31. Encapsulation efficiency and drug loading determination: Take 10 mL of the drug-loaded vesicle solution prepared by the optimal process in step S2, place it in a centrifugal ultrafiltration tube, set the centrifugation speed to 9000 r / min and the centrifugation time to 12 min, separate the free drug and drug-loaded vesicles by centrifugal ultrafiltration, collect the supernatant (containing free drug), add 10 mL of anhydrous ethanol to the centrifugal ultrafiltration tube, and sonicate for 5 min to completely rupture the vesicles, collect the vesicle rupture liquid (containing total drug), and determine the temozolomide concentration in the supernatant and vesicle rupture liquid respectively by high performance liquid chromatography (HPLC). The specific HPLC determination conditions are as follows: XB-C18 column (250 mm × 4.6 mm, 5 μm), mobile phase is acetonitrile / 0.1% ammonia (volume ratio 80:20), flow rate is 1.0 mL·min⁻¹, detection wavelength is 220 nm, column temperature is room temperature, injection volume is 20 μL, and each sample is measured in triplicate. Calculate the encapsulation efficiency and drug loading according to the following formula:

[0060] Encapsulation efficiency (EE, %) = (1 − W free / W total) × 100%;

[0061] Drug loading (DL, %) = (W total − W free) / (W total lipids + W total − W free) × 100%;

[0062] Wherein, Wfree represents the content of unencapsulated free drug (calculated by measuring the drug concentration in the supernatant × the volume of the supernatant using HPLC), Wtotal represents the total drug content in the drug-loaded system (calculated by measuring the drug concentration in the vesicle rupture fluid × the volume of the vesicle rupture fluid using HPLC), and Wtotal lipids represent the total carrier material content in the drug-loaded system (0.4 g in this example). The calculation results show that the average encapsulation efficiency of the three validation experiments was 88.4 ± 1.1%, and the average drug loading was 11.1 ± 0.4%, which meets the expected target.

[0063] S31. In vitro release characteristics determination: In vitro release experiments were conducted using a Franz diffusion cell. Healthy SD rats (weighing 200±20g) were selected, euthanized by cervical dislocation, and the abdominal skin was quickly peeled off to remove subcutaneous fat and connective tissue. The skin was rinsed with physiological saline and frozen at -20℃. Before use, the skin was thawed and rinsed three times with physiological saline. The treated isolated rat abdominal skin was fixed between the supply and receiving cells of the Franz diffusion cell, with the stratum corneum of the skin facing the supply cell, and the effective permeation area was 3.14cm2. 50mL of physiological saline containing 30% ethanol was added to the receiving cell as the receiving solution (to ensure that temozolomide has good solubility in the receiving solution). The stirring speed was set to 250r·min-1, and the experimental temperature was 32±0.5℃ (simulating human skin temperature). After equilibration for 30min, 10mL of the drug-loaded vesicle solution prepared by the optimal process was added to the supply cell, and the timing was started.

[0064] Samples were taken at 1h, 2h, 4h, 6h, 8h, 12h, and 24h, with 1mL of sample taken each time. Immediately after sampling, 1mL of fresh receiving fluid was added to the receiving pool to maintain a constant volume of receiving fluid. After filtration through a 0.22μm microporous membrane, the drug concentration was determined using the HPLC method described above. The cumulative drug release at each time point was calculated. Each time point was measured in triplicate, and the average value was taken. An in vitro release curve was plotted with sampling time as the x-axis and cumulative drug release as the y-axis. The results showed that the cumulative drug release of temozolomide vesicles within 24h was 78.3±2.1%, and the release curve exhibited obvious sustained-release characteristics. The first 4h was the rapid release phase, with a cumulative release of 45.2%, followed by a slow release phase. After 24h, a small amount of drug remained unreleased, indicating that the drug-loaded vesicles have good sustained-release properties, which can prolong the duration of drug action in vivo and reduce the frequency of administration.

[0065] S4. Vesicle purification and stability verification:

[0066] 50 mL of the drug-loaded vesicle solution prepared by the optimal process in step S2 was placed in a high-speed centrifuge. The centrifugation speed was set to 10000 r / min and the centrifugation time to 12 min. After centrifugation, the supernatant (containing free drug and unassembled PLGA) was discarded. 10 mL of purified water was added to the precipitate, and the mixture was gently resuspended by pipetting. The centrifugation was repeated twice to obtain purified drug-loaded vesicles. The purified vesicle solution was transferred to sterile centrifuge tubes and stored in a refrigerator at 4°C. Samples were taken at 0d, 7d, 14d, 21d, and 28d for stability testing.

[0067] The particle size and polydispersity index (PDI) of the vesicles were determined by dynamic light scattering (DLS) under the following conditions: temperature 25℃, scattering angle 90°, and three parallel measurements for each sample. The morphology of the vesicles was observed by transmission electron microscopy (TEM). The vesicle solution was diluted to an appropriate concentration, dropped onto a copper grid, stained with phosphotungstic acid, and then allowed to dry naturally before being observed and photographed under TEM. The stability test results showed that the particle size of the drug-loaded vesicles remained between 200-300 nm and the PDI remained between 0.20-0.25 within 28 days, with no significant changes. TEM observation showed that the vesicles were uniform in morphology, spherical or near-spherical, with intact vesicle walls, no aggregation or rupture, and no drug leakage was observed. This indicates that the drug-loaded vesicles have good long-term stability and meet the requirements for formulation storage and clinical application.

[0068] Example 2

[0069] A method for improving the drug-loading performance of vesicles involves using temozolomide as a model drug and polylactic acid (PLA) as the vesicle carrier material. Drug-loaded nanoparticles are prepared using a combination of thin-film dispersion-ultrasound and emulsification-solvent evaporation methods. Process parameters are optimized using response surface methodology. The specific steps are as follows:

[0070] S1: System optimization of drug delivery process:

[0071] Weigh 0.6g PLA and 0.1g temozolomide, and place them in a 50mL round-bottom flask at a mass ratio of 6:1. Add 25mL anhydrous ethanol as the organic solvent. Place the flask on a magnetic stirrer and stir at 250rpm for 40min at room temperature until PLA and temozolomide are completely dissolved, forming a homogeneous and transparent mixed solution. Fix the round-bottom flask on a rotary evaporator, set the rotation temperature to 35℃, the rotation speed to 70rpm, and the vacuum degree to 0.07MPa, and continue rotary evaporation for 40min to completely remove anhydrous ethanol until a uniform and dense honeycomb film forms on the inner wall of the round-bottom flask. Add 12mL of [unspecified solvent] to the flask. Using PBS buffer (pH=7.4) as the hydration solvent, the flask was placed in a 37°C constant temperature water bath shaker at a shaking frequency of 120 rpm for 60 min to allow the membrane to fully hydrate and detach, forming a homogeneous hydration solution. During this process, the flask was gently shaken once every 15 min to ensure complete hydration. The hydration solution was then transferred to 50 mL centrifuge tubes and placed in an ultrasonic cell disruptor. The ultrasonic power was set to 250 W, and the ultrasonic mode was pulsed (4 s sonication followed by 2 s intervals) for 20 min to promote the initial uniform dispersion of temozolomide molecules in the hydration solution. During sonication, the centrifuge tubes were kept in an ice bath to prevent drug degradation due to excessive temperature. After sonication, 0.12 g of Span 80 was added to the hydration solution as an emulsifier, and the mixture was stirred on a magnetic stirrer at room temperature and 350 rpm for 15 min to form a stable primary emulsion. The primary emulsion was then mixed with 72 mL of... Mix the PBS buffer (primary emulsion to aqueous phase volume ratio 1:6), transfer it to a high-speed disperser, set the emulsification speed to 3500 r / min, and stir for 15 min to form a stable oil-in-water (O / W) emulsion system. Transfer the emulsion to a 100 mL beaker, place it on a magnetic stirrer, and stir continuously at room temperature and 250 r / min for 5 h to allow the residual anhydrous ethanol in the emulsion to slowly evaporate. As the solvent evaporates, PLA gradually self-assembles into vesicle structures, and temozolomide is uniformly encapsulated inside the vesicles. Finally, uniformly dispersed and morphologically uniform drug-loaded nanoparticles (crude drug-loaded vesicles) are obtained and stored at 4°C for later use.

[0072] S2. Process parameter optimization based on response surface methodology:

[0073] S21. Identify influencing factors. Based on the results of previous single-factor experiments, select three core factors that significantly affect the drug loading performance (encapsulation efficiency and drug loading) of temozolomide vesicles: drug-to-carrier ratio (A), sonication time (B), and hydration time (C). Each factor has three gradient levels, specifically: A1=1:4, A2=1:6, A3=1:8; B1=15min, B2=20min, B3=25min; C1=40min, C2=50min, C3=60min.

[0074] S22. Response surface methodology: The Box-Behnken experimental design method was adopted, with the encapsulation efficiency (Y1, %) and drug loading (Y2, %) of temozolomide as the two core response values. Seventeen parallel experiments were designed, with each experiment repeated three times. The average of the three experimental results was taken as the final experimental data to ensure the reliability of the experimental data.

[0075] S23. Model Establishment and Parameter Selection: Multiple regression analysis was performed on the experimental data using Design-Expert 12.0 software to establish a quadratic polynomial mathematical model between the response value and the investigated factors. Analysis of variance verified that the model had a good fit (R²≥0.95) and was statistically significant (P<0.0001). The model was used to analyze the influence of each investigated factor and its interaction on drug loading performance. Combined with ridge analysis and model optimization, the optimal combination of drug loading process parameters was selected as follows: drug-to-carrier ratio 1:6 (A2), ultrasonic time 20 min (B2), and hydration time 50 min (C2). At this time, the model predicted an encapsulation rate of 86.5% and a drug loading of 10.6%. To verify the reliability of the model, three verification experiments were conducted according to this optimal parameter combination. The actual measured encapsulation rate was 86.3±1.2%, and the drug loading was 10.5±0.3%. The relative errors between the predicted and actual values ​​were 0.23% and 0.95%, respectively, both ≤5%, indicating that the optimal process parameter combination has good reliability and repeatability.

[0076] S3. Precise determination of drug loading capacity:

[0077] S31. Encapsulation efficiency and drug loading were determined using the same centrifugation and ultrafiltration method as in Example 1 to separate free drug from drug-loaded vesicles. The drug concentration was determined using the same HPLC conditions, and the encapsulation efficiency and drug loading were calculated using the same formula. Each sample was measured in triplicate. The results showed that the average encapsulation efficiency of the three verification experiments was 86.3±1.2%, and the average drug loading was 10.5±0.3%, which met the expected targets.

[0078] S32. In vitro release characteristics determination: The in vitro release experiment was conducted using the same Franz diffusion cell method as in Example 1, with completely identical experimental conditions. Each time point was measured in parallel three times, and the average value was used to plot the in vitro release curve. The results showed that the cumulative drug release of temozolomide vesicles within 24 hours was 75.8±2.3%, and the release curve showed obvious sustained-release characteristics. The cumulative release reached 42.5% in the first 4 hours, and then entered a slow release phase, indicating that the drug-loaded vesicles have good sustained-release performance and can meet the needs of clinical drug use.

[0079] S4. Vesicle purification and stability verification:

[0080] The drug-loaded vesicle solution was purified by gel filtration chromatography. The specific procedure was as follows: Sephadex G-50 dextran gel was used as the chromatography medium, and a chromatography column (1.5cm×30cm) was packed. The chromatography column was equilibrated with PBS buffer (pH=7.4) until the pH of the eluent was consistent with that of the equilibration buffer. 10mL of the drug-loaded vesicle solution prepared by the optimal process in step 2 was loaded onto the top of the chromatography column. PBS buffer was used as the eluent, and the elution rate was controlled at 1mL / min. One tube of eluent was collected every 5mL. The drug concentration in each tube of eluent was determined by HPLC. The elution peak with the higher drug concentration (target elution peak) was collected, which is the purified drug-loaded vesicle solution.

[0081] The purified vesicle solution was stored at 4°C. Samples were taken at 0, 7, 14, 21, and 28 days. The particle size and PDI of the vesicles were determined by DLS, and the morphology of the vesicles was observed by TEM. The stability test results showed that the particle size of the drug-loaded vesicles remained between 250 and 350 nm and the PDI remained between 0.22 and 0.24 within 28 days, with no significant changes. TEM observation showed that the vesicles were uniform in morphology, spherical or near-spherical, with intact vesicle walls, no aggregation or rupture, and no drug leakage was observed. The stability met the requirements for formulation storage and clinical application.

[0082] Comparative experiment

[0083] To further verify the method of this invention, a comparative experiment was set up. Temozolomide vesicles were prepared using the traditional single-film dispersion method without process optimization or response surface methodology parameter screening. The specific steps are as follows:

[0084] Preparation of drug-loaded vesicles: Accurately weigh 0.4 g of PLGA and 0.1 g of temozolomide, and place them in a 50 mL round-bottom flask at a mass ratio of 4:1. Add 20 mL of anhydrous ethanol and stir at room temperature for 30 min until completely dissolved. Fix the round-bottom flask on a rotary evaporator and rotary evaporate at 40 °C and 80 r / min for 30 min to remove anhydrous ethanol and form a thin film. Add 10 mL of purified water to the flask and incubate in a constant temperature water bath at 37 °C for 45 min. After hydration, sonicate for 15 min (power 300 W) to obtain the drug-loaded vesicle solution. Do not perform emulsification-solvent evaporation treatment and use it directly as the drug-loaded vesicle sample.

[0085] Drug loading performance determination: The encapsulation efficiency and drug loading of drug-loaded vesicles were determined by ultraviolet spectrophotometry (detection wavelength 220 nm). The in vitro release characteristics were determined by the same method as in Example 1. Each sample was measured in parallel 3 times and the average value was taken.

[0086] Stability verification: The drug-loaded vesicle solution was stored in a refrigerator at 4°C. Samples were taken at 0, 7 and 14 days. The particle size and PDI of the vesicles were determined by DLS method, and the morphology of the vesicles was observed by TEM to detect the stability of the vesicles.

[0087] The drug loading performance was determined by ultraviolet spectrophotometry. Comparative experimental results showed that the temozolomide vesicles prepared by the traditional single thin-film dispersion method had an average encapsulation efficiency of 65.2±2.3%, an average drug loading of 7.8±0.5%, and a cumulative drug release of 92.1±3.2% in vitro over 24 hours, indicating poor sustained-release effect. Stability testing showed that after 7 days of storage, the particle size of the vesicles increased significantly, and the PDI rose above 0.45. TEM observation showed that the vesicles exhibited obvious aggregation and rupture, resulting in drug leakage and poor stability.

[0088] Compared with traditional methods, Example 1 of this invention improves the encapsulation efficiency by more than 35%, the drug loading by more than 43%, and reduces the cumulative drug release over 24 hours by about 14%, demonstrating significantly better sustained-release performance and stability than traditional methods. Example 2 of this invention improves the encapsulation efficiency by more than 32% and the drug loading by more than 34%. Through the above examples and comparative experiments, it is demonstrated that this invention, by synergistically combining the thin-film dispersion-ultrasound method with the emulsification-solvent evaporation method and optimizing process parameters using response surface methodology, can effectively improve the drug loading performance of vesicles, overcome the defects of traditional single processes, and has significant technical advantages.

[0089] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present technical solution are within the protection scope of the present invention.

Claims

1. A method for improving the drug-carrying capacity of vesicles, characterized in that, Includes the following steps: S1. System optimization of drug loading process: Drug-loaded nanoparticles are prepared by combining thin film dispersion-ultrasound method and emulsification-solvent evaporation method. First, the vesicle carrier material and drug are dissolved and then formed into a uniform thin film by rotary evaporation. Aqueous solvent is added and incubated to obtain hydrated liquid. Then, the hydrated liquid is ultrasonically treated to promote drug dispersion. Subsequently, an emulsifier is added to form a primary emulsion and mixed with the aqueous phase for emulsification. The organic solvent is slowly evaporated by stirring to realize the self-assembly of the carrier material to form vesicle structure, and finally drug-loaded nanoparticles with uniform drug dispersion are obtained. S2. Based on the response surface methodology, the experimental design was carried out. Based on the drug-loaded nanoparticles prepared in step S1, the drug-to-carrier ratio, ultrasonic time, and emulsification rate were selected as the factors to be investigated. Combined with the Box-Behnken experimental design, the drug encapsulation efficiency and drug loading were used as the response values. The interaction between the factors was analyzed by establishing a mathematical model, and the optimal combination of drug loading process parameters was selected. S3. Temozolomide, a commonly used antitumor drug in clinical practice, was selected as the model drug. High performance liquid chromatography combined with centrifugation and ultrafiltration was used to accurately determine the core performance indicators of the drug-loaded vesicles, including drug encapsulation efficiency and drug loading. At the same time, an in vitro release experiment was conducted using a Franz diffusion cell to determine the in vitro drug release characteristics of the vesicles and to comprehensively evaluate the drug loading effect. S4. The drug-loaded vesicle solution prepared by the optimal process selected in step S2 is purified to remove unencapsulated free drug, unassembled carrier material and other impurities from the system. After purification, the vesicle solution is stored under low temperature conditions. The particle size and polydispersity index of the vesicles are measured periodically and the vesicle morphology is observed to verify the long-term stability of the drug-loaded vesicles and ensure that they do not aggregate, rupture and leak drugs during storage.

2. The method for improving the drug loading performance of vesicles according to claim 1, characterized in that, The specific operation of the combined thin-film dispersion-ultrasound method and emulsification-solvent evaporation method described in step S1 is as follows: First, a suitable vesicle carrier material is selected, and the carrier material and drug are dissolved together in an organic solvent at a preset ratio. The mixed solution is placed in a rotary evaporator and evaporated under preset temperature and speed conditions to completely remove the organic solvent until a uniform and dense honeycomb film is formed on the inner wall of the container. A preset hydration solvent is added to the container, and it is incubated in a 37°C constant temperature water bath for a preset time to allow the film to fully hydrate and obtain a uniform hydrated liquid. Subsequently, the hydrated liquid is placed in an ultrasonic cell disruptor and ultrasonically treated at a preset power for a preset time. The ultrasonic cavitation effect destroys the membrane structure and promotes the initial uniformity of drug molecules in the hydrated liquid. After dispersion and ultrasonic treatment, an appropriate amount of emulsifier is added to the hydrated liquid and stirred evenly to form a stable primary emulsion. The primary emulsion is then mixed with water in a preset volume ratio and stirred at a preset emulsification speed to form a stable oil-in-water (O / W) emulsion system. Finally, the emulsion is placed in a magnetic stirrer and stirred continuously at room temperature and a preset stirring speed for a preset time to allow the residual organic solvent in the emulsion to slowly evaporate. As the solvent evaporates, the carrier material gradually self-assembles to form a vesicle structure, and the drug is uniformly encapsulated inside the vesicles, ultimately obtaining uniformly dispersed and morphologically uniform drug-loaded nanoparticles, i.e., crude drug-loaded vesicles. During the emulsification process, an appropriate amount of stabilizer can be added as needed to improve the stability of the emulsion and prevent vesicle aggregation.

3. The method for improving the drug loading performance of vesicles according to claim 2, characterized in that, The carrier material is one or more of polylactic acid, polyglycolic acid, or polylactic acid-glycolic acid copolymer; the organic solvent is anhydrous ethanol; the rotary evaporator is used at a temperature of 30-50°C and a rotation speed of 50-100 r / min, with the evaporation time sufficient to completely remove the organic solvent and form a uniform honeycomb film; the hydration solvent is purified water, PBS buffer, or 0.9% physiological saline; the hydration incubation time is 30-60 min to ensure full hydration of the film and formation of a uniform film. The hydrated liquid is the standard; the ultrasonic cell disruptor has an ultrasonic power of 200-400W and an ultrasonic time of 5-30min; the volume ratio of the primary emulsion to the aqueous phase is 1:3-1:8, the emulsification speed is 1000-5000r / min, and the emulsification time is based on the formation of a stable emulsion; the evaporation time of the organic solvent is 1-6h, and the stirring speed is 100-300r / min; the emulsifier is one or more of Tween 80 and Span 80; the stabilizer is one or more of mannitol and sucrose.

4. The method for improving the drug loading performance of vesicles according to claim 1, characterized in that, The response surface methodology described in step S2 employs a Box-Behnken experimental design. The specific ranges of the factors under investigation are as follows: drug-to-carrier ratio of 1:2 to 1:10, sonication time of 5 to 30 min, emulsification rate of 1000 to 5000 r / min, organic solvent evaporation time of 1 to 6 h, and hydration time of 30 to 60 min.

5. The method for improving the drug loading performance of vesicles according to claim 1, characterized in that, The specific determination conditions for the high performance liquid chromatography method described in step S3 are as follows: the chromatographic column is XB-C18 (250mm×4.6mm, 5μm), the mobile phase is acetonitrile / 0.1% ammonia (volume ratio 80:20), the flow rate is 1.0mL·min-1, the detection wavelength is 220nm, the column temperature is room temperature, and the injection volume is 20μL; the specific conditions for the centrifugal ultrafiltration method are as follows: the centrifugation speed is 8000-10000r / min, the centrifugation time is 10-15min, and the ultrafiltration membrane pore size is 0.22μm.

6. The method for improving the drug loading performance of vesicles according to claim 1, characterized in that, The encapsulation efficiency and drug loading in step S3 are calculated using the following formulas: Encapsulation efficiency (EE, %) = (1 − W free / W total) × 100%; Drug loading (DL, %) = (W total − W free) / (W total lipids + W total − W free) × 100%; Wherein, Wfree represents the content of unencapsulated free drug, calculated by determining the drug concentration in the supernatant using high-performance liquid chromatography (HPLC) and combining it with the solution volume; Wtotal represents the total drug content in the drug-loaded system, calculated by determining the drug concentration in the vesicle fragmentation fluid using HPLC and combining it with the solution volume; and Wtotal represents the total carrier material content in the drug-loaded system, which is the mass of carrier material added during the experiment minus the mass of unassembled carrier material.

7. The method for improving the drug loading performance of vesicles according to claim 1, characterized in that, The specific steps for the in vitro release experiment using the Franz diffusion cell in step S3 are as follows: Isolated rat abdominal skin was fixed onto a diffusion chamber to determine the effective permeation area. Drug-loaded vesicle solution was added to the drug delivery chamber, and a pre-prepared receiving solution was added to the receiving chamber. An in vitro release experiment was conducted under pre-prepared stirring speed and constant temperature conditions. Samples were taken at pre-prepared time points and an equal volume of receiving solution was added. After the samples were filtered through a filter membrane, the cumulative drug release was determined by HPLC. An in vitro release curve was plotted to comprehensively evaluate the drug sustained-release performance of the vesicles.

8. The method for improving the drug loading performance of vesicles according to claim 7, characterized in that, The specific conditions for the in vitro release experiment described in step S3 are as follows: the effective permeation area of ​​the Franz diffusion cell is 3.14 cm². 2 The receiving solution was physiological saline containing 30% ethanol. The stirring speed was 250 r·min, the experimental temperature was 32±0.5℃, and the sampling time points were 1h, 2h, 4h, 6h, 8h, 12h, and 24h. The sample volume was 1mL each time. After sampling, an equal volume of fresh receiving solution was added immediately. The sample was filtered through a 0.22μm microporous membrane and then detected by high performance liquid chromatography.

9. A method for improving the drug loading performance of vesicles according to any one of claims 1, characterized in that, The purification method described in step S4 uses either centrifugation or gel filtration chromatography. The specific conditions for the centrifugation method are: centrifugation speed of 8000-12000 r / min, centrifugation time of 10-15 min, and multiple centrifugations to ensure that impurities are completely removed; The specific conditions for the gel filtration chromatography method are as follows: Sephadex G-50 dextran gel is used as the chromatography medium, PBS buffer is used as the elution buffer, the target elution peak is collected, and the purified drug-loaded vesicles are obtained. The low-temperature refrigeration condition is 4°C, and the storage time is not less than 28 days.

10. A method for improving the drug loading performance of vesicles according to any one of claims 1, characterized in that, The model drug can also be other commonly used clinical antitumor drugs such as paclitaxel, docetaxel, and doxorubicin.