Preparation method of pH / ROS double-response drug-loaded microspheres, microneedle system and method for optimizing microneedle system

Abstract

The invention provides a preparation method of pH / ROS double-response drug-loaded microspheres, a microneedle system and a method for optimizing the microneedle system, and belongs to the technical field of crossing of biomedical engineering and intelligent materials. The preparation method of the pH / ROS double-response drug-loading microspheres comprises the following steps: mixing chitosan, 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate and dialdehyde polyethylene glycol, emulsifying, and carrying out chemical crosslinking, so as to obtain the pH / ROS double-response drug-loading microspheres. According to the pH / ROS dual-response drug-loading microsphere provided by the invention, targeted precise drug release of an inflammation site can be realized; the core-shell structure layered microneedle system has a shell layer-transition layer-core layer three-dimensional structure, so that sequential controlled release can be realized; according to the method for optimizing the microneedle system, reverse optimization of the preparation process can be achieved, and an individualized treatment scheme can be predicted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering and smart materials, and in particular to a method for preparing pH / ROS dual-responsive drug-loaded microspheres, a microneedle system, and a method for optimizing the microneedle system. Background Art

[0002] In the field of transdermal drug delivery, traditional microneedle systems generally have the following problems: single-factor response limitation: existing technologies mostly rely on a single pH response mechanism, which cannot distinguish between the differences between physiological and pathological microenvironments, resulting in nonspecific drug release, which can easily cause systemic toxicity in clinical applications; structural homogeneity defects: traditional homogeneous drug-loaded microneedles have a significant burst release effect, which is difficult to meet the long-term treatment needs of chronic inflammation; uncontrollable preparation process: the microsphere encapsulation efficiency is limited by empirical parameter regulation, and the drug loading between batches fluctuates by more than ±15%, affecting the consistency of treatment.

[0003] Therefore, there is an urgent need to provide a drug delivery carrier that can specifically release and has strong sustained-release performance to solve the problems existing in existing drug delivery carriers. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of pH / ROS dual-responsive drug-loaded microspheres, a microneedle system and a method for optimizing the microneedle system. The pH / ROS dual-responsive drug-loaded microspheres can achieve targeted and precise drug release at inflammatory sites, the core-shell structure layered microneedle system can achieve time-controlled release, and the method for optimizing the microneedle system can achieve reverse optimization of the preparation process and prediction of individualized treatment plans.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing pH / ROS dual-responsive drug-loaded microspheres, comprising the following steps:

[0007] Chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol are mixed, emulsified, and chemically cross-linked to obtain the pH / ROS dual-responsive drug-loaded microspheres.

[0008] Preferably, the preparation method of the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester comprises the following steps:

[0009] 4-hydroxyphenylboronic acid pinacol ester is dissolved in dimethyl sulfoxide, activated, added with chitosan, reacted, and freeze-dried to obtain the chitosan modified with the 4-hydroxyphenylboronic acid pinacol ester.

[0010] Preferably, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide and N-hydroxysuccinimide are added during the activation; the activation time is 0.5 to 1 hour; the mass percentages of 4-hydroxyphenylboronic acid pinacol ester, chitosan, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide, and N-hydroxysuccinimide are 1 to 5:1 to 4:0.4 to 0.8:0.2 to 0.4; the reaction time is 10 to 14 hours; the freeze-drying temperature is -75 to -85°C, and the freeze-drying time is 70 to 75 hours.

[0011] Preferably, the mass percentage of the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol is 1-4:0.2-0.8:2-10; the emulsification speed is 1400-1600 r / min, and the emulsification time is 0.5-1.5 h; the chemical crosslinking temperature is 10-20° C., and the chemical crosslinking time is 5-10 min.

[0012] The present invention also provides a core-shell structured layered microneedle system, comprising a core layer, a transition layer and a shell layer;

[0013] The preparation method of the core layer comprises the following steps: mixing the pH / ROS dual-responsive drug-loaded microspheres prepared by the preparation method with polyvinyl alcohol, methacrylated hyaluronic acid, methacrylated gelatin, aminosulfonated cellulose, pyrogallic acid, and cerium oxide, and subjecting the mixture to a freeze-thaw cycle to form microneedles to obtain the core layer;

[0014] The preparation method of the aminosulfonated cellulose comprises the following steps: mixing aminosulfonic acid and N,N-dimethylformamide, adding cellulose, reacting, and freeze-drying to obtain the aminosulfonated cellulose.

[0015] Preferably, the mass ratio of aminosulfonic acid, N,N-dimethylformamide and cellulose is 0.04-0.06:0.01-0.03:0.1-0.3; the reaction temperature is 75-85°C, and the reaction time is 1-2 hours; the freeze-drying temperature is -75--85°C, and the freeze-drying time is 70-75 hours.

[0016] Preferably, the mass percentage of the pH / ROS dual-responsive drug-loaded microspheres to polyvinyl alcohol, methacrylated hyaluronic acid, methacrylated gelatin, aminosulfonated cellulose, pyrogallic acid, and cerium oxide is 30-40:1-4:1-4:10-20:1-4:0.05-0.2:0.05-0.2; an initiator and a catalyst are added during the mixing, and the added amounts of the initiator and catalyst are both 0.1-0.5% of the mass of the mixed solution; the initiator is ammonium persulfate, and the catalyst is tetramethylethylenediamine.

[0017] Preferably, the preparation method of the transition layer is the same as that of the core layer, and the mass percentage of pH / ROS dual-responsive drug-loaded microspheres to polyvinyl alcohol, methacrylated hyaluronic acid, methacrylated gelatin, aminosulfonated cellulose, pyrogallic acid, and cerium oxide in the transition layer is 15-20:1-3:1-3:8-12:1-3:0.1-0.3:0.1-0.3;

[0018] The shell layer is polyvinyl alcohol;

[0019] The preparation method of the shell layer comprises the following steps: dripping a polyvinyl alcohol solution into a microneedle mold, and subjecting the microneedle mold to a freeze-thaw cycle to form a needle tip shell, thereby obtaining the shell layer; the freeze-thaw cycle of the shell layer is the same as that of the core layer;

[0020] The number of freeze-thaw cycle treatments is 3 to 5 times, and the freeze-thaw cycle treatment method is: freezing at -25 to -15°C for 1 to 2 hours, and thawing at 20 to 30°C for 0.5 to 1 hour.

[0021] The present invention also provides a method for optimizing a core-shell structured layered microneedle system based on deep reinforcement learning, comprising the following steps:

[0022] (1) Extracting and collecting the DICOM data image features of the pH / ROS dual-responsive drug-loaded microspheres, the maximum release rate, half-life, and mechanical strength of the core-shell structured layered microneedle system;

[0023] (2) Constructing a deep deterministic policy gradient algorithm framework based on the Actor-Critic algorithm;

[0024] (3) The state space is defined as a 12-dimensional process parameter set, and the action space is defined as the structural parameter adjustment and process condition control operation; a hybrid reward function is used to dynamically evaluate the drug release efficiency, mechanical strength and biocompatibility to guide model training. The reward function is shown in Formula I, and the loss function is the temporal difference error (TD-Error) combined with the KL divergence constraint. The learning rate is 1e -4 , training cycle 480~520 times;

[0025]

[0026] Among them, α+β+γ=1, δ is the penalty coefficient;

[0027] (4) Through transfer learning, the algorithm framework of deep deterministic policy gradient is jointly trained with small sample experimental data and COMSOL fluid simulation data. The double-delay deep deterministic policy gradient is improved by using the algorithm, and finally the three-dimensional response surface model containing the process parameter optimization strategy is output.

[0028] Preferably, the DICOM data image features of the Micro CT in step (1) include porosity, particle size distribution, and sphericity; the Actor network in the Actor-Critic algorithm in step (2) adopts a four-layer fully connected structure to process process parameters, and the Critic network in the Actor-Critic algorithm integrates a convolution module to process the input Micro CT DICOM data; the 12-dimensional process parameters in step (3) include drug loading, microsphere / microneedle size, encapsulation efficiency, drug loading efficiency, drug release rate, dissolution rate, swelling rate, degradation time, bioavailability, Young's modulus, surface charge and permeability coefficient, and the action space includes adjusting the drug release rate, changing the structure of the microneedle system, and adjusting the temperature and pressure of the processing conditions; the small sample experimental data in step (4) include the drug release curve obtained by experiment, the depth of microneedle penetration into the skin, and the dissolution rate of microspheres at different pH values; the COMSOL fluid simulation data are the drug release rate, the diffusion process of microspheres in the fluid, the mechanical force of microneedle penetrating the skin, and the fluid flow simulation under different solvent concentrations, temperatures, and pressures.

[0029] The beneficial effects of the present invention compared with the prior art are:

[0030] 1. The present invention provides a method for preparing pH / ROS dual-responsive drug-loaded microspheres. The present invention establishes a pH / ROS dual-cascade response mechanism by introducing bisaldehyde polyethylene glycol (DFPEG) and 4-hydroxyphenylboronic acid pinacol ester (4-CPBAPE), which can achieve targeted and precise drug release at inflammatory sites; the present invention also provides a core-shell structured layered microneedle system, which consists of a shell layer-transition layer-core layer. By gradiently controlling the laminar rhinology (180-520 μm) and microsphere density (0-40 wt%) of the three-dimensional system, time-controlled release can be achieved; at the same time, the present invention realizes reverse optimization of the preparation process and prediction of individualized treatment plans by developing a deep reinforcement learning model for multimodal data fusion.

[0031] 2. The present invention achieves precise drug release at inflammatory sites through the dynamic response of boronate bonds mediated by 4-CPBAPE and synergistically with chitosan-polyethylene glycol diacrylate (PEGDA)-bisaldehyde polyethylene glycol (DFPEG) microspheres. Combined with the AI-optimized layered microneedle structure, the time it takes for the peak blood drug concentration to be extended by 3-5 times, and the clinical efficacy is expected to increase. This system overcomes the problem of nonspecific release of traditional transdermal drug delivery and provides an innovative solution for the treatment of gout.

[0032] 3. This system can be mass-produced, and it is expected that 500-1000 microneedle patches (3×3 array) can be prepared in a single batch; it is suitable for transdermal precision treatment of inflammatory diseases such as myocardial infarction, gout, and rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 The following is a flow chart of the preparation process;

[0035] Figure 2 This is the SEM image of pH / ROS dual-responsive microspheres, where Figure 2 (A) is a 50-fold magnified image. Figure 2 (B) is the image magnified 100 times. Figure 2 (C) in the figure is a 500-fold magnified image. Figure 2 (D) in the figure is a 1000-fold magnified image;

[0036] Figure 3 This is the infrared spectroscopy characterization result of pH / ROS dual-responsive microspheres;

[0037] Figure 4 This is the standard curve of colchicine;

[0038] Figure 5 The SEM images of microspheres with different drug loadings are shown in Figure 2. Figure 5 (A) is an image of microspheres containing 3 mg of colchicine per ml of chitosan solution at 50x magnification; Figure 5 (B) is an image of microspheres containing 3 mg of colchicine per ml of chitosan solution at 100 times magnification; Figure 5 (C) is an image of microspheres containing 30 mg of colchicine per ml of chitosan solution at 50x magnification; Figure 5 (D) is an image of microspheres containing 30 mg of colchicine per ml of chitosan solution at 100 times magnification; Figure 5(E) is an image of microspheres containing 150 mg of colchicine per ml of chitosan solution at 50x magnification; Figure 5 (F) in the middle is an image of microspheres containing 150 mg of colchicine per ml of chitosan solution at 100 times;

[0039] Figure 6 is the drug release amount of microspheres at different drug loading levels;

[0040] Figure 7 is the drug encapsulation efficiency and drug loading rate of the microspheres at different drug loading amounts;

[0041] Figure 8 is the pH-responsive release result;

[0042] Figure 9 The result is ROS responsive release;

[0043] Figure 10 This is the result of pH / ROS dual-responsive synergistic release;

[0044] Figure 11 SEM images of microspheres under different conditions of process parameter optimization; Figure 11 (A) is the image of the microspheres in the optimal oil-water ratio (10:1) group at 50 times magnification. Figure 11 (B) is the image of the microspheres in the optimal oil-water ratio (10:1) group at 100 times magnification. Figure 11 (C) is the image of the microspheres in the optimal oil-water ratio (10:1) group at 500 times magnification. Figure 11 (D) in the middle is the image of the microspheres in the optimal oil-water ratio (10:1) group at 1000 times magnification; Figure 11 (E) is the image of the microspheres in the optimal chitosan concentration (2%) group at 50 times magnification. Figure 11 (F) is the image of the microspheres in the optimal chitosan concentration (2%) group at 100 times magnification. Figure 11 (G) is the image of the microspheres in the optimal chitosan concentration (2%) group at 500 times magnification. Figure 11 (H) in the middle is the image of microspheres in the optimal chitosan concentration (2%) group at 1000 times magnification; Figure 11 (I) is the image of the microspheres in the optimal acetic acid concentration (2%) group at 50 times magnification. Figure 11 (J) in the middle is the image of the microspheres in the optimal acetic acid concentration (2%) group at 100 times magnification. Figure 11 (K) in the figure is the image of the microspheres in the optimal acetic acid concentration (2%) group at 500 times magnification. Figure 11 (L) in the middle is the image of the microspheres in the optimal acetic acid concentration (2%) group at 1000 times magnification; Figure 11 (M) in the middle is the image of the best PEGDA (0.5%) group of microspheres at 50 times, Figure 11(N) in the figure is the image of the best PEGDA (0.5%) group of microspheres at 100 times magnification. Figure 11 (O) in the middle is the image of the best PEGDA (0.5%) group of microspheres at 500 times magnification. Figure 11 (P) in the middle is the image of microspheres in the optimal PEGDA (0.5%) group at 1000 times magnification;

[0045] Figure 12 This is the SEM image of the core-shell structure layered microneedle system; Figure 12 (A) is the appearance of the microneedle under a stereo microscope; Figure 12 (B) is an image magnified 50 times. Figure 12 (C) in the figure is an image magnified 100 times. Figure 12 (D) in the figure is an image magnified 500 times. Figure 12 (E) in the figure is an image magnified 1000 times;

[0046] Figure 13 This is a stereomicroscope image of a core-shell structured layered microneedle system, where the magnification refers specifically to the objective lens magnification. Figure 13 (A) 0.68x top-down image; Figure 13 (B) is a 1x image from a bird's-eye view. Figure 13 (C) is a 2x image from a bird’s-eye view. Figure 13 (D) in the figure is a 4x image viewed from above. Figure 13 (E) in the figure is a 2x side view image. Figure 13 (F) in the figure is a 4x side-view image;

[0047] Figure 14 The following are the Micro-CT imaging results of different microneedle material groups on the back and ankle of SD rats; Figure 14 (A) is a Micro-CT image of the GHP microneedle material group on the back cross section of the rat. Figure 14 (B) is the Micro-CT image of the GHP microneedle material group on the sagittal plane of the rat back. Figure 14 (C) is the Micro-CT image of the GHP microneedle material group on the coronal plane of the rat ankle; Figure 14 (D) is a Micro-CT image of the GHPS microneedle material group on the back cross section of the rat. Figure 14 (E) is the Micro-CT image of the GHPS microneedle material group on the sagittal plane of the rat back. Figure 14 (F) is the Micro-CT image of the GHPS microneedle material group on the coronal plane of the rat ankle; Figure 14 (G) is the Micro-CT image of the GHPSG microneedle material group on the back cross section of the rat. Figure 14(H) is the Micro-CT image of the GHPSG microneedle material group on the sagittal plane of the rat back. Figure 14 (I) is the Micro-CT image of the GHPSG microneedle material group on the coronal plane of the rat ankle; Figure 14 (J) is a Micro-CT image of the GHPSG-CeO2 microneedle material group on the back cross section of the rat. Figure 14 (K) is the Micro-CT image of the GHPSG-CeO2 microneedle material group on the sagittal plane of the rat back. Figure 14 (L) is the Micro-CT image of the GHPSG-CeO2 microneedle material group on the coronal plane of the rat ankle;

[0048] Figure 15 The effect of microneedle therapy on ankle swelling index in MSU-induced gout rats; Figure 15 (A) is the appearance of the ankle of the normal control group (Control) SD rats after modeling and the operation diagram of measuring the ankle circumference with cotton thread. Figure 15 (B) is the appearance of the ankle of the SD rats in the model group (Model) after modeling and the operation diagram of measuring the ankle circumference with cotton thread. Figure 15 (C) is the appearance of the ankle of SD rats in the microneedle treatment group (Therapy) after modeling and the operation diagram of measuring the ankle circumference with cotton thread. Figure 15 (D) is a statistical graph showing the changes in ankle joint swelling percentage over time in each group of rats. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] The present invention provides a method for preparing pH / ROS dual-responsive drug-loaded microspheres, comprising the following steps:

[0055] Chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol are mixed, emulsified, and chemically cross-linked to obtain the pH / ROS dual-responsive drug-loaded microspheres.

[0056] In the present invention, the preparation method of the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester preferably comprises the following steps: dissolving 4-hydroxyphenylboronic acid pinacol ester in dimethyl sulfoxide, activating, adding chitosan, reacting, and freeze-drying to obtain the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester; preferably, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide and N-hydroxysuccinimide are added during the activation; the activation is preferably accompanied by stirring, and the activation time is preferably 0.5 to 1 hour, more preferably 0.6 to 0.8 hours. More preferably, it is 0.7h; the activation temperature is preferably 20-30°C, more preferably 24-28°C, and more preferably 25°C; the reaction time is preferably 10-14h, and more preferably 12-13h; the reaction is accompanied by light-proof stirring; the mass percentage of 4-hydroxyphenylboronic acid pinacol ester, chitosan, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide, and N-hydroxysuccinimide is preferably 1-5:1-4:0.4-0.8:0.2-0.4, and more preferably 2 ~4:2~3:0.5~0.7:0.3, more preferably 3:2.5:0.6:0.3; the chitosan is preferably pretreated, and the pretreatment method is preferably: dissolving chitosan in an aqueous glacial acetic acid solution and stirring to obtain pretreated chitosan; the mass volume ratio of the chitosan to the aqueous glacial acetic acid solution is preferably 1~3g:90~100ml, more preferably 2g:95ml; the mass percentage of glacial acetic acid in the aqueous glacial acetic acid solution is preferably 1~3%, more preferably 2%; the stirring temperature is preferably 35~ 45 ℃, more preferably 36 to 44 ℃, more preferably 38 to 42 ℃, and further preferably 40 ℃; the stirring time is 1 to 1.5 h, more preferably 1.2 to 1.4 h, and further preferably 1.3 h; dialysis is preferably performed before freeze-drying; the freeze-drying temperature is preferably -75 to -85 ℃, more preferably -78 to -82 ℃, and further preferably -80 ℃; the freeze-drying time is preferably 70 to 75 h, more preferably 72 to 74 h, and further preferably 73 h.

[0057] In the present invention, the ROS is reactive oxygen species; the mass percentage of the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol is preferably 1-4:0.2-0.8:1-4, more preferably 1.6-3.4:0.3-0.6:1.5-3.5, more preferably 1.8-2.2:0.5:2-3.3, and even more preferably 2.0:0.5:3.0; the mixing is preferably carried out in the dark, the mixing is preferably accompanied by stirring, and the stirring time is preferably The emulsification time is preferably 25 to 35 minutes, more preferably 26 to 34 minutes, more preferably 28 to 32 minutes, and even more preferably 30 minutes; the emulsification preferably uses a Span80 / liquid paraffin composite emulsifier, and the volume ratio of the oil phase to the water phase in the composite emulsifier is preferably 8 to 12:1, more preferably 9 to 11:1, and even more preferably 10:1; the emulsification speed is preferably 1400 to 1600 r / min, more preferably 1450 to 1550 r / min, and even more preferably 1500 to 1600 r / min. preferably 1500r / min; the emulsification time is preferably 0.5-1.5h, more preferably 0.6-1.4h, more preferably 0.8-1.2h, and further preferably 1.0h; glutaraldehyde is preferably added during the chemical crosslinking, and the mass percentage of the glutaraldehyde is preferably 20-30%, more preferably 22-28%, more preferably 24-26%, and further preferably 25%; the temperature of the chemical crosslinking is preferably 10-20°C, more preferably 12-18°C, more preferably 14-16°C, and further preferably 15°C; the time of the chemical crosslinking is preferably 5-10min, more preferably 6-8min, and further preferably 7min; vacuum drying treatment is preferably performed after the chemical crosslinking; the temperature of the vacuum drying treatment is preferably 70-80°C, more preferably 72-78°C, more preferably 74-76°C, and further preferably 75°C; the time of the vacuum drying treatment is 8-12h, more preferably 9-11h, and further preferably 10h.

[0058] In the present invention, the pH / ROS dual-responsive drug-loaded microspheres have a drug loading of 5-15% and an encapsulation efficiency of ≥80%; they have a dual-responsive mechanism of a pH-sensitive Schiff base structure and a ROS-responsive phenylboronic acid ester bond; in a slightly acidic environment (pH 5-6, H2O2 concentration >50 μM), the 2-h cumulative release rate is ≥90%; in an alkaline environment (pH ≥8, H2O2 ≤10 uM), the 2-h release rate is <20%.

[0059] The present invention also provides a core-shell structured layered microneedle system, comprising a core layer, a transition layer and a shell layer;

[0060] The preparation method of the core layer comprises the following steps: mixing the pH / ROS dual-responsive drug-loaded microspheres prepared by the preparation method with polyvinyl alcohol, methacrylated hyaluronic acid, methacrylated gelatin, aminosulfonated cellulose, pyrogallic acid, and cerium oxide, and subjecting the mixture to freeze-thaw cycle treatment to form microneedles to obtain the core layer.

[0061] In the present invention, the preparation method of aminosulfonated cellulose preferably comprises the following steps: mixing aminosulfonic acid and N,N-dimethylformamide, adding cellulose, reacting, and freeze-drying to obtain the aminosulfonated cellulose; the mass ratio of aminosulfonic acid, N,N-dimethylformamide and cellulose is preferably 0.04-0.06:0.01-0.03:0.1-0.3, more preferably 0.05:0.02:0.2, and further preferably 6:1.5; the reaction temperature is preferably 75-85°C, more preferably 76-82°C, and further preferably 80°C; the reaction time is preferably 1-2 hours, and further preferably 1.5 hours; the freeze-drying temperature is preferably -75--85°C, more preferably -78--82°C, and further preferably -80°C; and the freeze-drying time is preferably 70-75 hours, more preferably 72-74 hours, and further preferably 73 hours.

[0062] In the present invention, the mass ratio of the pH / ROS dual-responsive drug-loaded microspheres to polyvinyl alcohol, methacrylated hyaluronic acid, methacryloylated gelatin, sulfamate-esterified cellulose, pyrogallic acid, and cerium oxide is preferably 30-40:1-4:1-4:10-20:1-4:0.05-0.2:0.05-0.2, more preferably 32-38:2-3:2-3:12-18:2-3:0.10-0.15:0.10-0.15, further preferably 34-36:2.5:2.5:14-16:2.5:0.12-0.14:0.12-0.14, and even more preferably 35:2.5:2. 5:15:2.5:0.13:0.13; the mixing temperature is preferably 35-45°C, more preferably 36-44°C, more preferably 38-42°C, and further preferably 40°C; an initiator and a catalyst are preferably added during the mixing, and the addition amount of the initiator and catalyst is preferably 0.1-0.5% of the mass of the mixed solution, more preferably 0.2-04%, and further preferably 0.3%; the initiator is preferably ammonium persulfate, and the catalyst is preferably tetramethylethylenediamine; the number of freeze-thaw cycle treatments is preferably 3-5 times, more preferably 4 times; the freeze-thaw cycle treatment method is preferably: freezing at -25--15°C for 1-2 hours , thawed at 20-30℃ for 0.5-1h, more preferably -20--10℃ for 1.5h, thawed at 22-28℃ for 0.6-0.8h, more preferably -15℃ for 1.5h, thawed at 24-26℃ for 0.7h, and further preferably -15℃ for 1.5h, thawed at 25℃ for 0.7h; the surface of the core layer is preferably coated with polypropylene pyrrolidone to form a top layer; the mass percentage of polypropylene pyrrolidone is preferably 4-6%, more preferably 5%; the preparation method of the transition layer is preferably the same as that of the core layer, and the pH / ROS dual-responsive drug-loaded microspheres in the transition layer are mixed with polyvinyl alcohol PVA, methacrylated hyaluronic acid HAMA, methacrylated hyaluronic acid The mass percentage of acylated gelatin GelMA, aminosulfonated cellulose SAEC, pyrogallic acid PGA, and oxidized CeO2 is preferably 15-20:1-3:1-3:8-12:1-3:0.1-0.3:0.1-0.3, more preferably 16-18:2:2:9-11:2:0.2:0.2, and further preferably 17:2:2:9-11:2:0.2:0.2; the shell layer is preferably polyvinyl alcohol; the preparation method of the shell layer preferably includes the following steps: dripping polyvinyl alcohol solution into the microneedle mold, freeze-thaw cycle treatment, forming a needle tip shell, and obtaining the shell layer; the freeze-thaw cycle treatment of the shell layer is preferably the same as that of the core layer.

[0063] In the present invention, the core layer as the needle body has a height of 500-1000 μm, a bottom diameter of 100-200 μm, and an array density of 100-200 needles / cm 2 , puncture force ≥ 0.5N / needle. The shell layer contains a directional nanofiber structure with a tensile modulus ≥ 50MPa and a swelling rate ≤ 20% (measured by immersion in pure water for 48 hours). Micro-CT detection shows that the core-shell material interface bonding strength is > 2MPa. The thickness of the core layer in the core-shell structure layered microneedle system is 280-320μm, the thickness of the transition layer is 480-520μm, and the thickness of the shell layer is 180-220μm. The 24h sustained release rate of the transition layer is 30-50%.

[0064] The present invention also provides a method for optimizing a core-shell structured layered microneedle system based on deep reinforcement learning, comprising the following steps:

[0065] (1) Extracting and collecting the DICOM data image features of the pH / ROS dual-responsive drug-loaded microspheres, the maximum release rate, half-life, and mechanical strength of the core-shell structured layered microneedle system;

[0066] (2) Constructing a deep deterministic policy gradient algorithm framework based on the Actor-Critic algorithm;

[0067] (3) The state space is defined as a 12-dimensional process parameter set, and the action space is defined as the structural parameter adjustment and process condition control operation; a hybrid reward function is used to dynamically evaluate the drug release efficiency, mechanical strength and biocompatibility to guide model training. The reward function is shown in Formula I, and the loss function is the temporal difference error (TD-Error) combined with the KL divergence constraint. The learning rate is 1e -4 , training cycle 480~520 times;

[0068]

[0069] Among them, α+β+γ=1, δ is the penalty coefficient;

[0070] (4) Through transfer learning, the algorithm framework of deep deterministic policy gradient is jointly trained with small sample experimental data and COMSOL fluid simulation data. The double-delay deep deterministic policy gradient is improved by using the algorithm, and finally the three-dimensional response surface model containing the process parameter optimization strategy is output.

[0071] In the present invention, the DICOM data image features of the Micro CT in step (1) preferably include porosity, particle size distribution, and sphericity; the extraction of the maximum release rate, half-life, and mechanical strength is preferably obtained by performing Savitzky-Golay smoothing on the release kinetics curve; the Actor network in the Actor-Critic algorithm in step (2) preferably adopts a four-layer fully connected structure to process process parameters, and the Actor network preferably has 3 hidden layers and 256-128-64-3 nodes; the Critic network in the Actor-Critic algorithm preferably integrates a convolution module to process the input Micro DICOM data of CT; the 12-dimensional process parameters in step (3) include drug loading, microsphere / microneedle size, encapsulation efficiency, drug loading efficiency, drug release rate, dissolution rate, swelling rate, degradation time, bioavailability, Young's modulus, surface charge and permeability coefficient, and the action space includes adjusting the drug release rate, changing the structure of the microneedle system, and adjusting the temperature and pressure of the processing conditions; the training cycle is preferably 500 times; the small sample experimental data in step (4) include the drug release curve obtained by experiment, the depth of microneedle penetration into the skin, and the dissolution rate of microspheres at different pH values; the COMSOL fluid simulation data are the drug release rate, the diffusion process of microspheres in the fluid, the mechanical force of microneedle penetrating the skin, and the fluid flow simulation at different solvent concentrations, temperatures, and pressures.

[0072] Example 1

[0073] A method for preparing pH / ROS dual-responsive drug-loaded microspheres, comprising the following steps:

[0074] (1) Dissolve 2.5 g of chitosan (degree of deacetylation ≥ 95%) in 95 ml of 1% by mass glacial acetic acid solution and stir at 45°C for 1 h to obtain pretreated chitosan;

[0075] (2) 3 g of 4-hydroxyphenylboronic acid pinacol ester was dissolved in dimethyl sulfoxide, 0.6 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide and 0.3 g of N-hydroxysuccinimide were added, and the mixture was stirred at 30° C. for 0.5 h, and the mixture was added dropwise to 4 g of pretreated chitosan obtained in step (1), stirred in the dark for 14 h, dialyzed with a 3500D dialysis bag, and freeze-dried at -85° C. for 70 h to obtain the chitosan modified with the 4-hydroxyphenylboronic acid pinacol ester;

[0076] (3) Under light-proof conditions, chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol were mixed in a mass percentage of 4.0:0.8:6.0, stirred for 35 minutes, emulsified at 1600 r / min using a Span80 / liquid paraffin composite emulsifier (the volume ratio of the oil phase to the water phase was 12:1) for 0.5 hours, chemically cross-linked at 20°C with 30% by mass glutaraldehyde for 5 minutes, and vacuum dried at 70°C for 12 hours to obtain the pH / ROS dual-responsive drug-loaded microspheres.

[0077] Example 2

[0078] A method for preparing pH / ROS dual-responsive drug-loaded microspheres, comprising the following steps:

[0079] (1) Dissolve 2 g of chitosan (degree of deacetylation ≥ 95%) in 100 ml of 2% by mass glacial acetic acid solution and stir at 40°C for 1 h to obtain pretreated chitosan;

[0080] (2) 2.4 g of 4-hydroxyphenylboronic acid pinacol ester was dissolved in 24 mL of dimethyl sulfoxide (DMSO) (dissolution concentration was about 0.1 g / mL), 0.48 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide (EDC·HI) and 0.24 g of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at 25° C. for 0.8 h to form an activation solution; the activation solution was then added dropwise to 2 g of pretreated chitosan prepared in step (1), stirred in the dark for 12 h, dialyzed with a 3500D dialysis bag, and freeze-dried at -80° C. for 72 h to obtain the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester;

[0081] (3) Under light-proof conditions, chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol were mixed in a mass percentage of 2.0:0.5:3.0, stirred for 30 minutes, emulsified at 1500 r / min using a Span80 / liquid paraffin composite emulsifier (the volume ratio of the oil phase to the water phase was 10:1) for 1 hour, chemically cross-linked at 15°C with 25% by mass glutaraldehyde for 8 minutes, and vacuum dried at 80°C for 8 hours to obtain the pH / ROS dual-responsive drug-loaded microspheres.

[0082] Example 3

[0083] A method for preparing pH / ROS dual-responsive drug-loaded microspheres, comprising the following steps:

[0084] (1) Dissolve 1.8 g of chitosan (degree of deacetylation ≥ 95%) in 100 ml of 1% by mass glacial acetic acid solution and stir at 35°C for 1.5 h to obtain pretreated chitosan;

[0085] (2) 2.16 g of 4-hydroxyphenylboronic acid pinacol ester was dissolved in 21.6 mL of dimethyl sulfoxide (DMSO) (dissolution concentration was about 0.1 g / mL), 0.432 g of EDC-HI and 0.216 g of NHS were added, and stirred at 20° C. for 1 h. The mixture was added dropwise to 1 g of pretreated chitosan obtained in step (1), stirred in the dark for 10 h, dialyzed with a 3500D dialysis bag, and freeze-dried at -75° C. for 75 h to obtain the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester;

[0086] (3) Under light-proof conditions, chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol were mixed in a mass percentage of 1.0:0.2:2.0, stirred for 25 minutes, emulsified at 1400 r / min using a Span80 / liquid paraffin composite emulsifier (the volume ratio of the oil phase to the water phase was 8:1) for 1.5 hours, chemically cross-linked at 10°C with 20% by mass glutaraldehyde for 10 minutes, and vacuum dried at 75°C for 10 hours to obtain the pH / ROS dual-responsive drug-loaded microspheres.

[0087] Example 4

[0088] A core-shell structured layered microneedle system comprising a core layer, a transition layer and a shell layer;

[0089] 0.05 g of aminosulfonic acid and 0.02 g of N,N-dimethylformamide were mixed, 0.2 g of cellulose was added, the mixture was reacted at 80° C. for 1.5 h, and freeze-dried at -80° C. for 72 h to obtain the aminosulfonic acid esterified cellulose SAEC;

[0090] The preparation method of the core layer is as follows: at 40° C., the pH / ROS dual-responsive drug-loaded microspheres prepared in Example 1 are mixed with polyvinyl alcohol (PVA), methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GelMA), the aminosulfonated cellulose (SAEC) obtained above, pyrogallic acid (PGA), and cerium oxide (CeO2) in a mass percentage of 35:2:2:15:2:0.2:0.2, ammonium persulfate and tetramethylethylenediamine are added (the total amount of ammonium persulfate and tetramethylethylenediamine added is 0.3% of the mass of the mixed solution), and the mixture is treated four times by a freeze-thaw cycle of freezing at -20° C. for 2 h and thawing at 25° C. for 1 h to form microneedles to obtain the core layer, and coating with 5% by mass of polypropylene pyrrolidone to form a top layer;

[0091] The transition layer was prepared according to the preparation method of the core layer, except that the mass percentage of pH / ROS dual-responsive drug-loaded microspheres, polyvinyl alcohol (PVA), methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GelMA), aminosulfonated cellulose (SAEC), pyrogallic acid (PGA), and cerium oxide (CeO2) was adjusted to 18:2:2:10:2:0.2:0.2.

[0092] The polyvinyl alcohol solution is dripped into the microneedle mold, and the mold is subjected to freeze-thaw cycle treatment (the same as the freeze-thaw cycle treatment of the core layer) to form a needle tip shell to obtain a shell layer.

[0093] Example 5

[0094] A core-shell structured layered microneedle system comprising a core layer, a transition layer and a shell layer;

[0095] 0.04 g of aminosulfonic acid was mixed with 0.01 N, N-dimethylformamide, 0.1 g of cellulose was added, the mixture was reacted at 85° C. for 1 h, and freeze-dried at -75° C. for 75 h to obtain the aminosulfonated cellulose SAEC;

[0096] The core layer is prepared by mixing the pH / ROS dual-responsive drug-loaded microspheres prepared in Example 1 with polyvinyl alcohol (PVA), methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GelMA), aminosulfonated cellulose (SAEC), pyrogallic acid (PGA), and cerium oxide (CeO2) in a mass percentage of 30:4:1:20:1:0.05:0.05 at 35° C., adding ammonium persulfate and tetramethylethylenediamine (the total amount of ammonium persulfate and tetramethylethylenediamine is 0.1% of the mass of the mixed solution), and subjecting the microspheres to a freeze-thaw cycle of freezing at -25° C. for 1.5 h and thawing at 20° C. for 0.8 h for 5 times to form microneedles, thereby obtaining the core layer, and coating the top layer with 6% by mass of polypropylene pyrrolidone;

[0097] The transition layer was prepared according to the preparation method of the core layer, except that the mass percentage of pH / ROS dual-responsive drug-loaded microspheres, polyvinyl alcohol (PVA), methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GelMA), aminosulfonated cellulose (SAEC), pyrogallic acid (PGA), and cerium oxide (CeO2) was adjusted to 15:1:1:12:3:0.3:0.1.

[0098] The polyvinyl alcohol solution is dripped into the microneedle mold, and the mold is subjected to freeze-thaw cycle treatment (the same as the freeze-thaw cycle treatment of the core layer) to form a needle tip shell to obtain a shell layer.

[0099] Example 6

[0100] A core-shell structured layered microneedle system comprising a core layer, a transition layer and a shell layer;

[0101] 0.06 g of aminosulfonic acid was mixed with 0.03 N, N-dimethylformamide, 0.3 g of cellulose was added, the mixture was reacted at 75° C. for 2 h, and the sample was freeze-dried at -85° C. for 70 h to obtain the aminosulfonic acid esterified cellulose SAEC;

[0102] The core layer is prepared by mixing the pH / ROS dual-responsive drug-loaded microspheres prepared in Example 1 with polyvinyl alcohol (PVA), methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GelMA), aminosulfonated cellulose (SAEC), pyrogallic acid (PGA), and cerium oxide (CeO2) in a mass ratio of 40:1:4:10:4:0.1:0.1 at 45° C., adding ammonium persulfate and tetramethylethylenediamine (the total amount of ammonium persulfate and tetramethylethylenediamine is 0.5% of the mass of the mixed solution), and subjecting the microspheres to a freeze-thaw cycle of freezing at -15° C. for 1 hour and thawing at 30° C. for 0.5 hour for three times to form microneedles and obtain the core layer, and coating the microspheres with 4% by mass of polypropylene pyrrolidone to form a top layer.

[0103] The transition layer was prepared according to the preparation method of the core layer, except that the mass percentage of pH / RROS dual-responsive drug-loaded microspheres, polyvinyl alcohol (PVA), methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GelMA), aminosulfonated cellulose (SAEC), pyrogallic acid (GA), and cerium oxide (CeO2) was adjusted to 20:3:3:8:1:0.1:0.3;

[0104] The polyvinyl alcohol solution is dripped into the microneedle mold and subjected to freeze-thaw cycle treatment (the same as the freeze-thaw cycle treatment of the core layer) to form a needle tip shell to obtain a shell layer.

[0105] Example 7

[0106] A method for optimizing a core-shell layered microneedle system based on deep reinforcement learning comprises the following steps:

[0107] (1) ResNet-50 was used to extract the DICOM data image features (porosity, particle size distribution, and sphericity) of the pH / ROS dual-responsive drug-loaded microspheres prepared in Example 1, and the release kinetic parameters (maximum release rate from 0 to 72 h, half-life) and mechanical strength parameters of the microneedle system were analyzed using a TCN network.

[0108] (2) A deep deterministic policy gradient model based on the improved actor-critic algorithm was established, in which: the actor network used a four-layer fully connected structure to process process parameters; the critic network integrated a convolution module to process 1024×1024 pixel Micro CT images; the multi-source input data included: Micro CT image features, FTIR spectra (4000-500 cm-1), rheological parameters, process parameters (emulsification speed, cross-linking time, freezing cycle), and biocompatibility data (CCK-8 cell viability);

[0109] (3) The state space is defined as a 12-dimensional process parameter set, and the action space is defined as the structural parameter adjustment and process condition control operation; a hybrid reward function is used to dynamically evaluate the drug release efficiency, mechanical strength and biocompatibility to guide model training. The reward function is shown in Formula I, and the loss function is the temporal difference error (TD-Error) combined with the KL divergence constraint. The learning rate is 1e -4 , training cycle 480~520 times;

[0110]

[0111] Among them, α+β+γ=1, δ is the penalty coefficient;

[0112] (4) Build a dataset for algorithm training, including small sample experimental data and COMSOL simulation data. The experimental data covers drug release curves, microneedle penetration depth, and microsphere dissolution rate under different pH conditions; the simulation data covers drug release behavior under different solvent concentrations, temperatures, and pressures, the diffusion characteristics of microspheres in fluids, and the mechanical response and local fluid flow state during microneedle penetration. These two types of data together form the basis for mapping system characteristics and environmental responses, which are used for subsequent transfer learning and strategy optimization model training.

[0113] Test Example 1 Microsphere Performance Test

[0114] 1-1 Scanning electron microscopy (SEM) characterization

[0115] Sample preparation: Take appropriate amounts of pH / ROS dual-responsive drug-loaded microsphere samples prepared in Examples 1, 2, and 3 and place them in a desiccator for vacuum drying for 24 h to completely remove moisture. The dried microsphere samples are evenly dispersed on a double-sided conductive carbon tape to ensure that the microspheres are evenly dispersed and not piled up. Use an ion sputtering instrument to sputter-plate gold on the sample surface to a coating thickness of 10 nm. Sputtering parameters: current 15 mA, sputtering time 60 s; vacuum degree: 1×10 -2Pa; the prepared sample was mounted on the sample stage of a JSM-7500F scanning electron microscope; the following imaging parameters were set: accelerating voltage: 5 kV, working distance: 8 mm, emission current: 10 μA, and vacuum was applied to 3 × 10 -4 Pa, adjust the sample position, first find an area suitable for observation at low magnification (×50); increase the magnification to ×50, ×100, ×500, and ×1000 in sequence, take clear images of the microsphere surface and internal structure, and randomly select at least 10 microspheres for observation in different fields of view to ensure the representativeness of the results. Figure 2 shown.

[0116] 1.2 Fourier transform infrared spectroscopy (FT-IR) analysis

[0117] Sample preparation: The pH / ROS dual-responsive drug-loaded microsphere sample prepared in Example 1 was ground into a fine powder; 2 mg of the sample powder was mixed with 200 mg of spectral KBr (purchased from MacLean Reagent) and ground thoroughly until uniform; a tablet press was used to press the sample into a transparent thin sheet (about 0.5 mm thick) at a pressure of 10 MPa; at the same time, the following control samples were prepared as KBr tablets: ① 2% chitosan, ② 4-hydroxyphenylboronic acid pinacol ester, ③ 0.5% polyethylene glycol diacrylate, and ④ 8% dialdehyde polyethylene glycol.

[0118] FT-IR measurement: Thermo Scientific iS10 infrared spectrometer was used for measurement, and the following measurement parameters were set: Scan range: 4000-400 cm -1 , resolution: 4cm -1 , Scan number: 32 times, Detector: DTGS (deuterated triethylene glycol sulfate), Beam splitter: KBr. First, measure the background signal (air or pure KBr plate), measure the infrared spectrum of each sample in turn, and use OMNIC software to perform baseline correction and smoothing. The results are as follows Figure 3 shown.

[0119] The microsphere test results showed that FT-IR detected C=O stretching vibration peaks (1679, 1619 cm -1 ), CO stretching vibration peaks (1354, 1299 cm -1 ), the absorption peak of C=N double bond (1543cm -1 ), borate characteristic peak (1720cm -1 )(iS10, resolution 4cm -1 ).

[0120] 1.3 Drug loading performance analysis

[0121] Colchicine standard curve determination: Prepare analytically pure colchicine standard and ultrapure water, accurately weigh an appropriate amount of colchicine standard and dissolve it in water to prepare a 0.1 mg / mL stock solution, then dilute it in sequence to obtain a standard series of working solutions of 0.00, 0.0125, 0.025, 0.0375 and 0.05 mg / mL; then turn on the ultraviolet spectrophotometer to preheat for 20 minutes, and zero the instrument with a blank solution; then determine the maximum absorption wavelength of colchicine (possibly in the range of 245-350 nm), and measure the absorbance of each concentration standard solution in sequence at this wavelength, obtaining values of 0.068, 0.8488, 1.5399, 2.2759 and 2.71 respectively; finally, draw a standard curve with concentration as the horizontal axis and absorbance as the vertical axis and perform linear regression analysis. The standard curve is as follows: Figure 4 As shown, the regression equation y = 53.7736x + 0.1452 and the correlation coefficient R are obtained. 2 =0.99197, indicating a good linear relationship. This standard curve can be used for subsequent determination of colchicine samples of unknown concentrations. The absorbance of the released samples was measured at the maximum absorption wavelength of the drug using an ultraviolet spectrophotometer (Shimadzu UV-2600).

[0122] Sample preparation: Pretreated chitosan was prepared according to the method in Example 1, and colchicine was added to the pretreated chitosan to prepare chitosan solutions containing 3 mg / ml, 30 mg / ml and 150 mg / ml colchicine, respectively; pH / ROS dual-responsive drug-loaded microsphere samples were then prepared according to the method in Example 1 and placed in a dryer under vacuum for 24 h to completely remove moisture; 10 mg of the dried sample was taken and placed in a dialysis bag (MWCO = 3500 Da), and the dialysis bag was placed in a centrifuge tube containing 30 mL of pH 6.5 + 3 mM H2O2 release medium, and constant temperature oscillation was performed at 37 ± 0.5 ° C and 100 rpm. At the preset time points (0, 1 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 360 min), 2 mL of the shaken sample was placed in a cuvette to obtain a mixed solution. The drug concentration in the mixed solution release medium was determined by UV spectrophotometry. The electron microscopy results of the prepared pH / ROS microspheres at the above three drug loading levels are shown as follows: Figure 5 As shown, the release effect is as Figure 6 and Figure 7 shown.

[0123] The results showed that the encapsulation efficiency of pH / ROS microspheres was 81% and the drug loading efficiency was 2% when the dosage was 3 mg / ml; the encapsulation efficiency was 91% and the drug loading efficiency was 23% when the dosage was 3 mg / ml; and the encapsulation efficiency was 71% and the drug loading efficiency was 29% when the dosage was 3 mg / ml.

[0124] Test Example 2 In vitro performance verification

[0125] The drug-loaded microspheres prepared in Example 1 were placed in release media simulating different physiological environments, namely, PBS buffers at pH 5.0, pH 6.5, and pH 7.4, and PBS buffer at pH 7.4 containing different concentrations of H2O2 (0 μM, 25 μM, 50 μM, and 100 μM). The specific steps are as follows:

[0126] Weigh 10 mg of drug-loaded microspheres and place them in a dialysis bag (MWCO = 3500Da), place the dialysis bag in a centrifuge tube containing 30 mL of release medium, and oscillate at a constant temperature of 37 ± 0.5 ° C and 100 rpm. At the preset time points (0, 1 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 360 min), take 2 mL of the shaken sample and place it in a cuvette to obtain a mixed solution. The drug concentration in the mixed solution release medium was determined by ultraviolet spectrophotometry, and the cumulative release amount was calculated according to formula II, and the cumulative release rate was calculated according to formula III. Each group of experiments was measured in parallel 3 times. The results are shown in Figure 2. Figures 8 to 10 shown.

[0127] Cumulative release (M t )=Ct×V+∑(C i ×V), formula II;

[0128] Among them, C t is the drug concentration measured after sampling at time t, V is the volume of the receiving cavity, C i is the concentration at each previous time point, v is the sampling volume;

[0129] Cumulative release rate (%) = (M t / M0)×100%, formula III;

[0130] Among them, M0 is the theoretical loading amount of the drug in the microneedle layer.

[0131] 2.1 pH-responsive release results

[0132] The effects of different pH conditions on the release behavior of drug-loaded microspheres were investigated in the absence of H₂O₂. The results showed that lower pH values resulted in faster drug release. Cumulative release rates over 360 min were 85.3±2.1%, 78.6±1.7%, and 65.7±1.2% at pH 5.0, pH 6.5, and pH 7.4, respectively.

[0133] 2.2 ROS responsive release results

[0134] The effects of different H2O2 concentrations on the release behavior of drug-loaded microspheres were investigated at pH 7.4. The results showed that higher H2O2 concentrations resulted in faster drug release. The cumulative release rates over 360 min were 89.7±1.2%, 86.5±1.8%, 76.2±2.0%, 61.2%±2.4%, and 27.3±2.5% in release media containing 0mM, 1mM, 2mM, 3mM, and 4mM H2O2, respectively.

[0135] 2.3pH / ROS dual-response synergistic release results

[0136] To evaluate the synergistic effect of dual stimulation of pH and ROS, the drug-loaded microspheres were placed under release conditions of pH 6.5+3mM H2O2 and compared with single pH response conditions (pH 6.5+0mM H2O2) and single ROS response conditions (pH 7.4+3mM H2O2).

[0137] The results showed that under the dual-response conditions, the cumulative release rate over 360 min reached 94.5±2.3%, an increase of 15.9% compared with the single pH response group (78.6±1.7%) and an increase of 18.3% compared with the single ROS response group (76.2±2.0%).

[0138] Experimental Example 3 Optimization Effect

[0139] 3.1 Microsphere size variation coefficient optimization experiment

[0140] Determination of initial coefficient of variation: 50 mg was randomly sampled from the pH / ROS dual-responsive drug-loaded microspheres prepared under the initial process conditions (prepared according to the method in Example 1, initial conditions: chitosan concentration 1%, acetic acid concentration 1%, oil-water ratio 8:1, PEGDA concentration 0.3%); the microsphere particle size distribution was determined using a laser particle size analyzer (Malvern Mastersizer 3000): dispersion medium: 0.5% Tween 80 aqueous solution, stirring speed: 1500 rpm, ultrasonic treatment: 30 s pre-dispersion, light shielding rate: 10%, three batches of samples were measured, and the measurement was repeated three times for each batch. The initial coefficient of variation was recorded as 17.3%.

[0141] Coefficient of variation CV (%) = (standard deviation / average particle size) × 100%, Formula IV;

[0142] The orthogonal experimental design method was used to optimize the microsphere preparation process parameters, specifically:

[0143] Factor A: chitosan concentration gradient (1.0%, 2.0%, 3.0%); Factor B: acetic acid concentration gradient (1.0%, 2.0%, 3.0%); Factor C: oil-water volume ratio gradient (8:1, 10:1, 12:1); Factor D: PEGDA concentration gradient (0.3%, 0.5%, 0.7%). An L9(34) orthogonal table was designed to conduct 9 groups of experiments. Three batches of microspheres were prepared under each experimental condition. The particle size distribution and coefficient of variation of the microspheres prepared under each condition were determined using the above method.

[0144] Determination of the optimal conditions (coefficient of variation reduced to 4.1%): The main factors affecting the uniformity of microsphere particle size were determined by variance analysis, and the optimal process parameter combination was determined: optimal chitosan concentration: 2.0%, optimal acetic acid concentration: 2.0%, optimal oil-water ratio: 10:1, and optimal PEGDA concentration: 0.5%. Using the optimal process parameters, 5 batches of microspheres were continuously prepared according to the method in Example 1. The particle size distribution was repeatedly measured, and the coefficient of variation after optimization was calculated. The coefficient of variation before and after optimization was analyzed using the F test. The SEM results are shown in Figure 2. Figure 11 shown.

[0145] The results showed that the coefficient of variation of microsphere particle size decreased from 17.3% to 4.1%, and there was a significant difference in the coefficient of variation before and after optimization (p < 0.01).

[0146] 3.2 Characterization of core-shell microneedles

[0147] Sample preparation: pH / ROS dual-responsive drug-loaded microspheres were prepared according to the method in Example 1 using the optimized process conditions obtained in 3.1. Core-shell layered microneedle systems were prepared according to the method in Example 4. Ten batches of samples were prepared, with 5 microneedle arrays prepared in each batch. The morphology of the microneedles was observed using SEM and stereomicroscope. The morphology of the microneedles under SEM and stereomicroscope is shown in Figure 2. Figure 12 and Figure 13 shown.

[0148] The results showed that the prepared microneedle array was neatly arranged, with sharp tips and an intact overall structure, without any noticeable breakage or collapse. The microneedle surface was smooth, the core-shell structure was clearly layered, and the drug-loaded microspheres were evenly distributed with no apparent agglomeration. Compared to microneedles prepared under unoptimized process conditions, the optimized microneedles in this invention exhibited improved molding consistency and structural stability, laying a good foundation for subsequent transdermal delivery performance testing.

[0149] Test Case 4 Model Training

[0150] Step 1: Dataset Preparation

[0151] MicroCT imaging (DICOM format) and drug release profile data were collected for the following experimental groups: GH (methacryloyl-hyaluronic acid) with a gradient addition level (1 wt%, 2 wt%, 3 wt%, 4 wt%), GHP (polyvinyl alcohol 1799) with a gradient addition level (1 wt%, 2 wt%, 3 wt%, 4 wt%), GHPS (cellulose sulfamate with a gradient addition level (1 wt%, 2 wt%, 3 wt%, 4 wt%), GHPSG (pyrogallic acid) with a gradient addition level (0.05 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%), and GHPSG-CeO2 with a gradient addition level (0.05 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%). The data were classified based on the experimental group (GH, GHP, GHPS, GHPSG, GHPSG-CeO2). The imaging data and drug release data were used as input features for training and testing, respectively. The data were randomly divided into 80% training set and 20% test set.

[0152] Step 2: Image data preprocessing

[0153] After denoising, image enhancement and normalization of the MicroCT images, key structural features of microspheres and microneedles can be extracted, including morphological features (such as porosity, surface area, volume, aspect ratio), texture features (such as gray-level co-occurrence matrix features, edge density), size features (such as diameter / length, particle distribution), surface features (such as surface roughness) and three-dimensional structure analysis (such as surface curvature, three-dimensional connectivity). Figure 14 shown.

[0154] Step 3: Model training

[0155] The Deep Deterministic Policy Gradient (DDPG) algorithm is used to perform joint training with small sample experimental data and simulation data. The hyperparameters are set to: learning rate 3e -4 The batch size is 16, and the number of patients with early stopping is 15. The small sample experimental data includes experimentally obtained drug release curves, the depth of microneedle penetration into the skin, and the dissolution rate of microspheres at different pH values. The COMSOL fluid simulation data includes the drug release rate, the diffusion process of microspheres in the fluid, the mechanical force of microneedle penetration into the skin, and fluid flow simulation at different solvent concentrations, temperatures, and pressures.

[0156] Step 4: Model Evaluation

[0157] Using R from Python 2 (release rate prediction) and F1-score (cytotoxicity classification) to evaluate the performance of the model. The evaluation process usually involves using appropriate evaluation metrics such as R 2and F1-score), and are calculated using existing statistical learning software (such as Python's scikit-learn). For regression tasks (such as release rate prediction), R 2 To measure the degree of model fit; for classification tasks (such as cytotoxicity classification), use F1-score to balance the precision and recall of the model. The relevant code is as follows:

[0158] from sklearn.metrics import r2_score, f1_score

[0159] #For regression tasks

[0160] r2=r2_score(y_true, y_pred) #true value y_true and predicted value y_pred

[0161] #For classification tasks

[0162] f1 = f1_score(y_true, y_pred, average = 'binary') # true value y_true and predicted value y_pred

[0163] The results showed that the release rate prediction R 2 The F1-score of the cytotoxicity classification was 0.91.

[0164] Experimental Example 5 Animal Model Verification

[0165] (1) Experimental animals

[0166] Adult male SD rats weighing 200-250g were randomly divided into three groups: control, model, and treatment groups, with six rats in each group. The control group consisted of normal rats injected with an equal volume of saline; the model group (experimental group 1) consisted of rats with an acute gout model who received no treatment; and the treatment group (experimental group 2) consisted of rats with an acute gout model who received microneedle therapy.

[0167] (2) Preparation of acute gout rat model

[0168] Dissolve 0.1g of sodium urate (MSU) crystals in 10mL of sterile saline to obtain a 25mg / mL MSU crystal suspension. Prepare a 1mL sterile syringe, a 25G needle, a sterile operating table, 75% alcohol, and sterile gloves. The modeling steps are as follows:

[0169] Before the experiment, the rats were adaptively fed for 7 days with free access to food and water. They were fasted but not allowed to drink water for 12 hours before modeling. The rats were fixed in a rat fixator to fully expose the right ankle joint. The skin of the right ankle joint area was disinfected with 75% alcohol. In the control group, 0.2 mL of sterile saline was drawn with a 1 mL syringe and injected into the right ankle joint cavity with a 25G needle. In the model group and treatment group, 0.2 mL of MSU crystal suspension (25 mg / mL) was drawn with a 1 mL syringe and injected into the right ankle joint cavity with a 25G needle. After the injection, the injection site was gently massaged to ensure that the MSU crystals were fully dispersed in the joint cavity. The rats were returned to their cages and allowed to move freely and have free access to food and water.

[0170] (3) Microneedle treatment plan

[0171] The core-shell structured layered microneedles containing pH / ROS dual-responsive drug-loaded microspheres prepared in Example 4 were prepared. The microneedle patch was disinfected by irradiation with ultraviolet light for 30 minutes before use; the first microneedle treatment was performed on the second day after modeling (24 hours after injection of MSU crystals).

[0172] Rats were anesthetized with 3% sodium pentobarbital (45 mg / kg) via intraperitoneal injection. After confirming that the rats were fully anesthetized, the hair around the right ankle joint (approximately 2 cm × 2 cm area) was shaved. The skin in the shaved area was disinfected with 75% alcohol and allowed to dry completely. The microneedle patch was pressed against the most swollen area of the ankle joint. Medical tape was used to secure the microneedle patch to ensure full contact between the patch and the skin. A thin layer of gauze was wrapped around the outer layer of the microneedle patch for protection. The rats were returned to their cages to prevent them from biting the patch.

[0173] (4) Ankle joint measurement method

[0174] The ankle joints of the rats were measured using a digital vernier caliper (0.01 mm accuracy) and a soft, non-stretchable measuring tape (1 mm accuracy) at 6 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days after injection. Ankle joint swelling rate was calculated according to Formula V. Ankle joint swelling rate (%) = [(peripheral circumference or diameter of the affected side in the experimental group - peripheral circumference or diameter of the control group) / peripheral circumference or diameter of the control group] × 100%, Formula V.

[0175] The ankle circumference measurement method is as follows: fix the rat on the operating table so that the ankle joint is naturally extended; wrap a soft measuring tape around the most swollen part of the ankle joint; keep the measuring tape close to the skin but not compress it; record the measurement value, repeat the measurement three times for each rat, and take the average value; at the same time, measure the ankle circumference of the left side (non-injected side) as a control.

[0176] The ankle joint diameter was measured by using a digital vernier caliper to measure the anterior-posterior diameter of the ankle joint (sagittal position); measuring the lateral and medial diameter of the ankle joint (coronal position); repeating the measurement three times in each direction and taking the average value; and measuring the left (non-injected) ankle joint diameter as a control.

[0177] The results are as follows Figure 15 shown.

[0178] The results showed that after 7 days of treatment, the ankle swelling of the rats in the treatment group decreased by 81.7±2.5%, and the ankle swelling of the rats in the model group decreased by 35.5±1.8%.

[0179] It can be seen from the above embodiments that the present invention provides a method for preparing pH / ROS dual-responsive drug-loaded microspheres, a microneedle system, and a method for optimizing the microneedle system. The microspheres provided by the present invention can achieve targeted and precise drug release at inflammatory sites, and the core-shell structure layered microneedle system provided can achieve time-controlled release. In addition, the present invention realizes reverse optimization of the preparation process and prediction of individualized treatment plans by developing a deep reinforcement learning model for multimodal data fusion.

[0180] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing pH / ROS dual-responsive drug-loaded microspheres, characterized in that: The steps include: Chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol are mixed, emulsified, and chemically cross-linked to obtain the pH / ROS dual-responsive drug-loaded microspheres.

2. The preparation method according to claim 1, characterized in that The preparation method of the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester comprises the following steps: 4-hydroxyphenylboronic acid pinacol ester is dissolved in dimethyl sulfoxide, activated, added with chitosan, reacted, and freeze-dried to obtain the chitosan modified with the 4-hydroxyphenylboronic acid pinacol ester.

3. The preparation method according to claim 2, characterized in that During the activation, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide and N-hydroxysuccinimide are added; the activation time is 0.5 to 1 hour; the mass percentages of 4-hydroxyphenylboronic acid pinacol ester, chitosan, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide, and N-hydroxysuccinimide are 1 to 5:1 to 4:0.4 to 0.8:0.2 to 0.4; the reaction time is 10 to 14 hours; the freeze-drying temperature is -75 to -85°C, and the freeze-drying time is 70 to 75 hours.

4. The preparation method according to claim 1, characterized in that The mass percentages of the chitosan modified with 4-hydroxyphenylboronic acid pinacol ester, polyethylene glycol diacrylate, and dialdehyde polyethylene glycol are 1-4:02-0.8:2-10; the emulsification speed is 1400-1600 r / min, and the emulsification time is 0.5-1.5 h; the chemical crosslinking temperature is 10-20° C., and the chemical crosslinking time is 5-10 min.

5. A core-shell structured layered microneedle system, characterized in that: Includes core layer, transition layer and shell layer; The preparation method of the core layer comprises the following steps: mixing the pH / ROS dual-responsive drug-loaded microspheres prepared by the preparation method according to any one of claims 1 to 4 with polyvinyl alcohol, methacrylated hyaluronic acid, methacrylated gelatin, aminosulfonated cellulose, pyrogallic acid, and cerium oxide, and subjecting the mixture to a freeze-thaw cycle to form microneedles to obtain the core layer; The preparation method of the aminosulfonated cellulose comprises the following steps: mixing aminosulfonic acid and N,N-dimethylformamide, adding cellulose, reacting, and freeze-drying to obtain the aminosulfonated cellulose.

6. The core-shell structured layered microneedle system according to claim 5, characterized in that: The mass ratio of aminosulfonic acid, NN-dimethylformamide and cellulose is 0.04-0.06:0.01-0.03:0.1-0.3; the reaction temperature is 75-85°C, and the reaction time is 1-2 hours; the freeze-drying temperature is -75--85°C, and the freeze-drying time is 70-75 hours.

7. The core-shell structured layered microneedle system according to claim 5, characterized in that: The mass ratio of the pH / ROS dual-responsive drug-loaded microspheres to polyvinyl alcohol, methacrylated hyaluronic acid, methacrylated gelatin, aminosulfonated cellulose, pyrogallic acid, and cerium oxide is 30-40:1-4:1-4:10-20:1-4:0.05-0.2:0.05-0.2; an initiator and a catalyst are added during the mixing, and the added amounts of the initiator and catalyst are both 0.1-0.5% of the mass of the mixed solution; the initiator is ammonium persulfate, and the catalyst is tetramethylethylenediamine.

8. The core-shell structured layered microneedle system according to claim 5, characterized in that: The preparation method of the transition layer is the same as that of the core layer, and the mass percentage of pH / ROS dual-responsive drug-loaded microspheres to polyvinyl alcohol, methacrylated hyaluronic acid, methacrylated gelatin, aminosulfonated cellulose, pyrogallic acid, and cerium oxide in the transition layer is 15-20:1-3:1-3:8-12:1-3:0.1-0.3:0.1-0.3; The shell layer is polyvinyl alcohol; The preparation method of the shell layer comprises the following steps: dripping a polyvinyl alcohol solution into a microneedle mold, and subjecting the microneedle mold to a freeze-thaw cycle to form a needle tip shell, thereby obtaining the shell layer; the freeze-thaw cycle of the shell layer is the same as that of the core layer; The number of freeze-thaw cycle treatments is 3 to 5 times, and the freeze-thaw cycle treatment method is: freezing at -25 to -15°C for 1 to 2 hours, and thawing at 20 to 30°C for 0.5 to 1 hour.

9. A method for optimizing a core-shell layered microneedle system based on deep reinforcement learning, characterized in that: The steps include: (1) Extracting and collecting the DICOM data image features of the Micro CT of the pH / ROS dual-responsive drug-loaded microspheres according to any one of claims 1 to 4, and the maximum release rate, half-life, and mechanical strength of the core-shell structure layered microneedle system according to any one of claims 5 to 8; (2) Constructing a deep deterministic policy gradient algorithm framework based on the Actor-Critic algorithm; (3) The state space is defined as a 12-dimensional process parameter set, and the action space is defined as the structural parameter adjustment and process condition control operation; a hybrid reward function is used to dynamically evaluate the drug release efficiency, mechanical strength and biocompatibility to guide model training. The reward function is shown in Formula I, and the loss function is the temporal difference error combined with the KL divergence constraint. The learning rate is 1e -4 , training cycle 480~520 times; Among them, α+β+γ=1, δ is the penalty coefficient; (4) Through transfer learning, the algorithm framework of deep deterministic policy gradient is jointly trained with small sample experimental data and COMSOL fluid simulation data. The double-delay deep deterministic policy gradient is improved by using the algorithm, and finally the three-dimensional response surface model containing the process parameter optimization strategy is output.

10. The method according to claim 9, characterized in that In step (1), the DICOM data image features of the Micro CT include porosity, particle size distribution, and sphericity; in step (2), the Actor network in the Actor-Critic algorithm uses a four-layer fully connected structure to process process parameters, and the Critic network in the Actor-Critic algorithm integrates a convolution module to process the input Micro CT DICOM data; in step (3), the 12-dimensional process parameters include drug loading, microsphere / microneedle size, encapsulation efficiency, drug loading efficiency, drug release rate, dissolution rate, swelling rate, degradation time, bioavailability, Young's modulus, surface charge and permeability coefficient, and the action space includes adjusting the drug release rate, changing the structure of the microneedle system, and adjusting the temperature and pressure of the processing conditions; in step (4), the small sample experimental data include the drug release curve obtained by experiment, the depth of microneedle penetration into the skin, and the dissolution rate of microspheres at different pH values; the COMSOL fluid simulation data includes the drug release rate, the diffusion process of microspheres in the fluid, the mechanical force of microneedle penetration into the skin, and the fluid flow simulation under different solvent concentrations, temperatures, and pressures.

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