Temperature-sensitive hydrogel loaded with Chinese herbal medicine vesicles for regulating immunity and preparation method of temperature-sensitive hydrogel

Abstract

The invention relates to a Chinese herbal medicine vesicle-loaded temperature-sensitive hydrogel for regulating immunity and a preparation method thereof, and belongs to the technical field of biological medicines. The raw materials are wide in source, methacrylated gelatin (GelMA) has good biocompatibility, poly (N-isopropylacrylamide) (PNIPAM) and GelMA hydrogel are combined, meanwhile, lycium barbarum leaf source extracellular vesicle-like particles (LBL-EVLPs) are introduced, certain thermal responsiveness is achieved, the local immune microenvironment can be adjusted, and oxidative stress reaction can be effectively relieved. The temperature-sensitive hydrogel loaded with the Chinese herbal medicine vesicles provided by the invention has multiple effects of promoting tissue repair, removing reactive oxygen species (ROS), regulating immunity and the like, and has the advantages of simple preparation, low cost and the like.

Description

Technical Field

[0001] The present invention relates to a temperature-sensitive hydrogel loaded with Chinese herbal medicine vesicles for regulating immunity and a preparation method thereof, belonging to the field of biomedical technology. Background Art

[0002] During the process of tissue regeneration, the dynamic regulation of the inflammatory response is a key factor determining the repair effect. Moderate inflammation can clear pathogens and initiate repair signals, but excessive or persistent inflammatory responses may hinder the regeneration process. Research has shown that the excessive accumulation of reactive oxygen species (ROS) not only induces a strong inflammatory response but also leads to impaired immune regulation, resulting in a local chronic inflammatory state, thereby preventing tissue regeneration. Therefore, how to solve a series of problems such as up-regulated inflammatory response, oxidative stress, and immune disorders during tissue regeneration has become a major clinical challenge. In recent years, the cross-development of materials science and biomedicine has provided new ideas for solving the above problems. However, there is still room for optimization in the mechanical properties and multi-functional synergy (such as immune coordination) of existing research systems.

[0003] Chinese herbal medicine vesicles are derived from natural plants. Their component substances and functional activities are closely related to the source plants, and they have multiple biological effects and good biocompatibility. In recent years, related research has been continuously promoted. Wolfberry leaves are the dried young leaves of the Solanaceae plant Lycium barbarum Lycium barbarum L., which has the effects of tonifying deficiency and replenishing essence, clearing heat, quenching thirst, dispelling wind and improving eyesight. China is rich in wolfberry resources. As the main by-product in the production process of wolfberry medicinal materials, wolfberry leaves are only used as tea in small amounts and are largely discarded, and their medicinal value has not been fully utilized. Modern research shows that wolfberry leaves are rich in polysaccharides, alkaloids, and flavonoid components, and have good biological activities in aspects such as antioxidant and blood sugar lowering. However, there are few research reports on wolfberry leaves at present, and the extraction of wolfberry leaf plant vesicles is still in the blank stage.

[0004] Therefore, the present invention designs to introduce a thermally expandable polymer into a gelatin-based hydrogel to prepare a temperature-sensitive hydrogel loaded with Chinese herbal medicine vesicles for regulating immunity, which not only has certain thermal responsiveness but also can scavenge local reactive oxygen species and improve the local immune microenvironment. This design provides a more potential treatment strategy for tissue regeneration disorders. Summary of the Invention

[0005] Aiming at the defects existing in the above prior art, the present invention provides a temperature-sensitive hydrogel loaded with Chinese herbal medicine vesicles for regulating immunity and a preparation method thereof. Poly(N-isopropylacrylamide) (PNIPAM) is introduced into methacrylated gelatin (GelMA). While having certain thermal response performance and enhanced mechanical properties, extracellular vesicle-like particles derived from wolfberry leaves (LBL-EVLPs) are introduced into it, endowing it with properties such as antioxidant and immune regulation, and effectively improving the local microenvironment.

[0006] The present invention solves the technical problems through the following technical solutions: First, a thermosensitive hydrogel loaded with Chinese herbal medicine vesicles for regulating immunity is proposed, which includes the following components: methacrylated gelatin (GelMA); N-isopropylacrylamide (NIPAM); N,N'-methylenebisacrylamide; photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959); Chinese herbal medicine vesicles. Among them, the mass fraction of methacrylated gelatin is 5%-10%, the mass fraction of N-isopropylacrylamide is 5%-25%, the mass fraction of N,N'-methylenebisacrylamide is 0.35%-0.5%, the photoinitiator is selected from at least one of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), and the concentration of Chinese herbal medicine vesicles is 1×10 3 -1×10 7 cells / mL.

[0007] In the above ratio, the Chinese herbal medicine vesicles are extracellular vesicle-like particles derived from wolfberry leaves. Further, the preparation method of the Chinese herbal medicine vesicles includes the following steps: First step, soak wolfberry leaves in PBS buffer at 4°C, and then break the cell walls and grind to obtain a wolfberry leaf grinding solution; Second step, perform differential centrifugation on the wolfberry leaf grinding solution, and resuspend the precipitate obtained by centrifugation in PBS buffer at 4°C to obtain a crude suspension containing extracellular vesicle-like particles derived from wolfberry leaves; Third step, add the crude suspension to the upper layer of the sucrose density gradient solution, and then centrifuge. Take the solution between the 30%-45% density layers, add PBS buffer at 4°C for mixing, and after mixing evenly, centrifuge twice to obtain extracellular vesicle-like particles derived from wolfberry leaves.

[0008] In the first step of the above preparation method of Chinese herbal medicine vesicles, the mass-volume ratio of wolfberry leaves to PBS buffer is 300g:200mL - 300g:400mL, and the soaking time is 8h.

[0009] The differential centrifugation in the second step includes the following steps (1), Centrifuge the wolfberry leaf grinding solution at a temperature of 4°C and a centrifugal force of 4000g for 1 - 1.5h, take the supernatant to obtain the first centrifugate, and the centrifuge tube used for centrifugation is an ultracentrifuge tube; (2), Centrifuge the first centrifugate at a temperature of 4°C and a centrifugal force of 10000g for 1 - 1.5h, take the supernatant to obtain the second centrifugate; (3), Centrifuge the second centrifugate at a temperature of 4°C and a centrifugal force of 100000g for 1.5 - 2h to obtain a precipitate; (4) Resuspend the precipitate in PBS buffer at a temperature of 4°C to obtain a crude suspension containing extracellular vesicle-like particles derived from wolfberry leaves.

[0010] In the third step, the volume of the PBS buffer is 500 μL and the temperature is 4°C; the concentrations of the sucrose density gradient solutions are 15%, 30%, 45% and 60% respectively. Centrifugation means centrifuging in an ultracentrifuge tube for 1.5 - 2 h at a temperature of 4°C and a centrifugal force of 150,000 g. The conditions for the secondary centrifugation are: centrifuging in an ultracentrifuge tube for 1.5 - 2 h at a temperature of 4°C and a centrifugal force of 150,000 g.

[0011] Secondly, a preparation method of a temperature-sensitive hydrogel loaded with Chinese herbal medicine vesicles for regulating immunity is proposed, including the following steps. Step 1: Dissolve gelatin in phosphate buffer, then add methacrylic anhydride dropwise and continuously stir. Add an equal amount of phosphate buffer to terminate the reaction. The solution obtained after dialysis with deionized water is frozen overnight at -80°C and then freeze-dried to obtain a hydrogel precursor. Step 2: Dissolve the hydrogel precursor in water, add a photoinitiator, and after dissolution, perform ultraviolet cross-linking to obtain a GelMA hydrogel. Step 3: Dissolve N-isopropylacrylamide, N,N'-methylenebisacrylamide, and a photoinitiator in water to obtain a NIPAM pre-gel solution. Step 4: Freeze the GelMA hydrogel overnight and then freeze-dry it. Then, soak it in a mixture of the NIPAM pre-gel solution and extracellular vesicle-like particles derived from wolfberry leaves at low temperature and in vacuum. After soaking, perform ultraviolet cross-linking again to obtain a temperature-sensitive hydrogel loaded with Chinese herbal medicine vesicles for immune regulation.

[0012] Further, in Step 1, the gelatin is type A gelatin, the stirring time is 2 h, dialysis is performed for 5 days using a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, the dialysis temperature is 50°C; the freeze-drying time is 24 h, and the mass fraction of the hydrogel precursor is 10%.

[0013] The photoinitiator in Step 2 is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone Irgacure2959, and the mass fraction is 0.5%.

[0014] In Step 3, the mass fraction of N-isopropylacrylamide is 15%, the mass fraction of N,N'-methylenebisacrylamide is 0.5%, and the photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure2959), and the mass fraction is 0.5%.

[0015] In step 4, the freeze-drying time is 24 h, the total volume of the mixed solution is 1 mL, and the vacuum soaking time is 1 h. The concentration of extracellular vesicle-like particles derived from wolfberry leaves is 1×10 3 -1×10 7 particles / mL.

[0016] In the present invention, poly(N-isopropylacrylamide) (PNIPAM) is introduced into methacrylated gelatin (GelMA), which can not only make up for the deficiency of poor mechanical properties of NIPAM-based hydrogels, but also achieve contraction and drug release at body temperature. At the same time, extracellular vesicle-like particles (LBL-EVLPs) derived from natural Chinese herbal medicine wolfberry leaves are introduced to ensure safety while endowing it with antioxidant and immunomodulatory properties, thereby improving the local microenvironment. The extracellular vesicle-like particles derived from natural Chinese herbal medicine wolfberry leaves are obtained through a series of steps such as soaking wolfberry leaves in PBS buffer, grinding with a blender, high-speed centrifugation, ultra-high-speed centrifugation, sucrose gradient centrifugation purification, and resuspension in PBS buffer. They exhibit certain biological activities in terms of antioxidant and immunomodulatory effects, and at the same time promote the transformation of the wolfberry industry from "single fruit use" to "whole-plant high-value utilization", achieving a double breakthrough in resource efficiency increase and product innovation.

[0017] The raw materials of the present invention are widely sourced. GelMA hydrogel has certain biocompatibility. Combining poly(N-isopropylacrylamide) (PNIPAM) with GelMA and introducing extracellular vesicle-like particles (LBL-EVLPs) derived from wolfberry leaves not only has certain thermoresponsiveness, but also can regulate the immune microenvironment at the wound site and effectively alleviate the oxidative stress response. Its beneficial effects include multiple effects such as promoting tissue repair, scavenging reactive oxygen species (ROS), and immunomodulation, and it has the advantages of simple preparation and low cost. Description of the Drawings

[0018] Figure 1 It is the area shrinkage rate of the GP-x hydrogel in Example 1.

[0019] Figure 2 It is the stress-strain curve A) and compression modulus B) of the GP-x hydrogel in Example 1.

[0020] Figure 3 It is the transmission electron microscope detection result of extracellular vesicle-like particles derived from wolfberry leaves (scale = 100 nm).

[0021] Figure 4 It is the particle size distribution of extracellular vesicle-like particles derived from wolfberry leaves.

[0022] Figure 5 It is the fluorescence quantitative analysis of Live / Dead staining to evaluate the biocompatibility of the LBL-GP hydrogel.

[0023] Figure 6 For DCFH-DA staining fluorescence quantitative analysis and evaluation to evaluate the ability of the LBL-GP hydrogel to scavenge ROS.

[0024] Figure 7 Flow cytometry analysis of RAW264.7 cells treated with LPS (control), IL-4 (positive control), GP, and LBL-GP hydrogel. Specific implementation manners

[0025] The following examples evaluated the thermoresponsive behavior and mechanical properties of the hydrogel. At the same time, some in vitro studies were carried out to evaluate the cytocompatibility, antioxidant capacity, and the effect on macrophage polarization of the hydrogel. All reagents used in the experiments were commercially available and will not be elaborated.

[0026] A1: In terms of temperature response, freeze-dried GelMA was soaked in different concentrations of NIPAM to observe the shrinkage behavior of the GP-x hydrogel, where GP represents the temperature-sensitive hydrogel and x represents the concentration of NIPAM, which were 5%, 15%, and 25% respectively. The hydrogel was placed on a heating table at 37 °C to simulate body temperature, and the area shrinkage rate of the hydrogel was evaluated; A2: In terms of mechanical properties, the freeze-dried GelMA hydrogel soaked in different concentrations of NIPAM was made into a cylinder, and the sample was compressed at a speed of 2 mm / min using an Instron-5944 mechanical tester. Preferably, the final concentration of NIPAM is 15%; A3: In terms of cytotoxicity, after co-culturing NIH / 3T3 cells with GP and LBL-GP hydrogels for corresponding times, where LBL-GP represents the immune-regulation plant Chinese herbal medicine vesicle temperature-sensitive hydrogel, detection was carried out using a Live / Dead cell staining kit according to the operating procedures. A4: In terms of antioxidant, NIH / 3T3 cells were seeded in a 24-well plate. After co-culturing with GP and LBL-GP hydrogels in an oxidative environment (H2O2) for 24 h, they were incubated with DCFH-DA (10 µM, ROS fluorescence probe) for 20 min. The fluorescence of DCFH-DA was observed under a fluorescence microscope to evaluate the intracellular ROS level.

[0027] A5: In terms of the effect on macrophage polarization, mouse macrophages RAW264.7 were seeded in a 6-well plate. After treatment according to different treatment methods, the cells were rinsed with PBS and labeled with CD86-APC and CD206-FITC antibodies, and flow cytometry was used for analysis. The specific treatment methods are shown in the following examples.

[0028] Example 1 In this example, GelMA hydrogels soaked in 3 different NIPAM concentrations (5%, 15%, 25%) were prepared, and the preparation method is as follows: Preparation of GelMA hydrogel: After dissolving gelatin in phosphate buffer solution (PBS), methacrylic anhydride was slowly added dropwise with continuous stirring, and then PBS was added again to terminate the reaction. The solution obtained after dialysis with deionized water was frozen at -80 °C overnight and then freeze-dried. The freeze-dried hydrogel precursor was dissolved in water, and the photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) was added. After complete dissolution, it was added to a mold and cross-linked with ultraviolet light to obtain GelMA hydrogel; Preparation of NIPAM pre-gel solution: N-isopropylacrylamide (NIPAM) with concentrations of 5%, 15%, and 25% was dissolved in water respectively with N,N'-methylenebisacrylamide and the photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) to obtain NIPAM pre-gel solutions with corresponding concentrations; Preparation of thermosensitive hydrogel (GP): After the cross-linked GelMA hydrogel was frozen at -80 °C overnight again, it was freeze-dried for 24 h, and then soaked in NIPAM pre-gel solutions with different concentrations at low temperature and under vacuum. Among them, the soaking volume was 1 mL. After soaking for 1 h, it was cross-linked again to obtain GP-x hydrogel, where x represents the concentration of NIPAM.

[0029] Thermal responsiveness of hydrogel: The GP hydrogel was placed on a heating platform at 37 °C to simulate body temperature to evaluate the change in the area shrinkage rate of the hydrogel with the concentration of NIPAM, and the change in the area shrinkage rate of each GP-x hydrogel was quantified; Mechanical properties of hydrogel: Compression tests were performed on each group of samples respectively to further characterize the mechanical strength of the hydrogels soaked in different NIPAM concentrations.

[0030] As Figure 1 shown, the higher the concentration of NIPAM monomer, the smaller the area shrinkage rate of the hydrogel. The area shrinkage rates of GP-x hydrogels from large to small are GP-5, GP-15, and GP-25. At the same time, Figure 2 it is shown that the ultimate stress of the hydrogel increases with the increase in the concentration of NIPAM monomer, and the compression modulus of GP-x hydrogel also increases with the increase in the concentration of NIPAM monomer. The mechanical strength of GP-15 hydrogel is higher than that of GP-5 hydrogel.

[0031] Example 2 In this example, taking GP-15 as an example, a thermosensitive hydrogel (LBL-GP) loaded with Chinese herbal medicine vesicles with antioxidant and immunomodulatory abilities was prepared. The method is roughly the same as that in Example 1, and the differences are as follows: Extraction of extracellular vesicle-like particles from wolfberry leaves: extracellular vesicle-like particles from wolfberry leaves (LBL-EVLPs) were isolated from homogenized wolfberry leaves by differential and density gradient centrifugation; the specific steps are as follows: First step: Wash 300 g of wolfberry leaves with ultrapure water and put them into a clean blender. Then add 200 mL of PBS buffer at a temperature of 4°C to the container of the blender, soak for 8 h, and then turn on the blender to grind thoroughly for 3 min to obtain a wolfberry leaf grinding solution. Second step: Perform differential centrifugation on the wolfberry leaf grinding solution obtained in the first step. The differential centrifugation includes the following steps: Step 1: Centrifuge the wolfberry leaf grinding solution obtained in the first step at a temperature of 4°C and a centrifugal force of 4000 g for 1 - 1.5 h, and take the supernatant to obtain the first centrifugate. Step 2: Centrifuge the first centrifugate obtained in Step 1 at a temperature of 4°C and a centrifugal force of 10000 g for 1 - 1.5 h, and take the supernatant to obtain the second centrifugate. Step 3: Centrifuge the second centrifugate obtained in Step 2 at a temperature of 4°C and a centrifugal force of 100000 g for 1.5 - 2 h to obtain a precipitate. Step 4: Resuspend the precipitate obtained in Step 3 in PBS buffer at a temperature of 4°C to obtain a crude suspension containing extracellular vesicle-like particles from wolfberry leaves. Third step: Add the crude suspension obtained in the second step to the upper layer of a sucrose density gradient solution with concentrations of 15%, 30%, 45%, and 60% from top to bottom. Then, centrifuge in an ultracentrifuge tube at a temperature of 4°C and a centrifugal force of 150000 g for 1.5 - 2 h. Take the solution between the 30% - 45% density layers, then add PBS buffer at a temperature of 4°C for mixing. After mixing evenly, centrifuge in an ultracentrifuge tube at a temperature of 4°C and a centrifugal force of 150000 g for 1.5 - 2 h to obtain extracellular vesicle-like particles from wolfberry leaves.

[0032] Resuspend the extracellular vesicle-like particles from wolfberry leaves obtained after secondary centrifugation in the third step in 500 μL of PBS buffer at a temperature of 4°C to obtain a suspension of extracellular vesicle-like particles from wolfberry leaves.

[0033] Characterize the morphology of the extracellular vesicle-like particles from wolfberry leaves using a transmission electron microscope. The results are as Figure 3 shown. The extracellular vesicle-like particles from wolfberry leaves prepared in this example have a typical round double-membrane structure.

[0034] Perform nanoparticle tracking analysis on the extracellular vesicle-like particles from wolfberry leaves. The detection results are as Figure 4As shown, the average diameter of the extracellular vesicle-like particles derived from wolfberry leaves prepared in this example was 137.81 ± 10.31 nm, and the particle size distribution was uniform.

[0035] The suspension of extracellular vesicle-like particles derived from wolfberry leaves was stored in a -80 °C refrigerator.

[0036] Preparation of LBL-GP hydrogel: After the GelMA hydrogel was cross-linked by ultraviolet light and stored overnight at -80 °C, it was freeze-dried for 24 h, and then soaked at low temperature and in vacuo in a mixed solution of 1 mL of NIPAM pre-gel solution and the suspension of extracellular vesicle-like particles derived from wolfberry leaves. The concentration of NIPAM was 15%, and the concentration of the suspension of extracellular vesicle-like particles derived from wolfberry leaves was 1×10 3 -1×10 7 cells / mL. After soaking for 1 h, it was cross-linked by ultraviolet light again to obtain LBL-GP hydrogel, and further characterization and application were carried out.

[0037] Comparative Example 1 A thermosensitive hydrogel (GP) was prepared in this comparative example. The preparation method was as follows: The GelMA hydrogel after ultraviolet cross-linking was frozen overnight again and then freeze-dried. Then it was soaked at low temperature and in vacuo in a 15% NIPAM pre-gel solution. The preparation methods of the GelMA hydrogel and the 15% NIPAM pre-gel solution were the same as those in Example 2. The soaking volume was 1 mL, and the soaking time was 1 h. After soaking, it was cross-linked by ultraviolet light again to obtain GP hydrogel, and further characterization and application were carried out.

[0038] The experimental results are as follows: As Figure 5 shown, based on the materials obtained in Example 2 and Comparative Example 1, the cytotoxicity of the hydrogel was evaluated by the Live / Dead experiment. First, NIH / 3T3 cells were seeded in 24-well plates and co-cultured with GP and LBL-GP hydrogels respectively. After culturing for 24 h, 48 h, and 72 h, the hydrogels were gently washed with PBS, and then stained using the Live / Dead detection kit according to the operating procedures. The results showed that the survival rate of NIH / 3T3 cells was higher after culturing on the LBL-GP hydrogel for 72 h.

[0039] As Figure 6As shown, based on the materials obtained in Example 2 and Comparative Example 1, to evaluate the ability of the LBL-GP hydrogel to scavenge ROS, NIH / 3T3 cells were first seeded in a 24-well plate and co-cultured with GP and the LBL-GP hydrogel in an oxidative environment (H2O2) for 24 h, and then incubated with DCFH-DA for 20 min. The fluorescence of DCFH-DA was observed under a fluorescence microscope to evaluate the intracellular ROS level. Quantitative analysis of the fluorescence intensity showed that the fluorescence intensity of the LBL-GP + H2O2 group was significantly lower than that of the H2O2 group and the GP + H2O2 group.

[0040] As Figure 7 Shown, based on the materials obtained in Example 2 and Comparative Example 1, to evaluate the ability of the LBL-GP hydrogel to promote macrophage polarization, mouse macrophages RAW264.7 were first seeded in a 6-well plate. Cells treated with 40 ng / mL interleukin-4 (IL-4) were used as the positive control group, and cells treated with 1 μg / mL lipopolysaccharide (LPS) were used as the negative control. RAW264.7 cells were first treated with 1 μg / mL LPS and then co-cultured with GP and the LBL-GP hydrogel for 24 h respectively. Subsequently, the cells were rinsed with PBS and labeled with CD86-APC and CD206-FITC antibodies and analyzed by flow cytometry. The results showed that, compared with the GP group, the proportion of CD86-positive cells effectively decreased after treatment with the LBL-GP hydrogel, and the proportion of CD206-positive cells also increased significantly, indicating that the LBL-GP group could better promote the transformation of macrophages from the M1 type (CD86) to the M2 type (CD206) and had an immunomodulatory effect.

[0041] In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by equivalent replacement or equivalent transformation fall within the protection scope required by the present invention.

Claims

1. A temperature-sensitive hydrogel of Chinese herbal medicine-loaded vesicles for immunomodulation, characterized in that: It includes the following components: methacrylated gelatin (GelMA); N-isopropylacrylamide (NIPAM); N,N'-methylenebisacrylamide; photoinitiator; Chinese herbal medicine vesicles; wherein, the mass fraction of methacrylated gelatin is 5% - 10%, the mass fraction of N-isopropylacrylamide is 5% - 25%, the mass fraction of N,N'-methylenebisacrylamide is 0.35% - 0.5%, the photoinitiator is selected from at least one of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959); the concentration of Chinese herbal medicine vesicles is 1×10 3 -1×10 7 per mL.

2. The temperature-sensitive hydrogel of the Chinese herbal medicine vesicle for regulating immunity according to claim 1, wherein: The Chinese herbal medicine vesicles are extracellular vesicle-like particles derived from wolfberry leaves. The preparation method of the Chinese herbal medicine vesicles includes the following steps. First step: Soak wolfberry leaves in PBS buffer at 4°C, and then break the cell walls and grind them to obtain a wolfberry leaf grinding solution. Second step: Perform differential centrifugation on the wolfberry leaf grinding solution. The precipitate obtained by centrifugation is resuspended in PBS buffer at 4°C to obtain a crude suspension containing extracellular vesicle-like particles derived from wolfberry leaves. Third step: Add the crude suspension to the upper layer of a sucrose density gradient solution, and then perform centrifugation. Take the solution in the density layer of 30%-45%, add PBS buffer at 4°C for mixing. After mixing evenly, perform secondary centrifugation to obtain extracellular vesicle-like particles derived from wolfberry leaves.

3. The thermosensitive hydrogel of the Chinese herbal medicine vesicle for regulating immunity according to claim 2, wherein: In the first step, the mass-volume ratio of wolfberry leaves to PBS buffer is 300g:200mL - 300g:400mL, and the soaking time is 8h.

4. The thermosensitive hydrogel of the Chinese herbal medicine vesicle for regulating immunity according to claim 2, wherein: The differential centrifugation in the second step includes the following steps. (1) Centrifuge the wolfberry leaf grinding solution at a temperature of 4°C and a centrifugal force of 4000g for 1 - 1.5h, and take the supernatant to obtain the first centrifugate. The centrifuge tube used for centrifugation is an ultracentrifuge tube. (2) Centrifuge the first centrifugate at a temperature of 4°C and a centrifugal force of 10000g for 1 - 1.5h, and take the supernatant to obtain the second centrifugate. (3) Centrifuge the second centrifugate at a temperature of 4°C and a centrifugal force of 100000g for 1.5 - 2h to obtain a precipitate. (4) Resuspend the precipitate in PBS buffer at a temperature of 4°C to obtain a crude suspension containing extracellular vesicle-like particles derived from wolfberry leaves.

5. The temperature-sensitive hydrogel of the Chinese herbal medicine vesicle for regulating immunity according to claim 2, characterized in that: In the third step, the volume of PBS buffer is 500μL and the temperature is 4°C; the concentrations of the sucrose density gradient solution are 15%, 30%, 45% and 60% respectively. Centrifugation means centrifuging in an ultracentrifuge tube at a temperature of 4°C and a centrifugal force of 150000g for 1.5 - 2h. The conditions for the secondary centrifugation are: centrifuging in an ultracentrifuge tube at a temperature of 4°C and a centrifugal force of 150000g for 1.5 - 2h.

6. A preparation method of a temperature-sensitive hydrogel loaded with Chinese herbal medicine vesicles for regulating immunity, characterized in that: It includes the following steps. Step one: Dissolve gelatin in phosphate buffer, then add methacrylic anhydride dropwise, continuously stir, and then add an equal amount of phosphate buffer to terminate the reaction. The solution obtained after dialysis with deionized water is frozen overnight at -80°C and then lyophilized to obtain a hydrogel precursor. Step two: Dissolve the hydrogel precursor in water, add a photoinitiator, and after dissolution, perform ultraviolet crosslinking to obtain a GelMA hydrogel. Step three: Dissolve N-isopropylacrylamide, N,N'-methylenebisacrylamide, and a photoinitiator in water to obtain a NIPAM pre-gel solution. Step four: Freeze the GelMA hydrogel overnight and then lyophilize it. Then, soak it at low temperature and in vacuum in a mixture of the NIPAM pre-gel solution and extracellular vesicle-like particles derived from wolfberry leaves. After soaking, perform ultraviolet crosslinking again to obtain an immune-regulating Chinese herbal medicine vesicle-loaded thermosensitive hydrogel.

7. The preparation method of the temperature-sensitive hydrogel of Chinese herbal medicine-loaded vesicles for regulating immunity according to claim 6, characterized in that: In the first step, the gelatin is type A gelatin, the stirring time is 2 h, dialysis is carried out for 5 days using a dialysis bag with a molecular weight cut-off of 8 - 14 kDa, and the dialysis temperature is 50 °C; the freeze-drying time is 24 h, and the mass fraction of the hydrogel precursor is 10%.

8. The preparation method of the Chinese herbal medicine-loaded vesicle thermosensitive hydrogel for regulating immunity according to claim 6, wherein: In the second step, the photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone Irgacure 2959, and the mass fraction is 0.5%.

9. The preparation method of the Chinese herbal medicine-loaded vesicle thermosensitive hydrogel for regulating immunity according to claim 6, characterized in that: In the third step, the mass fraction of N-isopropylacrylamide is 15%, the mass fraction of N,N'-methylenebisacrylamide is 0.5%, and the photoinitiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), and the mass fraction is 0.5%.

10. The preparation method of the temperature-sensitive hydrogel of Chinese herbal medicine vesicle for regulating immunity according to claim 6, characterized in that: In step 4, the freeze-drying time is 24 h, the total volume of the mixed solution is 1 mL, and the vacuum soaking time is 1 h. The concentration of the extracellular vesicle-like particle suspension derived from wolfberry leaves is 1×10 3 -1×10 7 cells / mL.

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