Extracellular vesicle complex lyophilized powder, preparation method and application thereof

Abstract

This invention provides an exovesicle composite lyophilized powder, its preparation method, and its application, relating to the field of biopharmaceutical technology. The preparation method of the composite lyophilized powder provided by this invention includes: incubating exovesicles in a carboxymethyl chitosan solution to obtain a precursor solution; mixing and incubating a catechin solution with the precursor solution to obtain an intermediate solution; and lyophilizing a mixture of a trehalose solution and the intermediate solution to obtain the exovesicle composite lyophilized powder. This invention utilizes the intermolecular forces between carboxymethyl chitosan, catechin, and trehalose to encapsulate exovesicles, which helps improve the structural and functional stability of the exovesicles during the lyophilization process.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to an exovesicle composite lyophilized powder, its preparation method, and its application. Background Technology

[0002] The physical structure of external vesicles is easily damaged during the freeze-drying process, leading to the inactivation of the biologically active intracellular substances they carry. In order to improve the structural and functional stability of external vesicles, freeze-drying protectants are usually used to protect them. For example, Chinese patent CN117769353A provides a freeze-drying protectant composition including trehalose and sucrose, and Chinese patent CN119464200A provides a freeze-drying protectant including polyvinyl alcohol, dextran, trehalose, mannitol, vitamin C, hydroxytetrahydropyrimidine, and superoxide dismutase.

[0003] However, the protection mechanism of mono- or binary composite freeze-drying protection systems based on trehalose mainly involves reducing mechanical damage by forming filling and / or vitrification on the surface of the protected material. However, due to the extremely fragile lipid membrane structure on the surface of the outer vesicles, the trehalose-based protective layer is difficult to provide sufficient and effective protection. During freeze-drying, the outer vesicles are prone to rupture, leading to leakage of contents. This makes it impossible to effectively maintain the integrity and functional activity of the vesicle membrane on the surface of the outer vesicles. Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0004] The purpose of this invention is to provide an exovesicle composite freeze-dried powder, its preparation method, and its application. The exovesicles are encapsulated using the intermolecular forces between carboxymethyl chitosan, catechin, and trehalose, which helps to improve the structural and functional stability of the exovesicles during freeze-drying and transportation.

[0005] In a first aspect, the present invention provides a method for preparing an exovesicle composite lyophilized powder, comprising: incubating exovesicles in a carboxymethyl chitosan solution to obtain a precursor solution; mixing and incubating a catechin solution with the precursor solution to obtain an intermediate solution; and lyophilizing a mixture of a trehalose solution and the intermediate solution to obtain an exovesicle composite lyophilized powder; wherein the mass ratio of carboxymethyl chitosan to catechin is (1-10):1.

[0006] Optionally, the source of the external vesicles includes bacteria or cells, preferably the bacteria include Akkermansia myxophilus or Escherichia coli, and preferably the cells include plant cells or animal cells; and / or, the external vesicles include drug-loaded or drug-free vesicles, and the drug includes chemical drugs or biological drugs.

[0007] Optionally, the mass ratio of the external vesicles to carboxymethyl chitosan is 1:(50-200); and / or, the external vesicles are incubated in a carboxymethyl chitosan solution at -4℃ to 4℃; and / or, incubated in a carboxymethyl chitosan solution for 20 min to 60 min; and / or, the external vesicle stock solution is mixed with the carboxymethyl chitosan solution for incubation, preferably the concentration of the external vesicle stock solution is 0.2 mg / mL to 1.0 mg / mL.

[0008] Optionally, the catechin includes one of epigallocatechin gallate, epicatechin gallate, epigallocatechin, and epicatechin; and / or, the precursor solution is added dropwise to the catechin solution and mixed and incubated; and / or, the catechin solution and the precursor solution are mixed and incubated at -4℃ to 4℃ and / or in the dark; and / or, the catechin solution and the precursor solution are mixed and incubated for 10 min to 30 min; and / or, the mass ratio of carboxymethyl chitosan to the catechin is (5-7):1.

[0009] Optionally, the mass ratio of trehalose to carboxymethyl chitosan is (8-12):1, preferably 10:1; and / or, the trehalose solution is mixed with the intermediate solution at -4℃ to 4℃; and / or, the trehalose solution is ultrasonically mixed with the intermediate solution; and / or, the trehalose solution is mixed with the intermediate solution for 1 min to 15 min; and / or, the solution is pre-frozen at -50℃ to -90℃ and then freeze-dried.

[0010] Secondly, the present invention also provides an exovesicle composite lyophilized formulation prepared by any of the above-mentioned optional preparation methods.

[0011] Thirdly, the present invention also provides an application of the extravesicular vesicle complex lyophilized formulation prepared by any of the above-mentioned optional preparation methods, the application including the preparation of a drug for treating or preventing colitis.

[0012] The method for preparing an exovesicle composite lyophilized powder provided by this invention has at least one of the following beneficial technical effects compared with the prior art: 1. By utilizing the amphoteric polysaccharide backbone on the surface of carboxymethyl chitosan to anchor on the lipid membrane surface of the outer vesicle, and at the same time, catechins form a hydrophobic anti-aggregation barrier on the surface of carboxymethyl chitosan and outer vesicles through π-π stacking via the pyrogallol structure. Trehalose can be used as a small molecule filler to penetrate into the surface pores and improve the compactness of the outer vesicle surface coating layer through hydrogen bonding crosslinking. 2. By gradient-induced distribution and assembly of carboxymethyl chitosan, catechin and trehalose on the surface of the outer vesicle, it helps to improve the uniformity of encapsulation and surface density of the outer vesicle. By utilizing electrostatic complexation and hydrogen bonding to form a semi-interpenetrating network structure on the surface of the outer vesicle, it can effectively replace water molecules to maintain the fluidity of the vesicle membrane during the freeze-drying process, thereby improving the structural and functional stability of the outer vesicle during the freeze-drying process. Attached Figure Description

[0013] Figure 1 A flowchart illustrating a method for preparing an exovesicle composite lyophilized powder according to the present invention; Figure 2 This is a comparison chart of the particle size of the exovesicle composite powder prepared in Examples 1 to 2 and Comparative Examples 1 to 2 of the present invention with that of the fresh exovesicles prepared in Preparation Example 1. Figure 3 This is a comparison chart of NO levels in cells after intervention with the extravesicle composite powder prepared in Examples 1 to 2 and Comparative Examples 1 to 2 of the present invention and the fresh extravesicles prepared in Preparation Example 1; Figure 4 This is a comparison chart showing the levels of anti-inflammatory factors in cells after intervention with the extravesicle composite powder prepared in Examples 1 to 2 and Comparative Examples 1 to 2 of the present invention and the fresh extravesicles prepared in Preparation Example 1. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0015] See Figure 1 This invention provides a method for preparing an exovesicle composite lyophilized powder, comprising the following steps: S1. The outer vesicles are incubated in a carboxymethyl chitosan solution to obtain a precursor solution; S2. Mix the catechin solution with the precursor solution and incubate to obtain the intermediate solution; S3. After mixing the trehalose solution and the intermediate solution, freeze-dry the mixture to obtain the exovesicle composite freeze-dried powder.

[0016] In fact, during the preparation of the vesicle composite lyophilized powder, carboxymethyl chitosan is anchored to the lipid membrane surface of the vesicles through electrostatic and hydrogen bonding interactions. At the same time, catechins form a dense anti-aggregation layer on the periphery of the vesicles through π-π stacking and hydrophobic interactions, which can effectively resist mechanical stress during the lyophilization process and prevent membrane fusion of the vesicles. In addition, trehalose can act as a small molecule filler to penetrate into the network pores of the anti-aggregation layer and replace water molecules to bind with phospholipid heads through hydrogen bonds, which helps maintain membrane fluidity.

[0017] In some embodiments, the source of the extravesicles used in step S1 includes bacteria or cells. In fact, an extravesicle is a membrane-like vesicle with a lipid membrane structure, actively secreted by bacteria or cells, and encapsulates biomolecules such as proteins and nucleic acids; therefore, extravesicles possess a variety of functional activities. Specifically, the source bacteria of the extravesicles can be one of *Akermansia myxophilus* or *Escherichia coli*, and the source cells can be one of plant cells or animal cells. Indeed, it is well known in the art that the functional activity of extravesicles is related to their source.

[0018] Furthermore, the exovesicles used in step S1 can be obtained using extraction and purification methods commonly used in the art. For example, after culturing the source bacteria or source cells, purification and separation can be performed by means of centrifugation, filtration, etc. The obtained exovesicles can be stored at 4°C. In some embodiments, drugs can also be loaded into the exoves using loading methods commonly used in the art. Specifically, the drugs can include one of chemical drugs and biological drugs.

[0019] In some embodiments, the mass ratio of outer vesicles to carboxymethyl chitosan in step S1 is 1:(50-200). In practice, after mixing the outer vesicles and carboxymethyl chitosan, the carboxymethyl chitosan can be anchored to the surface of the outer vesicles via electrostatic interactions and hydrogen bonds. Furthermore, the carboxymethyl chitosan used is food-grade to suit the application of the freeze-dried powder in food. Further, the mass ratio of outer vesicles to carboxymethyl chitosan can also be any ratio or value within the range of 1:(50-200), such as 1:(50-150), 1:(100-200), 1:(80-120), 1:(70-85), 1:80, and is not limited to the values ​​listed above. Other unlisted values ​​within the range of 1:(50-200) can also achieve the purpose of this invention.

[0020] In some embodiments, step S1 involves incubating the exovesicles in a carboxymethyl chitosan solution at an environment of -4°C to 4°C. In fact, incubation at low temperatures helps maintain the activity and structural integrity of the exovesicles, preventing rupture and inactivation. Specifically, incubation can be performed in an ice bath. Furthermore, the exovesicles can be pre-suspended in PBS to prepare an exovesicle stock solution, which is then mixed with the carboxymethyl chitosan solution for incubation. This helps improve the uniformity of mixing between the exovesicles and carboxymethyl chitosan.

[0021] In some embodiments, the solvent for the carboxymethyl chitosan solution used in step S1 includes PBS buffer, which helps maintain a stable pH environment during exovesicle incubation and avoids structural damage to the exovesicles. Furthermore, when mixing the exovesicle stock solution with the carboxymethyl chitosan solution, the concentration of the exovesicle stock solution used is 0.2 mg / mL-1.0 mg / mL, specifically ensuring that the exoves are thoroughly mixed in the stock solution.

[0022] In some embodiments, the exovesicles can be incubated in a carboxymethyl chitosan solution for 20-60 minutes in step S1. In practice, when considering the incubation time, it is necessary to ensure that the carboxymethyl chitosan has sufficient time to anchor on the surface of the exovesicles through electrostatic and hydrogen bonding interactions, while avoiding excessive incubation time that could damage the structure of the exovesicles. Specifically, the incubation time can be any sub-interval within the range of 20-60 minutes, such as 20-50 minutes, 30-50 minutes, 20-40 minutes, etc., or any specific time value within the range, such as 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, and is not limited to the ranges and values ​​listed above. Other unlisted ranges or values ​​within the 20-60 minute range can also achieve the purpose of this invention.

[0023] In some embodiments, the catechins used in step S2 include one of epigallocatechin gallate, epicatechin gallate, epigallocatechin, and epicatechin. In fact, when the precursor solution is mixed with the catechin solution, the pyrogallol structure on the catechin surface can form a hydrophobic anti-aggregation protective barrier through π-π stacking, effectively improving the membrane fluidity of the exovesicles and their resistance to mechanical stress during freeze-drying.

[0024] In some embodiments, the precursor solution can be added dropwise to the catechin solution in step S2 for mixing and incubation. In fact, the solids dispersed in the precursor solution include outer vesicles and carboxymethyl chitosan anchored to the surface of the outer vesicle membrane via electrostatic and hydrogen bonding interactions. Therefore, when the precursor solution is added dropwise to the catechin solution, the catechin can be uniformly modified on the surface of the outer vesicles. Furthermore, the carboxymethyl chitosan and catechin in the precursor solution can co-modify and form an interwoven protective layer.

[0025] In some embodiments, in step S2, the catechin solution and the precursor solution can be mixed and incubated at -4℃ to 4℃ and / or in a light-protected environment. Preferably, the mixing and incubation can be carried out in a low-temperature, light-protected environment, which helps to improve the structural integrity and activity of the outer vesicles. Specifically, the precursor solution can be added dropwise to the catechin solution in an ice bath and in a light-protected environment, and after the addition is completed, the mixture can be incubated for 10 min to 30 min. Similarly, the incubation time can be any sub-interval within the range of 10 min to 30 min, such as 10 min to 20 min, 20 min to 30 min, etc., or any specific time value within the range, such as 10 min, 15 min, 20 min, 25 min, 30 min, and is not limited to the ranges and values ​​listed above. Other unlisted ranges or values ​​within the range of 10 min to 30 min can also achieve the purpose of this invention.

[0026] In some embodiments, when the precursor solution is added dropwise to the catechin solution in step S2, the mass ratio of carboxymethyl cellulose to catechin can be controlled to be (1-10):1. Further, the mass ratio of carboxymethyl cellulose to catechin can also be any ratio range or value within the range of (1-10):1, such as (1-5):1, (5-10):1, (2-6):1, 5:1, 7:1, and is not limited to the values ​​listed above. Other unlisted values ​​within the range of (1-10):1 can also achieve the purpose of this invention. Preferably, the mass ratio of carboxymethyl cellulose to catechin can be (5-7):1.

[0027] In some embodiments, trehalose and the intermediate solution can be mixed at 4°C to 4°C in step S3. In practice, during the mixing process, trehalose can penetrate into the pores of the protective network formed by carboxymethyl chitosan and catechins on the surface of the outer vesicles, and bind to the phospholipid heads on the surface of the outer vesicles through hydrogen bonding. This not only improves the fluidity of the outer vesicle surface membrane but also enhances the density and uniformity of the protective layer on the outer vesicle surface. Specifically, trehalose and the intermediate solution can be mixed in an ice bath environment.

[0028] In some embodiments, the trehalose solution and the intermediate solution may be ultrasonically mixed for 1-15 minutes in step S3. In fact, ultrasonic mixing helps improve the mixing uniformity of the trehalose solution and the intermediate solution, while also promoting the penetration of trehalose onto the surface of the outer vesicles. Furthermore, the mass ratio of trehalose to carboxymethyl chitosan can be (8-12):1, or any ratio range or value within the range of (8-12):1, such as (8-10):1, (10-12):1, (9-11):1, 10:1, and is not limited to the values ​​listed above. Other unlisted values ​​within the range of (8-12):1 can also achieve the purpose of this invention.

[0029] Preparation Example 1: A method for preparing an exovesicle stock solution, comprising: preparing *Akermansia muciniphila* (… Akkermansia muciniphila AKK) with 1×10 6 CFU / mL was inoculated into BHI medium and anaerobically cultured at 37°C for 48 h. The supernatant was separated by centrifugation. The supernatant was filtered through 2 μm, 1.2 μm, 0.45 μm, and 0.22 μm filter membranes. After ultracentrifugation at 150,000 × g for 2 h at 4°C, the precipitate was collected and resuspended in sterile PBS to obtain the exovesicle stock solution.

[0030] Example 1: A method for preparing an exovesicle composite lyophilized powder, comprising the following steps: S1. Dissolve carboxymethyl chitosan (CMCS, BR grade) in sterile PBS at pH 7.4 to prepare a 1% (w / v) carboxymethyl chitosan solution. Mix the stock solution of the outer vesicles prepared in Preparation Example 1 with the carboxymethyl chitosan solution and adjust the mass ratio of outer vesicles to carboxymethyl chitosan to 1:80. Incubate in an ice bath for 30 min to obtain the precursor solution. S2. Epigallocatechin gallate (EGCG) was dissolved in pre-cooled sterile deionized water to prepare a catechin solution with a concentration (w / v) of 0.2%. Under light-protected conditions and an ice bath, the precursor solution was added dropwise to the catechin solution, controlling the mass ratio of carboxymethyl chitosan to epigallocatechin gallate in the precursor solution to be 7:1. After the addition was completed, the solution was incubated for 15 min to obtain the intermediate solution. S3. Trehalose (D-trehalose dihydrate) was dissolved in sterile PBS at pH 7.4 to prepare a trehalose solution. The trehalose solution was mixed with the intermediate solution, and the mass ratio of trehalose to carboxymethyl chitosan was adjusted to 10:1. After ultrasonic mixing in an ice bath for 5 minutes, the mixture was pre-frozen at -80℃ and freeze-dried to obtain the exovesicle composite freeze-dried powder.

[0031] Example 2: A method for preparing an exovesicle composite lyophilized powder, which differs from Example 1 in that the mass ratio of carboxymethyl chitosan to epigallocatechin gallate in the precursor solution in step S2 is 5:1.

[0032] Comparative Example 1: A method for preparing an exovesicle composite lyophilized powder, which differs from Example 1 in that step S2 is omitted. In step S3, the trehalose solution is mixed with the precursor solution obtained in S1, and the mass ratio of trehalose to carboxymethyl chitosan is adjusted to 10:1. After ultrasonic mixing in an ice bath environment for 5 minutes, the mixture is pre-frozen at -80°C and lyophilized to obtain the exovesicle composite lyophilized powder.

[0033] Comparative Example 2: A method for preparing an exovesicle composite lyophilized powder, which differs from Example 1 in that steps S1 and S2 are not performed. In step S3, the exovesicle mother liquor obtained in Example 1 is mixed with trehalose solution, and the mass ratio of exovesicles to trehalose is adjusted to 1:800. After ultrasonic mixing in an ice bath environment for 5 minutes, the mixture is pre-frozen at -80°C and lyophilized to obtain the exovesicle composite lyophilized powder.

[0034] Particle size analysis: The exovesicle composite lyophilized powders prepared in Examples 1 to 2, Comparative Examples 1 to 2, and the fresh exovesicles prepared in Preparation Example 1 were dissolved and diluted 200-fold with PBS buffer. The particle size of the exovesicles in each group of lyophilized powders was analyzed using a NanoSight NS300 system. The main peak particle size (Mode particle size, the particle size value corresponding to the highest particle concentration) was used as the evaluation index. The results are as follows: Figure 2 As shown.

[0035] from Figure 2 As can be seen, the Mode particle size of the lyophilized vesicle composite powders prepared in Comparative Examples 1 and 2 was significantly increased compared to the fresh vesicles in Preparation Example 1. This indicates that the lyophilized vesicle composite powders prepared in Comparative Examples 1 and 2 underwent severe aggregation and / or fusion after reconstitution, resulting in an increase in the vesicle particle size. In contrast, the Mode particle size of the vesicles in Examples 1 and 2 after reconstitution was not statistically different from that of the fresh vesicles. Therefore, this invention demonstrates that by constructing a ternary co-assembly system using carboxymethyl chitosan, catechin, and trehalose, it can effectively maintain the particle size stability of vesicles and inhibit aggregation and fusion between vesicles.

[0036] Cellular experiments: Mouse macrophages (RAW cells) were seeded in complete medium containing 10% FBS and cultured at 37°C and 5% CO2. When the cell confluence reached 80%, cells in the logarithmic growth phase were harvested and cultured at 2 × 10⁻⁶ cells / year. 5Cells were seeded at a density of 250 μL of culture medium in each well of a 48-well plate and cultured until the cells reached 60% confluence. After discarding the culture medium, the cells were randomly divided into a control group, a model group, and an intervention group. The control group was cultured with LPS-free medium, while the model and intervention groups were cultured with medium containing 1 μg / mL LPS. The intervention group was supplemented with different concentrations of extracellular vesicle reconstitution solution (Examples 1-2, Comparative Examples 1-2, and Preparation Example 1: 5 μg / mL and 10 μg / mL). After culturing for 24 h, the NO and inflammatory factor levels in the cell supernatant were detected using a NO kit, a TNF-α kit, and an IL-6 kit. The results are shown below. Figure 3 and Figure 4 As shown.

[0037] from Figure 3 As can be seen, the NO level in the model group and the intervention group cells was significantly increased, indicating that the LPS-induced RAW macrophage validation model was successfully constructed. However, the NO release of the cells corresponding to Comparative Example 1 and Comparative Example 2 was not significantly different from that of the model group, indicating that the lyophilized powder of the exovesicles prepared in Comparative Example 1 and Comparative Example 2 did not show anti-inflammatory activity after reconstitution. In contrast, the NO release of the cells corresponding to Example 1 and Example 2 was not statistically different from that of Preparation Example 1, indicating that the lyophilized powder of the exovesicles prepared in Example 1 and Example 2 could show anti-inflammatory activity comparable to that of fresh exovesicles after reconstitution. In addition, both Example 1 and Example 2 showed stable NO inhibition at 5 μg / mL and 10 μg / mL.

[0038] from Figure 4 As can be seen, the levels of IL-6 and TNF-α in the model group were significantly increased compared to the blank group, further demonstrating the successful construction of the LPS-induced RAW macrophage validation model. Furthermore, the reconstituted lyophilized vesicle compound powders prepared in Examples 1 and 2 significantly inhibited the release of inflammatory factors such as TNF-α and IL-6, with a significantly greater reduction than the fresh vesicles in Example 1. In contrast, the lyophilized vesicle compound powders in Comparative Examples 1 and 2 did not exhibit anti-inflammatory activity. Figure 3 The results were consistent with those in the previous studies. This is because the outer vesicles could not be adequately protected during the freeze-drying process, resulting in damage to the outer vesicle structure and leakage of contents.

[0039] from Figures 2 to 4Based on Examples 1 to 2 and Comparative Examples 1 to 2, it can be seen that in Comparative Example 2, when trehalose is used alone for freeze-drying protection of outer vesicles, trehalose cannot form a stable three-dimensional network structure on the surface of the outer vesicles, and its ability to protect the lipid bilayer is limited. As a result, the outer vesicles are prone to fusion and rupture during the freeze-drying process. In Comparative Example 1, when carboxymethyl chitosan and trehalose are used in combination, due to the lack of a hydrophobic anti-aggregation layer provided by catechins, the carboxymethyl chitosan layer is prone to collapse during the freeze-drying dehydration process, and it is impossible to maintain an orderly protective structure.

[0040] Therefore, in Examples 1 and 2 of this application, by using carboxymethyl chitosan, catechins and trehalose to form a protective layer on the surface of the outer vesicles, the morphological integrity of the outer vesicles during the freeze-drying process can be maintained, effectively preventing leakage of contents. Moreover, after the freeze-dried powder is reconstituted, the outer vesicles can still be normally taken up by cells and release anti-inflammatory active factors. At the same time, catechins themselves have certain antioxidant activity, which can synergistically enhance the anti-inflammatory activity of the composite freeze-dried powder with the outer vesicles.

[0041] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for preparing an exovesicle composite lyophilized powder, characterized in that, include: The outer vesicles were incubated in a carboxymethyl chitosan solution to obtain a precursor solution; The intermediate solution was obtained by mixing and incubating the catechin solution with the precursor solution. Trehalose solution and intermediate solution were mixed and freeze-dried to obtain exovesicle composite freeze-dried powder; wherein the mass ratio of carboxymethyl chitosan to catechin was (1-10):

1.

2. The preparation method according to claim 1, characterized in that: The source of the external vesicles includes bacteria and cells, preferably the bacteria include Akkermansia myxophilus and Escherichia coli, and preferably the cells include plant cells and animal cells; and / or, the external vesicles include drug-loaded or drug-free vesicles, and the drug includes chemical drugs and biological drugs.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the external vesicles to carboxymethyl chitosan is 1:(50-200); and / or, the external vesicles are incubated in a carboxymethyl chitosan solution at -4℃ to 4℃; and / or, incubated in a carboxymethyl chitosan solution for 20min-60min; and / or, the external vesicle stock solution is mixed with the carboxymethyl chitosan solution for incubation, preferably the concentration of the external vesicle stock solution is 0.2mg / mL-1.0mg / mL.

4. The preparation method according to claim 1, characterized in that: The catechin includes one of epigallocatechin gallate, epicatechin gallate, epigallocatechin, and epicatechin; and / or, the precursor solution is added dropwise to the catechin solution and mixed and incubated; and / or, the catechin solution and the precursor solution are mixed and incubated at -4℃ to 4℃ and / or in the dark; and / or, the catechin solution and the precursor solution are mixed and incubated for 10 min to 30 min; and / or, the mass ratio of the carboxymethyl chitosan to the catechin is (5-7):

1.

5. The preparation method according to claim 1, characterized in that: The mass ratio of trehalose to carboxymethyl chitosan is (8-12):1, preferably 10:1; and / or, the trehalose solution is mixed with the intermediate solution at -4℃ to 4℃; and / or, the trehalose solution is ultrasonically mixed with the intermediate solution. And / or, mix the trehalose solution with the intermediate solution for 1 min to 15 min; And / or, freeze-dry after pre-freezing at -50℃ to -90℃.

6. An exovesicle composite lyophilized formulation prepared by the preparation method according to any one of claims 1 to 5.

7. The application of an exocapsule composite lyophilized formulation prepared by the preparation method according to any one of claims 1 to 5, characterized in that, The applications include the preparation of drugs for the treatment or prevention of colitis.

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