Exosome hydrogel for regulating immune microenvironment of cholangiocarcinoma and preparation method thereof

Abstract

The application relates to the technical field of biological medicine, and particularly discloses an exosome hydrogel for regulating an immune microenvironment of cholangiocarcinoma and a preparation method thereof. The hydrogel is formed by in-situ crosslinking after mixing of A liquid and B liquid, the A liquid contains thiolated chitosan, TIMP2-rich exosomes, trehalose, mannitol, glycine, mannose and an active oxygen-sensitive crosslinking regulator, and the B liquid contains oxidized hyaluronic acid, fucoidan sulfate and a CSF-1R inhibitor. The preparation method is as follows: TIMP2-rich exosomes are induced and prepared first, then A liquid and B liquid are prepared after being prepared with a protection liquid, the two liquids are synchronously output for in-situ gelation during use, and the hydrogel can also be prepared into a freeze-dried powder preparation. The hydrogel regulates the immune microenvironment of cholangiocarcinoma through multi-target synergistic action, has an active oxygen response release characteristic, can improve the concentration of active ingredients in a tumor region, reduces the whole-body exposure risk, the preparation process effectively retains the biological activity of the exosomes, the preparation has good stability, and is suitable for the requirement of clinical local drug delivery.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to an exosome hydrogel that regulates the immune microenvironment of cholangiocarcinoma and its preparation method. Background Technology

[0002] Cholangiocarcinoma is a common malignant tumor of the digestive system, characterized by its early insidious nature and high malignancy. Most patients are diagnosed after the opportunity for radical surgical treatment has passed, and the clinical benefits of traditional radiotherapy and chemotherapy are limited. Immune microenvironment dysregulation is a crucial mechanism in the development and progression of cholangiocarcinoma. Regulating the polarization state of tumor-associated macrophages and reshaping the immunosuppressive microenvironment can effectively enhance the anti-tumor immune response, providing a feasible pathway for interventional treatment of cholangiocarcinoma. Exosomes and small molecule immunomodulators are commonly used immunomodulatory active substances, but conventional systemic administration methods have limitations such as low accumulation efficiency at the tumor site and high risk of systemic exposure.

[0003] Currently, hydrogel formulations that can be used for local delivery of immunomodulatory active substances mostly adopt a single active component design, which makes it difficult to achieve synergistic immunomodulatory effects across multiple pathways. Furthermore, the release behavior of conventional gel systems lacks microenvironment responsiveness, and the local retention time and controlled release precision are insufficient. This makes it impossible to effectively control the systemic exposure while increasing the concentration of active ingredients in the tumor area, and thus fails to meet the practical application requirements for immune microenvironment regulation in cholangiocarcinoma. Summary of the Invention

[0004] To address the issues of limited active components, local controlled-release delivery, and difficulty in balancing the effective concentration in the tumor region with the safety of systemic exposure in existing immunomodulatory hydrogels, this application provides an exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma and its preparation method.

[0005] In a first aspect, this application provides an exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma, employing the following technical solution:

[0006] An exosome hydrogel that regulates the immune microenvironment of cholangiocarcinoma is formed by in-situ cross-linking of solution A and solution B in a volume ratio of 0.9:1 to 1.1:1.

[0007] The A solution comprises, by mass parts: 1.5 to 2.5 parts thiolated chitosan, 0.05 to 0.5 parts TIMP2-enriched exosomes, 2 to 5 parts trehalose, 1 to 3 parts mannitol, 0.1 to 0.5 parts glycine, 0.1 to 0.5 parts mannan, and 0.05 to 0.2 parts reactive oxygen species-sensitive crosslinking regulator;

[0008] The B solution comprises, by mass, 1.0 to 2.0 parts of sodium periodate aldehyde-modified oxidized hyaluronic acid, 0.1 to 0.5 parts of brown algae polysaccharide sulfate, and 0.08 to 0.1 parts of CSF-1R small molecule inhibitor.

[0009] By employing the above-mentioned technical solution, immunomodulatory active substances acting at different levels are co-loaded into a two-component in-situ hydrogel system. TIMP2-enriched exosomes can improve the conditions for immune cell infiltration by regulating tumor matrix remodeling, fucoidan sulfate can directly interfere with macrophage polarization signaling pathways, and CSF-1R small molecule inhibitors can inhibit the survival and recruitment of tumor-associated macrophages. The three types of active ingredients work synergistically through different targets to intervene in the immunosuppressive microenvironment of cholangiocarcinoma through multiple pathways. At the same time, the hydrogel can form a semi-solid gel entity in situ at the drug administration site, prolonging the residence time of active substances in the tumor area, reducing systemic circulation distribution, helping to increase the local concentration of action and reduce the potential risks of systemic exposure.

[0010] Preferably, the particle size of the TIMP2-enriched exosomes is 80 nm to 150 nm, and the TIMP2 protein expression level in the exosomes is 2 to 3 times that of ordinary bone marrow mesenchymal stem cell exosomes.

[0011] By adopting the above technical solutions, the exosome particle size can be controlled within an appropriate range, ensuring that it can successfully penetrate the interstitial space to reach the tumor microenvironment, while avoiding rapid clearance due to excessively small particle size or interception by the reticuloendothelial system due to excessively large particle size. The enriched TIMP2 protein can improve the dense fibrotic state of the tumor matrix by regulating the activity of matrix metalloproteinases, thus providing a favorable microenvironment for the infiltration of effector immune cells.

[0012] Preferably, the degree of thiol substitution of the thiolized chitosan is 5% to 15%, and the molecular weight is 100kDa to 150kDa; the degree of oxidation of the sodium periodate aldehyde-modified oxidized hyaluronic acid is 10% to 30%, and the molecular weight is 80kDa to 120kDa.

[0013] By adopting the above technical solutions, both natural polysaccharide matrices have good biocompatibility and degradability. The thiol and aldehyde groups can undergo covalent cross-linking under mild physiological conditions without the need for additional initiators, which is suitable for the encapsulation requirements of bioactive substances. By controlling the degree of substitution and molecular weight of polysaccharides, the cross-linking density, mechanical strength and in vivo degradation rate of the gel can be flexibly adjusted to match the retention time requirements of different drug delivery scenarios.

[0014] Preferably, the molecular weight of the brown algae polysaccharide sulfate ester is 5kDa to 50kDa, and the degree of sulfate substitution is 10% to 30%; the reactive oxygen species-sensitive crosslinking regulator is 3,3'-dithiodipropionylhydrazine; and the CSF-1R small molecule inhibitor is BLZ945.

[0015] By employing the above technical solutions, sulfated fucoidan can exert an immunomodulatory effect by acting on pattern recognition receptors on the surface of macrophages, downregulating the activation level of propolarization-related signaling pathways; the acylhydrazide groups at both ends of the 3,3'-dithiodipropionylhydrazide molecule can covalently bind to aldehyde groups, and the disulfide bond structure in the middle can be broken in response to the highly reactive oxygen species in the tumor microenvironment, serving as a functional unit for constructing a responsive cross-linking network; BLZ945 can specifically act on CSF-1 receptor tyrosine kinase, blocking downstream survival signal transduction and reducing the number and functional activity of pro-inflammatory macrophages in tumor tissue.

[0016] Secondly, this application provides a method for preparing exosome hydrogels that regulate the immune microenvironment of cholangiocarcinoma, using the following technical solution:

[0017] A method for preparing an exosome hydrogel that regulates the immune microenvironment of cholangiocarcinoma includes the following steps:

[0018] S1. Exosome preparation: Bone marrow mesenchymal stem cells were induced to enrich TIMP2 protein in exosomes. Serum-free conditioned medium was collected after induction. Cells and cell debris were removed by differential centrifugation. The supernatant was then separated by ultracentrifugation to obtain exosome precipitate. The exosome precipitate was resuspended in buffer to obtain TIMP2-enriched exosome suspension derived from bone marrow mesenchymal stem cells.

[0019] S2. Preparation of protective solution: Add trehalose, mannitol and glycine to the TIMP2 enriched exosome suspension obtained in step S1, and stir until completely dissolved to obtain exosome protective solution.

[0020] S3, Preparation of Solution A: Under low temperature conditions, thiolized chitosan is added to the exosome protection solution and stirred until completely dissolved. Then, mannan and reactive oxygen species-sensitive crosslinking regulator are added in sequence and stirred until evenly dispersed. After adjusting the pH value, the solution is sterilized and filtered to obtain Solution A.

[0021] Preparation of S4 and B solutions: Oxidized hyaluronic acid and sulfated fucoidan were dissolved in phosphate buffer and stirred until completely dissolved to obtain a polysaccharide mixed solution; CSF-1R small molecule inhibitor was pre-dissolved in a trace amount of dimethyl sulfoxide and added dropwise to the obtained polysaccharide mixed solution, stirred and mixed evenly, and after adjusting the pH value, sterilized and filtered to obtain solution B.

[0022] S5. In-situ gelation: Liquid A and liquid B are placed in separate containers and mixed synchronously. After the two liquids come into contact, a dynamic covalent cross-linked network is formed, and gelation is completed under physiological temperature conditions.

[0023] By adopting the above technical solution, a process route of independent preparation of two components and cross-linking during use is employed. Exosomes and the thiol matrix component are stored in solution A, while the aldehyde matrix and small molecule inhibitor are stored in solution B. In the separate state, each component is in a stable environment, preventing premature cross-linking reactions and avoiding interference between different active substances, effectively extending the shelf life of the formulation. During use, only simultaneous output of the two solutions is required for rapid in-situ gelation, adapting to the operational needs of clinical local drug delivery, improving the convenience of drug delivery and the uniformity of gelation. This fully meets the practical needs of various clinical treatment scenarios and ensures the effectiveness of clinical applications.

[0024] Preferably, in step S1, when the bone marrow mesenchymal stem cells are cultured to a confluence of 70% to 80%, the culture medium is replaced with a serum-free medium containing 4.5 μmol / L to 5.5 μmol / L GF-β receptor inhibitor, and the culture is continued for 46 to 50 hours before the conditioned medium is collected. The differential centrifugation parameters are 300×g for 9 to 11 minutes, 2000×g for 9 to 11 minutes, and 10000×g for 28 to 32 minutes, respectively. The ultracentrifugation parameters are 9800×g to 10200×g for 68 to 72 minutes.

[0025] By employing the above-mentioned technical solution, the relevant signaling pathways in stem cells are blocked using TGF-β receptor inhibitors, and the transcription and protein expression levels of the TIMP2 gene are upregulated. This allows the exosomes secreted by the cells to naturally accumulate target proteins during their formation process, without the need for gene editing or viral transfection. The preparation process is simple and has higher biosafety. Multi-stage differential centrifugation is used to gradually remove cell debris, large vesicles, and protein aggregates, and then ultracentrifugation is used to enrich the exosome particles, which can ensure the purity and homogeneity of the product and maintain the stability of the exosome membrane structure and the activity of its contents.

[0026] Preferably, in step S3, the low temperature condition is 3°C to 5°C, the stirring rate is 50 rpm to 80 rpm, and the pH value is adjusted to 6.0 to 6.5; the exosomes are pre-dispersed in the protective system and then mixed with thiolated chitosan.

[0027] By adopting the above technical solution, the ternary protection system composed of trehalose, mannitol and glycine can replace the bound water on the surface of exosome membranes through hydroxyl hydrogen bonding, forming a stable protective layer on the membrane surface. At the same time, it stabilizes the spatial conformation of membrane proteins and avoids protein denaturation and inactivation. Combined with the preparation conditions of low temperature and low speed stirring, the damage to the exosome structure caused by molecular thermal motion and shear force is reduced. The process sequence of protection before mixing can avoid direct contact between exosomes and highly reactive aldehydes, preventing membrane proteins from being covalently modified and losing their biological activity.

[0028] Preferably, in step S4, the dropping rate of the CSF-1R small molecule inhibitor is 0.5 mL / min to 1 mL / min, the stirring rate during the dropping process is 30 rpm to 50 rpm, and stirring continues for 10 min to 15 min after the dropping is completed, and the pH value is adjusted to 7.2 to 7.4.

[0029] By adopting the above technical solution, the hydrophobic small molecule inhibitor can be uniformly dispersed in the aqueous polysaccharide system through slow drop-addition and low-speed stirring, avoiding excessive local concentration that could lead to aggregation or precipitation, and maintaining the homogeneity and storage stability of the system.

[0030] Preferably, in step S5, after the two liquids come into contact, the thiol group and the aldehyde group undergo a Schiff base reaction, and disulfide bonds are formed between the thiol groups through oxidation. The reactive oxygen-sensitive crosslinking regulator participates in the aldehyde group crosslinking reaction, and together they form a dynamic covalent crosslinking network containing Schiff base bonds and disulfide bonds. The gelation time is 60s to 120s, and the gelation temperature is 36℃ to 38℃.

[0031] By adopting the above technical solution, Schiff base bonds and disulfide bonds together constitute the dynamic covalent cross-linked network of the gel. Among them, Schiff base bonds have pH-responsive characteristics and can undergo reversible dissociation under the weakly acidic conditions of the tumor microenvironment. Disulfide bonds can be reduced and broken by the high concentration of reactive oxygen species in the tumor region. The introduction of reactive oxygen species-sensitive cross-linking agents further increases the number of disulfide bonds in the network and enhances the gel's responsiveness to the microenvironment. The gel degrades as the cross-linking bonds break down, simultaneously releasing various encapsulated active ingredients, achieving a sequential release behavior that matches the characteristics of the tumor microenvironment.

[0032] Preferably, the process also includes a freeze-drying preparation step, wherein liquid A and liquid B are freeze-dried separately to obtain a freeze-dried powder preparation, which is then reconstituted with water for injection to the original volume before use, and then mixed into a gel at a volume ratio of 0.9:1 to 1.1:1.

[0033] By adopting the above technical solution, freeze drying can remove free water in the system, reduce the molecular diffusion rate, inhibit the chemical degradation of active substances and the proliferation of microorganisms, significantly improve the storage stability of the preparation, extend the shelf life, and facilitate room temperature storage and long-distance transportation. In clinical use, it can be reconstituted with water for injection to restore the original solution state, and after mixing in proportion, it can be gelled in situ. The operation is flexible and simple, and it is suitable for different clinical application scenarios.

[0034] In summary, this application has the following beneficial effects:

[0035] 1. Because this application uses an in-situ hydrogel system that co-delivers TIMP2-enriched exosomes and CSF-1R small molecule inhibitors, combined with the immunomodulatory effect of brown algae polysaccharide sulfate, it regulates macrophage polarization and intervenes in the immune microenvironment of cholangiocarcinoma through multi-target synergistic effects. Due to the local retention and sustained-release characteristics of the hydrogel, it achieves the effect of increasing the concentration of active ingredients in the tumor area and reducing the potential risk of systemic exposure.

[0036] 2. In this application, 3,3'-dithiodipropionylhydrazine is preferably used as a reactive oxygen species sensitive crosslinking regulator. Combined with the double crosslinking network containing Schiff base bonds and disulfide bonds formed by thiolated chitosan and oxidized hyaluronic acid, the sequential release of active ingredients and the improved delivery accuracy are achieved due to the responsiveness of crosslinking bonds to reactive oxygen species in the tumor microenvironment.

[0037] 3. The method of this application, through the process step of pre-preparing exosome protective solution, uses a ternary protective system composed of trehalose, mannitol and glycine to stabilize the exosome phospholipid membrane structure, and then prepares solution A and solution B separately in steps, thus achieving the effect of reducing the loss of exosome activity and preserving its biological function during the preparation process.

[0038] 4. The method of this application enriches TIMP2 protein in exosomes by inducing bone marrow mesenchymal stem cells with TGF-β receptor inhibitors, and combines it with a separation and purification process of multi-stage differential centrifugation combined with ultracentrifugation. Therefore, it can achieve the effect of preparing high-abundance target protein exosomes without gene editing.

[0039] 5. In this application, a two-component freeze-dried formulation is preferred, in which solution A and solution B are prepared separately as freeze-dried powders. Before use, they can be reconstituted with water for injection and mixed into a gel. Due to the stability advantage of freeze-dried formulations, the storage period is extended and it is suitable for various clinical use scenarios. Attached Figure Description

[0040] Figure 1 This is a flowchart of a method for preparing exosome hydrogels that regulate the immune microenvironment of cholangiocarcinoma, as provided in this application;

[0041] Figure 2 This is a comparison chart of the cumulative release rate test results of hydrogel reactive oxygen species in response to release over 48 hours between the embodiments and comparative examples provided in this application;

[0042] Figure 3 This is a comparison chart of the exosome bioactivity retention rate measurement results of the various embodiments and comparative examples provided in this application;

[0043] Figure 4 This is a comparison chart of the evaluation results of the in vitro macrophage polarization regulation effects of the various embodiments and comparative examples provided in this application. Detailed Implementation

[0044] The present application will be further described in detail below with reference to embodiments and comparative examples. Unless otherwise specified, the experimental methods used below are conventional methods. Unless otherwise specified, the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels or prepared according to literature methods.

[0045] Technical Concept: Current interventions targeting the immune microenvironment of cholangiocarcinoma often employ systemic administration of active substances, which has limitations such as low accumulation efficiency at the tumor site and high risk of systemic exposure. Existing locally delivered hydrogel formulations mostly employ a single active component design, intervening in the immune microenvironment through only a single pathway, resulting in limited synergistic regulatory effects. Furthermore, conventional gels often utilize non-responsive cross-linking systems, whose release behavior cannot match the characteristics of the tumor microenvironment, making it difficult to control systemic exposure while increasing local drug concentration. In addition, exosome-based bioactive substances are sensitive to the preparation environment; conventional mixing processes easily cause membrane structure damage and protein denaturation, leading to a significant loss of bioactivity and affecting actual efficacy.

[0046] This application employs a two-component in-situ cross-linked hydrogel architecture, combining TIMP2-enriched exosomes, fucoidan sulfate, and a CSF-1R small molecule inhibitor for loading. Through multi-target synergistic effects, it achieves multi-pathway regulation of the immune microenvironment. A reactive oxygen species-sensitive cross-linking regulator is introduced to construct a dynamic covalent cross-linked network containing Schiff base bonds and disulfide bonds, enabling the gel to respond to tumor microenvironment characteristics and achieve sequential release of active ingredients. Simultaneously, a process route of pre-preparing exosome protective solution is adopted, combined with the method of independent preparation of the two components and mixing them into a gel during use, to reduce the loss of exosome activity during preparation and to balance local delivery efficiency and formulation storage stability.

[0047] Example 1: This example provides an exosome hydrogel that regulates the immune microenvironment of cholangiocarcinoma, which is formed by in-situ crosslinking of solution A and solution B in a 1:1 volume ratio.

[0048] By mass fraction, solution A contains 2.0 parts of thiolated chitosan, 0.275 parts of TIMP2-enriched exosomes, 3.5 parts of trehalose, 2 parts of mannitol, 0.3 parts of glycine, 0.3 parts of mannan, and 0.125 parts of reactive oxygen species-sensitive crosslinking regulator; solution B contains 1.5 parts of sodium periodate aldehyde-modified oxidized hyaluronic acid, 0.3 parts of brown algae polysaccharide sulfate, and 0.09 parts of CSF-1R small molecule inhibitor.

[0049] Among them, the particle size of TIMP2-enriched exosomes was 115 nm, and the expression level of TIMP2 protein in the exosomes was 2.5 times that of ordinary bone marrow mesenchymal stem cell exosomes; the degree of thiol substitution of thiol-modified chitosan was 10%, and the molecular weight was 125 kDa; the degree of oxidation of sodium periodate aldehyde-modified oxidized hyaluronic acid was 20%, and the molecular weight was 100 kDa; the molecular weight of brown algae polysaccharide sulfate ester was 27.5 kDa, and the degree of sulfate substitution was 20%; the reactive oxygen species sensitive crosslinking regulator was 3,3'-dithiodipropionylhydrazine; and the CSF-1R small molecule inhibitor was BLZ945.

[0050] The above-mentioned method for preparing exosome hydrogels that regulate the immune microenvironment of cholangiocarcinoma includes the following steps:

[0051] S1. Exosome preparation: Bone marrow mesenchymal stem cells were induced to enrich TIMP2 protein in exosomes. Serum-free conditioned medium was collected after induction. Cells and cell debris were removed by differential centrifugation. The supernatant was then separated by ultracentrifugation to obtain exosome precipitate. The exosome precipitate was resuspended in buffer to obtain a suspension of TIMP2-enriched exosomes derived from bone marrow mesenchymal stem cells.

[0052] When bone marrow mesenchymal stem cells reached 75% confluence, the culture medium was replaced with serum-free medium containing 5 μmol / L LTGF-β receptor inhibitor, and cultured for another 48 hours before collecting the conditioned medium. The differential centrifugation parameters were 300×g for 10 min, 2000×g for 10 min, and 10000×g for 30 min, respectively, and the ultracentrifugation parameters were 10000×g for 70 min. The resuspension buffer used was 20 mmol / L LMES buffer, and all centrifugation operations were performed at low temperature.

[0053] S2. Preparation of protective solution: Add trehalose, mannitol and glycine to the TIMP2 enriched exosome suspension obtained in step S1, and stir until completely dissolved to obtain exosome protective solution.

[0054] The stirring rate was 60 rpm and the stirring time was 12 min. This protective system maintains the stability of the exosome phospholipid membrane structure by regulating osmotic pressure, thereby reducing the loss of activity during subsequent cross-linking processes.

[0055] S3, Preparation of Solution A: Under low temperature conditions, thiolated chitosan is added to the exosome protection solution and stirred until completely dissolved. Then, mannan and reactive oxygen species-sensitive crosslinking regulator are added in sequence and stirred until evenly dispersed. After adjusting the pH value, the solution is sterilized and filtered to obtain Solution A.

[0056] The low-temperature conditions were 4℃, the stirring rate was 65 rpm, the dissolution stirring time for thiolized chitosan was 35 min, and stirring continued for 15 min after the subsequent addition of components; the pH value was adjusted to 6.2; a 0.22 μm sterile filter membrane was used for sterilization filtration; exosomes were pre-dispersed in the protective system before being mixed with thiolized chitosan, and direct contact between exosomes and aldehydes was avoided throughout the process.

[0057] Preparation of S4 and B solutions: Sodium periodate aldehyde-modified oxidized hyaluronic acid and brown algae polysaccharide sulfate were dissolved in phosphate buffer and stirred until completely dissolved to obtain a polysaccharide mixed solution; CSF-1R small molecule inhibitor was pre-dissolved in a trace amount of dimethyl sulfoxide and added dropwise to the obtained polysaccharide mixed solution, stirred and mixed evenly, and after adjusting the pH value, sterilized and filtered to obtain solution B.

[0058] The CSF-1R small molecule inhibitor was added at a rate of 0.75 mL / min with a stirring rate of 40 rpm. After the addition was completed, stirring was continued for 12.5 min. The pH was adjusted to 7.3. A 0.22 μm sterile filter membrane was used for sterile filtration. The phosphate buffer concentration was 15 mmol / L. The final volume percentage of dimethyl sulfoxide did not exceed 0.1%, and it was non-cytotoxic.

[0059] S5. In-situ gelation: Liquid A and liquid B are placed in separate containers and mixed synchronously. After the two liquids come into contact, a dynamic covalent cross-linked network is formed, and gelation is completed under physiological temperature conditions.

[0060] In this process, after the two liquids come into contact, the thiol groups and aldehyde groups undergo a Schiff base reaction, and disulfide bonds are formed between the thiol groups through oxidation. The reactive oxygen species-sensitive crosslinking regulator participates in the aldehyde group crosslinking reaction, and together they form a dynamic covalent crosslinking network containing Schiff base bonds and disulfide bonds. The gelation time is 90s and the gelation temperature is 37℃. This dynamic crosslinking network can respond to the reactive oxygen species level in the tumor microenvironment and realize the sequential release of active molecules.

[0061] S6. Preparation of lyophilized formulation: Liquid A and liquid B are lyophilized separately to obtain lyophilized powder formulations. Before use, they are reconstituted with water for injection to the original volume and then mixed into a gel at a volume ratio of 1:1.

[0062] The freeze-drying process involved a pre-freezing temperature of -45℃, a pre-freezing time of 5 hours, a sublimation drying time of 28 hours, and a desorption drying time of 8 hours. The component concentration and cross-linking properties of the solution after reconstitution were not significantly different from those before freeze-drying.

[0063] Example 2: This example provides an exosome hydrogel that regulates the immune microenvironment of cholangiocarcinoma, which is formed by in-situ crosslinking of solution A and solution B in a volume ratio of 0.9:1.

[0064] By mass fraction, solution A contains 1.5 parts thiolated chitosan, 0.05 parts TIMP2 enriched exosomes, 2 parts trehalose, 1 part mannitol, 0.1 parts glycine, 0.1 parts mannan, and 0.05 parts reactive oxygen species sensitive crosslinking regulator; solution B contains 1.0 part sodium periodate aldehyde-modified oxidized hyaluronic acid, 0.1 part brown algae polysaccharide sulfate, and 0.08 parts CSF-1R small molecule inhibitor.

[0065] Among them, the particle size of TIMP2-enriched exosomes was 80 nm, and the expression level of TIMP2 protein in the exosomes was twice that of ordinary bone marrow mesenchymal stem cell exosomes; the degree of thiol substitution of thiol-modified chitosan was 5%, and the molecular weight was 100 kDa; the degree of oxidation of sodium periodate aldehyde-modified oxidized hyaluronic acid was 10%, and the molecular weight was 80 kDa; the molecular weight of brown algae polysaccharide sulfate ester was 5 kDa, and the degree of sulfate substitution was 10%; the reactive oxygen species sensitive crosslinking regulator was 3,3'-dithiodipropionylhydrazine; and the CSF-1R small molecule inhibitor was BLZ945.

[0066] The above-mentioned method for preparing exosome hydrogels that regulate the immune microenvironment of cholangiocarcinoma includes the following steps:

[0067] S1. Exosome preparation: Bone marrow mesenchymal stem cells were induced to enrich TIMP2 protein in exosomes. Serum-free conditioned medium was collected after induction. Cells and cell debris were removed by differential centrifugation. The supernatant was then separated by ultracentrifugation to obtain exosome precipitate. The exosome precipitate was resuspended in buffer to obtain a suspension of TIMP2-enriched exosomes derived from bone marrow mesenchymal stem cells.

[0068] When bone marrow mesenchymal stem cells reached 70% confluence, the culture medium was replaced with serum-free medium containing 4.5 μmol / L LTGF-β receptor inhibitor, and cultured for another 46 h before collecting the conditioned medium. The differential centrifugation parameters were 300×g for 9 min, 2000×g for 9 min, and 10000×g for 28 min, respectively, and the ultracentrifugation parameters were 9800×g for 68 min. The resuspension buffer used was 10 mmol / L LMES buffer, and all centrifugation operations were performed at low temperature.

[0069] S2. Preparation of protective solution: Add trehalose, mannitol and glycine to the TIMP2 enriched exosome suspension obtained in step S1, and stir until completely dissolved to obtain exosome protective solution.

[0070] The stirring rate was 50 rpm and the stirring time was 10 min. This protective system maintains the stability of the exosome phospholipid membrane structure by regulating osmotic pressure, thereby reducing the loss of activity during subsequent cross-linking processes.

[0071] S3, Preparation of Solution A: Under low temperature conditions, thiolated chitosan is added to the exosome protection solution and stirred until completely dissolved. Then, mannan and reactive oxygen species-sensitive crosslinking regulator are added in sequence and stirred until evenly dispersed. After adjusting the pH value, the solution is sterilized and filtered to obtain Solution A.

[0072] The low-temperature conditions were 3℃, the stirring rate was 50 rpm, the dissolution and stirring time of thiolated chitosan was 30 min, and stirring was continued for 10 min after subsequent addition of components; the pH value was adjusted to 6.0; sterile filtration was carried out using a 0.22 μm sterile filter membrane; exosomes were pre-dispersed in the protective system before being mixed with thiolated chitosan, and direct contact between exosomes and aldehydes was avoided throughout the process.

[0073] Preparation of S4 and B solutions: Sodium periodate aldehyde-modified oxidized hyaluronic acid and brown algae polysaccharide sulfate were dissolved in phosphate buffer and stirred until completely dissolved to obtain a polysaccharide mixed solution; CSF-1R small molecule inhibitor was pre-dissolved in a trace amount of dimethyl sulfoxide and added dropwise to the obtained polysaccharide mixed solution, stirred and mixed evenly, and after adjusting the pH value, sterilized and filtered to obtain solution B.

[0074] The CSF-1R small molecule inhibitor was added at a rate of 0.5 mL / min with a stirring rate of 30 rpm. After the addition was completed, stirring was continued for 10 min. The pH was adjusted to 7.2. A 0.22 μm sterile filter membrane was used for sterile filtration. The phosphate buffer concentration was 10 mmol / L. The final volume percentage of dimethyl sulfoxide did not exceed 0.1%, and it was non-cytotoxic.

[0075] S5. In-situ gelation: Liquid A and liquid B are placed in separate containers and mixed synchronously. After the two liquids come into contact, a dynamic covalent cross-linked network is formed, and gelation is completed under physiological temperature conditions.

[0076] In this process, after the two liquids come into contact, the thiol groups and aldehyde groups undergo a Schiff base reaction, and disulfide bonds are formed between the thiol groups through oxidation. The reactive oxygen species-sensitive crosslinking regulator participates in the aldehyde group crosslinking reaction, and together they form a dynamic covalent crosslinking network containing Schiff base bonds and disulfide bonds. The gelation time is 60s and the gelation temperature is 36℃. This dynamic crosslinking network can respond to the reactive oxygen species level in the tumor microenvironment and realize the sequential release of active molecules.

[0077] S6. Preparation of lyophilized formulation: Liquid A and liquid B are lyophilized separately to obtain lyophilized powder formulations. Before use, they are reconstituted with water for injection to the original volume and then mixed into a gel at a volume ratio of 0.9:1.

[0078] The freeze-drying process involved a pre-freezing temperature of -40℃, a pre-freezing time of 4 hours, a sublimation drying time of 24 hours, and a desorption drying time of 6 hours. The component concentration and cross-linking properties of the solution after reconstitution were not significantly different from those before freeze-drying.

[0079] Example 3: This example provides an exosome hydrogel that regulates the immune microenvironment of cholangiocarcinoma, which is formed by in-situ crosslinking of solution A and solution B in a volume ratio of 1.1:1.

[0080] By mass fraction, solution A contains 2.5 parts thiolated chitosan, 0.5 parts TIMP2 enriched exosomes, 5 parts trehalose, 3 parts mannitol, 0.5 parts glycine, 0.5 parts mannan, and 0.2 parts reactive oxygen species sensitive crosslinking regulator; solution B contains 2.0 parts sodium periodate aldehyde-modified oxidized hyaluronic acid, 0.5 parts brown algae polysaccharide sulfate, and 0.1 parts CSF-1R small molecule inhibitor.

[0081] Among them, the particle size of TIMP2-enriched exosomes was 150 nm, and the expression level of TIMP2 protein in the exosomes was 3 times that of ordinary bone marrow mesenchymal stem cell exosomes; the degree of thiol substitution of thiol-modified chitosan was 15%, and the molecular weight was 150 kDa; the degree of oxidation of sodium periodate aldehyde-modified oxidized hyaluronic acid was 30%, and the molecular weight was 120 kDa; the molecular weight of brown algae polysaccharide sulfate ester was 50 kDa, and the degree of sulfate substitution was 30%; the reactive oxygen species sensitive crosslinking regulator was 3,3'-dithiodipropionylhydrazine; and the CSF-1R small molecule inhibitor was BLZ945.

[0082] The above-mentioned method for preparing exosome hydrogels that regulate the immune microenvironment of cholangiocarcinoma includes the following steps:

[0083] S1. Exosome preparation: Bone marrow mesenchymal stem cells were induced to enrich TIMP2 protein in exosomes. Serum-free conditioned medium was collected after induction. Cells and cell debris were removed by differential centrifugation. The supernatant was then separated by ultracentrifugation to obtain exosome precipitate. The exosome precipitate was resuspended in buffer to obtain a suspension of TIMP2-enriched exosomes derived from bone marrow mesenchymal stem cells.

[0084] When bone marrow mesenchymal stem cells reached 80% confluence, the culture medium was replaced with serum-free medium containing 5.5 μmol / L LTGF-β receptor inhibitor, and cultured for another 50 h before collecting the conditioned medium. The differential centrifugation parameters were 300×g for 11 min, 2000×g for 11 min, and 10000×g for 32 min, respectively, and the ultracentrifugation parameters were 10200×g for 72 min. The resuspension buffer used was 25 mmol / L LMES buffer, and all centrifugation operations were performed at low temperature.

[0085] S2. Preparation of protective solution: Add trehalose, mannitol and glycine to the TIMP2 enriched exosome suspension obtained in step S1, and stir until completely dissolved to obtain exosome protective solution.

[0086] The stirring rate was 70 rpm and the stirring time was 15 min. This protective system maintains the stability of the exosome phospholipid membrane structure by regulating osmotic pressure, thereby reducing the loss of activity during subsequent cross-linking processes.

[0087] S3, Preparation of Solution A: Under low temperature conditions, thiolated chitosan is added to the exosome protection solution and stirred until completely dissolved. Then, mannan and reactive oxygen species-sensitive crosslinking regulator are added in sequence and stirred until evenly dispersed. After adjusting the pH value, the solution is sterilized and filtered to obtain Solution A.

[0088] The low-temperature conditions were 5℃, the stirring rate was 80 rpm, the dissolution and stirring time of thiolated chitosan was 40 min, and stirring was continued for 20 min after subsequent addition of components; the pH value was adjusted to 6.5; sterile filtration was carried out using a 0.22 μm sterile filter membrane; exosomes were pre-dispersed in the protective system before being mixed with thiolated chitosan, and direct contact between exosomes and aldehydes was avoided throughout the process.

[0089] Preparation of S4 and B solutions: Sodium periodate aldehyde-modified oxidized hyaluronic acid and brown algae polysaccharide sulfate were dissolved in phosphate buffer and stirred until completely dissolved to obtain a polysaccharide mixed solution; CSF-1R small molecule inhibitor was pre-dissolved in a trace amount of dimethyl sulfoxide and added dropwise to the obtained polysaccharide mixed solution, stirred and mixed evenly, and after adjusting the pH value, sterilized and filtered to obtain solution B.

[0090] The CSF-1R small molecule inhibitor was added at a rate of 1 mL / min, with a stirring rate of 50 rpm during the addition process, followed by stirring for 15 min after the addition was completed. The pH was adjusted to 7.4. A 0.22 μm sterile filter membrane was used for sterile filtration. The phosphate buffer concentration was 20 mmol / L. The final volume percentage of dimethyl sulfoxide did not exceed 0.1%, and it was non-cytotoxic.

[0091] S5. In-situ gelation: Liquid A and liquid B are placed in separate containers and mixed synchronously. After the two liquids come into contact, a dynamic covalent cross-linked network is formed, and gelation is completed under physiological temperature conditions.

[0092] In this process, after the two liquids come into contact, the thiol groups and aldehyde groups undergo a Schiff base reaction, and disulfide bonds are formed between the thiol groups through oxidation. The reactive oxygen species-sensitive crosslinking regulator participates in the aldehyde group crosslinking reaction, and together they form a dynamic covalent crosslinking network containing Schiff base bonds and disulfide bonds. The gelation time is 120s and the gelation temperature is 38℃. This dynamic crosslinking network can respond to the reactive oxygen species level in the tumor microenvironment and realize the sequential release of active molecules.

[0093] S6. Preparation of lyophilized formulation: Liquid A and liquid B are lyophilized separately to obtain lyophilized powder formulations. Before use, they are reconstituted with water for injection to the original volume and then mixed into a gel at a volume ratio of 1.1:1.

[0094] The freeze-drying process involved a pre-freezing temperature of -50℃, a pre-freezing time of 6 hours, a sublimation drying time of 36 hours, and a desorption drying time of 10 hours. The component concentration and cross-linking properties of the reconstituted solution were not significantly different from those before freeze-drying.

[0095] Comparative Example 1: The only difference between this comparative example and Example 1 is that the TIMP2-enriched exosomes in solution A are replaced with an equal mass fraction of ordinary bone marrow mesenchymal stem cell exosomes. All other raw material components, amounts, and preparation steps are consistent with Example 1.

[0096] Comparative Example 2: The only difference between this comparative example and Example 1 is that the CSF-1R small molecule inhibitor BLZ945 is not added to solution B. The other raw material components, dosages and all preparation steps are the same as in Example 1.

[0097] Comparative Example 3: The only difference between this comparative example and Example 1 is that the reactive oxygen species-sensitive crosslinking regulator 3,3'-dithiodipropionylhydrazine is not added to solution A. The other raw material components, dosages and all preparation steps are consistent with those of Example 1.

[0098] Comparative Example 4: The only difference between this comparative example and Example 1 is that the S2 protective solution preparation step is omitted, and the TIMP2 enriched exosome suspension obtained in step S1 is used directly for subsequent preparation of solution A without the addition of trehalose, mannitol and glycine. The remaining raw material components, dosages and all preparation steps are consistent with Example 1.

[0099] Comparative Example 5: This comparative example differs from Example 1 only in that the brown algae polysaccharide sulfate in solution B is replaced with an equal mass fraction of unsulfated common fucoidan, wherein the molecular weight is the same as in Example 1, and the remaining raw material components, amounts and all preparation steps are the same as in Example 1.

[0100] Comparative Example 6: This comparative example uses a conventional sodium alginate-calcium chloride ion crosslinking hydrogel system. The gel matrix is ​​replaced with 1.5 parts sodium alginate and 0.1 parts calcium chloride. The active component is only an equal mass of ordinary bone marrow mesenchymal stem cell exosomes. No CSF-1R small molecule inhibitor, reactive oxygen species sensitive crosslinking regulator, or exosome ternary protection system is added. The preparation process adopts the direct mixing crosslinking method, in which exosomes are mixed with sodium alginate solution and then calcium chloride solution is added to solidify into a gel. All other operating conditions are consistent with those in Example 1.

[0101] I. Hydrogel reactive oxygen species (ROS) responsive release performance test, test standard: Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0931, Dissolution and Release Determination Method; Three aliquots of hydrogel samples prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were taken, with each sample containing the same total mass of BLZ945 and TIMP2 after conversion. The release test was conducted using the dialysis bag method, with the dialysis bag having a molecular weight cutoff of 8000 Da to 14000 Da. The release media were set as phosphate buffer containing 10 μmol / L ROS and phosphate buffer containing 1 μmol / L ROS, respectively, corresponding to ROS in the tumor microenvironment and normal physiological environment. The release medium volume was 200 mL, the release temperature was set to 37℃, and the stirring rate was set to 50 rpm. 2 mL of the release solution was collected at time points of 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, and fresh release medium of the same temperature and volume was added simultaneously. The BLZ945 content in the samples at each time point was detected by high-performance liquid chromatography (HPLC), and the TIMP2 protein content in the samples at each time point was detected by enzyme-linked immunosorbent assay (ELISA). The cumulative release rate of the two active ingredients at different time points was calculated, and cumulative release curves were plotted to compare the differences in release rate and release behavior among the samples under different reactive oxygen species concentrations.

[0102] II. Determination of exosome bioactivity retention rate, test standard: GB / T42348-2023 "Particle size analysis - Particle tracking analysis (PTA)"; Take three aliquots of hydrogel samples prepared in Examples 1 to 3 and Comparative Examples 1 to 6. Exosomes were recovered 24 hours after preparation. An equal volume of phosphate buffer was added to each group of hydrogels, and the samples were placed at 37℃ until complete degradation. Exosomes were then extracted from the degradation solution using differential centrifugation combined with ultracentrifugation. The differential centrifugation parameters were 300×g for 10 min, 2000×g for 10 min, and 10000×g for 10 min, respectively. Centrifuge at 10000×g for 70 min for 30 min. Use freshly prepared exosomes enriched with the same dose of TIMP2 as a positive control. Western blotting was used to detect the expression level and biological activity of TIMP2 protein in exosomes. Particle tracking analysis was used to detect the particle size distribution and particle concentration of exosomes. Annexin V staining combined with flow cytometry was used to detect the integrity of the exosome phospholipid membrane. The TIMP2 activity value of the positive control was taken as 100%. The activity retention rate of exosomes in each group of samples was calculated to compare the protective effects of different preparation processes and gel systems on the biological activity of exosomes.

[0103] III. Evaluation of the in vitro macrophage polarization regulation effect, test standard: GB / T16886.5-2017 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test"; take three portions each of the hydrogel extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 6, isolate and culture mouse bone marrow-derived macrophages according to the standard requirements, prepare cell suspensions and adjust the cell concentration to 1×10⁻⁶. 6 Cells were seeded at a density of 1 / mL in 24-well culture plates, with a cell suspension volume of 1 mL per well. Complete culture medium containing 100 ng / mL interleukin-4 was added to induce macrophages to M2 polarization. Hydrogel extracts of each group were added simultaneously. A blank control group and a simple M2 induction group were set up as controls. The cells were cultured at 37℃ in a constant temperature incubator containing 5% carbon dioxide for 48 h. After culture, the cell supernatant was collected, and the secretion of M2 characteristic cytokines such as interleukin-10 and transforming growth factor β was detected by enzyme-linked immunosorbent assay. Adherent cells were collected, and the positive expression rate of M2 macrophage surface markers such as CD163 and CD206 was detected by flow cytometry. The mRNA expression level of M2 polarization-related genes such as arginase 1 and mannose receptor in cells was detected by real-time quantitative polymerase chain reaction. The inhibitory effect of each group of samples on macrophage M2 polarization was comprehensively evaluated from three levels: cytokines, surface markers, and related genes.

[0104] Table 1: Results of the cumulative release rate of reactive oxygen species from hydrogels after 48 hours

[0105]

[0106] Note: Data are expressed as mean ± standard deviation, n=3; cumulative release rate is the percentage of the mass of active ingredient released at each time point relative to the total load of that active ingredient in the sample; high reactive oxygen species (ROS) conditions simulate the tumor microenvironment, and low ROS conditions simulate the normal physiological environment.

[0107] Table 2: Results of Exosome Bioactivity Retention Rate Measurement

[0108]

[0109] Note: Data are expressed as mean ± standard deviation, n=3; TIMP2 protein activity retention rate is calculated with TIMP2 activity of freshly prepared exosome samples of the same group as 100%; exosome phospholipid membrane integrity is detected by flow cytometry with annexin V staining, and the higher the positive rate, the more intact the membrane structure; the lower the particle size dispersion index, the better the exosome uniformity and the lower the degree of aggregation.

[0110] Table 3: Evaluation results of macrophage polarization regulation effect in vitro

[0111] Note: Data are expressed as mean ± standard deviation, n=3; CD163 is a characteristic surface marker of M2 macrophages, and the lower the positive rate, the stronger the M2 polarization inhibition effect; IL-10 is a characteristic secreted cytokine of M2 macrophages, and the lower the secretion level, the better the polarization inhibition effect; the relative expression level of Arg-1 mRNA was normalized with the expression level of the M2-induced group as 1.00, and the lower the value, the weaker the expression of M2 polarization-related genes.

[0112] As can be seen from Examples 1-3 and Comparative Example 1, and Table 3, the enrichment level of TIMP2 protein in exosomes directly affects the regulatory effect of hydrogel on macrophage M2 polarization. The TIMP2 protein content of exosomes from ordinary bone marrow mesenchymal stem cells is limited and cannot fully intervene in the polarization process of tumor-associated macrophages. This also shows that TIMP2 enrichment is one of the core foundations for hydrogel to play a role in regulating the immune microenvironment.

[0113] As can be seen from Examples 1-3 and Comparative Example 2, and Table 3, the CSF-1R small molecule inhibitor is an important component of the entire immune regulation system. Without this component, the hydrogel cannot inhibit the survival and function maintenance of M2 macrophages by blocking the CSF-1-related pathway, and the overall polarization inhibition effect will decline significantly, which also confirms the practical value of dual-target synergistic regulation.

[0114] As can be seen from Examples 1-3 and Comparative Example 3, and Tables 1 and 3, the reactive oxygen species-sensitive crosslinking regulator is the core component for constructing the responsive release system. Without this component, the crosslinking network of the gel cannot respond to the high reactive oxygen species characteristics of the tumor microenvironment and will break down. The active ingredients are difficult to be preferentially released at the tumor site, and the subsequent immunomodulatory effect will also be affected.

[0115] Based on Examples 1-3 and Comparative Example 4, and in conjunction with Tables 2 and 3, it can be seen that the pre-protection system composed of trehalose-mannitol and glycine can effectively maintain the phospholipid membrane structure and protein bioactivity of exosomes. After the protection step is removed, membrane damage and protein denaturation occur in exosomes during subsequent stirring and cross-linking processes, ultimately leading to a simultaneous decrease in the bioactivity and immunomodulatory function of the hydrogel.

[0116] Based on Examples 1-3 and Comparative Example 5, and in conjunction with Table 3, it can be seen that the sulfation modification of fucoidan is the key structural basis for its immunomodulatory effect. Unsulfated ordinary fucoidan is difficult to effectively interfere with the signaling pathways related to M2 polarization, and its inhibitory effect on macrophage polarization is significantly weakened, indicating that the sulfate group directly participates in the relevant immunomodulatory process.

[0117] Based on Examples 1-3 and Comparative Example 6, and in conjunction with Tables 1 to 3, it can be seen that conventional ion-crosslinked hydrogel systems lack both environmentally responsive release capabilities and effective protection of exosomes. Furthermore, the active component design is singular, resulting in significant deficiencies in multiple dimensions, including release behavior, exosome activity retention, and immunomodulatory effects. This demonstrates the comprehensive design advantages of the dynamic covalent crosslinking system proposed in this scheme.

[0118] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma, characterized in that: It is formed by in-situ crosslinking of liquid A and liquid B in a volume ratio of 0.9:1 to 1.1:1; The A solution comprises, by mass parts: 1.5 to 2.5 parts thiolated chitosan, 0.05 to 0.5 parts TIMP2-enriched exosomes, 2 to 5 parts trehalose, 1 to 3 parts mannitol, 0.1 to 0.5 parts glycine, 0.1 to 0.5 parts mannan, and 0.05 to 0.2 parts reactive oxygen species-sensitive crosslinking regulator; The B solution comprises, by mass, 1.0 to 2.0 parts of sodium periodate aldehyde-modified oxidized hyaluronic acid, 0.1 to 0.5 parts of brown algae polysaccharide sulfate, and 0.08 to 0.1 parts of CSF-1R small molecule inhibitor.

2. The exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma according to claim 1, characterized in that: The TIMP2-enriched exosomes have a particle size of 80 nm to 150 nm, and the TIMP2 protein expression level in the exosomes is 2 to 3 times that of ordinary bone marrow mesenchymal stem cell exosomes.

3. The exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma according to claim 1, characterized in that: The thiolized chitosan has a thiol substitution degree of 5% to 15% and a molecular weight of 100kDa to 150kDa; the sodium periodate aldehyde-modified oxidized hyaluronic acid has an oxidation degree of 10% to 30% and a molecular weight of 80kDa to 120kDa.

4. The exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma according to claim 1, characterized in that: The molecular weight of the brown algae polysaccharide sulfate ester is 5kDa to 50kDa, and the degree of sulfate substitution is 10% to 30%; the reactive oxygen species sensitive crosslinking regulator is 3,3'-dithiodipropionylhydrazine; and the CSF-1R small molecule inhibitor is BLZ945.

5. A method for preparing an exosome hydrogel that regulates the immune microenvironment of cholangiocarcinoma, characterized in that, An exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma as described in any one of claims 1-4 comprises the following steps: S1. Exosome preparation: Bone marrow mesenchymal stem cells were induced to enrich TIMP2 protein in exosomes. Serum-free conditioned medium was collected after induction. Cells and cell debris were removed by differential centrifugation. The supernatant was then separated by ultracentrifugation to obtain exosome precipitate. The exosome precipitate was resuspended in buffer to obtain TIMP2-enriched exosome suspension derived from bone marrow mesenchymal stem cells. S2. Preparation of protective solution: Add trehalose, mannitol and glycine to the TIMP2 enriched exosome suspension obtained in step S1, and stir until completely dissolved to obtain exosome protective solution. S3, Preparation of Solution A: Under low temperature conditions, thiolized chitosan is added to the exosome protection solution and stirred until completely dissolved. Then, mannan and reactive oxygen species-sensitive crosslinking regulator are added in sequence and stirred until evenly dispersed. After adjusting the pH value, the solution is sterilized and filtered to obtain Solution A. Preparation of S4 and B solutions: Oxidized hyaluronic acid and sulfated fucoidan were dissolved in phosphate buffer and stirred until completely dissolved to obtain a polysaccharide mixed solution; CSF-1R small molecule inhibitor was pre-dissolved in a trace amount of dimethyl sulfoxide and added dropwise to the obtained polysaccharide mixed solution, stirred and mixed evenly, and after adjusting the pH value, sterilized and filtered to obtain solution B. S5. In-situ gelation: Liquid A and liquid B are placed in separate containers and mixed synchronously. After the two liquids come into contact, a dynamic covalent cross-linked network is formed, and gelation is completed under physiological temperature conditions.

6. The method for preparing an exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma according to claim 5, characterized in that: In step S1, when the bone marrow mesenchymal stem cells are cultured to a confluence of 70% to 80%, the culture medium is replaced with serum-free medium containing 4.5 μmol / L to 5.5 μmol / L LTGF-β receptor inhibitor. The culture is continued for 46 to 50 hours before the conditioned medium is collected. The differential centrifugation parameters are 300×g for 9 to 11 minutes, 2000×g for 9 to 11 minutes, and 10000×g for 28 to 32 minutes. The ultracentrifugation parameters are 9800×g to 10200×g for 68 to 72 minutes.

7. The method for preparing an exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma according to claim 5, characterized in that: In step S3, the low temperature conditions are 3°C to 5°C, the stirring rate is 50 rpm to 80 rpm, and the pH value is adjusted to 6.0 to 6.5; the exosomes are pre-dispersed in the protective system and then mixed with thiolated chitosan.

8. The method for preparing an exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma according to claim 5, characterized in that: In step S4, the CSF-1R small molecule inhibitor is added at a rate of 0.5 mL / min to 1 mL / min, with a stirring rate of 30 rpm to 50 rpm during the addition process. After the addition is completed, stirring continues for 10 min to 15 min, and the pH value is adjusted to 7.2 to 7.

4.

9. The method for preparing an exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma according to claim 5, characterized in that: In step S5, after the two liquids come into contact, the thiol groups and aldehyde groups undergo a Schiff base reaction, and disulfide bonds are formed between the thiol groups through oxidation. The reactive oxygen-sensitive crosslinking regulator participates in the aldehyde group crosslinking reaction, and together they form a dynamic covalent crosslinking network containing Schiff base bonds and disulfide bonds. The gelation time is 60s to 120s, and the gelation temperature is 36℃ to 38℃.

10. The method for preparing an exosome hydrogel for regulating the immune microenvironment of cholangiocarcinoma according to claim 5, characterized in that: It also includes a freeze-drying preparation step, in which liquid A and liquid B are freeze-dried separately to obtain freeze-dried powder preparations. Before use, they are reconstituted with water for injection to the original volume and then mixed into a gel at a volume ratio of 0.9:1 to 1.1:1.

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