Kiwi fruit extracellular vesicle supported injectable hydrogel with active oxygen scavenging capacity as well as preparation method and application of kiwi fruit extracellular vesicle supported injectable hydrogel

Abstract

The invention discloses kiwi fruit extracellular vesicle supported injectable hydrogel with active oxygen scavenging capacity as well as a preparation method and application of the kiwi fruit extracellular vesicle supported injectable hydrogel, and belongs to the cross technical field of biomedical materials and clinical medicine. Comprising the following raw materials: water-soluble chitosan (CS), polyvinyl alcohol (PVA), an active oxygen responsive chain segment construction macromonomer (Linker), a cross-linking agent and a drug-containing kiwi fruit extracellular vesicle. The injectable hydrogel is prepared by preparing the raw materials into an aqueous solution and carrying out a cross-linking reaction, and aims to optimize the postoperative recovery process and prevent the occurrence of abdominal adhesion by integrating a multi-effect treatment strategy of wound healing, inflammatory reaction control and tissue regeneration promotion.

Description

Technical Field

[0001] The present invention belongs to the cross-technical field of biomedical materials and clinical medicine, and in particular relates to a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging capability, and a preparation method and application thereof. Background Art

[0002] Abdominal adhesions are one of the most common complications after abdominal surgery and can lead to serious diseases, including adhesive intestinal obstruction, female infertility, chronic abdominal pain, etc. These complications not only affect the patient's health and quality of life, but also increase the difficulty and risk of reoperation. It is reported that the incidence of abdominal adhesions after abdominal surgery is 67% to 93%, and the incidence after open gynecological surgery is as high as 97%. At present, in addition to improving surgical techniques, drug therapy and biomaterial barriers are the most common methods of prevention. Although local or systemic drug therapy, such as anti-inflammatory drugs, has been used to prevent postoperative abdominal adhesions, their preventive effect is also significantly limited due to their rapid metabolism in the body. In addition, the clinical effects of these barrier materials are not ideal due to problems such as insufficient tissue adhesion, rapid degradation, and short retention time in the body.

[0003] Studies have found that hydrogel barrier materials are widely used in the prevention of postoperative abdominal adhesions due to their excellent coverage, flexibility for irregular wounds, and ability to accelerate wound healing. The research focus of these hydrogels is to improve the physical barrier properties, including achieving ideal viscoelasticity, enhancing wet tissue adhesion, enhancing hydrophilicity, and improving biodegradability. However, improving the physical barrier properties of hydrogels alone is not enough to completely prevent adhesions, and it is also necessary to promote the normal healing of postoperative wounds. There is increasing evidence that excessive inflammation and oxidative stress play an important role in the formation of abdominal adhesions. When the abdominal cavity is traumatic or surgically injured, a variety of inflammatory cells, including macrophages and neutrophils, migrate to the injured area, leading to overexpression of inflammatory cytokines. At the same time, the injured area undergoes oxidative stress due to the excessive production of reactive oxygen species (such as H2O2, ·OH), which further leads to an imbalance in the fibrinolytic system and collagen deposition, ultimately leading to the formation of abdominal adhesions. Therefore, the development of hydrogels that can effectively scavenge reactive oxygen species and have anti-inflammatory effects is of great significance for the prevention of postoperative abdominal adhesions. Summary of the invention

[0004] In view of the above technical problems, the present invention proposes a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability, as well as a preparation method and application thereof. The kiwifruit extracellular vesicles containing drugs are loaded into the injectable hydrogel, aiming to optimize the postoperative recovery process and prevent the occurrence of abdominal adhesions by integrating a multi-effect treatment strategy of wound healing, inflammatory response control, and tissue regeneration promotion, so as to provide patients with a more effective treatment plan.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention:

[0007] A kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability, comprising the following raw materials:

[0008] Water-soluble chitosan (CS), polyvinyl alcohol (PVA), reactive oxygen species-responsive chain segments to construct macromonomers (Linker), cross-linkers, and kiwifruit extracellular vesicles containing drugs;

[0009] The structural formula of the macromolecular monomer constructed by the active oxygen responsive segment is:

[0010]

[0011] The synthesis steps of the active oxygen responsive segment to construct the macromolecular monomer are:

[0012] (1) Add 10 mL of thioglycolic acid and 5 mL of acetone, then fill with hydrogen chloride gas and react at room temperature for 4 h;

[0013] (2) extracting and evaporating the solution in step (1) to obtain product 1;

[0014] (3) adding the product 1 obtained in step (2) to 10 mL of a tetrahydrofuran solution (1 mol / L) containing lithium aluminum hydride, and reacting at room temperature overnight;

[0015] (4) extracting and evaporating the solution in step (3) to obtain product 2;

[0016] (5) The product 2 of step (4) was mixed with 1 g of hydrogenated PMDA, 10 mL of DMF was added, and the mixture was reacted at room temperature for 12 h to obtain a macromolecular monomer for constructing an active oxygen responsive chain segment. The synthetic route is as follows:

[0017]

[0018] Beneficial effects: The present invention uses water-soluble chitosan, polyvinyl alcohol, and active oxygen responsive chain segments to construct macromolecular monomers as the main raw materials, uses genipin as a biological cross-linking agent, and adds kiwifruit extracellular vesicles loaded with drugs to obtain an injectable hydrogel. Among them, the function of Linker in the hydrogel is mainly to remove active oxygen and reduce inflammation; chitosan (CS) has excellent biocompatibility and stability, and has the characteristics of sterilization, hemostasis, absorption of wound exudate, reduction of scar formation and biodegradability; polyvinyl alcohol (PVA) has high intermolecular adhesion, as well as good biocompatibility and degradability. In addition, polyvinyl alcohol also has the characteristics of moisturizing, softness and lubrication. These characteristics enable PVA to improve the brittleness of chitosan and increase viscosity when combined with chitosan; the natural plant source of PDEVs (extracellular vesicles in plant cells) gives them the characteristics of non-immunogenicity and high yield, which provides new possibilities for solving the challenges encountered by MDEVs (extracellular vesicles in animal cells) in drug delivery. Therefore, the present invention loads drug-containing kiwifruit extracellular vesicles into injectable hydrogels, which can optimize the postoperative recovery process and prevent the occurrence of abdominal adhesions by integrating a multi-effect treatment strategy of wound healing, inflammatory response control, and tissue regeneration promotion.

[0019] Optionally, the concentration of the drug-containing kiwifruit extracellular vesicles is 5 to 10 mg / mL, preferably 5 mg / mL.

[0020] Furthermore, the drug includes at least one of puerarin, quercetin, and methyldeferoxamine.

[0021] Furthermore, the drug is quercetin.

[0022] Optionally, the cross-linking agent is genipin, and its concentration is 0.01 g / mL.

[0023] Optionally, the mass ratio of the water-soluble chitosan to polyvinyl alcohol is: (1-2):1, preferably 1.5:1;

[0024] The mass ratio of the water-soluble chitosan and the active oxygen responsive chain segment to construct the macromolecular monomer is 2:1.

[0025] The second technical solution of the present invention:

[0026] A method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability comprises the following steps:

[0027] The polyvinyl alcohol solution and the water-soluble chitosan solution were mixed, and then the linker solution was added thereto, reacted overnight, and then dialyzed, centrifuged and freeze-dried in sequence to obtain PVA-Linker-CS powder;

[0028] The PVA-Linker-CS powder is dissolved in distilled water, and the drug-containing kiwifruit extracellular vesicles and genipin are added, stirred evenly, and allowed to stand to obtain the kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability.

[0029] Optionally, the concentrations of the polyvinyl alcohol solution and the water-soluble chitosan solution are both 10 mg / mL; and / or

[0030] The concentration of the linker solution is 200 to 300 mg / mL, preferably 200 mg / mL.

[0031] Optionally, the dosage ratio of the PVA-Linker-CS powder, the drug-containing kiwifruit extracellular vesicles and genipin is: (0.2-0.4) g: 100-200 μL: (50-150) μL; wherein, in this ratio, the volume of genipin is preferably 50 μL, 100 μL, 150 μL; more preferably 100 μL.

[0032] Furthermore, the dosage ratio of the PVA-Linker-CS powder, the drug-containing kiwifruit extracellular vesicles and genipin is: 0.3 g: 200 μL: 100 μL.

[0033] The third technical solution of the present invention:

[0034] A kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability is used in the preparation of wound dressings, anti-inflammatory and antibacterial materials, or materials for preventing peritoneal mucosal adhesion.

[0035] Compared with the prior art, the present invention has the following advantages and technical effects:

[0036] The present invention uses water-soluble chitosan, polyvinyl alcohol, and active oxygen responsive chain segments to construct macromolecular monomers as main raw materials, uses genipin as a biological cross-linking agent, and adds kiwifruit extracellular vesicles loaded with drugs to obtain an injectable hydrogel. That is, the present invention loads kiwifruit extracellular vesicles containing drugs into injectable hydrogels, aiming to optimize the postoperative recovery process and prevent the occurrence of abdominal adhesions by integrating a multi-effect treatment strategy of wound healing, inflammatory response control, and tissue regeneration promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0038] Figure 1 The synthetic technology roadmap of PVA-Linker-CS powder in Example 1 of the present invention;

[0039] Figure 2 Figure 1 is a diagram of the injectable hydrogel before and after gelation of Example 1 of the present invention;

[0040] Figure 3 This is a SEM image of the injectable hydrogel of Example 1 of the present invention;

[0041] Figure 4 The NMR images (a) and IR images (b) of PVA, CS and PVA-Linker-CS in Example 1 of the present invention are shown;

[0042] Figure 5 The following are the wound healing conditions of mice in the model group, control group, blank gel group, and drug-added group 9 days after surgery. (a) is a simulation of wound healing in mice; (b) is an experimental diagram of wound healing in mice; (c) is a diagram of the recovery ratio of wounds in mice; (d) is a diagram of the recovery ratio of each group 9 days after surgery; (e) is a H&E and Masson staining diagram of wound healing in mice;

[0043] Figure 6 and Figure 7 The immunofluorescence images and relative fluorescence intensity bar graphs of wound healing of mice in the model group, control group, blank gel group and drug-added group 9 days after surgery;

[0044] Figure 8 The diagrams show the tissue adhesion between the peritoneum and intestines of mice in the model group, control group, blank gel group, and drug-added group 14 days after surgery, where (a) shows the tissue adhesion between the peritoneum and intestines of mice; (b) shows the H&E and Masson staining of the tissue adhesion between the peritoneum and intestines of mice; (c) shows the adhesion score diagram; and (d) shows the percentage of collagen loss.

[0045] Fig. 9 and Fig.10 The immunofluorescence images and relative fluorescence intensity bar graphs of abdominal adhesions in the model group, control group, blank glue group and drug-added group 14 days after surgery. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] The embodiment of the present invention discloses a kiwifruit extracellular vesicle-loaded injectable hydrogel with reactive oxygen species (ROS) scavenging capability, the raw materials comprising:

[0052] Water-soluble chitosan, polyvinyl alcohol, active oxygen responsive chain segments to construct macromolecular monomers, drug-containing kiwifruit extracellular vesicles and genipin (cross-linker).

[0053] In some preferred embodiments, the mass ratio of water-soluble chitosan to polyvinyl alcohol is (1-2):1; preferably 1.5:1.

[0054] In some preferred embodiments, water-soluble chitosan, polyvinyl alcohol, and active oxygen responsive segment-constructing macromolecular monomers need to be separately prepared to obtain aqueous solutions of a certain concentration before the reaction;

[0055] The concentration of the macromolecular monomer solution (linker) for constructing the active oxygen responsive chain segment is (200-300) mg / mL, and more preferably 200 mg / mL.

[0056] The embodiment of the present invention also provides a method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel having reactive oxygen species (ROS) scavenging capability, comprising the following steps:

[0057] 1) heating and dissolving polyvinyl alcohol in distilled water to form a polyvinyl alcohol solution;

[0058] 2) mixing the polyvinyl alcohol aqueous solution obtained in step 1) with the dissolved water-soluble chitosan solution, and reacting for 1 hour;

[0059] 3) gradually adding the linker solution to the mixed solution obtained in step 2) and reacting for 12 hours;

[0060] 4) dialyzing, centrifuging and freeze-drying the solution obtained in step 3) in sequence to form PVA-Linker-CS powder;

[0061] 5) dissolving the PVA-Linker-CS powder obtained in step 4) in distilled water, and adding the kiwifruit extracellular vesicles containing the drug;

[0062] 6) Genipin is added to the solution obtained in step 5) and the solution is allowed to stand to form an injectable hydrogel.

[0063] In some preferred embodiments, the drug contained in the drug-containing kiwifruit extracellular vesicles is any one of puerarin, quercetin, and methyldeferoxamine, more preferably quercetin.

[0064] In some preferred embodiments, the concentration of the kiwifruit extracellular vesicles containing the drug is 5-10 mg / mL, more preferably 5 mg / mL.

[0065] In addition, the present invention also provides a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability for use in the preparation of wound dressings, anti-inflammatory and antibacterial materials, or materials for preventing abdominal mucosal adhesion.

[0066] Unless otherwise specified, the "room temperature" in the present invention refers to 20-30° C. The preparation process of kiwifruit extracellular vesicles used in the following examples and comparative examples is as follows:

[0067] First, after the kiwifruit skin is peeled, the kiwifruit juice is obtained by repeated squeezing, and then filtered multiple times with gauze to remove coarse impurities;

[0068] Then, the filtered juice was centrifuged: first at 1000×g for 10 minutes, then at 3000×g for 10 minutes, then at 10000×g for 30 minutes, and finally at 20000×g for 30 minutes to gradually remove larger particulate matter;

[0069] Finally, the supernatant was centrifuged at 120,000 × g for 2 hours to enrich kiwifruit extracellular vesicles to obtain kiwifruit extracellular vesicles.

[0070] 5 mg / mL kiwifruit extracellular vesicles containing quercetin are prepared by adding 5 mg of quercetin to 1 ml of kiwifruit extracellular vesicles and stirring the mixture evenly.

[0071] Quercetin was purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd. (Shanghai), polyvinyl alcohol (PVA) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai), and water-soluble chitosan (CS) was purchased from Shandong Haidebei Biotechnology Co., Ltd. (Shandong).

[0072] The technical solution of the present invention is further illustrated by the following embodiments.

[0073] Example 1

[0074] A method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel with reactive oxygen species (ROS) scavenging ability, comprising the following steps:

[0075] like Figure 1 As shown, first, 1 g of polyvinyl alcohol is added to a beaker of 100 mL of distilled water and heated to dissolve, and then 1 g of water-soluble chitosan is added to another beaker of 100 mL of distilled water to dissolve, and the dissolved polyvinyl alcohol solution and the water-soluble chitosan solution are mixed at a mass ratio of polyvinyl alcohol to water-soluble chitosan of 1:1.5, and then Linker solution (the concentration of Linker solution is 200 mg / mL) is added thereto, and the solution is slowly added dropwise at a mass ratio of water-soluble chitosan to Linker of 2:1, and the reaction is allowed to react overnight, followed by dialysis for 48 days, centrifugation and freeze-drying for 72 hours, and finally PVA-Linker-CS powder is formed.

[0076] Then, PVA-Linker-CS (0.3 g) was heated and dissolved in 1 mL of distilled water. After stirring evenly, 200 μL of kiwifruit extracellular vesicles containing quercetin (the concentration of kiwifruit extracellular vesicles containing quercetin was 5 mg / mL) was added. After half an hour, 100 μL (0.01 g / mL) of genipin was added, stirred evenly, and allowed to stand to obtain an injectable hydrogel, achieving the expected results.

[0077] Figure 2 Figure 1 is a diagram of the injectable hydrogel before and after gelation of Example 1 of the present invention; Figure 2 It can be seen that the hydrogel after gelation is light blue.

[0078] Figure 3This is a SEM image of the injectable hydrogel of Example 1 of the present invention; as can be seen from the figure, the prepared hydrogel has a network structure, which helps to enhance its mechanical strength and stability while maintaining sufficient flexibility to adapt to different physiological environments.

[0079] Figure 4 : This is the NMR infrared image of PVA, CS and PVA-Linker-CS in Example 1 of the present invention; it can be seen from the figure that PVA-Linker-CS shows unique characteristic peaks compared with individual CS and PVA, which indicates that Linker has successfully bonded CS and PVA together.

[0080] Example 2

[0081] A method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel with reactive oxygen species (ROS) scavenging ability, comprising the following steps:

[0082] First, add 1 g of polyvinyl alcohol into a beaker containing 100 mL of distilled water and heat to dissolve, then add 1 g of water-soluble chitosan into another beaker containing 100 mL of distilled water to dissolve, mix the dissolved polyvinyl alcohol and water-soluble chitosan in a mass ratio of 1:2, and then add Linker solution (the concentration of Linker solution is 200 mg / mL) thereto, slowly add dropwise in a mass ratio of water-soluble chitosan to Linker of 2:1, react overnight, dialyze for 48 days, centrifuge and freeze-dry for 72 hours, and finally form PVA-Linker-CS powder.

[0083] Then, PVA-Linker-CS (0.3 g) was heated and dissolved in 1 mL of distilled water. After stirring evenly, 200 μL of kiwifruit extracellular vesicles containing quercetin (the concentration of kiwifruit extracellular vesicles containing quercetin was 5 mg / mL) was added. After half an hour, 100 μL (0.01 g / mL) of genipin was added, stirred evenly, and allowed to stand to obtain an injectable hydrogel.

[0084] Example 3

[0085] A method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel with reactive oxygen species (ROS) scavenging ability, comprising the following steps:

[0086] First, 1 g of polyvinyl alcohol was added to a beaker containing 100 mL of distilled water and heated to dissolve. Then, 1 g of water-soluble chitosan was added to another beaker containing 100 mL of distilled water to dissolve. The dissolved polyvinyl alcohol and water-soluble chitosan were mixed at a mass ratio of 1:1. Then, Linker solution (the concentration of Linker solution was 200 mg / mL) was added thereto. The solution was slowly added dropwise at a weight ratio of 2:1 between water-soluble chitosan and Linker. The mixture was allowed to react overnight, dialyzed for 48 days, centrifuged and freeze-dried for 72 hours, and PVA-Linker-CS powder was finally formed.

[0087] Then, PVA-Linker-CS (0.3 g) was heated and dissolved in 1 mL of distilled water. After stirring evenly, 200 μL of kiwifruit extracellular vesicles containing quercetin (the concentration of kiwifruit extracellular vesicles containing quercetin was 5 mg / mL) was added. After half an hour, 100 μL (0.01 g / mL) of genipin was added, stirred evenly, and allowed to stand to obtain an injectable hydrogel.

[0088] Comparative Example 1

[0089] The difference from Example 1 is that the kiwifruit extracellular vesicles containing quercetin are replaced with an equal volume of distilled water. The other steps are the same as those in Example 1.

[0090] Comparative Example 2

[0091] The difference from Example 1 is that the kiwifruit extracellular vesicles are not loaded with drug quercetin, and the other steps are consistent with Example 1.

[0092] Effect verification

[0093] Test 1

[0094] The injectable hydrogel prepared in Example 1 was used to evaluate the wound healing of the injectable hydrogel using the back wound injury of Kunming mice as an animal model. The detailed steps are as follows:

[0095] Fifteen female Kunming mice (Henan Sikebes Biotechnology Co., Ltd.) of similar size and weighing 20 g to 25 g were selected, and all operations on animals were in accordance with NIH guidelines.

[0096] The mice were raised for 10 days before surgery and randomly divided into four groups, with 5 mice in each group, namely, the first group was the model group (did not receive any intervention, control), the second group was the control group (Comparative Example 1, PVA-CS@HA), the third group was the blank gel group (Comparative Example 2, PVA-CS@HA-kiwi), and the fourth group was the drug-added group (Example 1, PVA-CS@HA-kiwi-Drug).

[0097] The required materials must be exposed to ultraviolet light for half an hour. The experiment was carried out in a sterile environment. First, 0.25-0.35 mL of chloral hydrate solution (5% w / v) was injected intraperitoneally to weigh and anesthetize the mice. The back was depilated, and then a punch was used to injure the back of the mice. The materials required for the experiment were loaded. The model group did not receive any intervention.

[0098] After the experiment, the mice were placed in a clean cage. Then, pictures were taken at the specified time. All mice were killed 9 days after the operation, the wound site on the back was cut, rinsed with PBS, and then immersed in tissue cell fixative (4% PFA), and finally embedded in paraffin to make paraffin sections with a thickness of 4.0 μm. The sections were then stained with eosin H&E and Masson for pathological analysis. And VEGF, CD31, CD68, IL-6, TNF-α, α-SMA, IL-10, and IL-1β were determined by immunofluorescence staining.

[0099] like Figure 5 As shown, the wound recovery in the drug-added group was the best, and the wound recovery in the model group was the worst. This indicates that kiwifruit extracellular vesicle-loaded injectable hydrogels with reactive oxygen species (ROS) scavenging ability can eliminate inflammation and promote wound healing.

[0100] Furthermore, all groups were labeled by immunofluorescence for inflammation assessment, including VEGF, CD31, CD68, IL-6, TNF-α, α-SMA, IL-10, and IL-1β. Figure 6 and Figure 7 As shown, in the model group, a large number of inflammatory factors, IL-6, TNF-α, α-SMA, and IL-1β, appeared at the wound site. In the control group and the blank gel group, the inflammatory factors decreased. In the drug-added group, there were almost no inflammatory factors. This shows that the effect of the drug-added group is the best. In addition, a large number of growth factors, VEGF, CD31, CD68, and IL-10 were observed in the drug-added group. In the control group and the blank gel group, there were a small amount of growth factors, and in the model group, there were almost no growth factors. This shows that the kiwifruit extracellular vesicle-loaded injectable hydrogel with reactive oxygen species (ROS) scavenging ability described in the present invention has a better effect in promoting wound healing.

[0101] The relative fluorescence intensity of the above growth factors and inflammatory factors was calculated using Image software.

[0102] Test 2

[0103] Kunming mice were purchased from Henan Swedish Biological Co., Ltd. and cultured in the Experimental Animal Center of the Affiliated Hospital of the Health Science Center of Hebei University of Engineering. The animal experiment protocol was approved by the Animal Experiment Ethics Committee of the Affiliated Hospital of Hebei University of Engineering. The anti-adhesion mouse model after laparotomy [20-30g, 5-6 weeks old] was used for in vivo anti-adhesion evaluation. The abdominal wall injury model was established according to the previous method using a triple-blind method. Three groups (n=3) were randomly selected from the experimental animals, namely the model group, the control group, the blank glue group and the drug-added group. The abdominal cavity was exposed by making a U-shaped incision along the midline of the abdominal wall. A wound was cut in the peritoneum with a scalpel. The model group was only rinsed with normal saline, while the control group, the blank glue group and the drug-added group completely covered the wound site. Finally, the abdominal incision was closed by suturing layer by layer to complete the closure of the abdominal cavity. All surgical procedures were performed under sterile conditions. Cefuroxime (300 mg / Kg) was given once a day for 3 consecutive days after surgery, and the wound was wiped with iodine twice a day for several consecutive days to prevent postoperative infection. After euthanasia on postoperative day 14, an overdose of pentobarbital was injected via the tail vein, followed by pathological analysis and immunofluorescence testing.

[0104] like Figure 8 As shown, the model group had a large area of ​​tissue adhesion between the peritoneum and the intestine. In the control group and the blank gel group, a small area of ​​adhesion occurred, and the drug-added group showed the best anti-adhesion effect, with almost no adhesion. This shows that the kiwifruit extracellular vesicle-loaded injectable hydrogel with reactive oxygen species (ROS) scavenging ability can resist inflammation and promote tissue regeneration, preventing the occurrence of abdominal adhesion.

[0105] Furthermore, all groups were labeled by immunofluorescence to assess inflammation, e.g. Fig. 9 and Fig.10 As shown, in the model group, a large amount of inflammatory factors CD86, TNF-α, IL-6, and α-SMA were observed in the wound tissue. In the control group and the blank gel group, the inflammatory factors decreased. In the drug-added group, there were almost no inflammatory factors. This shows that the effect of the drug-added group is the best. In addition, a large amount of growth factors VEGF, CD31, CD206, and IL-4 were observed in the drug-added group, while only a small amount of growth factors VEGF, CD31, CD206, and IL-4 were found in the control group and the blank group, and there was almost no growth factor VEGF, CD31, CD206, and IL-4 in the model group, which shows that the kiwifruit extracellular vesicle-loaded injectable hydrogel with reactive oxygen species (ROS) scavenging ability described in the present invention has a better effect of promoting tissue regeneration.

[0106] The relative fluorescence intensity of the above growth factors and inflammatory factors was calculated using Image software.

[0107] In summary, the present invention loads drug-containing kiwifruit extracellular vesicles into injectable hydrogels, aiming to optimize the postoperative recovery process and prevent the occurrence of abdominal adhesions by integrating a multi-effect therapeutic strategy of wound healing, inflammatory response control, and tissue regeneration promotion.

[0108] The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability, characterized in that: Including the following ingredients: Water-soluble chitosan, polyvinyl alcohol, reactive oxygen species-responsive segments were used to construct macromonomers, cross-linkers, and drug-containing kiwifruit extracellular vesicles; The structural formula of the macromolecular monomer constructed by the active oxygen responsive segment is:

2. The kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability according to claim 1, characterized in that: The concentration of the drug-containing kiwifruit extracellular vesicles is 5-10 mg / mL.

3. The kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability according to claim 2, characterized in that: The drug includes at least one of puerarin, quercetin and methyldeferoxamine.

4. The kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability according to claim 3, characterized in that: The drug is quercetin.

5. The kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability according to claim 1, characterized in that: The mass ratio of the water-soluble chitosan to the polyvinyl alcohol is: (1-2):1; The mass ratio of the water-soluble chitosan and the active oxygen responsive chain segment to construct the macromolecular monomer is 2:

1.

6. A method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability, characterized in that: The following steps are involved: The polyvinyl alcohol solution and the water-soluble chitosan solution were mixed, and then the macromolecular monomer solution of the active oxygen responsive segment was added thereto, reacted overnight, and then dialyzed, centrifuged and freeze-dried in sequence to obtain PVA-Linker-CS powder; The PVA-Linker-CS powder is dissolved in distilled water, and kiwi fruit extracellular vesicles containing drugs and genipin are added, stirred evenly, and allowed to stand to obtain the kiwi fruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability as described in any one of claims 1 to 5.

7. The method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability according to claim 6, characterized in that: The concentrations of the polyvinyl alcohol solution and the water-soluble chitosan solution are both 10 mg / mL; and / or The concentration of the linker solution is 200-300 mg / mL.

8. The method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability according to claim 6, characterized in that: The dosage ratio of the PVA-Linker-CS powder, the drug-containing kiwifruit extracellular vesicles and genipin is: (0.2-0.4) g: 100-200 μL: (50-150) μL.

9. The method for preparing a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability according to claim 8, characterized in that: The dosage ratio of the PVA-Linker-CS powder, the drug-containing kiwifruit extracellular vesicles and genipin is: 0.3 g: 200 μL: 100 μL.

10. Use of a kiwifruit extracellular vesicle-loaded injectable hydrogel with active oxygen scavenging ability as claimed in any one of claims 1 to 5 in the preparation of wound dressings, anti-inflammatory and antibacterial materials or materials for preventing peritoneal mucosal adhesion.

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