Injectable, tissue-adhesive exosome controlled-release hydrogel microspheres and preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to an injectable, tissue-adhesive exosome controlled-release hydrogel microsphere and its preparation method and application. The exosome controlled-release hydrogel microsphere is composed of the following components: 2-furanmethylamine-modified hyaluronic acid, terminal maleimide-modified polyethylene glycol, dopamine-modified hyaluronic acid and exosomes secreted by stem cells. Compared with the prior art, the hydrogel microspheres of the present invention are simple to prepare and low in cost; the hydrogel microspheres of the present invention can be administered by injection, which is convenient to use; the hydrogel microspheres of the present invention can effectively adhere to tissues near the injection site, thereby achieving effective release of SC-sEVs at the target site of action; the hydrogel microspheres of the present invention can achieve long-term release of SC-sEVs, and improve the efficiency of SC-sEVs in treating tendon diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an injectable, tissue-adhesive exosome controlled-release hydrogel microsphere and a preparation method and application thereof. Background Art

[0002] Exosomes are membrane vesicles with a diameter of 30–150 nm secreted by cells. They contain RNA, DNA, proteins and other substances of maternal cells and are important media for intercellular material communication. In recent years, extracellular vesicles derived from stem cells (pluripotent stem cells, mesenchymal stem cells, etc.) have shown great potential for replacing stem cell therapy. Extensive studies have shown that exosomes secreted by stem cells can regulate cell homeostasis, delay cell aging, inhibit tissue inflammation, etc. by delivering stem cell substances to recipient cells, thereby achieving therapeutic functions such as promoting tissue repair and alleviating disease symptoms. In view of the rich therapeutic functions of stem cell exosomes and their good biosafety, they are extremely valuable for clinical translational applications. However, how to achieve the long-term effect of stem cell exosomes at the treatment site is still a key technical issue that hinders its translational application.

[0003] Hydrogel materials provide potential carriers for the long-term controlled release of exosomes. Hydrogel is a hydrophilic cross-linked polymer with good biocompatibility. The long-term controlled release of the loaded substance can be achieved by optimizing parameters such as skeleton material, degradation rate, and cross-linking density. In recent years, researchers have developed a variety of exosome controlled release hydrogel materials (CN202110203774.4, CN201810643146.6, etc.). However, these hydrogels face problems such as complex material preparation, difficulty in administering drugs through minimally invasive methods, high reactivity of cross-linking methods, and difficulty in effective adhesion at the site of action. Therefore, it is urgent to design a new type of hydrogel carrier to achieve long-term controlled release of stem cell exosomes at the treatment site. Summary of the invention

[0004] In order to solve the problems faced by the hydrogels mentioned in the above-mentioned prior art, such as complex material preparation, difficulty in administering drugs through minimally invasive methods, high reactivity of cross-linking methods, and difficulty in effectively adhering to the site of action, the present invention provides an injectable, tissue-adhesive exosome controlled-release hydrogel microsphere and a preparation method and application thereof, so as to improve the efficiency of stem cell exosomes in promoting tissue damage repair and inflammatory disease treatment, and provide a more practical administration form for the transformation application of stem cell exosomes.

[0005] The present invention discloses an injectable, tissue-adhesive exosome controlled-release hydrogel microsphere, which can be administered in a non-invasive manner to achieve long-term controlled release of exosomes at the site of action.

[0006] In the present invention, water-soluble polymers (hyaluronic acid and polyethylene glycol) with good biocompatibility and FDA approval are used to prepare hydrogel microspheres through a mild Diels-Alder reaction. At the same time, dopamine-modified hyaluronic acid can achieve the adhesion of hydrogel microspheres on the tissue surface and the long-term release of exosomes through non-covalent bonding such as hydrogen bonding and π-π interaction with tissue or exosome surface proteins. In addition, hydrogel microspheres can be passed through a syringe needle, so they can be administered in a non-invasive manner by injection.

[0007] Tendinopathy is a common musculoskeletal disease, usually caused by overuse of muscle fibers and repeated strong pulling, which leads to degenerative changes in tendon collagen fibers. Currently, relevant studies have shown that exosomes secreted by stem cells can effectively relieve tendon pain and inflammation, and are a potential therapeutic factor for tendinopathy. However, how to ensure the long-term release of stem cell-derived exosomes in the tendon area is a key technical challenge in the treatment of tendinopathy. Only by forming a good and strong adhesion interface with the tissue can the hydrogel microspheres be stably adhered to the surface of the tendon tissue and prevented from falling off or moving during muscle movement. Therefore, the present invention further uses the prepared hydrogel microspheres with good tissue adhesion properties for the long-term release of stem cell exosomes in the tendon area to relieve tendinopathy symptoms.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The present invention first provides an injectable, tissue-adhesive exosome controlled-release hydrogel microsphere, which is composed of the following components:

[0010] 2-furylamine-modified hyaluronic acid, structural formula I, abbreviated as FHA;

[0011] Maleimide-terminated polyethylene glycol, structural formula II, abbreviated as Mal-PEG-Mal;

[0012] Dopamine-modified hyaluronic acid, structural formula III, abbreviated as DAHA; and,

[0013] Exosomes secreted by stem cells, abbreviated as SC-sEVs.

[0014]

[0015]

[0016] Among the above-mentioned exosome controlled-release hydrogel microsphere components, FHA and Mal-PEG-Mal are mainly used to form hydrogel microspheres through Diels-Alder cross-linking reaction; DAHA mainly achieves adhesion of hydrogel microspheres to the tissue interface and prolongs the sustained release time of exosomes through multiple non-covalent bonds mediated by dopamine groups with tissue proteins, polysaccharides and other molecules; stem cell exosomes mainly play a therapeutic role in diseases.

[0017] In one embodiment of the present invention, in the hydrogel microspheres, the mass concentration of hyaluronic acid modified with 2-furanylmethylamine is 1% to 5%, the mass concentration of polyethylene glycol modified with terminal maleimide is 1% to 10%, the mass concentration of hyaluronic acid modified with dopamine is 1% to 5%, and the particle concentration of exosomes secreted by stem cells is 1×10 9 / mL~5×10 11 / mL.

[0018] In one embodiment of the present invention, in the hydrogel microspheres, the mass concentration fraction of hyaluronic acid modified with 2-furanylmethylamine is 2.5%, the mass concentration fraction of polyethylene glycol modified with terminal maleimide is 5%, the mass concentration fraction of hyaluronic acid modified with dopamine is 1.5%, and the particle concentration of exosomes secreted by stem cells is 1×10 9 / mL~5×10 11 / mL.

[0019] In one embodiment of the present invention, the modification rate of 2-furylmethylamine in the hyaluronic acid modified with 2-furylmethylamine is 20% to 80% of the total number of hyaluronic acid carboxyl groups, preferably 40% to 60%.

[0020] In one embodiment of the present invention, the molecular weight of the maleimide-terminated polyethylene glycol is 1 KDa to 10 KDa, preferably 2 KDa.

[0021] In one embodiment of the present invention, in the dopamine-modified hyaluronic acid, the modification rate of dopamine is 20% to 80% of the total number of carboxyl groups of the hyaluronic acid, preferably 60%.

[0022] In one embodiment of the present invention, the preparation method of the 2-furanylmethylamine-modified hyaluronic acid (structural formula I, FHA) is:

[0023] At room temperature, hyaluronic acid (mass average molecular weight 100KDa) was fully dissolved in 4-morpholineethanesulfonic acid (MES) buffer (pH = 6.0) to prepare a solution with a mass fraction of 1%. Subsequently, a certain mass of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) was added to the solution and stirred at room temperature. After fully reacting for 30 minutes, a certain mass of 2-furanmethylamine was added, and the reaction solution was stirred at room temperature for 24 hours. Subsequently, the reaction solution was added to anhydrous ethanol at a volume ratio of 1:5 for precipitation. The precipitate was dissolved again with water, dialyzed at room temperature and freeze-dried to obtain the substance shown in structural formula I.

[0024] In the preparation process of the 2-furanmethylamine-modified hyaluronic acid, the modification rate of 2-furanmethylamine in the final product is controlled by adjusting the feeding amounts of DMTMM and 2-furanmethylamine.

[0025] In one embodiment of the present invention, the maleimide-terminated polyethylene glycol (structural formula II, Mal-PEG-Mal) can be obtained through commercial channels.

[0026] In one embodiment of the present invention, the preparation method of the dopamine-modified hyaluronic acid (structural formula III, DAHA) is:

[0027] At room temperature, hyaluronic acid (mass average molecular weight 400KDa) was fully dissolved in 4-morpholineethanesulfonic acid (MES) buffer (pH = 6.0) to prepare a solution with a mass fraction of 1%. Subsequently, a certain mass of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) was added to the solution and stirred at room temperature. After fully reacting for 30 minutes, a certain mass of dopamine hydrochloride was added, and the reaction solution was stirred at 4°C for 24 hours. Subsequently, the reaction solution was added to anhydrous ethanol at a volume ratio of 1:5 for precipitation. The precipitate was dissolved again with water, dialyzed at 4°C and freeze-dried to obtain the substance shown in structural formula III.

[0028] The modification rate of dopamine in the final product can be controlled by adjusting the feed amounts of DMTMM and dopamine hydrochloride.

[0029] In one embodiment of the present invention, the collection and purification method of the stem cell secreted exosomes (SC-sEVs) is as follows:

[0030] Under standard conditions (37°C, 5% CO2), pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells, etc.), mesenchymal stem cells, mesenchymal stem cells induced by pluripotent stem cells, etc. are expanded in cell culture bottles using serum-free medium. The cell culture medium is collected and subjected to differential centrifugation to remove cells, cell debris, etc. SC-sEVs are obtained by purification methods such as ultracentrifugation and tangential flow filtration. The collected stem cell exosomes are identified using transmission electron microscopy, immunoblotting, nanoflow cytometry, etc.

[0031] The present invention further provides a method for preparing the injectable, tissue-adhesive exosome controlled-release hydrogel microspheres, which is as follows:

[0032] Under sterile conditions, a certain amount of FHA, Mal-PEG-Mal and DAHA are fully dissolved in a biocompatible medium to prepare a hydrogel precursor solution, and the solution is fully mixed with a biocompatible medium suspension of SC-sEVs to obtain a hydrogel precursor solution;

[0033] Subsequently, the hydrogel precursor solution was added dropwise to mineral oil containing a certain concentration of Span80 at 37°C according to a certain volume ratio, and stirred at 300 rpm / min for a certain period of time to obtain hydrogel microspheres loaded with SC-sEVs;

[0034] The hydrogel microspheres were centrifuged and precipitated at 4°C, and washed with ether to remove residual mineral oil and surfactant Span 80. The hydrogel microspheres were allowed to stand under sterile conditions at room temperature to allow the ether to evaporate, and finally the hydrogel microspheres loaded with SC-sEVs were obtained, which were injectable, tissue-adhesive exosome controlled-release hydrogel microspheres.

[0035] In one embodiment of the present invention, the biocompatible medium is physiological saline, physiological buffer or cell culture medium.

[0036] In one embodiment of the present invention, the volume ratio of the hydrogel precursor solution containing SC-sEVs to mineral oil is 1:1000 to 1:20, preferably 1:100.

[0037] In one embodiment of the present invention, the volume fraction of Span 80 is 0.1% to 10%, preferably 2.5%.

[0038] In one embodiment of the present invention, the stirring time is 2 h to 24 h, preferably 7 h.

[0039] The present invention further provides a kit containing the injectable, tissue-adhesive exosome controlled-release hydrogel microspheres. The kit consists of two parts: injectable, tissue-adhesive exosome controlled-release hydrogel microspheres loaded in a disposable syringe and physiological saline.

[0040] In one embodiment of the present invention, the disposable syringe is 1 mL, the capacity of the injectable, tissue-adhesive exosome controlled-release hydrogel microspheres is 250 μL, and the loading amount of pluripotent stem cell-derived exosomes is 1×10 11 / mL, the volume of normal saline is 500μL.

[0041] The present invention further provides the use of the injectable, tissue-adhesive exosome controlled-release hydrogel microspheres in the preparation of a drug for treating tendon diseases.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] 1. The hydrogel microspheres loaded with SC-sEVs disclosed in the present invention are simple to prepare and low in cost;

[0044] 2. The hydrogel microspheres loaded with SC-sEVs disclosed in the present invention can be administered by injection, which is convenient to use;

[0045] 3. The hydrogel microspheres loaded with SC-sEVs disclosed in the present invention can effectively adhere to the tissue near the injection site, thereby achieving the effective release of SC-sEVs at the target site;

[0046] 4. The SC-sEVs-loaded hydrogel microspheres disclosed in the present invention can achieve long-term release of SC-sEVs and improve the efficiency of SC-sEVs in treating tendon diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 , the general view of hydrogel microspheres;

[0048] Figure 2 , particle size distribution of hydrogel microspheres;

[0049] Figure 3 , rate curve of controlled release of iMSC-sEVs from hydrogel microspheres;

[0050] Figure 4 , Representative images of HE staining of tendon tissue after 4 weeks of treatment with hydrogel microspheres controlled release of iMSC-sEVs;

[0051] Figure 5 , Results of static weight-bearing test and hind-foot withdrawal threshold test in SD rats after 2 and 4 weeks of treatment with hydrogel microspheres controlled-release iMSC-sEVs. DETAILED DESCRIPTION

[0052] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0053] Example 1

[0054] Preparation of FHA with 50% 2-furylmethylamine modification rate

[0055] At room temperature, 1g of hyaluronic acid (Mw 100KDa) was fully dissolved in 100mL of MES (pH=6.0, 0.1M) buffer to prepare a solution with a mass fraction of 1%. Subsequently, 0.56g of DMTMM was added to the solution, and after stirring at room temperature for 30min, 0.36g of 2-furanmethylamine was added. After stirring at room temperature for 24h, the reaction solution was added to anhydrous ethanol at a volume ratio of 1:5 for precipitation. The precipitate was dissolved again in water, dialyzed at room temperature, and freeze-dried to obtain 0.78g of white flocculent FHA. The results of H NMR characterization showed that the modification rate of 2-furanmethylamine was 49.52% of the total carboxyl groups in HA.

[0056] Example 2

[0057] Preparation of DAHA with a dopamine modification rate of 60%

[0058] At room temperature, 1g of hyaluronic acid (mass average molecular weight 400KDa) was fully dissolved in 100mL of MES buffer (pH=6.0, 0.1M) to form a solution with a mass fraction of 1%. Subsequently, 0.70g of DMTMM was added to the solution and stirred at room temperature. After 30min, 0.82g of dopamine hydrochloride was added, and the reaction solution was stirred at 4°C for 24h. Subsequently, the reaction solution was added to anhydrous ethanol at a volume ratio of 1:5 for precipitation. The precipitate was dissolved again with water, dialyzed at 4°C and freeze-dried to obtain 0.77g of white flocculent DAHA. The results of hydrogen nuclear magnetic resonance spectroscopy showed that the modification rate of dopamine was 60.4% of the total amount of hyaluronic acid carboxyl groups.

[0059] Example 3

[0060] Extraction of exosomes from induced pluripotent stem cells (iMSC-sEVs)

[0061] According to the method reported in the literature, gradient centrifugation combined with ultracentrifugation was used to extract sEVs from the supernatant of iMSCs culture. First, the supernatant of iMSCs culture was collected and centrifuged at 300g for 10 minutes at 4°C to remove dead cells. Then the precipitate was discarded and centrifuged at 2000g for 10 minutes at 4°C to remove apoptotic bodies and cell debris. Then the precipitate was discarded and centrifuged at 10000g for 30 minutes at 4°C. The supernatant was collected and then filtered through a 0.22μm sterile filter membrane (Millipore, USA) and centrifuged at 100000g for 120 minutes at 4°C; then the supernatant was discarded, and the liquid at the bottom of the collection tube was resuspended with PBS to about 1mL, and it was repeatedly blown and collected in the same centrifuge tube. Finally, PBS solution was added and centrifuged again at 100000g for 120 minutes at 4°C. The resulting precipitate was sEVs, which was collected in a sterile EP tube using PBS.

[0062] Example 4

[0063] Preparation of injectable, tissue-adhesive exosome-controlled release hydrogel microspheres (iMSC-sEVs-loaded hydrogel microspheres)

[0064] Under sterile conditions, 0.125 g FHA (prepared in Example 1), 0.25 g Mal-PEG-Mal (commercially available, such as SinoPEG), and 0.075 g DAHA (prepared in Example 2) were fully dissolved in 4.1 mL of saline and mixed with 2.5 mL iMSC-sEVs (2×10 11 / mL) of physiological saline solution to prepare a hydrogel precursor solution. Subsequently, 553.8g of mineral oil and 14.2g of Span80 were added to a 1L beaker, and the double-blade stirring paddle was fully stirred and heated to 37°C. At 300rpm, 5mL of hydrogel precursor solution was added dropwise to the mineral oil and stirred for 6h. Subsequently, the mineral oil was centrifuged at 4°C and 3000rcf. The centrifuged hydrogel microspheres were soaked and rinsed with ether to remove residual mineral oil and Span 80. The hydrogel microspheres were allowed to stand under sterile conditions at room temperature to allow the ether to evaporate, and finally hydrogel microspheres loaded with SC-sEVs were obtained.

[0065] The free water on the surface of the hydrogel microspheres was absorbed by sterile filter paper and loaded into syringes of different volumes for later use.

[0066] Example 5

[0067] Particle size distribution analysis of hydrogel microspheres

[0068] The hydrogel microspheres loaded with SC-sEVs were prepared by the method of Example 4, and fluorescently labeled BSA was added to the hydrogel microspheres. The hydrogel microspheres were dispersed in ultrapure water and dropped onto a glass slide. Fluorescence confocal microscopy was used to perform fluorescence imaging of the hydrogel microspheres, and the particle size distribution of the microspheres was statistically analyzed. The experimental results showed that the particle size distribution range of the prepared hydrogel microspheres was 20 μm to 100 μm, with an average particle size of 65.69 ± 27.39 μm (see Figure 4). Figure 1 and attached Figure 2 ).

[0069] Example 6

[0070] Characterization of the controlled release ability of hydrogel microspheres of SC-sEVs

[0071] 200 μL of the hydrogel microspheres prepared in Example 4 were dispersed in 1 mL of normal saline and released at 37°C under sterile conditions. During the release process, the hydrogel microspheres were centrifuged every 24 hours to obtain the supernatant. Subsequently, the particle concentration of iMSC-sEVs in the hydrogel microspheres was detected by nanoflow cytometry to evaluate the sustained release of exosomes from the hydrogel microspheres. The results showed that the hydrogel microspheres prepared in Example 4 can achieve sustained release of iMSC-sEVs for up to 14 days (see Appendix). Figure 3 ).

[0072] Example 7

[0073] Characterization of local tissue adhesion ability of hydrogel microspheres

[0074] Extract the Achilles tendon tissue of the pig's hind limbs, apply 100 μL of hydrogel microspheres containing FITC-BSA to the Achilles tendon tissue, and let it stand for 10 minutes. Subsequently, the Achilles tendon tissue adhered with hydrogel microspheres (prepared in Example 4) was immersed in physiological saline and violently shaken. Subsequently, the Achilles tendon tissue was taken out, and the FITC-BSA fluorescence was excited by a 480nm LED flashlight to observe the residual hydrogel on the surface of the Achilles tendon, and compared with the hydrogel microsphere component without DAHA. The experimental results confirmed that after soaking and violent shaking, the hydrogel microspheres prepared in Example 4 can still firmly adhere to the surface of the Achilles tendon tissue, while the hydrogel microspheres without DAHA fall off in large quantities during the soaking and shaking process.

[0075] Furthermore, 50 μL of the hydrogel microspheres containing fluorescence prepared in Example 4 and the hydrogel microspheres containing fluorescence-labeled DAHA components were injected into the Achilles tendon gap of the left / right hind limbs of SD rats. The Achilles tendon and its surrounding tissues were surgically exposed at 7d and 14d after injection, respectively, and 480nm LED flashlight was used to observe the adhesion of the injected hydrogel to the Achilles tendon of the rat. The experimental results showed that the hydrogel microspheres of Example 4 can adhere well to the Achilles tendon and surrounding tissues, while the hydrogel microspheres without DAHA have less retention at 14d.

[0076] 8. Evaluation of SC-sEVs-loaded hydrogel microspheres in relieving tendinopathy symptoms in rats

[0077] First, a tendinopathy model was established by injecting 100 μL of 4% carrageenan solution into the right quadriceps tendon of rats through a 27-gauge needle. Starting one week after modeling, 100 μL of iMSC-sEVs hydrogel microspheres prepared in Example 4 were administered around the right quadriceps tendon of rats every week for intervention, and a sham operation group and a PBS intervention group were set up as controls. The above experimental group and the control group were successfully injected around the quadriceps tendon of rats under ultrasound guidance. Next, the pain-relieving effect of the hydrogel microspheres prepared in Example 4 was evaluated by pain-related behavioral experiments. The results of the static weight bearing (SWB) and Hind-paw withdrawal threshold (PWT) experiments showed that compared with the PBS intervention group, the hydrogel microspheres loaded with stem cell exosomes could significantly relieve the pain of rats with tendinopathy after 4 weeks of intervention. After 4 weeks of intervention, the rats were killed and samples were taken in parallel, and H&E staining was performed on the lesions of the quadriceps tendon of the rats to evaluate the abnormalities of the tendon tissue. H&E staining results showed that the quadriceps tendon in the hydrogel microsphere group was more regular and continuous than that in the PBS group, and had less inflammatory cell infiltration and angiogenesis.

[0078] The Pain Withdrawal Threshold (PWT) test is often used to evaluate the reflex pain level of animals induced by external stimuli. The specific experimental method is as follows: Place the rat in a transparent cage where it can move freely, and place a mirror under the transparent cage to facilitate observation of the rat's sole; Before the formal test begins, let the rat fully adapt to the transparent cage environment for half an hour to one hour to eliminate the influence of factors such as rat fright; Next, use the probe of the von-Frey electronic analgesia meter to vertically apply force to the central part of the rat's sole (intensity is about 0 to 100 grams); During the gradual force application process, if the rat is observed to lift its hind paw for the first time or to swing its leg, lick, etc., it is considered positive, and the current stimulation pressure value PWT is recorded in grams (g); For each rat, three tests are performed at the same time point. The average of the three test results is taken as the PWT at that time point.

[0079] The static weight bearing experiment (SWB) is to measure the weight-bearing level between the lower limbs of rats in a resting state to evaluate whether the weight-bearing posture of the lower limbs is balanced, which indirectly reflects the pain level of the lower limbs; if the weight-bearing level of one lower limb is significantly reduced, it indicates that the rat has pain in the lower limb on that side; before the formal experiment, let the rats fully adapt to the static weight bearing instrument environment for half an hour to one hour to eliminate the influence of factors such as rats being frightened; when the rats are in a relatively quiet state as a whole, the lower limbs stand stably and act on the pressure sensor at the base of the instrument, and the value read on the instrument is the force of the lower limbs on both sides, in grams (g); further, the SWB index is calculated by the following formula: SWB index = right lower limb force / bilateral lower limb force (left lower limb force + right lower limb force); for each rat, three tests are performed at the same time point. The average of the three test results is taken as the SWB result at that time point.

[0080] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An injectable, tissue-adhesive exosome controlled-release hydrogel microsphere, characterized in that: It is composed of the following components: 2-furylamine modified hyaluronic acid, as shown in structural formula I; Maleimide-terminated polyethylene glycol, as shown in structural formula II; Dopamine-modified hyaluronic acid, represented by structural formula III; and Exosomes secreted by stem cells; In the exosome controlled-release hydrogel microsphere component, 2-furanylmethylamine-modified hyaluronic acid and terminal maleimide-modified polyethylene glycol are used to form hydrogel microspheres through Diels-Alder cross-linking reaction; dopamine-modified hyaluronic acid achieves adhesion of hydrogel microspheres to the tissue interface and prolongs the sustained release time of exosomes through multiple non-covalent bonding effects between tissue proteins and polysaccharide molecules mediated by dopamine groups; stem cell exosomes are used to exert the therapeutic function of diseases; In the hydrogel microspheres, the mass concentration of hyaluronic acid modified with 2-furanylmethylamine is 1% to 5%, the mass concentration of polyethylene glycol modified with terminal maleimide is 1% to 10%, the mass concentration of hyaluronic acid modified with dopamine is 1% to 5%, and the particle concentration of exosomes secreted by stem cells is 1×10 9 / mL~5×10 11 / mL.

2. The injectable, tissue-adhesive exosome controlled-release hydrogel microspheres according to claim 1, characterized in that: In the hydrogel microspheres, the mass concentration of hyaluronic acid modified with 2-furanylmethylamine was 2.5%, the mass concentration of polyethylene glycol modified with terminal maleimide was 5%, the mass concentration of hyaluronic acid modified with dopamine was 1.5%, and the particle concentration of exosomes secreted by stem cells was 1×10 9 / mL~5×10 11 / mL.

3. The injectable, tissue-adhesive exosome controlled-release hydrogel microspheres according to claim 1, characterized in that: The modification rate of 2-furylmethylamine in the hyaluronic acid modified with 2-furylmethylamine is 20% to 80% of the total number of hyaluronic acid carboxyl groups; The molecular weight of the maleimide-modified polyethylene glycol is 1KDa to 10KDa; In the dopamine-modified hyaluronic acid, the modification rate of dopamine is 20% to 80% of the total number of carboxyl groups of the hyaluronic acid.

4. A method for preparing an injectable, tissue-adhesive exosome controlled-release hydrogel microsphere according to any one of claims 1 to 3, characterized in that: The details are as follows: Under sterile conditions, a certain amount of 2-furylmethylamine-modified hyaluronic acid, terminal maleimide-modified polyethylene glycol, and dopamine-modified hyaluronic acid are fully dissolved in a biocompatible medium to prepare a hydrogel precursor solution, and the solution is fully mixed with a biocompatible medium suspension of exosomes secreted by stem cells to obtain a hydrogel precursor solution; The hydrogel precursor solution is added dropwise to mineral oil containing a certain concentration of Span 80 according to a certain volume ratio, and stirred for a certain period of time to obtain hydrogel microspheres loaded with stem cell exosomes; The hydrogel microspheres were centrifuged and washed with ether to remove residual mineral oil and surfactant Span 80. The hydrogel microspheres were allowed to stand at room temperature under sterile conditions to allow the ether to evaporate, and finally SC-sEVs-loaded hydrogel microspheres were obtained, which were injectable, tissue-adhesive exosome controlled-release hydrogel microspheres.

5. The method for preparing an injectable, tissue-adhesive exosome controlled-release hydrogel microsphere according to claim 4, characterized in that: The biocompatible medium is physiological saline, physiological buffer or cell culture medium.

6. The method for preparing an injectable, tissue-adhesive exosome controlled-release hydrogel microsphere according to claim 4, characterized in that: The volume ratio of the hydrogel precursor solution to the mineral oil is 1:1000 to 1:20; The volume fraction of Span 80 is 0.1% to 10%.

7. A kit containing the injectable, tissue-adhesive exosome controlled-release hydrogel microspheres according to any one of claims 1 to 3, characterized in that: The kit consists of two parts: injectable, tissue-adhesive exosome controlled-release hydrogel microspheres loaded in a disposable syringe and normal saline.

8. The kit according to claim 7, characterized in that The disposable syringe is 1 mL, the capacity of the injectable, tissue-adhesive exosome controlled-release hydrogel microspheres is 250 μL, and the loading amount of pluripotent stem cell-derived exosomes is 1×10 11 / mL, the volume of normal saline is 500μL.

9. Use of the injectable, tissue-adhesive exosome controlled-release hydrogel microspheres according to any one of claims 1 to 3 in the preparation of a medicament for treating tendon diseases.

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