Hydrogel for exosome delivery, method for preparing same, and use thereof

Abstract

The present invention relates to the technical field of hydrogels, and in particular, to a hydrogel for exosome delivery, a method for preparing same, and use thereof. The hydrogel for exosome delivery prepared in the present invention integrates various functions, features good biocompatibility, injectability, self-healing, tissue adhesion, exosome binding and other properties, and is thus beneficial to maintaining the integrity of a hydrogel scaffold and the integration of the hydrogel and the host tissue. The hydrogel can be further prepared into an exosome-hydrogel system, which is beneficial to the retention and long-term sustained or controlled release of the exosome at a lesion and continuous exertion of healing effects, so as to improve the tissue regeneration quality and efficiency.

Description

A hydrogel for exosome delivery and its preparation and application Technical Field

[0001] The present invention relates to the technical field of hydrogels, and in particular to a hydrogel for exosome delivery and its preparation and application. Background Art

[0002] Clinically, there is a huge demand for repairing critical-size tissue defects, but its treatment still faces severe challenges. Existing therapies, such as autologous tissue transplantation and cell-based therapies, have many limitations, including limited donor tissue, potential immune rejection, the risk of tumorigenesis, high costs, and ethical issues. In recent years, therapeutic strategies based on extracellular vesicles, especially exosomes, have been proposed as new methods to promote tissue regeneration. This is because they contain rich components such as proteins, nucleic acids, and lipids, have the potential to serve as effective drug delivery vehicles, and have many advantages, such as low immunogenicity, easy cross-biological barriers, and easy engineering modification to obtain special functions.

[0003] In existing exosome-based and engineered exosome-based therapies, exosomes are typically mixed with phosphate-buffered saline and injected into the desired treatment area via a syringe and needle. Disadvantages of this approach include rapid diffusion of exosomes through body fluids, difficulty accumulating at the affected site, low bioavailability, and the need for multiple dosing. There are also reports on the use of hydrogels to encapsulate exosomes in therapeutics. Hydrogels are hydrophilic, porous, and flexible materials composed of three-dimensional polymer networks with high water content that closely mimic the extracellular matrix. Over the past few decades, hydrogels have attracted significant research attention due to their promising applications in tissue engineering. However, existing hydrogels typically suffer from low mechanical strength and are prone to rupture when subjected to external tension. They also lack tissue adhesion, hindering good tissue integration and prone to migration from the defect area. Furthermore, due to a lack of exosome binding sites, exosomes are released too quickly, making sustained therapeutic efficacy difficult. There is an urgent need to develop functionally optimized hydrogels for exosome delivery. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention aims to provide a hydrogel for exosome delivery, and its preparation and application. The hydrogel for exosome delivery prepared by the present invention integrates multiple functions, exhibits excellent biocompatibility, and exhibits injectable, self-healing, tissue-adhesive, and exosome-binding properties, which facilitate the maintenance of the integrity of the hydrogel scaffold and the integration of the hydrogel with the host tissue. Furthermore, the resulting exosome-hydrogel system facilitates the retention of exosomes in the affected area and their long-term sustained release, facilitating the sustained therapeutic effect and improving the quality and efficiency of tissue regeneration.

[0005] The hydrogel for exosome delivery of the present invention is composed of specific hydrazide-grafted hyaluronic acid HA-A and aldehyde-modified hyaluronic acid HA-C, and has self-healing, tissue adhesion and exosome binding properties to meet the expected requirements of exosome delivery and tissue defect regeneration and repair treatment in clinical settings.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first object of the present invention is to provide a hydrogel for exosome delivery, wherein the hydrogel for exosome delivery is a HA-AC hydrogel, which is obtained by mixing hydrazide-grafted hyaluronic acid (abbreviated as "HA-A") and aldehyde-modified hyaluronic acid (abbreviated as "HA-A").

[0008] In one embodiment of the present invention, the molar ratio of the hydrazide-grafted hyaluronic acid to the aldehyde-modified hyaluronic acid is 1-2:1-3.

[0009] A second object of the present invention is to provide a method for preparing a hydrogel for exosome delivery, comprising the following steps:

[0010] The hydrazide-grafted hyaluronic acid and the aldehyde-modified hyaluronic acid were mixed and reacted to prepare the HA-AC hydrogel.

[0011] In one embodiment of the present invention, the preparation of hydrazide-grafted hyaluronic acid comprises the following steps:

[0012] After dissolving hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 1-hydroxy-benzotriazole are added and mixed for reaction, and then ADH is added, mixed for reaction, and then post-processed to obtain hydrazide-grafted hyaluronic acid.

[0013] In one embodiment of the present invention, the molar ratio of hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 1-hydroxy-benzotriazole and ADH is 1:2-6:2-6:10-30.

[0014] In one embodiment of the present invention, during the reaction, the temperature is room temperature and the reaction time is 12 to 36 hours.

[0015] In one embodiment of the present invention, the post-treatment is deep dialysis using deionized water (molecular cut-off of 10,000 MWCO) to purify the reaction product.

[0016] In one embodiment of the present invention, the preparation of aldehyde-modified hyaluronic acid comprises the following steps:

[0017] HA is dissolved and reacted with sodium periodate. After the reaction is completed, the reaction is stopped and post-processed to obtain aldehyde-modified hyaluronic acid.

[0018] In one embodiment of the present invention, the molar ratio of HA to sodium periodate is 0.5-1.5:0.5-1.5; during the reaction, the temperature is room temperature and the reaction time is 4-8 hours.

[0019] In one embodiment of the present invention, the post-treatment is deep dialysis using deionized water (molecular cut-off of 10,000 MWCO) to purify the reaction product.

[0020] The third object of the present invention is to provide a use of a hydrogel for exosome delivery in the preparation of an exosome-hydrogel system.

[0021] The fourth object of the present invention is to provide an exosome-hydrogel system obtained by mixing exosomes, hydrazide-grafted hyaluronic acid and aldehyde-modified hyaluronic acid.

[0022] A fifth object of the present invention is to provide a method for preparing an exosome-hydrogel system, comprising the following steps:

[0023] The exosomes are mixed with hydrazide-grafted hyaluronic acid, and then mixed with aldehyde-modified hyaluronic acid and reacted to obtain an exosome-hydrogel system.

[0024] In one embodiment of the present invention, the usage ratio of exosomes, hydrazide-grafted hyaluronic acid, and aldehyde-modified hyaluronic acid is 10-100 μg:0.5 mL:0.5 mL.

[0025] The sixth object of the present invention is to provide an application of an exosome-hydrogel system in the preparation of a bone defect repair drug.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The exosome delivery hydrogel of the present invention exhibits excellent biocompatibility and possesses multiple practical functions that meet clinical needs, including injectability, self-healing, tissue adhesion, and exosome binding properties. It is of great significance for the regeneration and repair of lacunar, anatomically complex, and irregular tissue defects. The exosome delivery hydrogel prepared using the present invention improves the efficiency of encapsulating, retaining, delivering, and releasing exosomes to target tissue sites, optimizing the hydrogel's potential for tissue engineering applications.

[0028] (1) The hydrogel for exosome delivery of the present invention can be directly gelled in situ at the tissue defect site, the reaction is mild and rapid, no additional cross-linking reagents need to be introduced, and it is non-toxic and harmless.

[0029] (2) Exosomes can be premixed with hydrazide-grafted hyaluronic acid before use, which is convenient for preoperative preparation. The hydrogel precursor solution has a certain fluidity and is easy to fill and store, which facilitates industrial production.

[0030] (3) The hydrogel for exosome delivery of the present invention is injectable, easy to operate, and conforms to the concept of minimally invasive treatment.

[0031] (4) The initial hydrogel solution can fill defects of any shape and can fit tightly with the defective tissue, which is of great significance for filling defects of complex morphology tissues.

[0032] (5) The hydrogel for exosome delivery of the present invention can adhere to the tissue while forming a gel, preventing the hydrogel from dislocating, and seamlessly connects with the host tissue to form a perfect integration.

[0033] (6) The hydrogel for exosome delivery of the present invention can heal itself after being damaged by external forces, which is beneficial to maintaining the integrity of the hydrogel scaffold.

[0034] (7) The hydrogel for exosome delivery of the present invention has exosome binding sites, which is beneficial to the retention of exosomes in the affected area and long-term sustained release, and is beneficial to the continued exertion of therapeutic effects.

[0035] (8) The hydrogel for exosome delivery of the present invention has good biocompatibility and strong adjustability, and the hydrogel preparation and application parameters can be customized for different tissue application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 is a diagram showing the working principle of the hydrogel for exosome delivery of the present invention;

[0037] FIG2 is an NMR diagram of HA-A;

[0038] Figure 3 is a FITR graph of HA-C;

[0039] FIG4 is a schematic diagram of the storage modulus and loss modulus of HA-AC hydrogels with different concentrations;

[0040] FIG5 is a schematic diagram of the gelation time of HA-AC hydrogels with different concentrations;

[0041] FIG6 is a schematic diagram of the swelling rate of HA-AC hydrogels with different concentrations;

[0042] FIG7 is a schematic diagram of HA-AC hydrogel injected into PBS solution;

[0043] FIG8 is a schematic diagram of rheological testing of the self-healing properties of HA-AC hydrogel;

[0044] FIG9 is a schematic diagram of a macroscopic image of the self-healing performance of HA-AC hydrogel;

[0045] FIG10 is a scanning electron micrograph of HA-AC hydrogel tissue adhesion;

[0046] FIG11 is a schematic diagram of the HA-AC hydrogel tissue adhesion mechanics test;

[0047] FIG12 is a schematic diagram of cell activity in co-culture of HA-AC hydrogel and stem cells;

[0048] FIG13 is a schematic diagram of apoptosis levels of cells co-cultured with HA-AC hydrogel and stem cells;

[0049] FIG14 is a schematic diagram of the distribution of exosomes in HA-AC hydrogel;

[0050] FIG15 is a schematic diagram of the release curve of exosomes in HA-AC hydrogel;

[0051] FIG16 is a schematic diagram of the exosome-hydrogel system promoting the regeneration and repair of bone defects. DETAILED DESCRIPTION

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

[0053] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0054] In the following embodiments, the exosomes can be exosomes or engineered exosomes derived from any cell with regenerative properties.

[0055] Example 1

[0056] This embodiment provides a hydrogel for exosome delivery, an exosome-hydrogel system, and a preparation method thereof.

[0057] (S1) Preparation of hydrazide-grafted hyaluronic acid (HA-A): Hyaluronic acid (HA) was dissolved in MES buffer (pH = 5.3), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT) were added to activate HA for 2 hours. Next, ADH was added to the mixed solution, and the pH was adjusted to 6.8 every 30 minutes for 4 hours. The reaction was then continued for 24 hours without pH adjustment. After completion of the reaction, the reaction product was purified by deep dialysis against deionized water (molecular weight cutoff 10,000 MWCO) to obtain hydrazide-grafted hyaluronic acid.

[0058] Among them, HA:EDC:HOBT:ADH=1:4:4:20 (molar ratio).

[0059] Aldehydated hyaluronic acid (HA-C) was prepared by dissolving HA in deionized water and reacting it with sodium periodate (NaIO4) in the dark for 6 hours at a molar ratio of HA:NaIO4 = 1:1. The reaction was then terminated by the addition of 1% (final concentration) of ethylene glycol. Following completion of the reaction, the product was thoroughly purified by dialysis against deionized water (molecular weight cutoff 10,000 MWCO) in the dark to yield aldehydated hyaluronic acid.

[0060] Exosome preparation: Bone marrow mesenchymal stem cells were expanded in a conventional culture medium and then cultured for 48 hours in an exosome-free culture medium (37°C, 5% CO2 incubator). The cell culture medium was collected and exosomes were isolated from the culture medium by continuous centrifugation. The continuous centrifugation steps are as follows:

[0061] 1) Centrifuge the collected liquid at 300g for 10 minutes at 4°C, discard the precipitate, and collect the supernatant;

[0062] 2) The collected supernatant was centrifuged at 2000 g for 20 minutes at 4°C, the precipitate was discarded, and the supernatant was collected;

[0063] 3) The collected supernatant was centrifuged at 10,000 g for 30 minutes at 4°C, the precipitate was discarded, and the supernatant was collected;

[0064] 4) Filter the supernatant using a 0.22 μm filter, then centrifuge at 100,000 × g for 90 minutes at 4°C in an ultracentrifuge, discard the supernatant, and retain the precipitate;

[0065] 5) The precipitate was washed with PBS and ultracentrifuged again at 100,000 g for 90 minutes. The resulting precipitate was resuspended in a small amount of PBS to obtain exosomes.

[0066] (S2) The hydrazide-grafted hyaluronic acid prepared in step (S1) and the aldehyde-modified hyaluronic acid are mixed in a volume ratio of 1:1 to obtain a hydrogel for exosome delivery: HA-AC hydrogel.

[0067] Furthermore, 50 μg of exosomes were mixed with 0.5 mL of hydrazide-grafted hyaluronic acid (mass fraction 2%), and then with 0.5 mL of aldehyde-modified hyaluronic acid (mass fraction 2%), and then cross-linked to obtain an exosome-hydrogel system.

[0068] Performance Analysis:

[0069] HA-AC reaction principle: As shown in Figure 1, HA-A and HA-C can self-assemble and cross-link through the interaction between ADH and aldehyde groups in a mild and rapid reaction, forming dynamically reversible C=N covalent bonds, thereby achieving self-healing properties; in addition, aldehyde groups can combine with amine groups on tissues or exosomes.

[0070] Characterization of HA-A: Purified HA-A was dissolved in D2O and analyzed by nuclear magnetic resonance (NMR). As shown in Figure 2, the appearance of the characteristic peak of ADH indicated the successful synthesis of HA-A.

[0071] Characterization of HA-C: The purified HA-C was characterized by Fourier transform infrared spectroscopy (FTIR). As shown in Figure 3, 1716 cm -1 The characteristic peak at indicated the successful synthesis of HA-C.

[0072] Storage modulus and loss modulus detection of HA-AC hydrogel: The time sweep oscillation test of HA-AC hydrogels with different concentrations was carried out in a rotational rheometer in CD mode at 1 Hz and 1% strain. As shown in Figure 4, the storage modulus of HA-AC hydrogel increased with the increase of solid content, and the solid content of 3% could exceed 10,000 Pa, indicating that HA-AC hydrogel has excellent mechanical properties and stability.

[0073] HA-AC hydrogel gelation time: The gelation time of HA-AC hydrogels at different concentrations was tested using the vial tilt method. Specifically, HA-AC hydrogel was added to a glass vial and allowed to stand at 37°C for a period of time. The remaining standing time was recorded when the HA-AC hydrogel solution stopped flowing after the vial was tilted. As shown in Figure 5, the gelation time of HA-AC hydrogel was inversely proportional to its concentration. As the concentration increased from 0.5% to 3%, the gelation time of HA-AC hydrogel decreased from over 25 minutes to under 30 seconds.

[0074] HA-AC hydrogel swelling rate test: HA-AC hydrogels with different concentrations were immersed in DPBS (pH = 7.4) at 37 ° C for 24 hours. The initial weight of the sample (W0) and the weight of the sample after swelling (W1) were recorded to calculate the swelling rate using the following equation: Swelling rate (%) = (W1 / W0-1) * 100%. As shown in Figure 6, the swelling rate of HA-AC hydrogel is proportional to its concentration. Hydrogels with higher solid content show a larger swelling ratio. When the concentration changes from 0.5% to 3%, the swelling ratio ranges from about 7% to about 60%. Taking into account the mechanical properties, gelation time and swelling rate, a concentration of 2% was selected for subsequent experiments.

[0075] Injectability of HA-AC hydrogel: HA-AC hydrogel was injected into PBS solution, and it was found that HA-AC hydrogel had excellent injectability (as shown in FIG7 ).

[0076] Rheological Characterization of the Self-Healing Behavior of HA-AC Hydrogels: HA-AC hydrogels were subjected to oscillation sweep tests in a rotational rheometer over a strain range of 1% to 10,000%. The damaged HA-AC hydrogels were then allowed to self-heal for 15 minutes, and the self-healing effect was characterized by a time oscillation sweep test. As shown in Figure 8, the hydrogel structure was destroyed with increasing strain, as evidenced by G" being greater than G'. After allowing the damaged HA-AC hydrogels to self-heal for 15 minutes, the time oscillation sweep test revealed that the damaged HA-AC hydrogels had recovered their initial G' / G", demonstrating significant self-healing properties.

[0077] Macroscopic observation of the self-healing ability of HA-AC hydrogel: HA-AC hydrogels, gelled into sheets and dyed with different colors, were cut into two halves and then attempted to reconnect after swapping. Figure 9 shows a macroscopic image of the self-healing properties of HA-AC. The bisected HA-AC hydrogel was swapped and then attempted to reassemble. The reassembled hydrogel sheet showed a blurred boundary at the healing interface, re-forming a new, complete sheet.

[0078] Scanning electron microscopy observation of the microstructure of HA-AC hydrogel: The HA-AC hydrogel was freeze-dried and broken, and then the surface was sprayed with gold. Next, the sample was photographed by scanning electron microscopy (Zeiss, Germany). In order to evaluate the integration of HA-AC hydrogel and tissue, HA-AC hydrogel was applied to the tissue and gelled in situ. After freeze-drying and breaking and gold spraying, the sample was observed by scanning electron microscopy. As shown in Figure 10, in order to evaluate the excellent tissue adhesion and integration ability of HA-AC hydrogel, the adhesion interface of hydrogel to tissue was observed using scanning electron microscopy. The seamless contact between HA-AC hydrogel and tissue confirmed their strong adhesion and integration.

[0079] HA-AC hydrogel tissue adhesion testing: Tissue adhesion was tested using a lap shear test. Fresh skin (10 mm × 10 mm) was adhered to a glass slide using cyanoacrylate glue. The HA-AC hydrogel was evenly applied to the skin surface. A second skin-attached glass slide was placed skin-to-skin on the first glass slide. After in situ gelation at 37°C, the bonded test samples were subjected to tensile mechanical testing using a universal testing machine. The tissue adhesion strength of the HA-AC hydrogel was determined by dividing the maximum tensile stress by the bonded area. As shown in Figure 11, the results show that the HA-AC hydrogel exhibited higher adhesion strength than both the HAMA hydrogel and commercial fibrin glue. When the HA-AC hydrogel was loaded with exosomes (multifunctional exosome-delivered hydrogel), the adhesion strength decreased slightly, likely due to the occupancy of some aldehyde sites by the exosomes. However, the multifunctional exosome-delivered hydrogel still exhibited stronger adhesion than both the HAMA and fibrin glue.

[0080] Cell viability assays for HA-AC hydrogel co-culture with stem cells: MSCs were incubated with HA-A, HA-C, and the resulting HA-AC hydrogel using a transwell model to assess the cytocompatibility of the HA-AC hydrogel. Cell viability was assessed using a CCK-8 assay at designated time points: 1, 3, and 5 days. As shown in Figure 12, the results demonstrate that neither the hydrogel precursors HA-A and HA-C nor the resulting HA-AC hydrogel compromised cell viability.

[0081] Apoptosis testing of stem cells co-cultured with HA-AC hydrogels: MSCs were cultured on the surface of HA-AC hydrogels, with cells seeded on a well plate serving as a control. After 3 days, apoptosis was measured using flow cytometry. As shown in Figure 13, the results showed that the apoptosis rate of stem cells cultured on the HA-AC hydrogel surface did not change significantly compared to the control group.

[0082] Exosome distribution in the multifunctional exosome delivery hydrogel: Exosomes were first mixed into a HA-A hydrogel solution and then gelled with an equal amount of HA-C solution. Subsequently, the reconstructed sample was scanned using a confocal laser scanning microscope. As shown in Figure 14, the results show that the red fluorescent-labeled exosomes were uniformly distributed throughout the HA-AC hydrogel.

[0083] Exosome release profiles from the multifunctional exosome delivery hydrogel: Equal amounts of exosomes were added to the HA-AC hydrogel precursor solution and the HAMA hydrogel precursor solution, respectively. After gelation, the samples were immersed in PBS buffer. At different time points, 10 μL of PBS was collected and supplemented with an equal amount of fresh PBS. Exosome release was detected using a BCA protein assay kit. As shown in Figure 15, compared with HAMA, the exosomes encapsulated in the HA-AC hydrogel (multifunctional exosome delivery hydrogel) had a smoother release profile and a longer duration, which may be attributed to the binding of the exosomes to the aldehyde groups in the hydrogel.

[0084] Micro-CT evaluation of the regenerative and repair effects of a hydrogel / exosome system (multifunctional exosome-delivering hydrogel) on diabetic bone defects: Four-week-old male Sprague Dawley rats were fed a high-fat diet for four weeks and then injected intraperitoneally with streptozotocin at a dose of 30 mg / kg. One week later, rats with blood glucose levels consistently above 16.7 mM were considered diabetic. Subsequently, the diabetic rats were randomly divided into three groups: 1) Balnk group, 2) Gel group, and 3) Gel-Exo group. Under general anesthesia, the surgical site was shaved and disinfected. A 3 mm diameter and 2.5 mm deep bone defect was created near the medial epicondyle of each rat using a drill. The defects were then fully filled with the different hydrogels, and the surgical site was sutured layer by layer. Four weeks later, the rats were sacrificed, and the femurs were removed and fixed in 10% neutral formalin. The samples were scanned and analyzed using a μCT50 (Scanco Medical, Switzerland) at 70 kVp, 114 μA, and 15 μm resolution. The defect area was selected as the region of interest (ROI) for quantitative analysis of bone volume / tissue volume (BV / TV) and bone mineral density (BMD). As shown in Figure 16, compared with the control group (Blank), the HA-AC hydrogel group (Gel) formed more new bone, and the multifunctional exosome-delivered hydrogel group (Gel-Exo) showed the best bone regeneration effect, indicating that the prepared multifunctional exosome-delivered hydrogel has good potential for tissue engineering applications.

[0085] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the explanations 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. A hydrogel for exosome delivery, characterized in that, The hydrogel for exosome delivery is HA-A-C hydrogel, which is obtained by mixing hydrazide-grafted hyaluronic acid and aldehyde-functionalized hyaluronic acid.

2. The hydrogel for exosome delivery according to claim 1, wherein, The molar ratio of the hydrazide-grafted hyaluronic acid to the aldehyde-functionalized hyaluronic acid is 1-2:1-3.

3. A method for preparing a hydrogel for exosome delivery according to any one of claims 1 to 2, characterized in that, It includes the following steps: Mix the hydrazide-grafted hyaluronic acid and the aldehyde-functionalized hyaluronic acid and react to prepare the HA-A-C hydrogel.

4. The preparation method of a hydrogel for exosome delivery according to claim 3, characterized in that, The preparation of the hydrazide-grafted hyaluronic acid includes the following steps: Dissolve hyaluronic acid, add N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 1-hydroxy-benzotriazole and mix them for reaction, then add ADH, mix them for reaction and perform post-treatment to obtain the hydrazide-grafted hyaluronic acid; Among them, the molar ratio of hyaluronic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 1-hydroxy-benzotriazole and ADH is 1:2-6:2-6:10-30; During the reaction process, the temperature is room temperature and the time is 12-36 h.

5. The preparation method of a hydrogel for exosome delivery according to claim 3, wherein, The preparation of the aldehyde-functionalized hyaluronic acid includes the following steps: Dissolve HA and react with sodium periodate, stop the reaction after the reaction is completed, and perform post-treatment to obtain the aldehyde-functionalized hyaluronic acid; Among them, the molar ratio of the HA to the sodium periodate is 0.5-1.5:0.5-1.5; during the reaction process, the temperature is room temperature and the time is 4-8 h.

6. Use of a hydrogel for exosome delivery as described in any one of claims 1-2 in the preparation of an exosome-hydrogel system.

7. An exosome-hydrogel system, characterized in that, It includes exosomes and the HA-A-C hydrogel as described in any one of claims 1-2.

8. A method for preparing the exosome-hydrogel system according to claim 7, characterized in that, It includes the following steps: Mix the exosomes with the hydrazide-grafted hyaluronic acid, and then mix them with the aldehyde-functionalized hyaluronic acid and react to obtain the exosome-hydrogel system.

9. The preparation method of an exosome-hydrogel system according to claim 8, characterized in that, The dosage ratio of exosomes, hydrazide-grafted hyaluronic acid, and aldehyde-functionalized hyaluronic acid is 10-100 μg:0.5 mL:0.5 mL.

10. Use of an exosome-hydrogel system as described in claim 7 in the preparation of a drug for bone defect repair.

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