Platelet-rich plasma exosome-containing hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to platelet-rich plasma exosome hydrogel as well as a preparation method and application thereof. According to the hydrogel containing the platelet-rich plasma exosome, the platelet-rich plasma exosome is dispersed in modified ethylene glycol chitosan hydrogel, and the mass ratio of the platelet-rich plasma exosome to the modified ethylene glycol chitosan hydrogel is 1: (1000-2000). The CBGCTS hydrogel disclosed by the invention is used for repairing skin wounds, is beneficial to wound healing, can be used as a support of PRP-exo exosomes, and can overcome the defects caused by pure application of the exosomes. The CBGCTS (at) EXO hydrogel obtained by combining the exosome and the CBGCTS gel can be used for effectively accelerating skin wound healing by promoting reepithelialization, deposition and remodeling of an STZ-induced diabetic wound of a diabetic rat model, enhancing angiogenesis and neuronal inward growth and reducing inflammation, so that the healing of the skin wound is accelerated. Therefore, a novel non-invasive application method of the exosome with practical value is provided for skin repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular, relates to a platelet-rich plasma exosome hydrogel and a preparation method and application thereof. Background Art

[0002] The treatment of chronic refractory wounds has always been a hot topic of widespread concern around the world. The mainstream view is that reducing wound inflammatory response and improving vascular regeneration and reconstruction are the key to promoting the healing of chronic refractory wounds. Chronic refractory wounds are difficult to manage and often require coordinated interventions of multiple methods, which poses a huge challenge to clinicians. At present, conventional treatments for chronic refractory wounds include debridement, wound dressings, lesion decompression, anti-infection measures, peripheral vascular disease management, and strict blood sugar control. New treatments are also being developed, but the treatment effect of chronic refractory wounds is still not ideal. Therefore, the development of more effective treatments for chronic refractory wounds has become a key clinical challenge.

[0003] Platelet-rich gel (PRP gel) is a fibrin glue formed by adding thrombin and / or calcium to platelet-rich plasma (PRP). It has been used clinically to treat diabetic refractory wounds. However, under physiological conditions, fibrin glue is easily degraded by proteases in the wound, resulting in rapid clearance of bioactive molecules in the gel by the body. Recent studies have shown that stem cell-derived exosomes have similar biological effects to stem cells and can be used as a substitute for stem cells in bone tissue repair, thereby avoiding many risks such as mutation tumorigenesis and immune rejection caused by direct use of stem cells. At present, the research on the use of exosomes to repair wounds mainly uses exosomes through injection, but this method will cause the exosomes to be quickly cleared in the body, and the repair of full-thickness skin defects is a long-term process. Therefore, how to regulate the release rate and release cycle of exosomes at the defect site to match it well with the skin growth cycle, improve the bioavailability of exosomes, and achieve the optimal therapeutic effect is a key issue that needs to be solved when using exosomes for wound repair.

[0004] With the increasing demand for regenerative medicine, biodegradable hydrogels can effectively deliver cells / drugs for wound healing and soft tissue reconstruction, and have attracted more and more attention from researchers. Biodegradable hydrogels have a typical three-dimensional network structure and a high cell / drug encapsulation rate. They can load bioactive molecules such as exosomes into the hydrogels and accelerate the healing of diabetic wounds by inducing angiogenesis. Among them, self-healing hydrogels have the characteristics of minimally invasive local targeted drug delivery, personalized plasticity, diffusivity and in situ gelation, which are significantly better than preformed hydrogels. They have also attracted great interest from researchers due to their excellent moisturizing properties, flexible physicochemical regulation, good biocompatibility, simple operation and non-invasive drug delivery.

[0005] Therefore, the synergistic effect of platelet-rich plasma-derived exosomes and self-healing hydrogel is utilized to deliver PRP-derived exosomes to the wound area and promote wound repair, in order to provide a new solution for the treatment of chronic and refractory wounds. Summary of the invention

[0006] The purpose of the present invention is to provide a platelet-rich plasma exosome hydrogel and a preparation method and application thereof, so that the prepared hydrogel can have good therapeutic effects while having good biocompatibility, self-healing properties, antibacterial properties and tissue adhesion when used for skin wound repair treatment.

[0007] To this end, the present invention provides the following technical solutions.

[0008] The first aspect of the present invention provides a platelet-rich plasma exosome hydrogel, wherein the platelet-rich plasma exosome hydrogel has platelet-rich plasma exosomes dispersed in a modified ethylene glycol chitosan hydrogel, and the mass ratio of the platelet-rich plasma exosomes to the modified ethylene glycol chitosan hydrogel is 1:(1000-2000).

[0009] The second aspect of the present invention provides a method for preparing the platelet-rich plasma exosome hydrogel as described above, the preparation method comprising the following steps:

[0010] S1: Extraction of exosomes from platelet-rich plasma;

[0011] S2: resuspending the platelet-rich plasma exosomes in PBS to obtain a platelet-rich plasma exosome suspension;

[0012] S3: adding modified ethylene glycol chitosan to the platelet-rich plasma exosome suspension and stirring evenly, and obtaining the platelet-rich plasma exosome hydrogel CBGCTS@EXO after solidification.

[0013] As a preferred embodiment of the present invention, S1 includes:

[0014] S11: Platelet-rich plasma was obtained by fractional centrifugation using human whole blood as raw material;

[0015] S12: obtaining a concentrate by differential centrifugation of the obtained platelet-rich plasma;

[0016] S13: The concentrated solution is subjected to ultracentrifugation to obtain platelet-rich plasma exosomes.

[0017] As a preferred embodiment of the present invention, S11 includes:

[0018] S111: Centrifuge human whole blood at 160 g for 10 min, take the upper layer, and obtain the plasma containing platelets;

[0019] S112: centrifuging the platelet-containing plasma at 250 g for 15 min to obtain the upper plasma and platelet precipitate;

[0020] S113: resuspending the obtained platelets in plasma and mixing them to obtain platelet-rich plasma.

[0021] As a preferred embodiment of the present invention, S12 includes:

[0022] S121: centrifuging the obtained platelet-rich plasma at 250 g for 15 min to obtain precipitated microspheres;

[0023] S122: resuspending and activating the precipitated microspheres, first centrifuging at 4°C, 300 g for 10 min, and then centrifuging at 4°C, 2000 g for 20 min to obtain a primary supernatant;

[0024] S123: filtering the obtained primary supernatant, and centrifuging the obtained filtrate at 4°C and 4000g for 30-50 min to obtain a secondary supernatant;

[0025] S124: Concentrate and wash the obtained secondary supernatant to obtain a concentrated solution.

[0026] As a preferred embodiment of the present invention, S13 includes:

[0027] S131: ultracentrifuging the obtained concentrated solution at 4°C and 10,000 g for 70 min to obtain an ultracentrifugation supernatant;

[0028] S132: ultracentrifuging the obtained ultracentrifugation supernatant at 4°C and 100,000 g for 60 min, and collecting the precipitate, which is the platelet-rich plasma exosomes;

[0029] S133: The obtained platelet-rich plasma exosomes are purified, centrifuged, sterilized, concentrated, and stored at -80°C for later use.

[0030] As a preferred embodiment of the present invention, S3 includes:

[0031] S31: mixing hydroxyethyl glycol chitosan and 4-carboxybenzaldehyde modified polyethylene glycol, and then grinding to obtain modified glycol chitosan;

[0032] S32: Dissolve the modified ethylene glycol chitosan in the platelet-rich plasma exosome suspension, and after solidification, obtain the platelet-rich plasma exosome hydrogel CBGCTS@EXO.

[0033] As a preferred embodiment of the present invention, in step S21,

[0034] The mass ratio of the hydroxyethyl glycol chitosan to the 4-carboxybenzaldehyde modified polyethylene glycol is 1:(0.5-2);

[0035] The preparation method of the 4-carboxybenzaldehyde modified polyethylene glycol comprises the following steps:

[0036] i) dissolving polyethylene glycol and 4-carboxybenzaldehyde in an organic solvent, and adding dicyclohexylcarbodiimide to react;

[0037] ii) adding ether to the reaction product for precipitation, and then washing the precipitate with ether once or multiple times to obtain 4-carboxybenzaldehyde-modified polyethylene glycol.

[0038] As a preferred embodiment of the present invention, in step i), the mass ratio of the polyethylene glycol, 4-carboxybenzaldehyde solution and dicyclohexylcarbodiimide is: (6-7): (0.5-2): (1.5-3.5), preferably 6.52:0.98:2.

[0039] As a preferred embodiment of the present invention, in step i), the organic solvent is selected from any one of tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, or a mixture of two or more thereof.

[0040] As a preferred embodiment of the present invention, in step i), the reaction conditions are: the temperature is room temperature, and the reaction time is 12 to 24 hours.

[0041] The third aspect of the present invention provides a product for promoting skin wound repair and / or replacing skin mucosa, wherein the product comprises the platelet-rich plasma exosome hydrogel as described above.

[0042] The fourth aspect of the present invention provides a use of the platelet-rich plasma exosome hydrogel as described above or the product as described above in the preparation of a medicament for promoting skin repair.

[0043] By means of the above technical solution, the present invention has at least the following advantages:

[0044] The CBGCTS@EXO hydrogel of the present invention is formed by dispersing platelet-rich plasma exosomes EXO in CBGCTS gel. On the one hand, the hydrogel can utilize the advantages of platelet-rich plasma-derived exosomes in promoting healing and serving as a natural carrier for drug delivery. On the other hand, it can utilize the adjustable biodegradation rate and anti-enzymatic properties of the self-healing hydrogel to achieve the effect of transporting PRP-derived exosomes to the wound area to promote wound repair, thereby providing a new direction for the treatment of chronic and refractory wounds.

[0045] The self-repairing chitosan hydrogel CBGCTS prepared by the present invention is conducive to wound healing and can be used as a scaffold for PRP-exo exosomes, which can overcome the shortcomings of simply using exosomes. The CBGCTS@EXO hydrogel obtained by combining exosomes and hydrogels can effectively accelerate skin wound healing by promoting re-epithelialization, deposition and remodeling of diabetic wounds in STZ-induced diabetic rat models, as well as enhancing angiogenesis, neuronal ingrowth and reducing the occurrence of inflammation, thereby providing a novel non-invasive application method of exosomes with practical value for skin repair.

[0046] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The preparation of EXO and CBGCTS@EXO hydrogel of the present invention and the schematic diagram of the principle of treating diabetic wounds are shown, wherein: (A): preparation of EXO; (B): preparation of CBGCTS@EXO hydrogel; (C): diabetic rats; (D): full-thickness skin damage in diabetic rats; (E): CBGCTS@EXO hydrogel in treating full-thickness skin damage in diabetes.

[0048] Figure 2 The figure shows the schematic diagram of the preparation and characterization principle of CBGCTS@EXO hydrogel, wherein A(1): structural formula of hydroxyethyl glycol chitosan (GCTS); A(2): structural formula of 4-carboxybenzaldehyde-modified polyethylene glycol (CBPEG); A(3): characterization and particle size analysis of PRP-derived exosomes EXO; A(4): CBGCTS@EXO hydrogel; B: SEM images of the surface topological structures of the three CBGCTS and CBGCTS@EXO hydrogels obtained in Examples 1-3.

[0049] Figure 3The self-healing performance and anti-enzymatic performance test of CBGCTS@EXO hydrogel are shown, where (A): the self-healing process of the self-healing hydrogel (the control group is gelatin); (B): the rheometer detects the G' and G" of the CBGCTS@EXO hydrogel and the control group hydrogel; (C): the stress change of CBGCTS@EXO hydrogel from 1% to 1000%, G' and (D): Changes in G' and G" of CBGCTS@EXO hydrogel when the critical strain changes between 1% and 1000%, and 3 cycles are detected; (E): Sustained release curve of total protein of CBGCTS@EXO and EXO-Gauze; (F): CBGCTS@EXO and EXO are stored in a sieve; (G): CBGCTS@EXO and EXO are placed in phospholipase, centrifuged at 3000 rpm, the supernatant is taken, concentrated and observed using TEM and tested by WB; (H): Electron micrograph of EXO; (I): Electron micrograph of CBGCTS@EXO; (J): WB image of CBGCTS@EXO and EXO.

[0050] Figure 4 The chemotactic effect of CBGCTS@EXO hydrogel on stem cells is shown, where: (A): CBGCTS@EXO hydrogel in the sieve for 24-72 hours; (B): Chemotactic effect of hydrogel extract and control group (PBS) on HUMSCs; (CE): Statistics of the number of HUMSCs chemotacticed by hydrogel extract at different time periods (C: extract-24h, D: extract-48h, E: extract-72h).

[0051] Figure 5 The figure shows the establishment of a full-thickness skin injury model and the wound healing process of diabetic rats, including: (A): establishment of a type 2 diabetic rat model; (B) preparation of a full-thickness skin injury model of type 2 diabetic rats, and photography and tissue extraction were performed 7, 14, and 21 days after surgery; (C): photos of the wounds of diabetic rats in each group on the 7th, 14th, and 21st days; (D): diagram of the wound healing pattern of diabetic rats in each group; (E): wound healing rates of diabetic rats in each group on the 7th, 14th, and 21st days.

[0052] Figure 6 The H&E staining and Masson staining of the wounds of diabetic rats treated with each material on the 7th, 14th and 21st days are shown, among which, (AC): H&E staining of the wounds of diabetic rats treated with each material (7 days, 14 days, 21 days); (DF): Masson staining of the wounds of diabetic rats treated with each material (7 days, 14 days, 21 days); (G): Changes in the length of the wounds of diabetic rats treated with each material; (H): Changes in the proportion of collagen fiber positive areas of diabetic rats treated with each material (the yellow dotted line is the length of the wound). DETAILED DESCRIPTION

[0053] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Hydrogels are highly cross-linked, water-swollen networks of hydrophilic polymers that are similar in structure to the natural extracellular matrix and have been used in the field of regeneration for decades. However, with advances in materials science, hydrogels can now be delivered through a standard needle and then gelled in situ. Self-healing polymer hydrogels provide a way to minimally invasively treat, fill complex tissue defects, and induce regeneration of damaged parts of the body. Compared with preformed materials, self-healing materials have the following obvious advantages: the associated implantation cost is lower, the patient's discomfort after use is significantly reduced, the loaded active ingredients are easily encapsulated and released through more complex dynamics, and they can take the shape of cavities, which is conducive to tissue regeneration.

[0055] The materials of polymer hydrogels can be either natural or synthetic. Naturally occurring polymers such as chitosan, alginate, hyaluronic acid, collagen and gelatin are inherently biodegradable. However, because these natural polymers often have significant differences between batches and are potentially immunogenic, their use is often limited. Other synthetic polymers, such as polyethylene glycol (PEG), polyacrylamide (PAM), polyvinyl alcohol (PVA) and polymethyl methacrylate (PMMA), have unlimited potential for synthesizing self-healing polymer hydrogels due to their strong mechanical properties, customizable structures and low immunogenicity, but due to the lack of specific biological functions, they must be specially treated to induce the desired response effect in vivo. Their combination form is either a copolymer composed of multiple main chain groups cross-linked together, or a polymer composed of interpenetrating networks (IPNs) constructed by the combination of oligomer chains within the polymer scaffold. This construction method allows the hydrogel material to be precisely modified to highlight the best performance of each of its components.

[0056] Newly developed self-healing hydrogels can better control the spatial variation of physical properties and loaded drug concentrations. The optimization of polymer hydrogel dressing design will largely rely on the progress of basic science and the in-depth understanding of the wound healing dynamics of complex biological systems. Self-healing polymer hydrogels have two main uses in wound healing therapy. One is to serve as a tissue scaffold to impart mechanical stability in the surrounding environment and promote tissue ingrowth; the other is as a drug delivery system, which can be designed to release more complex kinetics of effective drug components. Self-healing polymer hydrogel scaffolds can provide a regenerative template or matrix to which cells can better adhere and proliferate to damaged tissues after injury. These materials can be delivered by themselves or with pre-encapsulated cell populations. In the former case, surviving cells of the surrounding tissue migrate from the periphery into the material and interact within the scaffold to reconstruct the ideal tissue at the implantation site. The latter case is largely carried out through similar mechanisms, with the important difference that the delivered cells can interact with the native cell populations within the wound and release cytokines or other therapeutic secretions to facilitate tissue regeneration in the wound.

[0057] The following is a detailed description with specific embodiments:

[0058] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0059] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0060] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0062] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0063] Example 1: Preparation of CBGCTS@EXO hydrogel-1

[0064] 1. Preparation of CBGCTS

[0065] 6.52 g of polyethylene glycol (PEG-4000, purchased from Sinopharm Chemical Reagent Co., Ltd.) and 0.98 g of 4-carboxybenzaldehyde were dissolved in 100 mL of tetrahydrofuran, 2 g of dicyclohexylcarbodiimide was added under mechanical stirring, and then reacted at room temperature for 18 hours in a helium atmosphere to obtain a mixed solution. The obtained mixed solution was added to ether and mixed thoroughly, and then precipitated. The obtained precipitate was dried to obtain a white solid 4-carboxybenzaldehyde modified polyethylene glycol (hereinafter referred to as CBPEG, with a structural formula as shown below) Figure 2 A (2)). The obtained white solid CBPEG was dissolved in tetrahydrofuran, then added to ether for precipitation three times, and finally dried to obtain purified CBPEG powder. 1 g of hydroxyethyl glycol chitosan (hereinafter referred to as GCTS, with a structural formula as shown in FIG. Figure 2 As shown in A(1), 2 g CBPEG powder was purchased from Wako Co., Ltd., Japan, and sterilized by cobalt 60, and then ground by mortar and pestle to obtain the product modified ethylene glycol chitosan-1, referred to as CBGCTS-1.

[0066] 2. Preparation of platelet-rich plasma exosomes

[0067] Whole blood from healthy volunteers aged 21-45 years was used as raw material for the extraction and preparation of platelet-rich plasma exosomes. The specific process is as follows: after sampling the whole blood, it was placed in an anticoagulation tube containing citrate dextrose solution A (ACD-A). Before preparation, the stored whole blood was divided into 50mL centrifuge tubes, and 40mL of whole blood was placed in each tube. First, it was centrifuged at 160g for 10min. The whole blood after centrifugation was layered from top to bottom into a plasma layer containing platelets, a white blood cell layer, and a red blood cell layer. The plasma layer containing platelets was carefully aspirated and transferred to a new centrifuge tube. Then it was centrifuged at 250g for 15min to obtain the upper plasma and platelet precipitate, and the upper plasma was slowly aspirated, and finally 4mL of plasma was retained in each tube. The precipitated platelets were resuspended and mixed with the retained 4mL plasma to obtain platelet-rich plasma PRP.

[0068] The obtained PRP was centrifuged at 250g for 15min to obtain PRP precipitated microspheres. The obtained PRP microspheres were washed three times with PBS, resuspended and activated with PBS to obtain activated PRP. The activated PRP was then centrifuged at different speeds step by step. Unless otherwise specified, all subsequent centrifugations were performed at 4°C, i.e., first centrifuged at 300g for 10min, then centrifuged at 2000g for 20min, the activated platelets and residual cell debris were removed, and the obtained supernatant was transferred to a new centrifuge tube. The collected supernatant was filtered with a 0.22μm needle filter, and the filtrate was transferred to a 15mL Amicon Ultra-15 ultrafiltration centrifuge tube (Merck-Millipore) and centrifuged at 4000g for 45min. The obtained supernatant was concentrated to 250μL, and then washed three times with PBS to obtain a concentrated solution. The obtained concentrate was transferred to an ultracentrifuge tube containing 30% sucrose / heavy water and ultracentrifuged at 10000g for 70min to obtain an ultracentrifugation supernatant. The obtained ultracentrifugation supernatant was ultracentrifuged at 100000g for 60min, and the precipitate was taken, which was platelet-rich plasma exosomes PRP-Exos (hereinafter referred to as EXO). The obtained EXO was resuspended in PBS and ultracentrifuged again, and repeated 3 times to remove residual protein to obtain purified EXO. The purified EXO was washed with PBS and ultracentrifuged to obtain the EXO precipitate, which was resuspended in PBS and sterilized by filtration with a 0.22μm filter. The sterilized filtrate was ultracentrifuged at 4000g for 45min, and the obtained supernatant was concentrated to 250μL, and finally transferred to a sterile 1.5mL EP tube and stored in a -80℃ refrigerator for later use.

[0069] 3. Preparation of CBGCTS@EXO hydrogel-1

[0070] 100 μg of platelet-rich plasma exosomes were resuspended in 900 μl of PBS to obtain a platelet-rich plasma exosome suspension. 150 mg of CBGCTS-1 powder was added to the obtained platelet-rich plasma exosome suspension and mixed evenly at room temperature. After solidification, a platelet-rich plasma exosome hydrogel was prepared, referred to as CBGCTS@EXO hydrogel-1.

[0071] Example 2: Preparation of CBGCTS@EXO hydrogel-2

[0072] The only difference between this example and Example 1 is that in the process of preparing CBGCTS in step 1, 1 g of GCTS and 1 g of CBPEG powder are sterilized by cobalt 60 and then ground by mortar and pestle to obtain the product modified ethylene glycol chitosan-2, referred to as CBGCTS-2. The rest is consistent with Example 1.

[0073] Example 3: Preparation of CBGCTS@EXO hydrogel-3

[0074] The only difference between this example and Example 1 is that in the process of preparing CBGCTS in step 1, 2 g GCTS and 1 g CBPEG powder are sterilized by cobalt 60 and then ground by mortar and pestle to obtain the product modified polyethylene glycol chitosan-3, referred to as CBGCTS-3. The rest is consistent with Example 1.

[0075] like Figure 2 As shown in Figure B, the microstructures of the CBGCTS-1 hydrogel, CBGCTS-2 hydrogel, CBGCTS-3 hydrogel, CBGCTS@EXO hydrogel-1, CBGCTS@EXO hydrogel-2, and CBGCTS@EXO hydrogel-3 sponges obtained in Examples 1-3 were observed by scanning electron microscopy.

[0076] Example 4: Performance testing of CBGCTS@EXO hydrogel

[0077] 1. Self-healing of hydrogel

[0078] The CBGCTS@EXO hydrogel-2 prepared in Example 2 was used as the experimental group, and a circular defect with a diameter of 10 mm was made in the CBGCTS@EXO hydrogel-2. The gelatin gel was used as the control group, and a circular defect with a diameter of 10 mm was made on the gelatin gel in the same way. Photos were taken at 0 min, 10 min, 20 min, 30 min, and 40 min after the circular defect was made to record the self-healing of each group of hydrogels. The results are shown in Figure 3 (A).

[0079] 2. Rheological and strain properties of hydrogels

[0080] The storage modulus (G') and loss modulus (G") of the CBGCTS@EXO hydrogel-2 prepared in Example 2 at room temperature were measured using a DHR-2-TA rheometer. The G' and G" of the CBGCTS@EXO hydrogel under vibration strain conditions ranging from 1% to 1000% were tested using a DHR-2-TA rheometer. The CBGCTS@EXO hydrogel in the self-healing process was quantitatively analyzed using a DHR-2-TA rheometer. By keeping the temperature and frequency constant at 25°C and 1Hz, respectively, the strain amplitude effect of the CBGCTS@EXO hydrogel from 1% (every 100s interval) to 1000% (every 100s interval) was evaluated. The results are shown in Figure 3 (B)-(D).

[0081] 3. Protein release ability of hydrogel

[0082] 100 μg of exosomes EXO were dropped onto gauze (20×20 mm) to prepare EXO gauze, which was used as the EXO-Gauze group; the CBGCTS@EXO hydrogel prepared in Example 2 was used as the CBGCTS@EXO group; there were 3 samples in each group.

[0083] EXO gauze and CBGCTS@EXO hydrogel were immersed in PBS solution and placed in a 37°C incubator. Samples were taken at 2, 4, 8, 12, 24, 36, 48, 60 and 72 hours after incubation. The total protein content of the samples was determined using the BCA protein quantitative analysis kit, and the protein sustained release curve was drawn. The results are shown in Figure 3 (E).

[0084] 4. In vitro enzymatic hydrolysis experiment

[0085] Phospholipase (S10227-1mg, Shanghai Yuanye Biotechnology Co., Ltd.) was used to simulate the destructive effect of enzymes in diabetic skin wounds on exosomes EXO, and the effect of CBGCTS@EXO hydrogel on exosomes EXO after the action of phospholipase was detected. The specific operation diagram is shown in Figure 3 (F)-(G), namely: first, the CBGCTS@EXO hydrogel-2 prepared in Example 2 and 2 ml of exosome suspension were placed in a 500-mesh sieve respectively, and then the whole was placed in a phospholipase solution with an enzyme concentration of 0.15 IU / ml and placed at 4°C for 24 hours. The suspension in the sieve was collected with a pipette, and the supernatant was collected by centrifugation at 3000 rpm for 5 minutes. The supernatant was placed on a copper mesh, negatively stained with 3% phosphotungstic acid (pH 6.8) for 2 minutes, and the EXO morphology was observed by TEM. The expression of exosome surface marker proteins CD9, CD81, and Tsg101 was identified by Western Blot, and the results are shown in Figure 3 (H)-(J).

[0086] 5. Safety evaluation

[0087] (1) Preparation of hydrogel extract

[0088] 1 g CBGCTS hydrogel, 1 g CBGCTS@EXO hydrogel and 100 μg exosome EXO sample prepared in Example 2 were immersed in 100 ml α-MEM medium and incubated at 37°C for 24, 48 and 72 h. The α-MEM medium was replaced every day. The hydrogel extracts were collected at 24, 48 and 72 h and labeled as CBGCTS-extract-24, CBGCTS-extract-48, CBGCTS extract-72, EXO extract-24, EXO extract-48, EXO extract-72, CBGCTS@EXO extract-24, CBGCTS@EXO extract-48 and CBGCTS@EXO extract-72, respectively.

[0089] (2) In vitro chemotaxis of mesenchymal stem cells

[0090] Human umbilical cord mesenchymal stem cells (purchased from Shanghai Kanglang Biotechnology Co., Ltd.) were cultured in α-MEM culture medium without fetal bovine serum for 12 hours, and then separated by trypsin digestion to obtain a single cell suspension. α-MEM culture medium (500 μL) and 50 μL of the above extracts were added to a 24-well plate. Human umbilical cord blood mesenchymal stem cells (1×10 5 Cells) were seeded in Transwell implants with a polycarbonate membrane filter with a pore size of 8 μm. Transwell migration was carefully performed in a 24-well plate at 37°C for 12 h. After 12 h, human umbilical cord blood mesenchymal stem cells (HUMSCs) that had not migrated across the Transwell filter were gently wiped with a cotton swab. HUMSCs were fixed with 4% paraformaldehyde solution for 30 min and stained with 2-(4-aminophenyl)-6-indoleamine dihydrochloride. The migrated cells were observed using a fluorescence microscope (Nikon, Japan) and processed using Image-Pro Plus software. The results are shown in Figure 4 (B)-(E).

[0091] Example 5: Experimental study on skin wound healing by CBGCTS@EXO hydrogel

[0092] 60 male SD rats (weighing 280-320 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. All rats were housed in a room temperature (25°C), 12-hour light / dark cycle, SPF environment, and free access to water and food. All animal experimental protocols in this application were approved by the Ethics Committee of Peking University Hospital of Stomatology.

[0093] To induce diabetes, rats were fed a high sucrose and high fat diet for 10 weeks after 1 week of dietary conditioning, and then STZ (35 mg / kg, Sigma) was injected intraperitoneally at weeks 10 and 11. Fasting blood glucose and body weight levels were monitored weekly. In rats with fasting blood glucose levels of >11 mmol / L and sustained for more than 4 weeks, diabetes was considered to be successfully induced, thereby constructing a diabetic model rat.

[0094] Before surgery, the model rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg). The dorsal hair was shaved and the skin was disinfected, after which a full-thickness wound with a diameter of 20 mm was formed on the upper back of the rat. The model rats with full-thickness wounds were randomly divided into four groups, namely, the Control control group, the CBGCTS@EXO group, the EXO group, and the CBGCTS group, with 15 rats in each group. The full-thickness wounds of the above four groups of rats were covered with NS gauze (Control group), CBGCTS@EXO hydrogel-2 prepared in Example 2, EXO, and CBGCTS-2 gel, respectively. Afterwards, a wrapping bandage (Urgostrapping, URGO) was used to fix the wound and dressing. The dressing was changed twice a week as described above. The wound healing process was recorded on days 0, 7, 14, and 21. The wound area was calculated using Image J software, and then the wound closure rate was calculated: wound closure rate = (A0-A t ) / A0. A0 is the initial wound area, A t is the wound area after surgery. Figure 5 .

[0095] The wound areas of the four groups of rats were excised at 1 week, 2 weeks, and 3 weeks after surgery, including healthy skin 0.5 cm beyond the edge of the wound area, and analyzed by histopathology. The excised skin tissue was fixed in 4% paraformaldehyde, gradually dehydrated, embedded in paraffin and cut into 4 μm thick sections. The paraffin sections were stained with H&E and Masson, and then observed using MIDI-pannoramic (3DHistech, Hungary). The results are shown in Figure 6 .

[0096] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A platelet-rich plasma exosome hydrogel, characterized in that: The platelet-rich plasma exosomes hydrogel contains platelet-rich plasma exosomes dispersed in a modified ethylene glycol chitosan hydrogel, and the mass ratio of the platelet-rich plasma exosomes to the modified ethylene glycol chitosan hydrogel is 1:(1000-2000).

2. The method for preparing platelet-rich plasma exosome hydrogel according to claim 1, characterized in that: The preparation method comprises the following steps: S1: Extraction of exosomes from platelet-rich plasma; S2: resuspending the platelet-rich plasma exosomes in PBS to obtain a platelet-rich plasma exosome suspension; S3: adding modified ethylene glycol chitosan to the platelet-rich plasma exosome suspension and stirring evenly, and obtaining the platelet-rich plasma exosome hydrogel CBGCTS@EXO after solidification.

3. The preparation method according to claim 2, characterized in that: S1 includes: S11: Platelet-rich plasma was obtained by fractional centrifugation using human whole blood as raw material; S12: obtaining a concentrate by differential centrifugation of the obtained platelet-rich plasma; S13: The concentrated solution is subjected to ultracentrifugation to obtain platelet-rich plasma exosomes.

4. The preparation method according to claim 3, characterized in that: S11 includes: S111: Centrifuge human whole blood at 160 g for 10 min, take the upper layer, and obtain the plasma containing platelets; S112: centrifuging the platelet-containing plasma at 250 g for 15 min to obtain the upper plasma and platelet precipitate; S113: resuspending the obtained platelets in plasma and mixing them to obtain platelet-rich plasma.

5. The preparation method according to claim 3, characterized in that: S12 includes: S121: centrifuging the obtained platelet-rich plasma at 250 g for 15 min to obtain precipitated microspheres; S122: resuspending and activating the precipitated microspheres, first centrifuging at 4°C, 300 g for 10 min, and then centrifuging at 4°C, 2000 g for 20 min to obtain a primary supernatant; S123: filtering the obtained primary supernatant, and centrifuging the obtained filtrate at 4°C and 4000g for 30-50 min to obtain a secondary supernatant; S124: Concentrate and wash the obtained secondary supernatant to obtain a concentrated solution.

6. The preparation method according to claim 3, characterized in that: S13 includes: S131: ultracentrifuging the obtained concentrated solution at 4°C and 10,000 g for 70 min to obtain an ultracentrifugation supernatant; S132: ultracentrifuging the obtained ultracentrifugation supernatant at 4°C and 100,000 g for 60 min, and collecting the precipitate, which is the platelet-rich plasma exosomes; S133: The obtained platelet-rich plasma exosomes are purified, centrifuged, sterilized, concentrated, and stored at -80°C for later use.

7. The preparation method according to claim 2, characterized in that S3 include: S31: mixing hydroxyethyl glycol chitosan and 4-carboxybenzaldehyde modified polyethylene glycol, and then grinding to obtain modified glycol chitosan; S32: Dissolve the modified ethylene glycol chitosan in the platelet-rich plasma exosome suspension, and after solidification, obtain the platelet-rich plasma exosome hydrogel CBGCTS@EXO.

8. The preparation method according to claim 7, characterized in that: In step S31, The mass ratio of the hydroxyethyl glycol chitosan to the 4-carboxybenzaldehyde modified polyethylene glycol is 1:(0.5-2); The preparation method of the 4-carboxybenzaldehyde modified polyethylene glycol comprises the following steps: i) dissolving polyethylene glycol and 4-carboxybenzaldehyde in an organic solvent, and adding dicyclohexylcarbodiimide to react; ii) adding ether to the reaction product for precipitation, and then washing the precipitate with ether once or multiple times to obtain 4-carboxybenzaldehyde-modified polyethylene glycol.

9. A product for promoting skin wound repair and / or replacing skin and mucous membranes, characterized in that: The product comprises the platelet-rich plasma exosome hydrogel according to claim 1.

10. Use of the platelet-rich plasma exosome hydrogel according to claim 1 or the product according to claim 9 in the preparation of a medicament for promoting skin repair.

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