Preparation method of exosome-loaded hydrogel, exosome-loaded hydrogel and use thereof

Abstract

The embodiments of the present disclosure disclose a method for preparing an exosome-loaded hydrogel, an exosome-loaded hydrogel, and its uses. A specific embodiment of the method includes: oxidizing hyaluronic acid; mixing oxidized hyaluronic acid and methacrylic anhydride under preset environmental conditions to obtain a first mixed solution; dialyzing and freeze-drying the first mixed solution to obtain a target oxidized hyaluronic acid; adding the target oxidized hyaluronic acid to an exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution; mixing the exosome-loaded oxidized hyaluronic acid solution with polylysine to obtain an exosome-loaded hydrogel, wherein the mass ratio of the target oxidized hyaluronic acid to polylysine in the exosome-loaded hydrogel is in the range of 1 to 2. This embodiment can improve the mechanical properties and controllable degradability of the exosome-loaded hydrogel, thereby improving the applicability of the exosome-loaded hydrogel and the retention capacity and activity of the exosomes.

Description

Technical Field

[0001] Embodiments of the present disclosure relate to the technical field of biomedical materials, and particularly to a method for preparing an exosome-loaded hydrogel, an exosome-loaded hydrogel, and uses thereof. Background Art

[0002] As a three-dimensional hydrophilic polymer network, a hydrogel can be used as a drug delivery system to provide a moist treatment environment, promote tissue growth, and control the release of exosomes. Currently, when preparing a hydrogel loaded with exosomes, the commonly used method is to add exosomes derived from human umbilical vein endothelial cells (HUVECs-Exos) to a gelatin methacrylate (GelMA) hydrogel to prepare a hydrogel that can be used as a wound dressing.

[0003] However, it has been found in practice that when preparing an exosome-loaded hydrogel in the above manner, the following technical problems often exist:

[0004] The mechanical properties of the gelatin methacrylate hydrogel are relatively low, and the controllable degradability is poor, resulting in poor retention ability and activity of the exosomes loaded in the gelatin methacrylate hydrogel. Moreover, the gelatin methacrylate hydrogel has a relatively large viscosity, resulting in poor sprayability of the gelatin methacrylate hydrogel, thereby causing low applicability of the gelatin methacrylate hydrogel.

[0005] The above information disclosed in this background art section is only used to enhance the understanding of the background of the inventive concept, and thus, it may include information that does not form the prior art known to those of ordinary skill in the art in this country. Summary of the Invention

[0006] This content part of the present disclosure is used to briefly introduce the concepts, which will be described in detail in the following detailed implementation section. This content part of the present disclosure is not intended to identify the key features or essential features of the claimed technical solution, nor is it intended to limit the scope of the claimed technical solution.

[0007] Some embodiments of the present disclosure propose a method for preparing an exosome-loaded hydrogel, an exosome-loaded hydrogel, and uses thereof to solve the technical problems mentioned in the above background art section.

[0008] In a first aspect, some embodiments of the present disclosure provide a method for preparing a loaded exosome hydrogel, the method comprising: oxidizing hyaluronic acid to obtain oxidized hyaluronic acid; mixing the oxidized hyaluronic acid and methacrylic anhydride under preset environmental conditions to obtain a first mixed solution; subjecting the first mixed solution to dialysis freeze-drying to obtain a target oxidized hyaluronic acid, wherein the target oxidized hyaluronic acid is oxidized hyaluronic acid grafted with a methacrylate bond; adding the target oxidized hyaluronic acid to an exosome solution to obtain a loaded exosome oxidized hyaluronic acid solution; mixing the loaded exosome oxidized hyaluronic acid solution with polylysine to obtain a loaded exosome hydrogel, wherein the mass ratio of the target oxidized hyaluronic acid to polylysine in the loaded exosome hydrogel ranges from 1 to 2.

[0009] Optionally, the oxidizing hyaluronic acid to obtain oxidized hyaluronic acid includes: dissolving hyaluronic acid in water to obtain a hyaluronic acid solution, wherein the percentage concentration of the hyaluronic acid solution is 1%; mixing sodium periodate with the hyaluronic acid solution to obtain a second mixed solution, wherein the mass ratio of the sodium periodate to the hyaluronic acid is 3:5; mixing absolute ethanol with the second mixed solution to obtain a third mixed solution, wherein the solution ratio of the absolute ethanol to the second mixed solution is 1:100; subjecting the third mixed solution to dialysis freeze-drying to obtain oxidized hyaluronic acid.

[0010] Optionally, the preset environmental conditions are: the pH value range of the mixed solution is 8-9, and the environmental temperature is 4 degrees Celsius; and the mixing the oxidized hyaluronic acid and methacrylic anhydride under the preset environmental conditions to obtain a first mixed solution includes: dissolving the oxidized hyaluronic acid in distilled water to obtain an oxidized hyaluronic acid solution, wherein the concentration of the oxidized hyaluronic acid solution is 1%; in an environment with an environmental temperature of 4 degrees Celsius, mixing the oxidized hyaluronic acid solution and methacrylic anhydride for a preset duration to obtain a first mixed solution, wherein the volume ratio of the oxidized hyaluronic acid solution to the methacrylic anhydride is 100:2, and within a preset time period corresponding to the preset duration, the following steps also need to be performed: real-time detecting the pH value of the mixed solution through a pH value detection device; in response to determining that the pH value of the mixed solution is not within the pH value range, adding a sodium hydroxide solution to the mixed solution to make the pH value of the mixed solution within the pH value range, wherein the mixed solution is a solution mixed with the oxidized hyaluronic acid solution and the methacrylic anhydride.

[0011] Optionally, before adding the target oxidized hyaluronic acid to the exosome solution to obtain the exosome-loaded oxidized hyaluronic acid solution, the method further includes: isolating exosomes from umbilical cord stem cells to obtain stem cell exosomes; adding the stem cell exosomes to a phosphate buffer solution to obtain an exosome solution, wherein the ratio of the stem cell exosomes to the phosphate buffer solution is 50 micrograms: 100 milliliters.

[0012] Optionally, isolating exosomes from umbilical cord stem cells to obtain stem cell exosomes includes: centrifuging umbilical cord stem cells through a centrifuge device to obtain cell supernatant; centrifuging the cell supernatant through the centrifuge device to obtain exosome spheroids; precipitating the exosome spheroids with the phosphate buffer solution to obtain stem cell exosomes.

[0013] Optionally, adding the target oxidized hyaluronic acid to the exosome solution to obtain the exosome-loaded oxidized hyaluronic acid solution includes: determining the concentration of the hyaluronic acid solution according to the hydrogel application scenario information and the preset concentration of the polylysine solution; adding the target oxidized hyaluronic acid to the exosome solution to obtain the exosome-loaded oxidized hyaluronic acid solution corresponding to the concentration of the hyaluronic acid solution.

[0014] Optionally, determining the concentration of the hyaluronic acid solution according to the hydrogel application scenario information and the preset concentration of the polylysine solution includes: determining the exosome release rate according to the hydrogel application scenario information; determining the concentration of the hyaluronic acid solution according to the exosome release rate and the preset concentration of the polylysine solution.

[0015] Optionally, mixing and reacting the exosome-loaded oxidized hyaluronic acid solution with polylysine to obtain an exosome-loaded hydrogel includes: filling the exosome-loaded oxidized hyaluronic acid solution into an empty spray bottle through a filling device to obtain a hyaluronic acid spray bottle; filling a polylysine solution with a preset concentration into an empty spray bottle through the filling device to obtain a polylysine spray bottle; simultaneously pressing the hyaluronic acid spray bottle and the polylysine spray bottle so that the exosome-loaded oxidized hyaluronic acid solution sprayed from the hyaluronic acid spray bottle is mixed with the polylysine solution sprayed from the polylysine spray bottle at a preset spraying position to obtain a hydrogel mixed solution; performing photocuring treatment on the hydrogel mixed solution to obtain an exosome-loaded hydrogel.

[0016] In a second aspect, some embodiments of the present disclosure provide an exosome-loaded hydrogel, and the exosome-loaded hydrogel solution is prepared by the exosome-loaded hydrogel preparation method described in any implementation manner of the first aspect above.

[0017] In a third aspect, some embodiments of the present disclosure provide a use of the exosome-loaded hydrogel described in any implementation manner of the second aspect above, wherein the exosome-loaded hydrogel is used for treating skin wounds.

[0018] The above various embodiments of the present disclosure have the following beneficial effects: Through the method for preparing an exosome-loaded hydrogel according to some embodiments of the present disclosure, the mechanical properties and controllable degradability of the exosome-loaded hydrogel can be improved, thereby improving the applicability of the exosome-loaded hydrogel and the retention ability and activity of exosomes. Specifically, the reasons for the poor sprayability and applicability of gelatin methacrylate (hydrogel) are as follows: The mechanical properties of the gelatin methacrylate hydrogel are low, and the controllable degradability is poor, resulting in poor retention ability and activity of the exosomes loaded in the gelatin methacrylate hydrogel. Moreover, the viscosity of the gelatin methacrylate hydrogel is large, resulting in poor sprayability of the gelatin methacrylate hydrogel, thereby causing low applicability of the gelatin methacrylate hydrogel. Based on this, in the method for preparing an exosome-loaded hydrogel according to some embodiments of the present disclosure, hyaluronic acid is oxidized to obtain oxidized hyaluronic acid; the oxidized hyaluronic acid and methacrylic anhydride are mixed and reacted under preset environmental conditions to obtain a first mixed solution; the first mixed solution is subjected to dialysis and freeze-drying treatment to obtain target oxidized hyaluronic acid, wherein the target oxidized hyaluronic acid is oxidized hyaluronic acid grafted with methacrylate bonds; the target oxidized hyaluronic acid is added to an exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution; the exosome-loaded oxidized hyaluronic acid solution and polylysine are mixed and reacted to obtain an exosome-loaded hydrogel, wherein the mass ratio range of the target oxidized hyaluronic acid to polylysine in the exosome-loaded hydrogel is 1 to 2. Because the biocompatibility and moisturizing property of the obtained target oxidized hyaluronic acid are improved by oxidizing hyaluronic acid and grafting methacrylate bonds, the possibility of preparing a spray-type exosome-loaded hydrogel is increased. Also, because the antibacterial polylysine and the target oxidized hyaluronic acid having aldehyde groups and methacrylate bonds are combined, the exosome-loaded hydrogel obtained includes Schiff base bonds, thereby improving the antibacterial performance of the exosome-loaded hydrogel while improving the mechanical properties and controllable degradability of the exosome-loaded hydrogel, and further improving the exosome retention ability and activity of the exosome-loaded hydrogel. Thus, the mechanical properties and controllable degradability of the exosome-loaded hydrogel can be improved, thereby improving the applicability of the exosome-loaded hydrogel and the retention ability and activity of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In combination with the accompanying drawings and with reference to the following specific embodiments, the above and other features, advantages and aspects of the various embodiments of the present disclosure will become more apparent. Throughout the drawings, the same or similar reference numerals denote the same or similar elements. It should be understood that the drawings are schematic and the elements and elements are not necessarily drawn to scale.

[0020] Figure 1 is a flowchart of some embodiments of a method for preparing a loaded exosome hydrogel according to the present disclosure;

[0021] Figure 2 is the experimental result of identifying stem cell exosomes;

[0022] Figure 3 is an infrared spectrogram for characterizing hyaluronic acid, oxidized hyaluronic acid, and target oxidized hyaluronic acid respectively;

[0023] Figure 4 is a nuclear magnetic resonance characterization diagram for characterizing hyaluronic acid, oxidized hyaluronic acid, and target oxidized hyaluronic acid respectively;

[0024] Figure 5 is a scanning electron micrograph of a loaded exosome hydrogel generated at different ratios of target oxidized hyaluronic acid to polylysine;

[0025] Figure 6 is the experimental data of the rheological properties of the loaded exosome hydrogel;

[0026] Figure 7 is the experimental data of the self-healing performance of the loaded exosome hydrogel;

[0027] Figure 8 is the experimental data of the swelling performance of the loaded exosome hydrogel;

[0028] Figure 9 is the experimental data of the antibacterial performance of the loaded exosome hydrogel;

[0029] Figure 10 is the experimental data of the release of exosomes from the loaded exosome hydrogel;

[0030] Figure 11 is the experimental data of the cytotoxicity and proliferation of the loaded exosome hydrogel;

[0031] Figure 12 is the experimental data of the cell migration of the loaded exosome hydrogel;

[0032] Figure 13 is the experimental data of the angiogenesis of the loaded exosome hydrogel;

[0033] Figure 14 is the experimental data of the treatment of infectious full-thickness skin wounds with the loaded exosome hydrogel;

[0034] Figure 15 These are the toxicity experiment data of the exosome-loaded hydrogel. Specific Embodiments

[0035] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although some embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided to more thoroughly and completely understand the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for illustrative purposes and are not used to limit the protection scope of the present disclosure.

[0036] In addition, it should be noted that for ease of description, only parts related to the relevant invention are shown in the drawings. Without conflict, the embodiments in the present disclosure and the features in the embodiments can be combined with each other.

[0037] It should be noted that the concepts such as "first" and "second" mentioned in the present disclosure are only used to distinguish different devices, modules or units, and are not used to limit the order or interdependence relationship of the functions performed by these devices, modules or units.

[0038] It should be noted that the modifications of "one" and "multiple" mentioned in the present disclosure are illustrative rather than restrictive. Those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0039] The names of the messages or information exchanged between multiple devices in the embodiments of the present disclosure are only for illustrative purposes and are not used to limit the scope of these messages or information.

[0040] The present disclosure will be described in detail below with reference to the drawings and in conjunction with the embodiments.

[0041] Figure 1 Flow 100 of some embodiments of the method for preparing an exosome-loaded hydrogel according to the present disclosure is shown. The method for preparing the exosome-loaded hydrogel includes the following steps:

[0042] Step 101, oxidize hyaluronic acid to obtain oxidized hyaluronic acid.

[0043] In some embodiments, hyaluronic acid can be oxidized to obtain oxidized hyaluronic acid.

[0044] In some optional implementation manners of some embodiments, hyaluronic acid can be oxidized to obtain oxidized hyaluronic acid through the following steps:

[0045] Step 1: Dissolve hyaluronic acid (HA, Mw 200,000 - 400,000) in water to obtain a hyaluronic acid solution. The molecular structural formula of hyaluronic acid can be: The percentage concentration of the hyaluronic acid solution is 1%. As an example, 1 gram of hyaluronic acid can be dissolved in 100 milliliters of water to obtain a hyaluronic acid solution.

[0046] Step 2: Mix and react sodium periodate with the above hyaluronic acid solution to obtain a second mixed solution. The mass ratio of the above sodium periodate to the above hyaluronic acid can be 3:5. In practice, sodium periodate can be added to the above hyaluronic acid solution, and the mixture is reacted under indoor light - avoiding conditions for a preset mixing duration to obtain a second mixed solution. The above preset mixing duration can be the duration preset for completing the reaction of sodium periodate with the hyaluronic acid solution. The range of the above preset mixing duration can be 4.5 - 6 hours.

[0047] Step 3: Mix and react absolute ethanol with the above second mixed solution to obtain a third mixed solution. The solution ratio of the above absolute ethanol to the above second mixed solution can be 1:100. In practice, absolute ethanol can be added to the above second mixed solution to terminate the reaction for 1 hour to obtain a third mixed solution.

[0048] Step 4: Perform dialysis and freeze - drying on the above third mixed solution to obtain oxidized hyaluronic acid. In practice, first, the above third mixed solution can be dialyzed through deionized water and a dialysis bag to obtain wet oxidized hyaluronic acid. The dialysis duration can be 3 - 5 days. The above deionized water needs to be replaced at least 3 times during the dialysis period. Then, the above wet oxidized hyaluronic acid is vacuum freeze - dried by a vacuum freeze - dryer to obtain oxidized hyaluronic acid (OHA). The molecular formula structure of oxidized hyaluronic acid can be

[0049] As an example, first, 1 gram of hyaluronic acid can be dissolved in 100 milliliters of water to obtain a hyaluronic acid solution. Then, 2.8 mM (millimoles per liter) of sodium periodate can be added to the hyaluronic acid solution, and the mixture is reacted for 5 hours under room - temperature and light - avoiding conditions to obtain a second mixed solution. After that, 1 milliliter of absolute ethanol can be added to the second mixed solution to terminate the reaction for 1 hour to obtain a third mixed solution. Finally, the third mixed solution is dialyzed and vacuum freeze - dried to obtain oxidized hyaluronic acid (OHA).

[0050] Step 102: Mix and react oxidized hyaluronic acid and methacrylic anhydride under preset environmental conditions to obtain a first mixed solution.

[0051] In some embodiments, the above-mentioned oxidized hyaluronic acid and methacrylic anhydride can be mixed and reacted under preset environmental conditions to obtain a first mixed solution. Among them, the above-mentioned preset environmental conditions can be the conditions of the environment where the above-mentioned oxidized hyaluronic acid and methacrylic anhydride are located during the mixing reaction, which are preset in advance.

[0052] Optionally, the above-mentioned preset environmental conditions can be: the pH value range of the first mixed solution is 8-9. The environmental temperature is 4 degrees Celsius.

[0053] Optionally, the above-mentioned oxidized hyaluronic acid and methacrylic anhydride can be mixed and reacted under preset environmental conditions through the following steps to obtain a first mixed solution:

[0054] In the first step, the above-mentioned oxidized hyaluronic acid is dissolved in distilled water to obtain an oxidized hyaluronic acid solution. Among them, the concentration of the above-mentioned oxidized hyaluronic acid solution can be 1%.

[0055] In the second step, in an environment with an environmental temperature of 4 degrees Celsius, the above-mentioned oxidized hyaluronic acid solution and methacrylic anhydride are mixed for a preset duration to obtain a first mixed solution. Among them, the volume ratio of the above-mentioned oxidized hyaluronic acid solution to methacrylic anhydride can be 100:2. The above-mentioned preset duration can be a preset duration in advance. For example, the above-mentioned preset duration can be 10-15 hours. During the preset time period corresponding to the above-mentioned preset duration, the following sub-steps are executed:

[0056] In the first sub-step, the pH value of the mixed solution is detected in real time through a pH value detection device. Among them, the above-mentioned preset time period can be: the time interval from the time when methacrylic anhydride is added to the current time is the time period corresponding to the preset duration. The above-mentioned pH value detection device can be a pH meter. The pH value of the above-mentioned mixed solution can be the pH value of the mixed solution.

[0057] In the second sub-step, in response to determining that the detected pH value of the mixed solution is not within the above-mentioned pH value range, sodium hydroxide solution is added to the mixed solution to make the pH value of the mixed solution within the above-mentioned pH value range. Among them, the above-mentioned mixed solution can be a solution mixed with the above-mentioned oxidized hyaluronic acid solution and methacrylic anhydride. The concentration of the above-mentioned sodium hydroxide solution is 5 mol / L.

[0058] As an example, first, 1 gram of oxidized hyaluronic acid can be dissolved in 100 milliliters of distilled water to obtain an oxidized hyaluronic acid solution. Then, 2 milliliters of methacrylic anhydride is added to the obtained oxidized hyaluronic acid solution. After that, the pH value of the solution mixed with methacrylic anhydride and oxidized hyaluronic acid is detected in real time. When the detected pH value of the solution is not within the above-mentioned pH value range, 2 milliliters of sodium hydroxide solution is added to adjust the pH value of the solution to be within the above-mentioned pH value range. Detection is stopped after 12 hours.

[0059] Step 103: Perform dialysis and lyophilization on the first mixed solution to obtain the target oxidized hyaluronic acid.

[0060] In some embodiments, the above-mentioned first mixed solution can be subjected to dialysis and lyophilization to obtain the target oxidized hyaluronic acid. Among them, the above-mentioned target oxidized hyaluronic acid can be oxidized hyaluronic acid grafted with methacrylate bonds. In practice, the above-mentioned first mixed solution can be subjected to dialysis and lyophilization by a vacuum freeze dryer to obtain the target oxidized hyaluronic acid (OHAMA). Among them, the molecular formula structure of the target oxidized hyaluronic acid can be

[0061] Thus, the biocompatibility and moisture retention of hyaluronic acid can be improved by oxidation and grafting methacrylate bonds.

[0062] Step 104: Add the target oxidized hyaluronic acid to the exosome solution to obtain a loaded exosome oxidized hyaluronic acid solution.

[0063] In some embodiments, the above-mentioned target oxidized hyaluronic acid can be added to the exosome solution to obtain a loaded exosome oxidized hyaluronic acid solution. Among them, the above-mentioned exosome solution can be: a solution with stem cell exosomes as the solute. In practice, the target oxidized hyaluronic acid can be added to the exosome solution to obtain a loaded exosome oxidized hyaluronic acid solution with a preset hyaluronic acid solution concentration. Among them, the above-mentioned preset hyaluronic acid solution concentration can be the concentration of the loaded exosome oxidized hyaluronic acid solution set in advance. For example, the above-mentioned preset hyaluronic acid solution concentration can be 6% or 12%.

[0064] In some optional implementation manners of some embodiments, the target oxidized hyaluronic acid can be added to the exosome solution through the following steps to obtain a loaded exosome oxidized hyaluronic acid solution:

[0065] First step: Determine the concentration of the hyaluronic acid solution according to the hydrogel application scenario information and the preset concentration of the polylysine solution. Among them, the above hydrogel application scenario information can characterize the application scenario of the prepared exosome-loaded hydrogel. The above hydrogel application scenario information can include, but is not limited to, the application type. The above application type can be, but is not limited to, one of the following: acute infectious injury, chronic wound repair. The above concentration of the hyaluronic acid solution can be the concentration of the oxidized hyaluronic acid solution loaded with exosomes. In practice, the concentration of the hyaluronic acid solution can be determined according to the application type, the preset concentration of the polylysine solution, and the preset application concentration comparison table. Among them, the above preset application concentration comparison table can be a relationship comparison table of the application type, the preset concentration of the polylysine solution, and the concentration of the hyaluronic acid solution obtained in advance through experiments. In the above preset application concentration comparison table, each preset application type and preset polylysine solution concentration correspond to a preset hyaluronic acid solution concentration.

[0066] In some alternative implementation manners of some embodiments, the concentration of the hyaluronic acid solution can be determined according to the hydrogel application scenario information through the following steps:

[0067] Step 1: Determine the exosome release rate according to the hydrogel application scenario information. Among them, the above exosome release rate can be the release rate of exosomes. In practice, the preset exosome release rate corresponding to the application type included in the above hydrogel application scenario information can be determined as the exosome release rate. Among them, the above preset exosome release rate can be a preset exosome release rate.

[0068] In the process of adopting technical solutions to solve the technical problems in the background technology, there is often another technical problem: due to different patient constitutions and different injury situations, the required exosome release rates for wound repair are also different. How to improve the adaptability of the exosome release rate of the exosome-loaded hydrogel to the patient's wound to improve the wound repair performance of the exosome-loaded hydrogel. In response to the above technical problem 2, the conventional solution is generally: according to the type of the wound, determine the exosome release rate corresponding to the wound type through experiments. However, the above conventional solution still has the following problems: for the same wound type, for patients of different ages and different constitutions, the rate of promoting exosome release by the patients themselves is also different. Determining the exosome release rate only according to the wound type results in a low adaptability of the exosome release rate of the exosome-loaded hydrogel to the patient's wound, thereby causing a low wound repair performance of the exosome-loaded hydrogel.

[0069] Facing the above technical problem 2, combined with the artificial intelligence R & D technology owned by the cooperative college, the following solution can be adopted:

[0070] In practice, the exosome release rate can be determined by the hydrogel application scenario information and a preset release rate determining device. Among them, the above-mentioned preset release rate determining device can be a device preset for determining the exosome release rate. The above-mentioned preset release rate determining device can be a computing device storing an application program corresponding to the method for determining the exosome release rate. The above-mentioned method for determining the exosome release rate includes:

[0071] In the first step, in response to detecting the preparation user login information, an application scenario information input interface is displayed. Among them, the above-mentioned preparation user login information can be the information for the preparation user to log in to the application scenario information input system. For example, the above-mentioned preparation user login information can be "1". The above-mentioned preparation user can be a user who needs to prepare a hydrogel loaded with exosomes. The application scenario information input system can be a system for the preparation user to input the text of the application scenario of the hydrogel loaded with exosomes. The above-mentioned application scenario information input interface can be an interface for the preparation user to input the text of the application scenario of the hydrogel loaded with exosomes. The above-mentioned application scenario information input interface can include, but is not limited to, an information input text box and an information confirmation control. The above-mentioned information input text box can be used for the preparation user to input the application scenario text information. The above-mentioned information confirmation control can be a control for the preparation user to confirm the input application scenario text information. The above-mentioned application scenario text information can be the text describing the hydrogel loaded with exosomes. The application scenario text information can include, but is not limited to, the text corresponding to the patient's age, the type of injury scenario, the duration of the injury, and the wound area. For example, the above-mentioned application scenario text information can be "The patient is 20 years old, has suppurative infection during the postoperative incision care, which has lasted for 3 days, and the wound size is 5 cm."

[0072] In the second step, in response to detecting a selection operation acting on the above-mentioned information confirmation control, the application scenario text information corresponding to the above-mentioned information input text box is determined as the to-be-processed application scenario information. Among them, the above-mentioned selection operation can be at least one of, but not limited to, the following: click, slide, hover.

[0073] In the third step, feature extraction processing is performed on the above-mentioned to-be-processed application scenario information to obtain application scenario feature information. In practice, the above-mentioned execution subject can perform feature extraction processing on the above-mentioned to-be-processed application scenario information through a preset text feature extraction method to obtain application scenario feature information. Among them, the above-mentioned preset text feature extraction method can be a method preset for extracting text features. For example, the above-mentioned preset text feature extraction method can be a text feature extraction method based on a neural network.

[0074] Fourth step: Input the above application scenario feature information into the pre-trained patient health type generation model to obtain the patient health type. Among them, the above patient health type generation model can be a classification model that takes the application scenario feature information as the input and the patient health type as the output. The above classification model can be a support vector machine or a recurrent neural network. The above patient health type can represent the health status of the standard patient. The above patient health type can be, but is not limited to, one of the following: very healthy, good, average, poor.

[0075] Fifth step: Input the above application scenario feature information into the pre-trained wound type generation model to obtain the wound type. Among them, the above wound type generation model can be a classification model that takes the application scenario feature information as the input and the wound type as the output. The above wound type can represent the degree of injury of the wound. The above wound type can be, but is not limited to, one of the following: severely injured, generally injured, slightly injured.

[0076] Sixth step: In response to determining that the above hydrogel application scenario information meets the preset first application type condition, determine the preset first patient health weight as the first weight and the preset first wound weight as the second weight. Among them, the above preset first application type condition can be: the application type included in the above hydrogel application scenario information is acute infectious injury. The above preset first patient health weight can represent the degree of influence of the patient health type on the exosome release rate. The above preset first wound weight can represent the degree of influence of the wound type on the exosome release rate. The sum of the above preset first patient health weight and the above preset first wound weight can be 1. It should be noted that when the above hydrogel application scenario information meets the preset first application type condition, the preset first patient health weight is less than the preset first wound weight. For example, the preset first patient health weight is 0.3 and the preset first wound weight is 0.7.

[0077] Seventh step: In response to determining that the above hydrogel application scenario information meets the preset second application type condition, determine the preset second patient health weight as the first weight and the preset second wound weight as the second weight. Among them, the above preset first application type condition can be: the application type included in the above hydrogel application scenario information is chronic wound repair. The above preset second patient health weight can represent the degree of influence of the patient health type on the exosome release rate. The above preset second wound weight can represent the degree of influence of the wound type on the exosome release rate. The sum of the above preset second patient health weight and the above preset second wound weight can be 1. It should be noted that when the above hydrogel application scenario information meets the preset second application type condition, the preset second patient health weight is greater than or equal to the preset second wound weight. For example, the preset first patient health weight is 0.5 and the preset first wound weight is 0.5.

[0078] In the eighth step, determine the first score as the product of the preset health score corresponding to the above patient health type and the above first weight. Wherein, the above preset health score can be a score for the influence of the preset patient health type on the exosome release rate.

[0079] In the ninth step, determine the second score as the product of the preset wound score corresponding to the above wound type and the above second weight. Wherein, the above preset wound score can be a score for the influence of the preset wound type on the exosome release rate.

[0080] In the tenth step, determine the scene score as the sum of the above first score and the above second score. Wherein, the above scene score can be a score corresponding to the application scenario of the exosome-loaded. Through preliminary experiments, different scene scores correspond to different exosome release rates to achieve better repair performance of the exosome-loaded.

[0081] In the eleventh step, determine the exosome release rate as the preset exosome release rate corresponding to the above scene score. Wherein, the preset exosome release rate can be the exosome release rate obtained through experiments.

[0082] The above technical solution and its related content are an inventive point of an embodiment of the present disclosure, which solves the technical problem that "for the same wound type, for patients of different ages and constitutions, the rate of the patient's own promotion of exosome release is also different. Determining the exosome release rate only based on the wound type results in a low degree of adaptation between the exosome release rate of the exosome-loaded hydrogel and the patient's wound, thereby causing a low wound repair performance of the exosome-loaded hydrogel". The factors that cause the low wound repair performance of the exosome-loaded hydrogel are often as follows: for the same wound type, for patients of different ages and constitutions, the rate of the patient's own promotion of exosome release is also different. Determining the exosome release rate only based on the wound type results in a low degree of adaptation between the exosome release rate of the exosome-loaded hydrogel and the patient's wound, thereby causing a low wound repair performance of the exosome-loaded hydrogel. If the above factors are solved, the effect of improving the wound repair performance of the exosome-loaded hydrogel can be achieved. To achieve this effect, in the method for preparing the exosome-loaded hydrogel according to some embodiments of the present disclosure, when determining the exosome release rate, not only the application type is considered, but also the patient's own health condition, the scene and duration of the wound injury, and the speed of wound recovery are comprehensively considered, and the proportion of each influencing factor on the exosome is flexibly adjusted, thereby improving the degree of adaptation between the exosome release rate of the exosome-loaded hydrogel and the patient's wound. Thus, the wound repair performance of the exosome-loaded hydrogel can be improved.

[0083] Step 2: Determine the concentration of the hyaluronic acid solution according to the above exosome release rate and the preset concentration of the polylysine solution. In practice, the concentration of the hyaluronic acid solution can be determined according to the exosome release rate, the preset concentration of the polylysine solution, and a preset rate-concentration comparison table. The preset rate-concentration comparison table can be a relationship comparison table of the exosome release rate, the preset concentration of the polylysine solution, and the concentration of the hyaluronic acid solution obtained through experiments in advance. In the preset rate-concentration comparison table, each preset exosome release rate and preset polylysine solution concentration correspond to a preset hyaluronic acid solution concentration.

[0084] Step 2: Add the above-mentioned target oxidized hyaluronic acid to the exosome solution to obtain a loaded exosome oxidized hyaluronic acid solution corresponding to the concentration of the above-mentioned hyaluronic acid solution.

[0085] Optionally, before step 104, the following steps can also be performed:

[0086] Step 1: Isolate exosomes from umbilical cord stem cells to obtain stem cell exosomes.

[0087] Step 2: Add the above-mentioned stem cell exosomes to a phosphate (PBS) buffer solution to obtain an exosome solution. The ratio of the above-mentioned stem cell exosomes to the above-mentioned phosphate buffer solution is 50 micrograms: 100 milliliters.

[0088] Optionally, exosomes can be isolated from umbilical cord stem cells through the following steps to obtain stem cell exosomes:

[0089] Step 1: Centrifuge umbilical cord stem cells through a centrifuge device to obtain cell supernatant. The centrifuge device can be a high-speed centrifuge. The cell supernatant can be the supernatant of umbilical cord stem cells. In practice, umbilical cord stem cells can be centrifuged through a high-speed centrifuge to remove large membrane vesicles to obtain cell supernatant. The respective operating parameters of the high-speed centrifuge are: centrifugal force 10000g (acceleration due to gravity), temperature 4 degrees Celsius, and operating duration 30 minutes.

[0090] Step 2: Centrifuge the above-mentioned cell supernatant through the above-mentioned centrifuge device to obtain exosome spheroids. The exosome spheroids can be spheroids with exosomes. In practice, the above-mentioned cell supernatant can be centrifuged through the above-mentioned high-speed centrifuge to remove the cell supernatant to obtain exosome spheroids. The respective operating parameters of the high-speed centrifuge are: centrifugal force 120000g (acceleration due to gravity), temperature 4 degrees Celsius, and operating duration 70 minutes.

[0091] Step 3: Precipitate the above-mentioned exosome spheroids with a phosphate buffer solution to obtain stem cell exosomes.

[0092] Optionally, the stem cell exosomes can also be identified by the following methods:

[0093] In the first step, the stem cell exosomes are identified by Western blot (WB). As Figure 2 shown in (a), the exosome-specific enriched proteins CD9, CD81, and Alix are present, and the Golgi protein Gm130 is absent.

[0094] In the second step, the stem cell exosomes are identified by transmission electron microscopy. As Figure 2 shown in (b), the stem cell exosomes are cup-shaped under transmission electron microscopy.

[0095] In the third step, the stem cell exosomes are identified by nanoparticle tracking analysis (NTA). As Figure 2 shown in (c), the diameter of the exosome particle size is 30-150 nanometers.

[0096] Step 105, mixing the exosome-loaded oxidized hyaluronic acid solution with polylysine for a reaction to obtain an exosome-loaded hydrogel.

[0097] In some embodiments, the above exosome-loaded oxidized hyaluronic acid solution can be mixed with polylysine (EPL) for a reaction to obtain an exosome-loaded hydrogel. Among them, the mass ratio range of the target oxidized hyaluronic acid to polylysine in the above exosome-loaded hydrogel is 1-2. As an example, a 6% exosome-loaded oxidized hyaluronic acid solution can be mixed with a 6% polylysine solution for a reaction to obtain a fourth mixed solution. Among them, the volume ratio of the exosome-loaded oxidized hyaluronic acid solution to the polylysine solution is 1:1. Then, the above fourth mixed solution is subjected to ultraviolet light irradiation treatment with an ultraviolet lamp to obtain an exosome-loaded hydrogel.

[0098] In the process of adopting the technical solution to solve the technical problems in the background technology, there are often the following technical problems 3: The existing hydrogel is in a gel state, and smearing operation is required during use. During the smearing process, insufficient disinfection is carried out, resulting in a high risk of secondary wound infection, and some exosomes will remain on the smearing tool, reducing the antibacterial effect of the exosome-loaded hydrogel.

[0099] Facing the above technical problem 3, combined with the advantages of good moisturizing and biocompatibility of the exosome-loaded hydrogel, the following solutions can be adopted:

[0100] In some alternative implementations of some embodiments, the above-mentioned exosome-loaded oxidized hyaluronic acid solution can be mixed with polylysine through the following steps to obtain an exosome-loaded hydrogel:

[0101] First step, fill the above-mentioned exosome-loaded oxidized hyaluronic acid solution into an empty spray bottle through a filling device to obtain a hyaluronic acid spray bottle. Among them, the above-mentioned filling device can be a device for filling a liquid into an empty spray bottle. For example, the above-mentioned filling device can be a filling machine.

[0102] Second step, fill a polylysine solution with a preset concentration into an empty spray bottle through a filling device to obtain a polylysine spray bottle. Among them, the preset concentration can be a pre-set concentration. For example, the preset concentration can be 3% or 6%.

[0103] Third step, simultaneously press the above-mentioned hyaluronic acid spray bottle and the above-mentioned polylysine spray bottle so that the exosome-loaded oxidized hyaluronic acid solution sprayed from the above-mentioned hyaluronic acid spray bottle is mixed with the polylysine solution sprayed from the above-mentioned polylysine spray bottle at a preset spraying position to obtain a hydrogel mixed solution. Among them, the above-mentioned preset spraying position can be a position where an exosome-loaded hydrogel is required to be formed. As an example, the above-mentioned preset spraying position can be the position of a wound to be repaired.

[0104] Fourth step, perform photocuring treatment on the above-mentioned hydrogel mixed solution to obtain an exosome-loaded hydrogel. In practice, the above-mentioned hydrogel mixed solution can be subjected to photocuring treatment by an ultraviolet lamp to obtain an exosome-loaded hydrogel.

[0105] The above technical solution and its related content, as an inventive point of an embodiment of the present disclosure, solve the technical problem that "existing hydrogels are in a gel state, requiring a smearing operation during use. During the smearing process, insufficient disinfection leads to a relatively high risk of secondary wound infection, and some exosomes are left on the smearing tool, reducing the antibacterial effect of the exosome-loaded hydrogel." The factors that lead to a relatively high risk of secondary wound infection and a reduced antibacterial effect of the exosome-loaded hydrogel are often as follows: Existing hydrogels are in a gel state, requiring a smearing operation during use. During the smearing process, insufficient disinfection leads to a relatively high risk of secondary wound infection, and some exosomes are left on the smearing tool, reducing the antibacterial effect of the exosome-loaded hydrogel. If the above factors are solved, the risk of secondary wound infection can be reduced, and the antibacterial effect of the exosome-loaded hydrogel can be improved. To achieve this effect, in the method for preparing an exosome-loaded hydrogel according to some embodiments of the present disclosure, by separately preparing a spray solution of an exosome-loaded oxidized hyaluronic acid solution and polylysine at a certain concentration ratio, because EPL and OHAMA can bind through the formation of Schiff base bonds and form a gel through photocuring under the action of a photoinitiator, when the two solutions are mixed in the form of a spray, a gel state can be formed under the irradiation of natural light to repair the wound. Thus, the exosome-loaded hydrogel can be applied without the user performing a smearing operation, thereby reducing the risk of secondary wound infection and improving the antibacterial effect of the exosome-loaded hydrogel.

[0106] To prove the effect of the exosome-loaded hydrogel prepared by the above method for preparing an exosome-loaded hydrogel, the following experimental methods and experimental results are provided.

[0107] Experiment 1

[0108] Hyaluronic acid, oxidized hyaluronic acid, and target oxidized hyaluronic acid were respectively characterized by infrared spectroscopy to obtain infrared spectra as shown in Figure 3 shown. Among them, the three infrared spectra shown in Figure 3 correspond to hyaluronic acid, oxidized hyaluronic acid, and target oxidized hyaluronic acid in sequence from top to bottom.

[0109] Experiment 2

[0110] Hyaluronic acid, oxidized hyaluronic acid, and target oxidized hyaluronic acid were respectively characterized by nuclear magnetic resonance hydrogen spectroscopy to obtain nuclear magnetic characterizations as shown in Figure 4 shown.

[0111] Experiments 1 and 2 can show that the target oxidized hyaluronic acid contains groups with aldehyde groups and methacrylate bonds.

[0112] Experiment 3

[0113] According to the different mass ratios of the target oxidized hyaluronic acid to polylysine, the experiments were divided into 4 groups. In the first group of exosome-loaded hydrogels, the concentrations of the exosome-loaded oxidized hyaluronic acid solution and the polylysine solution were 6% and 6% respectively. In the second group of exosome-loaded hydrogels, the concentrations of the exosome-loaded oxidized hyaluronic acid solution and the polylysine solution were 8% and 6% respectively. In the third group of exosome-loaded hydrogels, the concentrations of the exosome-loaded oxidized hyaluronic acid solution and the polylysine solution were 10% and 6% respectively. In the fourth group of exosome-loaded hydrogels, the concentrations of the exosome-loaded oxidized hyaluronic acid solution and the polylysine solution were 12% and 6% respectively. The four groups of exosome-loaded hydrogels were respectively freeze-dried under vacuum for 24 hours. After sputtering and gold plating, the gel structures of the four groups of exosome-loaded hydrogels were observed by a scanning electron microscope (SEM), as Figure 5 shown. Figure 5 It shows that the higher the content of the target oxidized hyaluronic acid in the exosome-loaded hydrogel, the better the mechanical properties of the exosome-loaded hydrogel.

[0114] Experiment 5

[0115] The dynamic rheological properties of the exosome-loaded hydrogel were detected by a 25-mm parallel plate rheometer at 37°C. The exosome-loaded hydrogel was at 100 rad / s (radians per second) with a strain amplitude of 1% at 0.1 minute at 37°C. A strain sweep of the exosome-loaded hydrogel was performed from 0.1% to 10% at 37°C at an angular frequency of 1 rad / s, as Figure 6 shown. Figure 6 It can be shown that in the frequency sweep, the exosome-loaded hydrogel exhibited good mechanical stability.

[0116] Experiment 6

[0117] As Figure 7 shown, after cutting a methylene blue-stained exosome-loaded hydrogel and an unstained exosome-loaded hydrogel and recombining them along the cross-section, after 10 minutes, the two exosome-loaded hydrogels fused at the fracture ends to form one exosome-loaded hydrogel. After picking up one end of the exosome-loaded hydrogel, the exosome-loaded hydrogel did not fall off, indicating that the exosome-loaded hydrogel can self-heal into a complete hydrogel after being physically damaged.

[0118] Experiment 7

[0119] According to the different mass ratios of the target oxidized hyaluronic acid to polylysine, the experiments were divided into 4 groups. In the first group of exosome-loaded hydrogels, the concentrations of the exosome-loaded oxidized hyaluronic acid solution and the polylysine solution were 3% and 3% respectively. In the second group of exosome-loaded hydrogels, the concentrations of the exosome-loaded oxidized hyaluronic acid solution and the polylysine solution were 4% and 3% respectively. In the third group of exosome-loaded hydrogels, the concentrations of the exosome-loaded oxidized hyaluronic acid solution and the polylysine solution were 5% and 3% respectively. In the fourth group of exosome-loaded hydrogels, the concentrations of the exosome-loaded oxidized hyaluronic acid solution and the polylysine solution were 6% and 3% respectively. The initial weights of the four groups of exosome-loaded hydrogels were recorded respectively, placed in PBS solution, and carried out at 37 °C. They were taken out and weighed every 30 minutes until the gel began to degrade, and the swelling ratio was calculated. Among them, the swelling ratio was calculated by the following method:

[0120] First, the difference between the current weight and the initial weight was determined as the weight difference.

[0121] Then, the percentage of the ratio of the above weight difference to the initial weight was determined as the swelling ratio. As Figure 8 shown, the four groups of exosome-loaded hydrogels all had good swelling properties, and with the increase of the concentration of the target oxidized hyaluronic acid, the exosome hydrogels were crosslinked more tightly and the swelling ratio decreased. It should be noted that Figure 8 the concentrations of the exosome-loaded oxidized hyaluronic acid solutions corresponding to the broken lines from top to bottom in

[0122] Experiment 8

[0123] The experiment was divided into 5 groups, namely the control group (Control), the ampicillin group (Ampicilin 20 μg / mL), the OHAMA group, the EPL group, and the exosome-loaded hydrogel group. 10-6 Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA) were spread on the culture dishes, and at the same time, the corresponding drugs or gels of the 5 groups were spread on the culture dishes and incubated for 12 hours. The growth of bacteria on the culture dishes was observed and recorded to verify the antibacterial effect of EPL. As Figure 9 shown, no colonies were formed in the groups with EPL added, while ampicillin could only inhibit Escherichia coli and partially inhibit Staphylococcus aureus, and had no obvious inhibitory effect on methicillin-resistant Staphylococcus aureus. Thus, it can be shown that EPL has good antibacterial properties.

[0124] Experiment 9

[0125] The experiment was divided into two groups. Hydrogel loaded with 50 micrograms of exosomes was prepared and placed in a transwell-24 well plate. Then, 200 microliters of PBS (pH = 7.4) solution and 200 microliters of solution with pH = 6.0 were added to the two groups of 24 well plates respectively. The liquid in the two groups of well plates except the hydrogel was collected every 12 hours, and the corresponding solution was added. After 5 days, the collected liquid was subjected to fluorescence quantification. The release rate and cumulative release amount of exosomes from the hydrogel were detected, as Figure 10 shown. It can be shown that the exosome-loaded hydrogel added with PBS solution has the function of sustained and controlled release of exosomes.

[0126] Experiment 10

[0127] CCK-8 cell experiments were carried out. First, we "digested" the adherent NIH-3T3 cells in the logarithmic phase by adding trypsin. Then, the digested cells were transferred to the centrifuge tube of an ultracentrifuge, and the ultracentrifuge was run at a speed of 1000 revolutions per minute for 3 minutes. Then, the supernatant in the centrifuge tube was discarded, and the cells were resuspended with 1 milliliter of serum-containing medium. After that, a cell counter was used to count the cells, and the cell suspension was diluted according to the counting results. When the cell suspension was diluted to 30,000 cells per milliliter, three 96 well plates were taken, a blank control was set, and three experimental groups were set, with six replicates in each group. 100 microliters of the diluted cell suspension was added to each well, and 200 microliters of PBS buffer was added to the edge wells to reduce the influence of evaporation. After 12 hours of inoculation, when the cells adhered well, the medium in each well was aspirated. Then, a medium without drugs was added to the blank group, and media containing only hydrogel, only exosomes, and a combination of hydrogel and exosomes were added to the three experimental groups respectively. Immediately afterwards, it was placed in an incubator for culture. After the drug treatment for the corresponding time, the medium in each well was aspirated again, and a medium containing CCK-8 was added, with CCK-8 accounting for 1 / 10 of the volume of the cell medium. After placing it in the incubator for 45 minutes, the 96 well plate was taken out and placed in an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 450 nanometers. The detection results are as Figure 11 shown, where Figure 11 the corresponding experimental groups from left to right are medium (control group), medium containing only hydrogel (hydrogel group), medium containing only exosomes (exosome group), and medium containing a combination of hydrogel and exosomes (exosome-loaded hydrogel group). By comparing the exosome-loaded hydrogel group with the control group, it can be shown that the exosome hydrogel not only has good biocompatibility but also has a good effect on promoting cell proliferation.

[0128] Experiment 11

[0129] The steps of the cell migration experiment are as follows:

[0130] Step 1: Digest NIH-3T3 cells in the logarithmic growth phase into single-cell suspensions with trypsin and inoculate them in a 6-well culture plate.

[0131] Step 2: Seed 100,000 cells per well. The final total volume of the medium in each well can be 2 mL. Incubate the cells in a 37 °C, 5% carbon dioxide incubator for 24 hours.

[0132] Step 3: The next day, use a 200-μL pipette tip against a ruler to make scratches.

[0133] Step 4: Rinse the cells 3 times with PBS buffer to remove the scratched cells, add serum-free medium, and take pictures under a 4× microscope, denoted as 0 h.

[0134] Step 5: Divide the experimental groups into serum-free medium (control group), and add serum-free medium plus hydrogel (hydrogel group), serum-free medium plus exosomes (exosome group), and serum-free medium plus exosome-loaded hydrogel (exosome-loaded hydrogel group) respectively.

[0135] Step 6: Sample and take pictures at the 24-hour and 48-hour time points respectively. After opening the pictures with Image J software, randomly draw 6 to 8 horizontal lines to calculate the cell migration rate. Among them, the cell migration rate (wound healing rate) = (initial scratch area - scratch area at a certain time) / initial scratch area. As Figure 12 shown, after 24 h and 48 h, compared with the control group, the remaining areas of all experimental groups decreased, and the remaining area of the exosome-loaded hydrogel group was the smallest, with a significant difference (P < 0.001). It can be shown that the exosome-loaded hydrogel has a good effect on promoting cell migration.

[0136] Experiment 12

[0137] The steps of the angiogenesis experiment are as follows:

[0138] Step 1: Select human umbilical vein endothelial cells (HUVEC), passage them to the 4th generation, and make them reach 80% confluence within 24 hours.

[0139] Step 2: Thaw Matrigel. Take out Matrigel from the -80 °C refrigerator and place it in the 4 °C refrigerator until it thaws. Use a pre-cooled pipette to mix Matrigel until it is in a uniform state.

[0140] Step 3: Dilute Matrigel with culture medium to 10 mg / mL. Place the completely thawed Matrigel on ice and invert it 3 - 5 times to mix the contents. Take a 96-well plate and add 50 μL of Matrigel to each well.

[0141] Step 4: Transfer the 96-well plate to the cell culture incubator and incubate it at 37 °C for 30 minutes to form a gel for the basement membrane.

[0142] Step 5: Prepare three working solutions, namely, cell culture medium containing 10% FBS (Fetal Bovine Serum) (control group), FBS cell culture medium plus hydrogel (hydrogel group), FBS cell culture medium plus exosomes (exosome group), and FBS cell culture medium plus exosome-loaded hydrogel (exosome-loaded hydrogel group).

[0143] Step 6: Digest, centrifuge, and count the HUVEC cells that have reached 80% confluence in the T25 flask.

[0144] Step 7: Resuspend the cells in the prepared culture medium and working solutions to obtain a single-cell suspension with a concentration of 450,000 cells per milliliter.

[0145] Step 8: Add 100 μL of the cell suspension containing the sample to be tested (containing 45,000 cells) to each well of a 96-well plate. Place the 96-well plate in an incubator at 37 °C, 5% CO₂, and 90% humidity for 6 hours, then take a photo for observation. As Figure 13 shown, after 6 hours of experimental treatment, a small number of tube-like structures were observed in the control group, while obvious tubular structures were observed in the exosome group and the exosome-loaded hydrogel group. Use the ImageJ plugin Angiogenesis Analyzer to quantitatively analyze the taken photos and analyze the blood vessel length. The data results are as Figure 13 shown. As can be seen from Figure 13 , the blood vessel length in the exosome-loaded hydrogel group is the longest, indicating that the exosome-loaded hydrogel has a good promoting effect on angiogenesis.

[0146] Experiment 13

[0147] Step 1: Adaptively feed SPF-grade C57 / BL6 male mice at 5 weeks old and weighing 28.5 ± 2 g for one week at room temperature of 25 °C and humidity of 60%. After observing no physiological abnormalities, establish a mouse wound model. The specific modeling steps are as follows:

[0148] First, weigh the mice and anesthetize them with isoflurane.

[0149] Second, fix the anesthetized mice in the prone position, use a hair clipper to remove the hair on the back, and disinfect the skin of the surgical area with iodophor.

[0150] Then, use a puncher to create a full-thickness skin injury wound with a diameter of about 7 mm in the area surrounded by the back neck and above the scapulae on both sides of the mice, reaching the fascia.

[0151] Afterwards, a pre-sterilized 3-mm thick circular organosilica gel (inner diameter 10 mm, outer diameter 15 mm) was sutured and fixed to the wound with nylon sutures to prevent non-pathological contraction of the wound.

[0152] In the second step, the experiment was divided into four groups: a control group, a group using hydrogel, a group using exosomes, and a group using exosome-loaded hydrogel. For the four groups of experiments, the curative effect was observed and the dressing was changed on days 0, 3, 7, 10, and 14 respectively. The natural process of healing was observed for about 14 days, as Figure 14 (a) shows. In addition, tissue sections were fixed and immunostained on days 7 and 14 respectively to observe the treatment effect, as Figure 14 (b) shows. Figure 14 It can be shown that the wound healing effect of the exosome-loaded hydrogel is the best. Toxicity tests were performed on the four groups of experiments on day 14. Among them, the experimental data of blood routine and blood biochemistry are as Figure 15 shown. It can be seen from Figure 15 that there is no obvious difference between each experimental group and the control group, indicating that the hydrogel and exosomes have good biocompatibility.

[0153] The above-described various embodiments of the present disclosure have the following beneficial effects: Through the method for preparing a loaded exosome hydrogel according to some embodiments of the present disclosure, the mechanical properties and controllable degradability of the loaded exosome hydrogel can be improved, thereby improving the applicability of the loaded exosome hydrogel and the retention ability and activity of the exosomes. Specifically, the reasons for the poor sprayability and applicability of gelatin methacrylate (GelMA) hydrogels are as follows: The mechanical properties of GelMA hydrogels are low, and the controllable degradability is poor, resulting in poor retention ability and activity of the exosomes loaded in the GelMA hydrogels. In addition, the GelMA hydrogels have high viscosity, resulting in poor sprayability, thus reducing the applicability of the GelMA hydrogels. Based on this, in the method for preparing a loaded exosome hydrogel according to some embodiments of the present disclosure, hyaluronic acid is oxidized to obtain oxidized hyaluronic acid; the oxidized hyaluronic acid and methacrylic anhydride are mixed and reacted under preset environmental conditions to obtain a first mixed solution; the first mixed solution is subjected to dialysis and freeze-drying to obtain target oxidized hyaluronic acid, wherein the target oxidized hyaluronic acid is oxidized hyaluronic acid grafted with methacrylate bonds; the target oxidized hyaluronic acid is added to an exosome solution to obtain a loaded exosome oxidized hyaluronic acid solution; the loaded exosome oxidized hyaluronic acid solution and polylysine are mixed and reacted to obtain a loaded exosome hydrogel, wherein the mass ratio of the target oxidized hyaluronic acid to the polylysine in the loaded exosome hydrogel ranges from 1 to 2. By oxidizing hyaluronic acid and grafting methacrylate bonds, the biocompatibility and moisture retention of the obtained target oxidized hyaluronic acid are improved, thereby increasing the possibility of preparing a spray-type loaded exosome hydrogel. Moreover, by combining antibacterial polylysine with the target oxidized hyaluronic acid having aldehyde groups and methacrylate bonds, Schiff base bonds are formed in the obtained loaded exosome hydrogel. Therefore, while improving the antibacterial performance of the loaded exosome hydrogel, the mechanical properties and controllable degradability of the loaded exosome hydrogel are enhanced, and further, the exosome retention ability and activity of the loaded exosome hydrogel are improved. Thus, the mechanical properties and controllable degradability of the loaded exosome hydrogel can be improved, thereby improving the applicability of the loaded exosome hydrogel and the retention ability and activity of the exosomes.

[0154] The present disclosure also provides a loaded exosome hydrogel. The loaded exosome hydrogel solution is prepared by the above steps 101-105.

[0155] The present disclosure also provides a use of the loaded exosome hydrogel. The loaded exosome hydrogel can be used for treating skin wounds.

[0156] The above description is only some preferred embodiments of the present disclosure and an explanation of the applied technical principles. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above inventive concept. For example, the technical solutions formed by mutually replacing the above features with the technical features (but not limited to) disclosed in the embodiments of the present disclosure that have similar functions.

Claims

1. A method for preparing an exosome-loaded hydrogel, comprising: performing oxidation treatment on hyaluronic acid to obtain oxidized hyaluronic acid; The oxidized hyaluronic acid and methacrylic anhydride are mixed and reacted under preset environmental conditions to obtain a first mixed solution; Performing a dialysis and freeze-drying treatment on the first mixed solution to obtain a target oxidized hyaluronic acid, wherein the target oxidized hyaluronic acid is an oxidized hyaluronic acid grafted with a methacrylate bond; adding the target oxidized hyaluronic acid into an exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution; The exosome-loaded oxidized hyaluronic acid solution is mixed with poly-lysine to obtain an exosome-loaded hydrogel, wherein the mass ratio of the target oxidized hyaluronic acid to the poly-lysine in the exosome-loaded hydrogel is in the range of 1 to 2, and the exosome-loaded oxidized hyaluronic acid solution is mixed with poly-lysine to obtain an exosome-loaded hydrogel, comprising: Filling the exosome-loaded oxidized hyaluronic acid solution into an empty spray bottle by a filling device to obtain a hyaluronic acid spray bottle; Filling a poly-lysine solution of a preset concentration into an empty spray bottle by a filling device to obtain a poly-lysine spray bottle; Press the hyaluronic acid spray bottle and the polylysine spray bottle simultaneously, so that the exosome-loaded oxidized hyaluronic acid solution sprayed from the hyaluronic acid spray bottle and the polylysine solution sprayed from the polylysine spray bottle are mixed at a preset spraying position to obtain a hydrogel mixed solution; The hydrogel mixed solution is subjected to photocuring treatment to obtain an exosome-loaded hydrogel.

2. The method according to claim 1, wherein: The step of subjecting hyaluronic acid to oxidation to obtain oxidized hyaluronic acid comprises: Dissolving hyaluronic acid in water to obtain a hyaluronic acid solution, wherein the concentration of the hyaluronic acid solution is 1%; Mixing sodium periodate with the hyaluronic acid solution to obtain a second mixed solution, wherein the mass ratio of the sodium periodate to the hyaluronic acid is 3:5; Mixing anhydrous ethanol with the second mixed solution to obtain a third mixed solution, wherein the solution ratio of the anhydrous ethanol to the second mixed solution is 1:100; The third mixed solution is dialyzed and freeze-dried to obtain oxidized hyaluronic acid.

3. The method according to claim 1, wherein: The preset environmental conditions are: the pH value of the mixed solution ranges from 8 to 9, and the ambient temperature is 4 degrees Celsius; The step of mixing the oxidized hyaluronic acid and methacrylic anhydride under preset environmental conditions to obtain a first mixed solution comprises: dissolving the oxidized hyaluronic acid in distilled water to obtain an oxidized hyaluronic acid solution, wherein the concentration of the oxidized hyaluronic acid solution is 1%; In an environment with an ambient temperature of 4 degrees Celsius, the oxidized hyaluronic acid solution and methacrylic anhydride are mixed for a preset time to obtain a first mixed solution, wherein the volume ratio of the oxidized hyaluronic acid solution to the methacrylic anhydride is 100:2, and within a preset time period corresponding to the preset time, the following steps are further performed: The pH value of the mixed solution is detected in real time by a pH value detection device; In response to determining that the pH value of the mixed solution is not within the pH value range, a sodium hydroxide solution is added to the mixed solution to make the pH value of the mixed solution within the pH value range, wherein the mixed solution is a solution mixed with the oxidized hyaluronic acid solution and the methacrylic anhydride.

4. The method according to claim 1, wherein: Before adding the target oxidized hyaluronic acid to the exosome solution to obtain the exosome-loaded oxidized hyaluronic acid solution, the method further comprises: Exosomes are isolated from umbilical cord stem cells to obtain stem cell exosomes; The stem cell exosomes are added into a phosphate buffer solution to obtain an exosome solution, wherein the ratio of the stem cell exosomes to the phosphate buffer solution is 50 μg:100 ml.

5. The method according to claim 4, wherein: The method of isolating exosomes from umbilical cord stem cells to obtain stem cell exosomes comprises: Centrifuge the umbilical cord stem cells using a centrifuge device to obtain a cell supernatant; Centrifuging the cell supernatant using the centrifuge device to obtain exosome spheroids; The exosome spheroids are precipitated by using the phosphate buffer solution to obtain stem cell exosomes.

6. The method according to claim 1, wherein: The step of adding the target oxidized hyaluronic acid into an exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution comprises: Determine the concentration of the hyaluronic acid solution according to the hydrogel application scenario information and the preset poly-lysine solution concentration; The target oxidized hyaluronic acid is added to the exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution corresponding to the concentration of the hyaluronic acid solution.

7. The method according to claim 6, wherein: The method of determining the concentration of the hyaluronic acid solution according to the hydrogel application scenario information and the preset poly-lysine solution concentration includes: Determine the exosome release rate based on the hydrogel application scenario information; The concentration of the hyaluronic acid solution is determined according to the exosome release rate and the preset poly-lysine solution concentration.

8. An exosome-loaded hydrogel, wherein: The exosome-loaded hydrogel is prepared by the method for preparing the exosome-loaded hydrogel according to any one of claims 1 to 7.

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