Preparation method of exosome-loaded hydrogel, exosome-loaded hydrogel and application of exosome-loaded hydrogel

Abstract

The embodiment of the invention discloses a preparation method of exosome-loaded hydrogel, the exosome-loaded hydrogel and application of the exosome-loaded hydrogel. A specific embodiment of the method comprises the following steps: carrying out oxidation treatment on hyaluronic acid; carrying out mixed reaction on oxidized hyaluronic acid and methacrylic anhydride under a preset environmental condition to obtain a first mixed solution; performing dialysis and freeze-drying treatment on the first mixed solution to obtain target oxidized hyaluronic acid; adding the target oxidized hyaluronic acid into the exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution; the exosome-loaded oxidized hyaluronic acid solution and polylysine are subjected to a mixed reaction, the exosome-loaded hydrogel is obtained, and the mass ratio of target oxidized hyaluronic acid to polylysine in the exosome-loaded hydrogel ranges from 1 to 2. According to the embodiment, the mechanical property and controllable degradability of the exosome-loaded hydrogel can be improved, so that the applicability of the exosome-loaded hydrogel and the retention capacity and activity of the exosome are improved.

Description

Technical Field

[0001] The embodiments of the present disclosure relate to the technical field of biomedical materials, and specifically 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, hydrogel can be used as a drug delivery system to provide a moist therapeutic environment, promote tissue growth and control the release of exosomes. At present, when preparing hydrogels carrying exosomes, the commonly used method is to add exosomes derived from human umbilical vein endothelial cells (HUVECs-Exos) to gelatin methacryloyl (GelMA) hydrogel to prepare a hydrogel that can be used for wound dressings.

[0003] However, in practice, it is found that when the above method is used to prepare the hydrogel loaded with exosomes, the following technical problems often occur:

[0004] Gelatin methacryloyl hydrogel has low mechanical properties and poor controllable degradability, resulting in poor retention capacity and activity of exosomes loaded with gelatin methacryloyl hydrogel. In addition, the viscosity of gelatin methacryloyl hydrogel is relatively high, resulting in poor sprayability of gelatin methacryloyl hydrogel, thus causing low applicability of gelatin methacryloyl hydrogel.

[0005] The above information disclosed in this Background section is only for enhancement of understanding of the background of the inventive concept and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art. Summary of the invention

[0006] The content of this disclosure is used to introduce concepts in a brief form, which will be described in detail in the detailed implementation section below. The content of this disclosure is not intended to identify the key features or essential features of the technical solution claimed for protection, nor is it intended to limit the scope of the technical solution claimed for protection.

[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 technology section.

[0008] In a first aspect, some embodiments of the present disclosure provide a method for preparing an exosome-loaded 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; dialyzing and freeze-drying 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 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.

[0009] Optionally, the above-mentioned oxidation treatment of 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 above-mentioned hyaluronic acid solution to obtain a second mixed solution, wherein the mass ratio of the sodium periodate to the above-mentioned hyaluronic acid is 3:5; mixing anhydrous ethanol with the above-mentioned second mixed solution to obtain a third mixed solution, wherein the solution ratio of the above-mentioned anhydrous ethanol to the above-mentioned second mixed solution is 1:100; dialyzing and freeze-drying the above-mentioned third mixed solution to obtain oxidized hyaluronic acid.

[0010] Optionally, the preset environmental conditions are: the pH value of the mixed solution is in the range of 8 to 9, and the ambient temperature is 4 degrees Celsius; and the above-mentioned oxidized hyaluronic acid and methacrylic anhydride are mixed under the preset environmental conditions to obtain a first mixed solution, including: dissolving the above-mentioned oxidized hyaluronic acid in distilled water to obtain an oxidized hyaluronic acid solution, wherein the concentration of the above-mentioned oxidized hyaluronic acid solution is 1%; in an environment with an ambient temperature of 4 degrees Celsius, the above-mentioned 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 above-mentioned oxidized hyaluronic acid solution to the above-mentioned methacrylic anhydride is 100:2, and within a preset time period corresponding to the above-mentioned preset time period, the following steps are also 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 above-mentioned mixed solution is not within the above-mentioned pH value range, sodium hydroxide solution is added to the mixed solution so that the pH value of the mixed solution is within the above-mentioned pH value range, wherein the above-mentioned mixed solution is a solution mixed with the above-mentioned oxidized hyaluronic acid solution and the above-mentioned 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 comprises: 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 μg:100 ml.

[0012] Optionally, the above-mentioned separation of exosomes from umbilical cord stem cells to obtain stem cell exosomes includes: centrifuging the umbilical cord stem cells by a centrifuge device to obtain a cell supernatant; centrifuging the above-mentioned cell supernatant by the above-mentioned centrifuge device to obtain exosome spheroids; and precipitating the above-mentioned exosome spheroids using the above-mentioned phosphate buffer solution to obtain stem cell exosomes.

[0013] Optionally, the above-mentioned target oxidized hyaluronic acid is added to the exosome solution to obtain the exosome-loaded oxidized hyaluronic acid solution, comprising: determining the concentration of the hyaluronic acid solution according to the hydrogel application scenario information and the preset poly-lysine solution concentration; adding the above-mentioned target oxidized hyaluronic acid to the exosome solution to obtain the exosome-loaded oxidized hyaluronic acid solution corresponding to the above-mentioned hyaluronic acid solution concentration.

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

[0015] Optionally, the exosome-loaded oxidized hyaluronic acid solution is mixed with polylysine 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 polylysine solution of a preset concentration into an empty spray bottle by a filling device to obtain a polylysine spray bottle; pressing the hyaluronic acid spray bottle and the polylysine spray bottle at the same time to mix the exosome-loaded oxidized hyaluronic acid solution sprayed from the hyaluronic acid spray bottle and the polylysine solution sprayed from the polylysine spray bottle at a preset spray position to obtain a hydrogel mixed solution; and performing photocuring 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, wherein the exosome-loaded hydrogel solution is prepared by the exosome-loaded hydrogel preparation method described in any implementation of the first aspect.

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

[0018] The above-mentioned embodiments of the present disclosure have the following beneficial effects: through the preparation method of the exosome-loaded hydrogel of 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 capacity and activity of the exosomes. Specifically, the reasons for the poor sprayability and applicability of gelatin methacryloyl hydrogel are: the mechanical properties of gelatin methacryloyl hydrogel are low and the controllable degradability is poor, resulting in poor retention capacity and activity of exosomes loaded by gelatin methacryloyl hydrogel, and the viscosity of gelatin methacryloyl hydrogel is large, resulting in poor sprayability of gelatin methacryloyl hydrogel, thereby resulting in low applicability of gelatin methacryloyl hydrogel. Based on this, in some embodiments of the present disclosure, the method for preparing exosome-loaded hydrogel comprises: 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; dialyzing and freeze-drying the first mixed solution to obtain a target oxidized hyaluronic acid, wherein the target oxidized hyaluronic acid is an oxidized hyaluronic acid grafted with methacrylate bonds; adding the target oxidized hyaluronic acid to an exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution; The exosome-loaded oxidized hyaluronic acid solution is mixed with polylysine to obtain an exosome-loaded hydrogel, wherein the mass ratio of the target oxidized hyaluronic acid to the polylysine in the above-mentioned exosome-loaded hydrogel is in the range of 1 to 2. Because the biocompatibility and moisture retention of the obtained target oxidized hyaluronic acid are improved by oxidizing and grafting methacrylate bonds on hyaluronic acid, the possibility of preparing a spray-type exosome-loaded hydrogel is improved, and because the antibacterial polylysine is combined with the target oxidized hyaluronic acid having an aldehyde group and a methacrylate bond, the exosome-loaded hydrogel includes a Schiff base bond, thereby improving the antibacterial property of the exosome-loaded hydrogel while improving the mechanical properties and controllable degradability of the exosome-loaded hydrogel, thereby improving the exosome retention capacity and activity of the exosome-loaded hydrogel, thereby improving 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 exosome-loaded hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and other features, advantages and aspects of the embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the accompanying drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and that components and elements are not necessarily drawn to scale.

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

[0021] Figure 2 This is the result of an experiment to identify stem cell exosomes;

[0022] Figure 3 are infrared spectra for characterizing hyaluronic acid, oxidized hyaluronic acid and target oxidized hyaluronic acid respectively;

[0023] Figure 4 These are NMR characterization images for characterizing hyaluronic acid, oxidized hyaluronic acid and target oxidized hyaluronic acid respectively;

[0024] Figure 5 is the scanning electron microscopy image of the exosome-loaded hydrogel generated at different ratios of target oxidized hyaluronic acid and poly-lysine;

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

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

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

[0028] Fig. 9 It is the experimental data of antibacterial properties of exosome-loaded hydrogel;

[0029] Fig.10 is the experimental data of exosome release from exosome-loaded hydrogel;

[0030] Fig.11 It is the cytotoxicity and proliferation experimental data of exosome-loaded hydrogel;

[0031] Fig.12 This is the experimental data of cell migration of exosome-loaded hydrogels;

[0032] Fig.13 This is the experimental data of angiogenesis of exosome-loaded hydrogels;

[0033] Fig.14 It is the experimental data of exosome-loaded hydrogel for the treatment of infected full-thickness skin wounds;

[0034] Fig.15 These are the toxicity test data of exosome-loaded hydrogels. DETAILED DESCRIPTION

[0035] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not intended to limit the scope of protection of the present disclosure.

[0036] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure 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 of the functions performed by these devices, modules or units.

[0038] It should be noted that the modifications of "one" and "plurality" mentioned in the present disclosure are illustrative rather than restrictive, and 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 used 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 accompanying drawings and in conjunction with embodiments.

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

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

[0043] In some embodiments, hyaluronic acid may be subjected to oxidation treatment to obtain oxidized hyaluronic acid.

[0044] In some optional implementations of some embodiments, hyaluronic acid may be oxidized by the following steps to obtain oxidized hyaluronic acid:

[0045] In the first step, hyaluronic acid (HA, Mw 200000-400000) is dissolved in water to obtain a hyaluronic acid solution. The molecular structure 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] In the second step, sodium periodate is mixed with the above-mentioned hyaluronic acid solution to obtain a second mixed solution. The mass ratio of the above-mentioned sodium periodate to the above-mentioned hyaluronic acid can be 3:5. In practice, sodium periodate can be added to the above-mentioned hyaluronic acid solution, and the mixture is mixed and reacted for a preset mixing time under indoor light-proof conditions to obtain a second mixed solution. The above-mentioned preset mixing time can be a pre-set time for completing the reaction of sodium periodate and hyaluronic acid solution. The above-mentioned preset mixing time can range from 4.5 to 6 hours.

[0047] The third step is to mix anhydrous ethanol with the second mixed solution to obtain a third mixed solution. The ratio of anhydrous ethanol to the second mixed solution can be 1:100. In practice, anhydrous ethanol can be added to the second mixed solution to terminate the reaction for 1 hour to obtain the third mixed solution.

[0048] The fourth step is to perform a dialyzed freeze-drying treatment on the third mixed solution to obtain oxidized hyaluronic acid. In practice, first, the third mixed solution can be dialyzed through deionized water and a dialysis bag to obtain wet oxidized hyaluronic acid. The dialysis time can be 3 to 5 days. The deionized water needs to be replaced at least 3 times during the dialysis period. Then, the 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 ml 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 5 hours under the condition of room temperature and protection from light can be obtained to obtain a second mixed solution. Afterwards, 1 ml of absolute ethanol can be added to the second mixed solution to perform a termination reaction for 1 hour to obtain a third mixed solution. Finally, the third mixed solution is dialyzed and vacuum-freezed to obtain oxidized hyaluronic acid (OHA).

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

[0051] In some embodiments, the oxidized hyaluronic acid and methacrylic anhydride may be mixed and reacted under preset environmental conditions to obtain a first mixed solution, wherein the preset environmental conditions may be pre-set environmental conditions in which the oxidized hyaluronic acid and methacrylic anhydride are mixed and reacted.

[0052] Optionally, the preset environmental conditions may be: the pH value of the first mixed solution is in the range of 8 to 9, and 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 to obtain a first mixed solution by the following steps:

[0054] In the first step, the oxidized hyaluronic acid is dissolved in distilled water to obtain an oxidized hyaluronic acid solution, wherein the concentration of the oxidized hyaluronic acid solution may be 1%.

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

[0056] In the first sub-step, the pH value of the mixed solution is detected in real time by a pH value detection device. The preset time period may be a time period corresponding to the preset time period from the time when methacrylic anhydride is added to the current time. The pH value detection device may be a pH value meter. The pH value of the mixed solution may be the pH value of the mixed solution.

[0057] In a second sub-step, in response to determining that the pH value of the detected 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. The mixed solution may be a solution containing the oxidized hyaluronic acid solution and methacrylic anhydride. The concentration of the 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 are 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 pH value of the detected solution is not within the above pH value range, 2 milliliters of sodium hydroxide solution are added to adjust the pH value of the solution so that it is within the above pH value range. Stop the detection after 12 hours.

[0059] Step 103, dialyzing and freeze-drying the first mixed solution to obtain target oxidized hyaluronic acid.

[0060] In some embodiments, the first mixed solution can be dialyzed and freeze-dried to obtain the target oxidized hyaluronic acid. Wherein, the target oxidized hyaluronic acid can be an oxidized hyaluronic acid grafted with a methacrylate bond. In practice, the first mixed solution can be dialyzed and freeze-dried by a vacuum freeze dryer to obtain the target oxidized hyaluronic acid (OHAMA). Wherein, the molecular formula structure of the target oxidized hyaluronic acid can be

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

[0062] Step 104, adding the target oxidized hyaluronic acid into the exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution.

[0063] In some embodiments, the target oxidized hyaluronic acid can be added to the exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution. Wherein, the exosome solution can be: a solution in which the solute is a stem cell exosome. In practice, the target oxidized hyaluronic acid can be added to the exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution with a preset hyaluronic acid solution concentration. Wherein, the preset hyaluronic acid solution concentration can be a pre-set concentration of the exosome-loaded oxidized hyaluronic acid solution. For example, the preset hyaluronic acid solution concentration can be 6% or 12%.

[0064] In some optional implementations of some embodiments, the target oxidized hyaluronic acid can be added to the exosome solution to obtain the exosome-loaded oxidized hyaluronic acid solution by the following steps:

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

[0066] In some optional implementations of some embodiments, the concentration of the hyaluronic acid solution may be determined according to the hydrogel application scenario information by the following steps:

[0067] Step 1, according to the hydrogel application scenario information, determine the exosome release rate. Wherein, the exosome release rate may be the exosome release rate. In practice, the preset exosome release rate corresponding to the application type included in the hydrogel application scenario information may be determined as the exosome release rate. Wherein, the preset exosome release rate may be a pre-set exosome release rate.

[0068] In the process of adopting technical solutions to solve the technical problems in the background technology, the following problems are often accompanied. Technical Problem 2: The patient's physique is different, the injury situation is different, and the exosome release rate required for wound repair is also different. How to improve the compatibility of the exosome release rate of the exosome hydrogel loaded with exosomes and the patient's wound to improve the wound repair performance of the exosome hydrogel loaded with exosomes. For the above technical problem 2, the conventional solution is generally: according to the type of wound, the exosome release rate of the corresponding wound type is determined experimentally. However, the above conventional solution still has the following problems: for the same wound type, for patients of different age groups and different physiques, the patient's own rate of promoting exosome release is also different. The exosome release rate is determined only according to the wound type, resulting in a low compatibility of the exosome release rate of the exosome hydrogel loaded with exosomes and the patient's wound, thereby causing the wound repair performance of the exosome hydrogel loaded with exosomes to be low.

[0069] In the face of the above technical problem 2, combined with the artificial intelligence research and development technology owned by the partner institutions, the following solutions can be adopted:

[0070] In practice, the exosome release rate can be determined by the hydrogel application scenario information and the preset release rate determination device. The preset release rate determination device may be a pre-set device for determining the exosome release rate. The preset release rate determination device may be a computing device storing an application corresponding to the method for determining the exosome release rate. The method for determining the exosome release rate includes:

[0071] The first step is to display the application scenario information input interface in response to detecting the preparation user login information. The preparation user login information may be information for the preparation user to log in to the application scenario information input system. For example, the preparation user login information may be "1". The preparation user may be a user who needs to prepare an exosome-loaded hydrogel. The application scenario information input system may be a system for the preparation user to input text of the application scenario of the exosome-loaded hydrogel. The application scenario information input interface may be an interface for the preparation user to input text of the application scenario of the exosome-loaded hydrogel. The application scenario information input interface may include but is not limited to an information input text box and an information confirmation control. The information input text box may be used for the preparation user to input the application scenario text information. The information confirmation control may be a control for the preparation user to confirm the input application scenario text information. The application scenario text information may be a text describing the exosome-loaded hydrogel. The application scenario text information may include but is not limited to text corresponding to the patient's age, injury scenario type, injury duration, and wound area. For example, the application scenario text information may be "The patient is 20 years old, and the surgical incision has been purulently infected during maintenance for 3 days, and the wound size is 5 cm."

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

[0073] The third step is to perform feature extraction processing on the above-mentioned application scenario information to be processed to obtain application scenario feature information. In practice, the above-mentioned execution subject can perform feature extraction processing on the above-mentioned application scenario information to be processed by 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 pre-set method 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] In the fourth step, the application scenario feature information is input into the pre-trained patient health type generation model to obtain the patient health type. The patient health type generation model can be a classification model that takes the application scenario feature information as input and the patient health type as output. The classification model can be a support vector machine or a recurrent neural network. The patient health type can be a standard patient's health status. The patient health type can be, but is not limited to, one of the following: very healthy, good, average, poor.

[0075] In the fifth step, the application scenario feature information is input into the pre-trained wound type generation model to obtain the wound type. The wound type generation model can be a classification model that takes the application scenario feature information as input and the wound type as output. The wound type can characterize the degree of injury of the wound. The wound type can be, but is not limited to, one of the following: severe injury, general injury, and minor injury.

[0076] In the sixth step, in response to determining that the above-mentioned hydrogel application scenario information meets the preset first application type condition, the preset first patient health weight is determined as the first weight, and the preset first wound weight is determined as the second weight. Among them, the above-mentioned preset first application type condition can be: the application type included in the above-mentioned hydrogel application scenario information is acute infectious injury. The above-mentioned preset first patient health weight can characterize the degree of influence of the patient's health type on the exosome release rate. The above-mentioned preset first wound weight can characterize the degree of influence of the wound type on the exosome release rate. The sum of the above-mentioned preset first patient health weight and the above-mentioned preset first wound weight can be 1. It should be noted that when the above-mentioned 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] In the seventh step, in response to determining that the above-mentioned hydrogel application scenario information meets the preset first application type condition, the preset second patient health weight is determined as the first weight, and the preset second wound weight is determined as the second weight. Among them, the above-mentioned preset first application type condition can be: the application type included in the above-mentioned hydrogel application scenario information is chronic wound repair. The above-mentioned preset second patient health weight can characterize the degree of influence of the patient's health type on the exosome release rate. The above-mentioned preset second wound weight can characterize the degree of influence of the wound type on the exosome release rate. The sum of the above-mentioned preset second patient health weight and the above-mentioned preset second wound weight can be 1. It should be noted that when the above-mentioned 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, the product of the preset health score corresponding to the patient's health type and the first weight is determined as the first score. The preset health score may be a score of the effect of the patient's health type on the exosome release rate.

[0079] In the ninth step, the product of the preset wound score corresponding to the wound type and the second weight is determined as the second score. The preset wound score may be a score of the effect of the wound type on the exosome release rate.

[0080] In the tenth step, the sum of the first score and the second score is determined as the scenario score. The scenario score may be a score corresponding to the application scenario of the loaded exosomes. After preliminary experiments, different scenario scores correspond to different exosome release rates to achieve better repair performance of the loaded exosomes.

[0081] In the eleventh step, the preset exosome release rate corresponding to the above scenario score is determined as the exosome release rate. The preset exosome release rate may be an exosome release rate obtained through experiments.

[0082] The above technical scheme and its related contents, as an inventive point of an embodiment of the present disclosure, solve the technical problem that "for the same wound type, for patients of different age groups and different physiques, the rate at which the patient promotes the release of exosomes is different. The exosome release rate is determined only according to the wound type, resulting in a low compatibility between the exosome release rate of the exosome-loaded hydrogel and the patient's wound, thereby causing the wound repair performance of the exosome-loaded hydrogel to be low". The factors that lead to the low wound repair performance of the exosome-loaded hydrogel are often as follows: for the same wound type, for patients of different age groups and different physiques, the rate at which the patient promotes the release of exosomes is different. The exosome release rate is determined only according to the wound type, resulting in a low compatibility between the exosome release rate of the exosome-loaded hydrogel and the patient's wound, thereby causing the wound repair performance of the exosome-loaded hydrogel to be low. If the above factors are solved, the effect of improving the wound repair performance of the exosome-loaded hydrogel can be achieved. In order to achieve this effect, the preparation method of the exosome-loaded hydrogel in some embodiments of the present disclosure, when determining the exosome release rate, not only takes into account the application type, but also comprehensively considers the patient's own health status, the wound injury scene and duration, and the speed at which the wound needs to recover, and flexibly adjusts the proportion of various influencing factors on the exosomes, thereby improving the compatibility of the exosome release rate of the exosome-loaded hydrogel with the patient's wound, thereby improving the wound repair performance of the exosome-loaded hydrogel.

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

[0084] In the second step, the target oxidized hyaluronic acid is added into the exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution corresponding to the concentration of the hyaluronic acid solution.

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

[0086] The first step is to isolate exosomes from umbilical cord stem cells to obtain stem cell exosomes.

[0087] The second step is to add the above stem cell exosomes into a phosphate buffer solution (PBS) to obtain an exosome solution, wherein the ratio of the above stem cell exosomes to the above phosphate buffer solution is 50 micrograms: 100 milliliters.

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

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

[0090] Step 2, centrifuge the cell supernatant by the centrifugal device to obtain exosome spheroids. The exosome spheroids may be spheroids with exosomes. In practice, the cell supernatant may be centrifuged by the high-speed centrifuge to remove the cell supernatant to obtain exosome spheroids. The operating parameters corresponding to the high-speed centrifuge are: centrifugal force 120000g (gravitational acceleration), temperature 4 degrees Celsius, and operating time 70 minutes.

[0091] Step three, using a phosphate buffer solution to precipitate the exosome spheroids to obtain stem cell exosomes.

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

[0093] In the first step, Western blot (WB) was used to identify stem cell exosomes. Figure 2 As shown in (a), exosomes are specifically enriched in proteins CD9, CD81, and Alix, but do not contain the Golgi protein Gm130.

[0094] The second step is to identify stem cell exosomes by transmission electron microscopy. Figure 2 As shown in (b), transmission electron microscopy revealed that stem cell exosomes were in the form of a cup-disc structure.

[0095] The third step is to identify stem cell exosomes by nanoparticle tracking analysis (NTA). Figure 2 As shown in (c), the diameter of exosome particles is 30 to 150 nanometers.

[0096] Step 105, mixing the exosome-loaded oxidized hyaluronic acid solution with poly-lysine to obtain an exosome-loaded hydrogel.

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

[0098] In the process of adopting technical solutions to solve the technical problems in the background technology, the following problems are often accompanied. Technical problem three: the existing hydrogel is in a gel state and needs to be smeared during use. The application process is not fully disinfected, resulting in a high risk of secondary injury and infection of the wound, and some exosomes will remain on the application tool, resulting in a reduction in the antibacterial effect of the exosome-loaded hydrogel.

[0099] In the face of the above technical problem three, combined with the advantages of good moisturizing and biocompatibility of exosome-loaded hydrogel, the following solution can be adopted:

[0100] In some optional implementations of some embodiments, the exosome-loaded oxidized hyaluronic acid solution and poly-lysine may be mixed and reacted to obtain an exosome-loaded hydrogel by the following steps:

[0101] In the first step, the exosome-loaded oxidized hyaluronic acid solution is filled into an empty spray bottle by a filling device to obtain a hyaluronic acid spray bottle. The filling device may be a device for filling a liquid into an empty spray bottle. For example, the filling device may be a filling machine.

[0102] The second step is to fill the polylysine solution of the preset concentration into the empty spray bottle by a filling device to obtain a polylysine spray bottle. The preset concentration may be a pre-set concentration. For example, the preset concentration may be 3% or 6%.

[0103] The third step is to press the hyaluronic acid spray bottle and the polylysine spray bottle at the same time, 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 preset spraying position may be a pre-set position where the exosome hydrogel needs to be loaded. As an example, the preset spraying position may be the position of a wound that needs to be repaired.

[0104] The fourth step is to perform a photocuring treatment on the hydrogel mixed solution to obtain an exosome-loaded hydrogel. In practice, the hydrogel mixed solution can be photocured by an ultraviolet lamp to obtain an exosome-loaded hydrogel.

[0105] The above technical scheme and its related contents, as an inventive point of an embodiment of the present disclosure, solve the technical problem that "the existing hydrogel is in a gel state, and needs to be smeared during use. It is not sufficiently disinfected during the smearing process, resulting in a high risk of secondary injury and infection of the wound, and some exosomes will remain on the smearing tool, resulting in a reduction in the antibacterial effect of the exosome-loaded hydrogel". The factors that lead to a high risk of secondary injury and infection of the wound and a reduction in the antibacterial effect of the exosome-loaded hydrogel are often as follows: the existing hydrogel is in a gel state, and needs to be smeared during use. It is not sufficiently disinfected during the smearing process, resulting in a high risk of secondary injury and infection of the wound, and some exosomes will remain on the smearing tool, resulting in a reduction in the antibacterial effect of the exosome-loaded hydrogel. If the above factors are solved, the risk of secondary injury and infection of the wound can be reduced and the antibacterial effect of the exosome-loaded hydrogel can be improved. In order to achieve this effect, in some embodiments of the present disclosure, the preparation method of the loaded exosome hydrogel is prepared by respectively preparing the loaded exosome oxidized hyaluronic acid solution and poly-lysine into a spray solution with a certain concentration ratio. Because EPL and OHAMA can combine by forming Schiff base bonds and form a gel by 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 daily light to repair wounds. Therefore, the loaded exosome hydrogel can be applied without the user having to perform an application operation, thereby reducing the risk of secondary injury and infection of the wound and improving the antibacterial effect of the loaded exosome hydrogel.

[0106] In order to demonstrate the effect of the exosome-loaded hydrogel prepared by the above-mentioned exosome-loaded hydrogel preparation method, the following experimental methods and experimental results are provided.

[0107] Experiment 1

[0108] Hyaluronic acid, oxidized hyaluronic acid and target oxidized hyaluronic acid were characterized by infrared spectroscopy, and infrared spectra were obtained, such as Figure 3 As shown. Among them, Figure 3 The three infrared spectra shown in the figure correspond to hyaluronic acid, oxidized hyaluronic acid and target oxidized hyaluronic acid from top to bottom.

[0109] Experiment 2

[0110] Hyaluronic acid, oxidized hyaluronic acid and target oxidized hyaluronic acid were characterized by nuclear magnetic resonance spectroscopy, and nuclear magnetic resonance characterization was obtained, such as Figure 4 shown.

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

[0112] Experiment 3

[0113] According to the different mass ratios of the target oxidized hyaluronic acid and polylysine, the experiment was divided into 4 groups. The concentrations of the exosome oxidized hyaluronic acid solution and the polylysine solution in the first group of exosome-loaded hydrogels were 6% and 6%, respectively. The concentrations of the exosome oxidized hyaluronic acid solution and the polylysine solution in the second group of exosome-loaded hydrogels were 8% and 6%, respectively. The concentrations of the exosome oxidized hyaluronic acid solution and the polylysine solution in the third group of exosome-loaded hydrogels were 10% and 6%, respectively. The concentrations of the exosome oxidized hyaluronic acid solution and the polylysine solution in the fourth group of exosome-loaded hydrogels were 12% and 6%, respectively. The four groups of exosome-loaded hydrogels were freeze-dried under vacuum for 24 hours, and after sputtering gold plating, the gel structures of the four groups of exosome-loaded hydrogels were observed by scanning electron microscope (SEM), as shown Figure 5 shown. Figure 5 This indicates that the higher the content of 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 hydrogels at 37°C were tested using a 25 mm parallel plate rheometer. The exosome-loaded hydrogels were subjected to a strain sweep of 100 rad / s (radians / second) at 37°C for 0.1 min-strain amplitude of 1% at 37°C. The exosome-loaded hydrogels were subjected to a strain sweep from 0.1% to 10% at an angular frequency of 1 rad / s at 37°C, as shown in FIG. 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] like Figure 7 As shown, a piece of methylene blue-stained exosome-loaded hydrogel and a piece of unstained exosome-loaded hydrogel were cut and reassembled along the cross section. After 10 minutes, the two exosome-loaded hydrogels fused at the broken ends to form a piece of exosome-loaded hydrogel. After clamping 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 target oxidized hyaluronic acid and polylysine, the experiment was divided into 4 groups. The concentrations of the exosome oxidized hyaluronic acid solution and the polylysine solution in the first group of exosome-loaded hydrogels were 3% and 3%, respectively. The concentrations of the exosome oxidized hyaluronic acid solution and the polylysine solution in the second group of exosome-loaded hydrogels were 4% and 3%, respectively. The concentrations of the exosome oxidized hyaluronic acid solution and the polylysine solution in the third group of exosome-loaded hydrogels were 5% and 3%, respectively. The concentrations of the exosome oxidized hyaluronic acid solution and the polylysine solution in the fourth group of exosome-loaded hydrogels were 6% and 3%, respectively. The initial weights of the four groups of exosome-loaded hydrogels were recorded, placed in PBS solution, and carried out at 37°C. The gels were taken out and weighed every 30 minutes until the gel began to degrade, and the swelling rate was calculated. Among them, the swelling rate was calculated by the following method:

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

[0121] Then, the percentage of the ratio of the above weight difference to the initial weight is determined as the swelling rate. Figure 8 As shown, the four groups of exosome-loaded hydrogels all have good swelling properties, and as the concentration of target oxidized hyaluronic acid increases, the cross-linking of the exosome hydrogels becomes tighter and the swelling rate decreases. It should be noted that Figure 8 The broken lines in the figure correspond to the concentrations of the exosome-loaded oxidized hyaluronic acid solution of 3%, 4%, 5%, and 6% from top to bottom.

[0122] Experiment 8

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

[0124] Experiment 9

[0125] The experiment was divided into two groups. Exosome-loaded hydrogels loaded with 50 micrograms of exosomes were prepared and placed in transwell-24-well plates. Then, 200 microliters of PBS (pH = 7.4) solution and 200 microliters of pH = 6.0 solution were added to the two groups of 24-well plates respectively. The liquid except the hydrogel in the two groups of wells was collected every 12 hours, and the corresponding solution was added. After 5 days, the collected liquid was quantified by fluorescence. The release rate and cumulative release amount of hydrogel exosomes were detected, such as Fig.10 As shown. This shows that the exosome-loaded hydrogel added with PBS solution has the function of slow-controlled release of exosomes.

[0126] Experiment 10

[0127] CCK-8 cell experiment was performed. 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 the ultracentrifuge and the ultracentrifuge was run at 1000 rpm for 3 minutes. Then, the supernatant of the centrifuge tube was discarded and resuspended with 1 ml of serum-containing medium. After that, the cells were counted using a counter and the cell suspension was diluted according to the counting results. When the cell suspension was diluted to 30,000 cells / ml, three 96-well plates were taken, a blank control and three experimental groups were set up, and 6 replicates were set up in each group. 100 microliters of diluted cell suspension were added to each well, and 200 microliters of PBS buffer were added to the edge wells to reduce the effect of evaporation. After 12 hours of inoculation, the cells were well attached to the wall, and the culture medium of each well was aspirated. Then, the blank group was added with medium without drugs, and the three experimental groups were added with medium containing only hydrogel, medium containing only exosomes, and medium combined with hydrogel and exosomes, respectively. Then, the cells were placed in an incubator for incubation. After the drug treatment time, the culture medium of each well was aspirated again, and a culture medium containing CCK-8 was added. CCK-8 accounted for 1 / 10 of the volume of the cell culture medium. After 45 minutes of incubation, the 96-well plate was taken out and placed in an ELISA reader to detect the absorbance at 450 nm. The test results were as follows: Fig.11 As shown, Fig.11 The corresponding experimental groups from left to right are culture medium (control group), culture medium containing only hydrogel (hydrogel group), culture medium containing only exosomes (exosome group), culture medium combining 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 in promoting cell proliferation.

[0128] Experiment 11

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

[0130] In the first step, NIH-3T3 cells in the logarithmic growth phase were digested with trypsin to form a single cell suspension, which was then inoculated into a 6-well culture plate.

[0131] In the second step, 10w / well of cells are plated, and the final total amount of culture medium in each well can be 2 ml. The cells are cultured in a 37°C, 5% carbon dioxide incubator for 24 hours.

[0132] The third step is to use a 200 μl pipette tip to scratch the surface with a ruler the next day.

[0133] Step 4: Wash the cells three times with PBS buffer, remove the scratched cells, add serum-free culture medium, take pictures under a 4x microscope, and record it as 0h.

[0134] In the fifth step, the experimental groups were divided into serum-free medium (control group), 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).

[0135] Step 6: Take samples and take photos at 24 hours and 48 hours. After opening the pictures with Image J software, randomly draw 6 to 8 horizontal lines to calculate the cell migration rate. Cell migration rate (wound healing rate) = (initial scratch area - scratch area at the moment) / initial scratch area. Fig.12 As shown in the figure, after 24h and 48h, compared with the control group, the remaining area of ​​all experimental groups was reduced, and the remaining area of ​​the exosome-loaded hydrogel group was the smallest, and the difference was significant (P<0.001), which shows that the exosome-loaded hydrogel has a good effect in promoting cell migration.

[0136] Experiment 12

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

[0138] In the first step, human umbilical vein endothelial cells (HUVEC) were selected, passaged to the 4th generation, and allowed to reach 80% confluence within 24 hours.

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

[0140] The third step is to dilute the matrix gel to 10 mg / mL with culture medium, put the completely thawed matrix gel on ice, turn it over 3 to 5 times to mix the contents, take a 96-well plate, and add 50 μl of matrix gel to each well.

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

[0142] The fifth step was to prepare three working solutions: 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: HUVEC cells reaching 80% confluence in the T25 flask were digested, centrifuged, and counted.

[0144] Step 7: Resuspend the cells in the prepared culture medium and working solution to a concentration of 45w cells per ml of single cell suspension.

[0145] Step 8: Add 100 μl of cell suspension (containing 4.5w cells) containing the sample to be tested to each well of the 96-well plate. Place the 96-well plate in an incubator at 37 degrees Celsius, 5% carbon dioxide, and 90% humidity. Take photos and observe after 6 hours. Fig.13 As shown in the figure, after 6 hours of experimental treatment, a small amount of tubular structures were observed in the control group, while obvious tubular structures were observed in the exosome group and the exosome-loaded hydrogel group. The taken photos were quantitatively analyzed using the ImageJ plug-in AngiogenesisAnalyzer to analyze the blood vessel length. The data results are shown in the figure below. Fig.13 As shown, from Fig.13 It can be seen that the blood vessels in the exosome-loaded hydrogel group are the longest, which shows that the exosome-loaded hydrogel has a good promoting effect on angiogenesis.

[0146] Experiment 13

[0147] In the first step, 5-week-old SPF-grade C57 / BL6 male mice weighing 28.5±2 grams were fed adaptively for one week at room temperature of 25 degrees Celsius and humidity of 60%. After no physiological abnormalities were observed, a mouse trauma model was established. The specific modeling steps are as follows:

[0148] First, the mice were weighed and anesthetized with isoflurane.

[0149] Secondly, the anesthetized mice were fixed in a prone position, the back hair was removed using a shaver, and the skin in the surgical area was disinfected with iodine.

[0150] Then, a punch was used to create full-thickness skin injury wounds with a diameter of about 7 mm in the area surrounded by the back of the neck and above the shoulder blades on both sides, deep into the fascia.

[0151] Afterwards, a pre-sterilized 3-mm-thick annular silicone gel (10 mm inner diameter, 15 mm outer diameter) 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, namely the control group, the hydrogel group, the exosome group, and the exosome-loaded hydrogel group. For the four groups of experiments, the efficacy was observed and the dressing was changed on days 0, 3, 7, 10, and 14. The natural healing process was observed for about 14 days. Fig.14 (a) In addition, tissues were sliced ​​and fixed on the 7th and 14th days, and immunostained to observe the treatment effects, as shown in Fig.14 (b) as shown. Fig.14 It can be shown that the wound healing effect of exosome-loaded hydrogel is the best. Toxicity tests were performed on the four groups of experiments on the 14th day. Among them, the experimental data of blood routine and blood biochemistry are as follows Fig.15 As shown. Fig.15 It can be seen that there is no significant difference between the experimental groups and the control group, which shows that the hydrogel and exosomes have good biocompatibility.

[0153] The above-mentioned embodiments of the present disclosure have the following beneficial effects: through the preparation method of the exosome-loaded hydrogel of 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 capacity and activity of the exosomes. Specifically, the reasons for the poor sprayability and applicability of gelatin methacryloyl hydrogel are: the mechanical properties of gelatin methacryloyl hydrogel are low and the controllable degradability is poor, resulting in poor retention capacity and activity of exosomes loaded by gelatin methacryloyl hydrogel, and the viscosity of gelatin methacryloyl hydrogel is large, resulting in poor sprayability of gelatin methacryloyl hydrogel, thereby resulting in low applicability of gelatin methacryloyl hydrogel. Based on this, in some embodiments of the present disclosure, the method for preparing exosome-loaded hydrogel comprises: 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; dialyzing and freeze-drying the first mixed solution to obtain a target oxidized hyaluronic acid, wherein the target oxidized hyaluronic acid is an oxidized hyaluronic acid grafted with methacrylate bonds; adding the target oxidized hyaluronic acid to an exosome solution to obtain an exosome-loaded oxidized hyaluronic acid solution; and The exosome oxidized hyaluronic acid solution is mixed with polylysine to obtain an exosome-loaded hydrogel, wherein the mass ratio of the target oxidized hyaluronic acid to the polylysine in the exosome-loaded hydrogel is in the range of 1 to 2. Because the biocompatibility and moisture retention of the target oxidized hyaluronic acid obtained by oxidation and grafting methacrylate bonds to hyaluronic acid are improved, the possibility of preparing a spray-type exosome-loaded hydrogel is improved, and because the antibacterial polylysine is combined with the target oxidized hyaluronic acid having an aldehyde group and a methacrylate bond, the exosome-loaded hydrogel obtained includes a Schiff base bond, thereby improving the antibacterial properties of the exosome-loaded hydrogel while improving the mechanical properties and controllable degradability of the exosome-loaded hydrogel, thereby improving the exosome retention capacity and activity of the exosome-loaded hydrogel, thereby improving 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 exosome-loaded hydrogel.

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

[0155] The present disclosure also provides a use of an exosome-loaded hydrogel, wherein the exosome-loaded hydrogel can be used for skin wound treatment.

[0156] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. 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 a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the embodiments of the present disclosure (but not limited to) and the technical solutions formed.

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 and poly-lysine are mixed and reacted 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.

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.

9. A use of the exosome-loaded hydrogel as claimed in claim 8, wherein: The exosome-loaded hydrogel is used for treating skin wounds.

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