Exosome hydrogel and methods of making and uses thereof

Abstract

The application embodiment provides an exosome hydrogel, a preparation method and application thereof. The exosome hydrogel is prepared by cross-linking of exosomes, oxidized hyaluronic acid and carboxymethyl chitosan; wherein, in the exosome hydrogel, the final concentration of the exosomes is 20-5000 mu g / mL, the final concentration of the oxidized hyaluronic acid is 1-200 mg / mL, and the final concentration of the carboxymethyl chitosan is 10-500 mg / mL. The application embodiment is based on the prepared exosomes, and the hydrogel is prepared by cross-linking of the oxidized hyaluronic acid, the carboxymethyl chitosan and the exosomes. The hydrogel has the properties of injectability, adhesion, self-healing, ROS / glucose response and excellent mechanical performance, and can be used for accelerating the healing of chronic wounds.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, specifically to an exosome hydrogel, its preparation method, and its uses. Background Technology

[0002] Chronic wounds typically refer to skin barrier defects that fail to heal within four weeks or longer. Instead of undergoing the typical wound repair phase, they become inflammatory, entering a vicious cycle of inflammation and tissue destruction. Chronic wounds, as a classic case in the field of dermatological wound care, have received widespread attention from researchers and healthcare professionals.

[0003] Currently, hydrogels used as wound dressings have become an effective means of wound healing. Hydrogels not only create an anti-inflammatory environment conducive to modulating immune responses but also improve exosome stability and control and sustain exosome release. However, traditional hydrogels have poor properties, such as lack of tissue adhesion, unsatisfactory mechanical and degradation properties, and no self-healing ability, limiting their biomedical applications. In recent years, there has been increasing interest in stimulus-responsive hydrogels because they can modulate under the influence of external stimuli (including temperature, pH, ROS, and glucose). Considering the high ROS and high glucose microenvironment of chronic wounds (such as diabetic wounds), developing a microenvironment-responsive hydrogel is considered an effective strategy. Summary of the Invention

[0004] The purpose of this application is to provide an exosome hydrogel, its preparation method, and its uses.

[0005] To achieve the above objectives, the embodiments of this application propose the following technical solutions:

[0006] In a first aspect, embodiments of this application provide an exosome hydrogel, which is prepared by cross-linking exosomes, oxidized hyaluronic acid and carboxymethyl chitosan;

[0007] In the exosome hydrogel, the final concentration of the exosomes is 20-5000 μg / mL, the final concentration of oxidized hyaluronic acid is 1-200 mg / mL, and the final concentration of carboxymethyl chitosan is 10-500 mg / mL.

[0008] As one embodiment, the method for preparing the exosomes includes:

[0009] Prepare a cell culture medium containing a culture medium that includes trace elements;

[0010] Human umbilical cord mesenchymal stem cells were placed in a cell culture medium to obtain a cell culture medium containing human umbilical cord mesenchymal stem cells.

[0011] A 3D bioreactor was used to culture cell culture medium containing human umbilical cord mesenchymal stem cells in a 3D environment, and the supernatant was collected.

[0012] After differential centrifugation, human umbilical cord mesenchymal stem cell exosomes were obtained from the supernatant.

[0013] As one embodiment, the cell culture medium containing culture medium includes:

[0014] Take ore powder and dissolve it in a dissolving solution to obtain a mixture;

[0015] After filtration, the mixture is added to an alternating strong magnetic field. After shearing, aging, filtration and purification in the alternating strong magnetic field, a metal ion solution is obtained.

[0016] The metal ion solution was diluted with a diluent to obtain a diluted metal ion solution;

[0017] The diluted metal ion solution was sterilized and filtered to obtain the culture medium;

[0018] The culture medium is added dropwise to the cell culture medium to obtain a cell culture medium containing the culture medium;

[0019] The ore powder is selected from at least one of zinc ore powder, mica powder, vermiculite powder, coal ore powder, celestite powder, and maifanite powder.

[0020] In one embodiment, the solution is a mixed solution of dilute hydrochloric acid, dilute sulfuric acid, and oxalic acid; the volume ratio of the dilute hydrochloric acid, dilute sulfuric acid, and oxalic acid is 3:5:2.

[0021] The diluent is a 0.9% sodium chloride solution; the magnetic field strength of the alternating strong magnetic field is 1×10⁻⁶. 3～4 Gauss.

[0022] In one embodiment, the supernatant is centrifuged at differential speed to obtain human umbilical cord mesenchymal stem cell exosomes, comprising:

[0023] Centrifuge the supernatant at 300×g for 10-15 minutes at room temperature.

[0024] Centrifuge the supernatant from the previous step at 2000×g at room temperature for 20-30 min; then centrifuge at 10000×g at 4℃ for 20-30 min, and filter to obtain human umbilical cord mesenchymal stem cell exosomes.

[0025] In one embodiment, the degree of oxidation of the oxidized hyaluronic acid is 5%-50%, preferably 20%-40%.

[0026] In one embodiment, the trace element is selected from at least one of iron, magnesium, zinc, manganese, titanium, calcium, selenium, vanadium, chromium, nickel, cobalt, molybdenum, copper, and potassium; the 3D bioreactor is a 3D Flotrix miniSpin bioreactor.

[0027] In one embodiment, the final concentration of exosomes in the exosome hydrogel is 30-2500 μg / mL, the final concentration of oxidized hyaluronic acid is 25-75 mg / mL, and the final concentration of carboxymethyl chitosan is 75-300 mg / mL.

[0028] Secondly, embodiments of this application provide a method for preparing exosome hydrogels as described in the first aspect, the preparation method comprising:

[0029] Carboxymethyl chitosan was dissolved in PBS solution to obtain carboxymethyl chitosan PBS solution;

[0030] Oxidized hyaluronic acid was dissolved in PBS solution to obtain oxidized hyaluronic acid PBS solution;

[0031] Exosomes were uniformly dispersed in a carboxymethyl chitosan PBS solution to obtain a mixture;

[0032] The mixture was thoroughly mixed with oxidized hyaluronic acid PBS solution to obtain exosome hydrogel.

[0033] Thirdly, embodiments of this application propose the use of the exosome hydrogel as described in the first aspect in the preparation of dressings for treating or accelerating the healing of chronic wounds.

[0034] The embodiments of this application have at least the following beneficial effects:

[0035] This application employs a cell culture medium containing trace elements, combined with 3D culture, to culture human umbilical cord mesenchymal stem cells. The 3D culture containing trace elements alters the survival environment of stem cells, reduces cytoskeletal tension, increases the expression of various genes, and influences stem cell differentiation, achieving the production of high concentrations and high activity of exosomes in a minimal culture medium volume. Furthermore, in addition to the physical and mechanical changes in the culture environment, the 3D culture containing trace elements also affects the overall hypoxic state of stem cells, altering the content of proteins and nucleic acids within the exosomes. These trace element-carrying exosomes participate in the regulation of intercellular and intercellular microenvironments, modulating the immune environment, controlling the expression of inflammation-related genes, and enhancing the anti-inflammatory activity of exosomes.

[0036] This application describes the preparation of a hydrogel based on exosomes, which is cross-linked with oxidized hyaluronic acid, carboxymethyl chitosan, and exosomes. This hydrogel exhibits injectability, adhesiveness, self-healing properties, ROS / glucose responsiveness, and excellent mechanical properties, suggesting its potential for accelerating the healing of chronic wounds.

[0037] Additional aspects and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of flow cytometry analysis of cell surface markers on human umbilical cord mesenchymal stem cells in Example 2;

[0039] Figure 2 This is a schematic diagram showing the differentiation capacity of human umbilical cord mesenchymal stem cells in Example 2; A represents osteogenic differentiation, and B represents adipogenic differentiation.

[0040] Figure 3 This is a schematic diagram of the exosome property detection results in Example 4; A is the result of the nanoparticle tracking analyzer, B is the particle size result, C is the potential result, D is the transmission electron microscopy result, a is 3D-TE-Exo, b is 3D-Exo, and E is the protein blotting result.

[0041] Figure 4 This is a schematic diagram of the detection results of oxidized hyaluronic acid in Example 5; A is the FT-IR result, B is... 1 H NMR results;

[0042] Figure 5 This is a schematic diagram of the rheological results of the OHA-CMCS@Exo hydrogel in Example 6. A is the frequency scan, B is the continuous strain scan, and C is the self-healing result.

[0043] Figure 6 This is a schematic diagram showing the adhesion and self-healing properties of the OHA-CMCS@Exo hydrogel in Example 6;

[0044] Figure 7 The diagram below shows the cell proliferation test results for Example 7; A represents the proliferation results of HUVECs cells at different concentrations of 3D-TE-Exo; B represents the proliferation results of HaCaT cells at different concentrations of 3D-TE-Exo; C represents the proliferation results of HUVECs cells at different concentrations of 3D-Exo; and D represents the proliferation results of HaCaT cells at different concentrations of 3D-Exo.

[0045] Figure 8This is a schematic diagram of the biocompatibility results of OHA-CMCS@Exo hydrogel in Example 7; A shows the proliferation results of HUVECs cells induced by OHA-CMCS@Exo hydrogel, and B shows the proliferation results of HaCaT cells induced by OHA-CMCS@Exo hydrogel.

[0046] Figure 9 This is a schematic diagram of the ROS removal results of the OHA-CMCS@Exo hydrogel in Example 8; A is the result of laser confocal microscopy, B is the quantitative result of laser confocal microscopy, a is HUVECs cells, and b is HaCaT cells;

[0047] Figure 10 This is a schematic diagram showing the results of OHA-CMCS@Exo hydrogel regulating macrophage phenotype in Example 8;

[0048] Figure 11 This is a schematic diagram showing the results of OHA-CMCS@Exo hydrogel accelerating the healing of diabetic wounds in Example 9. Detailed Implementation

[0049] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0050] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0051] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of this application. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0052] The following explanations of some of the terms and materials used in this embodiment will be provided to facilitate understanding by those skilled in the art.

[0053] 3D-TE-Exo: This refers to the human umbilical cord mesenchymal stem cell exosomes prepared in this embodiment.

[0054] OHA-CMCS@Exo: This refers to the exosome hydrogel prepared in this embodiment.

[0055] The exosome hydrogels of this application, their preparation methods, and their uses will be described in detail below.

[0056] First, let me explain the exosome hydrogel of this embodiment.

[0057] Exosome hydrogel

[0058] This embodiment provides an exosome hydrogel, which is prepared by cross-linking exosomes, oxidized hyaluronic acid and carboxymethyl chitosan;

[0059] In the exosome hydrogel, the final concentration of the exosomes is 20-5000 μg / mL, the final concentration of oxidized hyaluronic acid is 1-200 mg / mL, and the final concentration of carboxymethyl chitosan is 10-500 mg / mL.

[0060] It is understood that this embodiment is based on exosomes prepared using a specific method, and a hydrogel is prepared by cross-linking oxidized hyaluronic acid, carboxymethyl chitosan, and exosomes. This hydrogel exhibits injectability, adhesiveness, self-healing properties, ROS / glucose responsiveness, and excellent mechanical properties, suggesting its potential for accelerating the healing of chronic wounds.

[0061] Preferably, in the exosome hydrogel, the final concentration of the exosomes is 30-2500 μg / mL, the final concentration of oxidized hyaluronic acid is 25-75 mg / mL, and the final concentration of carboxymethyl chitosan is 75-300 mg / mL.

[0062] The preparation method of the above-mentioned exosomes will be further described below.

[0063] Specifically, the methods for preparing exosomes include:

[0064] (1) Prepare a cell culture medium containing a culture medium including trace elements.

[0065] In step (1), a cell culture medium containing culture medium is prepared to provide a culture basis for subsequent 3D culture. The culture medium includes trace elements, which participate in the 3D culture process and change the survival environment of stem cells. At the same time, the trace elements can reduce the cytoskeleton tension in stem cells and increase the expression of multiple genes together with the subsequent 3D culture, thereby achieving the production of high concentration and high activity exosomes in the smallest culture medium volume.

[0066] The trace element is selected from at least one of iron, magnesium, zinc, manganese, titanium, calcium, selenium, vanadium, chromium, nickel, cobalt, molybdenum, copper, and potassium.

[0067] It is understood that the trace elements in this embodiment can be obtained by adding mineral powder.

[0068] (2) Human umbilical cord mesenchymal stem cells were placed in a cell culture medium to obtain a cell culture medium containing human umbilical cord mesenchymal stem cells.

[0069] In step (2), human umbilical cord mesenchymal stem cells are placed in the cell culture medium obtained in step (1) to facilitate subsequent 3D culture steps.

[0070] (3) The cell culture medium containing human umbilical cord mesenchymal stem cells was cultured in a 3D bioreactor and the supernatant was collected.

[0071] In this step, a 3D bioreactor is used to culture stem cells containing trace elements. As mentioned earlier, the combination of trace element-containing 3D culture and the culture alters the survival environment of stem cells, reduces cytoskeleton tension, increases the expression of multiple genes, and influences the differentiation state of stem cells, achieving the production of high concentrations and high activity of exosomes in a minimal culture medium volume. Simultaneously, in addition to the physical and mechanical changes in the culture environment, trace element-containing 3D culture also affects the overall hypoxic state of stem cells, which alters the content of proteins and nucleic acids in exosomes, resulting in more significant anti-inflammatory, antioxidant, cell proliferation-promoting, and migration-enhancing effects.

[0072] Based on the combined involvement of 3D culture and trace elements, the generated exosomes carrying trace elements can better participate in the regulation of intercellular and intercellular microenvironments, regulate the immune environment, control the expression of inflammation-related genes, and improve the anti-inflammatory activity of exosomes.

[0073] (4) After differential centrifugation, human umbilical cord mesenchymal stem cell exosomes were obtained from the supernatant.

[0074] This step is to remove dead cells and their cell debris, thereby obtaining exosomes with higher purity.

[0075] Based on the above steps, the exosomes prepared in this embodiment have higher purity and better biological effects, such as anti-inflammatory, antioxidant, cell proliferation, cell migration, and angiogenesis, compared to exosomes extracted from conventionally cultured human umbilical cord mesenchymal stem cells. Furthermore, the exosomes prepared in this embodiment have a higher number of particles, higher biological activity, and are rich in trace elements, exhibiting a more significant anti-inflammatory effect, suggesting their potential use in accelerating the healing of chronic wounds.

[0076] The following will further explain the relevant steps of the above preparation method.

[0077] In step (1), the preparation of the cell culture medium containing the culture medium includes:

[0078] (1.1) Take the ore powder and dissolve it in the dissolving solution to obtain a mixture;

[0079] (1.2) After the mixture is filtered, it is added to an alternating strong magnetic field. After shearing, aging and filtration in the alternating strong magnetic field, a metal ion solution is obtained.

[0080] (1.3) Dilute the metal ion solution with a diluent to obtain a diluted metal ion solution;

[0081] (1.4) After sterilizing and filtering the diluted metal ion solution, a culture medium is obtained;

[0082] (1.5) Add the culture medium dropwise to the cell culture medium to obtain a cell culture medium containing the culture medium;

[0083] The ore powder is selected from at least one of zinc ore powder, mica powder, vermiculite powder, coal ore powder, celestite powder, and maifanite powder.

[0084] The ore powder in this embodiment can be made by crushing the corresponding ore; the ore powder is 200-250 mesh.

[0085] In step (1.1), the dissolving solution is a mixed solution of dilute hydrochloric acid, dilute sulfuric acid and oxalic acid, which can better dissolve the ore powder and allow trace elements to remain in the solution better.

[0086] In this embodiment, an alternating strong magnetic field is used to shear the mixture, which can enhance the reactivity of trace elements by changing the molecular structure and energy state in the solution. This is beneficial for the trace elements to better alter the survival environment of stem cells during subsequent culture, reduce cytoskeleton tension in stem cells, and increase the expression of multiple genes, thereby achieving the production of high concentrations and high activity of exosomes in a minimal culture medium volume, thus improving reaction efficiency and product quality.

[0087] In a preferred embodiment, the volume ratio of the dilute hydrochloric acid, dilute sulfuric acid, and oxalic acid is 3:5:2.

[0088] In one embodiment, the diluent is a 0.9% sodium chloride solution.

[0089] In one implementation, the magnetic field strength of the alternating strong magnetic field is 1×10⁻⁶. 3～4 Gauss.

[0090] Exemplarily, the step of preparing a cell culture medium containing culture medium includes:

[0091] (1.1) Using ore powders, including 20 parts zinc ore powder, 15 parts mica powder, 15 parts vermiculite powder, 20 parts coal ore powder, 15 parts celestite powder, and 15 parts maifanite powder, all were placed in a mixing tank. At a temperature of 120–150℃ and a pressure of 3–6 atmospheres, the volume of the solution was twice the total mass of the ore powders. The entire solution was passed into the mixing tank and stirred at 100 rpm for 4 hours, then boiled at 150℃ for 4 hours to dissolve the various metal ions in the solution, thus obtaining a mixed solution.

[0092] The solution is: 3% dilute hydrochloric acid: 10% dilute sulfuric acid: 15% oxalic acid = 3:5:2 (v / v).

[0093] (1.2) The resulting mixture was filtered through a large amount of activated carbon and then flowed into an alternating strong magnetic field. Driven by the alternating strong magnetic field, it was sheared and mixed using a high-energy ultrasonic field for 4 hours at a magnetic field strength of 1×10⁻⁶. 3～4 Gaussian, after aging (e.g., standing in an aging tank for 2 hours) and filtration (0.45μm polyethersulfone filtration), yields a metal ion solution.

[0094] (1.3) The volume of the diluent is 50 times the total mass of the ore powder. The above metal ion solution is diluted with the diluent to obtain a diluted metal ion solution; wherein the diluent is a 0.9% sodium chloride solution.

[0095] (1.4) Sterilize with ozone at a concentration of 5 ppm for 10 minutes, then irradiate with ultraviolet light for 3 hours, and then filter through a 0.45 μm microporous membrane to obtain the culture medium (or trace element solution).

[0096] (1.5) Add the culture medium dropwise to DMEM cell culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Each 500 mL of DMEM cell culture medium contains 0.17 mL of culture medium. Shake well and store at 4°C for later use.

[0097] In this embodiment, the cell culture medium can be conventional DMEM cell culture medium, that is, DMEM cell culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0098] In this embodiment, after obtaining the supernatant, the supernatant needs to be differentially centrifuged to obtain the final human umbilical cord mesenchymal stem cell exosomes.

[0099] In step (4), the supernatant is centrifuged at differential speed to obtain human umbilical cord mesenchymal stem cell exosomes, including:

[0100] (4.1) Centrifuge the supernatant at 300×g for 10-15 min at room temperature;

[0101] (4.2) Centrifuge the supernatant after the previous centrifugation at a speed of 2000×g at room temperature for 20-30 min; then centrifuge at a speed of 10000×g at 4℃ for 20-30 min, and filter to obtain human umbilical cord mesenchymal stem cell exosomes.

[0102] In step (4.1), the purpose of centrifugation is to remove cells and cell debris. In step (4.2), the supernatant after centrifugation in the previous step is centrifuged at 2000×g at room temperature for 20 minutes to further remove dead cells and cell debris. Then, it is centrifuged at 10000×g at 4℃ for 30 minutes to further remove any remaining cell debris. The exosomes that are visible to the naked eye at the bottom of the centrifuge tube are referred to as 3D-TE-Exo in this embodiment.

[0103] As one implementation method, the 3D bioreactor used in this embodiment is a 3D Flotrix miniSpin bioreactor.

[0104] In one embodiment, the degree of oxidation of the oxidized hyaluronic acid is 5%-50%, preferably 20%-40%.

[0105] For example, a method for preparing oxidized hyaluronic acid includes:

[0106] Add 1.25 mL of 10.72% (w / v) NaIO4 solution dropwise to 50 mL of 5% (w / v) hyaluronic acid solution, and react in the dark at 25 °C and 600 rpm for 3.5 h. Add 1.25 mL of ethylene glycol to the solution, continue stirring for 15 min, dialyze using a dialysis bag (molecular weight cutoff of 1000), and freeze-dry the solution to obtain oxidized hyaluronic acid.

[0107] In summary, the Schiff base in the hydrogel provided in this embodiment can respond to the high ROS and high glucose at the diabetic wound site, regulate the macrophage phenotype, regulate the inflammatory microenvironment, alleviate oxidative stress, and accelerate the healing of chronic wounds.

[0108] Next, the preparation method of the above-mentioned exosome hydrogel will be explained.

[0109] Preparation method

[0110] This embodiment provides a method for preparing exosome hydrogels, specifically including:

[0111] Carboxymethyl chitosan was dissolved in PBS solution to obtain carboxymethyl chitosan PBS solution;

[0112] Oxidized hyaluronic acid was dissolved in PBS solution to obtain oxidized hyaluronic acid PBS solution;

[0113] Exosomes were uniformly dispersed in a carboxymethyl chitosan PBS solution to obtain a mixture;

[0114] The mixture was thoroughly mixed with oxidized hyaluronic acid PBS solution to obtain exosome hydrogel.

[0115] For example, 1.25 mL of 10.72% (w / v) NaIO4 solution was added dropwise to 50 mL of 5% (w / v) hyaluronic acid solution, and the reaction was carried out at 25°C and 600 rpm in the dark for 3.5 h. Then, 1.25 mL of ethylene glycol was added to the solution, and the mixture was stirred for another 15 min. The solution was dialyzed using a dialysis bag (molecular weight cutoff of 1000), and the solution was lyophilized to obtain oxidized hyaluronic acid (OHA).

[0116] A 20 mg / mL OHA solution and a 20 mg / mL carboxymethyl chitosan (CMCS) solution were prepared using pH 7.4 PBS. 3D-TE-Exo was added to the CMCS solution and mixed thoroughly. Equal volumes of the two solutions were then mixed to obtain the 3D-TE-Exo hydrogel (or OHA-CMCS@Exo).

[0117] Next, the uses of the above-mentioned exosome hydrogel will be explained.

[0118] use

[0119] Based on the description in the first aspect, this embodiment uses a cell culture medium containing trace elements and combines it with 3D culture to culture human umbilical cord mesenchymal stem cells. The 3D culture containing trace elements alters the survival environment of stem cells, reduces cytoskeleton tension, increases the expression of various genes, and influences the differentiation state of stem cells, achieving the production of high concentrations and high activity of exosomes in a minimal culture medium volume. Simultaneously, in addition to the physical and mechanical changes in the culture environment, the 3D culture containing trace elements also affects the overall hypoxic state of stem cells, which alters the content of proteins and nucleic acids within the exosomes. These trace element-carrying exosomes participate in the regulation of intercellular and intercellular microenvironments, modulate the immune environment, control the expression of inflammation-related genes, and enhance the anti-inflammatory activity of exosomes.

[0120] Meanwhile, the exosomes prepared in this embodiment exhibit higher purity and superior biological effects, such as anti-inflammatory, antioxidant, cell proliferation, cell migration, and angiogenesis effects. Furthermore, the exosomes prepared in this embodiment have a higher number of particles, higher biological activity, and are rich in trace elements, thus possessing a more significant anti-inflammatory effect.

[0121] Therefore, in hydrogels derived from exocrine mechanisms, Schiff bases can respond to high ROS and high glucose levels at diabetic wound sites, regulate macrophage phenotypes, modulate the inflammatory microenvironment, alleviate oxidative stress, and accelerate the healing of chronic wounds.

[0122] In other words, the exosome hydrogel of this embodiment can be used to prepare dressings for treating or accelerating the healing of chronic wounds.

[0123] For example, chronic wounds include common chronic wounds such as diabetic wounds, infected wounds, or frostbite wounds.

[0124] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustration / explanation only and are not intended to limit the scope of the present application.

[0125] Unless otherwise specified, all materials, reagents, and instruments used in the following embodiments are commercially available. Specifically, the ore powder used in this embodiment is 200-250 mesh, and the carboxymethyl chitosan was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., model C832672.

[0126] Example 1

[0127] Preparation of cell culture medium rich in trace elements

[0128] (1) Using ore powders, including 20 parts zinc ore powder, 15 parts mica powder, 15 parts vermiculite powder, 20 parts coal ore powder, 15 parts celestite powder, and 15 parts maifanite powder, all were placed in a mixing tank. At a temperature of 130℃ and a pressure of 5 atmospheres, the volume of the solution was twice the total mass of the ore powders. The entire solution was passed into the mixing tank and stirred at 100 rpm for 4 hours, then boiled at 150℃ for 4 hours to dissolve the various metal ions in the solution, thus obtaining a mixed solution.

[0129] The solution is: 3% dilute hydrochloric acid: 10% dilute sulfuric acid: 15% oxalic acid = 3:5:2 (v / v).

[0130] (2) After the mixture is filtered through a large amount of activated carbon, it flows into an alternating strong magnetic field. Under the driving force of the alternating strong magnetic field and a high-energy ultrasonic field, it is sheared and mixed for 4 hours. The magnetic field strength is 1×10⁻⁶. 3 Gaussian, after aging and filtration, yields a metal ion solution.

[0131] (3) The volume of the diluent is 50 times the total mass of the ore powder. The above metal ion solution is diluted with the diluent to obtain a diluted metal ion solution; wherein the diluent is a 0.9% sodium chloride solution.

[0132] (4) Sterilize with ozone at a concentration of 5 ppm for 10 minutes, then irradiate with ultraviolet light for 3 hours, and then filter through a 0.45 μm microporous membrane to obtain the culture medium.

[0133] (5) Add the culture medium dropwise to DMEM cell culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Each 500 mL of DMEM cell culture medium contains 0.17 mL of culture medium. Shake well and store at 4°C for later use.

[0134] After preparing the culture medium, the concentrations of major trace elements in the trace metal element injection solution were determined using an Agilent 7500 ICP-MS. ICP-MS analysis showed that the concentrations of Fe, Mg, Zn, Mn, and Se in the metal element culture medium were 1920 mg / L, 1790 mg / L, 606 mg / L, 154 mg / L, and 21.1 mg / L, respectively.

[0135] Example 2

[0136] 3D cultured exosomes carrying trace elements secretion

[0137] In this embodiment, human umbilical cord mesenchymal stem cells were cultured in a 3D Flotrix miniSpin bioreactor at a concentration of 2.5 × 10⁻⁶. 6 The culture medium used was the trace element-rich cell culture medium of Example 1. The culture medium containing human umbilical cord mesenchymal stem cells was transferred to a 3D Flotrix miniSpin bioreactor. The conditions were 35 rpm, 10 min, stop for 1 h, repeated multiple times, and cultured continuously for 3D until the stem cells proliferated for 8-10 generations. The stem cell species were identified by flow cytometry and other methods.

[0138] Test results are as follows Figure 1 As shown; Figure 1 This diagram illustrates flow cytometry analysis of surface markers on human umbilical cord mesenchymal stem cells. The surface markers CD73, CD90, and CD105 were strongly positive, while the surface markers CD14, CD19, CD34, CD45, and HLA-DR were negative. Figure 1 ).at the same time, Figure 2 A schematic diagram of the differentiation capacity of human umbilical cord mesenchymal stem cells is also shown, with 2A representing osteogenic differentiation and 2B representing adipogenic differentiation. The results indicate that the human umbilical cord mesenchymal stem cells prepared in this embodiment possess the characteristics and multi-directional differentiation potential of stem cells.

[0139] Example 3

[0140] Large-scale extraction and preparation of exosomes by ultra-high differential centrifugation

[0141] The serum-free mesenchymal stem cell supernatant (prepared in Example 2) collected in advance was subjected to differential ultracentrifugation. The first centrifugation was performed at 300×g at room temperature for 10 min to remove cells and cell debris. The supernatant from the previous step was then centrifuged again at 2000×g at room temperature for 20 min to further remove dead cells and cell debris. Finally, it was centrifuged at 10000×g at 4℃ for 30 min to further remove any remaining cell debris. Exosomes were visible as precipitates at the bottom of the centrifuge tubes, denoted as 3D-TE-Exo. 3D cultured exosomes without trace elements served as a control, denoted as 3D-Exo.

[0142] The exosomes prepared in Example 3 will be subjected to relevant tests or performance evaluations.

[0143] Example 4

[0144] Exosome detection

[0145] 3D-TE-Exo and 3D-Exo were identified using a nanoparticle tracking analyzer, particle size, potential, transmission electron microscopy, and Western blotting, respectively.

[0146] Test results are as follows Figure 3 As shown, Figure 3 A- Figure 3 In equation E, A represents the results from the nanoparticle tracking analyzer, B represents the particle size result, C represents the potential result, D represents the transmission electron microscopy result, a represents 3D-TE-Exo, b represents 3D-Exo, and E represents the protein blotting result. According to... Figure 3 The test results showed that the particle size of both exosomes was 30-150 nm. Figure 3 A). Compared to 3D-Exo, 3D-TE-Exo has a higher particle count ( Figure 3 B). The number of 3D-Exo particles is 9.6*10. 10 The number of 3D-TE-Exo particles is 1.9*10. 12 Compared with traditional 2D culture, 3D culture containing trace elements alters the survival environment of stem cells, reduces cytoskeleton tension in stem cells, increases the expression of multiple genes, affects the differentiation state of stem cells, and achieves the production of high concentrations and high activity of exosomes in the smallest culture medium volume.

[0147] Specifically, both types of exosomes exhibit a negative charge. Figure 3 C). Under electron microscopy, the two types of exosomes exhibit cup-shaped and disc-shaped vesicle-like structures. Figure 3 D). Western blot analysis revealed two typical exosome biomarkers: TSG101, CD63, and CD9. Figure 3 E).

[0148] Example 5

[0149] Synthesis of carrier materials

[0150] Add 1.25 mL of 10.72% (w / v) NaIO4 solution dropwise to 50 mL of 5% (w / v) hyaluronic acid solution, and react at 25 °C and 600 rpm in the dark for 3.5 h. Add 1.25 mL of ethylene glycol to the solution, continue stirring for 15 min, dialyze using a dialysis bag (molecular weight cutoff of 1000), and freeze-dry the solution to obtain oxidized hyaluronic acid (OHA).

[0151] Please see Figure 4 , Figure 4 The results of oxidized hyaluronic acid detection are shown in Figure A, where A is the FT-IR result and B is the result of FT-IR. 1 1H NMR results. (Based on FT-IR) Figure 4 A) and 1 H NMR ( Figure 4 B) The results showed that OHA was successfully prepared.

[0152] Example 6

[0153] Hydrogel construction and performance evaluation

[0154] This embodiment includes an evaluation of the hydrogel's construction, rheology, adhesion, injectability, and self-healing properties.

[0155] (1) Construction of hydrogel

[0156] A 20 mg / mL OHA solution and a 20 mg / mL carboxymethyl chitosan (CMCS, degree of substitution ≥80%) solution were prepared using pH 7.4 PBS. A certain amount of 3D-TE-Exo was added to the CMCS solution and mixed thoroughly. The two solutions were then mixed in equal volumes to obtain a 3D-TE-Exo hydrogel (OHA-CMCS@Exo). The amount of 3D-TE-Exo added was 1% of the hydrogel volume.

[0157] (w / v).

[0158] (2) Rheology

[0159] The storage modulus (G′) and loss modulus (G″) of the hydrogel were measured using a rheometer at 37 °C. Frequency scanning was performed in the linear viscoelastic region at a constant strain of 0.5% from 0.01 to 1 Hz.

[0160] Among them, G′ reveals the elastic properties of the hydrogel, and G″ reveals the viscous properties of the hydrogel. Figure 5 The test results Figure 5 The rheological results of the OHA-CMCS@Exo hydrogel are shown. A is the frequency scan, B is the continuous strain scan, and C is the self-healing result.

[0161] Combination Figure 5 The test results show that within the 0.01-1Hz range, G′ in each group is higher than G″, with no crossover marks. Figure 5 A) indicates that the hydrogel is stable and has good elasticity. The oscillation of G″ may be related to the dynamic Schiff base breakage and recombination in the gel network. Under high strain conditions, G″ is greater than G′, indicating that the hydrogel network structure has loosened or even been destroyed, losing its integrity and mechanical stability. When the strain amplitude returns to low strain, G″ and G′ immediately return to their original values ​​(A). Figure 5 B). Specifically, the self-healing results also demonstrate the excellent self-healing properties of the hydrogel. Figure 5 C).

[0162] (3) Adhesion and self-healing

[0163] This embodiment uses items made of different materials, including glass, polypropylene centrifuge tubes, paper, polyvinyl chloride syringes, and stainless steel weighing spoons, to evaluate the adhesion of the OHA-CMCS@Exo hydrogel.

[0164] like Figure 6 The results show that the test results demonstrate the good adhesion and self-healing properties of the OHA-CMCS@Exo hydrogel.

[0165] Example 7

[0166] Cell proliferation

[0167] This study investigated the effects of different concentrations of two exosomes on cell proliferation using a CCK-8 assay. HUVECs and HaCaT cells were cultured at 5 × 10⁻⁶ cells per cell line. 3 Cells were seeded at a density of [number] wells in 96-well plates, and different concentrations of exosomes were added. Incubation was continued for 1 and 3 days. The culture medium was then discarded, and cells were incubated with 100 μL of serum-free medium containing 10% (v / v) CCK-8 reagent. The absorbance of the plates was measured at 490 nm using a microplate reader (n=6). Wells without any added exosomes served as a control group.

[0168] Figure 7 The diagram shows the results of cell proliferation. A represents the proliferation of HUVECs cells with different concentrations of 3D-TE-Exo; B represents the proliferation of HaCaT cells with different concentrations of 3D-TE-Exo; C represents the proliferation of HUVECs cells with different concentrations of 3D-Exo; and D represents the proliferation of HaCaT cells with different concentrations of 3D-Exo.

[0169] The results showed that increasing concentrations of both exosomes significantly promoted the proliferation of HUVECs and HaCaT cells. Figure 7 It is worth noting that, at the same concentration, 3D-TE-Exo has a more significant cell proliferation-promoting effect than 3D-Exo.

[0170] Simultaneously, the cytotoxicity of OHA-CMCS@Exo hydrogel to HUVECs and HaCaT cells was evaluated using the CCK-8 assay. HUVECs and HaCaT cells were inoculated at 5 × 10⁻⁶ cells per cell line. 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and induced with 200 μmol H2O2 for 24 h. Different samples were added, and incubation continued for 1 and 3 days. Then, the culture medium was discarded, and cells were incubated with 100 μL of serum-free medium containing 10% (v / v) CCK-8 reagent. The absorbance of the plates was measured at 490 nm using a microplate reader (n=6). Wells without any exosomes were used as control groups.

[0171] Figure 8 The biocompatibility results of OHA-CMCS@Exo hydrogel are shown. A shows the proliferation effect of OHA-CMCS@Exo hydrogel on HUVECs cells, and B shows the proliferation effect of OHA-CMCS@Exo hydrogel on HaCaT cells.

[0172] The results showed that 3D-Exo, 3D-TE-Exo, and OHA-CMCS@Exo hydrogels did not significantly inhibit cell proliferation within 1 and 3 days. Figure 8 Conversely, 3D-Exo, 3D-TE-Exo, and OHA-CMCS@Exo hydrogels reversed the effects of H2O2, enhanced cell activity, and promoted the proliferation of HUVECs and HaCaT cells, demonstrating the good biocompatibility of OHA-CMCS@Exo hydrogels.

[0173] Example 8

[0174] OHA-CMCS@Exo hydrogel's anti-inflammatory mechanism

[0175] (1) ROS removal

[0176] This embodiment utilizes DCFH-DA to detect the ROS scavenging ability of OHA-CMCS@Exo hydrogel. HUVECs and HaCaT cells were tested at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 12-well plates. Cells were induced with 200 μmol H₂O₂ for 24 h. Cells were co-cultured with the hydrogel extract for 24 h. They were then incubated with 10 μmol / L DCFH-DA for 30 min. Results were obtained using a laser scanning confocal microscope.

[0177] Figure 9 The results of ROS removal by OHA-CMCS@Exo hydrogel are shown. A is the result of laser confocal microscopy, B is the quantitative result of laser confocal microscopy, a is HUVECs cells, and b is HaCaT cells.

[0178] The results are as follows Figure 9 As shown, the OHA-CMCS@Exo hydrogel significantly reduced the ROS level in cells, indicating that the OHA-CMCS@Exo hydrogel has the property of scavenging excess ROS.

[0179] (2) Macrophage phenotype

[0180] This embodiment uses immunofluorescence staining to detect the phenotype of macrophages. RAW 264.7 at 2×10⁻⁶... 5 Cells were seeded at a density of 10 cells / well in 6-well plates and cultured for 12 h. Cells were induced with 1 μg / mL LPS for 24 h. Cells were co-cultured with hydrogel extract for 24 h. Next, cells were fixed, permeabilized, and blocked. Cells were incubated overnight at 4 °C with CD68 (1:500) and CD206 (5 μg / mL), followed by incubation with fluorescent secondary antibody at 25 °C for 1 h. Cell nuclei were co-incubated with DAPI dye for 10 min. Results were obtained using a laser scanning confocal microscope.

[0181] Figure 10 The results of OHA-CMCS@Exo hydrogel modulating macrophage phenotype are shown.

[0182] like Figure 10 As shown, OHA-CMCS@Exo hydrogel induces macrophages to transform from the pro-inflammatory M1 type to the anti-inflammatory M2 type, exerting an anti-inflammatory effect. Besides the physical and mechanical changes in the culture environment, 3D culture containing trace elements also affects the overall hypoxic state of stem cells, which alters the content of proteins and nucleic acids in exosomes. These trace element-carrying exosomes participate in the regulation of intercellular and intercellular microenvironments, modulate the immune environment, control the expression of inflammation-related genes, and enhance the anti-inflammatory activity of exosomes.

[0183] Example 9

[0184] OHA-CMCS@Exo hydrogel accelerates chronic wound healing.

[0185] This example uses a diabetic wound as an example. Male SD rats were selected as experimental subjects for skin wound healing experiments. A type 1 diabetic SD rat model was established by intraperitoneal injection of 60 mg / kg streptozotocin (citrate buffer, pH 4.5). The type 1 diabetic rat model was considered successfully established when the fasting blood glucose of the rats remained above 16.7 mmol / L for one week. The rats were anesthetized by inhalation, and the hair on their backs was shaved. Rats with intact skin were selected to establish the wound model. The hair on the backs was shaved, and a full-thickness wound model with a diameter of 8 mm was established on the backs. The 36 successfully modeled SD rats were randomly divided into 3 groups of 12 each and housed in separate cages. They were named the model group, the OHA-CMCS@Exo group (0.5 mL), and the positive group, respectively. The model group was not treated, and the positive group was covered with commercially available Tegadron™ dressing (3M, USA). To evaluate the healing ability of the hydrogel, optical images of the wound at different times were recorded with a camera to calculate the wound healing rate, and body weight was also recorded. The wound contraction rate (%) was determined using ImageJ software. Six healthy SD rats were used as a control group.

[0186] Among them, the healing process of diabetic wounds, such as Figure 11 As shown; Figure 11 The test results showed that the wounds of rats in all groups contracted over time. The model group had the lowest wound healing rate and showed signs of scab formation. Compared with the model group, the OHA-CMCS@Exo hydrogel treatment group showed significantly faster wound contraction and the highest healing rate.

[0187] The results showed that OHA-CMCS@Exo hydrogel had a better healing-promoting effect than other groups (model and positive groups).

[0188] In summary, the exosomes prepared in this embodiment exhibit higher purity and superior biological effects, such as anti-inflammatory, antioxidant, cell proliferation, cell migration, and angiogenesis effects. Furthermore, the exosomes prepared in this embodiment have a higher particle count, higher biological activity, and are rich in trace elements, resulting in a more significant anti-inflammatory effect. Therefore, in the hydrogel prepared based on exosomes, Schiff bases can respond to the high ROS and high glucose levels at diabetic wound sites, regulate macrophage phenotype, modulate the inflammatory microenvironment, alleviate oxidative stress, and accelerate the healing of chronic wounds.

[0189] The technical solutions provided by the embodiments of this application have been described in detail above. Specific examples have been used in this document to illustrate the principles and implementation methods of the embodiments of this application. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. An exosome hydrogel, characterized in that, The exosome hydrogel was prepared by cross-linking exosomes, oxidized hyaluronic acid and carboxymethyl chitosan. In the exosome hydrogel, the final concentration of the exosomes is 20-5000 μg / mL, the final concentration of oxidized hyaluronic acid is 1-200 mg / mL, and the final concentration of carboxymethyl chitosan is 10-500 mg / mL.

2. The exosome hydrogel according to claim 1, characterized in that, The method for preparing the exosomes includes: Prepare a cell culture medium containing a culture medium that includes trace elements; Human umbilical cord mesenchymal stem cells were placed in a cell culture medium to obtain a cell culture medium containing human umbilical cord mesenchymal stem cells. A 3D bioreactor was used to culture cell culture medium containing human umbilical cord mesenchymal stem cells in a 3D environment, and the supernatant was collected. After differential centrifugation, human umbilical cord mesenchymal stem cell exosomes were obtained from the supernatant.

3. The exosome hydrogel according to claim 2, characterized in that, The cell culture medium containing the culture medium includes: Take ore powder and dissolve it in a dissolving solution to obtain a mixture; After filtration, the mixture is added to an alternating strong magnetic field. After shearing, aging, and filtration in the alternating strong magnetic field, a metal ion solution is obtained. The metal ion solution was diluted with a diluent to obtain a diluted metal ion solution; The diluted metal ion solution was sterilized and filtered to obtain the culture medium; The culture medium is added dropwise to the cell culture medium to obtain a cell culture medium containing the culture medium; The ore powder is selected from at least one of zinc ore powder, mica powder, vermiculite powder, coal ore powder, celestite powder, and maifanite powder.

4. The exosome hydrogel according to claim 3, characterized in that, The solution is a mixed solution of dilute hydrochloric acid, dilute sulfuric acid and oxalic acid; the volume ratio of the dilute hydrochloric acid, dilute sulfuric acid and oxalic acid is 3:5:

2. The diluent is a 0.9% sodium chloride solution; the magnetic field strength of the alternating strong magnetic field is 1×10⁻⁶. 3～4 Gauss.

5. The exosome hydrogel according to claim 1, characterized in that, The supernatant was centrifuged at differential speed to obtain human umbilical cord mesenchymal stem cell exosomes, comprising: Centrifuge the supernatant at 300×g for 10-15 minutes at room temperature. Centrifuge the supernatant from the previous step at 2000×g at room temperature for 20-30 min; then centrifuge at 10000×g at 4℃ for 20-30 min, and filter to obtain human umbilical cord mesenchymal stem cell exosomes.

6. The exosome hydrogel according to claim 1, characterized in that, The degree of oxidation of the oxidized hyaluronic acid is 5%-50%, preferably 20%-40%.

7. The preparation method according to claim 1, characterized in that, The trace elements are selected from at least one of iron, magnesium, zinc, manganese, titanium, calcium, selenium, vanadium, chromium, nickel, cobalt, molybdenum, copper, and potassium; the 3D bioreactor is a 3D Flotrix miniSpin bioreactor.

8. The preparation method according to claim 1, characterized in that, In the exosome hydrogel, the final concentration of the exosomes is 30-2500 μg / mL, the final concentration of oxidized hyaluronic acid is 25-75 mg / mL, and the final concentration of carboxymethyl chitosan is 75-300 mg / mL.

9. A method for preparing an exosome hydrogel as described in any one of claims 1-8, characterized in that, The preparation method includes: Carboxymethyl chitosan was dissolved in PBS solution to obtain carboxymethyl chitosan PBS solution; Oxidized hyaluronic acid was dissolved in PBS solution to obtain oxidized hyaluronic acid PBS solution; Exosomes were uniformly dispersed in a carboxymethyl chitosan PBS solution to obtain a mixture; The mixture was thoroughly mixed with oxidized hyaluronic acid PBS solution to obtain exosome hydrogel.

10. Use of the exosome hydrogel according to any one of claims 1-8 in the preparation of dressings for treating or accelerating the healing of chronic wounds.

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