Mussel mucoprotein loaded extracellular vesicle hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of hydrogel, in particular to mussel mucoprotein loaded extracellular vesicle hydrogel as well as a preparation method and application thereof. The hydrogel is prepared by adopting a mode of combining human umbilical cord mesenchymal stem cell exosome Exo and mussel mucoprotein MAP; the method has the following advantages: 1, the wound repair effect is improved: the biological activity of the exosome Exo and the adhesion performance of the mussel mucin MAP are utilized to jointly promote the wound repair process, so that the repair effect is improved; 2, the adhesion performance is enhanced: the strong adhesion of the mussel adhesive protein MAP is utilized to ensure that the hydrogel can be tightly attached to the wound surface in a wet environment, and the dressing is prevented from falling off and infection risk; and 3, multifunctionality is realized: the composite hydrogel not only has the basic functions of promoting healing, resisting bacteria and the like, but also can transmit genetic information through exosome Exo, so that tissue regeneration and skin barrier repair are further promoted.

Description

Technical Field

[0001] The invention relates to the technical field of hydrogels, and in particular to a mussel mucin-loaded extracellular vesicle hydrogel and a preparation method and application thereof. Background Art

[0002] Extracellular vesicles are a type of nano-sized particles that are secreted or released by cells into the extracellular microenvironment and have a double lipid membrane structure. These vesicles are widely present in various body fluids (such as blood, lymph, saliva, urine, semen, milk, etc.) and cell culture supernatants, and play an important role in multiple biological processes such as intercellular communication, cell migration, angiogenesis, and immune regulation. Extracellular vesicles can be classified according to their size, source, and formation mechanism. They mainly include: exosomes Exo, microvesicles, and apoptotic bodies, among which exosomes Exo are the most widely used.

[0003] Although Exo has excellent regenerative therapeutic effects, when it is used as a carrier of bioactive molecules, it may face problems of low delivery efficiency and poor stability when directly applied. It is necessary to develop a carrier system that can efficiently and stably deliver Exo to improve its therapeutic effect in wound repair.

[0004] In addition, traditional wound repair materials have shortcomings in terms of adhesion, healing promotion effect, and ease of use. In particular, the adhesion performance of some wound repair materials may decrease in a humid environment, causing the dressing to fall off and increasing the risk of infection. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies in the prior art. The present invention provides a preparation method and application of a mussel mucin-loaded extracellular vesicle hydrogel. The mussel mucin-loaded extracellular vesicle hydrogel has the advantages of good adhesion, good healing promoting effect, convenience in use, and the like.

[0006] The purpose of the present invention is achieved through the following technical solutions: A mussel mucin-loaded extracellular vesicle hydrogel, which combines human umbilical cord mesenchymal stem cell exosomes Exo with mussel mucin MAP.

[0007] A method for preparing a mussel mucin-loaded extracellular vesicle hydrogel comprises the following preparation steps: Step (1): dissolving 0.7-1.7 parts by weight of mussel mucin and 0.5-1.5 parts by weight of ectoine carboxylic acid in 100 parts by weight of PBS buffer solution, and adjusting the pH of the mussel mucin solution to 4.0 dropwise with acetic acid to obtain a mussel mucin solution, wherein ectoine carboxylic acid can effectively protect the mussel mucin; Step (2): adding 2-4 parts by weight of polyethylene glycol diglycidyl ether and 3-5 parts by weight of N-hydroxysuccinimide to the mucin solution obtained in step (1), and reacting for 30-60 minutes; Step (3): adding 1.5-3 parts by weight of human umbilical cord mesenchymal stem cell exosomes to the mucin solution obtained in step (2) and stirring evenly; Step (4): Add 4-8 parts by weight of trehalose to the solution obtained in step (3), stir evenly, and obtain a mussel mucin-loaded extracellular vesicle hydrogel, wherein the trehalose further protects the stability of the entire system.

[0008] The concentration of PBS buffer is 0.01M.

[0009] Wherein, the method for preparing human umbilical cord mesenchymal stem cell exosomes in step (3) comprises: Step (a): 100 mg of umbilical cord mesenchymal stem cells of passage 4-5 were cultured until 60%-70% confluence, and then replaced with serum-free medium. After further culture for 48-72 hours, the supernatant of the cell culture medium was collected, and the supernatant was centrifuged at 4°C and 300g for 10-20 minutes to remove the mesenchymal stem cells; Step (b): centrifuging the cell supernatant at 4°C, 2000 g for 15-25 min to further remove dead cells and impurities, and collecting the supernatant; continuing to centrifuge at 4°C, 10000 g for 25-35 min to remove cell debris, and collecting the supernatant; Step (c): The supernatant obtained in step (b) was centrifuged at 4°C, 100,000 g for 60-90 min, the supernatant was discarded to obtain the exosome precipitate, 1 mL of sterile ice-cold PBS was added for resuspending, and then filtered with a 0.22 μm filter membrane to obtain human umbilical cord mesenchymal stem cell exosomes.

[0010] Wherein, the serum-free culture medium consists of α-MEM and platelet lysate.

[0011] Preferably, the serum-free medium specifically comprises the following components (calculated by volume): inorganic salts: 200 mg / L calcium chloride, 97.67 mg / L magnesium sulfate, 400 mg / L potassium chloride, 2200 mg / L sodium bicarbonate, 6800 mg / L sodium chloride, 122 mg / L anhydrous disodium hydrogen phosphate; amino acids: 126 mg / L L-alanine-L-glutamine, 100 mg / L L-arginine hydrochloride, 31.3 mg / L L-cysteine ​​hydrochloride monohydrate, 292 mg / L L-glutamine, 42 mg / L-histidine hydrochloride monohydrate, 52 mg / L-isoleucine, 52 mg / L-leucine, 72.5 mg / L-lysine hydrochloride, 15 mg / L-methionine, 32 mg / L-phenylalanine, 48 mg / L-threonine, 51 mg / L-tyrosine disodium salt dihydrate, 1 0mg / L tryptophan, 46mg / L alanine, 50mg / L glycine; 60mg / L vitamins; 40mg / L ribonucleotides; 41mg / L deoxyribonucleotides; 1000mg / L glucose; 0.2mg / L zinc sulfate; 110mg / L sodium pyruvate; 10mg / L phenol red; 10ug / L growth factor; 1.7ug / L endothelial growth factor; 50ug / L transforming growth factor; 0.27ug / L basic fibroblast factor; 11ug / L vascular endothelial growth factor.

[0012] The application of the mussel mucin-loaded extracellular vesicle hydrogel is to apply the mussel mucin-loaded extracellular vesicle hydrogel to wound repair.

[0013] When the mussel mucin-loaded extracellular vesicle hydrogel is applied to wound repair, 1-3 parts by weight of SOD 1,2-hexanediol embedding material is added dropwise during the reaction of step (2), so that SOD can be stably present in the system of the present invention, further increasing the wound repair effect of the present invention.

[0014] Preferably, the 1,2-hexanediol embedding material of SOD is obtained by drying 3-6 parts by weight of 1,2-hexanediol at 65° C. in a forced air drying oven to remove moisture, adding 1 part by weight of SOD, and mixing in a shaker at -20° C. until coagulation.

[0015] The mussel mucin MAP of the present invention is a single protein with high purity, extracted from the foot gland of marine mussels (such as blue mussel, thick-shell mussel, green mussel, etc.), and has the functions of promoting cell adhesion and crawling, promoting wound healing, inhibiting itching, broad-spectrum adhesion, forming a water-resistant protective film, etc. The present invention not only further improves the stability and adhesion performance of mussel mucin by multi-faceted modification of mussel mucin, but also can effectively and stably combine with the human umbilical cord mesenchymal stem cell exosomes prepared by the present invention, giving full play to the biological activity of exosomes Exo.

[0016] Beneficial effects of the present invention: The present invention prepares hydrogels from human umbilical cord mesenchymal stem cell exosomes and mussel mucin, which has the following advantages: 1. Improving the wound repair effect: utilizing the biological activity of exosomes Exo and the adhesion properties of mussel mucin MAP to jointly promote the wound repair process and improve the repair effect; 2. Enhancing adhesion properties: utilizing the strong adhesion of mussel mucin MAP to ensure that the hydrogel can fit tightly to the wound in a moist environment to prevent the dressing from falling off and the risk of infection; 3. Realizing multifunctionality: The composite hydrogel not only has basic functions such as promoting healing and antibacterial, but may also transmit genetic information through exosomes Exo, further promoting tissue regeneration and skin barrier repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the Exo particle size distribution diagram; Figure 2 It is a transmission electron microscope image; Figure 3 This is a picture of the cell scratch experiment; Figure 4 This is the adhesion test diagram; Figure 5 This is a picture of the repair effect of infected wounds in rats; DETAILED DESCRIPTION

[0018] The present invention is further described in conjunction with the following examples.

[0019] Example 1

[0020] A mussel mucin-loaded extracellular vesicle hydrogel, which combines human umbilical cord mesenchymal stem cell exosomes Exo with mussel mucin MAP.

[0021] A method for preparing a mussel mucin-loaded extracellular vesicle hydrogel comprises the following preparation steps: Step (1): dissolving 0.7 parts by weight of mussel mucin and 0.5 parts by weight of ectoine in 100 parts by weight of PBS buffer solution, and adjusting the pH of the mussel mucin solution to 4.0 dropwise with acetic acid to obtain a mussel mucin solution; Step (2): adding 2 parts by weight of polyethylene glycol diglycidyl ether and 3 parts by weight of N-hydroxysuccinimide to the mucin solution obtained in step (1), and reacting for 30 minutes; Step (3): adding 1.5 parts by weight of human umbilical cord mesenchymal stem cell exosomes to the mucin solution obtained in step (2) and stirring evenly; Step (4): Add 4 parts by weight of trehalose to the solution obtained in step (3), and stir evenly to obtain a mussel mucin-loaded extracellular vesicle hydrogel.

[0022] The concentration of PBS buffer is 0.01M.

[0023] Wherein, the method for preparing human umbilical cord mesenchymal stem cell exosomes in step (3) comprises: Step (a): 100 mg of umbilical cord mesenchymal stem cells of passage 4-5 were cultured until 60% confluence, and then replaced with serum-free medium. After further culture for 48 hours, the supernatant of the cell culture medium was collected, and the supernatant was centrifuged at 4°C and 300g for 10 minutes to remove the mesenchymal stem cells; Step (b): centrifuging the cell supernatant at 4°C, 2000 g for 15 min to further remove dead cells and impurities, and collecting the supernatant; continuing to centrifuge at 4°C, 10000 g for 25 min to remove cell debris, and collecting the supernatant; Step (c): The supernatant obtained in step (b) was centrifuged at 4°C, 100,000 g for 60 min, the supernatant was discarded to obtain the exosome precipitate, 1 mL of sterile ice-cold PBS was added for resuspending, and then filtered with a 0.22 μm filter membrane to obtain human umbilical cord mesenchymal stem cell exosomes.

[0024] Wherein, the serum-free culture medium consists of α-MEM and platelet lysate.

[0025] The serum-free medium specifically includes the following components (calculated by volume): inorganic salts: 200 mg / L calcium chloride, 97.67 mg / L magnesium sulfate, 400 mg / L potassium chloride, 2200 mg / L sodium bicarbonate, 6800 mg / L sodium chloride, 122 mg / L anhydrous disodium hydrogen phosphate; amino acids: 126 mg / L L-alanine-L-glutamine, 100 mg / L L-arginine hydrochloride, 31.3 mg / L L-cysteine ​​hydrochloride monohydrate, 292 mg / L L-glutamine, 42 mg / L-histidine hydrochloride monohydrate, 52 mg / L-isoleucine, 52 mg / L-leucine, 72.5 mg / L-lysine hydrochloride, 15 mg / L-methionine, 32 mg / L-phenylalanine, 48 mg / L-threonine, 51 mg / L-tyrosine disodium salt dihydrate, 1 0mg / L tryptophan, 46mg / L alanine, 50mg / L glycine; 60mg / L vitamins; 40mg / L ribonucleotides; 41mg / L deoxyribonucleotides; 1000mg / L glucose; 0.2mg / L zinc sulfate; 110mg / L sodium pyruvate; 10mg / L phenol red; 10ug / L growth factor; 1.7ug / L endothelial growth factor; 50ug / L transforming growth factor; 0.27ug / L basic fibroblast factor; 11ug / L vascular endothelial growth factor.

[0026] The application of the mussel mucin-loaded extracellular vesicle hydrogel is to apply the mussel mucin-loaded extracellular vesicle hydrogel to wound repair.

[0027] Example 2

[0028] A mussel mucin-loaded extracellular vesicle hydrogel, which combines human umbilical cord mesenchymal stem cell exosomes Exo with mussel mucin MAP.

[0029] A method for preparing a mussel mucin-loaded extracellular vesicle hydrogel comprises the following preparation steps: Step (1): dissolving 1 part by weight of mussel mucin and 1 part by weight of ectoine in 100 parts by weight of PBS buffer solution, and adjusting the pH of the mussel mucin solution to 4.0 dropwise with acetic acid to obtain a mussel mucin solution; Step (2): adding 3 parts by weight of polyethylene glycol diglycidyl ether and 4 parts by weight of N-hydroxysuccinimide to the mucin solution obtained in step (1), and reacting for 40 minutes; Step (3): adding 2 parts by weight of human umbilical cord mesenchymal stem cell exosomes to the mucin solution obtained in step (2) and stirring evenly; Step (4): Add 6 parts by weight of trehalose to the solution obtained in step (3), stir evenly, and obtain a mussel mucin-loaded extracellular vesicle hydrogel.

[0030] The concentration of PBS buffer is 0.01M.

[0031] Wherein, the method for preparing human umbilical cord mesenchymal stem cell exosomes in step (3) comprises: Step (a): 100 mg of umbilical cord mesenchymal stem cells of passage 4-5 were cultured until 65% confluence, and then replaced with serum-free medium. After further culture for 62 h, the supernatant of the cell culture medium was collected and centrifuged at 4°C and 300 g for 15 min to remove the mesenchymal stem cells; Step (b): centrifuging the cell supernatant at 4°C, 2000 g for 20 min to further remove dead cells and impurities, and collecting the supernatant; continuing to centrifuge at 4°C, 10000 g for 30 min to remove cell debris, and collecting the supernatant; Step (c): The supernatant obtained in step (b) was centrifuged at 4°C, 100,000 g for 70 min, the supernatant was discarded to obtain the exosome precipitate, 1 mL of sterile ice-cold PBS was added for resuspending, and then filtered with a 0.22 μm filter membrane to obtain human umbilical cord mesenchymal stem cell exosomes.

[0032] Wherein, the serum-free culture medium consists of α-MEM and platelet lysate.

[0033] The serum-free medium specifically includes the following components (calculated by volume): inorganic salts: 200 mg / L calcium chloride, 97.67 mg / L magnesium sulfate, 400 mg / L potassium chloride, 2200 mg / L sodium bicarbonate, 6800 mg / L sodium chloride, 122 mg / L anhydrous disodium hydrogen phosphate; amino acids: 126 mg / L L-alanine-L-glutamine, 100 mg / L L-arginine hydrochloride, 31.3 mg / L L-cysteine ​​hydrochloride monohydrate, 292 mg / L L-glutamine, 42 mg / L-histidine hydrochloride monohydrate, 52 mg / L-isoleucine, 52 mg / L-leucine, 72.5 mg / L-lysine hydrochloride, 15 mg / L-methionine, 32 mg / L-phenylalanine, 48 mg / L-threonine, 51 mg / L-tyrosine disodium salt dihydrate, 1 0mg / L tryptophan, 46mg / L alanine, 50mg / L glycine; 60mg / L vitamins; 40mg / L ribonucleotides; 41mg / L deoxyribonucleotides; 1000mg / L glucose; 0.2mg / L zinc sulfate; 110mg / L sodium pyruvate; 10mg / L phenol red; 10ug / L growth factor; 1.7ug / L endothelial growth factor; 50ug / L transforming growth factor; 0.27ug / L basic fibroblast factor; 11ug / L vascular endothelial growth factor.

[0034] The application of the mussel mucin-loaded extracellular vesicle hydrogel is to apply the mussel mucin-loaded extracellular vesicle hydrogel to wound repair.

[0035] During the reaction of step (2), 2 parts by weight of SOD embedded in 1,2-hexanediol is added dropwise. The 1,2-hexanediol embedded in SOD is obtained by drying 4 parts by weight of 1,2-hexanediol in a forced air drying oven at 65°C to remove moisture, adding 1 part by weight of SOD, and mixing in a shaker at -20°C until condensation occurs.

[0036] Example 3

[0037] A mussel mucin-loaded extracellular vesicle hydrogel, which combines human umbilical cord mesenchymal stem cell exosomes Exo with mussel mucin MAP.

[0038] A method for preparing a mussel mucin-loaded extracellular vesicle hydrogel comprises the following preparation steps: Step (1): dissolving 1.5 parts by weight of mussel mucin and 1.5 parts by weight of ectoine in 100 parts by weight of PBS buffer solution, and adjusting the pH of the mussel mucin solution to 4.0 dropwise with acetic acid to obtain a mussel mucin solution; Step (2): adding 4 parts by weight of polyethylene glycol diglycidyl ether and 5 parts by weight of N-hydroxysuccinimide to the mucin solution obtained in step (1), and reacting for 60 minutes; Step (3): Add 3 parts by weight of human umbilical cord mesenchymal stem cell exosomes to the mucin solution obtained in step (2) and stir evenly; Step (4): Add 8 parts by weight of trehalose to the solution obtained in step (3), stir evenly, and obtain a mussel mucin-loaded extracellular vesicle hydrogel.

[0039] The concentration of PBS buffer is 0.01M.

[0040] Wherein, the method for preparing human umbilical cord mesenchymal stem cell exosomes in step (3) comprises: Step (a): 100 mg of umbilical cord mesenchymal stem cells of passage 4-5 were cultured until 70% confluence, and then replaced with serum-free medium. After further culture for 72 h, the supernatant of the cell culture medium was collected and centrifuged at 4°C and 300 g for 20 min to remove the mesenchymal stem cells; Step (b): centrifuging the cell supernatant at 4°C, 2000 g for 25 min to further remove dead cells and impurities, and collecting the supernatant; further centrifuging at 4°C, 10000 g for 35 min to remove cell debris, and collecting the supernatant; Step (c): The supernatant obtained in step (b) was centrifuged at 4°C, 100,000 g for 90 min, the supernatant was discarded to obtain the exosome precipitate, 1 mL of sterile ice-cold PBS was added for resuspending, and then filtered with a 0.22 μm filter membrane to obtain human umbilical cord mesenchymal stem cell exosomes.

[0041] Wherein, the serum-free culture medium consists of α-MEM and platelet lysate.

[0042] The serum-free medium specifically includes the following components (calculated by volume): inorganic salts: 200 mg / L calcium chloride, 97.67 mg / L magnesium sulfate, 400 mg / L potassium chloride, 2200 mg / L sodium bicarbonate, 6800 mg / L sodium chloride, 122 mg / L anhydrous disodium hydrogen phosphate; amino acids: 126 mg / L L-alanine-L-glutamine, 100 mg / L L-arginine hydrochloride, 31.3 mg / L L-cysteine ​​hydrochloride monohydrate, 292 mg / L L-glutamine, 42 mg / L-histidine hydrochloride monohydrate, 52 mg / L-isoleucine, 52 mg / L-leucine, 72.5 mg / L-lysine hydrochloride, 15 mg / L-methionine, 32 mg / L-phenylalanine, 48 mg / L-threonine, 51 mg / L-tyrosine disodium salt dihydrate, 1 0mg / L tryptophan, 46mg / L alanine, 50mg / L glycine; 60mg / L vitamins; 40mg / L ribonucleotides; 41mg / L deoxyribonucleotides; 1000mg / L glucose; 0.2mg / L zinc sulfate; 110mg / L sodium pyruvate; 10mg / L phenol red; 10ug / L growth factor; 1.7ug / L endothelial growth factor; 50ug / L transforming growth factor; 0.27ug / L basic fibroblast factor; 11ug / L vascular endothelial growth factor.

[0043] The application of the mussel mucin-loaded extracellular vesicle hydrogel is to apply the mussel mucin-loaded extracellular vesicle hydrogel to wound repair.

[0044] During the reaction of step (2), 3 parts by weight of SOD embedded in 1,2-hexanediol is added dropwise. The 1,2-hexanediol embedded in SOD is prepared by drying 4 parts by weight of 1,2-hexanediol at 65° C. in a forced air drying oven to remove moisture, adding 1 part by weight of SOD, and mixing in a shaker at -20° C. until coagulation occurs.

[0045] Comparative Example 1

[0046] The difference between this comparative example 1 and example 2 is that it only uses human umbilical cord mesenchymal stem cell exosomes Exo, and the preparation method of the human umbilical cord mesenchymal stem cell exosomes comprises: Step (a): 100 mg of umbilical cord mesenchymal stem cells of passage 4-5 were cultured until 65% confluence, and then replaced with serum-free medium. After further culture for 62 h, the supernatant of the cell culture medium was collected and centrifuged at 4°C and 300 g for 15 min to remove the mesenchymal stem cells; Step (b): centrifuging the cell supernatant at 4°C, 2000 g for 20 min to further remove dead cells and impurities, and collecting the supernatant; continuing to centrifuge at 4°C, 10000 g for 30 min to remove cell debris, and collecting the supernatant; Step (c): The supernatant obtained in step (b) was centrifuged at 4°C, 100,000 g for 70 min, the supernatant was discarded to obtain the exosome precipitate, 1 mL of sterile ice-cold PBS was added for resuspending, and then filtered with a 0.22 μm filter membrane to obtain human umbilical cord mesenchymal stem cell exosomes, i.e., Exo product.

[0047] Wherein, the serum-free culture medium consists of α-MEM and platelet lysate.

[0048] The serum-free medium specifically includes the following components (calculated by volume): inorganic salts: 200 mg / L calcium chloride, 97.67 mg / L magnesium sulfate, 400 mg / L potassium chloride, 2200 mg / L sodium bicarbonate, 6800 mg / L sodium chloride, 122 mg / L anhydrous disodium hydrogen phosphate; amino acids: 126 mg / L L-alanine-L-glutamine, 100 mg / L L-arginine hydrochloride, 31.3 mg / L L-cysteine ​​hydrochloride monohydrate, 292 mg / L L-glutamine, 42 mg / L-histidine hydrochloride monohydrate, 52 mg / L-isoleucine, 52 mg / L-leucine, 72.5 mg / L-lysine hydrochloride, 15 mg / L-methionine, 32 mg / L-phenylalanine, 48 mg / L-threonine, 51 mg / L-tyrosine disodium salt dihydrate, 1 0mg / L tryptophan, 46mg / L alanine, 50mg / L glycine; 60mg / L vitamins; 40mg / L ribonucleotides; 41mg / L deoxyribonucleotides; 1000mg / L glucose; 0.2mg / L zinc sulfate; 110mg / L sodium pyruvate; 10mg / L phenol red; 10ug / L growth factor; 1.7ug / L endothelial growth factor; 50ug / L transforming growth factor; 0.27ug / L basic fibroblast factor; 11ug / L vascular endothelial growth factor.

[0049] Comparative Example 2

[0050] The difference between this comparative example 2 and example 2 is that it only uses mussel mucin MAP, and it includes the following preparation steps: Step (1): dissolving 1 part by weight of mussel mucin and 1 part by weight of ectoine in 100 parts by weight of PBS buffer solution, and adjusting the pH of the mussel mucin solution to 4.0 dropwise with acetic acid to obtain a mussel mucin solution; Step (2): Add 3 parts by weight of polyethylene glycol diglycidyl ether and 4 parts by weight of N-hydroxysuccinimide to the mucin solution obtained in step (1), and react for 40 minutes to obtain a MAP product.

[0051] The present invention uses the MAP@Exo product of Example 2 and the Exo product of Comparative Example 1, and the MAP product of Comparative Example 2 to conduct experiments under the same conditions.

[0052] Experimental part:

[0053] 1. Exo particle size distribution

[0054] Figure 1 This is a diagram of the Exo particle size distribution. The nanoparticle tracking analysis technology (NTA) detects the Exo particle size distribution. The diameter of the Exo of the present invention is about 140nm.

[0055] 2. Transmission electron microscopy observation

[0056] Figure 2 The left picture shows the morphology of Exo under transmission electron microscope. It is a spherical vesicle with double membrane structure. The particle size is Figure 1 The results are consistent. The right picture shows the morphological characteristics of MAP-loaded Exo hydrogel. MAP is adhered to the periphery of Exo, and the particle size is not significantly different from Exo.

[0057] 3. Cell scratch test

[0058] Figure 3 This is a cell scratch test, which is used to study cell migration and repair ability. The control group (Control) used PBS solution instead. The cell migration rate after 24 hours: MAP@Exo (98%) > Exo (81%) > MAP (75%) > PBS (1%). This shows that MAP@Exo hydrogel promotes cell migration and repair better than single MAP and Exo.

[0059] 4 Adhesion test

[0060] Figure 4 This is an adhesion test diagram, which is used to study the adhesion ability of MAP@Exo hydrogel to different materials to verify that the hydrogel has strong adhesion and can fit closely to the wound surface to improve the treatment effect.

[0061] The left picture shows that MAP@Exo hydrogel is a solution with good fluidity under normal conditions.

[0062] The abdominal skin of a 1.5-month-old male Bama Xiang pig was used to simulate human skin. MAP@Exo hydrogel was applied only on one side of the outer surface of the pig skin. After 5 minutes, weights and different materials (PET, glass, PP, ceramic) were used to gently press on both sides of the pig skin. All materials can be effectively adhered without falling off. This shows that the hydrogel has good adhesion and can adhere to the pig skin. It also has a certain degree of adhesion on the other side of the pig skin, which also shows that the hydrogel has strong penetration ability and can penetrate the skin.

[0063] In addition, during the above-mentioned adhesion test, deionized water was drawn with a dropper and dripped onto the adhesion site between the pig skin and the material. There was no significant change in the adhesion performance, which also shows that MAP@Exo hydrogel has good water resistance and can achieve water-proof adhesion.

[0064] 5. Repair effect of infected wounds in rats

[0065] Figure 5 This is a diagram of the repair effect of infected wounds in rats. Experimental steps: Each rat was marked, weighed, and recorded. According to the calculation of 6 ml / Kg for rats weighing less than 300 g, 2.5% tribromoethanol solution was used for intraperitoneal push anesthesia. After the rat was anesthetized, it was placed on a fixed board with its ventral side facing down, and its limbs were fixed. The hair on both sides of its back was cut and the skin was prepared. During the experiment, the body temperature of the rat was maintained at (36.5±0.3)℃. The back of the rat was disinfected with 10% povidone iodine to prepare for surgery. Press a 10mm skin biopsy sampler on the hair-cut area on the back to form a circular incision, and then use a marker to outline the cut area. Use scissors to cut the epidermis of the marked area along the marked edge line and destroy the wound to an appropriate depth. 100μl of Staphylococcus aureus liquid (1×10 7 ~1×10 9 CFU / ml) was dripped onto the wound surface, and the wound was properly sealed with an indwelling needle film. Each rat was kept in an independent ventilation cage. After 24 hours, the surface infection of the wounds in each group was observed and photographed. After an obvious infection focus was formed on the wound, 100 μl of the experimental sample was directly dripped onto the skin defect wound with a pipette, and the wound was properly sealed with an indwelling needle film. The dressing was changed once a day.

[0066] The spirit, activity and diet of the experimental mice were observed every day. The wound healing and inflammation were observed on the 0th, 3rd, 7th, 10th and 14th days after the wound was repaired, including the color and gloss of the wound, the contraction shape, the amount and shape of the wound exudate, the hardness and swelling of the tissue around the wound, the percentage of granulation tissue and epithelial tissue in the wound volume, and local inflammation.

[0067] During the wound healing process, the wound area was measured on days 0, 3, 7, 10, and 14, and the wound healing rate was calculated. Wound healing rate (%) = (original wound area - unhealed wound area) / original wound area × 100%.

[0068] During the wound healing process, wound healing and inflammation were observed on days 0, 3, 7, 10, and 14, and wound exudate, inflammatory response, and wound area were recorded. H&E staining and cytokine detection.

[0069] Results evaluation: The wound was repeatedly infected with Staphylococcus aureus liquid of a certain concentration, forming a difficult-to-heal wound. On the third day, obvious suppuration and ulceration were observed on the wound, indicating that the infected wound model was successfully established. The single MAP, Exo, and MAP@Exo hydrogels were used to evaluate the repair effect of infected wounds.

[0070] The wounds in the PBS group did not heal after 14 days; the wounds in the MAP and Exo groups were completely healed after 14 days, but scars were formed; the wounds in the MAP@Exo hydrogel group were completely healed after 4 days, with the wounds fully restored and no scars formed.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A mussel mucin-loaded extracellular vesicle hydrogel, characterized in that: It combines human umbilical cord mesenchymal stem cell exosomes Exo with mussel mucin MAP.

2. A method for preparing a mussel mucin-loaded extracellular vesicle hydrogel, characterized in that: It includes the following preparation steps: Step (1): dissolving 0.7-1.7 parts by weight of mussel mucin and 0.5-1.5 parts by weight of ectoine in 100 parts by weight of PBS buffer solution, and adjusting the pH of the mussel mucin solution to 4.0 dropwise with acetic acid to obtain a mussel mucin solution; Step (2): adding 2-4 parts by weight of polyethylene glycol diglycidyl ether and 3-5 parts by weight of N-hydroxysuccinimide to the mucin solution obtained in step (1), and reacting for 30-60 minutes; Step (3): adding 1.5-3 parts by weight of human umbilical cord mesenchymal stem cell exosomes to the mucin solution obtained in step (2) and stirring evenly; Step (4): Add 4-8 parts by weight of trehalose to the solution obtained in step (3), stir evenly, and obtain a mussel mucin-loaded extracellular vesicle hydrogel.

3. The method for preparing a mussel mucin-loaded extracellular vesicle hydrogel according to claim 2, characterized in that: The method for preparing human umbilical cord mesenchymal stem cell exosomes in step (3) comprises: Step (a): 100 mg of umbilical cord mesenchymal stem cells of passage 4-5 were cultured until 60%-70% confluence, and then replaced with serum-free medium. After further culture for 48-72 hours, the supernatant of the cell culture medium was collected, and the supernatant was centrifuged at 4°C and 300g for 10-20 minutes to remove the mesenchymal stem cells; Step (b): centrifuging the cell supernatant at 4°C, 2000 g for 15-25 min to further remove dead cells and impurities, and collecting the supernatant; continuing to centrifuge at 4°C, 10000 g for 25-35 min to remove cell debris, and collecting the supernatant; Step (c): The supernatant obtained in step (b) was centrifuged at 4°C, 100,000 g for 60-90 min, the supernatant was discarded to obtain the exosome precipitate, 1 mL of sterile ice-cold PBS was added for resuspending, and then filtered with a 0.22 μm filter membrane to obtain human umbilical cord mesenchymal stem cell exosomes.

4. The method for preparing a mussel mucin-loaded extracellular vesicle hydrogel according to claim 3, characterized in that: The serum-free culture medium consists of α-MEM and platelet lysate.

5. An application of the mussel mucin-loaded extracellular vesicle hydrogel as claimed in claim 2, characterized in that: This mussel mucin-loaded extracellular vesicle hydrogel is used in wound repair.

6. The use of the mussel mucin-loaded extracellular vesicle hydrogel according to claim 2, characterized in that: When the mussel mucin-loaded extracellular vesicle hydrogel is applied to wound repair, 1-3 parts by weight of SOD 1,2-hexanediol embedding material is added dropwise during the reaction of step (2).

Images