Temperature-sensitive hydroxybutyl chitosan hydrogel loaded with exosome as well as preparation method and application of temperature-sensitive hydroxybutyl chitosan hydrogel

Abstract

The invention relates to the technical field of gel, in particular to exosome-loaded temperature-sensitive hydroxybutyl chitosan hydrogel as well as a preparation method and application thereof.The HBC-SH hydrogel is formed by self-assembly of hydroxybutyl chitosan and silk fibroin, and under the action of two components, the hydrogel is higher in gelation speed and better in mechanical property, so that the temperature-sensitive hydroxybutyl chitosan hydrogel can be used for preparing the hydrogel. According to the invention, Exo is wrapped in microspheres by adopting a microencapsulation technology, and then the microspheres are stably encapsulated in HBC-SH hydrogel, so that the residence time and the stability of the exosome are prolonged, the longer-time slow release is realized, and the wound repair effect is improved. HBC-SH hydrogel can simulate the environment of a natural extracellular matrix and promote cell adhesion, migration and proliferation, so that tissue repair is accelerated, genetic information can be transmitted through Exo, tissue regeneration is further promoted, growth factors and cell factors of Exo and bioactive components in hydroxybutyl chitosan and silk fibroin generate a synergistic effect, and the effect of promoting tissue regeneration is achieved. And wound repair, cartilage repair and nerve regeneration processes are promoted together.

Description

Technical Field

[0001] The present invention relates to the field of gel technology, and in particular to a temperature-sensitive hydroxybutyl chitosan hydrogel loaded with exosomes, and a preparation method and application thereof. Background Art

[0002] Traditional exosomes derived from bone marrow mesenchymal stem cells and adipose-derived mesenchymal stem cells may lead to tumor formation, immune rejection, and abnormal differentiation in stem cell therapy, making them unsuitable for clinical application. Exosomes (Exo) derived from human umbilical cord mesenchymal stem cells are tiny vesicles with a diameter of 30 to 150 nm surrounded by a phospholipid bilayer. They are important mediators of intercellular communication by delivering RNA and proteins to target cells. They have multiple biological functions, such as promoting cell proliferation, migration, and angiogenesis. They have been shown to participate in various pathophysiological processes and play a vital role in the occurrence and development of various diseases, such as promoting wound healing, anti-aging, and inhibiting scar formation.

[0003] As carriers of bioactive molecules, Exosomes have a short half-life, rapid clearance, and low targeting, limiting their retention and stability. Therefore, it is necessary to develop a carrier system that can efficiently and stably deliver Exosomes to enhance their therapeutic efficacy in wound repair. However, traditional wound repair materials may have deficiencies in biocompatibility, degradability, sustained release, mechanical properties, and healing-promoting effects, making them difficult to meet the requirements for stable encapsulation and long-term release of exosomes.

[0004] For example, Chinese patent application number CN202311415044 discloses a method for preparing a thermosensitive gel that can carry exosomes, which uses recombinant collagen cross-linking technology. However, the method has weak mechanical strength, poor sustained release, weak antibacterial properties, and poor wound repair effect, and cannot meet the needs of complex wound treatment. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies in the prior art and provide a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel with good mechanical strength, good sustained release, strong antibacterial properties, and good wound repair effect, as well as a preparation method and application thereof.

[0006] The objectives of the present invention are achieved through the following technical solutions: a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel, which is liquid at low temperatures and turns into a gel at body temperature. It is constructed by hydroxybutyl chitosan, silk fibroin and encapsulated exosomes, wherein the microencapsulated exosomes are human umbilical cord mesenchymal stem cell exosomes wrapped in a sodium alginate-chitosan multilayer shell.

[0007] Preferably, the exosome-loaded thermosensitive hydroxybutyl chitosan hydrogel contains 50-200 μg / mL of microencapsulated exosomes.

[0008] More preferably, the exosome-loaded thermosensitive hydroxybutyl chitosan hydrogel contains 100 μg / mL of microencapsulated exosomes.

[0009] A method for preparing a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel comprises the following steps: Step A: Preparation of thermosensitive hydroxybutyl chitosan hydrogel:

[0010] Dissolve 2-3 g of hydroxybutyl chitosan powder in 100 ml of deionized water and stir at 4°C for 20-40 min. After complete dissolution, repeatedly place the prepared hydroxybutyl chitosan solution at 4°C for 10-15 min and 37°C for 10-15 min to obtain a thermosensitive hydroxybutyl chitosan hydrogel, which is then kept at 4°C for later use. Step B: Preparation of thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel:

[0011] B1. Dissolve 0.2 g to 0.4 g of silk fibroin powder in 10 ml of deionized water, stirring at 20-25°C, and adjust the pH to 7.0-8.0 with 0.1 M NaOH solution while stirring to obtain a silk fibroin precursor solution. B2. At 4°C, add 10 ml of the silk fibroin precursor solution dropwise to 90 ml of the thermosensitive hydroxybutyl chitosan hydrogel and stir for 10-20 min. Then, adjust the pH to 5.5-6.5 with 0.1 M NaOH solution while stirring. Let the mixture stand to obtain a thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel (HBC-SF hydrogel for short). Store the mixture at 4°C for later use. Step C: Preparation of microencapsulated exosomes:

[0012] C1. At 4°C, add 1 mL of 100 μg / mL exosome suspension in PBS to 10 mL of chitosan solution, stir for 15-25 minutes, then add to 20 mL of sodium alginate solution and stir for 15-25 minutes. C2. After centrifugation, resuspend the precipitate with PBS and repeat step C1 to allow chitosan and sodium alginate to alternately adsorb on the surface of the exosomes to form a multilayer membrane structure, thereby obtaining an exosome microcapsule precursor solution; C3. Add 5 mL of calcium chloride solution to the exosome microcapsule precursor solution obtained in step C2, and incubate at 4°C for 15-25 minutes for cross-linking to form stable microcapsules. Wash the microcapsules with PBS buffer to remove unreacted chitosan and sodium alginate to obtain microencapsulated exosomes (abbreviated as Exo). Finally, resuspend the washed microencapsulated exosomes with PBS to obtain a microencapsulated exosome suspension. Step D: Preparation of thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes:

[0013] D1. Dilute the microencapsulated exosome suspension to 50–200 μg / mL with PBS buffer pre-cooled to 4°C. D2. Then, in a 4°C ice-water bath, the diluted microencapsulated exosome suspension was added dropwise to the thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, stirred at a speed below 200 rpm for 10–15 minutes, and allowed to stand. D3. The mixed solution obtained in D2 was kept at 37°C for 20–30 minutes. The sol was transformed into a solid gel, and the microcapsules were physically embedded in the gel network to obtain a thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes (abbreviated as HBC-SF+Exo hydrogel).

[0014] Wherein, the preparation method of hydroxybutyl chitosan in step A is: A1. Take 2 g of chitosan powder and alkalize it in 20 ml of 50% NaOH solution. Stir in a sealed container at 0°C in an ice bath for 20-28 h, filter, and squeeze out the excess alkali solution to obtain alkalized chitosan. 0.1–0.5 g of nanocellulose was dispersed in 20 ml of deionized water and treated with ultrasound to obtain a nanocellulose suspension; A2, the alkalized chitosan obtained in step A1 was added to 80 ml of a mixed solvent of isopropanol and distilled water in a volume ratio of 1:1, and then the nanocellulose suspension obtained in A1 was added and stirred at 4 ° C for 20-40min; A3. Add 1.0-1.1 g of sodium lauryl sulfate dropwise as an emulsifier and 40-42 ml of 1,2-butylene oxide as an etherifying agent. Stir and react at 60°C for 10-14 hours. After the reaction, adjust the pH to neutral. Dialyze at 4°C for 4 days and freeze-dry to obtain hydroxybutyl chitosan white powder.

[0015] In step C, the chitosan solution is prepared by adding 0.25 g of chitosan to 80 mL of 4°C PBS, stirring to dissolve, and then adding 0.1 M NaOH dropwise to adjust the pH to 6.5. Finally, 0.1 g of poloxamer F68 is added and the mixture is made up to 100 mL. The sodium alginate solution is prepared by dissolving 1.5 g of sodium alginate powder in 100 mL of 0.9% NaCl aqueous solution and stirring. The calcium chloride solution is prepared by dissolving 1.5 g of anhydrous CaCl2 powder in 100 mL of deionized water and stirring.

[0016] Wherein, in step C, step C1 is repeated three times.

[0017] Wherein, the preparation method of the exosome PBS suspension in step C is: When umbilical cord mesenchymal stem cells of passage 4 to 5 were cultured to 60% to 70% confluence, the medium was replaced with serum-free medium and cultured for 48-72 h. The supernatant of the cell culture medium was collected and centrifuged at 4°C, 300g for 10 min to remove the mesenchymal stem cells to obtain the cell supernatant. The supernatant was further centrifuged at 4°C, 2000g for 20 min to further remove dead cells and impurities, and the supernatant was collected. The supernatant was then centrifuged at 4°C, 10,000g for 30 min to remove cell debris and collect the supernatant. The supernatant was then filtered using a 0.22 μm filter membrane. The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 4°C, 100,000g for 70 min. The supernatant was removed and the exosome pellet was resuspended in PBS buffer to obtain an exosome PBS suspension, which was aliquoted and stored at -80°C.

[0018] 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 L-histidine hydrochloride monohydrate, 52 mg / L L-isoleucine, 52 mg / L L-leucine, 72.5 mg / L L-lysine hydrochloride, 15 mg / L L-methionine, 32 mg / L L-phenylalanine, 48 mg / L L-threonine, 51 mg / L L-tyrosine disodium salt dihydrate, 10 mg / L L-tryptophan, 46mg / L alanine, 50mg / L glycine; 60mg / L vitamins; 40mg / L ribonucleotides; 41mg / L deoxyribonucleotides; 1000mg / L D-glucose; 0.2mg / L zinc sulfate; 110mg / L sodium pyruvate; 10mg / L phenol red; 10ug / L growth factors; 1.7ug / L endothelial growth factor; 50ug / L transforming growth factor; 0.27ug / L basic fibroblast growth factor; 11ug / L vascular endothelial growth factor.

[0019] A thermosensitive hydroxybutyl chitosan hydrogel loaded with human umbilical cord mesenchymal stem cell exosomes for wound repair.

[0020] Silk fibroin (SF) is a natural, inactive biomacromolecule, a naturally occurring high-molecular-weight fibrous protein. It possesses unique structure and properties, including excellent biocompatibility, degradation properties, ease of processing and modification, and non-toxic and low immunogenic degradation products. Furthermore, silk fibroin materials exhibit significantly superior degradability and mechanical strength to other biomaterials. It can be used in tissue engineering and wound repair. Hydroxybutyl chitosan (HBC) is a derivative prepared by chemically modifying chitosan. It exhibits excellent biocompatibility, biodegradability, and thermosensitivity. The incorporation of nanocellulose further enhances the mechanical and sustained-release properties of this invention. By regulating temperature and pH, this invention achieves the self-assembly of hydroxybutyl chitosan and silk fibroin to form a thermosensitive gel. This dual-component system results in a faster gelation rate and improved mechanical properties.

[0021] The thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel is fluid. It is liquid at low temperatures but rapidly transforms into a gel at body temperature (20-37°C), forming a non-flowing liquid gel. In wound care, it maintains a liquid state at low temperatures for easy application, while forming a gel at body temperature to secure it to the wound surface. Specifically, the low-temperature gelation range is 2–8°C, allowing for smooth extrusion through a syringe. The gelation trigger temperature is ≥25°C. Rapid gelation at body temperature: at a physiological temperature of 37°C, a self-supporting gel forms within 20 ± 5 seconds.

[0022] The present invention has the following beneficial effects: The present invention uses hydroxybutyl chitosan and silk fibroin to self-assemble to form HBC-SH hydrogel. Under the action of the two components, its gelation speed is faster and its mechanical properties are better. The present invention also uses microencapsulation technology to encapsulate Exosomes in microspheres, which are then stably encapsulated in the HBC-SH hydrogel, extending the residence time and stability of the exosomes to achieve longer-term sustained release and improve the wound repair effect. The HBC-SH hydrogel can simulate the environment of the natural extracellular matrix, promoting cell adhesion, migration and proliferation, thereby accelerating tissue repair. The present invention can also further promote tissue regeneration by transmitting genetic information through Exo. The growth factors and cytokines of Exo work synergistically with the bioactive components in hydroxybutyl chitosan and silk fibroin to jointly promote wound repair, cartilage repair, and nerve regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Characterization and rheological results of hydroxybutyl chitosan hydrogel; Figure 2 This is the degradation rate test result of HBC-SF hydrogel; Figure 3 Detect particle size distribution for nanoparticle tracking analysis (NTA); Figure 4 for Exo transmission electron microscopy (TEM); Figure 5 for cytotoxicity assay (MTT assay); Figure 6 This is a diagram of a cell scratch test; Figure 7 Data statistics for cell scratch test; Figure 8 This is the result diagram of exosome release; Figure 9 The repair effect of infected wounds in rats; Figure 10 The data are statistical data of infectious wound healing in rats. DETAILED DESCRIPTION

[0024] The present invention is further described with reference to the following examples. Example 1

[0025] A thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel is liquid at low temperatures and gels at body temperature. The hydrogel is composed of hydroxybutyl chitosan, silk fibroin, and encapsulated exosomes. The microencapsulated exosomes are human umbilical cord mesenchymal stem cell exosomes encapsulated in a sodium alginate-chitosan multilayer shell.

[0026] A method for preparing a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel comprises the following steps:

[0027] Step A: Preparation of thermosensitive hydroxybutyl chitosan hydrogel:

[0028] Dissolve 2 g of hydroxybutyl chitosan powder in 100 ml of deionized water and stir at 4°C for 20 min. After complete dissolution, repeatedly place the prepared hydroxybutyl chitosan solution at 4°C for 10 min and 37°C for 10 min to obtain a thermosensitive hydroxybutyl chitosan hydrogel, which is then kept at 4°C for later use. Step B: Preparation of thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel:

[0029] B1. Dissolve 0.2 g of silk fibroin powder in 10 ml of deionized water, stirring at 20° C., and adjust the pH to 7.0 with 0.1 M NaOH solution while stirring to obtain a silk fibroin precursor solution. B2. At 4°C, 10 ml of the silk fibroin precursor solution was added dropwise to 90 ml of the thermosensitive hydroxybutyl chitosan hydrogel, stirred for 10 min, and then the pH was adjusted to 5.5 with 0.1 M NaOH solution under stirring. The mixture was allowed to stand to obtain a thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, which was stored at 4°C for later use. Step C: Preparation of microencapsulated exosomes:

[0030] C1. At 4°C, 1 mL of 100 μg / mL exosome suspension in PBS was added dropwise to 10 mL of chitosan solution, stirred for 15 minutes, and then added to 20 mL of sodium alginate solution, stirred for 15 minutes. C2. After centrifugation, resuspend the precipitate with PBS and repeat step C1 to allow chitosan and sodium alginate to alternately adsorb on the surface of the exosomes to form a multilayer membrane structure, thereby obtaining an exosome microcapsule precursor solution; C3. Add 5 mL of calcium chloride solution to the exosome microcapsule precursor solution obtained in step C2, and incubate at 4°C for 15 minutes for cross-linking to form stable microcapsules. Wash the microcapsules with PBS buffer to remove unreacted chitosan and sodium alginate to obtain microencapsulated exosomes. Finally, resuspend the washed microencapsulated exosomes with PBS to obtain a microencapsulated exosome suspension. Step D: Preparation of thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes:

[0031] D1. Dilute the microencapsulated exosome suspension to 50 μg / mL with PBS buffer pre-cooled to 4°C. D2. Then, in a 4°C ice water bath, the diluted microencapsulated exosome suspension was added dropwise to the thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, stirred at a speed below 200 rpm for 10 minutes, and allowed to stand. D3. The mixed solution obtained in D2 was kept at 37°C for 20 minutes, and the sol was transformed into a solid gel. The microcapsules were physically embedded in the gel network to obtain a thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes.

[0032] Wherein, the preparation method of hydroxybutyl chitosan in step A is: A1. Take 2 g of chitosan powder and alkalize it in 20 ml of 50% NaOH solution. Stir in a sealed container at 0°C in an ice bath for 20 h. Filter and squeeze out the excess alkali solution to obtain alkalized chitosan. 0.1 g of nanocellulose was dispersed in 20 ml of deionized water and treated with ultrasound to obtain a nanocellulose suspension; A2, the alkalized chitosan obtained in step A1 was added to 80 ml of a 1:1 volume ratio of isopropanol and distilled water mixed solvent, and then the nanocellulose suspension obtained in A1 was added and stirred at 4 ° C for 20 min; A3. Add 1.0 g of sodium lauryl sulfate dropwise as an emulsifier and 40 ml of 1,2-butylene oxide as an etherifying agent. Stir and react at 60°C for 10 hours. After the reaction, adjust the pH to neutral. Dialyze at 4°C for 4 days and freeze-dry to obtain hydroxybutyl chitosan white powder.

[0033] In step C, the chitosan solution is prepared by adding 0.25 g of chitosan to 80 mL of 4°C PBS, stirring to dissolve, and then adding 0.1 M NaOH dropwise to adjust the pH to 6.5. Finally, 0.1 g of poloxamer F68 is added and the mixture is made up to 100 mL. The sodium alginate solution is prepared by dissolving 1.5 g of sodium alginate powder in 100 mL of 0.9% NaCl aqueous solution and stirring. The calcium chloride solution is prepared by dissolving 1.5 g of anhydrous CaCl2 powder in 100 mL of deionized water and stirring.

[0034] Wherein, in step C, step C1 is repeated three times.

[0035] Wherein, the preparation method of the exosome PBS suspension in step C is: When umbilical cord mesenchymal stem cells of passage 4 to 5 were cultured to 60% confluence, the medium was replaced with serum-free medium and cultured for 48 h. The supernatant of the cell culture medium was collected and centrifuged at 4°C, 300g for 10 min to remove the mesenchymal stem cells to obtain the cell supernatant. The supernatant was further centrifuged at 4°C, 2000g for 20 min to further remove dead cells and impurities, and the supernatant was collected. The supernatant was then centrifuged at 4°C, 10,000g for 30 min to remove cell debris and collect the supernatant. The supernatant was then filtered using a 0.22 μm filter membrane. The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 4°C, 100,000g for 70 min. The supernatant was removed and the exosome precipitate was resuspended in PBS buffer to obtain an exosome PBS suspension, which was aliquoted and stored at -80°C.

[0036] A thermosensitive hydroxybutyl chitosan hydrogel loaded with human umbilical cord mesenchymal stem cell exosomes for wound repair. Example 2

[0037] A thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel is liquid at low temperatures and gels at body temperature. The hydrogel is composed of hydroxybutyl chitosan, silk fibroin, and encapsulated exosomes. The microencapsulated exosomes are human umbilical cord mesenchymal stem cell exosomes encapsulated in a sodium alginate-chitosan multilayer shell.

[0038] A method for preparing a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel comprises the following steps: Step A: Preparation of thermosensitive hydroxybutyl chitosan hydrogel:

[0039] Dissolve 2.5 g of hydroxybutyl chitosan powder in 100 ml of deionized water and stir at 4°C for 30 min. After complete dissolution, repeatedly place the prepared hydroxybutyl chitosan solution at 4°C for 15 min and 37°C for 15 min to obtain a thermosensitive hydroxybutyl chitosan hydrogel, which is then kept at 4°C for later use. Step B: Preparation of thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel:

[0040] B1. Dissolve 0.3 g of silk fibroin powder in 10 ml of deionized water at 22°C with stirring. Adjust the pH to 7.5 with 0.1 M NaOH solution while stirring to obtain a silk fibroin precursor solution. B2. At 4°C, 10 ml of the silk fibroin precursor solution was added dropwise to 90 ml of the thermosensitive hydroxybutyl chitosan hydrogel, stirred for 15 min, and then the pH was adjusted to 6.0 with 0.1 M NaOH solution under stirring. The mixture was allowed to stand to obtain a thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, which was stored at 4°C for later use. Step C: Preparation of microencapsulated exosomes:

[0041] C1. At 4°C, 1 mL of 100 μg / mL exosome suspension in PBS was added dropwise to 10 mL of chitosan solution, stirred for 20 minutes, and then added to 20 mL of sodium alginate solution, stirred for 20 minutes. C2. After centrifugation, resuspend the precipitate with PBS and repeat step C1 to allow chitosan and sodium alginate to alternately adsorb on the surface of the exosomes to form a multilayer membrane structure, thereby obtaining an exosome microcapsule precursor solution; C3. Add 5 mL of calcium chloride solution to the exosome microcapsule precursor solution obtained in step C2, and incubate at 4°C for 20 minutes for cross-linking to form stable microcapsules. Wash the microcapsules with PBS buffer to remove unreacted chitosan and sodium alginate to obtain microencapsulated exosomes. Finally, resuspend the washed microencapsulated exosomes with PBS to obtain a microencapsulated exosome suspension. Step D: Preparation of thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes:

[0042] D1. Dilute the microencapsulated exosome suspension to 100 μg / mL with PBS buffer pre-cooled to 4°C. D2. Then, in a 4°C ice water bath, the diluted microencapsulated exosome suspension was added dropwise to the thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, stirred at a speed below 200 rpm for 15 minutes, and allowed to stand. D3. The mixed solution obtained in D2 was kept at 37°C for 25 minutes, and the sol was transformed into a solid gel. The microcapsules were physically embedded in the gel network to obtain a thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes.

[0043] Wherein, the preparation method of hydroxybutyl chitosan in step A is: A1. Take 2 g of chitosan powder and alkalize it in 20 ml of 50% NaOH solution. Stir in a sealed container at 0°C in an ice bath for 24 h. Filter and squeeze out the excess alkali solution to obtain alkalized chitosan. 0.3 g of nanocellulose was dispersed in 20 ml of deionized water and ultrasonically treated to obtain a nanocellulose suspension; A2, the alkalized chitosan obtained in step A1 was added to 80 ml of a 1:1 volume ratio of isopropanol and distilled water mixed solvent, and then the nanocellulose suspension obtained in A1 was added and stirred at 4 ° C for 30 min; A3. Add 1.0 g of sodium lauryl sulfate dropwise as an emulsifier and 40 ml of 1,2-butylene oxide as an etherifying agent. Stir and react at 60°C for 12 hours. After the reaction, adjust the pH to neutral. Dialyze at 4°C for 4 days and freeze-dry to obtain hydroxybutyl chitosan white powder.

[0044] In step C, the chitosan solution is prepared by adding 0.25 g of chitosan to 80 mL of 4°C PBS, stirring to dissolve, and then adding 0.1 M NaOH dropwise to adjust the pH to 6.5. Finally, 0.1 g of poloxamer F68 is added and the mixture is made up to 100 mL. The sodium alginate solution is prepared by dissolving 1.5 g of sodium alginate powder in 100 mL of 0.9% NaCl aqueous solution and stirring. The calcium chloride solution is prepared by dissolving 1.5 g of anhydrous CaCl2 powder in 100 mL of deionized water and stirring.

[0045] Wherein, in step C, step C1 is repeated three times.

[0046] Wherein, the preparation method of the exosome PBS suspension in step C is: Umbilical cord mesenchymal stem cells of passage 4 to 5 were cultured to 65% confluence and replaced with serum-free medium. After continued culture for 60 h, the supernatant of the cell culture medium was collected and centrifuged at 4°C, 300g for 10 min to remove the mesenchymal stem cells to obtain the cell supernatant. The supernatant was further centrifuged at 4°C, 2000g for 20 min to further remove dead cells and impurities, and the supernatant was collected. The supernatant was then centrifuged at 4°C, 10,000g for 30 min to remove cell debris and collect the supernatant. The supernatant was then filtered using a 0.22 μm filter membrane. The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 4°C, 100,000g for 70 min. The supernatant was removed and the exosome pellet was resuspended in PBS buffer to obtain an exosome PBS suspension, which was aliquoted and stored at -80°C.

[0047] A thermosensitive hydroxybutyl chitosan hydrogel loaded with human umbilical cord mesenchymal stem cell exosomes for wound repair. Example 3

[0048] A thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel is liquid at low temperatures and gels at body temperature. The hydrogel is composed of hydroxybutyl chitosan, silk fibroin, and encapsulated exosomes. The microencapsulated exosomes are human umbilical cord mesenchymal stem cell exosomes encapsulated in a sodium alginate-chitosan multilayer shell.

[0049] A method for preparing a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel comprises the following steps: Step A: Preparation of thermosensitive hydroxybutyl chitosan hydrogel:

[0050] Dissolve 3 g of hydroxybutyl chitosan powder in 100 ml of deionized water and stir at 4°C for 40 min. After complete dissolution, repeatedly place the prepared hydroxybutyl chitosan solution at 4°C for 15 min and 37°C for 15 min to obtain a thermosensitive hydroxybutyl chitosan hydrogel, which is then kept at 4°C for later use. Step B: Preparation of thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel:

[0051] B1. Dissolve 0.4 g of silk fibroin powder in 10 ml of deionized water, stir at 25°C, and adjust the pH to 8.0 with 0.1 M NaOH solution while stirring to obtain a silk fibroin precursor solution. B2. At 4°C, 10 ml of the silk fibroin precursor solution was added dropwise to 90 ml of the thermosensitive hydroxybutyl chitosan hydrogel and stirred for 20 min. The pH was then adjusted to 6.5 with 0.1 M NaOH solution under stirring and the mixture was allowed to stand to obtain a thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, which was then stored at 4°C for later use. Step C: Preparation of microencapsulated exosomes:

[0052] C1. At 4°C, 1 mL of 100 μg / mL exosome suspension in PBS was added dropwise to 10 mL of chitosan solution, stirred for 25 minutes, and then added to 20 mL of sodium alginate solution, stirred for 25 minutes; C2. After centrifugation, resuspend the precipitate with PBS and repeat step C1 to allow chitosan and sodium alginate to alternately adsorb on the surface of the exosomes to form a multilayer membrane structure, thereby obtaining an exosome microcapsule precursor solution; C3. Add 5 mL of calcium chloride solution to the exosome microcapsule precursor solution obtained in step C2, and incubate at 4°C for 25 minutes for cross-linking to form stable microcapsules. Wash the microcapsules with PBS buffer to remove unreacted chitosan and sodium alginate to obtain microencapsulated exosomes. Finally, resuspend the washed microencapsulated exosomes with PBS to obtain a microencapsulated exosome suspension. Step D: Preparation of thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes:

[0053] D1. Dilute the microencapsulated exosome suspension to 180 μg / mL with PBS buffer pre-cooled to 4°C. D2. Then, in a 4°C ice water bath, the diluted microencapsulated exosome suspension was added dropwise to the thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, stirred at a speed below 200 rpm for 15 minutes, and allowed to stand. D3. The mixed solution obtained in D2 was kept at 37°C for 30 minutes, and the sol was transformed into a solid gel. The microcapsules were physically embedded in the gel network to obtain a thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes.

[0054] Wherein, the preparation method of hydroxybutyl chitosan in step A is: A1. Take 2 g of chitosan powder and alkalize it in 20 ml of 50% NaOH solution. Stir it in a sealed container under ice bath conditions for 28 h. Filter it and squeeze out the excess alkali solution to obtain alkalized chitosan. 0.5 g of nanocellulose was dispersed in 20 ml of deionized water and treated with ultrasound to obtain a nanocellulose suspension; A2, the alkalized chitosan obtained in step A1 was added to 80 ml of a 1:1 volume ratio of isopropanol and distilled water mixed solvent, and then the nanocellulose suspension obtained in A1 was added and stirred at 4 ° C for 40 min; A3. Add 1.1 g of sodium lauryl sulfate dropwise as an emulsifier and 42 ml of 1,2-butylene oxide as an etherifying agent. Stir and react at 60°C for 14 hours. After the reaction, adjust the pH to neutral. Dialyze at 4°C for 4 days and freeze-dry to obtain hydroxybutyl chitosan white powder.

[0055] In step C, the chitosan solution is prepared by adding 0.25 g of chitosan to 80 mL of 4°C PBS, stirring to dissolve, and then adding 0.1 M NaOH dropwise to adjust the pH to 6.5. Finally, 0.1 g of poloxamer F68 is added and the mixture is made up to 100 mL. The sodium alginate solution is prepared by dissolving 1.5 g of sodium alginate powder in 100 mL of 0.9% NaCl aqueous solution and stirring. The calcium chloride solution is prepared by dissolving 1.5 g of anhydrous CaCl2 powder in 100 mL of deionized water and stirring.

[0056] Wherein, in step C, step C1 is repeated three times.

[0057] Wherein, the preparation method of the exosome PBS suspension in step C is: Umbilical cord mesenchymal stem cells of passage 4 to 5 were cultured to 70% confluence and 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, 300g for 10 min to remove the mesenchymal stem cells to obtain the cell supernatant. The supernatant was further centrifuged at 4°C, 2000g for 20 min to further remove dead cells and impurities, and the supernatant was collected. The supernatant was then centrifuged at 4°C, 10,000g for 30 min to remove cell debris and collect the supernatant. The supernatant was then filtered using a 0.22 μm filter membrane. The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 4°C, 100,000g for 70 min. The supernatant was removed and the exosome pellet was resuspended in PBS buffer to obtain an exosome PBS suspension, which was aliquoted and stored at -80°C.

[0058] A thermosensitive hydroxybutyl chitosan hydrogel loaded with human umbilical cord mesenchymal stem cell exosomes for wound repair.

[0059] Experimental part:

[0060] 1. Characterization and rheological results of hydroxybutyl chitosan hydrogel

[0061] See Figure 1 , the sample of Example 2 was tested.

[0062] Figure A: (left) Silk fibroin precursor solution, (right) Silk fibroin precursor solution forming a gel after being placed at room temperature for 5 days; Figure B: The process of HBC-SF hydrogel transforming from fluid state at low temperature to gel state at 37°C; Figure C: Characterization results of HBC-SF hydrogel. HBC-SF hydrogel has obvious pore size and can be loaded with drugs; Figure D: Rheological test results. When G'>G", the HBC-SF hydrogel changes from liquid to colloid. The prepared HBC-SF hydrogel can gel in a relatively short time and can therefore be used in skin repair research.

[0063] 2. HBC-SF hydrogel degradation rate test results

[0064] See Figure 2 The prepared HBC-SF hydrogel showed a trend of gradual degradation over time. The degradation rate was the highest within 0-96 h, and began to decrease within 96-120 h. After 120 h, the degradation rate was the slowest and tended to be stable. The hydrogel could maintain continuous degradation within 7 days.

[0065] 3. Nanoparticle Tracking Analysis (NTA) to detect particle size distribution

[0066] See Figure 3 Nanoparticle tracking analysis (NTA) technology was used to detect the Exo particle size distribution of the sample in Example 2 of the present invention. The diameter of Exo was about 145 nm.

[0067] 4. Exo transmission electron microscopy (TEM)

[0068] See Figure 4 : The morphology of the sample of Example 2 of the present invention under the transmission electron microscope is a double-layer membrane structure spherical vesicle with a particle size of Figure 3 The results are consistent.

[0069] 5. Cytotoxicity test (MTT method)

[0070] See Figure 5 The cell survival rate of the HBC group was greater than that of the blank group, indicating that HBC promotes cell growth. The cell survival rate of the 100μg / ml Exo group was greater than that of the 10μg / ml Exo group, which was greater than that of the positive control group, indicating that Exo significantly promotes cell growth with increasing Exo concentration. Compared with the single component groups at the same concentration, the cell survival rate of the HBC-SF + Exo group was significantly improved. The HBC-SF + 100μg / ml Exo group had the highest cell survival rate.

[0071] 6. Cell scratch test

[0072] See Figure 6 and Figure 7 , Figure 6 This is the cell scratch test diagram. Figure 7 Data from a cell scratch test is presented. The cell scratch test is used to study cell migration and repair capabilities. The control group (Control) used PBS solution instead, and the other samples used samples obtained at different stages of Example 2. The 24-hour cell migration rates were: HBC-SF + Exo (94%) > Exo (93%) > PBS (63%) > HBC-SF (51%). This indicates that the HBC-SF + Exo hydrogel promotes cell migration and repair better than either HBC-SF or Exo alone.

[0073] 8. Exosome release results

[0074] See Figure 8 To investigate the release of exosomes from a thermosensitive hydroxybutyl chitosan hydrogel loaded with human umbilical cord mesenchymal stem cell exosomes, we monitored the sample obtained in Example 2 for 7 consecutive days. The results were plotted as a line graph to analyze the exosome release pattern. The exosome release rate curve demonstrated that the HBC-SF+Exo hydrogel sustained exosome release over a 7-day period.

[0075] 9. Rat infectious wound repair experiment

[0076] See Figure 9 and Figure 10 , Figure 9 The repair effect of infected wounds in rats, Figure 10 The data are statistical data for infected wound healing in rats. To investigate the effects of HBC-SF+Exo hydrogel on wound repair, we treated rat wounds using different samples obtained at different stages of Example 2. We recorded wound closure status on days 0, 7, and 14. Comparative analysis of wound photos from each group on days 0, 4, 7, and 14 was performed to assess wound healing efficacy. On day 7, the wound closure area in the NC group was smaller than that in the Exo and HBC-SF+Exo groups, with the HBC-SF group showing the largest wound closure area. This suggests that initial wound formation may be smaller, leading to faster wound closure later in life.

[0077] On day 14, wound closure rates in all groups approached 90%, with the HBC-SF+Exo group achieving the highest rate, reaching 94.85%. There was no significant difference in wound closure rates between the NC and HBC-SF groups, with healing rates of 90.82% in the NC group and 90.11% in the HBC-SF group. The HBC-SF group had a slightly lower healing rate than the NC group, likely due to the PBS solvent used in the blank group. The saline solution has an anti-inflammatory effect on wounds, leading to faster healing on day 14. The Exo group had a healing rate of 92%, lower than the HBC-SF+Exo group, indicating that hydrogel-loaded stem cell exosomes can promote wound closure and tissue regeneration.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions 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 solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel, characterized by: This exosome-loaded thermosensitive hydroxybutyl chitosan hydrogel is liquid at low temperatures and turns into a gel at body temperature. It is constructed from hydroxybutyl chitosan, silk fibroin and encapsulated exosomes, wherein the microencapsulated exosomes are human umbilical cord mesenchymal stem cell exosomes wrapped in a sodium alginate-chitosan multilayer shell.

2. The exosome-loaded thermosensitive hydroxybutyl chitosan hydrogel according to claim 1, characterized in that: The exosome-loaded thermosensitive hydroxybutyl chitosan hydrogel microencapsulated exosomes at a concentration of 50-200 μg / mL.

3. The exosome-loaded thermosensitive hydroxybutyl chitosan hydrogel according to claim 1, characterized in that: The exosome-loaded thermosensitive hydroxybutyl chitosan hydrogel microencapsulated exosomes at a concentration of 100 μg / mL.

4. The method for preparing a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel according to claim 1, wherein: It includes the following steps: Step A: Preparation of thermosensitive hydroxybutyl chitosan hydrogel: Dissolve 2-3 g of hydroxybutyl chitosan powder in 100 ml of deionized water and stir at 4°C for 20-40 min. After complete dissolution, repeatedly place the prepared hydroxybutyl chitosan solution at 4°C for 10-15 min and 37°C for 10-15 min to obtain a thermosensitive hydroxybutyl chitosan hydrogel, which is then kept at 4°C for later use. Step B: Preparation of thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel: B1. Dissolve 0.2 g to 0.4 g of silk fibroin powder in 10 ml of deionized water, stirring at 20-25° C., and adjust the pH to 7.0-8.0 with 0.1 M NaOH solution while stirring to obtain a silk fibroin precursor solution. B2. At 4°C, 10 ml of the silk fibroin precursor solution was added dropwise to 90 ml of the thermosensitive hydroxybutyl chitosan hydrogel and stirred for 10-20 min. The pH was then adjusted to 5.5-6.5 with 0.1 M NaOH solution under stirring and the mixture was allowed to stand to obtain a thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, which was then stored at 4°C for later use. Step C: Preparation of microencapsulated exosomes: C1. At 4°C, add 1 mL of 100 μg / mL exosome suspension in PBS to 10 mL of chitosan solution, stir for 15-25 minutes, then add to 20 mL of sodium alginate solution and stir for 15-25 minutes. C2. After centrifugation, resuspend the precipitate with PBS and repeat step C1 to allow chitosan and sodium alginate to alternately adsorb on the surface of the exosomes to form a multilayer membrane structure, thereby obtaining an exosome microcapsule precursor solution; C3. Add 5 mL of calcium chloride solution to the exosome microcapsule precursor solution obtained in step C2, and incubate at 4°C for 15-25 minutes for cross-linking to form stable microcapsules. Wash the microcapsules with PBS buffer to remove unreacted chitosan and sodium alginate to obtain microencapsulated exosomes. Finally, resuspend the washed microencapsulated exosomes with PBS to obtain a microencapsulated exosome suspension. Step D: Preparation of thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes: D1. Dilute the microencapsulated exosome suspension to 50–200 μg / mL with PBS buffer pre-cooled to 4°C. D2. Then, in a 4°C ice-water bath, the diluted microencapsulated exosome suspension was added dropwise to the thermosensitive hydroxybutyl chitosan-silk fibroin hydrogel, stirred at a speed below 200 rpm for 10–15 minutes, and allowed to stand. D3. The mixed solution obtained in D2 was kept at 37°C for 20-30 minutes. The sol was transformed into a solid gel, and the microcapsules were physically embedded in the gel network to obtain a thermosensitive hydroxybutyl chitosan hydrogel loaded with exosomes.

5. The method for preparing a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel according to claim 4, characterized in that: The preparation method of hydroxybutyl chitosan in step A is: A1. Take 2 g of chitosan powder and alkalize it in 20 ml of 50% NaOH solution. Stir in a sealed container at 0°C in an ice bath for 20-28 h, filter, and squeeze out the excess alkali solution to obtain alkalized chitosan. 0.1–0.5 g of nanocellulose was dispersed in 20 ml of deionized water and treated with ultrasound to obtain a nanocellulose suspension; A2, the alkalized chitosan obtained in step A1 was added to 80 ml of a mixed solvent of isopropanol and distilled water in a volume ratio of 1:1, and then the nanocellulose suspension obtained in A1 was added and stirred at 4 ° C for 20-40min; A3. Add 1.0-1.1 g of sodium lauryl sulfate dropwise as an emulsifier and 40-42 ml of 1,2-butylene oxide as an etherifying agent. Stir and react at 60°C for 10-14 hours. After the reaction, adjust the pH to neutral. Dialyze at 4°C for 4 days and freeze-dry to obtain hydroxybutyl chitosan white powder.

6. The method for preparing a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel according to claim 4, characterized in that: In step C, the chitosan solution is prepared by adding 0.25 g of chitosan to 80 mL of 4° C. PBS, stirring and dissolving, and then adding 0.1 M NaOH dropwise to adjust the pH to 6.

5. Finally, 0.1 g of poloxamer F68 is added and the mixture is made up to 100 mL. The sodium alginate solution is prepared by dissolving 1.5 g of sodium alginate powder in 100 mL of 0.9% NaCl aqueous solution and stirring. The calcium chloride solution is prepared by dissolving 1.5 g of anhydrous CaCl2 powder in 100 mL of deionized water and stirring.

7. The method for preparing a thermosensitive exosome-loaded hydroxybutyl chitosan hydrogel according to claim 4, characterized in that: The preparation method of the exosome PBS suspension in step C is: When umbilical cord mesenchymal stem cells of passage 4 to 5 were cultured to 60% to 70% confluence, the medium was replaced with serum-free medium and cultured for 48-72 h. The supernatant of the cell culture medium was collected and centrifuged at 4°C, 300g for 10 min to remove the mesenchymal stem cells to obtain the cell supernatant. The supernatant was further centrifuged at 4°C, 2000g for 20 min to further remove dead cells and impurities, and the supernatant was collected. The supernatant was then centrifuged at 4°C, 10,000g for 30 min to remove cell debris and collect the supernatant. The supernatant was then filtered using a 0.22 μm filter membrane. The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 4°C, 100,000g for 70 min. The supernatant was removed and the exosome pellet was resuspended in PBS buffer to obtain an exosome PBS suspension, which was aliquoted and stored at -80°C.

8. The thermosensitive hydroxybutyl chitosan hydrogel loaded with human umbilical cord mesenchymal stem cell exosomes as claimed in claim 1, which is used for wound repair.

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