A ROS-regulated hydrogel loaded with acid koumiss-derived exosomes, a preparation method thereof and application thereof in preparing burn drugs

Abstract

The application provides a ROS-regulated hydrogel loaded with acid koumiss-derived exosomes and a preparation method and application in preparation of burn drugs, and belongs to the technical field of burn drugs. The acid koumiss-derived exosomes (K-Exo) are wrapped by using silk fibroin peptides, and the obtained nano drug-loaded fibrous microcrystals can be used as a ROS-responsive nano drug-loaded sustained-release system of K-Exo. When the microenvironment of a burn site highly expresses ROS, the silk fibroin nano fiber microcrystals are dissolved, K-Exo is released, the ROS content and inflammatory cell infiltration at the damage site are effectively reduced, and the skin at the burn site is rapidly healed. The K-Exo is loaded into the GelMa hydrogel, and the obtained hydrogel shows broad-spectrum antioxidant and anti-inflammatory biological effects, as well as active regulation of the immune system, ROS-responsive controlled release, promotion of cell migration ability, nerve regeneration and blood vessel regeneration, so that the repair of large-area burn non-healing wounds can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of anti-burn drugs, in particular to a ROS-regulated hydrogel loaded with exosomes from sour koumiss and a preparation method thereof and application thereof in preparing burn drugs. BACKGROUND

[0002] Burn wound of skin is one of the diseases that is difficult to treat in skin injury, and has become a major challenge in clinical practice. Burn wound includes skin injury caused by exposure to heat, extreme cold, electric current, chemicals, radiation and friction, and can be divided into I~III degrees according to the depth and degree of damaged tissue. I degree burn usually occurs in the superficial layer of skin, and the hair germ is alive, which does not cause blisters and most of them can self-heal. II degree and above burns often need surgical intervention, and deep II degree burn and deep III degree burn wound often need longer time to heal, and even some cases cannot heal naturally. Based on the severity of burn and the urgency of treatment, the research and development of burn dressing are increasingly important.

[0003] With the continuous development of biomedical materials, more and more burn dressings play a role in the field of burn due to their good permeability, water absorption and antibacterial properties, etc., providing a good solution for burn repair. However, many researchers only focus on the antibacterial effect and the effect of promoting wound repair in the process of burn repair, often ignoring the regulation effect of implanted biomaterials on the microenvironment of the damaged site.

[0004] The microenvironment of large-area burn wound damage site accumulates a large amount of ROS, and the excessive accumulation of ROS produces strong oxidative stress. Strong local oxidative stress is the key to chronic inflammation, forming a vicious cycle and seriously hindering the regeneration and repair of skin. Inflammation prevents or delays the production of new blood vessels and epidermis, and seriously hinders the regeneration and reconstruction of epidermis. Therefore, regulating the inflammatory response and ROS content at the damage site is very important for successful repair of burn damaged skin. The implantation of biomaterials at the site of skin defect can activate the foreign body reaction produced by macrophages, and the high inflammatory response will inhibit the formation of epithelium. Macrophages exist in two phenotypes, M1 and M2. M1 macrophages secrete many inflammatory factors, prolong the inflammatory response at the damage site, inhibit re-epithelialization, and lead to difficult healing of the wound. M2 type macrophages are an important source of growth factors, secrete VEGF to induce vascular remodeling, accelerate the synthesis of collagen, fibronectin and other extracellular matrix, and thus promote the healing of burn wound.

[0005] Therefore, it is of great significance to seek an anti-inflammatory hydrogel dressing with ROS response for the treatment of large-area skin burn. SUMMARY

[0006] Therefore, the present application aims to provide a ROS-regulated hydrogel loaded with acid-koumiss-derived exosomes, a preparation method thereof and an application thereof in preparing burn drugs.

[0007] To achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0008] The present application provides a preparation method of a ROS-regulated hydrogel loaded with acid-koumiss-derived exosomes, comprising the following steps:

[0009] The acid koumiss is subjected to gradient centrifugation to obtain acid-koumiss-derived exosomes;

[0010] The acid-koumiss-derived exosomes, silk peptide, surfactant and phosphate buffer are mixed to perform β-fold self-assembly to obtain a nano-drug-loaded fiber microcrystal solution; the silk peptide has the sequence shown in SEQ ID No. 1;

[0011] The nano-drug-loaded fiber microcrystal solution and a GelMa solution are mixed to perform 3D printing to obtain the ROS-regulated hydrogel loaded with acid-koumiss-derived exosomes.

[0012] Preferably, the gradient centrifugation comprises:

[0013] The solution is sequentially subjected to first centrifugation at 3000 rpm for 10 min, second centrifugation at 5000 rpm for 30 min, third centrifugation at 12000 rpm for 60 min, fourth centrifugation at 35000 rpm for 60 min, fifth centrifugation at 70000 rpm for 60 min, and sixth centrifugation at 110000 rpm for 80 min, and the precipitate is collected;

[0014] The acid-koumiss-derived exosomes have a particle size of 30-150 nm.

[0015] Preferably, the method further comprises filtering the solution after the third centrifugation; after the fourth centrifugation, the obtained solution is passed through a 0.45 μm filter to collect the filtrate; after the fifth centrifugation, the obtained solution is passed through a 0.22 μm filter to collect the filtrate.

[0016] Preferably, the acid-koumiss-derived exosomes have a concentration of 1-20 mg / mL in the phosphate buffer;

[0017] The silk peptide has a concentration of 1-20 mg / mL in the phosphate buffer;

[0018] The surfactant is sorbitan laurate, and the volume fraction of the surfactant in the phosphate buffer is 0.1-10%.

[0019] Preferably, the self-assembly temperature of the beta-fold is 4-37 DEG C, and the time is 6-8 h.

[0020] The oscillation rate of the self-assembly of the beta-fold is 300-800 rpm.

[0021] Preferably, the width of the nanometer drug-loaded fiber microcrystal is 20-40 nm, and the length is 150-800 nm.

[0022] Preferably, the mass concentration of the GelMa solution is 5-15%.

[0023] The concentration of the nanometer drug-loaded fiber microcrystal in the nanometer drug-loaded fiber microcrystal solution is 10-20 mg / mL.

[0024] The volume of the nanometer drug-loaded fiber microcrystal solution is 0.1-20% of the volume of the GelMa solution.

[0025] Preferably, the parameters of the 3D printing include that the printing needle size is 0.4 mm, the printing speed is 1.2 mm / s, the air pressure is 0.15 MPa, the linear filling, and the corner is 90 degrees.

[0026] The application provides a ROS regulated hydrogel loaded with acid koumiss derived exosomes prepared by the preparation method.

[0027] The nanometer drug-loaded fiber microcrystal comprises acid koumiss derived exosomes and beta-folded silk fibroin peptides wrapping the acid koumiss derived exosomes.

[0028] The application provides an application of the ROS regulated hydrogel loaded with acid koumiss derived exosomes in preparing an anti-burn drug.

[0029] The application provides a preparation method of ROS-regulated hydrogel loaded with acid koumiss-derived exosomes, comprising the following steps: gradient centrifugation of acid koumiss to obtain acid koumiss-derived exosomes; mixing the acid koumiss-derived exosomes, silk fibroin peptides, a surfactant and a phosphate buffer, and performing beta-sheet self-assembly to obtain a nano drug-loaded fibrous microcrystal solution; the silk fibroin peptide has the sequence shown in SEQ ID No. 1; mixing the nano drug-loaded fibrous microcrystal solution with a GelMa solution, and performing 3D printing to obtain the ROS-regulated hydrogel loaded with acid koumiss-derived exosomes. Koumiss is a grassland health drink with high nutritional value and strong Mongolian characteristics, and is divided into raw milk and cooked milk. Raw milk refers to fresh horse milk, which is sweet and cool in nature, good at clearing heat in the gallbladder and stomach, and can treat throat and tooth diseases, and has the effects of tonifying deficiency, strengthening the body, moisturizing the skin, clearing heat and quenching thirst. Cooked milk refers to dairy products formed by natural fermentation of fresh horse milk by microorganisms such as lactic acid bacteria and yeast, which are used as a dietary therapy guided by tonifying theory in medical monographs such as Four Parts of Nectar, Mongolian Medicine Selection and Observer's Joy. Acid koumiss-derived exosomes (K-Exo) have anti-inflammatory effects, can promote the secretion of anti-inflammatory factor IL-10, and play the function of M2 macrophages, and can actively regulate the effect of inflammatory cells and ROS response. The repeated sequence of silk fibroin peptide (GAGAGSGA) 2 can be assembled into beta-sheet in the phosphate buffer, and the beta-sheet is orderly arranged and self-assembled into fibrous nano microcrystals. The nano beta-nanofiber microcrystals self-assembled by silk fibroin peptides can be oxidized by ROS to form irregularly coiled structures dissolved in water. The silk fibroin peptides in the application can encapsulate K-Exo, and the obtained nano drug-loaded fibrous microcrystals can be used as a ROS-responsive nano drug-loaded sustained-release system for K-Exo. When the microenvironment of the burn site highly expresses ROS, the silk fibroin peptide nano microcrystals are dissolved, and K-Exo is released, which can effectively reduce the ROS content and inflammatory cell infiltration at the damage site, and accelerate the rapid healing of the skin at the burn site. The application takes advantage of Mongolian medicine to load K-Exo into GelMa hydrogel, and through 3D printing technology, the shape of the hydrogel can be customized according to the different shapes and depths of skin damage. The hydrogel obtained by the application has broad-spectrum antioxidant and anti-inflammatory biological effects, and shows the functions of actively regulating the immune system, ROS response, promoting cell migration, nerve regeneration and blood vessel regeneration, so as to improve the repair effect of large-area burn non-healing wounds.

[0030] Meanwhile, the preparation method provided by the application is simple to operate and low in cost, and is conducive to realizing industrialized mass production. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a transmission electron microscope image of acid koumiss-derived exosomes;

[0032] Figure 2A particle size distribution diagram of the acid kumiss-derived exosomes;

[0033] Figure 3 A western blot (WB) detection diagram of the acid kumiss-derived exosomes;

[0034] Figure 4 A microstructure diagram of the nanometer drug-loaded fiber microcrystals, wherein A and C are microstructure diagrams at different magnifications, and B and D are partial enlarged views of A and C;

[0035] Figure 5 A physical diagram of GelMa@beta-C-K-Exo;

[0036] Figure 6 A cell compatibility test result of the GelMa@beta-C-K-Exo extraction solution;

[0037] Figure 7 The ability of GelMa@beta-C-K-Exo to scavenge free radicals;

[0038] Figure 8 The ability of GelMa@beta-C-K-Exo to scavenge intracellular reactive oxygen species, wherein A is a microscopic photograph of the positive control group, the negative control group and the extraction solution-72h scavenge intracellular reactive oxygen species, and B is a fluorescence intensity statistical diagram of the positive control group, the negative control group and the extraction solution-72h scavenge intracellular reactive oxygen species;

[0039] Figure 9 The ability of GelMa@beta-C-K-Exo to regulate inflammatory cells;

[0040] Figure 10 A wound healing rate statistical diagram at 3, 7, 10, 14, 17, 20, 23 and 27 days after surgery. DETAILED DESCRIPTION

[0041] The application provides a preparation method of a ROS-regulated hydrogel loaded with acid kumiss-derived exosomes, comprising the following steps:

[0042] The acid kumiss is subjected to gradient centrifugation to obtain acid kumiss-derived exosomes;

[0043] The acid kumiss-derived exosomes, the silk fibroin peptide, the surfactant and the phosphate buffer solution are mixed to perform beta-sheet self-assembly to obtain a nanometer drug-loaded fiber microcrystal solution; the silk fibroin peptide has the sequence shown in SEQ ID No. 1;

[0044] The nanometer drug-loaded fiber microcrystal solution and the GelMa solution are mixed to perform 3D printing to obtain the ROS-regulated hydrogel loaded with the acid kumiss-derived exosomes.

[0045] The acid kumiss is subjected to gradient centrifugation to obtain acid kumiss-derived exosomes. In the present application, the gradient centrifugation preferably comprises:

[0046] first centrifugation at 3000 rpm for 10 min, second centrifugation at 5000 rpm for 30 min, third centrifugation at 12000 rpm for 60 min, filtration paper; fourth centrifugation at 35000 rpm for 60 min, filtration of the filtrate through a 0.45 μm filter; fifth centrifugation at 70000 rpm for 60 min, filtration of the filtrate through a 0.22 μm filter; and sixth centrifugation at 110000 rpm for 80 min, collection of the precipitate.

[0047] In the present application, after the collection of the precipitate, the obtained precipitate is preferably washed with PBS buffer, and the number of washing is preferably 2 times. After the washing, the acid kumiss-derived exosomes obtained in the present application are preferably stored at -80 ℃ for standby use.

[0048] In the present application, the particle size of the acid kumiss-derived exosomes is preferably 30-150 nm, more preferably 50-120 nm, and further preferably 80-100 nm.

[0049] After obtaining the acid kumiss-derived exosomes, the acid kumiss-derived exosomes, silk fibroin peptides, surfactants and phosphate buffer are mixed to perform β-sheet self-assembly to obtain a nanometer drug-loaded fibrous microcrystal solution. In the present application, the silk fibroin peptide has the sequence shown in SEQ ID No. 1, and the SEQ ID No. 1 is specifically: GAGAGSGAGAGAGSGA, which is abbreviated as (GAGAGSGA)2. The nanometer fibrous microcrystal formed by the silk fibroin peptide (GAGAGSGA)2 is longer and wider than the silk fibroin peptide (GAGSGA)2 (having the sequence shown in SEQ ID No. 2, and the SEQ ID No. 2 is specifically GAGSGAGAGSGA), and is more stable than the silk fibroin peptide (GAGSGA)2, so a higher concentration of ROS is required to change its β-sheet structure into a random coil structure. A large area burn refers to a burn area greater than 30% of the total skin, and there is a high concentration of ROS at the burn wound site, which hinders the healing of the skin wound. This high concentration of ROS environment can change the silk fibroin peptide (GAGAGSGA)2 β-sheet self-assembled nanometer drug-loaded fibrous microcrystal from a fibrous crystal insoluble state to a dissolved state, thereby releasing K-Exo and clearing the high concentration of ROS in the large area burn wound. Therefore, the high concentration of ROS-responsive nanometer fibrous microcrystal formed by the silk fibroin peptide (GAGAGSGA)2 is suitable for rapid repair of large area burn wound damage.

[0050] In the present application, the surface active agent is preferably sorbitan laurate, and the volume fraction of the surface active agent in the phosphate buffer is preferably 0.1-10%, more preferably 1-5%. In the present application, the surface active agent promotes the self-assembly of the silk fibroin peptide (GAGSGA)2β-sheet.

[0051] In the present application, the pH value of the phosphate buffer is preferably 7.0. In the present application, the mixing method is preferably to dissolve the silk fibroin peptide in the phosphate buffer first, and then add the acid mare's milk-derived exosome and the surface active agent. In the present application, the concentration of the silk fibroin peptide in the phosphate buffer is preferably 1-20 mg / mL, more preferably 5-15 mg / mL, and further preferably 10 mg / mL; and the concentration of the acid mare's milk-derived exosome in the phosphate buffer is preferably 1-20 mg / mL, more preferably 10-20 mg / mL.

[0052] In the present application, the β-sheet self-assembly is preferably carried out under shaking conditions, and the temperature of the β-sheet self-assembly is preferably 2-10℃, and the time is preferably 6-8 h; the shaking rate of the β-sheet self-assembly is 300-800 rpm, more preferably 600 rpm. In the present application, the width of the nano-drug-loaded fibrous microcrystal is preferably 20-40 nm, more preferably 20-30 nm, and the length is preferably 150-800 nm, more preferably 150-500 nm, and more preferably 200-400 nm. After obtaining the nano-drug-loaded fibrous microcrystal, the present application preferably stores it at 4℃ for standby use. In the present application, the concentration of the nano-drug-loaded fibrous microcrystal in the nano-drug-loaded fibrous microcrystal solution is preferably 10-20 mg / mL, more preferably 15-20 mg / mL.

[0053] After obtaining the nano-drug-loaded fibrous microcrystal, the present application mixes the nano-drug-loaded fibrous microcrystal solution with the GelMa solution to carry out 3D printing, thereby obtaining the ROS-regulated hydrogel loaded with acid mare's milk-derived exosomes. In the present application, the mass concentration of the GelMa solution is preferably 5-15%, more preferably 5-10%, and the volume of the nano-drug-loaded fibrous microcrystal solution is preferably 0.1-20% of the volume of the GelMa solution, more preferably 1-15%, and further preferably 5-10%. The present application does not have special requirements for the mixing method, and any mixing method known to those skilled in the art can be used, such as stirring mixing.

[0054] In the present application, the parameters of the 3D printing preferably include: the printing needle size is 0.4 mm, the printing speed is 1.2 mm / s, the air pressure is 0.15 MPa, the linear filling, and the corner angle is 90 degrees.

[0055] The application provides a ROS-regulated hydrogel loaded with acid-koumiss-derived exosomes prepared by the preparation method, and the ROS-regulated hydrogel comprises a hydrogel matrix and nanometer drug-loaded fiber microcrystals loaded in the hydrogel matrix.

[0056] The nanometer drug-loaded fiber microcrystals comprise acid-koumiss-derived exosomes and beta-folded silk fibroin peptides wrapping the acid-koumiss-derived exosomes.

[0057] The application provides an application of the ROS-regulated hydrogel loaded with acid-koumiss-derived exosomes in preparation of an anti-burn medicament.

[0058] The ROS-regulated hydrogel loaded with acid-koumiss-derived exosomes, the preparation method and the application in preparation of an anti-burn medicament are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0059] Example 1

[0060] (1) 50 mL of acid koumiss (purchased from Inner Mongolia Masu Dairy Co., Ltd.) was taken into a centrifuge tube, and gradient centrifugation was performed, and the method was as follows:

[0061] 3000 rpm centrifugation for 10 min, 5000 rpm centrifugation for 30 min, 12000 rpm centrifugation for 60 min, 35000 rpm centrifugation for 60 min, 70000 rpm centrifugation for 60 min, 110000 rpm centrifugation for 80 min, and the precipitate after low-temperature centrifugation was gently washed with PBS for 2 times. The solution prepared at 12000 rpm was filtered through filter paper, the solution prepared at 35000 rpm was filtered through a 0.45 μm filter to collect the filtrate, the solution prepared at 70000 rpm was filtered through a 0.22 μm filter to collect the filtrate, and acid koumiss-derived exosomes (K-Exo) were obtained, which were stored at-80℃ after sub-packaging.

[0062] (2) Silk fibroin peptides (GAGAGSGA) 2 were dissolved in a phosphate buffer (PBS) to obtain a 2wt% silk fibroin peptide solution, K-Exo (concentration of 20 mg / mL) and dodecanoic acid sorbitol ester (volume fraction of 1%) were added to the silk fibroin peptide solution, and the ROS-responsive nanometer drug-loaded fiber microcrystals (beta-C-K-Exo) were prepared by shaking at 4℃ and 600 rpm for 6 h, and were stored at 4℃ for standby.

[0063] (3) 10 mL of a 5 wt% GelMa solution was mixed with 1 mL of a 10 mg / mL β-C-K-Exo solution, and a hydrogel was prepared using 3D printing technology. The 3D printing parameters were as follows: a printing needle size of 0.4 mm, a printing speed of 1.2 mm / s, an air pressure of 0.15 MPa, linear filling, and an angle of 90 degrees. A ROS-regulated hydrogel loaded with acid-kumiss-derived exosomes was obtained, and was named GelMa@β-C-K-Exo.

[0064] Structural characterization

[0065] (1) The acid-kumiss-derived exosomes K-Exo obtained in Example 1 were negatively stained with 3% phosphotungstic acid, and the submicrostructure of K-Exo was observed using a tungsten filament transmission electron microscope. The transmission electron microscope image of the acid-kumiss-derived exosomes is shown in Figure 1 It can be seen that the size of the exosomes is about 150 nm.

[0066] (2) The particle size and distribution of the extracted acid-kumiss-derived exosomes were analyzed using a particle size distribution / zeta potential instrument. The particle size distribution graph is shown in Figure 2 It can be seen that the particle size distribution of the acid-kumiss-derived exosomes is between 30 nm and 150 nm.

[0067] (3) Western blotting (WB) was used to detect the specific membrane proteins CD81, Alix, and TSG101 on the surface of K-Exo membranes. The results are shown in Figure 3 The specific membrane proteins CD81, Alix, and TSG101 on the surface of K-Exo membranes were positive, indicating that the vesicles separated by the application were exosomes.

[0068] (4) The microstructure of the nanometer drug-loaded fiber microcrystals (β-C-K-Exo) obtained in Example 1 is shown in Figure 4

[0069] Figure 4 In the figure, (A) and (C) are microstructure images at different magnifications, and (B) and (D) are partial enlarged views of (A) and (C). As can be seen from Figure 4 The length of the nanometer drug-loaded fiber microcrystals (β-C-K-Exo) is about 200-400 nm, and the width is about 20-30 nm.

[0070] (5) The actual photo of GelMa@β-C-K-Exo in Example 1 is shown in Figure 5

[0071] Performance test

[0072] ​​(1) The cell compatibility of the GelMa@β-C-K-Exo extract was evaluated by using a CCK-8 detection kit. Specifically, 1 g of GelMa@β-C-K-Exo hydrogel was immersed in 10 mL of cell culture solution DMEM, and the cell culture solution was taken out at 24, 48 and 72 h under the condition of 37°C, and was named as extract-24, extract-48 and extract-72, respectively. The cell culture solution DMEM was used as a control group. The results are shown in Figure 6 Figure 6 It can be seen that the GelMa@β-C-K-Exo extract has a proliferation effect on cells for 24, 48 and 72 h, and has a significant difference from the control group. It shows that the prepared GelMa@β-C-K-Exo has good cell compatibility.

[0073] (2) In vitro study on the broad-spectrum antioxidant and anti-inflammatory biological effects of GelMa@β-C-K-Exo

[0074] The hydrogel GelMa@β-C-K-Exo was incubated with hydroxyl radicals (OH), hydrogen peroxide (H2O2) and superoxide anion radicals (O2 - ) respectively, and the ability of the hydrogel to scavenge OH, H2O2 and O2 - was evaluated by using detection OH, H2O2 and O2 - kits for staining, respectively.

[0075] Specifically, NIH 3T3 cells were inoculated into a 6-well plate pretreated with 0.1% gelatin for 2 h at a density of 5×10 5 cells per well, and were cultured at 37°C overnight. Mouse embryonic fibroblasts NIH 3T3 were stimulated by hydrogen peroxide (100 μM) to express excess ROS in cells. The extract-72 accounting for 10% of the cell culture solution was added and incubated at 37°C for 20 min. The ability of the GelMa@β-C-K-Exo extract to scavenge intracellular ROS was analyzed by using intracellular ROS live cell staining technology. The ROS-sensitive fluorescent probe for detecting the content of ROS in living cells was 2',7'-dichlorofluorescein diacetate (DCFH-DA), and the detection concentration was 10 μM. After staining, the cells were washed twice with serum-free medium, observed and imaged by using a confocal microscope (Nikon A1, Japan), and then the green ROS fluorescence intensity in the cells was quantitatively analyzed by using NIS Element AR software (version 5.30.03), and statistical analysis was performed. Cells without H2O2 were used as a control group.

[0076] The ability of GelMa@β-C-K-Exo to scavenge free radicals is shown in Figure 7 ​As shown, GelMa@β-CK-Exo has a •OH scavenging ability of around 60%, and it also scavenges O2. - The ability to remove H2O2 is around 40%, and the ability to remove H2O2 is around 50%.

[0077] The ability of GelMa@β-CK-Exo extract to scavenge intracellular reactive oxygen species for 72 hours is as follows: Figure 8 As shown, A represents photomicrographs of the positive control group, negative control group, and the extract after 72 hours of scavenging intracellular reactive oxygen species (ROS). B represents a statistical graph of the fluorescence intensity of the positive control group, negative control group, and the extract after 72 hours of scavenging intracellular ROS. It can be seen that exogenous H2O2 stimulation of NIH 3T3 cells resulted in high ROS expression. The GelMa@β-CK-Exo extract after 72 hours significantly reduced ROS expression in NIH 3T3 cells compared to the positive control group, but showed no significant difference compared to the negative control group. This indicates that the GelMa@β-CK-Exo hydrogel effectively removes overexpressed ROS while retaining the reactive oxygen species necessary for normal cell growth.

[0078] (3) ROS (H2O2 represents ROS) responsiveness of drug-loaded nanofibers

[0079] The specific method is as follows: Prepare nano-drug-loaded microcrystals formed by the self-assembly of silk fibroin peptide (GAGAGS)2 in accordance with step (2) of Example 1. The difference is that silk fibroin peptide (GAGAGSGA)2 is replaced with silk fibroin peptide (GAGAGS)2, while the other parameters remain unchanged.

[0080] Different concentrations of H2O2 (1 µM, 10 µM, 100 µM, 1 mM, 10 mM, and 100 mM) were prepared. Drug-loaded nanocrystals (formed by self-assembly of (GAGAGS)2) and drug-loaded fiber nanocrystals (formed by self-assembly of (GAGAGSGA)2) were reacted with these different concentrations of H2O2 for 30 min, respectively. Then, thioflavin T (final concentration 10 µM) was added under light-protected conditions and reacted for another 10 min. The samples were detected using a microplate reader (Thermo Scientific, USA) with an excitation wavelength of approximately 440 nm. If an absorption peak was observed near the emission wavelength of 490 nm, it indicated that the drug-loaded nanocrystals were insoluble in H2O2 at that concentration and did not exhibit ROS responsiveness; if no absorption peak was observed near the emission wavelength of 490 nm, it indicated that the drug-loaded nanocrystals were soluble in H2O2 at that concentration and exhibited ROS responsiveness. The experimental results are shown in Table 1.

[0081] Table 1. ROS responsiveness of drug-loaded nanocrystals

[0082]

[0083] The results in Table 1 show that the hydrogen peroxide response concentration of the nanocrystalline formed by silk fibroin peptide (GAGAGS) 2 is above 100 µM, while the hydrogen peroxide response concentration of the nanocrystalline formed by silk fibroin peptide (GAGAGSGA) 2 is above 1 mM, indicating that the nanocrystalline formed by silk fibroin peptide (GAGAGSGA) 2 has ROS responsiveness only at high concentrations of ROS.

[0084] (4) Ability of GelMa@β-C-K-Exo to regulate inflammatory cells

[0085] The mouse monocyte macrophage leukemia cell (Raw264.7) cells were stimulated with lipopolysaccharide (LPS) to highly express cell inflammatory factors such as IL-6, PGE2 and TNF-α. Specifically, Raw264.7 cells were seeded into 6-well plates pretreated with 0.1% gelatin for 2 h at a density of 5×10 5 cells per well and incubated at 37 °C overnight. The RAW264.7 cells were stimulated with a DMEM solution containing LPS (100 ng / mL, purchased from Sigma Company) for 24 h. The extracted solution-72 of GelMa@β-C-K-Exo accounting for 10% of the cell culture solution was co-cultured with the LPS-stimulated Raw264.7 cells at 37 °C, and the cell supernatant was collected. The concentration of IL-6, PGE2 and TNF-α was detected according to the instructions of the ELISA kit (purchased from Sigma Company), and the effect of the extracted solution of the hydrogel dressing on the polarization of Raw264.7 cells and the expression amount of IL-6, PGE2 and TNF-α was evaluated. The positive control group was stimulated with LPS, and the extracted solution-72 was replaced with an equal volume of PBS. The negative control group was not stimulated with LPS, and no extracted solution-72 was added.

[0086] The ability of GelMa@β-C-K-Exo to regulate inflammatory cells is shown in Figure 9 It can be seen that the hydrogel extracted solution of GelMa@β-C-K-Exo (experimental group) can inhibit the high expression of PGE2, TNF-α and IL-6 inflammatory related cytokines stimulated by LPS, and has a significant difference compared with the positive control group.

[0087] (5) Evaluation of the repair effect of GelMa@β-C-K-Exo on deep Ⅱ degree burn skin damage model

[0088] On the basis of the research of the cell biology effect, the preparation process of GelMa@β-C-K-Exo is further optimized and designed, and the repair effect of GelMa@β-C-K-Exo on the deep Ⅱ degree burn skin damage model is evaluated. Adult male SD rats are selected, anesthetized, and the back is shaved. A 30% deep burn is made on the back of the total body surface area. The rats are randomly divided into 4 groups: burn group, K-Exo group, GelMa group and GelMa@β-C-K-Exo group (burn group: no treatment, sterile gauze dressing; K-Exo group: 5 mL of K-Exo with a concentration of 10 mg / mL, dropped on sterile gauze; GelMa group: 5 mL of GelMa hydrogel with a concentration of 5%; GelMa@β-C-K-Exo group: 5 mL of GelMa@β-C-K-Exo hydrogel), 30 rats in each group; after treatment, the wound healing is observed on days 3, 7, 10, 14, 17, 20, 23 and 27.

[0089] The wound healing rate statistics chart on days 3, 7, 10, 14, 17, 20, 23 and 27 after operation is shown in Figure 10 As can be seen from the chart, the treatment effect of the GelMa@β-C-K-Exo hydrogel group is the best on days 3, 7, 10, 14, 17, 20, 23 and 27 after operation.

[0090] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a ROS-regulated hydrogel loaded with exosomes derived from fermented mare's milk, characterized in that, Includes the following steps: Gradient centrifugation was performed on fermented mare's milk to obtain exosomes derived from fermented mare's milk; The exosomes derived from fermented mare's milk, silk fibroin peptides, surfactants, and phosphate buffer were mixed and subjected to β-sheet self-assembly to obtain a solution of drug-loaded nanofibers; the sequence of the silk fibroin peptides is shown in SEQ ID No.

1. The nano-drug-loaded fiber microcrystal solution was mixed with GelMa solution and 3D printed to obtain a ROS-regulated hydrogel loaded with exosomes derived from mare's milk. The particle size of the exosomes derived from fermented mare's milk is 30~150 nm; The surfactant is sorbitol dodecanoate, and the volume fraction of the surfactant in the phosphate buffer is 0.1-10%. The β-sheet self-assembly temperature is 4~37 ℃ and the time is 6~8 h; The oscillation rate of the β-fold self-assembly is 300~800 rpm; The width of the drug-loaded nanofibers is 20-40 nm and the length is 150-800 nm.

2. The preparation method according to claim 1, characterized in that, The gradient centrifugation includes: Centrifuge sequentially at 3000 rpm for 10 min, 5000 rpm for 30 min, 12000 rpm for 60 min, 35000 rpm for 60 min, 70000 rpm for 60 min, and 110000 rpm for 80 min, and collect the precipitate.

3. The preparation method according to claim 2, characterized in that, It also includes filtering the solution after the third centrifugation with filter paper; after the fourth centrifugation, the resulting solution is filtered through a 0.45 μm filter and the filtrate is collected; after the fifth centrifugation, the resulting solution is filtered through a 0.22 μm filter and the filtrate is collected.

4. The preparation method according to claim 1, characterized in that, The concentration of the exosomes derived from fermented mare's milk in phosphate buffer was 1-20 mg / mL; The concentration of the silk fibroin peptide in phosphate buffer is 1~20 mg / mL.

5. The preparation method according to claim 1, characterized in that, The mass concentration of the GelMa solution is 5-15%; The concentration of the drug-loaded fiber microcrystals in the solution is 10~20 mg / mL; The volume of the drug-loaded nanofiber microcrystal solution is 0.1~20% of the volume of the GelMa solution.

6. The preparation method according to claim 1, characterized in that, The 3D printing parameters include: a printing needle size of 0.4 mm, a printing speed of 1.2 mm / s, an air pressure of 0.15 MPa, linear infill, and a 90-degree rotation angle.

7. The ROS-regulated hydrogel loaded with exosomes derived from fermented mare's milk, prepared by the method according to any one of claims 1 to 6, is characterized in that... It includes a hydrogel matrix and drug-loaded nanofibers loaded in the hydrogel matrix; The drug-loaded nanofibers include exosomes derived from fermented mare's milk and β-sheeted silk peptides that encapsulate the exosomes derived from fermented mare's milk.

8. The application of the ROS-regulated hydrogel loaded with exosomes derived from mare's milk as described in claim 7 in the preparation of anti-burn drugs.

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