Silk fibroin hydrogel loaded with ginger extracellular vesicles and preparation method and application thereof

Abstract

The present invention provides a silk fibroin hydrogel carrying ginger exovesicles and a preparation method and application thereof, the preparation method comprising: adding hydrogen peroxide and horseradish peroxidase to a silk fibroin solution, and mixing thoroughly; then adding the ginger exovesicle solution to the silk fibroin solution to obtain a ginger exovesicle-silk fibroin mixed solution; standing to obtain a silk fibroin hydrogel carrying ginger exovesicles. The silk fibroin hydrogel carrying ginger exovesicles provided by the present invention can promote the M2 polarization of macrophages, is beneficial to the cartilage differentiation of bone marrow mesenchymal stem cells, and achieves the purpose of repairing cartilage damage.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to a silk fibroin hydrogel carrying ginger extracellular vesicles, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis is a common degenerative joint disease characterized by articular cartilage degeneration and is one of the main causes of disability in adults worldwide. According to statistics, more than 500 million people suffer from osteoarthritis worldwide, which brings huge economic burden to patients and society. Currently commonly used cartilage repair strategies, including microfracture, cartilage autotransplantation, cartilage allotransplantation, etc., are widely used in clinical practice, but they all have certain limitations and shortcomings. For example, microfracture may lead to poor defect filling and cartilage fibrosis; cartilage autotransplantation may have the problem of donor graft incompatibility due to the limitation of donor area; cartilage allotransplantation has the risk of immune rejection and infection, and it is difficult to match allogeneic tissue with autologous cartilage, which may lead to unbalanced biomechanical load and decreased joint bearing capacity. Therefore, new technologies and means are urgently needed to solve the problem of cartilage repair, and the development of new treatment strategies is crucial to solving clinical osteoarthritis-related problems.

[0003] The human immune regulation of osteoarthritis mainly activates macrophages, removes cell debris through the phagocytosis of macrophages, produces cytokines, proteases and reactive oxygen species, and forms an inflammatory microenvironment. Therefore, intervening in the immune regulation of macrophages and the inflammatory microenvironment is an effective means of treating osteoarthritis. Macrophages are a kind of natural immune cells with phagocytic function in the human body. They are mainly involved in the occurrence and development of various inflammatory pathologies and play an important role in the physiological environment of the human body. The role of macrophages is also applied to osteoarthritis. Macrophages are stimulated by specific pathogenic factors at various stages of inflammation and polarized into different subtypes (M1 macrophages or M2 macrophages), thereby secreting different products for subsequent reactions. M1 macrophages release a large number of proinflammatory cytokines, accelerate the catabolism of chondrocytes, and promote the development of osteoarthritis. On the contrary, M2 macrophages can secrete anti-inflammatory cytokines, effectively weaken the inflammatory response, protect chondrocytes, and alleviate the symptoms of osteoarthritis.

[0004] In recent years, exosomes have been widely used in osteoarthritis as an advanced cell-free treatment. Exosomes are a type of extracellular vesicles secreted by mother cells with a diameter of 100-150 nanometers. They have a phospholipid bilayer-based biological membrane structure and cytoplasmic components similar to mother cells, which enables them to exist stably in vivo. Previous studies have shown that the various bioactive components carried by exosomes (such as proteins, nucleic acids, and small molecule metabolites) can play an important role in the treatment of osteoarthritis by activating signal cascades in receptor cells. However, ginger exosomes still have many shortcomings, such as short joint residence time and low bioavailability, which may accelerate the clearance of ginger exosomes in the joint cavity.

[0005] Functional biomaterials, silk fibroin (SF) hydrogel, as a biomaterial based on natural biomacromolecules, has good biocompatibility and biodegradability, and can achieve long-term stable sustained release of extracellular vesicles. As a carrier of extracellular vesicles, it is used to improve its local delivery efficiency and has become a potential regenerative medicine strategy. Summary of the invention

[0006] In view of the defects of the prior art, the object of the present invention is to provide a silk fibroin hydrogel loaded with ginger extracellular vesicles and a preparation method and application thereof.

[0007] The technical solution of the present invention is: The first aspect of the present invention provides a method for preparing a silk fibroin hydrogel loaded with ginger extracellular vesicles, the preparation method comprising: Hydrogen peroxide and horseradish peroxidase are added to the silk fibroin solution and fully mixed; then the ginger exovesicle solution is added to the silk fibroin solution to obtain a ginger exovesicle-silk fibroin mixed solution; and the solution is allowed to stand to obtain a silk fibroin hydrogel loaded with ginger exovesicles.

[0008] The preparation method of the ginger exocyst comprises: Fresh ginger was washed, chopped, soaked in phosphate buffer, and ground to obtain ginger juice; the ginger juice was centrifuged at 1000 × g for 10 min, 3000 × g for 20 min, and 1 × 10 4 × g centrifuge for 40 min; take the supernatant and use an ultra-high speed centrifuge at 1.5 × 10 5 The pellet was centrifuged at 400 × g for 2 h, the supernatant was removed, and the precipitate was resuspended in phosphate buffer and transferred to 8 wt%, 15 wt%, 30 wt%, 45 wt%, and 60 wt% sucrose density gradient solutions at 1.5 × 10 5 × g for 2 h, and the bands between the 8%, 30%, and 45% layers were collected at 4°C, 1.5 × 10 5× g for 1 h to obtain ginger exosomes, which were redispersed in phosphate buffer and stored at −80° C. for subsequent use.

[0009] The preparation method of the silk fibroin solution comprises: Cut the silk cocoons into pieces and add them into boiling Na2CO3 solution to separate silk fibroin and sericin. Wash and dry the extracted silk fibroin and dissolve it in LiBr solution. After dissolution, dialyze it in deionized water for 2 days, changing the water 3-4 times a day. After dialysis, take out the silk fibroin solution and centrifuge it at 4 ℃, 8000-10000 rpm for 5-60 min. The supernatant is the silk fibroin protein solution.

[0010] The concentration of the silk fibroin solution is 1%-20% (w / v).

[0011] The horseradish peroxidase activity concentration is 1-100 U / mL, preferably 10-50 U / mL.

[0012] The concentration of hydrogen peroxide is 0.001%-1% (w / v), preferably 0.01%-0.1% (w / v).

[0013] The second aspect of the present invention provides a silk fibroin hydrogel loaded with ginger extracellular vesicles, and the preparation method of the silk fibroin hydrogel loaded with ginger extracellular vesicles is as described above.

[0014] The third aspect of the present invention provides the use of silk fibroin hydrogel loaded with ginger extracellular vesicles in the preparation of a product for treating osteoarthritis. The silk fibroin hydrogel loaded with ginger extracellular vesicles is as described above.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The silk fibroin hydrogel loaded with ginger exovesicles provided by the present invention, the presence of ginger exovesicles will induce the cross-linking process to form a hydrogel with a smaller internal pore size and a tighter three-dimensional network structure. This tight three-dimensional network structure can achieve the purpose of sustained release of ginger exovesicles, increase the joint residence time of ginger exovesicles, and improve bioavailability.

[0016] 2. The silk fibroin hydrogel loaded with ginger exosomes provided by the present invention can promote the polarization of macrophages to M2 macrophages to obtain M2 macrophages. The exosomes secreted by M2 macrophages can significantly promote the migration of bone marrow mesenchymal stem cells, with a migration rate of up to 74%; the exosomes secreted by M2 macrophages can promote the chondrogenic differentiation of bone marrow mesenchymal stem cells, thereby achieving the purpose of repairing cartilage damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 An electron microscope image of ginger exocysts provided in an embodiment of the present invention; Figure 2 A graph showing the particle size detection results of ginger exosomes provided in an embodiment of the present invention; Figure 3 Electron micrographs of the silk fibroin hydrogel and the silk fibroin hydrogel loaded with ginger extracellular vesicles provided in the embodiments of the present invention (SF-Gel-GEVs: silk fibroin hydrogel loaded with ginger extracellular vesicles; SF-Gel: silk fibroin hydrogel); Figure 4 A distribution diagram of ginger extracellular vesicles in a silk fibroin hydrogel carrying ginger extracellular vesicles provided in an embodiment of the present invention (SF-Gel-GEVs: silk fibroin hydrogel carrying ginger extracellular vesicles; GEVs: ginger extracellular vesicles); Figure 5 Schematic diagram of the results of ginger extracellular vesicles promoting macrophage polarization to M2 macrophages provided in the first experimental example of the present invention (M0+LPS: macrophages+lipopolysaccharide; M0+LPS+GEVs: macrophages+lipopolysaccharide+ginger extracellular vesicles); Figure 6 A schematic diagram of the results of exosomes secreted by M2 macrophages promoting the migration of bone marrow mesenchymal stem cells provided in the second experimental example of the present invention (M2-Exos: experimental group with added M2 macrophage exosomes; Control: blank control group without added M2 macrophage exosomes); Figure 7 Schematic diagram of the results of exosomes secreted by M2 macrophages promoting chondrogenic differentiation of bone marrow mesenchymal stem cells provided in the third experimental example of the present invention (M2-Exos: experimental group with added M2 macrophage exosomes; Control: blank control group without added M2 macrophage exosomes). DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to specific embodiments.

[0019] The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

[0020] Example This embodiment provides a silk fibroin hydrogel carrying ginger extracellular vesicles. The preparation method of the silk fibroin hydrogel carrying ginger extracellular vesicles comprises the following steps: 1) Preparation of ginger extracellular vesicles: Take fresh ginger, wash it thoroughly with deionized water, chop it, soak it in phosphate buffer, and grind it in a blender to obtain ginger juice; the ginger juice was successively heated at 4 °C at 1 × 10 3 × g for 10 min, 3000 × g for 20 min and 1×10 4 × g centrifuge for 40 min; take the supernatant and use an ultra-high speed centrifuge at 1.5 × 10 5 The pellet was centrifuged at 400 × g for 2 h, the supernatant was removed, and the precipitate was resuspended in phosphate buffer and transferred to 8 wt%, 15 wt%, 30 wt%, 45 wt%, and 60 wt% sucrose density gradient solutions at 1.5 × 10 5 × g for 2 h, and the bands between the 8%, 30%, and 45% layers were collected and heated at 4°C for 1.5 × 10 5 × g for 1 h to obtain ginger exosomes, which were redispersed in phosphate buffer and stored at −80 °C for subsequent use.

[0021] The ginger exosomes prepared in this example are as follows Figure 1 As shown in the transmission electron microscope, the ginger extracellular vesicles present a relatively uniform and regular spherical vesicle structure; the particle size distribution NTA results are as follows Figure 2 As shown, NTA showed that the average particle size of ginger extracellular vesicles was 107.3 nm.

[0022] 2) Preparation of silk fibroin solution: The silk cocoons were cut into pieces and added to a boiling Na2CO3 solution to separate silk fibroin and sericin. The extracted silk fibroin was washed and dried and then dissolved in a LiBr solution. After dissolution, it was dialyzed in deionized water for 2 days, with the water changed 3-4 times a day. After dialysis, the silk fibroin solution was taken out and centrifuged at 4°C, 8000-10000 rpm for 5-60 min to remove impurities in the silk fibroin solution. The silk fibroin solution was concentrated by drying and the concentration was determined to be 4% (w / v).

[0023] 3) Preparation of silk fibroin hydrogel loaded with ginger extracellular vesicles: Silk protein molecules contain tyrosine residues. Horseradish peroxidase can catalyze the oxidation of tyrosine residues in the presence of hydrogen peroxide. The active center of horseradish peroxidase contains an iron porphyrin cofactor. Under the action of hydrogen peroxide, iron ions are converted from Fe 3⁺ Oxidized to Fe 4⁺, forming a high-valent iron intermediate with strong oxidizing properties. The high-valent iron intermediate can oxidize tyrosine residues into dityrosine, which are connected by covalent bonds. This cross-linking effect makes the originally loose silk protein molecules connect to each other to form a three-dimensional network structure, thereby achieving gelation.

[0024] The ginger extracellular vesicles obtained in step 1) are fluorescently labeled with DiD fluorescent dye, and ginger extracellular vesicles-loaded silk fibroin hydrogel (SF-Gel-GEVs) are prepared using the ginger extracellular vesicles labeled with DiD.

[0025] Hydrogen peroxide and horseradish peroxidase are added to the silk fibroin solution and mixed thoroughly; then a ginger extracellular vesicle solution (containing ginger extracellular vesicles labeled with DiD) is added to the silk fibroin solution to obtain a ginger extracellular vesicle-silk fibroin mixed solution; the solution is allowed to stand to obtain a silk fibroin hydrogel loaded with ginger extracellular vesicles (SF-Gel-GEVs).

[0026] The activity concentration of horseradish peroxidase is 1-100 U / mL, preferably 10-50 U / mL.

[0027] The concentration of hydrogen peroxide is 0.001%-1% (w / v), preferably 0.01%-0.1% (w / v).

[0028] Referring to the operation of step 3), the silk fibroin hydrogel (SF-Gel) was prepared without adding the ginger extracellular vesicle solution, hydrogen peroxide and horseradish peroxidase were added to the silk fibroin solution, and the mixture was fully mixed; the obtained silk fibroin mixed solution was allowed to stand to obtain the silk fibroin hydrogel (SF-Gel).

[0029] The silk fibroin hydrogel (SF-Gel) and the silk fibroin hydrogel loaded with ginger extracellular vesicles (SF-Gel-GEVs) were frozen at -196°C and then freeze-dried. The surface of the freeze-dried samples was sprayed with gold and then electron microscopy images were taken. The electron microscopy results are shown in FIG. Figure 3 As shown in the figure, the internal pore size of SF-Gel-GEVs is smaller than that of SF-Gel, which indicates that the presence of ginger extracellular vesicles will induce the hydrogel to form a tighter microstructure.

[0030] Ginger exosomes were fluorescently labeled with DiD fluorescent dye, and SF-Gel-GEVs were prepared using DiD-labeled ginger exosomes. The distribution of DiD-labeled ginger exosomes in SF-Gel-GEVs was observed and recorded using a laser confocal microscope. Figure 4As shown, ginger extracellular vesicles can be evenly distributed in SF-Gel-GEVs, and the silk fibroin hydrogel carrying ginger extracellular vesicles has a tight three-dimensional network structure, which enables the ginger extracellular vesicles to be slowly released in SF-Gel-GEVs, which is beneficial to increase the joint residence time of ginger extracellular vesicles and improve their bioavailability, thereby alleviating the symptoms of osteoarthritis (silk fibroin exhibits green fluorescence and ginger extracellular vesicles exhibit red fluorescence).

[0031] Application Examples This application example provides an application of silk fibroin hydrogel loaded with ginger extracellular vesicles in the preparation of a product for treating osteoarthritis. The silk fibroin hydrogel loaded with ginger extracellular vesicles in this application example is provided by an embodiment.

[0032] First Experimental Example LPS (lipopolysaccharide): an inducer that can induce macrophage polarization to M1 macrophages.

[0033] M0: Macrophages that are not stimulated by any activation signals under in vitro experimental conditions and are in a resting state.

[0034] iNOS and CD206 are surface markers of M1 macrophages and M2 macrophages, respectively. M1 macrophages release a large amount of pro-inflammatory cytokines, accelerate the catabolism of chondrocytes, and promote the development of osteoarthritis. M2 macrophages can secrete anti-inflammatory cytokines, effectively weaken the inflammatory response, protect chondrocytes, and alleviate the symptoms of osteoarthritis.

[0035] This experiment mainly investigates whether ginger extracellular vesicles (GEVs) can promote M2 polarization of macrophages. After macrophages take up ginger extracellular vesicles, the polarization of macrophages is evaluated by immunofluorescence staining. Figure 5 As shown, in the M0+LPS group, iNOS expression increased and CD206 expression decreased, indicating that under the action of LPS, M0 polarized toward M1 macrophages; in the M0+LPS+GEVs group, iNOS expression decreased and CD206 expression increased, indicating that ginger extracellular vesicles can effectively inhibit the polarization of macrophages toward M1 macrophages and induce the polarization of macrophages toward M2 macrophages.

[0036] Second Experimental Example This experiment mainly explores the effect of M2 macrophages on the migration ability of bone marrow mesenchymal stem cells. The exosomes of M2 macrophages were added to the culture medium and cultured with bone marrow mesenchymal stem cells for 12 hours. M2-Exos: experimental group with added M2 macrophage exosomes; Control group is a blank control group without added M2-Exos. The experimental results are as follows Figure 6As shown, the experimental results showed that compared with the blank control group, the experimental group with the addition of M2 macrophage exosomes could significantly promote the migration of bone marrow mesenchymal stem cells, with a migration rate of up to 74%.

[0037] The third experimental example This experiment mainly explores whether M2 macrophages can promote the chondrogenic differentiation of bone marrow mesenchymal stem cells. The exosomes of M2 macrophages were added to the culture medium as chondrogenic inducing agents and cultured with bone marrow mesenchymal stem cells for 7 days before Alcian blue staining. The experimental results are as follows Figure 7 As shown, the experimental results showed that bone marrow mesenchymal stem cells that ingested M2 macrophage exosomes showed a more significant positive staining signal, indicating that M2 macrophage exosomes have a significant effect in promoting the chondrogenic differentiation of bone marrow mesenchymal stem cells.

[0038] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A method for preparing a silk fibroin hydrogel loaded with ginger extracellular vesicles, characterized in that: The preparation method comprises: Hydrogen peroxide and horseradish peroxidase are added to the silk fibroin solution and fully mixed; then the ginger exovesicle solution is added to the silk fibroin solution to obtain a ginger exovesicle-silk fibroin mixed solution; and the solution is allowed to stand to obtain a silk fibroin hydrogel loaded with ginger exovesicles.

2. The method for preparing the silk fibroin hydrogel carrying ginger extracellular vesicles according to claim 1, characterized in that: The preparation method of the ginger exocyst comprises: Take fresh ginger, wash it, chop it, soak it in phosphate buffer, grind it, and get ginger juice; The ginger juice was centrifuged at 1000×g for 10 min, 3000×g for 20 min and 1×10 4 × g for 40 min, take the supernatant, and use an ultra-high speed centrifuge at 1.5 × 10 5 The supernatant was removed and the pellet was resuspended in phosphate buffer and transferred to 8 wt%, 15 wt%, 30 wt%, 45 wt% and 60 wt% sucrose density gradient solutions at 1.5 × 10 5 × g for 2 h, and the bands between the 8%, 30%, and 45% layers were collected at 4°C, 1.5 × 10 5 × g for 1 hour to obtain ginger exosomes, which were redispersed in phosphate buffer and stored at -80°C for future use.

3. The method for preparing the silk fibroin hydrogel carrying ginger extracellular vesicles according to claim 1, characterized in that: The preparation method of the silk fibroin solution comprises the following steps: Cut the silk cocoons into pieces and add them into boiling Na2CO3 solution to separate silk fibroin and sericin. Wash and dry the extracted silk fibroin and dissolve it in LiBr solution. After dissolution, dialyze it in deionized water for 2 days, changing the water 3-4 times a day. After dialysis, centrifuge it at 4℃, 8000-10000 rpm for 5-60 min. The supernatant is the silk fibroin solution.

4. The preparation method according to claim 3, characterized in that: The concentration of the silk fibroin solution is 1%-20% (w / v).

5. The method for preparing the silk fibroin hydrogel carrying ginger extracellular vesicles according to claim 1, characterized in that: The horseradish peroxidase activity concentration is 1-100 U / mL.

6. The method for preparing the silk fibroin hydrogel carrying ginger extracellular vesicles according to claim 5, characterized in that: The horseradish peroxidase activity concentration is preferably 10-50 U / mL.

7. The method for preparing the silk fibroin hydrogel carrying ginger extracellular vesicles according to claim 1, characterized in that: The concentration of hydrogen peroxide is 0.001%-1% (w / v).

8. The method for preparing the silk fibroin hydrogel carrying ginger extracellular vesicles according to claim 7, characterized in that: The concentration of hydrogen peroxide is preferably 0.01%-0.1% (w / v).

9. A silk fibroin hydrogel loaded with ginger extracellular vesicles, characterized in that: The preparation is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the silk fibroin hydrogel loaded with ginger extracellular vesicles as claimed in claim 9 in preparing a product for treating osteoarthritis.

Images