Hydrogel for promoting wound healing and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to hydrogel for promoting wound healing and a preparation method and application thereof.The preparation method comprises the steps that firstly, coriander exosomes are extracted from coriander through a gradient centrifugation and sucrose density gradient method, then the coriander exosomes and a sodium alginate solution are mixed according to the mass-to-volume ratio of 0.5-2 mg: 1 mL, and a hydrogel solution is obtained; and mixing the exosome suspension with a calcium chloride solution according to a volume ratio of (0.5-2): 1, and carrying out crosslinking to obtain the coriander exosome-loaded hydrogel. The coriander exosome-loaded novel hydrogel provided by the invention has good viscoelasticity and biocompatibility, and can be applied to the surface of a skin wound as a dressing; meanwhile, the hydrogel has a coriander exosome slow-release effect, so that the exosome release time is prolonged, and the biological function is better played; by slowly releasing the coriander exosome, the hydrogel can relieve oxidative stress and inflammation of wounds and promote wound repair.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a hydrogel that promotes wound healing and a preparation method and application thereof. Background Art

[0002] The treatment of skin wounds such as large-area skin wounds, diabetic wounds and skin burns is still a major challenge in wound treatment. Skin healing requires a series of complex stages including hemostasis, inflammation, proliferation and remodeling, among which the inflammation stage is a necessary natural stage. If it lasts too long, chronic inflammatory wounds may form. Oxidative stress is closely related to inflammation, so controlling the redox homeostasis of the wound microenvironment is crucial for wound healing.

[0003] Existing studies have shown that exosomes from various plant sources have antioxidant, anti-inflammatory and wound healing effects. Plant exosomes are vesicles with a phospholipid bilayer structure. The main components are proteins, lipids, nucleic acids, etc. They can act as messengers for intercellular communication to regulate various physiological and pathological processes. Their characteristics are similar to mammalian exosomes, but they have lower immunogenicity and the potential for large-scale production. However, due to the shortcomings of poor targeting and easy removal, the direct application of plant exosomes on wounds is not ideal. Hydrogels are widely used in the field of medical dressings because of their ability to fill irregular wounds, maintain a moist environment, adjust mechanical properties and good biocompatibility. As a commonly used drug delivery system, hydrogels can achieve sustained release of drugs, reducing the need for repeated administration in clinical settings.

[0004] Based on this, the present invention develops and designs a new hydrogel loaded with plant exosomes to promote wound healing. Summary of the invention

[0005] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and proposes a hydrogel for promoting wound healing, and a preparation method and application thereof. By loading coriander exosomes extracted from coriander, a new hydrogel for promoting wound healing is prepared, which is expected to become a safer and more economical medical dressing in clinical wound treatment.

[0006] The technical solution of the present invention is:

[0007] The present invention provides a method for preparing a hydrogel for promoting wound healing, comprising the following steps:

[0008] Coriander exosomes were extracted from coriander by gradient centrifugation combined with sucrose density gradient method, and the coriander exosomes were mixed with sodium alginate solution at a mass volume ratio of 0.5-2 mg:1 mL to obtain an exosome suspension. The exosome suspension was then mixed with calcium chloride solution at a volume ratio of 0.5-2:1, and cross-linked to obtain a hydrogel loaded with coriander exosomes.

[0009] Furthermore, the mass volume ratio of the coriander exosomes mixed with the sodium alginate solution is 1 mg:1 mL.

[0010] Furthermore, the volume ratio of the exosome suspension to the calcium chloride solution is 1:1.

[0011] Furthermore, the sodium alginate solution is obtained by dissolving sodium alginate in PBS, and the concentration of the sodium alginate solution is 1.5-2.5% (w / v); the calcium chloride solution is obtained by dissolving calcium chloride in distilled water, and the concentration of the calcium chloride solution is 1.5-2.5% (w / v).

[0012] Preferably, the concentration of the sodium alginate solution is 2% (w / v), and the concentration of the calcium chloride solution is 2% (w / v).

[0013] The present invention also provides a method for extracting coriander exosomes, comprising the following steps:

[0014] (1) extracting distilled water and coriander in a mass ratio of 0.75 to 1.25:1 to obtain coriander juice;

[0015] (2) The coriander juice is subjected to gradient centrifugation in sequence, and the supernatant finally obtained is filtered to obtain a filtrate. The gradient centrifugation is performed in sequence:

[0016] First centrifugation: 4°C, 3000×g~3500×g for 20min~25min,

[0017] Second centrifugation: 4°C, 9500×g~10000×g for 100min~120min;

[0018] (3) The filtrate was centrifuged at 100,000 × g to 120,000 × g for 1.5 h to 2 h at 4 ° C, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS (0.01 M, pH = 7.4) to obtain a resuspension;

[0019] (4) transferring the resuspended liquid to a sucrose density gradient solution with mass fractions of 8%, 30%, 45%, and 60% from top to bottom, and centrifuging at 140,000 × g to 160,000 × g for 1.5 h to 2 h, and collecting the suspension between the 30% and 45% sucrose layers;

[0020] (5) After adding 8 to 12 times the volume of PBS to the suspension, the suspension was centrifuged at 100,000 × g to 120,000 × g for 45 min to 60 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS (0.01 M, pH = 7.4) and filtered to obtain purified coriander exosomes.

[0021] Furthermore, the volume of the appropriate amount of PBS involved in steps (3) and (5) is preferably sufficient to cover the precipitate.

[0022] Furthermore, the specific steps of the coriander exosome extraction method are:

[0023] (1) removing the roots of an appropriate amount of fresh coriander, washing and chopping it, adding it into a wall breaking machine to extract juice, then adding distilled water to extract the juice, and filtering with gauze to obtain coriander juice; the mass ratio of the distilled water to coriander is 1:1;

[0024] (2) centrifuging the coriander juice at 3000×g and 10000×g for 20 min and 120 min at 4°C, respectively, and filtering the supernatant obtained by the centrifugation through a 0.45 μm filter membrane to obtain a filtrate;

[0025] (3) The filtrate was centrifuged at 100,000 × g for 1.5 h at 4°C, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS (0.01 M, pH = 7.4) to obtain a resuspension; the volume of PBS used should be sufficient to cover the precipitate;

[0026] (4) Prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, respectively. Add an appropriate amount of 60% sucrose solution to the bottom of the centrifuge tube, and then slowly add equal volumes of 45%, 30%, and 8% sucrose solutions to the tube wall in sequence. The solution interface should be clear. Transfer the resuspension in step (3) to the upper layer of the 8% sucrose solution and centrifuge at 150,000 × g for 1.5 h. After centrifugation, collect the suspension between the 30% and 45% sucrose layers.

[0027] The volume of the sucrose solution is preferably greater than or equal to the volume of the resuspension in step (3).

[0028] (5) After adding 10 times the volume of PBS to the suspension, centrifuge at 100,000×g for 45 min at 4°C, discard the supernatant, resuspend the precipitate in an appropriate amount of PBS (0.01 M, pH = 7.4) and filter through a 0.22 μm filter membrane to obtain purified coriander exosomes; the volume of the appropriate amount of PBS added should be sufficient to cover the precipitate.

[0029] The present invention also provides a hydrogel loaded with coriander exosomes prepared by any of the preparation methods described above.

[0030] The present invention also provides the use of the above-mentioned hydrogel loaded with coriander exosomes and / or the hydrogel loaded with coriander exosomes prepared by any of the above-mentioned preparation methods in the preparation of a drug that promotes wound healing.

[0031] The present invention further provides a medicine for promoting wound healing, comprising the coriander exosome-loaded hydrogel prepared by any of the preparation methods and / or the coriander exosome-loaded hydrogel.

[0032] Furthermore, the medicine also includes a pharmaceutically acceptable dressing.

[0033] Beneficial effects of the present invention:

[0034] (1) The novel hydrogel loaded with coriander exosomes provided by the present invention has good viscoelasticity and biocompatibility, and can be applied as a dressing on the surface of skin wounds; at the same time, the hydrogel has a sustained release effect of coriander exosomes, prolongs the release time of the exosomes, and enables them to better exert their biological functions; by sustained release of coriander exosomes, the hydrogel can relieve wound oxidative stress and inflammation and promote wound repair.

[0035] (2) The coriander exosomes loaded by the hydrogel of the present invention present a typical saucer-shaped double-layer membrane structure with a particle size of about 150 nm. The extracted coriander exosomes contain 2.11×10 11 The invention discloses a method for extracting coriander exosomes, which can be taken up by skin cells, promote cell migration, resist oxidation and fight inflammation, and can effectively promote skin wound healing. The method for extracting coriander exosomes provided by the invention can obtain a higher yield and purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The microstructure of coriander exosomes provided by the present invention.

[0037] Figure 2 The particle size distribution of coriander exosomes provided by the present invention.

[0038] Figure 3 This is the uptake of coriander exosomes by human immortalized keratinocytes (HaCaT) in Experimental Example 1.

[0039] Figure 4 The proliferation and migration promoting effects of coriander exosomes on HaCaT cells in Experimental Example 1. A: MTT assay; B: scratch assay; C: scratch healing rate.

[0040] Figure 5 The effect of coriander exosomes on ROS, SOD, CAT, and GSH-Px in HaCaT cells stimulated by H2O2 in Experimental Example 1. A: ROS; B: SOD; C: CAT; D: GSH-Px.

[0041] Figure 6 The effect of coriander exosomes on the transcriptional levels of proinflammatory factors and MMP-1 in HaCaT cells stimulated by H2O2 in Experimental Example 1. A: TNF-α; B: IL-6; C: IL-1β; D: IL-8; E: NF-κB; F: MMP-1.

[0042] Figure 7 The microstructure of the hydrogel and exosome hydrogel in Experimental Example 2.

[0043] Figure 8 Rheological properties of the hydrogel and exosome hydrogel in Experimental Example 2. A: strain sweep; B: frequency sweep.

[0044] Fig. 9 The swelling properties of the hydrogel and exosome hydrogel in Experimental Example 2.

[0045] Fig.10 This is the sustained release curve of the exosome hydrogel in Experimental Example 2.

[0046] Fig.11 This is the hemolysis experiment of exosome hydrogel in Experimental Example 2.

[0047] Fig.12 The wound healing conditions of mice skin in different treatment groups in Experimental Example 3. A: wound change record; B: wound healing rate.

[0048] Fig.13 The activities of ROS, SOD, CAT, and GSH-Px in the skin of mice in different treatment groups in Experimental Example 3. A: ROS; B: SOD; C: CAT; D: GSH-Px.

[0049] Fig.14 The levels of TNF-α and IL-10 in the skin of mice in different treatment groups in Experimental Example 3. A: TNF-α; B: IL-10. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the embodiments and drawings, and the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods; the materials, reagents, etc. used are all available from commercial channels; and the equipment used are all conventional equipment.

[0052] Example 1

[0053] This embodiment provides a method for extracting coriander exosomes, and the steps are as follows:

[0054] (1) Remove the roots of 200 g fresh coriander, wash and chop it, add it to a wall breaking machine together with 200 mL of distilled water, and filter it with gauze to obtain coriander juice;

[0055] (2) centrifuging the coriander juice at 3000×g and 10000×g for 20 min and 120 min at 4°C, respectively, and filtering the supernatant through a 0.45 μm filter membrane to obtain a filtrate;

[0056] (3) The filtrate was centrifuged at 100,000 × g for 1.5 h at 4 °C, the supernatant was discarded, and the precipitate was resuspended in 0.5 mL PBS (0.01 M, pH = 7.4) to obtain a resuspension;

[0057] (4) Prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, respectively. Add an appropriate amount of 60% sucrose solution to the bottom of the centrifuge tube, and then slowly add equal volumes of 45%, 30%, and 8% sucrose solutions to the wall in sequence, with a clear interface between the solutions. Take a sucrose solution with a volume greater than or equal to the volume of the resuspension, transfer the resuspension to the upper layer of the 8% sucrose solution, and centrifuge at 150,000 × g for 1.5 h. Use a syringe to collect the suspension between the 30% and 45% sucrose layers.

[0058] (5) After adding 10 mL of PBS to the suspension, the mixture was centrifuged at 100,000 × g for 45 min, the supernatant was discarded, and the precipitate was resuspended in 1.5 mL of PBS and filtered through a 0.22 μm filter to obtain purified coriander exosomes.

[0059] The coriander exosomes extracted above were identified and characterized:

[0060] Coriander exosomes were negatively stained with 2% phosphotungstic acid and photographed by transmission electron microscopy (TEM). Figure 1 As shown in Figure 1, it is a typical "tray-like" structure with a double-layer membrane. Its diameter and concentration were determined by nanoparticle tracking analysis (NTA), as shown in Figure 1. Figure 2 As shown in the figure, the average particle size is 150.10±38.40nm. The number of vesicle particles in 1mL of coriander exosomes extracted by this method is 2.11×10 11 indivual.

[0061] Example 2

[0062] This embodiment provides a method for extracting coriander exosomes, and the steps are as follows:

[0063] (1) Remove the roots of 200 g fresh coriander, wash and chop it, add it to a wall breaking machine and squeeze out the juice together with 150 mL of distilled water, and filter it through gauze to obtain coriander juice;

[0064] (2) centrifuging the coriander juice at 3200×g and 9500×g for 25 min and 110 min at 4°C, respectively, and then filtering the supernatant through a 0.45 μm filter membrane to obtain a filtrate;

[0065] (3) The filtrate was centrifuged at 110,000 × g for 2 h at 4 °C, the supernatant was discarded, and the precipitate was resuspended in 0.5 mL PBS (0.01 M, pH = 7.4) to obtain a resuspension;

[0066] (4) Prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, respectively. Add an appropriate amount of 60% sucrose solution to the bottom of the centrifuge tube, and then slowly add equal volumes of 45%, 30%, and 8% sucrose solutions to the wall in sequence, with a clear interface between the solutions. Take a sucrose solution with a volume greater than or equal to the volume of the resuspension, transfer the resuspension to the upper layer of the 8% sucrose solution, and centrifuge at 140,000 × g for 2 h. Use a syringe to collect the suspension between the 30% and 45% sucrose layers.

[0067] (5) After adding 10 mL of PBS to the suspension, the mixture was centrifuged at 120,000 × g for 50 min, the supernatant was discarded, and the precipitate was resuspended in 1.5 mL of PBS and filtered through a 0.22 μm filter to obtain purified coriander exosomes.

[0068] Example 3

[0069] This embodiment provides a method for extracting coriander exosomes, and the steps are as follows:

[0070] (1) Remove the roots of 200 g fresh coriander, wash and chop it, add it to a wall breaking machine together with 250 mL of distilled water, and filter it through gauze to obtain coriander juice;

[0071] (2) centrifuging the coriander juice at 3500×g and 9800×g for 22 min and 100 min at 4°C, respectively, and then filtering the supernatant through a 0.45 μm filter membrane to obtain a filtrate;

[0072] (3) The filtrate was centrifuged at 120,000 × g for 1.7 h at 4 °C, the supernatant was discarded, and the precipitate was resuspended in 0.5 mL PBS (0.01 M, pH = 7.4) to obtain a resuspension;

[0073] (4) Prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, respectively. Add an appropriate amount of 60% sucrose solution to the bottom of the centrifuge tube, and then slowly add equal volumes of 45%, 30%, and 8% sucrose solutions to the wall in sequence, with a clear interface between the solutions. Take a sucrose solution with a volume greater than or equal to the volume of the resuspension, transfer the resuspension to the upper layer of the 8% sucrose solution, and centrifuge at 160,000 × g for 1.8 h. Use a syringe to collect the suspension between the 30% and 45% sucrose layers.

[0074] (5) The suspension was added with 10 mL of PBS and centrifuged at 110,000 × g for 60 min. The supernatant was discarded and the precipitate was resuspended in 1.5 mL of PBS and filtered through a 0.22 μm filter to obtain purified coriander exosomes.

[0075] Test Example 1

[0076] Taking the coriander exosomes extracted in Example 1 as an example, the efficacy of coriander exosomes was verified.

[0077] 1. Uptake of coriander exosomes by HaCaT cells

[0078] 800 μL of coriander exosomes were incubated with 8 μL PKH26 and 800 μL diluent C for 5 min, 1.6 mL of 1% bovine serum albumin was added, and the mixture was centrifuged at 100,000 × g for 1 h to remove the residual dye. After the exosomes were resuspended with PBS, the resuspended solution was transferred to a 100KD ultrafiltration tube and centrifuged at 4000 rpm for 20 min to obtain PKH26-labeled coriander exosomes.

[0079] HaCaT cells were cultured at a rate of 1×10 5 Each dish was inoculated in a confocal dish and cultured for 24 hours at 37°C and 5% CO2. Then, cilantro exosomes labeled with PKH26 were added and incubated for 0.5 hours or 6 hours. The cells were washed twice with PBS, fixed with 4% paraformaldehyde, and the cell membrane and nucleus were stained with DiO and DAPI, respectively, and observed under a laser confocal scanning microscope. Figure 3 As shown, compared with the control (0.5h), red fluorescence appeared in the cell membrane after 6h of incubation, indicating that HaCaT cells can take up coriander exosomes.

[0080] 2. Effects of coriander exosomes on the proliferation and migration of HaCaT cells

[0081] HaCaT cells were collected at 1×10 4 Each well was inoculated into a 96-well plate. After 24 h of culture, the medium was replaced with complete culture medium containing coriander exosomes (0, 5, 10, 20, 40, 60 μg / mL) and cultured for 12, 24, and 48 h. 10 μL of MTT reagent (5 mg / mL) was added to each well and cultured for 4 h. The culture was terminated, the culture medium in the well was removed, 200 μL of DMSO was added to each well to completely dissolve the crystals, and the OD570 was measured on a microplate reader. Figure 4 As shown in A, 12h or 24h of coriander exosome treatment had no significant effect on cell proliferation. After 48h of treatment, the viability of HaCaT cells was significantly enhanced under the treatment of coriander exosomes at concentrations of 10, 20, and 40μg / mL compared with the control group (0μg / mL).

[0082] HaCaT cells were cultured at 4 × 10 5The cells were inoculated into 6-well plates at a density of 100 μg / well. After the cells adhered to the wall to form a single cell layer, the original culture medium was removed, a 1 mL pipette tip was used to make a scratch, and the detached cells were washed with PBS. The control group was added with complete culture medium, and the experimental group was added with an equal amount of complete culture medium containing coriander exosomes (10, 20, 40 μg / mL) and continued to be cultured. The cell status at 0 and 24 h after the scratch was made was observed under an inverted microscope and photographed ( Figure 4 B), calculate the scratch healing rate ( Figure 4 C), healing rate = (S0-S t ) / S0×100%, where S0 is the initial scratch area, S t The scratch area at 24h. The results showed that 10-40μg / mL coriander exosomes promoted the migration of HaCaT cells.

[0083] 3. Antioxidant effects of coriander exosomes

[0084] HaCaT cells were cultured at 1×10 4 The cells were inoculated at a density of 1000 / well in a 96-well plate and cultured for 24 hours. The original culture medium was aspirated and replaced with fresh complete culture medium for the control and model groups, and complete culture medium containing coriander exosomes (10, 20, 40 μg / mL) for the experimental group. After incubation for 24 hours, the culture medium was discarded again, and fresh basal culture medium was added to the control group, and an equal amount of basal culture medium containing 200 μM H2O2 was added to the model and experimental groups. After 2 hours, each well was replaced with a DCFH-DA solution (10 μM) diluted with basal culture medium, incubated at 37°C for 20 minutes, and the fluorescence intensity was detected at 488 nm excitation wavelength / 525 nm emission wavelength to characterize the intracellular ROS level. Figure 5 As shown in A, H2O2 stimulation significantly increased the ROS of the model group cells, proving that the oxidative stress model was successfully established; while the ROS of the experimental group pre-incubated with coriander exosomes was significantly reduced, indicating that coriander exosomes have the effect of clearing intracellular ROS.

[0085] To characterize the effect of coriander exosomes on intracellular antioxidant enzymes, HaCaT cells were cultured at 4×10 5 The cells were inoculated into 6-well plates at a density of 100 cells / well, and the cells were intervened using the above grouping method. The cell suspension was collected and the effect of coriander exosomes on the activities of SOD, CAT, and GSH-Px was evaluated using a commercial kit. Figure 5 As shown in BD, coriander exosomes significantly increased the enzymatic activities of antioxidant enzymes SOD, CAT, and GSH-Px.

[0086] 4. Anti-inflammatory effects of coriander exosomes

[0087] HaCaT cells were cultured at 4 × 10 5The cells were inoculated into 6-well plates at a density of 100 cells / well, and the cells were intervened using the above grouping method. Total RNA was isolated using a cell RNA extraction kit. Real-time quantitative PCR was performed using an RT-qPCR kit. β-actin was used as an internal reference gene, and the threshold cycle (2 -ΔΔCt ) method to calculate the relative gene expression of inflammatory factors and matrix metalloproteinase 1 (MMP-1). Figure 6 As shown in the results, H2O2 caused a significant increase in the transcription levels of intracellular proinflammatory factors TNF-α, IL-6, IL-1β, IL-8, and NF-κB. MMP is expressed in keratinocytes and mainly acts on the degradation of collagen, while collagen deposition is closely related to wound healing. The results showed that MMP-1 in the model group showed an upward trend. The transcription levels of inflammatory factors and MMP-1 in the experimental group pretreated with coriander exosomes decreased to varying degrees, showing the potential to promote wound healing.

[0088] Example 4

[0089] This embodiment provides a method for preparing a hydrogel loaded with coriander exosomes, the steps are as follows:

[0090] (1) Preheat 50 mL of PBS to 40°C, add 1.0 g of sodium alginate powder into the solution while stirring, and stir for 1 h until the sodium alginate is completely dissolved to obtain a 2% (w / v) sodium alginate solution;

[0091] (2) Weigh 1.0 g of calcium chloride powder and dissolve it in 50 mL of distilled water to obtain a 2% (w / v) calcium chloride solution;

[0092] (3) Add 1 mg of coriander exosomes to every 1 mL of sodium alginate solution, mix well, then add 1 mL of calcium chloride solution and cross-link for 30 seconds to obtain a hydrogel loaded with coriander exosomes.

[0093] Test Example 2

[0094] The properties of the exosome hydrogel prepared in Example 4 were studied.

[0095] Blank hydrogel without coriander exosomes was prepared by adding 1 mL of calcium chloride solution to every 1 mL of sodium alginate solution and cross-linking for 30 s to obtain blank hydrogel.

[0096] (I) Microstructure

[0097] Using cryo-electron microscopy to observe the cross-sections of blank hydrogel and exosome hydrogel at different magnifications, it was found that both showed typical cross-linked porous structures with relatively uniform distribution ( Figure 7 ), and the pore sizes were measured to be 2.32±0.38μm and 0.70±0.06μm, respectively, indicating that the retention of exosomes reduced the pore size of the hydrogel microstructure.

[0098] (II) Rheology

[0099] The viscoelasticity and structural stability of the hydrogels were measured by rheometer. The samples were subjected to 0.1-1000% strain sweep test at a constant frequency of 10 rad / s at 37°C, and 1-100 rad / s frequency sweep test at a constant strain of 1%. Within the strain range of 0.1-10%, the blank hydrogel and the exosome hydrogel exhibited reversible elastic deformation, maintaining a relatively stable high storage modulus (G') and low loss modulus (G") ( Figure 8 A). During frequency sweep, G' is always higher than G" ( Figure 8 B). The results showed that both blank hydrogel and exosome hydrogel had primary elasticity, while the blank hydrogel had better viscoelasticity.

[0100] (III) Swelling effect

[0101] The hydrogel was dried at 37°C overnight, weighed, and placed in a PBS solution to swell. At a predetermined time point, the surface moisture of the hydrogel was removed and weighed. The swelling rate was calculated based on the initial mass. Swelling rate = (m t -m0) / m0×100%, where represents the initial hydrogel mass, represents the hydrogel mass at each time point, and the swelling curve is drawn according to the swelling rate. Fig. 9 As shown in the figure, the swelling ratios of the hydrogel and exosome hydrogel were both above 10 after 20 min, and the swelling ratio of the hydrogel was slightly higher than that of the exosome hydrogel, but there was no significant difference between the two. Good swelling property indicates that the hydrogel can quickly absorb wound tissue exudate.

[0102] (IV) Sustained release performance

[0103] The hydrogel containing 100 μg of exosomes was placed in a 12-well plate, and 500 μL PBS was added to immerse and swell. At the predetermined time point, 250 μL of supernatant was aspirated, and the protein concentration was measured by BCA protein kit. At the same time, 250 μL of fresh PBS was added to calculate the release rate of exosomes at each time point. Fig.10 As shown in Figure 2, exosomes can be released continuously for 3 days. The fitted release curve shows that the exosomes are released slowly in the first 6 hours, and then the release rate accelerates, reaching 74.13% at 48 hours.

[0104] (V) Biocompatibility

[0105] Prepare 4% (v / v) red blood cell suspension for later use. Add 0.5 mL of red blood cell suspension and 0.5 mg of hydrogel or exosome hydrogel to a 1.5 mL centrifuge tube, incubate at 37°C for 1 h, centrifuge at 2400 rpm for 5 min, and take 100 uL of supernatant to measure OD540. 0.1% Triton X-100 was used as a positive control, PBS as a negative control, and the hemolysis rate was calculated by the following formula: Hemolysis rate = (OD sample-OD negative) / (OD positive-OD negative) × 100%, where OD sample, OD negative, and OD positive are the OD540 of the sample, negative control, and positive control, respectively. Fig.11 As shown, the positive control 0.1% Triton X-100 group had severe hemolysis, and the hemolysis rate of the exosome hydrogel group was 0.75±0.27%, which was not statistically different from that of the PBS group.

[0106] Example 5

[0107] This embodiment provides a method for preparing a hydrogel loaded with coriander exosomes, the steps are as follows:

[0108] (1) Preheat 50 mL of PBS to 35°C, add 1.25 g of sodium alginate powder into the solution while stirring, and stir for 0.5 h until the sodium alginate is completely dissolved to obtain a 2.5% (w / v) sodium alginate solution;

[0109] (2) Weigh 1.25 g of calcium chloride powder and dissolve it in 50 mL of distilled water to obtain a 2.5% (w / v) calcium chloride solution;

[0110] (3) Add 0.5 mg of coriander exosomes to every 1 mL of sodium alginate solution, mix well, then add 1 mL of calcium chloride solution and cross-link for 30 seconds to obtain a hydrogel loaded with coriander exosomes.

[0111] Example 6

[0112] This embodiment provides a method for preparing a hydrogel loaded with coriander exosomes, the steps are as follows:

[0113] (1) Preheat 50 mL of PBS to 45°C, add 0.75 g of sodium alginate powder into the solution while stirring, and stir for 0.8 h until the sodium alginate is completely dissolved to obtain a 1.5% (w / v) sodium alginate solution;

[0114] (2) Weigh 0.75 g of calcium chloride powder and dissolve it in 50 mL of distilled water to obtain a 1.5% (w / v) calcium chloride solution;

[0115] (3) Add 2 mg of coriander exosomes to every 1 mL of sodium alginate solution, mix well, then add 1 mL of calcium chloride solution and cross-link for 30 seconds to obtain a hydrogel loaded with coriander exosomes.

[0116] Test Example 3

[0117] Taking the coriander exosome hydrogel prepared in Example 4 as an example, the performance of the new hydrogel was verified.

[0118] Effect of coriander exosome hydrogel on promoting skin wound healing in mice

[0119] 1. Wound healing effect of coriander exosome hydrogel

[0120] After 1 week of adaptive feeding of ICR mice (20-25g, 4-6 weeks old), all mice were divided into an untreated control group, a hydrogel group, and an exosome hydrogel group, with 12 mice in each group. After fasting for 12 hours, the mice were anesthetized by intraperitoneal injection of 1.25% tribromoethanol (0.2mL / 10g), the hair on the back of the mice was removed, and a 4mm diameter perforation was made on the back with a hole puncher. During the experiment, the mice were treated with different preparations every 4 days and covered and fixed with 3M transparent dressings. On days 0, 4, 8, and 12 after wound formation, wound photos were taken and the wound closure rate was calculated. Fig.12 As shown in A, on the 4th day, scabs had formed on the wound surface of the exosome hydrogel group, earlier than the control group and the hydrogel group. On the 12th day, the wound surface was almost invisible in the mice in the exosome hydrogel group, and the wound healed well. After further quantification of the wound healing rate, the data showed that on the 8th day, the wound healing rate of the exosome hydrogel group was significantly higher than that of the control group and the hydrogel group. On the 12th day, the average wound healing rates of each group were 88.24% (control group), 90.61% (hydrogel group), and 96.21% (exosome hydrogel group), respectively, indicating the good performance of exosome hydrogel in promoting wound healing ( Fig.12 B).

[0121] 2. Antioxidant effect of coriander exosome hydrogel

[0122] Blood was collected from each group of mice, and the serum was obtained by coagulation and centrifugation, and then the ROS level was measured. Fig.13 A shows that the ROS in the exosome hydrogel group was significantly lower than that in the control group and the hydrogel group; wound skin samples were collected and the contents of SOD, CAT, and GSH-Px in the skin homogenate supernatant were detected by a kit. The results showed that exosome hydrogel treatment can effectively increase the activity of antioxidant enzymes ( Fig.13 B) High antioxidant enzyme activity is beneficial to remove intracellular ROS and maintain redox balance.

[0123] 3. Anti-inflammatory effect of coriander exosome hydrogel

[0124] Wound skin samples from each group of mice were collected, and the levels of pro-inflammatory factor TNF-α and anti-inflammatory factor IL-10 were detected by ELISA kit. The results showed that the TNF-α level in the exosome hydrogel group was significantly reduced ( Fig.14 A), while the IL-10 level was significantly higher than that of the other two groups ( Fig.14 B), indicating that exosome hydrogel can effectively relieve wound inflammation and accelerate wound repair.

[0125] As can be seen from the above examples, the present invention provides a novel hydrogel for promoting wound healing and its application. The hydrogel loaded with coriander exosomes prepared by the present invention has good viscoelasticity and biocompatibility, and promotes skin wound healing by sustained release of coriander exosomes with cell migration promotion, anti-oxidation and anti-inflammatory effects. The present invention is of great significance for the development of new wound healing dressings in the clinical field.

[0126] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel for promoting wound healing, characterized in that: The following steps are involved: Coriander exosomes were extracted from coriander by gradient centrifugation combined with sucrose density gradient method, and the coriander exosomes were mixed with sodium alginate solution at a mass volume ratio of 0.5-2 mg:1 mL to obtain an exosome suspension. The exosome suspension was then mixed with calcium chloride solution at a volume ratio of 0.5-2:1, and cross-linked to obtain a hydrogel loaded with coriander exosomes.

2. The preparation method according to claim 1, characterized in that: The mass volume ratio of the coriander exosomes and the sodium alginate solution is 1 mg:1 mL.

3. The preparation method according to claim 1, characterized in that: The volume ratio of the exosome suspension to the calcium chloride solution is 1:

1.

4. The preparation method according to claim 1, characterized in that: The sodium alginate solution is obtained by dissolving sodium alginate in PBS, and the concentration of the sodium alginate solution is 1.5-2.5% (w / v); the calcium chloride solution is obtained by dissolving calcium chloride in distilled water, and the concentration of the calcium chloride solution is 1.5-2.5% (w / v).

5. The preparation method according to claim 1, characterized in that: The extraction of coriander exosomes comprises the following steps: (1) extracting distilled water and coriander in a mass ratio of 0.75 to 1.25:1 to obtain coriander juice; (2) The coriander juice is subjected to gradient centrifugation in sequence, and the supernatant finally obtained is filtered to obtain a filtrate. The gradient centrifugation is performed in sequence: First centrifugation: 4°C, 3000×g~3500×g for 20min~25min, Second centrifugation: 4°C, 9500×g~10000×g for 100min~120min; (3) The filtrate was centrifuged at 100,000 × g to 120,000 × g for 1.5 h to 2 h at 4 ° C, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS (0.01 M, pH = 7.4) to obtain a resuspension; (4) transferring the resuspended liquid to a sucrose density gradient solution with mass fractions of 8%, 30%, 45%, and 60% from top to bottom, and centrifuging at 140,000 × g to 160,000 × g for 1.5 h to 2 h, and collecting the suspension between the 30% and 45% sucrose layers; (5) After adding 8 to 12 times the volume of PBS to the suspension, the suspension was centrifuged at 100,000 × g to 120,000 × g for 45 to 60 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS and filtered to obtain purified coriander exosomes.

6. The preparation method according to claim 5, characterized in that: The following steps are involved: (1) removing the roots of an appropriate amount of fresh coriander, washing and chopping it, adding it into a wall breaking machine to extract juice, then adding distilled water to extract the juice, and filtering with gauze to obtain coriander juice; the mass ratio of the distilled water to coriander is 1:1; (2) centrifuging the coriander juice at 3000×g and 10000×g for 20 min and 120 min at 4°C, respectively, and filtering the supernatant obtained by the centrifugation through a 0.45 μm filter membrane to obtain a filtrate; (3) The filtrate was centrifuged at 100,000 × g for 1.5 h at 4°C, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS (0.01 M, pH = 7.4) to obtain a resuspension; the volume of PBS used should be sufficient to cover the precipitate; (4) Prepare sucrose solutions with mass fractions of 8%, 30%, 45%, and 60%, respectively. Add an appropriate amount of 60% sucrose solution to the bottom of the centrifuge tube, and then slowly add equal volumes of 45%, 30%, and 8% sucrose solutions to the tube wall in sequence. The solution interface should be clear. Transfer the resuspension in step (3) to the upper layer of the 8% sucrose solution and centrifuge at 150,000 × g for 1.5 h. After centrifugation, collect the suspension between the 30% and 45% sucrose layers. (5) After adding 10 times the volume of PBS to the suspension, centrifuge at 100,000 × g for 45 min at 4°C, discard the supernatant, resuspend the precipitate in an appropriate amount of PBS and filter through a 0.22 μm filter to obtain purified coriander exosomes.

7. A hydrogel loaded with coriander exosomes prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the coriander exosome-loaded hydrogel according to claim 7 and / or the coriander exosome-loaded hydrogel prepared by the preparation method according to any one of claims 1 to 6 in the preparation of a drug for promoting wound healing.

9. A drug for promoting wound healing, comprising the hydrogel loaded with coriander exosomes prepared by the preparation method according to any one of claims 1 to 6 and / or the hydrogel loaded with coriander exosomes according to claim 7.

10. The drug according to claim 9, characterized in that Pharmaceutically acceptable dressings are also included.

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