A hydrogel that promotes wound healing, its preparation method and application
By extracting and loading exosomes into a hydrogel, the problems of poor targeting of plant exosomes and insufficient drug delivery by hydrogels in wound treatment were solved, achieving a highly efficient wound healing effect with good biocompatibility and sustained-release properties.
Patent Information
- Application Number
- CN202411294543.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2025-10-31
- Estimated Expiration
- 2044-09-14
AI Technical Summary
In existing technologies, plant exosomes have poor targeting and are easily removed in wound treatment, resulting in unsatisfactory effects in treating skin wounds. Furthermore, hydrogels as drug delivery systems have shortcomings in terms of sustained drug release.
Cilantro exosomes were extracted from cilantro using gradient centrifugation combined with sucrose density gradient method, and then crosslinked with sodium alginate solution and calcium chloride solution to prepare a hydrogel loaded with cilantro exosomes, forming a novel hydrogel with a saucer-like bilayer membrane structure.
This hydrogel has good viscoelasticity and biocompatibility, and can release coriander exosomes in a sustained manner, prolonging their action time at the wound site, relieving oxidative stress and inflammation, promoting wound repair, improving the migration ability of skin cells, and promoting skin wound healing.
Smart Images
Figure CN119139219B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to a hydrogel that promotes wound healing, its preparation method, and its application. Background Technology
[0002] The treatment of large-area skin wounds, diabetic wounds, and burns remains a major challenge in wound care. Skin healing involves a complex series of stages, including hemostasis, inflammation, proliferation, and remodeling. The inflammatory stage is a necessary and natural process; if it persists for too long, it may lead to chronic inflammatory wounds. Oxidative stress is closely related to inflammation; therefore, controlling the redox homeostasis of the wound microenvironment is crucial for wound healing.
[0003] Existing studies have shown that exosomes from various plant sources possess antioxidant, anti-inflammatory, and wound-healing properties. Plant exosomes are vesicles with a phospholipid bilayer structure, mainly composed of proteins, lipids, and nucleic acids. They can act as messengers for intercellular communication, regulating 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 their poor targeting and susceptibility to clearance, direct application of plant exosomes to wounds is not ideal. Hydrogels are widely used in medical dressings because they can fill irregular wounds, maintain a moist environment, have adjustable mechanical properties, and good biocompatibility. As a commonly used drug delivery system, hydrogels can achieve sustained drug release, reducing the need for repeated drug administration in clinical settings.
[0004] Based on this, the present invention develops and designs a novel hydrogel loaded with plant exosomes to promote wound healing. Summary of the Invention
[0005] The purpose of this invention is to solve the above-mentioned problems in the prior art, and to propose a hydrogel that promotes wound healing, its preparation method and application. By loading coriander exosomes extracted from coriander, a novel hydrogel that promotes 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 this invention is:
[0007] This invention provides a method for preparing a hydrogel that promotes wound healing, comprising the following steps:
[0008] Cilantro exosomes were extracted from cilantro using a gradient centrifugation method combined with a sucrose density gradient method. The cilantro exosomes were mixed with sodium alginate solution at a mass-to-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 cilantro exosomes.
[0009] Furthermore, the mass-to-volume ratio of the coriander exosomes to 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] This invention also provides a method for extracting coriander exosomes, comprising the following steps:
[0014] (1) Extract cilantro juice by juicing distilled water and cilantro at a mass ratio of 0.75 to 1.25:1;
[0015] (2) The coriander juice was subjected to gradient centrifugation in sequence, and the supernatant was filtered to obtain the filtrate. The gradient centrifugation was performed as follows:
[0016] First centrifugation: Centrifuge at 3000×g~3500×g for 20min~25min at 4℃.
[0017] Second centrifugation: Centrifuge at 9500×g~10000×g for 100min~120min at 4℃;
[0018] (3) Centrifuge the filtrate at 100,000×g~120,000×g for 1.5h~2h at 4℃, discard the supernatant, and resuspend the precipitate in an appropriate amount of PBS (0.01M, pH=7.4) to obtain a resuspension;
[0019] (4) Transfer the resuspended solution to a sucrose density gradient solution with mass fractions of 8%, 30%, 45%, and 60% from top to bottom, and centrifuge at 140,000×g to 160,000×g for 1.5h to 2h, and collect the suspension between the 30-45% sucrose layers;
[0020] (5) Add 8 to 12 times the volume of PBS to the suspension and centrifuge at 100,000 to 120,000 g for 45 to 60 minutes at 4°C. Discard the supernatant, resuspend the precipitate in an appropriate amount of PBS (0.01 M, pH = 7.4) and filter to obtain purified coriander exosomes.
[0021] Furthermore, the appropriate volume of PBS involved in steps (3) and (5) should be sufficient to cover the precipitate.
[0022] Furthermore, the specific steps of the coriander exosome extraction method are as follows:
[0023] (1) Remove the roots from an appropriate amount of fresh coriander, wash it, chop it, add it to a blender to extract the juice, add distilled water to extract the juice, and filter it through gauze to obtain coriander juice; the mass ratio of distilled water to coriander is 1:1.
[0024] (2) Centrifuge the coriander juice at 3000×g and 10000×g at 4℃ for 20 min and 120 min respectively, and take the supernatant obtained at the end and filter it through a 0.45μm filter membrane to obtain the filtrate;
[0025] (3) Centrifuge the filtrate at 100,000 × g for 1.5 h at 4 °C, discard the supernatant, and resuspend the precipitate 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 along the wall in sequence. The solution interface should be distinct. Transfer the resuspension from 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 30-45% sucrose layers.
[0027] The volume of the sucrose solution should preferably be 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℃, discard the supernatant, resuspend the precipitate in an appropriate amount of PBS (0.01M, pH=7.4) and filter through a 0.22μm filter membrane to obtain purified coriander exosomes; the volume of the added PBS should be enough to cover the precipitate.
[0029] The present invention also provides a hydrogel loaded with coriander exosomes prepared by any of the above preparation methods.
[0030] The present invention also provides the application of the above-described hydrogel loaded with coriander exosomes and / or the hydrogel loaded with coriander exosomes prepared by any of the above preparation methods in the preparation of drugs that promote wound healing.
[0031] The present invention also provides a drug for promoting wound healing, comprising a hydrogel loaded with coriander exosomes prepared by any of the preparation methods described herein and / or the hydrogel loaded with coriander exosomes described herein.
[0032] Furthermore, the medicine also includes a pharmaceutically acceptable dressing.
[0033] The beneficial effects of this invention are:
[0034] (1) The novel hydrogel loaded with coriander exosomes provided by the present invention has good viscoelasticity and biocompatibility and can be used as a dressing on the surface of skin wounds; at the same time, the hydrogel has a sustained release effect of coriander exosomes, which prolongs the release time of exosomes and allows them to better exert their biological functions; by the sustained release of coriander exosomes, the hydrogel can relieve oxidative stress and inflammation of wounds and promote wound repair.
[0035] (2) The coriander exosomes loaded with hydrogel in this invention exhibit a typical saucer-like bilayer membrane structure with a particle size of approximately 150 nm. The extracted coriander exosomes contain 2.11 × 10⁻⁶ particles per milliliter. 11 The exosomes are granules that can be ingested by skin cells and have cell migration-promoting, antioxidant, and anti-inflammatory effects, effectively promoting skin wound healing. The method for extracting coriander exosomes provided by this invention can achieve high yield and purity. Attached Figure Description
[0036] Figure 1 The microstructure of coriander exosomes provided by this invention.
[0037] Figure 2 The particle size distribution of coriander exosomes provided by this invention.
[0038] Figure 3 This refers to the uptake of coriander exosomes by human immortalized keratinocytes (HaCaT) in Experiment Example 1.
[0039] Figure 4 The effects of coriander exosomes on the proliferation and migration of HaCaT cells in Experiment 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 Experiment Example 1. A: ROS; B: SOD; C: CAT; D: GSH-Px.
[0041] Figure 6 The effect of coriander exosomes on the transcriptional levels of pro-inflammatory factors and MMP-1 in HaCaT cells stimulated by H2O2 in Experiment 1. A: TNF-α; B: IL-6; C: IL-1β; D: IL-8; E: NF-κB; F: MMP-1.
[0042] Figure 7 The microstructures of the hydrogel and exosome hydrogel in Experiment Example 2 are shown.
[0043] Figure 8 Rheological properties of the hydrogel and exosome hydrogel in Experiment Example 2. A: Strain scan; B: Frequency scan.
[0044] Figure 9 The swelling properties of the hydrogel and exosome hydrogel in Experiment Example 2 are shown.
[0045] Figure 10 The sustained-release curve of the exosome hydrogel in Experiment Example 2 is shown.
[0046] Figure 11 This is the hemolysis experiment of the exosome hydrogel in Experiment Example 2.
[0047] Figure 12 This shows the wound healing status of mice in different treatment groups in Experiment 3. A: Wound change record; B: Wound healing rate.
[0048] Figure 13 The figures show the skin ROS, SOD, CAT, and GSH-Px activities of mice in different treatment groups in Experiment 3. A: ROS; B: SOD; C: CAT; D: GSH-Px.
[0049] Figure 14 The figures show the skin TNF-α and IL-10 levels in mice from different treatment groups in Experiment 3. A: TNF-α; B: IL-10. Detailed Implementation
[0050] The present invention will be further described below with reference to embodiments and accompanying drawings. The technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0051] Unless otherwise specified, the operating methods used in the following embodiments are all conventional operating methods; the materials and reagents used are all commercially available; and the equipment used is all conventional equipment.
[0052] Example 1
[0053] This embodiment provides a method for extracting coriander exosomes, the steps of which are as follows:
[0054] (1) Remove the roots from 200g of fresh coriander, wash it, chop it, and add it to a blender with 200mL of distilled water to extract the juice. Filter the juice through cheesecloth.
[0055] (2) Centrifuge the coriander juice at 3000×g and 10000×g at 4℃ for 20 min and 120 min respectively, and then filter the supernatant through a 0.45μm filter membrane to obtain the filtrate.
[0056] (3) Centrifuge the filtrate at 100,000 × g for 1.5 h at 4 °C, discard the supernatant, and resuspend the precipitate in 0.5 mL PBS (0.01 M, pH = 7.4) to obtain the 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 a centrifuge tube, and then slowly add equal volumes of 45%, 30%, and 8% sucrose solutions along the wall in sequence, ensuring a clear solution interface. 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. Collect the suspension between the 30-45% sucrose layers using a syringe.
[0058] (5) Add 10 mL of PBS to the suspension and centrifuge at 100000×g for 45 min. Discard the supernatant, resuspend the precipitate in 1.5 mL of PBS and filter through a 0.22 μm filter membrane 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 using a transmission electron microscope (TEM). Figure 1 As shown, it exhibits a typical "coffee saucer" structure with a double membrane. Its diameter and concentration were determined by nanoparticle tracking analysis (NTA), such as... Figure 2 As shown, the average particle size is 150.10 ± 38.40 nm. The number of vesicle particles in 1 mL of coriander exosomes extracted by this method is 2.11 × 10⁻⁶. 11 indivual.
[0061] Example 2
[0062] This embodiment provides a method for extracting coriander exosomes, the steps of which are as follows:
[0063] (1) Remove the roots from 200g of fresh coriander, wash it, chop it, and add it to a blender with 150mL of distilled water to extract the juice. Filter the juice through cheesecloth.
[0064] (2) Centrifuge the coriander juice at 3200×g and 9500×g at 4℃ for 25 min and 110 min respectively, and then filter the supernatant through a 0.45μm filter membrane to obtain the filtrate.
[0065] (3) Centrifuge the filtrate at 110000×g for 2h at 4℃, discard the supernatant, and resuspend the precipitate in 0.5mL PBS (0.01M, pH=7.4) to obtain the 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 a centrifuge tube, and then slowly add equal volumes of 45%, 30%, and 8% sucrose solutions along the wall in sequence, ensuring a clear solution interface. 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 hours. Collect the suspension between the 30-45% sucrose layers using a syringe.
[0067] (5) Add 10 mL of PBS to the suspension and centrifuge at 120000×g for 50 min. Discard the supernatant, resuspend the precipitate in 1.5 mL of PBS and filter through a 0.22 μm filter membrane to obtain purified coriander exosomes.
[0068] Example 3
[0069] This embodiment provides a method for extracting coriander exosomes, the steps of which are as follows:
[0070] (1) Remove the roots from 200g of fresh coriander, wash it, chop it, and add it to a blender with 250mL of distilled water to extract the juice. Filter the juice through cheesecloth.
[0071] (2) Centrifuge the coriander juice at 3500×g and 9800×g at 4℃ for 22 min and 100 min respectively, and then filter the supernatant through a 0.45μm filter membrane to obtain the filtrate.
[0072] (3) Centrifuge the filtrate at 120,000 × g for 1.7 h at 4 °C, discard the supernatant, and resuspend the precipitate in 0.5 mL PBS (0.01 M, pH = 7.4) to obtain the 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 a centrifuge tube, and then slowly add equal volumes of 45%, 30%, and 8% sucrose solutions along the wall in sequence, ensuring a clear solution interface. 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. Collect the suspension between the 30-45% sucrose layers using a syringe.
[0074] (5) Add 10 mL of PBS to the suspension and centrifuge at 110000×g for 60 min. Discard the supernatant, resuspend the precipitate in 1.5 mL of PBS and filter through a 0.22 μm filter membrane to obtain purified coriander exosomes.
[0075] Experimental Example 1
[0076] Taking the coriander exosomes extracted in Example 1 as an example, the efficacy of coriander exosomes was verified.
[0077] 1. HaCaT cell uptake of coriander exosomes
[0078] 800 μL of coriander exosomes were incubated with 8 μL of PKH26 and 800 μL of dilution buffer 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 residual dye. The exosomes were resuspended in PBS, and the resuspended solution was transferred to a 100 kDa ultrafiltration tube and centrifuged at 4000 rpm for 20 min to obtain PKH26-labeled coriander exosomes.
[0079] HaCaT cells were categorized at a rate of 1×10 5 Cells were seeded per confocal dish and cultured at 37°C and 5% CO2 for 24 hours. Then, co-incubated with PKH26-labeled coriander exosomes for 0.5 or 6 hours. After washing twice with PBS and fixing with 4% paraformaldehyde, the cell membrane and nucleus were stained with DiO2 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 processed at a rate of 1×10 4 Inoculate each well with one coriander exosome. After culturing for 24 hours, replace the medium with complete culture medium containing coriander exosomes (0, 5, 10, 20, 40, 60 μg / mL), and continue culturing for 12, 24, and 48 hours. Add 10 μL of MTT reagent (5 mg / mL) to each well and continue culturing for 4 hours. Terminate the culture, remove the culture medium from the wells, add 200 μL of DMSO to each well to completely dissolve the crystals, and measure the OD570 using a microplate reader. Figure 4 As shown in Figure A, treatment with coriander exosomes for 12 h or 24 h had no significant effect on cell proliferation. After 48 h of treatment, compared with the control group (0 μg / mL), the viability of HaCaT cells was significantly enhanced by treatment with coriander exosomes at concentrations of 10, 20, and 40 μg / mL.
[0082] HaCaT cells were fed at a rate of 4 × 10 5Cells were seeded at a density of 1 cell / well in 6-well plates. After adhering and forming a monolayer, the original culture medium was aspirated, and scratches were created using a 1 mL pipette tip. Exfoliated cells were then washed with PBS. The control group received complete culture medium, while the experimental groups received equal volumes of complete culture medium containing coriander exosomes (10, 20, and 40 μg / mL), and cultured continuously. Cell status was observed and photographed at 0 and 24 hours after scratch creation using an inverted microscope. 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 was measured over 24 hours. Results showed that 10-40 μg / mL coriander exosomes promoted the migration of HaCaT cells.
[0083] 3. Antioxidant effects of coriander exosomes
[0084] HaCaT cells were used at a rate of 1×10 4 Seeds were planted at a density of [number] cells / well in 96-well plates and cultured for 24 h. The original culture medium was aspirated, and the control and model groups were replaced with fresh complete culture medium. The experimental groups were replaced with complete culture medium containing coriander exosomes (10, 20, and 40 μg / mL), and incubated for 24 h. The culture medium was discarded again. Fresh basal culture medium was added to the control group, while an equal volume of basal culture medium containing 200 μM H2O2 was added to the model and experimental groups. After 2 h, each well was replaced with DCFH-DA solution (10 μM) diluted with basal culture medium. The cells were incubated at 37°C for 20 min, and fluorescence intensity was detected at an excitation wavelength of 488 nm / emission wavelength of 525 nm to characterize intracellular ROS levels. Figure 5 As shown in Figure A, H2O2 stimulation significantly increased the ROS in the model group cells, proving that the oxidative stress model was successfully established; while the ROS in 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 effects of coriander exosomes on intracellular antioxidant enzymes, HaCaT cells were sputtered at 4 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of [number] cells / well in 6-well plates, and the cells were treated using the grouping method described above. Cell suspensions were collected, and the effects of coriander exosomes on SOD, CAT, and GSH-Px activities were evaluated using a commercial kit. Figure 5 As shown in BD, coriander exosomes significantly increased the activity of antioxidant enzymes SOD, CAT, and GSH-Px.
[0086] 4. Anti-inflammatory effects of coriander exosomes
[0087] HaCaT cells were fed at a rate of 4 × 10 5Cells were seeded at a density of [number] cells / well in 6-well plates and treated using the grouping method described above. 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 control gene, and a comparison threshold cycle (2... -ΔΔCt The relative gene expression levels of inflammatory factors and matrix metalloproteinase 1 (MMP-1) were calculated using the [method name missing]. The results are as follows: Figure 6 As shown, H2O2 treatment significantly increased the transcriptional levels of intracellular pro-inflammatory cytokines TNF-α, IL-6, IL-1β, IL-8, and NF-κB. MMPs are expressed in keratinocytes and primarily act on collagen degradation, while collagen deposition is closely related to wound healing; the results showed an increasing trend in MMP-1 levels in the model group. In the experimental group pretreated with coriander exosomes, the transcriptional levels of inflammatory cytokines and MMP-1 decreased to varying degrees, indicating potential for promoting wound healing.
[0088] Example 4
[0089] This embodiment provides a method for preparing a hydrogel loaded with coriander exosomes, the steps of which are as follows:
[0090] (1) Preheat 50mL PBS to 40℃, add 1.0g sodium alginate powder while stirring, stir for 1h, and wait for the sodium alginate to dissolve completely to obtain a 2% (w / v) sodium alginate solution.
[0091] (2) Weigh 1.0g of calcium chloride powder and dissolve it in 50mL of distilled water to obtain a 2% (w / v) calcium chloride solution;
[0092] (3) Add 1 mg of coriander exosomes to each 1 mL sodium alginate solution, mix well, add 1 mL calcium chloride solution, crosslink for 30 s, and obtain a hydrogel loaded with coriander exosomes.
[0093] Experimental Example 2
[0094] The properties of the exosome hydrogel prepared in Example 4 were studied.
[0095] Preparation of blank hydrogels without added coriander exosomes: Add 1 mL of calcium chloride solution to every 1 mL of sodium alginate solution and crosslink for 30 s to obtain blank hydrogels.
[0096] (I) Microstructure
[0097] Cross-sections of blank hydrogels and exosome hydrogels at different magnifications were observed using cryo-electron microscopy. Both exhibited typical cross-linked porous structures with relatively uniform distribution. Figure 7 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 using a rheometer. Samples were subjected to strain scans of 0.1-1000% at a constant frequency of 10 rad / s at 37 °C, and frequency scans of 1-100 rad / s at a constant strain of 1%. Within the strain range of 0.1-10%, both the blank hydrogel and the exosome hydrogel exhibited reversible elastic deformation, maintaining relatively stable high storage modulus (G') and low loss modulus (G”). Figure 8 A). During frequency scanning, G' is always higher than G(). Figure 8 B). The results showed that both the blank hydrogel and the exosome hydrogel exhibited primary elasticity, while the blank hydrogel showed better viscoelasticity.
[0100] (III) Swelling effect
[0101] After drying the hydrogel at 37°C overnight, it was weighed, placed in PBS solution to swell, and the surface water of the hydrogel was removed and weighed again at predetermined time points. The swelling ratio was calculated based on the initial mass. Swelling ratio = (m t -m0) / m0×100%, where represents the initial hydrogel mass, represents the hydrogel mass at each time point, and a swelling curve is plotted based on the swelling ratio. For example... Figure 9 As shown, both hydrogel and exosome hydrogel exhibited swelling rates exceeding 10 after 20 minutes, with the hydrogel showing a slightly higher swelling rate than the exosome hydrogel, but the difference was not statistically significant. This good swelling property indicates that the hydrogel can rapidly absorb wound exudate.
[0102] (iv) Sustained-release performance
[0103] A hydrogel containing 100 μg of exosomes was placed in a 12-well plate, soaked and swollen with 500 μL of PBS, and 250 μL of supernatant was aspirated at predetermined time points. Protein concentration was measured using a BCA protein assay kit, and 250 μL of fresh PBS was added simultaneously. The exosome release rate at each time point was calculated. Figure 10 As shown, exosomes can be released continuously for 3 days. The fitted release curve shows that exosome release is slow in the first 6 hours, then the release rate accelerates, reaching 74.13% by 48 hours.
[0104] (v) Biocompatibility
[0105] Prepare a 4% (v / v) red blood cell suspension. Add 0.5 mL of the 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 μL of the supernatant to measure OD540. Use 0.1% Triton X-100 as a positive control and PBS as a negative control. The hemolysis rate is calculated using 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 values of the sample, negative control, and positive control, respectively. Figure 11 As shown, the positive control group (0.1% Triton X-100) showed severe hemolysis, while the hemolysis rate in the exosome hydrogel group was 0.75±0.27%, which was not statistically different from that in the PBS group.
[0106] Example 5
[0107] This embodiment provides a method for preparing a hydrogel loaded with coriander exosomes, the steps of which are as follows:
[0108] (1) Preheat 50mL of PBS to 35℃, add 1.25g of sodium alginate powder while stirring, stir for 0.5h until the sodium alginate is completely dissolved to obtain a 2.5% (w / v) sodium alginate solution.
[0109] (2) Weigh 1.25g of calcium chloride powder and dissolve it in 50mL of distilled water to obtain a 2.5% (w / v) calcium chloride solution;
[0110] (3) Add 0.5 mg of coriander exosomes to each 1 mL sodium alginate solution, mix well, add 1 mL calcium chloride solution, crosslink for 30 s, and 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 of which are as follows:
[0113] (1) Preheat 50mL PBS to 45℃, add 0.75g sodium alginate powder while stirring, stir for 0.8h until the sodium alginate is completely dissolved to obtain a 1.5% (w / v) sodium alginate solution.
[0114] (2) Weigh 0.75g of calcium chloride powder and dissolve it in 50mL of distilled water to obtain a 1.5% (w / v) calcium chloride solution;
[0115] (3) Add 2 mg of coriander exosomes to each 1 mL sodium alginate solution, mix well, add 1 mL calcium chloride solution, crosslink for 30 s, and obtain a hydrogel loaded with coriander exosomes.
[0116] Experimental Example 3
[0117] The performance of this novel hydrogel was verified using the coriander exosome hydrogel prepared in Example 4 as an example.
[0118] Effect of coriander exosome hydrogel on promoting skin wound healing in mice
[0119] 1. Wound healing effect of coriander exosome hydrogel
[0120] ICR mice (20-25g, 4-6 weeks old) were acclimatized for one week and then randomly divided into three groups: a control group (no treatment), 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 in the back using a punch. During the experiment, the mice were treated with different formulations every 4 days, and the wounds were covered and fixed with 3M transparent dressings. Wound photographs were taken on days 0, 4, 8, and 12 after wound formation, and the wound closure rate was calculated. Figure 12 As shown in Figure A, by day 4, the wound surface of the exosome hydrogel group had already scabbed over, earlier than the control group and the hydrogel group. By day 12, the wounds in the exosome hydrogel group were almost invisible, indicating good healing. Further quantification of wound healing rates showed that by day 8, the wound healing rate of the exosome hydrogel group was significantly higher than that of the control group and the hydrogel group. On day 12, the average wound healing rates of each group were 88.24% (control group), 90.61% (hydrogel group), and 96.21% (exosome hydrogel group), demonstrating the good wound healing properties of exosome hydrogel. Figure 12 B).
[0121] 2. Antioxidant effect of coriander exosome hydrogel
[0122] Blood was collected from each group of mice, and serum was obtained after coagulation and centrifugation, followed by measurement of ROS levels. Figure 13 A showed 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 using a kit. The results showed that exosome hydrogel treatment could effectively increase the activity of antioxidant enzymes (…). Figure 13 B). High antioxidant enzyme activity helps to clear intracellular ROS and maintain redox balance.
[0123] 3. Anti-inflammatory effects of coriander exosome hydrogel
[0124] Skin samples were collected from wounds of mice in each group, and the levels of pro-inflammatory TNF-α and anti-inflammatory IL-10 were detected using an ELISA kit. The results showed that the TNF-α level was significantly lower in the exosome hydrogel group. Figure 14 A), while IL-10 levels were significantly higher than in the other two groups ( Figure 14 B) indicates that exosome hydrogels can effectively relieve wound inflammation and accelerate wound repair.
[0125] As can be seen from the above embodiments, the present invention provides a novel hydrogel for promoting wound healing and its applications. The hydrogel loaded with coriander exosomes prepared by the present invention has good viscoelasticity and biocompatibility, and promotes skin wound healing by slowly releasing coriander exosomes with cell migration-promoting, antioxidant, and anti-inflammatory effects. The present invention is of great significance for the development of novel wound-healing dressings in the clinical field.
[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing a hydrogel that promotes wound healing, characterized in that, Includes the following steps: Cilantro exosomes were extracted from cilantro using a gradient centrifugation method combined with a sucrose density gradient method. The cilantro exosomes were mixed with sodium alginate solution at a mass-to-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 cilantro exosomes.
2. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the coriander exosomes to the sodium alginate solution was 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 was obtained by dissolving sodium alginate in PBS, and the concentration of the sodium alginate solution was 1.5-2.5% (w / v); the calcium chloride solution was obtained by dissolving calcium chloride in distilled water, and the concentration of the calcium chloride solution was 1.5-2.5% (w / v).
5. The preparation method according to claim 1, characterized in that, The extraction of coriander exosomes includes the following steps: (1) Extract cilantro juice by juicing distilled water and cilantro at a mass ratio of 0.75 to 1.25:1; (2) The coriander juice was subjected to gradient centrifugation in sequence, and the supernatant was filtered to obtain the filtrate. The gradient centrifugation was performed as follows: First centrifugation: Centrifuge at 3000×g~3500×g for 20min~25min at 4℃. Second centrifugation: Centrifuge at 9500×g~10000×g for 100min~120min at 4℃; (3) Centrifuge the filtrate at 100,000×g~120,000×g for 1.5h~2h at 4℃, discard the supernatant, and resuspend the precipitate in an appropriate amount of PBS (0.01M, pH=7.4) to obtain a resuspension; (4) Transfer the resuspended solution to a sucrose density gradient solution with mass fractions of 8%, 30%, 45%, and 60% from top to bottom, and centrifuge at 140,000×g to 160,000×g for 1.5h to 2h, and collect the suspension between the 30-45% sucrose layers; (5) Add 8 to 12 times the volume of PBS to the suspension and centrifuge at 100,000 to 120,000 g for 45 to 60 minutes at 4°C. Discard the supernatant, resuspend the precipitate in an appropriate amount of PBS, and filter to obtain purified coriander exosomes.
6. The preparation method according to claim 5, characterized in that, Includes the following steps: (1) Remove the roots from an appropriate amount of fresh coriander, wash it, chop it, add it to a blender to extract the juice, add distilled water to extract the juice, and filter it through gauze to obtain coriander juice; the mass ratio of distilled water to coriander is 1:
1. (2) Centrifuge the coriander juice at 3000×g and 10000×g at 4℃ for 20 min and 120 min respectively, and take the supernatant obtained at the end and filter it through a 0.45μm filter membrane to obtain the filtrate; (3) Centrifuge the filtrate at 100,000 × g for 1.5 h at 4 °C, discard the supernatant, and resuspend the precipitate 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 along the wall in sequence. The solution interface should be distinct. Transfer the resuspension from 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 30-45% sucrose layers. (5) After adding 10 times the volume of PBS to the suspension, centrifuge at 100,000×g for 45 min at 4℃, discard the supernatant, resuspend the precipitate in an appropriate amount of PBS and filter through a 0.22μm filter membrane to obtain purified coriander exosomes.
7. The hydrogel loaded with coriander exosomes prepared by the preparation method according to any one of claims 1-6.
8. The use of the hydrogel loaded with coriander exosomes according to claim 7 and / or the hydrogel loaded with coriander exosomes prepared by the preparation method according to any one of claims 1-6 in the preparation of a drug for promoting wound healing.
9. A medicament for promoting wound healing, comprising a hydrogel loaded with coriander exosomes prepared by any one of claims 1-6 and / or a hydrogel loaded with coriander exosomes as described in claim 7.
10. The medicament according to claim 9, characterized in that, It also includes pharmaceutically acceptable dressings.