Application of exosomes from rabdosia rubescens in preparation of drugs for promoting wound healing

Abstract

The present application relates to the field of biotechnology, in particular to the application of Radix Rabdosiae Rubescens derived exosomes in the preparation of a drug for promoting wound healing, wherein the prepared Radix Rabdosiae Rubescens derived exosomes are uniform in size, good in appearance, can promote cell proliferation, have good effects of promoting fibroblast migration and reducing scar formation; and a scar-free wound healing hydrogel containing Radix Rabdosiae Rubescens derived exosomes is also provided, wherein the hydrogel can encapsulate rEVs or ORI, can effectively increase the proportion of type III collagen in the new skin tissue, effectively reduce the formation of scars, effectively promote the healing of deep second-degree burn wounds, and effectively inhibit the MMT process of macrophages in the wound healing process, and has a wide application prospect in the field of scar prevention and treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of exosomes derived from *Rabdosia rubescens* in the preparation of drugs that promote wound healing. Background Technology

[0002] Rabdosia rubescens is a traditional Chinese herbal medicine widely cultivated in the Yellow River basin of my country. Its main active ingredient, oridonin (ORI), is a tetracyclic diterpenoid natural organic compound (C20H28O6, MW: 364.43 g / mol) that has been proven to have good anti-inflammatory effects. Studies have shown that ORI can inhibit the activation of the NF-κB and MAPK pathways, thereby inhibiting the release of various pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore, ORI can directly form a covalent bond with NLRP3, thereby inhibiting the assembly of inflammasomes and exhibiting effective anti-inflammatory activity. Whether ORI, as a potential candidate anti-inflammatory small molecule drug, can be used to alleviate excessive inflammatory responses in deep second-degree burn wounds warrants further investigation. On the other hand, although the pharmacological activity of traditional medicinal plants is mostly attributed to their small molecule compounds, recent studies have found that their exosomes may be another key factor mediating multi-target therapeutic effects.

[0003] Plant-derived exosomes (PDExos) are nanoscale (30-150 nm) bilayer lipid membrane vesicles secreted by medicinal plant cells. They are rich in proteins, nucleic acids, lipids, and various secondary metabolites, playing a crucial role in mediating intercellular communication and regulating target cell function. Due to their wide availability, high biocompatibility, and low immunogenicity, they are gradually becoming a cutting-edge research direction in the development of natural drugs. For example, exosome-like nanovesicles (TDNPs) extracted from turmeric rhizomes can significantly alleviate DSS-induced ulcerative colitis in mice by inhibiting the NF-κB signaling pathway, reducing the secretion of pro-inflammatory factors (such as TNF-α and IL-6), and promoting the release of anti-inflammatory factors (such as IL-10). Pueraria exosomes can be efficiently taken up by macrophages, and by downregulating their pro-inflammatory genes (IL-6, IL-1β, TNF-α, CD11c) and upregulating their anti-inflammatory genes (IL-10, YM1, CD206), macrophages are induced to polarize towards the M2 type.

[0004] Currently, there are no reports on the function of exosomes derived from *Rabdosia rubescens*. Our preliminary research has preliminarily confirmed that it may have the ability to inhibit macrophage-fibroblast transformation (MMT), and therefore may have the potential to reduce scarring in wound tissue. Further research into its effects on deep second-degree burns and exploration of its mechanism of action has significant theoretical implications and promising clinical applications. Summary of the Invention

[0005] The primary objective of this invention is to provide the application of exosomes derived from *Rabdosia rubescens* in the preparation of drugs that promote wound healing.

[0006] Preferably, the wound includes chronic wounds, acute trauma, surgical incisions, burns, or ulcers.

[0007] A second objective of this invention is to provide the use of exosomes derived from *Rabdosia rubescens* in the preparation of drugs that reduce scar formation.

[0008] Preferably, the scar includes hypertrophic scars, atrophic scars, or keloids.

[0009] Preferably, the exosomes derived from *Rabdosia rubescens* are prepared by the following method: Fresh *Rabdosia rubescens* leaves are picked, washed clean, and then washed twice more with PBS. The *Rabdosia rubescens* leaves and PBS are weighed at a mass-to-volume ratio of 1:3. The cell walls are broken, the obtained juice is filtered, leaf residue is removed, and centrifuged. The supernatant is balanced and centrifuged again to remove impurities of different sizes. The supernatant is filtered, and the filtered solution is transferred to a centrifuge tube and centrifuged. The supernatant is discarded, and the precipitate is resuspended in PBS. The precipitate is centrifuged again, impurities are washed away, the supernatant is discarded, and the precipitate is resuspended in PBS according to the amount of precipitate.

[0010] The third objective of this invention is to provide a scarless, wound-healing-promoting hydrogel containing exosomes derived from *Rabdosia rubescens*, wherein the hydrogel is made by mixing F127 and F68 in a ratio of 22.5%:2.5% and adding exosomes derived from *Rabdosia rubescens*.

[0011] A fourth objective of this invention is to provide the use of the hydrogel in the preparation of drugs that promote wound healing.

[0012] A fifth objective of this invention is to provide the application of the hydrogel in the preparation of medicaments that inhibit or reduce scar formation.

[0013] The sixth objective of this invention is to provide the application of the hydrogel in the preparation of medical materials for scar prevention.

[0014] The beneficial effects of this invention are: (1) This invention provides a method for preparing exosomes derived from *Rabdosia rubescens*, wherein the exosomes derived from *Rabdosia rubescens* are uniform in size with an average particle size of less than 150 nm. Under a transmission electron microscope, the exosomes derived from *Rabdosia rubescens* show good morphology, appearing as round vesicles, uniformly distributed in the solution, and can promote cell proliferation. Experimental results show that the ORI group did not have the ability to promote cell migration, while after 24 h of treatment with rEVs, the cell migration rate was significantly improved compared with the control group; rEVs have a good effect on promoting fibroblast migration.

[0015] (2) Compared with the control group, the ORI treatment group showed a significant increase in α-SMA positive cells, while the rEVs treatment group showed a significant decrease. This indicates that ORI can promote macrophage MMT, while rEVs can effectively inhibit macrophage MMT. Since excessive fibrosis is an important cause of scar formation during the wound healing and remodeling period, rEVs may have the potential to reduce scar formation by inhibiting macrophage MMT and have good hematologic safety.

[0016] (3) This invention provides a scarless wound-healing hydrogel containing exosomes derived from *Rabdosia rubescens*. The hydrogel can encapsulate rEVs or ORI. When applied to rabbit ear scar experiments, the results showed that the newly formed wound in the rEVs / cryoMNs group was thinner, possibly because rEVs inhibited excessive macrophage fibrosis, reducing scar formation. Compared to the scar control group, the blank microneedle and ORI microneedle groups failed to effectively alleviate the thickness of scar formation, and the scars were mainly composed of type I collagen. In contrast, the thickness of rabbit ear scars was significantly reduced in the rEVs microneedle group, and the scar tissue was mainly composed of green type III collagen. rEVs can effectively increase the proportion of type III collagen in newly formed skin tissue, effectively reducing scar formation.

[0017] (4) Wound healing photographs show that the spontaneous healing rate of deep second-degree burn wounds in the control group was very slow; the wound healing process in the blank microneedle group was similar to that in the control group, showing no obvious healing-promoting effect; while both drug-treated groups showed strong healing-promoting ability for deep second-degree burn wounds, especially the rEVs group, where the residual wound rate showed a significant difference from the control group and the blank microneedle group on day 4, and by day 13, the residual wound rate had dropped to below 3%. This result indicates that rEVs can effectively promote the healing of deep second-degree burn wounds. HE staining results show that the composition of the newly formed tissue is relatively simple, with no formation of skin appendages such as sweat glands and hair follicles. Masson staining shows that the newly formed skin tissue is mainly composed of collagen deposition, exhibiting the general characteristics of scars. Among them, the ORI microneedle group had the thickest collagen deposition, while the rEVs treatment group had less collagen deposition compared to the ORI treatment group. This result indicates that drug-treated microneedles can effectively promote the healing of deep second-degree burn wounds, and the presence of rEVs is beneficial to reducing scar formation.

[0018] (5) In the blank microneedle and ORI microneedle treatment groups, the number of macrophages and α-SMA expression were both relatively high, and no effect was shown in inhibiting the MMT process. However, in the rEVs group, macrophage expression was almost absent, and the α-SMA co-expressed with macrophages was also significantly reduced, indicating that rEVs can effectively inhibit the macrophage MMT process during wound healing, which may be the main reason for its effective inhibition of scar formation. Attached Figure Description

[0019] Figure 1 Particle size distribution analysis and morphological characterization of exosomes derived from Rabdosia rubescens; Note: (A) Particle size distribution of rEVs; (B) Transmission electron microscopy (TEM) image of rEVs, scale bar 200 nm.

[0020] Figure 2 Effects of rEVs on the viability of (A) L929 cells and (B) RAW264.7 cells.

[0021] Figure 3 Effects of ORI on the viability of (A) L929 cells and (B) RAW264.7 cells.

[0022] Figure 4 Preparation of F127 (22.5%) + F68 (2.5%) thermosensitive gel.

[0023] Figure 5 Physicochemical characterization of F127 / F68 thermosensitive gel; Note: (A) Thermogravimetric analysis of F127 / F68 thermosensitive hydrogels with different ratios; (B) FT-IR spectra of F127, F68, F127+F68 and ORI, rEVs, and ORI+rEVs loaded respectively.

[0024] Figure 6 Evaluation of the effects of ORI and rEVs on the proliferation and migration of L929 cells; Note: (A) Cell migration; (B) Quantitative graph of cell migration rate in each group. Data are expressed as mean ± SD, n=3, ns represents p>0.05. * This means p < 0.05. **** This represents the p<0.0001 vs. Control group, with a scale bar of 500 μm.

[0025] Figure 7 RAW264.7 cell immunofluorescence staining (red indicates macrophage marker F4 / 80, green indicates myofibroblast marker α-SMA); Note: (A) Representative image of RAW264.7 cell immunofluorescence staining; (B) Quantitative graph of mean fluorescence intensity of α-SMA. Data are expressed as mean ± SD, n=3. **** This represents the group p<0.0001 vs. cont.

[0026] Figure 8 Blood safety evaluation of different cryoMNs, n=3.

[0027] Figure 9 Establishment and treatment of rabbit ear scar model; (A) Flowchart of rabbit ear scar model establishment and treatment plan; (B) Healing status of rabbit ear wounds in different treatment groups from 1 to 14 days.

[0028] Figure 10 Sirius red staining results of rabbit ear wound tissue; scale bar: 200 μm, n=3, data are expressed as mean ± SD, ns represent p>0.05. *** This means p < 0.001. **** This represents p<0.0001 vs. the control group.

[0029] Figure 11 Establishment and treatment of a mouse model of deep second-degree burns; (A) Procedure and experimental protocol for establishing a mouse model of deep second-degree burns; (B) Wound healing status of mice in different treatment groups from day 1 to 13; (C) Residual wound rate at different time points; (n=3) Data are expressed as mean ± SD. ** This means p < 0.01. **** This represents p<0.0001 vs. the control group.

[0030] Figure 12 HE and Masson staining of newly formed skin tissue from mice on day 14, scale bar: 500 μm.

[0031] Figure 13 Immunofluorescence staining of wound tissue in mice on day 14 of treatment; (A) Immunofluorescence staining results of macrophage marker F4 / 80 (green) and myofibroblast marker α-SMA (red) in each treatment group, scale bar 100 μm; (B) Quantitative analysis of α-SMA fluorescence intensity in each group; (n=3) Data are expressed as mean ± SD. **** This represents p<0.0001 vs. the control group. Detailed Implementation

[0032] The scope of protection of the present invention will be described in detail below with reference to specific embodiments. It should be noted that the scope of protection of the present invention is not limited by the following embodiments.

[0033] Example 1: Extraction and Characterization of Exosomes from Rabdosia rubescens 1. Extraction of exosomes (rEVs) from Rabdosia rubescens Fresh leaves of *Rabdosia rubescens* were picked, washed thoroughly with running water, and then washed twice more with PBS. An appropriate amount of clean leaves was weighed into a blender, with 150 mL of PBS added for every 50 g of leaves. The blending process lasted 2 minutes. The resulting juice was filtered through a double-layer filter to remove leaf residue. The juice was then aliquoted into 50 mL centrifuge tubes, balanced, and centrifuged at 5000 g, 4 °C for 30 minutes. The supernatant was then balanced again and centrifuged at 8000 g, 4 °C for 45 minutes, 11000 g, 4 °C for 1 hour, and 50000 g, 4 °C for 1 hour to remove impurities of varying sizes. The supernatant was then filtered through a 0.45 μm filter. The filtered solution was transferred to an ultracentrifuge tube, precisely balanced, and centrifuged at 150000 g, 4 °C for 70 minutes. The supernatant was discarded, and the precipitate was resuspended in 20 mL of PBS. The solution was then centrifuged again at 150000 g, 4 °C for 70 minutes. After washing away impurities, discard the supernatant, add an appropriate amount of PBS to resuspend the precipitate, and store it in a -80 ℃ refrigerator for later use.

[0034] 2. Characterization of exosomes derived from Rabdosia rubescens (1) Detection of exosome size of *Rabdosia rubescens*: The solution containing exosomes from *Rabdosia rubescens* was ultrasonically dispersed and mixed at 4 ℃. 10 μL of the solution was added to 1 mL of PBS for dilution. Then, 10 μL of the diluted solution was added to 1 mL of PBS for further dilution. The solution was ultrasonically dissolved at 4 ℃ for 30 s and then added to the sample cell. The particle size of the sample was detected using a Malvern particle size analyzer. Each sample was tested three times.

[0035] (2) Morphological and distribution observation: The morphology and distribution of the extracted exosomes from *Rabdosia rubescens* were characterized by transmission electron microscopy (TEM).

[0036] The results are as follows Figure 1 As shown in Figure A, the exosomes derived from *Rabdosia rubescens* extracted by ultracentrifugation were uniform in size, with an average particle size below 150 nm. Under a transmission electron microscope, the exosomes from *Rabdosia rubescens* exhibited good morphology, appearing as round vesicles and uniformly distributed in the solution. Figure 1 B). This indicates that the present invention extracted exosomes from *Rabdosia rubescens*, and the extracted exosomes from *Rabdosia rubescens* had good morphology and uniform particle size.

[0037] 3. Evaluation of the cell compatibility of exosomes derived from Rabdosia rubescens The cytocompatibility of rEVs was evaluated by using different concentrations of exosomes derived from *Rabdosia rubescens* to promote the proliferation of mouse fibroblasts (L929) (ATCCCCL-1) and mouse monocyte-macrophage leukocytes (RAW264.7) (ATCC CCL-1).

[0038] (1) Plating: Take a 96-well plate and add the digested and counted L929 or RAW264.7 cell suspension to the wells (3×10⁻⁶ cells per well). 3 (100 μL / well, 1 cell / well) were placed in a cell culture incubator (37 °C, containing 5% CO2) and cultured overnight.

[0039] (2) Drug addition: The rEVs stock solution was diluted with PBS to 2 mg / mL, and then diluted with DMEM or 1640 complete medium to 5, 10, 20, 30, 40, 50, 60, 70, and 80 μg / mL. When the cell density in the 96-well plate reached 90%, the medium was discarded, and 100 μL of the corresponding concentration of rEVs solution was added. The control group was added with the same volume of DMEM or 1640 complete medium. Six replicates were made for each group, and the cells were placed in a cell culture incubator (37 ℃, containing 5% CO2) for 24 h.

[0040] (3) Add MTT: After incubating with the drug in a cell culture incubator for 24 h, discard the supernatant containing different concentrations of rEVs, add 100 μL of MTT solution (MTT to complete culture medium 1:9) to each well, and incubate in a cell culture incubator for 2.5-4 h under dark conditions.

[0041] (4) Measure the maximum absorbance and calculate the cell viability: Discard the MTT supernatant, add 150 μL of DMSO solution to each well, shake on a shaker for 5 min, measure the absorbance at 490 nm, and calculate the cell viability of each group based on the absorbance.

[0042] Figure 2 The effects of exosomes derived from *Rabdosia rubescens* on L929 cells ( Figure 2 A) and RAW264.7 cells ( Figure 2 B) Effect on proliferation: In the concentration range of 5-60 μg / mL, rEVs have a certain promoting effect on the proliferation of both types of cells, but the effect is not significant. Moreover, high concentrations of rEVs have an inhibitory effect on the proliferation of RAW264.7 cells. Therefore, the concentration of rEVs loaded in the subsequent preparation of microneedles was 40 μg / mL.

[0043] Example 2: Preparation and Characterization of Oridonin A Solution 1. Preparation of oridonin A (ORI) solution Weigh 2 mg of ORI powder and dissolve it in 10 mL of ddH2O. Sonicate for 3 min to disperse it evenly, and obtain a 0.2 mg / mL LORI solution. Seal and store at room temperature for later use.

[0044] 2. Evaluation of cell compatibility of ORI solution The cytocompatibility of ORI was evaluated by toxicity experiments on mouse mononuclear macrophage leukocytes (RAW264.7) and mouse fibroblasts (L929) with different concentrations of ORI solution.

[0045] (1) Digesting cells: When the RAW264.7 or L929 cells in the culture dish or culture flask have grown to 90% or more, digest the cells with PBS or trypsin and collect them into 15 mL centrifuge tubes. After centrifugation, add 3 mL of complete culture medium and resuspend the cell pellet.

[0046] (2) Counting: Take a cell counting chamber, sterilize and dry it, take a coverslip and place it on the counting chamber to cover it. Take 10 μL of cell suspension and drop it steadily and quickly along the coverslip onto one side of the counting chamber, allowing it to flow naturally into the counting chamber on one side. Count under a 10x microscope.

[0047] (3) Plate formation: Add 1×10⁻⁶ L929 or RAW264.7 cell suspension (after counting) to a 96-well plate. 4 (100 μL / well, 1 cell / well) and incubated overnight in a cell culture incubator (37 °C, containing 5% CO2).

[0048] (4) Adding drugs: Dilute the ORI solution with DMEM or 1640 complete medium to 0, 5, 10, 15, 20, 30, 50 and 60 μg / mL. When the cell density in the 96-well plate reaches 90%, discard the medium and add 100 μL of the corresponding concentration of ORI solution. For the 0 μg / mL group, add 100 μL of DMEM or 1640 complete medium. There are 6 replicates for each group. Place the cells in a cell culture incubator (37 ℃, containing 5% CO2) for 24 h.

[0049] (5) Add MTT: After incubating with the drug in a cell culture incubator for 24 h, discard the supernatant containing different concentrations of ORI, add 100 μL of MTT solution (MTT to complete culture medium 1:9) to each well, and incubate in a cell culture incubator for 2.5-4 h under dark conditions.

[0050] (6) Measure the maximum absorbance and calculate the cell viability: Discard the MTT supernatant, add 150 μL of DMSO solution to each well, shake on a shaker for 5 min, measure the absorbance at 490 nm, and calculate the cell viability of each group based on the absorbance.

[0051] Although ORI has anti-inflammatory effects, it is highly toxic to cells at high concentrations. Therefore, cytotoxicity experiments were conducted to test the cell compatibility of the ORI solution and determine the optimal ORI concentration loaded in cryo-microneedles. Figure 3 As shown, the viability of both L929 and RAW264.7 cells decreased to varying degrees with increasing ORI concentration. When the ORI concentration reached 30 μg / mL, the viability of L929 cells dropped below 50%. Figure 3 A), while ORI had a smaller effect on RAW264.7 cells ( Figure 3 (B) When the concentration reached 60 μg / mL, cell viability decreased to approximately 70%. The results show that when the ORI concentration was 10 μg / mL, it did not produce toxic effects on either type of cell. Therefore, we chose a concentration of 10 μg / mL as the ORI drug treatment concentration for subsequent experiments.

[0052] Example 3: Preparation and Characterization of F127 / F68 Thermosensitive Hydrogel 1. Preparation of F127 / F68 thermosensitive hydrogel Prepare a 20 mL screw-top glass sample vial. Weigh 3.375 g of F127 and 0.375 g of F68 into the vial, and add 11.25 mL of ddH2O. Place an ice box on a magnetic stirrer and place the sample vial in the ice box (4 ℃) and stir for 6 h. Then, let it stand overnight in a 4 ℃ refrigerator to remove air bubbles and store it in a 4 ℃ refrigerator for later use. When using, take it out and place it in a 32 ℃ oven for 5 min to allow it to solidify into a gel.

[0053] 2. Physicochemical characterization of thermosensitive gels with different ratios of F127 / F68 (1) Fourier transform infrared spectroscopy (FT-IR) scanning: Take 1 mL of F127 / F68 thermosensitive solution with different proportions into 5 mL EP tubes, place them in an oven at 32 ℃ for 5 min to heat up and solidify into a gel, place them in an ultra-low temperature freezer and freeze for at least 30 min, then transfer them to a vacuum freeze dryer to dry overnight to obtain F127 / F68 powder with different proportions, weigh them and seal them at 4 ℃; take a small amount of F127, F68, F127 / F68 powder with different proportions and an appropriate amount of potassium bromide into a mortar, dry the mixed powder under a tungsten lamp for 10 min, remove as much moisture as possible, grind and mix them, press them into thin sheets with a tablet press, and scan the infrared absorption spectrum of the samples with an FT-IR instrument.

[0054] (2) Thermogravimetric analysis: Thermogravimetric analysis was performed on F127 / F68 powders of different proportions using an SDT Q600 thermogravimetric analyzer. The thermogravimetric analyzer was preheated in advance. An appropriate amount of alumina was placed in the sample pan of the thermogravimetric analyzer. After leveling the sample pan, a small amount of F127, F68, and F127 / F68 powders of different proportions were placed in the other sample pan to ensure that the samples were placed stably and to avoid moving the samples during heating. Under nitrogen protection, the initial temperature was set to 30 ℃, the heating rate was 20 ℃ / min, and the measurement range was 0-800 ℃. After confirming that the parameters were correct, the thermogravimetric analyzer was started.

[0055] In previous studies, our research group experimentally investigated the mixing ratio of the two substances and their corresponding lower critical solution temperatures. Considering both ambient temperature and the mechanical strength of the hydrogel, we ultimately selected a mixing ratio of F127 (22.5%) + F68 (2.5%). Previous experiments revealed that the lower critical solution temperature of this hydrogel solution was 31.2 ± 0.4℃. We successfully prepared an F127 (22.5%) + F68 (2.5%) hydrogel solution using a low-temperature stirring method, and this solution successfully transitioned from a liquid to a gel state when the temperature reached the lower critical solution temperature. Figure 4 ).

[0056] Hydrogels of different proportions were freeze-dried into powder and then subjected to thermogravimetric analysis, such as... Figure 5 As shown in Figure A, as the temperature increases, F68 begins to lose mass at 200 °C, and F127 begins to lose mass near 400 °C. The hydrogel material with the ratio of F127 (22.5%) + F68 (2.5%) only undergoes weight change near 400 °C compared to the other two ratios, indicating that this ratio of hydrogel material has better thermal stability.

[0057] Next, we loaded ORI and rEVs onto the hydrogels of this ratio, respectively. The specific process is as follows: F127 and F68 were mixed at 4℃ in a ratio of 22.5%:2.5%. While mixing with magnetic stirring, oridonin A at a concentration of 10 μg / mL and exosomes from oridon at a concentration of 40 μg / mL were added to the mixed solution. After stirring evenly, the mixture was placed at room temperature. When the temperature rose to about 25℃, it spontaneously formed a gel, thus preparing the hydrogels loaded with ORI or rEVs, which were named ORI MNs group or rEVs MNs group, respectively. Here, MNs has the same meaning as cyroMNs in the following text and is used for subsequent experiments.

[0058] Fourier transform infrared analysis was performed on it. Figure 5 B), as can be seen from the results in the figure, the characteristic peaks of the raw materials were not changed after the addition of the two drugs, indicating that loading ORI and rEVs did not affect the properties of F127 and F68.

[0059] Example 4: In vitro experiments 1. Scratch assay to evaluate the effects of ORI and rEVs on the proliferation and migration of L929 cells. (1) Plating and grouping: Take 1 mL of digested and resuspended L929 cell suspension and plate it in 6-well plates (4×10⁻⁶). 5 1 cell / well, 1 mL / well), incubated overnight in a cell culture incubator (37 °C, containing 5% CO2).

[0060] (2) Scratching, drug addition, and photography: When the cell density in the well plate reaches about 90%, take a 200 μL pipette tip and slowly draw a straight and uniform line along the ruler at the bottom of the well plate. The line width of each group should be kept as consistent as possible. Then discard the supernatant of each well and add an appropriate amount of PBS (usually 1 mL) to each well for washing twice. When adding drugs, add 2 mL of fresh DMEM complete culture medium to the control group and 2 mL of 10 μg / mL ORI solution and 40 μg / mL rEVs solution diluted with DMEM complete culture medium to the drug group respectively. Take pictures under a microscope and record the scratch area of ​​each group at 0 h. Perform two replicates for each group.

[0061] (3) Photographs: The well plates were removed at 24 h and 48 h after drug administration, and photographed under a microscope. The area of ​​the scratched area in each group was observed and recorded.

[0062] (4) Calculate the mobility:

[0063] The results are as follows Figure 6 As shown in A and B, compared with the Control group, the ORI group did not have the ability to promote cell migration, while after 24 h of rEVs treatment, the cell migration rate was significantly improved compared with the control group; by 72 h of drug treatment, the cell migration rate of the rEVs-treated group reached about 70%, which was significantly higher than that of the control group. rEVs have a good effect on promoting fibroblast migration.

[0064] 2. Cell immunofluorescence staining assay to evaluate the effect of rEVs or ORI on the transformation of macrophages into myofibroblasts. The effects of rEVs or ORI on the transformation of RAW264.7 macrophages into myofibroblasts were evaluated using cellular immunofluorescence assays to assess the regulatory effects of the two drugs on the MMT process.

[0065] (1) Cell treatment: When the RAW264.7 cell density in the cell culture dish reaches more than 90%, discard the supernatant, add 4 mL of physiological saline to gently wash twice, and then add a certain volume of PBS to act for 2 min; use a 1 mL pipette tip to gently blow off the cells attached to the bottom of the culture dish, collect them in a 15 mL centrifuge tube, centrifuge and discard the supernatant, add 3 mL of DMEM to completely culture and resuspend the cell pellet.

[0066] (2) Counting: Take 10 μL of cell suspension and count the total number of cells on a counting plate.

[0067] (3) Grouping: The experimental groups were set as control group, TGF-β group, TGF-β+ ORI group, TGF-β+ rEVs group, and TGF-β+ ORI+ rEVs group, for a total of five groups.

[0068] (4) Plating: Soak 20 mm diameter cell slides in a small culture dish containing industrial alcohol for 10 min; pick up the slides with tweezers, dab them on gauze to absorb excess alcohol as much as possible, and burn off the alcohol on the slides over an alcohol lamp; slowly place the burned-off slides flat in a 12-well plate, and after the slides have cooled, transfer the counted cell suspension (1.8 × 10⁻⁶ cells / well) into the wells. 5 Add cells / well, 1 mL) to the slide; three replicates per group, and incubate in a cell culture incubator (37 ℃, containing 5% CO2) for 24 h.

[0069] (5) Adding drugs: When the cells adhere to the climbing slide and the density reaches 80%, discard the culture medium in the wells and add 1 mL of room temperature PBS to each well to wash gently twice; add 1 mL of DMEM complete culture medium and 10 ng / mL of TGF-β solution diluted with DMEM complete culture medium to the control group and the TGF-β group respectively; add 10 ng / mL of TGF-β solution diluted with DMEM to the TGF-β+ ORI group, the TGF-β+ rEVs group and the TGF-β+ ORI+ rEVs group respectively; place in a cell culture incubator (37 ℃, containing 5% CO2) and continue to culture for 24 h.

[0070] (6) Change of medicine: Discard the supernatant of TGF-β+ ORI group, TGF-β+ rEVs group and TGF-β+ ORI+ rEVs group, wash twice with 1 mL of room temperature PBS; add 10 μg / mL ORI solution, 40 μg / mL rEVs solution and 10 μg / mL ORI+ 40 μg / mL rEVs solution diluted with DMEM according to the group; incubate in a cell culture incubator for 24 h.

[0071] (7) Fixation: Discard the supernatant in the 12-well plate, gently wash twice with an appropriate amount of PBS solution (usually 1 mL), add an equal volume of 4% paraformaldehyde solution to each well to cover the slide, and let it stand for 10 minutes to fix; discard the paraformaldehyde, add 1 mL of PBS solution to each well, and gently wash 3 times.

[0072] (8) Blocking: Add 1 mL of 5% goat serum to each well to completely cover the plate, seal the plate with a sealing strip, and incubate in a cell culture incubator (37 ℃, containing 5% CO2) for 30 min.

[0073] (9) Primary antibody incubation: Remove the goat serum blocking solution from the wells, add a certain amount of primary antibody working solution prepared with antibody diluent to each well, and completely cover the slide. Seal the well plate with a sealing strip and incubate overnight at 4 ℃.

[0074] (10) Warming: Take out the 12-well plate, let it stand for 15 min to warm, recover the primary antibody in the well plate, and wash with TBST buffer and TBS buffer 1-3 times, 5 minutes each time.

[0075] (11) Incubate the secondary antibody: Add the secondary antibody (fluorescent) corresponding to the primary antibody to the well plate, completely cover the plate, and incubate at 37°C in the dark for 1 hour; recover the secondary antibody and wash it 1-3 times with TBST buffer and TBS buffer, 5 minutes each time.

[0076] (12) DAPI staining: Add 1 µg / mL DAPI working solution to each well and incubate at room temperature for 30 minutes in the dark; remove the DAPI working solution and wash with TBST buffer and TBS buffer 1-3 times, 5 minutes each time.

[0077] (13) Use tweezers to remove the slide, place it on a glass slide with anti-fluorescence attenuation mounting medium, and take a picture under a fluorescence microscope.

[0078] The results are as follows Figure 7 As shown, red represents the macrophage marker F4 / 80, and green represents the myofibroblast marker α-SMA. The results show that after TGF-β induction, the control group expressed a certain amount of α-SMA-positive cells, indicating the induction of macrophage MMT. Compared with the control group, the ORI treatment group showed a significant increase in α-SMA-positive cells, while the rEVs treatment group showed a significant decrease. This indicates that ORI can promote macrophage MMT, while rEVs can effectively inhibit macrophage MMT. Since excessive fibrosis is a major cause of scar formation during wound healing and remodeling, rEVs' inhibition of macrophage MMT may have the potential to reduce scar formation.

[0079] 3. Hemolysis test to evaluate the biocompatibility of different cryomicroneedles Grasp a normal mouse, trim the whiskers on both sides from the base, hold the mouse in an upside-down position with one hand, and wait for the eyeballs to become congested and bulging. Gently pull and slowly remove the eyeballs with forceps, collecting the flowing blood in a pre-prepared anticoagulant tube. Add 10 mL of physiological saline to the red blood cells and mix well. Centrifuge at 10000 g for 5 min, discard the supernatant, and resuspend the precipitate with 10 mL of physiological saline. Repeat the above operation until the supernatant after centrifugation is clear. Add 5 mL of physiological saline to resuspend the red blood cells in the precipitate again. Take 1 / 4 and 1 / 2 of F127 / F68 cryoMNs, ORI cryoMNs, and rEVs cryoMNs respectively and place them in centrifuge tubes. Add 800 μL of physiological saline to each tube to dissolve and mix the microneedles, and then add 200 μL of red blood cell suspension to each tube (800 μL of ddH2O or physiological saline + 200 μL of ddH2O were added to the positive control and negative control, respectively). After mixing the supernatant (μL of red blood cell suspension) by pipetting, incubate at room temperature for 4 h. Place each group in a centrifuge and centrifuge at 10000 g for 5 min. After centrifugation, remove the samples, arrange them neatly, and take photos for observation. Prepare a 96-well plate, add 100 μL of supernatant from each group to the 96-well plate, and set up three replicates for each group. Measure the absorbance of each well at 540 nm using a microplate reader and calculate the hemolysis rate.

[0080] The results are as follows Figure 8As shown, the hemolysis rate of different groups of frozen microneedles after co-incubation with red blood cells was less than 5%, indicating that different groups of frozen microneedles have good blood safety.

[0081] Example 5: Rabbit Ear Scar Model and Treatment 1. Establishment of a rabbit ear scar model Two New Zealand rabbits, each weighing approximately 2 kg, were purchased from the Experimental Animal Center of Xinxiang Medical University a week in advance for acclimatization. Scarring models were then constructed on the rabbits. Anesthesia was achieved by intravenous injection of 2% sodium pentobarbital (3 ml / kg) into the ear margin vein. After a few minutes, the corneal reflex was checked. Once anesthetized, a 10 mm biopsy puncturer was used to make a perforation on the ventral side of the ear, penetrating the dermis. The circular dermis above the cartilage was then carefully removed with forceps, and the perichondrium was meticulously exfoliated to expose the cartilage. Hemostasis was achieved by applying pressure with alcohol swabs, and the area was disinfected. Four holes were created in each rabbit's ear.

[0082] 2. Treatment of rabbit ear wounds by different MNs (1) The holes on the rabbit ears were randomly divided into 4 groups: blank group, F127 / F68 cryoMNs group, ORI cryoMNs group, and rEVs cryoMNs group, with 3 parallels in each group.

[0083] (2) On the first day after surgery, the corresponding cyroMNs patch was applied to the wound area of ​​the New Zealand rabbit ear according to the group. The patch was gently pressed for 30 seconds with pre-cooled tweezers to adhere to the wound area. No treatment was performed on the blank group.

[0084] (3) The rabbit ear wound area was treated in the same way as on the first day on the 1st, 4th, 7th and 14th days after surgery. After taking pictures and observing, the wound area of ​​each group was measured and recorded with calipers.

[0085] (4) On the 19th day after the operation, the New Zealand rabbits were anesthetized by intravenous injection of 2% sodium pentobarbital in the ear. The wound and new skin tissue were gently removed by cutting around the rabbit ear wound with a scalpel. A square filter paper was taken, and the removed wound tissue was spread out and laid flat on the filter paper. After folding and wrapping, it was fixed and stored at 4 ℃ in a specimen bottle containing 4% paraformaldehyde. Each group was marked for subsequent Sirius red staining.

[0086] The flowchart for the establishment and treatment of rabbit ear scar models is as follows: Figure 9 As shown in Figure A, different groups of cryotherapy microparticles were used to treat rabbit ear wounds on days 1, 4, 7, and 14 after modeling. Results showed that scabs formed on all wounds during the first 7 days of healing, and by day 14, the scabs had fallen off naturally and the wounds had completely healed. Figure 9(B) It can be observed that the new wounds in the rEVs cryoMNs group are thinner, which may be because rEVs inhibit excessive macrophage fibrosis and reduce scar formation.

[0087] 3. Sirius Red staining (1) Paraffin embedding and sectioning of skin tissue Remove the wound skin tissue from each specimen bottle, cut it to a certain size, place it in an embedding cassette, and label it. Dehydrate the tissue according to the following steps: overnight in 75% ethanol, 1.5 h in 85% ethanol, 1 h in 95% ethanol, 20 min in anhydrous ethanol I, 20 min in anhydrous ethanol II, and 20 min in a 1:1 mixture of anhydrous ethanol and xylene; clearing: 10 min in xylene I, 10 min in xylene II; paraffin infiltration: 1 h in paraffin I, 2 h in paraffin II, and 2 h in paraffin III. After completing the above dehydration, clearing, and paraffin infiltration steps, embed the tissue using a paraffin embedding machine. Then, cut thin sections with a thickness of 5 μm using a microtome, spread the sections, bake them, and then place them in a slide cassette for storage at room temperature for later use.

[0088] (2) Staining The paraffin sections were dewaxed and rehydrated according to the following steps: soaked in xylene I for 20 min, soaked in xylene II for 20 min, then soaked in anhydrous ethanol I for 10 min, soaked in anhydrous ethanol II for 10 min, then soaked in 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min, and then gently rinsed with running tap water; after rehydration, staining was performed with saturated Sirius red staining solution for 8 min; rinsing was performed with anhydrous ethanol for several minutes; clearing was performed by drying the sections in a 60 ℃ oven, then placing them in xylene I for 5 min, followed by xylene II for 5 min; mounting was performed using neutral resin, and after air drying, the sections were photographed and observed under a microscope.

[0089] The results are as follows Figure 10 As shown in Figure A, the normal rabbit ear cartilage layer has only a very thin layer of collagen, and polarized light results show that its main component is green type III collagen. In contrast, the scar control group formed a thicker deposit of tissue, with a significantly increased scar thickness. Polarized light showed that the scar tissue was mainly composed of red / yellow type I collagen. Compared with the scar control group, the blank microneedling and ORI microneedling groups failed to effectively alleviate the thickness of the scar formation, and the scar was mainly composed of type I collagen. However, in the rEVs microneedling group, the thickness of the rabbit ear scar formation was significantly reduced, and the scar tissue was mainly composed of green type III collagen. Figure 10(B) In normal skin tissue, type I collagen accounts for 80-90%, along with type III collagen (approximately 10-20%), maintaining the skin's resilience. In the early stages of skin injury, type III collagen is synthesized rapidly and in large quantities to fill the wound space. During wound reconstruction, type I collagen synthesis increases to replace type III collagen. In mature scar tissue, type I collagen can account for over 95%. This change in proportion makes scar tissue tougher and less elastic. The appropriate presence of type III collagen can alleviate this tendency to contracture to some extent because it increases tissue elasticity. In this experimental result, regenerated endothelial cells (rEVs) effectively increased the proportion of type III collagen in newly formed skin tissue, effectively reducing scar formation.

[0090] Example 6: Establishment and Treatment of a Mouse Model of Deep Second-Degree Burns 1. Establish a mouse model of deep second-degree burn wounds. (1) Establishment of a mouse model of deep second-degree burns: One week in advance, 30 male KM mice weighing about 30g were purchased from the Animal Center of Xinxiang Medical College and acclimatized. Then, a deep second-degree burn model was established in the mice. The mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital at a dose of 0.1 mL / 10 g. After the mice lost consciousness, the hair on the back of the mice was shaved off with an electric hair removal device. An appropriate amount of hair removal cream was applied evenly to the hairless area on the back of the mice. After the hair removal cream had been in effect for a period of time, the hair removal cream and residual hair were wiped off with gauze soaked in physiological saline, and the skin on the back of the mice was completely exposed. An iron rod with a circular head with a diameter of 1 cm was heated to 95 ℃ on an alcohol lamp. When the temperature was uniform and stable, the iron rod was placed flat on the back of the mice for 10 s with its own weight without applying additional pressure, thus forming a deep second-degree burn.

[0091] (2) Preparation of wounds: On the second day after the burns were caused, all mice were anesthetized with 0.3% sodium pentobarbital. The necrotic skin in the burn area was cut off along the edge of the burn area on the back of the mice with surgical scissors to create a deep second-degree burn wound in the mice.

[0092] 2. Treatment of deep second-degree burn wounds in mice with different cyroMNs (1) Thirty burned mice were randomly divided into five groups: blank group, F127 / F68 cryoMNs group, ORIcryoMNs group, rEVs cryoMNs group, and rEVs / ORI cryoMNs group, with six mice in each group.

[0093] (2) On the first day after surgery, according to the group, the corresponding cyroMNs patch was applied to the wound area on the back of the mouse. The cyroMNs patch was gently pressed for 30 seconds with pre-cooled tweezers to adhere to the wound area. The blank group was not treated.

[0094] (3) The mice were treated on the same day 1, 4, 7 and 14 days after surgery. The wounds were photographed and the wound area of ​​each group was measured and recorded with calipers. The residual wound rate of each group was calculated using the following formula:

[0095] A0 represents the area of ​​the initial wound, and At represents the residual wound area on postoperative days 1, 4, 7, and 13.

[0096] (4) On the 14th day after surgery, mice were euthanized by intraperitoneal injection of an overdose of anesthetic. The wound and new skin around the mouse wound were cut off. A square piece of filter paper was taken, and the removed wound tissue was spread out and laid flat on the filter paper. After being folded and wrapped, it was fixed and stored at 4 ℃ in a specimen bottle containing 4% paraformaldehyde. Each group was marked for subsequent HE, Masson staining and immunohistochemical analysis.

[0097] A deep second-degree burn model was established on the back of mice. After randomization into groups, the mice were treated with cryo-microneedles on days 0, 1, 4, 7, and 14, respectively. Figure 11 A) The healing status of mice in different treatment groups was observed and recorded, and the effects of different cryotherapy micro-particles on the healing of deep second-degree burn wounds in mice were evaluated by histological analysis. Wound healing photographs show ( Figure 11 In the control group, the spontaneous healing rate of deep second-degree burn wounds was very slow. By day 13, the residual area of ​​the wound was still over 20%, and the wound was still partially covered by necrotic scabs with no obvious new epithelial tissue. The wound healing process in the blank microneedle group was similar to that in the control group, showing no significant healing-promoting effect. However, both drug-treated groups showed strong healing-promoting abilities for deep second-degree burn wounds, especially the rEVs group. By day 4, the residual wound rate showed a significant difference from the control group and the blank microneedle group, and by day 13, the residual wound rate had dropped to below 3%. Figure 11 C). This result indicates that rEVs can effectively promote the healing of deep second-degree burn wounds.

[0098] 3. Staining experiment (1) Paraffin embedding and sectioning of skin tissue The specific steps are the same as in Example 5.

[0099] (2) Hematoxylin-eosin (H&E) staining The embedded paraffin sections were dewaxed and rehydrated according to the steps in Example 5, and then stained as follows: soaked in hematoxylin stain for 5 min, washed with water for 10 min, stained with differentiation solution for 3 s, washed with water to return to blue for 10 min, and soaked in eosin stain for 30 s; dehydrated: soaked in anhydrous ethanol I for 5 min, soaked in anhydrous ethanol II for 5 min, and soaked in anhydrous ethanol III for 5 min; cleared: soaked in xylene I for 5 min, and soaked in xylene II for 5 min; mounted: the slides were mounted with neutral resin, air-dried naturally, and then photographed and observed under a microscope.

[0100] (3) Masson staining After dewaxing and rehydrating the paraffin sections according to the steps in Example 5, they were soaked in potassium dichromate overnight, followed by gentle rinsing with running tap water; stained in iron hematoxylin solution for 5 min, followed by gentle rinsing with running tap water; differentiated in differentiation solution for 15 s, followed by gentle rinsing with running tap water; blued in blue solution for 5 min, followed by gentle rinsing with running distilled water for 1 min; soaked in Ponceau S and Acid Fuchsin solution for 8 min, followed by gentle rinsing with running tap water; soaked in phosphomolybdic acid solution for 2 min, then transferred to aniline blue solution for 5 min, followed by differentiation with 1% glacial acetic acid; the subsequent dehydration, clearing, and mounting steps were the same as in 1.7.2.4.

[0101] The results are as follows Figure 12 As shown, the control group still had significant tissue loss beneath the scab layer, while the blank microneedle group showed some new skin tissue formation beneath the scab layer. In the three drug-loaded microneedle treatment groups, wound healing was relatively complete, but HE staining results indicated that the new tissue composition was relatively simple, lacking the formation of skin appendages such as sweat glands and hair follicles. Masson staining revealed that the new skin tissue was mainly composed of collagen deposition, exhibiting typical characteristics of scarring. The ORI microneedle group showed the thickest collagen deposition, while the rEVs treatment group showed reduced collagen deposition compared to the ORI treatment group. These results indicate that drug-loaded microneedles can effectively promote the healing of deep second-degree burn wounds, and the presence of rEVs helps reduce scar formation.

[0102] (4) Immunofluorescence staining of tissues Paraffin sections were dewaxed and rehydrated according to the steps in Example 5, followed by endogenous peroxidase blocking: 3% hydrogen peroxide was added, and the sections were placed in a humidified chamber at room temperature for 10 min, then immersed in distilled water for 1 min; antigen retrieval: the sections were placed in antigen retrieval solution and subjected to antigen retrieval in a microwave oven at medium-high power for 4 min, then turned off for 4 min, followed by medium-low power for 8 min, cooled to room temperature, and washed 3 times with PBS for 3 min each time; permeabilization: 0.3% Triton was permeabilized at room temperature for 15 min; blocking: 3% BSA was added, and the sections were blocked at 37℃ for 30 min; primary antibody application: the blocking solution was gently shaken off, the fluid around the tissue was aspirated, and the primary antibody diluted with antibody diluent was added, and the sections were incubated at 4℃ overnight; primary antibody washing: the primary antibody was aspirated, and the sections were washed in PBS for 5 min, repeated 3 times; secondary antibody incubation: the sections were gently shaken off, and the fluorescently labeled secondary antibody diluted with antibody diluent was added, and the sections were incubated at room temperature in the dark for 1 h, then washed in PBS for 5 min. Wash 3 times, then gently shake dry; Stain with DAPI: Add diluted DAPI, incubate at room temperature in the dark for 15 min, wash 3 times with PBS for 5 min each time; Mount: Add one drop of anti-fluorescence quenching mounting medium to the tissue, cover with a coverslip, and place under a microscope for observation.

[0103] The results are as follows Figure 13 As shown, compared with normal mice, the number of macrophages and α-SMA expression were significantly increased in deep second-degree burn wounds, indicating that MMT (metabolism-mediated transformation) occurs extensively during the regeneration phase of burn wounds, which may be the main reason for excessive fibroblast proliferation and severe scar formation. In the blank microneedle and ORI microneedle treatment groups, the number of macrophages and α-SMA expression were also relatively high, failing to show any effect in inhibiting the MMT process. However, in the rEVs group, almost no macrophage expression was observed, and the α-SMA co-expressed with macrophages was also significantly reduced, indicating that rEVs can effectively inhibit the macrophage MMT process during wound healing, which may be the main reason for its effective inhibition of scar formation.

[0104] In summary, this invention provides a method for preparing exosomes derived from *Rabdosia rubescens*. The *Rabdosia rubescens*-derived exosomes are uniform in size, with an average particle size below 150 nm. Under transmission electron microscopy, the *Rabdosia rubescens*-derived exosomes exhibit good morphology, appearing as round vesicles, uniformly distributed in the solution, and can promote cell proliferation. Experimental results show that the ORI group did not promote cell migration, while after 24 h of treatment with rEVs, the cell migration rate was significantly increased compared to the control group; rEVs have a good effect on promoting fibroblast migration. Compared with the control group, the ORI-treated group showed a significant increase in α-SMA-positive cells, while the rEVs-treated group showed a significant decrease, indicating that ORI can promote macrophage MMT, while rEVs can effectively inhibit macrophage MMT. Since excessive fibrosis is a major cause of scar formation during the wound healing and remodeling period, rEVs' inhibition of macrophage MMT may have the potential to reduce scar formation and has good hematologic safety.

[0105] This invention also provides a scarless, wound-healing-promoting hydrogel containing exosomes derived from *Rabdosia rubescens*. This hydrogel can encapsulate recombinant human endothelial cells (rEVs) or oral recombinant collagen (ORI). When applied to rabbit ear scar experiments, the results showed that the newly formed wound in the rEVs / cryoMNs group was thinner, possibly because rEVs inhibited excessive macrophage fibrosis, reducing scar formation. Compared to the scar control group, the blank microneedle and ORI microneedle groups failed to effectively alleviate scar thickness, and the scars were mainly composed of type I collagen. In contrast, the rEVs microneedle group showed a significant reduction in the thickness of rabbit ear scars, and the scar tissue was mainly composed of green type III collagen. rEVs can effectively increase the proportion of type III collagen in newly formed skin tissue, effectively reducing scar formation. The wound healing photographs show that the spontaneous healing rate of deep second-degree burn wounds in the control group was very slow; the wound healing process in the blank microneedle group was similar to that of the control group, showing no obvious healing-promoting effect; while both drug-treated groups showed strong healing-promoting abilities for deep second-degree burn wounds, especially the rEVs group, where the residual wound rate showed a significant difference from the control and blank microneedle groups by day 4, and by day 13, the residual wound rate had dropped to below 3%. This result indicates that rEVs can effectively promote the healing of deep second-degree burn wounds. HE staining results show that the composition of the newly formed tissue is relatively simple, with no formation of skin appendages such as sweat glands and hair follicles. Masson staining shows that the newly formed skin tissue is mainly composed of collagen deposition, exhibiting the general characteristics of scarring. Among them, the ORI microneedle group had the thickest collagen deposition, while the rEVs treatment group had less collagen deposition compared to the ORI treatment group. This result indicates that drug-treated microneedles can effectively promote the healing of deep second-degree burn wounds, and the presence of rEVs helps reduce scar formation. In the blank microneedle and ORI microneedle treatment groups, the number of macrophages and α-SMA expression were both relatively high, failing to show any effect in inhibiting the MMT process. However, in the rEVs group, macrophage expression was almost absent, and the α-SMA co-expressed with macrophages was also significantly reduced, indicating that rEVs can effectively inhibit the macrophage MMT process during wound healing, which may be the main reason for its effective inhibition of scar formation.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of exosomes derived from Rabdosia rubescens in the preparation of drugs that promote wound healing.

2. The application as described in claim 1, characterized in that, The wounds include chronic wounds, acute trauma, surgical incisions, burns, or ulcers.

3. Application of exosomes derived from Rabdosia rubescens in the preparation of drugs that reduce scar formation.

4. The application as described in claim 3, characterized in that, The scars include hypertrophic scars, atrophic scars, or keloids.

5. The application as described in any one of claims 1-4, characterized in that, The exosomes derived from *Rabdosia rubescens* were prepared by the following method: Fresh *Rabdosia rubescens* leaves were picked, washed clean, and then washed twice with PBS. The leaves and PBS were weighed at a mass-to-volume ratio of 1:

3. The cell walls were broken, the resulting juice was filtered to remove leaf residue, centrifuged, and the supernatant was balanced and centrifuged again to remove impurities of different sizes. The supernatant was filtered, and the filtered solution was transferred to a centrifuge tube and centrifuged. The supernatant was discarded, and the precipitate was resuspended in PBS. The precipitate was centrifuged again, and impurities were washed away. The supernatant was discarded, and the precipitate was resuspended in PBS according to the amount of precipitate.

6. A scarless, wound-healing-promoting hydrogel containing exosomes derived from *Rabdosia rubescens*, characterized in that... The hydrogel is made by mixing F127 and F68 in a ratio of 22.5%:2.5%, and adding exosomes derived from *Rabdosia rubescens*.

7. The use of the hydrogel as described in claim 6 in the preparation of a wound-healing medicament.

8. The use of the hydrogel as described in claim 6 in the preparation of a drug for inhibiting or reducing scar formation.

9. The use of the hydrogel as described in claim 6 in the preparation of medical materials for scar prevention.

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