Rapamycin-induced extracellular vesicles and application thereof in preparation of drugs for promoting wound repair

Abstract

The invention relates to the technical field of biological medicines, in particular to rapamycin-induced extracellular vesicles and application thereof in preparation of a medicine for promoting wound repair. The rapamycin-induced extracellular vesicles can enhance the migration and tube forming ability of endothelial cells, so that angiogenesis is promoted; meanwhile, the proliferation and migration capabilities of macrophages can be inhibited, and the proportion of M1 type macrophages in a wound is reduced, so that the inflammatory response mediated by the macrophages is relieved; and the composition shows extraordinary effects in the aspect of accelerating wound repair. The biological activity of the extracellular vesicles induced by the rapamycin depends on the activation of a PI3K / Akt signal channel.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to rapamycin-induced extracellular vesicles and their application in the preparation of drugs for promoting wound repair. Background Art

[0002] As a complex multi-system metabolic disorder, poor postoperative wound healing and pathological healing in diabetes remain urgent clinical problems, which not only seriously affect the quality of life of patients, but also become an important risk factor for inducing long-term complications.

[0003] In the complex regulatory mechanism of diabetic wound healing, angiogenesis plays a key role. The formation of new blood vessels can effectively transport oxygen and nutrients to the wound area, promoting repair processes such as collagen synthesis, fibroblast proliferation, and re-epithelialization. At the same time, inflammatory injury is another core mechanism for the difficulty in healing diabetic wounds. Persistent inflammatory responses trigger a chain of pathological effects, including microcirculation perfusion disorders, cascading amplification of pro-inflammatory factors, explosive production of reactive oxygen species, and the risk of secondary infection caused by the formation of bacterial biofilms. It is worth noting that macrophages, as key executors of immune regulation, play a pivotal role in the inflammatory homeostasis of wound repair. Their phagocytic function and cytokine / growth factor secretion patterns precisely regulate inflammatory responses, fibrosis processes, and tissue regeneration mechanisms. Macrophages participate in the whole process of wound repair, accompanied by a transformation from the early pro-inflammatory M1 type to the late anti-inflammatory M2 type. Therefore, new strategies to stimulate angiogenesis and reduce macrophage-mediated inflammation have broad prospects in the treatment of diabetic wounds. Based on this, the present invention provides a rapamycin-induced extracellular vesicle and its application in the preparation of drugs for promoting wound repair. Summary of the Invention

[0004] The purpose of the present invention is to provide a rapamycin-induced extracellular vesicle and its application in the preparation of drugs for promoting wound repair.

[0005] On the one hand, the present invention provides the application of rapamycin-induced extracellular vesicles in the preparation of drugs for promoting wound repair.

[0006] Further, the way of promoting wound repair includes inhibiting the proliferation and migration ability of macrophages and reducing the expression of pro-inflammatory mediators including TNF-α, IL-1β, and iNOS.

[0007] Further, the way of promoting wound repair includes promoting the migration and tube formation of endothelial cells and increasing the level of VEGF.

[0008] Further, the way of promoting wound repair includes accelerating angiogenesis and / or reducing macrophage-mediated inflammation by using the PI3K / Akt signaling pathway.

[0009] Furthermore, the wound includes a diabetic wound.

[0010] Furthermore, the preparation steps of the rapamycin-induced extracellular vesicles include: adding rapamycin to a complete medium, culturing human umbilical cord blood mesenchymal stem cells, separating sEVs from the medium through gradient centrifugation steps, and then centrifuging again. The precipitate is resuspended in phosphate buffer to obtain rapamycin-induced extracellular vesicles.

[0011] Furthermore, the complete medium is an α-MEM medium supplemented with 10 wt.% fetal bovine serum and 1 wt.% penicillin-streptomycin.

[0012] Furthermore, the gradient centrifugation steps include: centrifuging the cell supernatant at 300×g for 10 min, 2000×g for 10 min, and 10000×g for 30 min at 4°C.

[0013] Furthermore, the re-centrifugation step is to re-centrifuge at 100000×g for 2 times at 4°C, each time for 70 min.

[0014] The beneficial effects of the present invention are as follows:

[0015] In the present invention, rapamycin-induced extracellular vesicles inhibit the proliferation and migration ability of macrophages and reduce the expression of pro-inflammatory mediators including TNF-α, IL-1β, and iNOS; at the same time, they can also promote the migration and tube formation of endothelial cells and increase the level of VEGF. In addition, in streptozotocin (STZ)-induced diabetic mice, rapamycin-induced extracellular vesicles show extraordinary efficacy in accelerating wound repair by promoting angiogenesis, reducing the percentage of M1 macrophages, and alleviating excessive inflammatory responses. The biological activity of rapamycin-induced extracellular vesicles depends on the PI3K / Akt signaling pathway, and the pro-angiogenic and anti-inflammatory effects of rapamycin-induced extracellular vesicles are weakened after the pathway is inhibited. Brief Description of the Drawings

[0016] Figure 1 It is a mechanism diagram of the present invention;

[0017] Figure 2Identification diagrams of hUCMSCs and rapamycin-induced sEVs; among them, (A) Morphological diagram of hUCMSCs showing spindle shape under light microscope, scale bar, 200 μm; (B-D) Diagrams showing the chondrogenic, adipogenic and osteogenic differentiation potential of hUCMSCs, scale bar, 200 μm; (E) Evaluation of hUCMSC surface markers including CD11b, CD45, CD105 and CD166 by flow cytometry; blue curve, isotype control; red curve, cell surface marker; (F) Analysis of the effect of different concentration gradients of 0.5 μM, 1 μM, 2 μM rapamycin on the proliferation ability of hUCMSCs induced by rapamycin, OD value measured at 450 nm; (G) TEM observation of the morphology of sEVs (sEVs1) and rapamycin-sEVs (sEVs2); scale bar, 100 nm; (H) Western blot analysis of surface markers (CD9, CD81, CD63); (I) Detection of the diameter and particle concentration of sEVs1 and sEVs2 by NTA method; (J-K) Scanning electron microscope (SEM) and atomic force microscope (AFM) of the hydrogel; mean ± standard deviation; NS, not significant, *P < 0.05; **P < 0.01;

[0018] Figure 3 Rapamycin-sEVs inhibit the proliferation of macrophages and the production of inflammatory cytokines; among them, (A) Macrophages phagocytosing sEVs can be seen under light microscope; scale bar, 100 μm; (B) Fluorescence micrograph of PKH-26-labeled sEVs (red); scale bar, 100 μm; (C) Ki67 immunofluorescence staining; scale bar, 100 μm; (D) Quantitative analysis of Ki67-positive cells (%) ; (E) Evaluation of the effect of rapamycin-sEVs on macrophage proliferation by CCK-8 method; OD value measured at 450 nm; (F) mRNA expression of inflammatory cytokines including TNF-α, IL-1β and iNOS; (G-H) Detection of M1 macrophages in different groups by flow cytometry and corresponding quantitative analysis; CD68, total macrophage marker; CD86, M1 macrophage marker; mean ± standard deviation; *P < 0.05; **P < 0.01;

[0019] Figure 4 Rapamycin-sEVs inhibit the migration of macrophages; among them, (A) Cell migration of macrophages treated differently was performed using the scratch assay; scale bar, 200 μm; (B) Transwell experiment to evaluate the effect of rapamycin-sEVs on macrophage migration ability; scale bar, 200 μm; (C-D) Quantitative analysis of the migration area and the number of migrating macrophages; mean ± standard deviation; *P < 0.05; **P < 0.01;

[0020] Figure 5Rapamycin-sEVs promote angiogenesis; among them, (A) HUVECs internalize rapamycin-sEVs labeled with PKH-26 (red), and confocal microscopy shows localization in the perinuclear region; scale bar, 50 μm; (B) CCK-8 assay is performed to evaluate the proliferation ability of HUVECs; (C) Scratch assay is used for cell migration of HUVECs with different treatments; scale bar, 200 μm; (D) Quantitative analysis of the migration area (mm 2 2); (E) Cell migration of HUVECs after crystal violet staining in the Transwell assay; scale bar, 100 μm; (F) Quantitative analysis of the number of migrated HUVECs; (G) Images of HUVECs tube formation; scale bar, 200 μm; (H-J) Quantitative analysis of tube formation, including nodes, junctions, and branches; mean ± standard deviation; NS, not significant, *P < 0.05; **P < 0.01;

[0021] Figure 6 Rapamycin-sEVs accelerate the closure of diabetic wounds in vivo; (A) Representative images of full-thickness defects (7 mm) in STZ-induced diabetic mice treated with PBS (control), hydrogel, hydrogel + sEVs1, and hydrogel + sEVs2 at 0, 4, 9, and 14 days postoperatively; (B-C) Quantification of wound area (mm2) and wound closure rate (%); mean ± standard deviation; *P < 0.05; **P < 0.01;

[0022] Figure 7 Rapamycin-sEV promotes epithelial regeneration and tissue repair; (A-B) H&E and Masson's trichrome staining of each treatment group on the 14th day after intervention; scale bar, 500 μm or 200 μm as shown in specific images; (C-E) Quantification of wound width (mm), epithelial thickness (μm), and percentage of collagen fibers (%); mean ± standard deviation; *P < 0.05; **P < 0.01;

[0023] Figure 8 Rapamycin-sEVs enhance vascularization while reducing macrophage-mediated inflammatory responses; among them, (A-B) Tissue immunofluorescence evaluation of M1 macrophage marker CD86 and angiogenesis marker CD31 staining; scale bar, 200 μm; (C, E) Quantitative analysis of CD86 and CD31 tissue immunofluorescence; (D, F) mRNA levels of inflammation-related factors (TNF-α, IL-1β, iNOS) and angiogenesis-related markers (PECAM-1 and VEGF); mean ± standard deviation; *P < 0.05; **P < 0.01;

[0024] Figure 9Blocking the PI3K / Akt pathway can eliminate the inhibition of macrophage proliferation and inflammatory factors mediated by rapamycin-sEVs; (A-B) Ki67 immunofluorescence staining of macrophages and the percentage of Ki67-positive cells (%) after treatment with the PI3K inhibitor PI103; Scale bar, 100 μm; (C) mRNA expression levels of inflammatory cytokines in macrophages receiving various treatments, including TNF-α, IL-1β, and iNOS; Mean ± standard deviation, *P<0.05; **P<0.01;

[0025] Figure 10 Blocking the PI3K / Akt pathway can weaken the inhibitory effect of rapamycin-sEVs on macrophage migration; Among them, (A) Scratch assay was used to evaluate the migration of macrophages treated with different treatments; Scale bar, 200 μm; (B) Transwell assay was used to evaluate the migration ability of macrophages after treatment with PI103; Scale bar, 100 μm; (C-D) Quantification of the migration area and the number of macrophages under different treatment interventions; Mean ± standard deviation; *P<0.05; **P<0.01;

[0026] Figure 11 Inhibiting the PI3K / Akt pathway impairs the stimulatory effect of rapamycin-sEVs on the migration and tube formation ability of HUVECs; Among them, (A) Scratch assay was used to detect the migration ability of HUVECs after treatment with PI103; Scale bar, 200 μm; (B) Quantitative analysis of the migration area (mm 2 );(C) Transwell assay was used to evaluate the migration of HUVECs. Scale bar, 100 μm; (D) Quantitative analysis of the number of migrated cells; (E) Tube formation assay was used to evaluate angiogenesis under different treatments. Scale bar, 400 μm; (F-H) Quantification of the number of nodes, junctions, and branches; Mean ± standard deviation; *P<0.05; **P<0.01.

[0027] Figure 12 The PI3K / Akt signaling pathway is responsible for rapamycin-sEV-induced diabetic wound repair. Among them, (A) Diabetic mice with full-thickness defects (7 mm) were treated with hydrogel, hydrogel + sEVs2, and hydrogel + sEVs2 + PI103 on days 0, 4, 9, and 14 after surgery; (B) Quantitative analysis of wound closure rate (%); (C) After treatment with PI103, the phosphorylation levels of PI3K and AKT, and the relative protein expression levels of wound skin tissues were detected by Western blotting; (D) Quantitative analysis by Western blotting; (E) Relative mRNA expression levels of inflammation-related factors (TNF-α and IL-1β) and angiogenesis-related markers (PECAM-1 and VEGF) after PI103 treatment. Mean ± standard deviation. *P<0.05; **P<0.01. Detailed implementation mode

[0028] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] The present invention provides the application of rapamycin-induced extracellular vesicles in the preparation of drugs for promoting wound repair.

[0030] The preparation steps of rapamycin-induced extracellular vesicles include:

[0031] Add different amounts of rapamycin (such as 1 μM) to the complete medium, and culture human umbilical cord blood mesenchymal stem cells (hUCMSCs) for 24 hours; the complete medium is α-MEM medium (Gibco, USA) added with 10 wt.% fetal bovine serum (Gibco, USA) and 1 wt.% penicillin-streptomycin (Gibco, USA).

[0032] Separate sEVs from the medium through gradient centrifugation steps, that is: the cell supernatant is centrifuged at 300×g for 10 min at 4°C, 2000×g for 10 min, and 10000×g for 30 min to remove cell debris.

[0033] Subsequently, centrifuge at 100000×g twice at 4°C for 70 min each time, and then resuspend the precipitate in phosphate buffer (PBS) to obtain rapamycin-induced extracellular vesicles.

[0034] Example 1. Identification of human umbilical cord blood mesenchymal stem cells and characterization of rapamycin-induced sEVs

[0035] Observed by transmission electron microscopy (JEM-1400), and photographed the morphological characteristics of sEVs; the micrographs showed the typical spindle-like morphology of human umbilical cord blood mesenchymal stem cells ( Figure 2 A); human umbilical cord blood mesenchymal stem cells showed various differentiation functions, including chondrogenic, adipogenic and osteogenic differentiation potentials ( Figure 2 B-D); flow cytometry data showed that the negative markers on the surface of human umbilical cord blood mesenchymal stem cells included CD11b and CD45, and the positive markers included CD105 and CD166 ( Figure 2 E).

[0036] Human umbilical cord blood mesenchymal stem cells were induced with rapamycin at different concentration gradients, including 0 μM, 0.5 μM, 1 μM, and 2 μM. It was found that rapamycin could inhibit the proliferation of human umbilical cord blood mesenchymal stem cells at a concentration up to 2 μM, while the proliferation ability of human umbilical cord blood mesenchymal stem cells was not significantly affected at concentrations of 0.5 μM and 1 μM( Figure 2 F).

[0037] The extracellular vesicles sEVs1 isolated from the supernatant of human umbilical cord blood mesenchymal stem cells without rapamycin were used as the control group. The extracellular vesicles sEVs2 were extracted from the supernatant of human umbilical cord blood mesenchymal stem cells induced with 1 μm rapamycin for 24 hours and then characterized by TEM. Similar to normal sEVs (sEVs1), sEVs2 showed a typical cup-shaped morphology with a double-membrane microstructure( Figure 2 G).

[0038] Western blot analysis revealed that the surface markers CD9, CD81, and CD63 of sEVs1 and sEVs2 were all positive compared with human umbilical cord blood mesenchymal stem cells( Figure 2 H). NTA showed that the size distributions of sEVs1 and sEVs2 both exhibited a single peak at approximately 150 - 200 nm( Figure 2 I).

[0039] These data confirmed the successful extraction of hUCMSC-derived sEVs from the conditioned medium. In addition, observation using scanning electron microscopy (SEM) and atomic force microscopy (AFM) clearly showed that the hydrogel exhibited an interconnected porous structure( Figure 2 J-K).

[0040] Example 2. Rapamycin-induced extracellular vesicles inhibit macrophage function

[0041] To determine whether rapamycin-induced extracellular vesicles could be phagocytosed and internalized into macrophages, PKH-26-labeled sEVs were co-cultured with macrophages, that is, sEVs were added to the macrophage culture medium, and the phagocytosis of sEVs by macrophages was observed.

[0042] The experimental procedures for sEV phagocytosis include: staining sEVs using a PKH-26 staining kit (MINI26, Sigma-Aldrich, Germany) according to the instructions. Dilute 100 μL of the sEV suspension with Diluent C (a reagent included in the kit), and stain with PKH-26 in the dark for 10 min. Add 1% bovine serum albumin (BSA) to terminate the staining reaction. Then, centrifuge the mixture at 100,000×g for 70 min at 4°C, and resuspend the pellet in cold PBS to obtain stained sEVs. Incubate macrophages or HUVECs with the labeled sEVs for 24 h. Then, fix the cells with 4% paraformaldehyde (PFA), stain with DAPI (4',6-diamidino-2-phenylindole, Sigma-Aldrich), and observe the stained cells using a fluorescence microscope.

[0043] Under the microscope, PKH-26-labeled sEVs were internalized by macrophages ( Figure 3 A). Confocal microscopy further revealed that rapamycin-induced extracellular vesicles were localized in the perinuclear region of macrophages ( Figure 3 B), confirming the internalization of sEVs by macrophages. Macrophages were treated with LPS (lipopolysaccharide), LPS + sEVs1, and LPS + sEVs2, respectively. Immunofluorescence staining showed that, compared with the LPS-treated group, LPS successfully increased the positive rate of macrophage Ki67, while both sEVs1 and sEVs2 decreased the proportion of Ki67 positivity ( Figure 3 C-D).

[0044] The CCK-8 assay confirmed that both sEVs1 and sEVs2 could inhibit the proliferation increased by LPS, and sEVs2 showed a stronger anti-proliferative effect than sEVs1 ( Figure 3 E). More importantly, rapamycin-induced extracellular vesicles inhibited the expression of LPS-stimulated inflammatory factors, including TNF-α, IL-1β, and iNOS ( Figure 3 F). Both sEVs1 and sEVs2 could reduce the percentage of pro-inflammatory M1 macrophages, and rapamycin-induced extracellular vesicles showed a more effective reduction than sEVs from human umbilical cord blood mesenchymal stem cells ( Figure 3 G-H).

[0045] In addition, a scratch test was also conducted. The steps included: (1) Seeding plates: Inoculate the cell suspension into a culture dish at an appropriate density, and place the cells in an incubator at 37°C and 5% CO2 until the cells reach 90%-100% confluence; (2) Scratching: Use a sterile pipette tip or a scriber to draw a straight line on the cell monolayer to form a scratch with the same width; (3) Washing: Wash with PBS to remove the cells and debris shed during scratching; (4) Continuing culture: Replace the medium with serum-free medium, put the cells back into the incubator, and at time points such as 0 hour, 24 hours, 48 hours, and 72 hours after scratching, use a microscope to photograph the scratched area; (5) Image analysis: Use image analysis software (Image J) to measure the scratch width and calculate the cell migration rate or healing rate.

[0046] After testing, the results showed that compared with the LPS treatment group, rapamycin-induced extracellular vesicles reduced macrophage migration ( Figure 4 A, C).

[0047] The steps of the Transwell experiment included: (1) Preparation of items: Transwell chambers (with porous membranes), cells, culture medium, PBS, methanol as a fixative, and crystal violet as a staining solution; (2) Cell preparation: Culture the cells in a culture flask until the logarithmic growth phase, digest the cells with trypsin, centrifuge and resuspend them in serum-free medium, and adjust the cell concentration; (3) Cell seeding: Add the cell suspension to the upper chamber of the Transwell chamber; add the medium containing 20% fetal bovine serum to the lower chamber to attract cell migration; (4) Cell culture: Place the Transwell chamber in an incubator at 37°C and 5% CO2 and culture for 24-48 hours, and adjust the specific time according to the cell type and experimental purpose; (5) Cell fixation and staining: Take out the chamber, wash with PBS, fix with methanol for 10-15 minutes; stain with crystal violet for 10-15 minutes, and wash with PBS to remove the excess dye; (6) Cell counting and analysis: Use a cotton swab to wipe off the non-migrated cells in the upper chamber, observe and photograph under a microscope; randomly select multiple fields of view to count the number of migrated or invaded cells, or use image analysis software for quantitative analysis.

[0048] The Transwell experiment confirmed that rapamycin-induced extracellular vesicles reduced the migration of macrophages induced by LPS ( Figure 4 B, D), so rapamycin-induced extracellular vesicles can inhibit the cell function of macrophages and the inflammatory mediators released by them.

[0049] Example 3. Rapamycin-induced extracellular vesicles promote angiogenesis

[0050] The tube formation experiment procedure includes: evaluating in vitro angiogenesis using the tube formation experiment. After Matrigel (basement membrane matrix, BD Biosciences) is thawed overnight at 4°C, 200 μL / well is added to a pre-cooled 24-well plate, and it is shaken evenly on ice to avoid gelation; then the plate is incubated at 37°C for 1 h to facilitate the gelation of Matrigel. HUVECs (1.5×10 5 cells / well) are seeded onto the 24-well plate and incubated with the medium. After 8 h, the capillary-like structures, including the number of nodes, junctions, and branches, are observed and counted under an inverted light microscope.

[0051] In the experimental group, sEVs1 or sEVs2 was added, while nothing was added in the control group.

[0052] Rapamycin-induced extracellular vesicles can also be internalized into HUVECs and localize around the nucleus ( Figure 5 A). Neither sEV1 nor sEVs2 affects the proliferation of HUVECs ( Figure 5 B), but the scratch assay and Transwell assay show that they can enhance the migration of HUVECs ( Figure 5 C-F). Similarly, sEVs2 shows stronger migratory ability than sEVs1.

[0053] The results of the tube formation assay show that compared with the control group, more tube structures, including nodes, connections, and branches, are detected in HUVECs treated with rapamycin-induced extracellular vesicles, indicating that rapamycin-induced extracellular vesicles enhance the tube formation ability of endothelial cells ( Figure 5 G-J). Therefore, these findings confirm the potential of rapamycin-induced extracellular vesicles in enhancing the biological functions of HUVECs.

[0054] Example 4: Rapamycin-induced extracellular vesicles accelerate the closure of diabetic wounds in vivo

[0055] Diabetic mice induced by streptozotocin (STZ) received full-thickness skin excision wounds to evaluate the therapeutic potential of rapamycin-sEV. The specific steps were as follows: All animal experiments were carried out with the approval of the Animal Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (2024-N(A)-0412). Female C57 / BL6 mice at 8 weeks of age were fed according to the Guidelines for the Care and Use of Laboratory Animals in China. The mice were injected with STZ solution (80 mg / kg) at two high doses before surgery. Mice with blood glucose levels above 16.7 mM were selected as diabetic mice. Under anesthesia, two wounds with a diameter of 7 mm were created on the back of each mouse using a puncher. The diabetic model mice were randomly divided into different groups, with 3 mice in each group; PBS (control group), hydrogel, hydrogel + sEVs1, hydrogel + sEVs2, or hydrogel + sEVs2 + PI103 were used to treat the wounds. 100 μl of the hydrogel containing 10 μg sEVs was evenly applied to the skin defect, and after drying, the mice were put back into the cage.

[0056] After model establishment, PI103 (phosphatidylinositol 3-kinase inhibitor 103, which can block the PI3K / AKT signaling pathway) with a concentration of 10 mg / 100 μl / kg was intraperitoneally injected daily; after surgery, the defect was covered with a skin patch (3M). On the 0th, 4th, 9th, and 14th days after surgery, the diabetic wounds were photographed with a digital camera to record the recovery outcome; the wound closure rate was calculated by P(%) = (A0 - Ad) / A0 × 100, where P(%) is the percentage of wound healing, A0 is the initial wound area on the 0th day, and Ad is the wound area at each time point.

[0057] From the images of the skin defects, there were no obvious differences between the control group (PBS) and the hydrogel group on the 4th, 9th, and 14th days after surgery. Compared with the hydrogel group, both the sEVs1 group and the sEVs2 group promoted the healing of diabetic wounds ( Figure 6 A). The wound repair ability of sEVs2 was better than that of sEVs1. Specific statistical data including wound area and wound closure rate confirmed this finding ( Figure 6 B-C).

[0058] In addition, the wound area treated with the hydrogel of rapamycin-induced extracellular vesicles remained free of erythema and edema, indicating its superior biosafety. In summary, these results suggest that rapamycin pretreatment promotes hUCMSC-sEV-mediated diabetic wound repair, which is effective and has sufficient safety in vivo.

[0059] Example 5: Rapamycin-induced extracellular vesicles promote epithelial regeneration and angiogenesis, while reducing macrophage-mediated inflammatory responses

[0060] After 14 days, the skin lesions on the backs of the mice were collected for subsequent staining analysis.

[0061] Hematoxylin and eosin (HE) staining showed that compared with the hydrogel treatment group, both the sEVs1 group and the sEVs2 group had better epithelialization and granulation formation processes in diabetic wounds, including narrower wound width, thicker epithelial layer, and more abundant epithelial structures ( Figure 7 A, C-D).

[0062] Masson staining revealed that rapamycin-induced extracellular vesicles, especially sEVs2, had thicker collagen fibers accompanied by extensive and well-arranged collagen deposition ( Figure 7 B, E), indicating that rapamycin-induced extracellular vesicles have strong extracellular matrix (ECM) remodeling potential.

[0063] In addition, immunofluorescence staining showed that the abundance of the specific marker CD86 of M1 macrophages in the sEVs1 and sEVs2 groups was higher than that in the control group and the hydrogel group ( Figure 8 A). IF analysis targeting CD31 showed that compared with the control group and the hydrogel group, rapamycin-sEV treatment increased dermal neovascularization at the wound site ( Figure 8 B). qPCR analysis of fresh skin lesions confirmed that compared with the control group and the hydrogel group, both the sEVs1 and sEVs2 groups expressed less TNF-α, IL-1β, and iNOS, and more PECAM-1 and VEGF simultaneously ( Figure 8 C-D). sEVs2 showed a stronger effect than sEVs1. Therefore, rapamycin-induced extracellular vesicles enhanced the epithelial regeneration and vascularization processes, while reducing macrophage-mediated inflammatory responses in diabetic skin defects.

[0064] Example 6. Blocking the PI3K / Akt pathway can eliminate rapamycin-sEV-mediated inhibition of macrophage activity and promotion of angiogenesis

[0065] In the rescue experiment, the potent inhibitor PI103 of the PI3K / Akt pathway was administered, and the stimulatory effect of sEVs2-induced macrophage proliferation was eliminated, which was demonstrated by immunofluorescence staining of the proliferation marker Ki67 ( Figure 9 A-B). The results showed that compared with the LPS + sEV2 group, PI103 treatment restored the expression of macrophage-related inflammatory mediators, including TNF-α, IL-1β, and iNOS ( Figure 9 C). Similarly, the scratch assay and Transwell assay showed that the rapamycin-sEV-mediated pro-migration effect on macrophages could be reversed by PI103 ( Figure 10 A-D). In addition, PI103 administration could effectively eliminate the enhanced migration of human umbilical vein endothelial cells (HUVECs) induced by rapamycin-sEV ( Figure 11A-D). PI103 can mitigate the promoting effect of rapamycin-induced extracellular vesicles on the tube formation ability of endothelial cells. Figure 11 E-H). In summary, rapamycin-induced extracellular vesicles exert biological effects on macrophages and endothelial cells through the PI3K / Akt signaling pathway.

[0066] Example 7. The PI3K / Akt signaling pathway is responsible for rapamycin-sEV-induced diabetic wound repair

[0067] To verify the above conclusion in vivo, we established a full-thickness skin wound model of diabetic mice again. The experimental procedures were the same as those in Example 4.

[0068] The results showed that blocking the PI3K / Akt signaling pathway could eliminate the effect of rapamycin-induced extracellular vesicles on accelerating skin wound healing on days 4, 9, and 14. Figure 12 A-B). In addition, Western blot experiments verified that the PI3K / Akt signaling pathway was effectively inhibited by PI103 in the skin defect tissues of diabetic mice. Figure 12 C-D). More importantly, in vivo experiments confirmed that compared with the hydrogel + sEVs2 group, after inhibiting the PI3K / Akt signaling pathway, the expressions of macrophage-related inflammatory factors TNF-α and IL-1β increased, while the levels of angiogenesis-related markers PECAM-1 and VEGF decreased. Figure 12 E). Generally speaking, these results revealed that rapamycin-induced extracellular vesicles promote diabetic wound healing depending on the activation of the PI3K / Akt signaling pathway.

[0069] Finally, it should be noted that the above examples are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. Use of rapamycin-induced extracellular vesicles in the preparation of a drug for promoting wound repair.

2. The application according to claim 1, characterized in that The way of promoting wound repair includes inhibiting the proliferation and migration ability of macrophages and reducing the expression of pro-inflammatory mediators including TNF-α, IL-1β and iNOS.

3. The application according to claim 1, characterized in that, The way of promoting wound repair includes promoting the migration and tube formation of endothelial cells and increasing the level of VEGF.

4. The application according to claim 1, wherein The way of promoting wound repair includes accelerating angiogenesis and / or reducing macrophage-mediated inflammation by using the PI3K / Akt signaling pathway.

5. The application according to claim 1, characterized in that, The wound includes diabetic wounds.

6. The application according to any one of claims 1-5, characterized in that, The preparation steps of the rapamycin-induced extracellular vesicles include: adding rapamycin to the complete medium, culturing human umbilical cord blood mesenchymal stem cells, separating sEVs from the medium by gradient centrifugation steps, and then centrifuging again. The precipitate is resuspended in phosphate buffer to obtain rapamycin-induced extracellular vesicles.

7. The application according to claim 4, wherein The complete medium is α-MEM medium supplemented with 10 wt.% fetal bovine serum and 1 wt.% penicillin-streptomycin.

8. The application according to claim 4, characterized in that, The gradient centrifugation steps include: centrifuging the cell supernatant at 300×g for 10 min, 2000×g for 10 min, and 10000×g for 30 min at 4°C.

9. The application according to claim 4, characterized in that, The re-centrifugation step is to centrifuge at 100000×g for 2 more times at 4°C, each time for 70 min.

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