Application of exosomes in the preparation of a preparation for treating diabetic wounds

Abstract

The present disclosure provides an application of exosomes in the preparation of a preparation for treating diabetic wounds, wherein the exosomes are mesenchymal stem cell exosomes with enhanced surface sLeX expression. According to the present disclosure, an application of exosomes in the preparation of a preparation for treating diabetic wounds can be provided, and the preparation has a good therapeutic effect on diabetic wounds.

Description

Technical Field

[0001] The present disclosure relates to the field of new biological materials, and particularly to the application of exosomes in the preparation of a preparation for treating diabetic wounds. Background Art

[0002] With the rapid development of society and economy, the incidence of diabetes has gradually increased. Refractory diabetic wounds such as diabetic foot ulcers, as serious and costly chronic complications, have attracted great attention.

[0003] In diabetic wounds, angiogenesis is reduced, resulting in insufficient capillary and blood vessel density, reduced transport of oxygen and nutrients, and ultimately delayed wound healing. Moreover, diabetic wounds are extremely prone to infection, and therapies that inhibit inflammation play a crucial role in wound healing. Therefore, in the clinical treatment of diabetic infected wounds, great importance should be attached to the research and development of drugs that can promote angiogenesis and relieve inflammation. Existing studies have shown that mesenchymal stem cells and exosomes secreted by mesenchymal stem cells can improve diabetes and its complications. Among them, compared with stem cells, exosome therapy is a cell-free therapy that can avoid immune rejection after stem cell transplantation. Since exosomes cannot self-replicate, the risk of tumor formation can be reduced, so the risk of using exosome therapy is lower.

[0004] However, due to the relatively low specificity of exosomes for target cells, the therapeutic effect of exosomes may be affected. Summary of the Invention

[0005] The present disclosure is completed in view of the above-mentioned existing technical situation, and its purpose is to provide the application of exosomes in the preparation of a preparation for treating diabetic wounds, and the preparation has a good therapeutic effect on diabetic wounds.

[0006] To this end, a first aspect of the present disclosure provides the application of exosomes in the preparation of a preparation for treating diabetic wounds, and the exosomes are mesenchymal stem cell exosomes with enhanced surface sLeX expression.

[0007] The applicant found that the expression of E-selectin in diabetic wounds is higher than that in normal non-diabetic wounds. In the first aspect of the present disclosure, exosomes (s-Exo) with enhanced surface sLeX (Sialyl Lewis-X) expression are applied in the preparation of a preparation for treating diabetic wounds. The exosomes can target human umbilical vein endothelial cells (HUVECs), and E-selectin can mediate the absorption of s-Exo by HUVECs, so that s-Exo can specifically bind to HUVECs, enhance the function of HUVECs stimulated by inflammation in vitro, thereby promoting angiogenesis and promoting the healing of diabetic wounds. Thus, according to the present disclosure, a preparation with a good therapeutic effect on diabetic wounds can be provided.

[0008] In the application related to the first aspect of the present disclosure, optionally, the preparation promotes the healing of diabetic wounds by promoting angiogenesis. Thus, it can contribute to the promotion of the healing of diabetic wounds.

[0009] In the application related to the first aspect of the present disclosure, optionally, the preparation is applied topically, and the preparation is directly applied to the wound or the area adjacent to the wound. Thus, it can contribute to the promotion of the healing of diabetic wounds.

[0010] In the application related to the first aspect of the present disclosure, optionally, the exosomes are obtained by the following steps: transducing mesenchymal stem cells with a lentiviral vector encoding FUT7 and CD63-P19; screening the successfully transduced mesenchymal stem cells; collecting the supernatant to obtain the exosomes. Thus, exosomes with enhanced expression of surface sLeX can be obtained.

[0011] In the application related to the first aspect of the present disclosure, optionally, the preparation further includes a hydrogel carrier, and the exosomes are distributed in the hydrogel carrier and the hydrogel carrier has a sustained release effect on the exosomes. Thus, the sustained release of exosomes by the hydrogel carrier can contribute to improving the therapeutic effect of the preparation on diabetic wounds.

[0012] In the application related to the first aspect of the present disclosure, optionally, the hydrogel carrier includes a first polymer and a second polymer, the first polymer is methacrylated gelatin, and the second polymer is polylysine methacrylate. In this case, a hydrogel carrier including methacrylated gelatin (GelMA) and polylysine methacrylate (PLMA) is used to carry exosomes, where GelMA has a porous structure suitable for the loading and slow release of exosomes, PLMA can improve the overall anti-inflammatory ability of the preparation, contribute to alleviating inflammation to promote the healing of diabetic wounds, and PLMA can further improve the sustained release function of the hydrogel carrier on exosomes.

[0013] In the application related to the first aspect of the present disclosure, optionally, the mass ratio of the first polymer to the second polymer is 1:2 to 15:2. The mass ratio of the first polymer to the second polymer is 1:2 to 15:2. In this case, it can contribute to obtaining a hydrogel carrier with high stability and an appropriate exosome release rate.

[0014] The second aspect of the present disclosure relates to a preparation for treating diabetic wounds, which is characterized by including exosomes, and the exosomes are mesenchymal stem cell exosomes with enhanced expression of surface sLeX.

[0015] In the preparation involved in the second aspect of the present disclosure, optionally, it further includes a hydrogel carrier, the exosomes are distributed in the hydrogel carrier, and the hydrogel carrier has a sustained-release effect on the exosomes. Thus, the sustained release of exosomes by the hydrogel carrier can help improve the therapeutic effect of the preparation on diabetic wounds.

[0016] In the preparation involved in the second aspect of the present disclosure, optionally, the hydrogel carrier includes a first polymer and a second polymer, the first polymer is methacrylated gelatin, and the second polymer is methacrylated polylysine. In this case, a hydrogel carrier including methacrylated gelatin (GelMA) and methacrylated polylysine (PLMA) is used to carry exosomes, where GelMA has a porous structure, which is suitable for the loading and slow release of exosomes. PLMA can improve the overall anti-inflammatory ability of the composite gel composition, help relieve inflammation to promote the healing of diabetic wounds, and PLMA can further improve the sustained-release function of the hydrogel carrier on exosomes.

[0017] According to the present disclosure, an application of exosomes in the preparation of a preparation for treating diabetic wounds can be provided, and the preparation has a good therapeutic effect on diabetic wounds. Description of the Drawings

[0018] Figure 1 It is a schematic diagram showing the high expression of endothelial injury-related genes in diabetic wounds in the embodiments of the present disclosure.

[0019] Figure 2 It is a schematic diagram showing the identification of MSCs and s-MSCs in the embodiments of the present disclosure.

[0020] Figure 3 It is a schematic diagram showing the identification results of Exo and s-Exo in the embodiments of the present disclosure.

[0021] Figure 4 It is a schematic diagram showing that LPS-stimulated HUVECs rapidly uptake s-Exo in the embodiments of the present disclosure.

[0022] Figure 5 It is a schematic diagram showing the characteristics of the hydrogel in the embodiments of the present disclosure.

[0023] Figure 6 It is a schematic diagram showing the biocompatibility and antibacterial activity of the hydrogel in the embodiments of the present disclosure.

[0024] Figure 7 It is a schematic diagram showing the healing of infected wounds of diabetic mice receiving different treatments in the embodiments of the present disclosure.

[0025] Figure 8It is a schematic diagram showing angiogenesis and inflammation in diabetic wounds in the embodiments of the present disclosure. Detailed implementation manners

[0026] Hereinafter, with reference to the accompanying drawings, the preferred embodiments of the present disclosure will be described in detail. In the following description, the same reference numerals are given to the same components, and repeated descriptions are omitted. In addition, the drawings are only schematic diagrams, and the ratio of the sizes of the components to each other or the shapes of the components may be different from the actual ones.

[0027] It should be noted that the terms "include" and "have" in the present disclosure and any variations thereof, for example, the processes, methods, systems, products or devices including or having a series of steps or units do not necessarily have to be limited to those steps or units clearly listed, but may include or have other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0028] The present disclosure relates to an application of exosomes in the preparation of a preparation for treating wounds. In particular, the present disclosure relates to an application of exosomes in the preparation of a preparation for treating diabetic wounds. The exosomes of the present disclosure have a good therapeutic effect on diabetic wounds, and applying exosomes in a preparation for treating diabetic wounds can help obtain a preparation with a good therapeutic effect on diabetic wounds. In the present disclosure, a diabetic wound refers to a chronic wound that is likely to form in the feet or other parts of a diabetic patient due to skin lesions, poor blood circulation, and decreased immune function. Due to the influence of diabetes, these wounds are often difficult to heal, prone to infection, and have more complications during the healing process. In the present disclosure, a wound can also be referred to as a wound surface.

[0029] The present disclosure also relates to a preparation for treating diabetic wounds. In the present disclosure, the preparation for treating diabetic wounds can be simply referred to as a preparation.

[0030] In addition, the present disclosure also relates to a composite gel composition that can promote wound healing. That is to say, the present disclosure can provide a composite gel composition that can promote wound healing. In particular, the composite gel composition of the present disclosure has a good therapeutic effect on diabetic wounds. In the present disclosure, the composite gel composition can be simply referred to as a composite gel, and can also be referred to as a composition, a complex, a hydrogel composition, or a composite hydrogel, etc.

[0031] Hereinafter, with reference to the accompanying drawings, the application of exosomes related to the present disclosure in the preparation of a preparation for treating wounds and the preparation including exosomes will be described in detail.

[0032] In the present disclosure, there is provided an application of exosomes in the preparation of a preparation for treating diabetic wounds. Among them, the exosomes can be mesenchymal stem cell exosomes with enhanced surface sLeX (Sialyl Lewis-X) expression (hereinafter simply referred to as s-Exo, also referred to as exosomes with overexpressed sLeX). sLeX is expressed by white blood cells and can bind to human umbilical vein endothelial cells (hereinafter simply referred to as HUVECs) expressing E-selectin under inflammatory stimulation. In this case, s-Exo can target HUVECs and enhance the function of HUVECs stimulated by inflammation in vitro, thereby promoting angiogenesis and alleviating inflammation, which helps to promote the healing of diabetic wounds. Specifically, the applicant of the present disclosure found that the expression of E-selectin in infected human and mouse wounds was significantly higher than that in normal non-diabetic wounds, reflecting the potential of E-selectin as a therapeutic target for diabetic wounds. E-selectin is highly expressed in HUVECs stimulated by inflammation and mediates the uptake of exosomes expressing sLeX by endothelial cells. s-Exo can specifically bind to HUVECs, thereby promoting its in vitro function and in vivo angiogenesis. Therefore, s-Exo can have a good therapeutic effect on diabetic wounds, and applying it in a preparation for treating diabetic wounds can obtain a preparation with a good therapeutic effect.

[0033] In some examples, the preparation can promote the healing of diabetic wounds by promoting angiogenesis. Thus, it can help to promote the healing of diabetic wounds.

[0034] In some examples, the exosomes can be exosomes derived from mesenchymal stem cells (MSCs). In some examples, the mesenchymal stem cells can be derived from any one of human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, placental mesenchymal stem cells, and dental pulp mesenchymal stem cells.

[0035] In some examples, preferably, the mesenchymal stem cells can be human umbilical cord mesenchymal stem cells (hucMSCs), and the exosomes can be exosomes derived from hucMSCs. In this case, the umbilical cord as a material source of mesenchymal stem cells and their exosomes has the advantages of reducing harm to the human body and being convenient for collection compared with other materials such as bone marrow and liver during collection, and the umbilical cord-derived mesenchymal stem cells have stronger proliferation and differentiation abilities. In some examples, hucMSCs and their exosomes can be obtained from fresh umbilical cord tissue of newborns.

[0036] In some examples, hucMSCs can be obtained by separating umbilical cord tissue. In some examples, human umbilical cord tissue can be cultured using a culture medium. In some examples, exosomes can be isolated after culturing human umbilical cord tissue using a culture medium. In some examples, the supernatant after culturing can be filtered using a filter to obtain exosomes.

[0037] In some examples, the exosomes can also be exosomes developed for different types of wounds. For example, the exosomes can be modified by genetic engineering means to obtain engineered exosomes with targeted design.

[0038] In some examples, the exosomes can target inflammatory vascular endothelial cells. Thereby, it can help promote wound recovery, especially the healing of diabetic wounds.

[0039] In some examples, the exosomes can be glycosylated to obtain s-Exo. For example, the outermost dense glycocalyx coating of the exosomes can be modified by glycoengineering technology to improve the targeting of the exosomes. In some examples, MSCs can be transduced with a lentiviral vector, and then cells stably expressing the transduction vector can be screened, thereby enabling the acquisition of predetermined exosomes.

[0040] In some examples, exosomes can be obtained through the following steps: transducing MSCs with a lentiviral vector; screening the successfully transduced MSCs; collecting the supernatant to obtain exosomes.

[0041] In some examples, the lentiviral vector can be a lentiviral vector encoding FUT7 (fucosyltransferase VII) and CD63-P19. Among them, P19 (19-mer peptide) is a short glycosylated domain derived from PSGL-1. The surface protein CD63 can serve as an exosome scaffold protein, and its larger extracellular loop can be used for genetic engineering and surface expression of functional components. The large extracellular loop of CD63 is fused with P19 to form CD63-P19. In this case, compared with using a lentiviral vector encoding only FUT7, which can only slightly increase the expression of sLeX on the cell surface, the co-expression of FUT7 and the sLeX protein carrier PSGL-1 can significantly increase the expression of sLeX on the cell surface. Transducing MSCs with a lentiviral vector encoding FUT7 and CD63-P19 can help obtain exosomes with enhanced surface expression of sLeX.

[0042] In some examples, puromycin screening can be used to obtain cells stably expressing the transduction vector. In some examples, the concentration of puromycin used for screening can be from 1 μg / mL to 3 μg / mL. In some examples, preferably, screening can be first performed with 3 μg / mL puromycin and then with 1 μg / mL puromycin for the maintenance concentration. Thus, it can help to obtain cells stably expressing the transduction vector. In some examples, other means can also be used to obtain targeted exosomes.

[0043] In some examples, exosomes can be used in combination with a hydrogel carrier. In other words, in some examples, the formulation can further include a hydrogel carrier. Among them, the exosomes can be distributed in the hydrogel carrier and the hydrogel carrier has a sustained-release effect on the exosomes. Thus, the sustained release of exosomes by the hydrogel carrier can help to improve the therapeutic effect of the formulation on diabetic wounds. In the present disclosure, the hydrogel carrier can be simply referred to as a hydrogel or also called a carrier. In some examples, the exosomes can be uniformly dispersed in the hydrogel carrier. Thus, it can help the hydrogel carrier to stably release exosomes within a certain period of time.

[0044] In some examples, the hydrogel carrier can include a first polymer. The first polymer can serve as the backbone structure of the hydrogel carrier, improving the overall stability of the hydrogel carrier and its ability to sustainably release exosomes. In some examples, preferably, the first polymer can be gelatin methacryloyl (GelMA, which can also be simply referred to as Gel). In this case, GelMA has a porous structure, which is suitable for the loading and slow release of exosomes; in addition, GelMA also has good biocompatibility and degradability.

[0045] In some examples, the hydrogel carrier can include a second polymer. The second polymer can at least provide functional effects for the hydrogel carrier. For example, the antibacterial ability of the hydrogel carrier can be increased by adding the second polymer. In some examples, preferably, the second polymer can be polylysine methacryloyl (PLMA, which can also be simply referred to as PL). In this case, PLMA can improve the overall antibacterial ability of the formulation, help to relieve inflammation to promote wound healing, especially the healing of diabetic wounds; in addition, PLMA can further improve the stability of the hydrogel carrier and its sustained-release function for repair factors. PLMA is the product of the methacrylation of ε-PL glycidyl methacrylate and introduces a double bond on the ε-PL molecule. ε-PL and its derivatives help to avoid the drug resistance and metal ion deposition caused by traditional antibacterial drugs or metal ions (such as Ag + )

[0046] In some examples, the hydrogel carrier may include a first polymer and a second polymer. In some examples, the first polymer may be crosslinked with the second polymer. In some examples, the first polymer may be crosslinked with the second polymer to form a three-dimensional network structure. In some examples, exosomes may be distributed in the three-dimensional network structure. In this case, it is beneficial to improve the stability of the hydrogel carrier and the sustained-release effect on exosomes.

[0047] In some examples, GelMA and PLMA are photopolymerizable hydrogels that can be crosslinked under ultraviolet light irradiation. In some examples, a photoinitiator can be added to promote the crosslinking of the first polymer and the second polymer. In other words, the hydrogel carrier may include a photoinitiator. Thereby, it can contribute to the crosslinking of the first polymer and the second polymer. In some examples, when the first polymer is GelMA and the second polymer is PLMA, the photoinitiator can be lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). In this case, LAP has excellent biocompatibility and low immunogenicity, and can contribute to the crosslinking of GelMA and PLMA under blue light. In some examples, GelMA and PLMA can be crosslinked under blue light with a wavelength of 405 nm.

[0048] In some examples, the concentration of the first polymer in the hydrogel carrier may be 5% w / v to 15% w / v. For example, the concentration of the first polymer may be 5% w / v, 6% w / v, 7% w / v, 8% w / v, 9% w / v, 10% w / v, 11% w / v, 12% w / v, 13% w / v, 14% w / v, or 15% w / v. In this case, selecting an appropriate concentration can help ensure the stability and biosafety of the hydrogel carrier. In some examples, preferably, the concentration of the first polymer in the hydrogel carrier may be 5% w / v. In this case, it can help obtain a hydrogel carrier with high stability and strong sustained-release effect for s-Exo.

[0049] In some examples, the concentration of the second polymer in the hydrogel carrier can be from 2% w / v to 10% w / v. For example, the concentration of the second polymer can be 2% w / v, 3% w / v, 4% w / v, 5% w / v, 6% w / v, 7% w / v, 8% w / v, 9% w / v, or 10% w / v. In this case, selecting an appropriate concentration can help improve the stability and antibacterial ability of the hydrogel carrier. By adjusting the concentration of the second polymer, the degradation rate of the hydrogel carrier can be adjusted. For example, when the second polymer has a relatively high concentration, it can result in a higher degree of crosslinking inside the hydrogel carrier, thereby reducing the swelling rate of the hydrogel carrier and decreasing the degradation rate of the hydrogel carrier, and further extending the release time. In some examples, preferably, the concentration of the second polymer in the hydrogel carrier can be 5% w / v. In this case, it can help obtain a hydrogel carrier with high stability and strong sustained-release effect for s-Exo.

[0050] In some examples, the mass ratio of the first polymer to the second polymer can be from 1:2 to 15:2. For example, the mass ratio of the first polymer to the second polymer can be 1:2, 2:2 (1:1), 3:2, 4:2, 5:2, 6:2, 7:2, 8:2, 9:2, 10:2, 11:2, 12:2, 13:2, 14:2, or 15:2. In this case, it can help obtain a hydrogel carrier with high stability and an appropriate exosome release rate. In some examples, preferably, the mass ratio of the first polymer to the second polymer can be 1:1. In this case, it can help obtain a hydrogel carrier with high stability and strong sustained-release effect for s-Exo.

[0051] In some examples, the formulation can further include pharmaceutically acceptable excipients. For example, the formulation can further include excipients such as diluents, thickeners, and / or antioxidants.

[0052] In some examples, preferably, the formulation can include a hydrogel carrier and exosomes. The hydrogel carrier includes a first polymer and a second polymer. The first polymer is methacrylated gelatin, the second polymer is methacrylated polylysine, and the exosomes are mesenchymal stem cell exosomes that enhance the expression of surface sLeX. Thus, according to the present disclosure, a formulation with good therapeutic effect on diabetic wounds can be provided.

[0053] As described above, the present disclosure also provides a formulation for treating diabetic wounds. The formulation has a good therapeutic effect on diabetic wounds. Among them, the formulation can be the formulation as described above, and the specific components and compositions can be referred to the previous description and will not be elaborated here.

[0054] In some examples, preferably, the preparation may include exosomes, and the exosomes are mesenchymal stem cell exosomes that enhance the expression of surface sLeX. Thus, a preparation with good therapeutic effects on diabetic wounds can be provided.

[0055] In some examples, preferably, the preparation may include a hydrogel carrier and exosomes. The hydrogel carrier includes a first polymer and a second polymer. The first polymer is methacrylated gelatin, and the second polymer is methacrylated polylysine. The exosomes are mesenchymal stem cell exosomes that enhance the expression of surface sLeX. Thus, a preparation with good therapeutic effects on diabetic wounds can be provided. In the present disclosure, the exosomes including the hydrogel carrier and exosomes may be referred to as a composite gel composition, or may also be simply referred to as a composite gel, composition, complex, hydrogel composition, or composite hydrogel, etc. That is, in the present disclosure, a composite gel composition for treating diabetic wounds can also be provided.

[0056] In some examples, the preparation can promote the healing of diabetic wounds by promoting angiogenesis. Since angiogenesis is very important for maintaining the blood supply required for skin repair, but angiogenesis is usually inhibited in chronic diabetic wounds, resulting in a decrease in capillary density, reducing the supply of nutrients and oxygen, and ultimately delaying wound healing. In this case, the preparation provided in the present disclosure can have good therapeutic effects on diabetic wounds by promoting angiogenesis to promote the healing of diabetic wounds.

[0057] In the present disclosure, the preparation can be a drug. In some examples, the preparation can be used in an external application manner. In some examples, the preparation can be directly applied to the wound or the area adjacent to the wound. Thus, it can help promote the healing of diabetic wounds. In some examples, the preparation can be an external dosage form such as a patch, gel, cream, or spray.

[0058] In some examples, preferably, the preparation can be applied as a wound dressing. For example, the preparation can be applied as a wound dressing for diabetic wounds. Thus, an easy-to-apply application method can be provided. In some examples, the preparation can be used by being carried on a microneedle dressing. In this case, it can help the preparation come into full contact with the wound, enabling the repair factors in the preparation to better act on the wound, thereby further improving the therapeutic effect of the preparation on the wound. In the present disclosure, the preparation as a wound dressing can also be simply referred to as a dressing or a patch.

[0059] In summary, according to the present disclosure, an application of exosomes in the preparation of a preparation for treating diabetic wounds can be provided, and the preparation has good therapeutic effects on diabetic wounds.

[0060] To further illustrate the present disclosure, the following provides a detailed description of the preparation for treating diabetic wounds provided by the present disclosure in conjunction with embodiments.

[0061] In the embodiments of the present disclosure, unless otherwise specified, the reagents and instruments used are all commercially available products.

[0062] [Embodiment]

[0063] Ethical Approval Statement All animal experiments were approved by the Institutional Animal Care and Use Committee of Qilu Hospital of Shandong University. This study involved human samples and was approved by the Ethics Committee of Qilu Hospital of Shandong University. All participants provided written informed consent for the collection and analysis of tissue samples.

[0064] Experimental operations involved

[0065] (1) scRNA-seq of wound samples

[0066] We obtained and re-analyzed the data based on the expression matrix. The data came from 12 wound samples provided by 9 participants, among which 5 had diabetic foot ulcers, 4 had non-diabetic foot wounds, with 1 providing 2 samples and another providing 3 samples. The data was analyzed using ScanPy. We first filtered low-quality cells and genes and performed double filtering using scrulet. After normalizing and logarithmically transforming the data, we determined the cell cycle and regressed it to eliminate its influence. After removing batch effects using the BBKNN method, PCA and UMAP dimensionality reduction were performed, and Leiden clustering analysis was used to identify different cell populations. Using the CellTypist software package, cell types were determined by annotation according to the previous human skin atlas. An independent t-test was used to evaluate the inter-group differences in gene expression.

[0067] (2) Preparation and transfection of lentiviral vectors

[0068] The exosomal scaffold protein CD63 has a large extracellular loop that can be fused with the potential sLeX vector P19 and the C-terminal luciferase reporter Nluc, thereby constructing the CD63-P19-Nluc protein vector. GeneChem Co., Ltd. (Shanghai, China) prepared a lentiviral vector co-expressing FUT7 and CD63-P19-Nluc. The concentrated and purified lentivirus was stored at -80 °C until use.

[0069] The method for inducing lentiviral infection of mesenchymal stem cells (MSCs) was carried out on passage 3 MSCs. The MSCs (3 - 5×10 4 cells / mL) were seeded into a T25 cell culture flask and cultured at 37 °C and 5% CO 2Cultured in a cell incubator until reaching a confluence of approximately 20%. Transfected MSCs with lentivirus overnight. After the transfected MSCs reached approximately 80% confluence and grew well, screened with puromycin (3 μg / mL; product number IA0530, Solarbio, Beijing, China) for 48 hours. Then further cultured the surviving MSCs in a medium containing puromycin (1 μg / mL). Changed the medium every 2 days and collected the cells. Detected surface markers, adipogenic and osteogenic induction of the transfected cells by flow cytometry (DxFLEX, Beckman Coulter, USA), and stained to determine the differentiation ability of s-MSCs (MSCs overexpressing sLeX). Evaluated the expression of sLeX on the surface of s-MSCs (Cat. No. 563526, BD Pharmingen, San Diego, USA) by flow cytometry.

[0070] (3) Detect E-selectin on the surface of HUVEC

[0071] HUVECs were cultured in endothelial cell medium (ECM; ScienCell, Carlsbad, CA, USA). Inoculated HUVECs into six-well plates, treated with negative control and 1 μg / mL LPS (MedChemExpress, Shanghai, China) for 4 hours, digested with trypsin, washed with PBS, and incubated with PE-conjugated mouse anti-human E-selectin (Cat. No. 322606, BioLegend, San Diego, CA, USA) in the dark for 30 minutes. Finally, detected the expression of E-selectin by flow cytometry.

[0072] (4) Preparation of hydrogel

[0073] Dissolved the LAP standard solution in PBS (0.25%, w / v) and stored it in the dark. Filtered the GelMA and PLMA solutions through a 0.22 μm pore filter to ensure sterility, and then mixed them with the LAP solution in different ratios to obtain 5% (w / v) GelMA (subsequently abbreviated as Gel), 5% (w / v) GelMA + 2% (w / v) PLMA (subsequently abbreviated as Gel / PL-2), and 5% (w / v) GelMA + 5% (w / v) PLMA (subsequently abbreviated as Gel / PL-5). Cross-linked the precursor solution under ultraviolet radiation for 15 s to form a hydrogel.

[0074] (5) Treat the wounds of diabetic mice with s-Exo@Gel / PL-5

[0075] 8-week-old C57BL / KsJ mice were intraperitoneally injected with 150 mg / kg streptozotocin (Cat. No. S0130, Sigma-Aldrich, St. Louis, MO, USA). Blood glucose levels were measured twice randomly, and mice with blood glucose levels reaching 16.7 mmol / L were considered diabetic and included in the experimental group. Mice were anesthetized with isoflurane (2%), the back hair was shaved off, and the subcutaneous skin was disinfected with 75% ethanol. A full-thickness wound (8 mm) was created on the dorsal skin using a skin biopsy punch. A mixed solution of Staphylococcus aureus and Escherichia coli (1×10 8 CFU / mL) was inoculated into the wound for 2 days. Subsequently, four mice were euthanized under anesthesia, and skin tissues were collected to determine the expression of E-selectin in infected diabetic wounds. The remaining mice were randomly divided into five groups (n = 6 per group) and were given respectively:

[0076] ① PBS

[0077] ② Exo (100 μg)

[0078] ③ s-Exo (100 μg), where s-Exo is exosomes secreted by s-MSCs.

[0079] ④ Gel / PL-5

[0080] ⑤ s-Exo@Gel / PL-5 (containing 100 μg s-Exo)

[0081] The day when the mice received dressing treatment was recorded as day 0. The dressings of groups ①, ②, and ③ were injected around the wound (a total of 100 μL, 25 μL at each site), the hydrogels of group ④ (Gel / PL-5) and group ⑤ (s-Exo@Gel / PL-5, containing 100 μg s-Exo) were dropped onto the wound surface (total dose of 100 μL), and cross-linked under ultraviolet irradiation for 15 s. The dressings were changed every 3 days. Wound photos were taken at fixed times and distances (calibrated with a ruler), and the wound area was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The calculation method of the remaining area was as follows: remaining area (%) = S t / S 0 ×100%, where S t represents the wound area at different times, and S 0 represents the initial wound area. On day 12, the mice were euthanized under anesthesia. The collected skin wound tissues were preserved for future use.

[0082] (6) In vivo toxicity test of the hydrogel

[0083] Peripheral blood was collected on the 12th day of hydrogel treatment. EDTA anticoagulant was added to a part of the blood to obtain whole blood for detecting blood indexes. Another part of the blood was centrifuged to obtain serum for detecting different protein markers. The heart, liver, kidneys and lungs were taken, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into hematoxylin and eosin (H&E) stained sections for histological analysis of hydrogel toxicity.

[0084] (7) Histology and immunohistochemical staining

[0085] Healing wound samples were collected on the 12th day of hydrogel treatment, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm thick sections. After deparaffinization, the samples were stained with H&E (Catalog No.: BA4907, Baisi, Wuhan, China) and Masson's trichrome staining (Catalog No.: G1006, Servicebio, China). To determine the angiogenesis and anti-inflammatory effects of the hydrogel, the samples were immunohistochemically stained with anti-CD31 antibody (Cat. No. ab182981, Abcam, Cambridge, UK), anti-α-SMA (Cat. No. ab7817, Abcam), anti-IL-1β (Cat. No. ab283818, Abcam) and anti-IL-6 (Cat. No. ab290735, Abcam) according to the manufacturer's instructions. The epidermal thickness and mean integrated optical density value were quantified using ImageJ Pro software.

[0086] (8) Detection of E-selectin expression in wound tissues by immunofluorescence staining

[0087] Human wound tissue specimens were taken from non-diabetic and diabetic patients. Wound tissues taken from humans and mice were fixed with 4% paraformaldehyde, embedded in paraffin, cut into 5-μm thick sections, deparaffinized, antigen repaired, blocked with 10% goat serum at room temperature for 1 hour, and incubated overnight at 4 °C with anti-CD31 (Cat. No. sc-376764, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-E-selectin (Cat. No. ab18981, Abcam). The next day, the sections were incubated with Alexa Fluor-labeled secondary antibody at room temperature for 1 hour. The nuclei were stained with 4,6′-diamidino-2-phenylindole at room temperature for 5 minutes. Finally, images were acquired using a fluorescence microscope.

[0088] (9) Statistical analysis

[0089] Data are presented as mean ± standard error of the mean (SEM) and analyzed using GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA, USA). Student's t-test was used to analyze differences between groups, and one-way analysis of variance (ANOVA) was used to analyze differences between three or more groups. All experiments were repeated at least three times. Statistical significance was defined as P < 0.05.

[0090] Analysis of experimental results

[0091] (1) scRNA-seq data indicate gene-driven endothelial injury

[0092] Figure 1 It is a schematic diagram showing the high expression of genes related to endothelial injury in diabetic wounds in the embodiments of the present disclosure. Figure 1 Part a is a schematic diagram of grouping the UMAP (uniform manifold approximate projection) dimensionality reduction map using Leiden clustering. Figure 1 Part b shows different cell types on the UMAP dimensionality reduction map after annotation based on the skin cell atlas. Figure 1 Part c is a schematic diagram of the expression of marker genes of two types of endothelial cells on the UMAP dimensionality reduction map. Figure 1 Part d is a schematic diagram of a heat map of the top 20 differentially expressed genes in endothelial cells of diabetic and non-diabetic patients. Figure 1 Part e is a schematic diagram of the expression of genes related to endothelial injury in endothelial cells of two groups of patients. Figure 1 Part f is a schematic diagram of representative immunofluorescence staining images of E-selectin expression in wounds of non-diabetic and diabetic patients, scale bar: 50 μm. Figure 1 Part g is a schematic diagram of representative immunofluorescence staining images of E-selectin expression in wounds of non-diabetic and diabetic mice, scale bar: 20 μm. In the drawings related to the embodiments of the present disclosure, ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0093] After filtering and processing the data, the cell cycle was identified and regressed. The final constructed matrix included 44,426 cells expressing 24,771 genes. Clustering after dimensionality reduction yielded 19 cell clusters (see Figure 1 part a), and were annotated as 13 different cell types (see Figure 1 part b). There are two types of endothelial cells in the dermis of healthy adults, namely vascular endothelial cells (VEC) and lymphatic endothelial cells (LEC), which express PECAM1 and LYVE1 respectively (seeFigure 1 Part c). We analyzed the gene expression of these two types of endothelial cells and found that the expression levels of endothelial injury-related genes, such as "SELE", "RPS4Y1", "NFKBIA", "C2CD4B", and "JUNB", were higher in the diabetic group than in the non-diabetic group (see Figure 1 Parts d and e).

[0094] The E-selectin adhesion molecule encoded by SELE is present in inflamed endothelial cells and is thought to promote the accumulation of blood leukocytes at the site of inflammation by mediating cell adhesion to the vascular endothelium. To design an E-selectin-targeted glycoengineered gel for treating diabetic wounds, we verified the expression of E-selectin, an expression product of vascular endothelium upon inflammatory activation. In human samples, E-selectin co-localized with the vascular endothelial marker CD31 in diabetic wounds, while E-selectin was not expressed in non-diabetic wounds ( Figure 1 f). Immunofluorescence staining of mouse model samples showed that E-selectin was highly expressed in the wounds of diabetic mice compared to non-diabetic mice ( Figure 1 g).

[0095] (2) Preparation and identification of MSCs, s-MSCs, Exo, and s-Exo

[0096] Figure 2 is a schematic diagram showing the identification of MSCs and s-MSCs in the embodiments of the present disclosure. Figure 2 Part a is a schematic diagram of the cell viability assay of MSCs and s-MSCs, Figure 2 Part b is a schematic diagram of detecting the expression of sLeX on the surface of s-MSC by flow cytometry. Figure 2 Parts c and d are schematic diagrams of Oil Red O and Alizarin Red staining of MSCs and s-MSCs after adipogenic and osteogenic induction, scale bar: 20 μm. Figure 2 Part e is a schematic diagram of identifying MSCs and s-MSCs by flow cytometry.

[0097] It was determined by the CCK-8 assay that lentiviral infection did not affect the viability of MSCs (see Figure 2 Part a). Flow cytometry showed that sLeX was highly expressed in s-MSCs (see Figure 2 Part b). Notably, the expression of FUT7 and CD63-P19-Nluc could significantly increase the expression of E-selectin on the surface of s-MSCs. To demonstrate the adipogenic differentiation ability of MSCs and s-MSCs, the cells were cultured in adipogenic medium for 3 weeks and stained with Oil Red O (see Figure 2Part c). To demonstrate the osteogenic differentiation ability of MSCs and s-MSCs, the cells were cultured in osteogenic medium for 4 weeks and then stained with alizarin red (see Figure 2 Part d). Flow cytometry was used to detect the surface marker expression of passage 5 MSCs and s-MSCs. MSCs and s-MSCs were positive for CD105 and CD73 expression and negative for CD34 and HLA-DR expression (see Figure 2 Part e). These findings confirmed the successful isolation of MSCs, and the cells transduced with the lentiviral vector retained the characteristics of mesenchymal stem cells.

[0098] Figure 3 is a schematic diagram showing the results of the identification of Exo and s-Exo in the examples of the present disclosure. Figure 3 Parts a and b in are schematic diagrams of the diameter and zeta potential of Exo and s-Exo measured by NTA. Figure 3 Part c in is a schematic diagram of the Western blot analysis of the endoplasmic reticulum marker Calnexin and exosome markers CD9, Alix, and TSG101 in cells. Figure 3 Part d in is a schematic diagram of observing the morphology of exosomes by transmission electron microscopy. Figure 3 Part e in is a schematic diagram of the Western blot analysis of sLeX expression. Figure 3 Part f in is a schematic diagram of the statistical analysis of the expression level of sLeX relative to GAPDH.

[0099] Exo and s-Exo were characterized by nanoparticle tracking analysis (NTA), immunoblotting, and transmission electron microscopy (TEM). NTA showed that the median diameters of Exo and s-Exo were 132.4 nm and 141.4 nm, respectively, both within the normal range (see Figure 3 Part a). The zeta potentials of Exo and s-Exo were -29.55 mV and -33.44 mV, respectively (see Figure 3 Part b). Western blotting confirmed the expression of exosome markers CD9, Alix, and TSG101, while the endoplasmic reticulum marker calnexin was not expressed in Exo and s-Exo (see Figure 3 Part c). TEM revealed the cup-shaped morphology and bilayer membrane structure of Exo and s-Exo (see Figure 3 Part d). Western blotting showed that the level of sLeX in s-Exo was higher than that in Exo (see Figure 3 Parts e and f).

[0100] (3) S-Exo has a strong binding ability to activated endothelial cells

[0101] Figure 4 It is a schematic diagram showing that LPS (lipopolysaccharide)-stimulated HUVECs rapidly uptake s-Exo. Figure 4 Part a in it is a schematic diagram of the expression of E-selectin on HUVECs after 4 hours of LPS stimulation. Figure 4 Part b in it is a schematic diagram of the quantification of cell Nluc expression after 4 hours of exosome treatment. Figure 4 Part c in it is a representative image of normal and LPS-activated HUVECs uptake of DIO-labeled Exo and s-Exo. Figure 4 Part d in it is a schematic diagram of a representative image of the HUVECs Transwell migration assay under different treatments, scale bar: 100 μm. Figure 4 Part e in it is a schematic diagram of the quantitative analysis of cell migration in the migration experiment. Figure 4 Part f in it is a schematic diagram of a representative image of the HUVECs cell scratch assay at 10 h and 20 h under different treatments, scale bar: 100 μm. Figure 4 Part g in it is a schematic diagram of the quantitative analysis of the migration area in the cell scratch experiment.

[0102] To verify the uptake of s-Exo by HUVECs cells, cells were first stimulated with LPS to determine the expression of E-selectin. Flow cytometry showed that after 4 h of exposure to LPS, the expression of E-selectin was significantly higher than that of the control group (see Figure 4 part a in it). The uptake rate of s-Exo by activated HUVECs (>3.8-fold) was significantly higher than that of non-activated HUVECs (see Figure 4 part b in it). Fluorescence microscopy showed that HUVECs could uptake Exo and s-Exo with and without LPS stimulation, while LPS-stimulated HUVECs could uptake more s-Exo (see Figure 4 part c in it).

[0103] The migration ability of four groups of HUVECs (LPS, LPS+Exo, and LPS+s-Exo) was measured using the scratch assay (see Figure 4 part d in it). HUVECs in the LPS+s-Exo group showed the highest migration rate (see Figure 4 part e in it), which was consistent with the results of the migration assay (see Figure 4 part f in it). Treated HUVECs were pre-cultured with negative control and LPS for 4 hours, then the cells were scratched, rinsed with PBS, and provided with fresh medium. The scratch assay showed that at 10 hours and 20 hours, HUVECs in the LPS+s-Exo group migrated more than those in the other three groups (see Figure 4 part g in it).

[0104] (4)Characterization of Gel, Gel / PL-2, and Gel / PL-5

[0105] Figure 5 It is a schematic diagram showing the characteristics of the hydrogel in the embodiments of the present disclosure. Figure 5 Part a shows a schematic diagram of the precursor solution gelling to form a hydrogel under ultraviolet irradiation. Figure 5 Part b shows schematic diagrams of various shapes obtained from the precursor solution under ultraviolet irradiation. Figure 5 Part c shows a schematic diagram of the swelling characteristics of the hydrogel. Figure 5 Part d shows a schematic diagram of the degradation characteristics of the hydrogel. Figure 5 Part e shows a schematic diagram of the s-Exo release curve. Figure 5 Part f shows a schematic diagram of the rheological behavior of the hydrogel. Figure 5 Part g shows a schematic diagram of the rheological analysis of the hydrogel under frequency scanning. Figure 5 Part h shows a schematic diagram of the three-dimensional reconstruction of PKH 67-labeled s-Exo in Gel / PL-5. Scale bar: 500 μm. Figure 5 Part i shows a schematic diagram of the SEM images of all hydrogels. Figure 5 Part j shows a schematic diagram of the average pore size and pore size distribution of each hydrogel.

[0106] Previous studies have shown that a 5% concentration of GelMA is suitable for wound healing. Therefore, we selected 5% (w / v) GelMA as the application concentration. In addition, we also selected 2% and 5% PLMA to determine the mechanical and antibacterial properties of the gel. To evaluate the transition of the hydrogel system from sol to gel, we conducted an inversion test. The solution showed flow characteristics after the test tube was inverted, but showed a rapid transition from sol to gel under ultraviolet radiation, and no fluidity was found after inversion (see Figure 5 part a). The hydrogel can be molded into various shapes (see Figure 5 part b). The swelling rate of Gel / PL-5 was lower than that of Gel and Gel / PL-2, indicating that the swelling ability of the hydrogel gradually decreases with the increase in crosslinking density (see Figure 5 part c). The degradation rate (measured every 2 days) showed that the degradation rate of Gel was faster than that of other hydrogels, and the degradation rate of Gel / PL-5 was significantly slower than that of low-concentration PL (see Figure 5 part d). The hydrogel was immersed in PBS to obtain the s-Exo release curve. Gel showed a rapid and explosive release of s-Exo in the first 3 days, while Gel / PL-5 continuously and stably released s-Exo (see Figure 5 part e).

[0107] Over time, the storage modulus (G′) values of the three groups of hydrogels continuously increased, exceeding the loss modulus (G″) values and finally reaching equilibrium, indicating that the precursor solution formed a solid hydrogel with a stable internal structure (see Figure 5 part f). As the oscillation frequency increased, the G′ of the three hydrogels was always higher than the corresponding G″ value, indicating a stable internal structure (see Figure 5 part g). Confocal microscopy imaging showed the successful loading and uniform three-dimensional distribution of s-Exo in Gel / PL-5 (see Figure 5 part h). Scanning electron microscope images showed the porous structure inside the three hydrogels (see Figure 5 part i). As the PL concentration increased, the internal crosslinking of the hydrogel was enhanced and the pore size of the hydrogel gradually decreased (see Figure 5 part j).

[0108] (5) Biocompatibility of the hydrogel

[0109] Figure 6 is a schematic diagram showing the biocompatibility and antibacterial activity of the hydrogel in the embodiments of the present disclosure. Figure 6 Part a in Figure 6 is a schematic diagram of the cell viability of HUVECs after incubating with the hydrogel extract for 48 h. Figure 6 Part b in Figure 6 is a schematic diagram of the hemolysis rate of the hydrogel, Figure 6 and part c in Figure 6 is a schematic diagram of the hematological properties of the hydrogel, with TritonX-100 as the positive control and PBS as the negative control.

[0110] Good biocompatibility is very important for the clinical application of wound dressings. We cultured HUVECs in conditioned media containing different concentrations of hydrogels for 48 h and found that all hydrogels did not affect cell viability (see Figure 6 part a). The hemolysis test showed that the hemolysis rate of the blood co-cultured with the hydrogel extract was less than 5% compared with the positive control group treated with Triton-X100, indicating that the hydrogel had good blood compatibility (see Figure 6parts b and c). After culturing in the hydrogel conditioned medium for 48 hours, the cytotoxicity was further determined by live / dead cell staining. Compared with the positive control group, fewer dead cells (red fluorescence) of HUVECs were observed in the hydrogel treatment group (see Figure 6 part d), indicating that Gel, Gel / PL-2, and Gel / PL-5 have good biosafety.

[0111] (6) In vitro antibacterial activity of the hydrogel

[0112] Diabetic wounds are usually accompanied by various bacterial infections, the most common of which are infections caused by Staphylococcus aureus and Escherichia coli. The antibacterial activity of Gel / PL is mainly attributed to PL, which contains a large number of positively charged amino groups that can interfere with the membrane potential of bacteria. Gel alone has no antibacterial properties, but as the concentration of PL increases, the antibacterial properties also increase, as evidenced by the gradual decrease in the number of bacterial colonies (see Figure 6 part e). The survival rates of Staphylococcus aureus and Escherichia coli in the Gel / PL group were 1.098% ± 0.8283% and 3.912% ± 0.5489% respectively (see Figure 6 part f). Live / dead staining detection showed that the bacteria in the control group and the gel group were mainly alive (green), while the bacteria in the Gel / PL-2 group and the Gel / PL-5 group were mainly dead (red, see Figure 6 part g). Therefore, the Gel / PL group has good antibacterial properties and can inhibit diabetic foot infections and promote diabetic wound healing.

[0113] (7) S-Exo@Gel / PL-5 can accelerate in vivo diabetic wound healing

[0114] Figure 7 is a schematic diagram showing the healing of infected wounds in diabetic mice receiving different treatments in the embodiments of the present disclosure. Figure 7 Part a is a schematic diagram of diabetic wound treatment. Figure 7 Part b is a schematic diagram of representative photographs of diabetic wounds and wound marks in the PBS, Exo, s-Exo, Gel / PL-5, and s-Exo@Gel / PL-5 groups at 0, 3, 6, 9, and 12 days after applying the medicine. Figure 7 Part c is a schematic diagram of the quantification of the remaining wound area in each group at 3, 6, 9, and 12 days. Figure 7 Part d is a schematic diagram of representative H&E staining images of wound tissues in each group at 12 days, scale bar: 500 μm. Figure 7 Part e is a schematic diagram of the quantification of the epithelial cell thickness in each group.

[0115] Figure 8It is a schematic diagram showing angiogenesis and inflammation in diabetic wounds in the embodiments of the present disclosure. Figure 8 Part a shows representative images of Masson's trichrome, CD31, α-SMA, IL-1β, and IL-6 staining of wound tissues in each group on the 12th day, scale bar: 500 μm. Figure 8 Parts b - e are schematic diagrams for quantitatively analyzing the average IOD (integrated optical density) of CD31, α-SMA, IL-1β, and IL-6 respectively.

[0116] We measured the effect of Gel / PL-5 in full-thickness diabetic wounds infected with Staphylococcus aureus and Escherichia coli. Figure 7 Part a shows the experimental schedule of wound infection and treatment. Figure 7 Part b shows representative images and healing rates of each group on the 0th, 3rd, 6th, 9th, and 12th days of treatment. The infected open wounds showed redness and swelling. The s-Exo@Gel / PL-5 group had the smallest remaining wound area (1.353%) compared with the PBS, Exo, s-Exo, and Gel / PL-5 groups (24.73%, 12.06%, 7.05%, and 16.45% respectively). The wound healing rate of the s-Exo group was faster than that of the Exo group, indicating that the surface expression of sLeX on exosomes promoted the healing of infected diabetic wounds.

[0117] After 12 days of treatment, re-epithelialization of the skin and generation of appendages were observed by H&E staining (see Figure 7 part d). The skin epithelium of the s-Exo@Gel / PL-5 group was the thickest, followed by the s-Exo group, which was superior to the PBS, Exo, and Gel / PL-5 groups (see Figure 7 part e). The wounds treated with s-Exo@Gel / PL-5 showed denser collagen fibers, thicker granulation tissue, and higher deposition of newly formed collagen (see Figure 8 part a).

[0118] On the 12th day after treatment, we evaluated the changes in angiogenesis by immunohistochemical staining of vascular markers (CD31 and α-SMA, see Figure 8 part a). The s-Exo@Gel / PL-5 treatment group showed more newly formed blood vessels, indicating that its angiogenesis effect was superior to other groups (see Figure 7 part b and Figure 8 part c). In addition, the expression of inflammatory cytokines (IL-1β and IL-6) was also evaluated (see Figure 8 parts d and e).

[0119] (8) Detect the toxicity of the hydrogel to the body

[0120] The hydrogel has no pathological effect on the blood components in vivo, and no damage or pathological changes are observed in the vital organs (heart, liver, lungs, and kidneys).

[0121] Summary

[0122] In this disclosure, we re-analyzed the single-cell gene expression profiles of chronic foot ulcers in diabetic and non-diabetic patients. The scRNA-seq results showed that the expressions of endothelial injury-related genes such as "SELE", "RPS4Y1", "NFKBIA", "C2CD4B", and "JUNB" were up-regulated in diabetic wounds compared with non-diabetic wounds, which reflected the vascular injury in diabetic wounds. We selected "SELE" as the key factor for exosome glycoengineering. E-selectin (expressed by the SELE gene) is essential for the adhesion of endothelial progenitor cells, endothelial cell homing in vitro, and neovascularization in vivo. We found that the expression of E-selectin was significantly higher in infected human and mouse wounds than in normal wounds, reflecting the potential of E-selectin as a target for diabetic wound healing. We confirmed that E-selectin was highly expressed in stimulated HUVECs and mediated the uptake of sLeX-expressing exosomes by endothelial cells. Cell and mouse experiments confirmed the expression of E-selectin in wound vascular endothelium, reflecting the importance of exosome glycosylation and the feasibility of the glycoengineered hydrogel involved in this disclosure.

[0123] We modified the outermost dense glycocalyx coating of exosomes using rational glycoengineering techniques to improve the targeting of exosomes. MSCs were transduced with lentiviral vectors encoding FUT7 and CD63-P19, screened with 3 μg / mL puromycin, and then maintained with 1 μg / mL puromycin to obtain cells stably expressing the transduction vectors. Our results showed that the surface expression of sLeX in the paracrine exosomes of s-MSCs was significantly increased. Under inflammatory stimulation, exosomes expressing sLeX (s-Exo) could specifically bind to HUVECs, thereby promoting their in vitro function and in vivo angiogenesis.

[0124] In this disclosure, we infected diabetic wounds with Staphylococcus aureus and Escherichia coli to determine the antibacterial effect of the hydrogel. Through bacterial colony counting and live / dead assays, we found that Gel / PL-5 containing 5% PL had better antibacterial ability than Gel / PL-2.

[0125] In vivo, we found that s-Exo@Gel / PL-5 had the best healing effect on infected wounds compared with other treatments. The wound healing rate, regenerated epidermal thickness, and collagen deposition rate indicated that the performance of exosomes was significantly improved after glycosylation and Gel / PL-5 encapsulation. The expressions of CD31 and α-SMA in the wound tissues of the s-Exo group were higher than those in the Exo group, indicating that the surface expression of sLeX in exosomes promoted angiogenesis in diabetic infected wounds. The s-Exo@Gel / PL-5 group showed better anti-inflammatory and angiogenic effects than the s-Exo group, indicating that the exosome-loaded hydrogel had a better therapeutic effect than exosomes alone. The levels of pro-inflammatory cytokines (IL-1β and IL-6) in the s-Exo@Gel / PL-5 group were the lowest, and the microvessel density was the highest. The results showed that s-Exo in Gel / PL-5 retained its function, and the combined effect of s-Exo@Gel / PL-5 was much greater than that of a single component.

[0126] Biosafety is the key to the clinical acceptance of hydrogel dressings. We tested the biocompatibility and toxicity of the hydrogels in vitro and in vivo and found that Gel and Gel / PL had good biocompatibility, which was demonstrated by CCK8 and live / dead staining assays after co-culturing HUVECs with hydrogel extracts for 48 hours. After co-culturing mouse blood with hydrogel extracts for 1 hour, we found that the hemolysis rate was significantly lower than that of the positive control group, indicating good blood compatibility of the hydrogels in vivo. We observed no obvious histological or hematological changes in the peripheral blood, heart, liver, kidney, and lung tissues of mice treated with the hydrogels, indicating that the hydrogels could improve diabetic wound healing and had no toxic effects in vivo.

[0127] Conclusion

[0128] The research of this disclosure shows that the enhanced expression of sLeX on the surface of exosomes can promote wound angiogenesis. The photo-polymerizable hydrogel Gel / PL-5 has excellent antibacterial activity, biocompatibility, and mechanical properties. Gel / PL-5 carrying s-Exo shows a sustained release of s-Exo, accelerating the healing of diabetic infected wounds. The newly developed s-Exo@Gel / PL-5 hydrogel is a novel dressing for treating diabetic infected wounds with broad application prospects.

[0129] In summary, this disclosure can provide an application of exosomes in the preparation of a preparation for treating diabetic wounds, and the preparation has a good therapeutic effect on diabetic wounds.

[0130] Although the present disclosure has been specifically described above in connection with the accompanying drawings and examples, it is to be understood that the above description does not limit the present disclosure in any way. Those skilled in the art may make modifications and variations to the present disclosure as needed without departing from the spirit and scope of the present disclosure, and such modifications and variations fall within the scope of the present disclosure.

Claims

1. An application of exosomes in preparing a preparation for treating diabetic wounds, characterized in that: The exosomes are mesenchymal stem cell exosomes that enhance the expression of surface sLeX. The preparation also includes a hydrogel carrier, the exosomes are distributed in the hydrogel carrier, and the hydrogel carrier has a sustained release effect on the exosomes. The hydrogel carrier includes a first polymer and a second polymer, the first polymer is methacryloyl gelatin, and the second polymer is methacryloyl polylysine, and the mass ratio of the first polymer to the second polymer is 1:

1.

2. The use according to claim 1, characterized in that: The preparation promotes the healing of diabetic wounds by promoting angiogenesis.

3. The use according to claim 1, characterized in that: The preparation is applied topically by applying the preparation directly to the wound or to a site adjacent to the wound.

4. The use according to claim 1, characterized in that: The exosomes are obtained by the following steps: Mesenchymal stem cells were transduced with lentiviral vectors encoding FUT7 and CD63-P19; Screening of successfully transduced mesenchymal stem cells; The supernatant was collected to obtain the exosomes.

5. A preparation for treating diabetic wounds, characterized in that: It includes exosomes, which are mesenchymal stem cell exosomes that enhance the expression of surface sLeX, and also includes a hydrogel carrier, in which the exosomes are distributed and the hydrogel carrier has a sustained release effect on the exosomes, and the hydrogel carrier includes a first polymer and a second polymer, the first polymer is methacryloyl gelatin, the second polymer is methacryloyl polylysine, and the mass ratio of the first polymer to the second polymer is 1:1.

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