Hydrogel carrier and its application in preparing preparation for treating diabetic wounds

Abstract

The present disclosure provides a hydrogel carrier and its application in preparing a preparation for treating diabetic wounds. The hydrogel carrier is used to carry a repair factor and has a sustained release effect on the repair factor. The hydrogel carrier includes a first polymer and a second polymer. The first polymer is methacryloyl gelatin and the second polymer is methacryloyl polylysine. According to the present disclosure, a hydrogel carrier that helps promote diabetic wound healing and its application in preparing a preparation for treating diabetic wounds can be provided.

Description

Technical Field

[0001] The present disclosure relates to the field of new biological materials, and in particular to a hydrogel carrier and an application thereof in preparing 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 have attracted great attention as serious and costly chronic complications. Reduced angiogenesis in diabetic wounds leads to insufficient capillary and vascular density, reduced transport of oxygen and nutrients, and ultimately delayed wound healing. In addition, diabetic wounds are highly susceptible to infection, and therapies that inhibit inflammation play a vital role in wound healing. Therefore, in the clinical treatment of diabetic infected wounds, special attention should be paid to the research and development of drugs that can promote angiogenesis and relieve inflammation.

[0003] 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 replicate themselves, the risk of tumor formation can be reduced, so the risk of using exosome therapy is lower. In addition, biomedical hydrogels have good biocompatibility, mechanical properties, and biodegradability, which can preserve exosomes and maintain their release. Hydrogels are usually used to deliver exosomes to the disease site for sustained release of exosomes.

[0004] However, general hydrogels usually have poor sustained-release effects on engineered exosomes, and general hydrogels can usually only provide sustained-release effects on repair factors and do not have the effect of promoting wound healing. Summary of the invention

[0005] The present disclosure is made in view of the above-mentioned prior art conditions, and its purpose is to provide a hydrogel carrier that helps to promote diabetic wound healing and its application in preparing a preparation for treating diabetic wounds.

[0006] To this end, the present disclosure provides an application of a hydrogel carrier in the preparation of a preparation for treating diabetic wounds, wherein the hydrogel carrier is used to carry a repair factor and has a sustained-release effect on the repair factor, and the hydrogel carrier comprises a first polymer and a second polymer, wherein the first polymer is methacryloyl gelatin, and the second polymer is methacryloyl polylysine.

[0007] In the first aspect of the present disclosure, a hydrogel carrier including methacryloyl gelatin (GelMA) and methacryloyl polylysine (PLMA) is used to carry the repair factor, wherein GelMA has a porous structure suitable for loading and slow release of the repair factor, PLMA can improve the overall anti-inflammatory ability of the preparation, help relieve inflammation to promote the healing of diabetic wounds, and PLMA can further improve the sustained release function of the hydrogel carrier for the repair factor. Thus, according to the present disclosure, a hydrogel carrier that helps promote the healing of diabetic wounds can be provided, and the application of the hydrogel carrier in the preparation of a preparation for treating diabetic wounds can obtain a preparation with a good therapeutic effect on diabetic wounds.

[0008] In the application involved in the first aspect of the present disclosure, optionally, the concentration of the first polymer in the hydrogel carrier is 5% w / v to 15% w / v. In this case, selecting a suitable concentration can help ensure the stability and biosafety of the hydrogel carrier.

[0009] In the application involved in the first aspect of the present disclosure, optionally, the concentration of the second polymer in the hydrogel carrier is 2% w / v to 10% w / v. In this case, selecting a suitable concentration can help improve the stability and antibacterial ability of the hydrogel carrier, and the degradation rate of the hydrogel carrier can be adjusted by adjusting the concentration of the second polymer. For example, when the second polymer has a relatively high concentration, the hydrogel carrier can have a higher degree of cross-linking, thereby reducing the swelling rate of the hydrogel carrier, reducing the degradation rate of the hydrogel carrier, and thus prolonging the administration time.

[0010] In the application involved in 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. In this case, it is helpful to obtain a hydrogel carrier with high stability and suitable repair factor release rate.

[0011] In the application involved in the first aspect of the present disclosure, optionally, the first polymer and the second polymer are cross-linked to form a three-dimensional network structure, and the exosomes are 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 of the repair factor.

[0012] In the application involved in the first aspect of the present disclosure, optionally, the hydrogel carrier further comprises a photoinitiator, and the photoinitiator promotes the cross-linking of the first polymer and the second polymer, thereby facilitating the cross-linking of the first polymer and the second polymer.

[0013] In the application involved in the first aspect of the present disclosure, optionally, the repair factor is an exosome, and the exosome is a mesenchymal stem cell exosome that enhances the expression of surface sLeX. The applicant found that the expression of E-selectin in diabetic wounds is higher than that in normal non-diabetic wounds. In this case, the repair factor is an exosome (s-Exo) that enhances the expression of surface sLeX (Sialyl Lewis-X). When the preparation is used to treat diabetic wounds, E-selectin can mediate the absorption of s-Exo by human umbilical vein endothelial cells (HUVECs), so that s-Exo can specifically bind to HUVECs, enhance the function of inflammatory-stimulated HUVECs in vitro, thereby promoting angiogenesis, and helping to further promote the healing of diabetic wounds.

[0014] In the application involved in the first aspect of the present disclosure, optionally, the preparation is applied in an external manner, and the preparation is directly applied to a wound or a part near a wound. Thus, it can help promote the healing of diabetic wounds. In the application involved in the first aspect of the present disclosure, optionally, the composite gel composition also includes pharmaceutically acceptable excipients.

[0015] The second aspect of the present disclosure relates to a hydrogel carrier, which is used to carry a repair factor and has a sustained release effect on the repair factor, and the hydrogel carrier includes a first polymer and a second polymer, the first polymer is methacryloyl gelatin, and the second polymer is methacryloyl polylysine. In the second aspect of the present disclosure, a hydrogel carrier that helps promote diabetic wound healing can be provided.

[0016] According to the present disclosure, a hydrogel carrier that helps promote diabetic wound healing and use of the hydrogel carrier in preparing a preparation for treating diabetic wounds can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram showing the high expression of endothelial injury-related genes in diabetic wounds in the embodiments of the present disclosure.

[0018] Figure 2 is a schematic diagram showing the identification of MSCs and s-MSCs in an embodiment of the present disclosure.

[0019] Figure 3 Schematic diagram showing the results of Exo and s-Exo identification in the embodiments of the present disclosure.

[0020] Figure 4 Schematic diagram showing the rapid uptake of s-Exo by LPS-stimulated HUVECs in an embodiment of the present disclosure.

[0021] Figure 5Schematic diagram showing the characteristics of hydrogels in embodiments of the present disclosure.

[0022] Figure 6 is a schematic diagram showing the biocompatibility and antibacterial activity of the hydrogel in the embodiments of the present disclosure.

[0023] Figure 7 Schematic diagram showing the healing of infected wounds in diabetic mice receiving different treatments in the embodiments of the present disclosure.

[0024] Figure 8 Schematic diagram showing angiogenesis and inflammation in diabetic wounds in an embodiment of the present disclosure. DETAILED DESCRIPTION

[0025] Hereinafter, the preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same symbols are assigned to the same components, and repeated descriptions are omitted. In addition, the accompanying drawings are only schematic diagrams, and the ratio of the dimensions of the components or the shapes of the components may be different from the actual ones.

[0026] It should be noted that the terms "including" and "having" and any variations thereof in the present disclosure, such as a process, method, system, product or device that includes or has a series of steps or units, are not necessarily limited to those steps or units clearly listed, but may include or have other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0027] The present disclosure relates to a hydrogel carrier, which can be used to carry a repair factor, and has a sustained release effect on the repair factor, which helps to promote wound healing. That is, the present disclosure can provide a hydrogel carrier that helps to promote wound healing. In the present disclosure, the repair factor refers to a substance that promotes the healing of wounds. In particular, the hydrogel carrier involved in the present disclosure can be used to carry the repair factor described later in the present disclosure, which will be described in detail later. In particular, the hydrogel carrier of the present disclosure is particularly helpful in promoting the healing of diabetic wounds. In the present disclosure, diabetic wounds refer to chronic wounds that are easily formed on the feet or other parts of diabetic patients due to skin lesions, poor blood circulation, and decreased immune function. These wounds are often difficult to heal and prone to infection due to the influence of diabetes, and there are many complications in the healing process. In the present disclosure, the hydrogel carrier may also be referred to as a hydrogel, or as a carrier. In the present disclosure, the wound may also be referred to as a wound surface.

[0028] The present disclosure also relates to the use of a hydrogel carrier in the preparation of a preparation for treating wounds. The hydrogel carrier has a good therapeutic effect on wounds, and the application of the hydrogel carrier in the preparation for treating wounds can help obtain a preparation with a good therapeutic effect on wounds. In particular, the present disclosure relates to the use of a hydrogel carrier in the preparation for treating diabetic wounds. The hydrogel carrier helps promote the healing of diabetic wounds, and the application of the hydrogel carrier in the preparation for treating diabetic wounds can obtain a preparation that helps promote the healing of diabetic wounds. In the present disclosure, the preparation for treating diabetic wounds may be referred to as a preparation.

[0029] The present disclosure also relates to a composite gel composition. The composite gel composition may include a hydrogel carrier and a repair factor, which helps to promote wound healing. In particular, the composite gel composition involved in the present disclosure has a good therapeutic effect on diabetic wounds. In the present disclosure, the composite gel composition may be referred to as a composite gel, or may be referred to as a composition, a complex, a hydrogel composition, or a composite hydrogel, etc.

[0030] Hereinafter, the hydrogel carrier and its application involved in the present disclosure will be described in detail with reference to the accompanying drawings.

[0031] In some examples, as described above, the hydrogel carrier can carry the repair factor. In some examples, the hydrogel carrier can have a sustained release effect on the repair factor. When applied, the hydrogel carrier can release the repair factor slowly, which can help promote wound healing.

[0032] In some examples, the repair factor can be evenly dispersed in the hydrogel carrier, thereby helping the hydrogel carrier to stably release the repair factor over a certain period of time.

[0033] In some examples, the hydrogel carrier may include a first polymer. The first polymer may serve as a skeleton structure of the hydrogel carrier to improve the overall stability of the hydrogel carrier and the ability to slowly release the repair factor. In some examples, preferably, the first polymer may be methacryloyl gelatin (GelMA, also referred to as Gel). In this case, GelMA has a porous structure, which is suitable for loading and slowly releasing the repair factor; in addition, GelMA also has good biocompatibility and degradability.

[0034] In some examples, the hydrogel carrier may include a second polymer. The second polymer may at least provide a functional effect for the hydrogel carrier. For example, the antibacterial ability of the hydrogel carrier may be increased by adding a second polymer. In some examples, preferably, the second polymer may be poly(methacryloyl-lysine) (PLMA, also referred to as PL). In this case, PLMA can improve the overall antibacterial ability of the composite gel composition, help relieve inflammation to promote wound healing, especially promote the healing of diabetic wounds; in addition, PLMA can further improve the stability of the hydrogel carrier and the sustained release function of the repair factor.

[0035] PLMA is the product of methacrylation of ε-PL glycidyl methacrylate, and a double bond is introduced into the ε-PL molecule. ε-PL and its derivatives help to avoid the effects of traditional antimicrobial drugs or metal ions (such as Ag). + ) produces drug resistance and metal ion deposition.

[0036] In some examples, the hydrogel carrier may include a first polymer and a second polymer. In some examples, the first polymer may be cross-linked with the second polymer. In some examples, the first polymer may be cross-linked with the second polymer to form a three-dimensional network structure. In some examples, the repair factor 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 of the repair factor. In some examples, GelMA and PLMA are photopolymerizable hydrogels that can be cross-linked under ultraviolet irradiation.

[0037] In some examples, a photoinitiator can be added to promote crosslinking of the first polymer and the second polymer. In other words, the hydrogel carrier can include a photoinitiator. Thus, it can help crosslink 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-trimethylbenzoylphosphonate (LAP). In this case, LAP has excellent biocompatibility and less immunogenicity, and can help GelMA and PLMA to crosslink under blue light. In some examples, GelMA and PLMA can be crosslinked under blue light with a wavelength of 405 nanometers.

[0038] In some examples, the concentration of the first polymer in the hydrogel carrier can be 5% w / v to 15% w / v. For example, the concentration of the first polymer can 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 a suitable 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 can be 5% w / v. In this case, it can help to obtain a hydrogel carrier with high stability and strong sustained release effect for s-Exo.

[0039] In some examples, the concentration of the second polymer in the hydrogel carrier can be 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 a suitable 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, the hydrogel carrier can have a higher degree of cross-linking inside, thereby reducing the swelling rate of the hydrogel carrier, reducing the degradation rate of the hydrogel carrier, and thus 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 to obtain a hydrogel carrier with high stability and strong sustained release effect for s-Exo.

[0040] In some examples, the mass ratio of the first polymer to the second polymer may be 1:2 to 15:2. For example, the mass ratio of the first polymer to the second polymer may 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 is possible to obtain a hydrogel carrier with high stability and a suitable release rate of the repair factor. In some examples, preferably, the mass ratio of the first polymer to the second polymer may be 1:1. In this case, it is possible to obtain a hydrogel carrier with high stability and a strong sustained release effect for s-Exo.

[0041] In some examples, the repair factor may include mesenchymal stem cells (MSCs) and / or mesenchymal stem cell-derived exosomes (Exo). In the present disclosure, mesenchymal stem cell-derived exosomes may be referred to as exosomes. In some examples, the repair factor may also include other substances that promote healing of wounds.

[0042] 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.

[0043] In some examples, preferably, the mesenchymal stem cells can be human umbilical cord mesenchymal stem cells (hucMSCs), and the exosomes can be hucMSCs-derived exosomes. In this case, the umbilical cord, as a source of mesenchymal stem cells and their exosomes, has the advantages of reducing harm to the human body and facilitating collection when collected compared to other materials such as bone marrow, liver, etc., and the mesenchymal stem cells derived from the umbilical cord have stronger proliferation and differentiation capabilities. In some examples, hucMSCs and their exosomes can be obtained from fresh umbilical cord tissue of newborns.

[0044] 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 culture can be filtered using a filter to obtain exosomes.

[0045] In some examples, preferably, the repair factor can be an exosome. In some examples, the exosome can be an exosome developed for different types of wounds. For example, the exosome can be modified by genetic engineering to obtain a targeted engineered exosome.

[0046] In some examples, exosomes can target inflammatory vascular endothelial cells, thereby helping to promote wound recovery, especially the healing of diabetic wounds.

[0047] In some examples, exosomes that enhance the expression of surface sLeX (Sialyl Lewis-X) (hereinafter referred to as s-Exo, also referred to as sLeX overexpressing exosomes) may be used. In other words, the preparation may include exosomes that enhance the expression of surface sLeX. sLeX is expressed by leukocytes and can bind to human umbilical vein endothelial cells (hereinafter referred to as HUVECs) expressing E-selectin under inflammatory stimulation. In this case, s-Exo is able to target HUVECs and enhance the function of inflammatory-stimulated HUVECs in vitro, thereby promoting angiogenesis and relieving 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 target for the treatment of diabetic wounds. E-selectin is highly expressed in inflammatory-stimulated HUVECs and mediates the absorption of sLeX-expressing exosomes by endothelial cells. s-Exo can specifically bind to HUVECs, thereby promoting its function in vitro and angiogenesis in vivo. Therefore, in the example where the formulation of the present disclosure includes s-Exo, the repair factor is able to have a good therapeutic effect on diabetic wounds.

[0048] In some examples, exosomes can be glycosylated to obtain s-Exo. For example, the outermost dense glycocalyx coating of exosomes can be modified by glycoengineering technology to improve the targeting of exosomes. In some examples, MSCs can be transduced with lentiviral vectors, and then cells that stably express the transduction vector can be screened to obtain predetermined exosomes.

[0049] In some examples, exosomes can be obtained by the following steps: transducing MSCs using a lentiviral vector; screening successfully transduced MSCs; and collecting the supernatant to obtain exosomes.

[0050] 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 glycosylation domain derived from PSGL-1, and the surface protein CD63 can be used as an exosome scaffold protein. Its large 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 the use of 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 vector 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 sLeX expression.

[0051] In some examples, puromycin screening can be used to obtain cells that stably express the transduction vector. In some examples, the concentration of puromycin used for screening can be 1 μg / mL to 3 μg / mL. In some examples, preferably, 3 μg / mL puromycin can be used for screening first, and then 1 μg / mL puromycin can be used for screening to maintain the concentration. Thus, it can help to obtain cells that stably express the transduction vector. In some examples, targeted exosomes can also be obtained by other means.

[0052] In some examples, the preparation may further include pharmaceutically acceptable excipients, such as diluents, thickeners, and / or antioxidants.

[0053] 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 methacryloyl gelatin, the second polymer is methacryloyl polylysine, and the exosomes are mesenchymal stem cell exosomes that enhance the expression of surface sLeX. Thus, according to the present disclosure, a preparation having a good therapeutic effect on diabetic wounds can be provided.

[0054] 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 reduced capillary density, reduced nutrition and oxygen supply, and ultimately delayed wound healing. In this case, the preparation provided by the present disclosure can promote the healing of diabetic wounds by promoting angiogenesis, which can have a good therapeutic effect on diabetic wounds.

[0055] In the present disclosure, the preparation can be a drug. In some examples, the preparation can be used in an external manner. In some examples, the preparation can be directly applied to a wound or a part adjacent to a 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, a gel, an ointment or a spray.

[0056] 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 application method that is easy to apply can be provided. In some examples, the preparation can be carried on a microneedle dressing for use. In this case, it can help the preparation to fully contact the wound, so that the repair factors in the preparation can better act on the wound, thereby further improving the therapeutic effect of the preparation on the wound. In the present disclosure, the preparation used as a wound dressing may also be referred to as a dressing or patch.

[0057] The hydrogel carrier involved in the present disclosure can not only carry the engineered exosomes described in the present disclosure, but also serve as a carrier of other repair factors. The hydrogel carrier involved in the present disclosure is used in the preparation of a preparation for treating diabetic wounds. On the one hand, it can release the repair factor slowly, and on the other hand, it can improve the overall anti-inflammatory ability of the preparation, which helps to relieve inflammation and promote the healing of diabetic wounds. In addition, the hydrogel carrier involved in the present disclosure can also be used in the preparation of a preparation for treating ordinary wounds or other wounds.

[0058] As mentioned above, the present disclosure also provides a composite gel composition. The composite gel composition has a good therapeutic effect on diabetic wounds, and applying the composite gel composition in a preparation for treating diabetic wounds can help obtain a preparation with a good therapeutic effect on diabetic wounds.

[0059] In some examples, preferably, the composite gel composition may include a hydrogel carrier and exosomes, 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 exosomes are mesenchymal stem cell exosomes that enhance the expression of surface sLeX. In this case, the composite gel composition has a good therapeutic effect on diabetic wounds, and applying the composite gel composition in a preparation for treating diabetic wounds can help obtain a preparation with a good therapeutic effect on diabetic wounds.

[0060] In summary, according to the present disclosure, a hydrogel carrier that helps promote diabetic wound healing and its use in preparing a preparation for treating diabetic wounds can be provided.

[0061] To further illustrate the present disclosure, the hydrogel carrier and its application provided by the present disclosure are described in detail below in conjunction with examples.

[0062] In the embodiments of the present disclosure, unless otherwise specified, the reagents and instruments used are all commercially available products.

[0063] [Example]

[0064] 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.

[0065] Experimental procedures involved

[0066] (1) scRNA-seq of wound samples

[0067] We acquired and reanalyzed data based on the expression matrix. Data were obtained from 12 wound samples provided by nine participants, five with diabetic foot ulcers and four with nondiabetic foot wounds, one of whom provided two samples and one provided three samples. Data were analyzed using ScanPy. We first filtered low-quality cells and genes and double-filtered using scrulet. After normalizing and log-transforming the data, we identified the cell cycle and regressed it to remove its effect. After removing batch effects using the BBKNN method, PCA and UMAP were performed for dimensionality reduction, and Leiden cluster analysis was used to identify different cell populations. Cell types were identified using the CellTypist package, which was annotated based on a previous human skin atlas. Intergroup differences in gene expression were assessed using independent t tests.

[0068] (2) Preparation and transfection of lentiviral vectors

[0069] The exosome 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 to construct the CD63-P19-Nluc protein vector. The lentiviral vector co-expressing FUT7 and CD63-P19-Nluc was prepared by Jikai Chemical Co., Ltd. (Shanghai, China). The concentrated and purified lentivirus was stored at -80°C until use.

[0070] The method of inducing mesenchymal stem cells (MSCs) to be infected with lentivirus was performed on MSCs at the third generation. MSCs (3-5×10 4 cells / mL) were inoculated into T25 cell culture flasks and cultured in a cell culture incubator at 37°C and 5% CO2 until a confluence of about 20% was reached. MSCs were transfected with lentivirus overnight. After the transfected MSCs reached about 80% confluence and grew well, they were screened with puromycin (3 μg / mL; Product No. IA0530, Solarbio, Beijing, China) for 48 hours. The surviving MSCs were then further cultured with a culture medium containing puromycin (1 μg / mL). The culture medium was changed and the cells were collected every 2 days. The transfected cells were used to detect surface markers, adipogenic and osteogenic induction by flow cytometry (DxFLEX, Beckman Coulter, USA), and the differentiation ability of s-MSCs (MSCs overexpressing sLeX) was determined by staining. The expression of sLeX (Cat. No. 563526, BD Pharmingen, San Diego, USA) on the surface of s-MSCs was estimated by flow cytometry.

[0071] (3) Detection of E-selectin on the surface of HUVEC

[0072] HUVECs were cultured in endothelial cell culture medium (ECM; ScienCell, Carlsbad, CA, USA). HUVECs were seeded into six-well plates, treated with negative control and 1 μg / mL LPS (MedChemExpress, Shanghai, China) for 4 hours, trypsinized, washed with PBS, and cultured in the dark for 30 minutes with PE-conjugated mouse anti-human E-selectin (Cat. No. 322606, BioLegend, San Diego, CA, USA). Finally, the expression of E-selectin was detected by flow cytometry.

[0073] (4) Preparation of hydrogel

[0074] The LAP standard solution was dissolved in PBS (0.25%, w / v) and stored in the dark. The GelMA and PLMA solutions were passed through a 0.22 μm pore filter to ensure sterility, and then mixed with the LAP solution in different proportions to obtain 5% (w / v) GelMA (hereinafter referred to as Gel), 5% (w / v) GelMA + 2% (w / v) PLMA (hereinafter referred to as Gel / PL-2), and 5% (w / v) GelMA + 5% (w / v) PLMA (hereinafter referred to as Gel / PL-5). The precursor solution was cross-linked under ultraviolet radiation for 15s to form a hydrogel.

[0075] (5) Treatment of wounds in diabetic mice with s-Exo@Gel / PL-5

[0076] Eight-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 to have diabetes and were included in the experimental group. The mice were anesthetized with isoflurane (2%), the dorsal hair was shaved, 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 CPU / mL) was inoculated into the wound for 2 days. Four mice were then 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 given:

[0077] ①PBS

[0078] ②Exo (100 μg)

[0079] ③s-Exo (100 μg), where s-Exo is the exosomes secreted by s-MSCs.

[0080] ④Gel / PL-5

[0081] ⑤s-Exo@Gel / PL-5 (containing 100μg s-Exo)

[0082] The day when the mice received the dressing treatment was recorded as day 0. The dressings of groups ①, ② and ③ were injected around the wound (100 μL in total, 25 μL at each site), and the hydrogels of group ④ (Gel / PL-5) and group ⑤ (s-Exo@Gel / PL-5, containing 100 μg s-Exo) were dripped on the wound surface (total dose of 100 μL) and cross-linked under ultraviolet light for 15 seconds. The dressing was changed every 3 days. Wound photos were taken at fixed time and distance (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 is as follows: Remaining area (%) = S t / S0×100%, where S t represents the wound area at different times, and S0 represents the initial wound area. On day 12, the mice were euthanized under anesthesia. The collected skin wound tissues were stored for future use.

[0083] (6) In vivo toxicity test of hydrogel

[0084] Peripheral blood was collected on the 12th day of hydrogel treatment. EDTA anticoagulant was added to part of the blood to obtain whole blood for blood index testing. The other part of the blood was centrifuged to obtain serum for the detection of different protein markers. The heart, liver, kidney, and lung were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into hematoxylin and eosin (H&E) stained sections for histological analysis of hydrogel toxicity.

[0085] (7) Histology and immunohistochemical staining

[0086] Healed 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 (Order No. BA4907, Baise, Wuhan, China) and Masson's trichrome (Order No. G1006, Servicebio, China). To determine the angiogenic 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 values ​​were quantified using ImageJPro software.

[0087] (8) Immunofluorescence staining to detect the expression of E-selectin in wound tissue

[0088] Human wound tissue specimens were obtained from non-diabetic and diabetic patients. Wound tissues from humans and mice were fixed with 4% paraformaldehyde, embedded in paraffin, cut into 5 μm thick sections, deparaffinized, antigen retrieved, blocked with 10% goat serum for 1 hour at room temperature, and incubated with anti-CD31 (Cat. No. sc-376764, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-E-selectin (Cat. No. ab18981, Abcam) at 4°C overnight. The next day, sections were incubated with AlexaFluor-labeled secondary antibodies for 1 hour at room temperature. Cell nuclei were stained with 4,6′-diamidino-2-phenylindole for 5 minutes at room temperature. Finally, images were collected using a fluorescence microscope.

[0089] (9) Statistical analysis

[0090] 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 set at P < 0.05.

[0091] Experimental results analysis

[0092] (1) scRNA-seq data indicate gene-driven endothelial injury

[0093] Figure 1Schematic diagram showing the high expression of endothelial injury-related genes in diabetic wounds in the embodiments of the present disclosure. Figure 1 Part a is a schematic diagram of using Leiden clustering to group the UMAP (uniform manifold approximate projection) dimensionality reduction map. Figure 1 Part b in the middle shows the 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 endothelial cell types on the UMAP dimensionality reduction map. Figure 1 Part (d) is a schematic diagram of the heat map of the top 20 differentially expressed genes in endothelial cells of diabetic and non-diabetic patients. Figure 1 Part e in the middle is a schematic diagram of the expression of endothelial injury-related genes in endothelial cells of the 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: 20 μm. In the figures involved in the examples of the present disclosure, ns means no significant difference, * means p<0.05, ** means p<0.01, *** means p<0.001.

[0094] After filtering and processing the data, the cell cycle was identified and regressed. The final matrix constructed included 44,426 cells expressing 24,771 genes. After dimensionality reduction, 19 cell populations were obtained (see Figure 1 ), and annotated into 13 different cell types (see Figure 1 There are two types of endothelial cells in the dermis of healthy adults, namely vascular endothelial cells (VECs) and lymphatic endothelial cells (LECs), which express PECAM1 and LYVE1, respectively (see Figure 1 We analyzed the gene expression of these two types of endothelial cells and found that endothelial injury-related genes, such as "SELE", "RPS4Y1", "NFKBIA", "C2CD4B" and "JUNB", were expressed at higher levels in the diabetic group than in the non-diabetic group (see Figure 1 (see parts d and e in the figure).

[0095] The E-selectin adhesion molecule encoded by SELE is present in endothelial cells upon inflammatory stimulation and is thought to promote the recruitment of blood leukocytes to sites of inflammation by mediating cell adhesion to the vascular endothelium. To design an E-selectin-targeted glycoengineered hydrogel for the treatment of diabetic wounds, we validated the expression of E-selectin, a product expressed by the vascular endothelium upon inflammatory activation. In human samples, E-selectin colocalized with the vascular endothelial marker CD31 in diabetic wounds, whereas 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 with non-diabetic mice ( Figure 1 g).

[0096] (2) Preparation and identification of MSCs, s-MSCs, Exo and s-Exo

[0097] Figure 2 is a schematic diagram showing the identification of MSCs and s-MSCs in an embodiment of the present disclosure. Figure 2 Part a in the middle is a schematic diagram of the cell viability assay of MSCs and s-MSCs. Figure 2 Part b is a schematic diagram of flow cytometry detection of sLeX expression on the surface of s-MSCs. Figure 2 Parts c and d in the middle are schematic diagrams of Oil Red O and Alizarin Red staining of MSCs and s-MSCs after adipogenesis and osteogenesis induction, scale bar: 20 μm. Figure 2 Part e in the middle is a schematic diagram of flow cytometry identification of MSCs and s-MSCs.

[0098] Lentiviral infection did not affect the viability of MSCs as determined by the CCK-8 assay (see Figure 2 Flow cytometry showed that sLeX was highly expressed in s-MSCs (see Figure 2 Part b). It is noteworthy that the expression of FUT7 and CD63-P19-Nluc can 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 2 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 d in the middle). Flow cytometry was used to detect the expression of surface markers of MSCs and s-MSCs at passage 5. MSCs and s-MSCs were positive for CD105 and CD73, and negative for CD34 and HLA-DR (see Figure 2These findings confirm that MSCs were isolated successfully and that cells transduced with the lentiviral vector retain the properties of mesenchymal stem cells.

[0099] Figure 3 Schematic diagram showing the results of Exo and s-Exo identification in the embodiments of the present disclosure. Figure 3 Parts a and b are schematic diagrams of the diameter and zeta potential of Exo and s-Exo determined by NTA. Figure 3 Part c is a schematic diagram of Western blot analysis of the endoplasmic reticulum marker Calnexin and exosome markers CD9, Alix and TSG101. Figure 3 Part d in the middle is a schematic diagram of the exosome morphology observed by transmission electron microscopy. Figure 3 Part e in the middle is a schematic diagram of Western blot analysis of sLeX expression. Figure 3 Part f is a schematic diagram of the statistical analysis of the expression level of sLeX relative to GAPDH.

[0100] Exo and s-Exo were characterized by nanoscale light 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 The zeta potentials of Exo and s-Exo are -29.55 mV and -33.44 mV, respectively (see Figure 3 Western blotting confirmed the expression of exosomal markers CD9, Alix, and TSG101, while the endoplasmic reticulum marker calnexin was not expressed in Exo and s-Exo (see Figure 3 TEM revealed the cup-shaped morphology and bilayer membrane structure of Exo and s-Exo (see Figure 3 Western blot showed that the level of sLeX in s-Exo was higher than that in Exo (see Figure 3 e and f).

[0101] (3) S-Exo has a strong binding affinity with activated endothelial cells

[0102] Figure 4 Schematic diagram showing the rapid uptake of s-Exo by HUVECs stimulated by LPS (lipopolysaccharide) in an embodiment of the present disclosure. Figure 4 Part a in the middle is a schematic diagram of the expression of E-selectin on HUVECs after LPS stimulation for 4 hours. Figure 4 Part b is a schematic diagram of the quantification of cellular Nluc expression after 4 hours of exosome treatment. Figure 4Part c is a representative image of the uptake of DIO-labeled Exo and s-Exo by normal and LPS-activated HUVECs. Figure 4 Part d in the middle is a schematic diagram of representative images of HUVECs Transwell migration assay under different treatments, scale bar: 100 μm. Figure 4 Part e is a schematic diagram of the quantitative analysis of cell migration in the migration experiment. Figure 4 Part f in the middle is a schematic diagram of representative images of the HUVECs cell scratch assay at different treatments for 10 h and 20 h, scale bar: 100 μm. Figure 4 Part g is a schematic diagram of the quantitative analysis of the migration zone in the cell scratch assay.

[0103] To verify the uptake of s-Exo by HUVECs cells, the 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 The uptake rate of s-Exo by activated HUVECs (>3.8 times) was significantly higher than that of non-activated HUVECs (see Figure 4 Fluorescence microscopy showed that HUVECs could take up both Exo and s-Exo with or without LPS stimulation, and LPS-stimulated HUVECs could take up more s-Exo (see Figure 4 (Part c).

[0104] The migration ability of four groups of HUVECs (LPS, LPS+Exo, and LPS+s-Exo) was determined by scratch assay (see Figure 4 HUVECs in the LPS+s-Exo group showed the highest migration rate (see Figure 4 e), which is consistent with the migration test results (see Figure 4 (f). The treated HUVECs were pre-incubated with negative control and LPS for 4 h, then the cells were scratched, rinsed with PBS, and supplied with new culture medium. The scratch assay showed that HUVECs in the LPS+s-Exo group migrated more than those in the other three groups at 10 and 20 h (see Figure 4 (g part).

[0105] (4) Characterization of Gel, Gel / PL-2, and Gel / PL-5

[0106] Figure 5 Schematic diagram showing the characteristics of hydrogels in embodiments of the present disclosure. Figure 5 Part a in the middle is a schematic diagram of the gelation of the precursor solution to form a hydrogel under ultraviolet irradiation. Figure 5Part b is a schematic diagram of the various shapes obtained by the precursor solution under ultraviolet irradiation. Figure 5 Part c is a schematic diagram of the swelling properties of the hydrogel. Figure 5 Part (d) is a schematic diagram of the degradation characteristics of the hydrogel. Figure 5 Part e is a schematic diagram of the s-Exo release curve. Figure 5 Part f is a schematic diagram of the rheological behavior of the hydrogel. Figure 5 Part g is a schematic diagram of the rheological analysis of the hydrogel under frequency sweep. Figure 5 Part h in the middle is 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 is a schematic diagram of the SEM images of all hydrogels. Figure 5 Part j in the middle is a schematic diagram of the average pore size and pore size distribution of each hydrogel.

[0107] Previous studies have shown that 5% concentration of GelMA is suitable for wound healing, so we chose 5% (w / v) GelMA as the applied 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 performed an inversion test. The solution showed flow properties after the test tube was inverted, but showed a rapid transition from sol to gel under UV irradiation, and no flowability was found after inversion (see Figure 5 Hydrogels can be molded into various shapes (see Figure 5 The swelling ratio of Gel / PL-5 was lower than that of Gel and Gel / PL-2, indicating that the swelling capacity of the hydrogel gradually decreased with the increase of cross-linking density (see Figure 5 The degradation rate (measured every 2 days) showed that Gel degraded faster than other hydrogels, and Gel / PL-5 degraded significantly slower than low-concentration PL (see Figure 5 The hydrogels were immersed in PBS to obtain the s-Exo release curve. Gel showed rapid and explosive s-Exo release in the first 3 days, while Gel / PL-5 released s-Exo continuously and steadily (see Figure 5 (see section e).

[0108] As time goes by, the storage modulus (G′) values ​​of the three groups of hydrogels increase continuously, exceeding the loss modulus (G″) value, and finally reaching equilibrium, indicating that the precursor solution forms a solid hydrogel with a stable internal structure (see Figure 5 As the oscillation frequency increases, the G′ of the three hydrogels is always higher than the corresponding G″ value, indicating that the internal structure is stable (see Figure 5Confocal microscopy imaging showed the successful loading and uniform three-dimensional distribution of s-Exo in Gel / PL-5 (see Figure 5 Scanning electron microscopy images show the porous structures inside the three hydrogels (see Figure 5 As the PL concentration increases, the internal cross-linking of the hydrogel is enhanced and the pore size of the hydrogel gradually decreases (see Figure 5 (middle part).

[0109] (5) Biocompatibility of hydrogels

[0110] 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 is a schematic diagram of the cell viability of HUVECs after incubation with the hydrogel extract for 48 h. Figure 6 Part b is a schematic diagram of the hemolysis rate of the hydrogel. Figure 6 Part c is a schematic diagram of the hematological properties of the hydrogel, TritonX-100 is the positive control, and PBS is the negative control. Figure 6 Part d is a schematic diagram of live / dead cell staining of HUVECs after incubation with hydrogel extracts for 48 hours. Scale bar: 100 μm. Figure 6 Parts e and f are photographs of Staphylococcus aureus and Escherichia coli cultured on agar plates after incubation with different hydrogels and schematic diagrams of the survival rates. Figure 6 Part (g) is a schematic diagram of live / dead staining of Staphylococcus aureus and Escherichia coli extracted after incubation with different hydrogels, scale bar: 20 μm.

[0111] Good biocompatibility is very important for the clinical application of wound dressings. We cultured HUVECs in conditioned medium containing different concentrations of hydrogels for 48 hours and found that all hydrogels did not affect cell viability (see Figure 6 The hemolysis test showed that the hemolysis rate of 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 has good blood compatibility (see Figure 6 b and c). After 48 h of culture in hydrogel-conditioned medium, cytotoxicity was further determined by live / dead cell staining. Fewer dead HUVECs (red fluorescence) were observed in the hydrogel-treated group compared with the positive control group (see Figure 6 This indicates that Gel, Gel / PL-2 and Gel / PL-5 have good biosafety.

[0112] (6) In vitro antibacterial activity of hydrogels

[0113] Diabetic wounds are often associated with multiple bacterial infections, the most common of which are caused by Staphylococcus aureus and Escherichia coli. The antimicrobial 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 antimicrobial properties, but as the concentration of PL increases, the antimicrobial properties also increase, as evidenced by the gradual decrease in the number of bacterial colonies (see Figure 6 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 Live / dead staining assay showed that the bacteria in the control group and gel group were mainly alive (green), while the bacteria in the Gel / PL-2 group and Gel / PL-5 group were mainly dead (red, see Figure 6 Therefore, the Gel / PL group has good antibacterial properties, which can inhibit diabetic foot infection and promote diabetic wound healing.

[0114] (7) S-Exo@Gel / PL-5 can accelerate diabetic wound healing in vivo

[0115] Figure 7 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 in the middle 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 application. Figure 7 Part c is a schematic diagram of the quantification of the remaining wound area of ​​each group on the 3rd, 6th, 9th and 12th days. Figure 7 Part d in the middle is a schematic diagram of the representative H&E staining images of the wound tissues of each group on the 12th day, scale bar: 500μm. Figure 7 Part e in the middle is a schematic diagram of the quantification of epithelial cell thickness in each group.

[0116] Figure 8 Schematic diagram showing angiogenesis and inflammation in diabetic wounds in an embodiment of the present disclosure. Figure 8 Part a in the middle is a representative image 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 be in the middle are schematic diagrams of the quantitative analysis of the average IOD (integrated optical density) of CD31, α-SMA, IL-1β, and IL-6.

[0117] We determined the efficacy of Gel / PL-5 in full-thickness diabetic wounds infected with Staphylococcus aureus and Escherichia coli. Figure 7 Part a shows the experimental timeline for wound infection and treatment. Figure 7 Part b shows representative images and healing rates of each group on days 0, 3, 6, 9, and 12 of treatment. The infected open wounds appeared red and swollen. 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 in the s-Exo group was faster than that in the Exo group, indicating that the expression of sLeX on the surface of exosomes promoted the healing of infected diabetic wounds.

[0118] After 12 days of treatment, skin re-epithelialization and appendage formation were observed by H&E staining (see Figure 7 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 The wounds treated with s-Exo@Gel / PL-5 showed denser collagen fibers, thicker granulation tissue, and higher neocollagen deposition (see Figure 8 (Part a).

[0119] On day 12 after treatment, we measured the expression of vascular markers (CD31 and α-SMA, see Figure 8 Immunohistochemical staining was performed to evaluate the changes in angiogenesis. The s-Exo@Gel / PL-5 treated group showed more new blood vessels, indicating that its angiogenesis effect was better than that of other groups (see Figure 7 Part b and Figure 8 In addition, the expression of inflammatory cytokines (IL-1β and IL-6) was also assessed (see Figure 8 (see parts d and e in the figure).

[0120] (8) Detection of hydrogel toxicity to the body

[0121] The hydrogel had no pathological effects on blood components in vivo, and no damage or pathological changes were observed in vital organs (heart, liver, lungs, and kidneys).

[0122] Summarize

[0123] In this disclosure, we reanalyzed single-cell gene expression profiles of chronic foot ulcers from diabetic and non-diabetic patients. scRNA-seq results showed that the expression of endothelial injury-related genes such as "SELE", "RPS4Y1", "NFKBIA", "C2CD4B" and "JUNB" were upregulated in diabetic wounds compared with non-diabetic wounds, reflecting vascular damage in diabetic wounds. We selected "SELE" as a key factor for exosome glycoengineering. E-selectin (expressed by the SELE gene) is required for endothelial progenitor cell adhesion, endothelial cell homing in vitro, and neovascularization in vivo. We found that the expression of E-selectin in infected human and mouse wounds was significantly higher than that in normal wounds, reflecting the potential of E-selectin as a target for diabetic wound healing. We confirmed that E-selectin is highly expressed in stimulated HUVECs and mediates 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 hydrogels involved in the present disclosure.

[0124] We modified the dense outermost glycocalyx coating of exosomes using rational glycoengineering technology to improve the targeting of exosomes. MSCs were transduced with lentiviral vectors encoding FUT7 and CD63-P19, selected with 3μg / mL puromycin, and then screened with 1μg / mL puromycin maintenance concentration to obtain cells that stably expressed the transduced vector. Our results showed that the surface expression of sLeX was significantly increased in paracrine exosomes of s-MSCs. Under inflammatory stimulation, exosomes expressing sLeX (s-Exo) can specifically bind to HUVECs, thereby promoting their function in vitro and angiogenesis in vivo.

[0125] In this disclosure, we infected diabetic wounds with Staphylococcus aureus and Escherichia coli to determine the antibacterial effect of the hydrogel. Through bacterial colony counts and live / dead tests, we found that Gel / PL-5 containing 5% PL had better antibacterial ability than Gel / PL-2.

[0126] In vivo, we found that s-Exo@Gel / PL-5 showed 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 expression of CD31 and α-SMA in the wound tissue of the s-Exo group was higher than that of 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 has better therapeutic effects than exosomes alone. The s-Exo@Gel / PL-5 group had the lowest levels of pro-inflammatory cytokines (IL-1β and IL-6) and the highest microvessel density. 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 the single component.

[0127] Biosafety is 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, as evidenced by CCK8 and live / dead staining assays after HUVECs were co-cultured with the hydrogel extracts for 48 h. After co-culture of mouse blood with the hydrogel extracts for 1 h, we found that the hemolysis rate was significantly lower than that of the positive control group, reflecting the good blood compatibility of the hydrogel in vivo. We observed no obvious histological or hematological changes in the peripheral blood, heart, liver, kidney, and lung tissues of hydrogel-treated mice, indicating that the hydrogels can both improve diabetic wound healing and have no toxic effects in vivo.

[0128] in conclusion

[0129] The research disclosed in this paper shows that the enhanced expression of sLeX on the surface of exosomes can promote wound angiogenesis. The photopolymerized hydrogel Gel / PL-5 has excellent antibacterial activity, biocompatibility and mechanical properties. Gel / PL-5 carrying s-Exo showed sustained release of s-Exo, accelerating the healing of diabetic infected wounds. The newly developed s-Exo@Gel / PL-5 hydrogel is a new type of dressing for treating diabetic infected wounds with broad application prospects.

[0130] In summary, the present disclosure can provide a hydrogel carrier that helps promote diabetic wound healing and use of the hydrogel carrier in preparing a preparation for treating diabetic wounds.

[0131] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and examples, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. Use of a hydrogel carrier in preparing a preparation for treating diabetic wounds, characterized in that: The hydrogel carrier is used to carry the repair factor and has a sustained release effect on the repair factor. The hydrogel carrier includes a first polymer and a second polymer. The first polymer is methacryloyl gelatin, and the second polymer is methacryloyl polylysine. The concentration of the first polymer in the hydrogel carrier is 5% w / v, and the concentration of the second polymer in the hydrogel carrier is 5% w / v. The repair factor is an exosome, and the exosome is a mesenchymal stem cell exosome with enhanced expression of surface sLeX. The mesenchymal stem cell exosome with enhanced expression of surface sLeX is obtained by the following method: Modifying the glycocalyx coating of the outermost layer of exosomes by glycoengineering technology to obtain the mesenchymal stem cell exosomes with enhanced expression of surface sLeX; or Mesenchymal stem cells are transduced with a lentiviral vector, cells that stably express the transduction vector are screened, and the supernatant is collected to obtain the mesenchymal stem cell exosomes with enhanced expression of surface sLeX.

2. The use according to claim 1, characterized in that: The first polymer and the second polymer are cross-linked to form a three-dimensional network structure, and the repair factor is distributed in the three-dimensional network structure.

3. The use according to claim 2, characterized in that: The hydrogel carrier also includes a photoinitiator that causes the first polymer to cross-link with the second polymer.

4. 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.

5. The use according to claim 1, characterized in that: The preparation also includes pharmaceutically acceptable excipients.

6. A hydrogel carrier, characterized in that: The hydrogel carrier is used to carry the repair factor and has a sustained release effect on the repair factor. The hydrogel carrier includes a first polymer and a second polymer. The first polymer is methacryloyl gelatin, and the second polymer is methacryloyl polylysine. The concentration of the first polymer in the hydrogel carrier is 5% w / v, and the concentration of the second polymer in the hydrogel carrier is 5% w / v. The repair factor is an exosome, and the exosome is a mesenchymal stem cell exosome with enhanced expression of surface sLeX. The mesenchymal stem cell exosome with enhanced expression of surface sLeX is obtained by the following method: Modifying the glycocalyx coating of the outermost layer of exosomes by glycoengineering technology to obtain the mesenchymal stem cell exosomes with enhanced expression of surface sLeX; or Mesenchymal stem cells are transduced with a lentiviral vector, cells that stably express the transduction vector are screened, and the supernatant is collected to obtain the mesenchymal stem cell exosomes with enhanced expression of surface sLeX.

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