An engineered small extracellular vesicle hydrogel enriched with pd-l1 and siglec-15, and preparation method and application thereof

Abstract

The present application relates to the technical field of biomedical materials, in particular to an engineered small extracellular vesicle hydrogel enriched with PD-L1 and Siglec-15, and a preparation method and application thereof. The engineered small extracellular vesicle hydrogel is simultaneously enriched with PD-L1 and Siglec-15, and comprises a hydrogel matrix and PDL1-Siglec15-sEVs loaded in the hydrogel matrix, wherein the PDL1-Siglec15-sEVs are small extracellular vesicles overexpressing PD-L1 and Siglec-15. Compared with natural small extracellular vesicles and traditional wound treatment strategies, the engineered small extracellular vesicle hydrogel of the present application can realize more precise immune microenvironment regulation at the local wound by enriching PD-L1 and Siglec-15 in the vesicles, moderately inhibit excessive inflammatory response, promote the phenotype transformation of macrophages to a phenotype conducive to tissue regeneration, help smooth transition from the inflammatory stage to the proliferation and reconstruction stage, and the hydrogel as a carrier can provide a moist healing environment and a three-dimensional scaffold structure, significantly prolong the residence time of the engineered small extracellular vesicles at the local wound, and realize slow release.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an engineered small extracellular vesicle hydrogel enriched with PD-L1 and Siglec-15, its preparation method, and its application. Background Technology

[0002] As one of the largest organs in the human body, the skin performs multiple functions, including barrier, defense, sensation, and regulation. Acute and chronic skin wounds caused by various reasons are very common in clinical practice, including mechanical trauma, surgical incisions, burns, diabetic foot ulcers, venous or arterial lower extremity ulcers, pressure sores, and radiation-induced skin injuries. Some wounds, due to factors such as insufficient local blood supply, persistent infection, metabolic abnormalities, or underlying systemic diseases, are prone to developing into chronic wounds that are difficult to heal or do not heal for a long time, causing severe physical and mental suffering and economic burden to patients.

[0003] Currently, clinical wound treatment mainly relies on debridement and routine dressing changes, moist wound dressings, negative pressure wound therapy, various growth factor-containing gels or dressings, skin or skin substitute transplantation, and cell therapy such as stem cells. Routine dressings primarily serve to isolate and absorb exudates, but their ability to repair deep or difficult-to-heal wounds is limited. While growth factor-containing preparations can promote cell proliferation and angiogenesis to some extent, their short half-life, easy inactivation, and insufficient local retention time limit their actual efficacy and require frequent use. Cell therapy can promote tissue repair through various mechanisms, but it faces challenges such as limited cell sources, strict requirements for preservation and transportation, insufficient in vivo survival and homing efficiency, and potential safety risks, hindering large-scale implementation. Furthermore, many existing treatment strategies focus on simply accelerating wound closure, lacking precise control over the local immune and inflammatory response, often neglecting improvements in wound healing quality, such as scarring, collagen fiber arrangement, and skin appendage regeneration.

[0004] Small extracellular vesicles (SMEVVs) are a type of cell-secreting nanovesicle that has attracted much attention in recent years. They possess good biocompatibility and low immunogenicity, and can carry various bioactive molecules such as proteins, nucleic acids, and lipids. They are considered important mediators for the "paracrine effect" of cells such as stem cells. Studies have confirmed that natural SMEVVs derived from mesenchymal stem cells play a positive role in promoting cell migration, angiogenesis, collagen deposition, and epithelialization in wounds, representing a promising cell-free therapeutic strategy. However, the composition of natural SMEVVs is complex, and the content and ratio of functional molecules are difficult to control precisely. Their ability to regulate the local immune microenvironment of wounds is limited, making it difficult to achieve targeted and controllable immunomodulation, especially in suppressing excessive inflammation, inducing an immune state conducive to tissue regeneration, and promoting the regeneration of skin appendages.

[0005] PD-L1 and Siglec-15 are immunomodulatory molecules that have attracted much attention in the fields of tumor immunology and inflammatory immunology in recent years. Moderate PD-L1 signaling can inhibit T cell overactivation and reduce tissue damage by interacting with the PD-1 receptor; Siglec-15 is closely related to the regulation of innate immune cell function. Both play important roles in regulating the intensity of inflammatory responses, influencing macrophage polarization, and maintaining immune homeostasis. Effectively enriching and presenting PD-L1 and Siglec-15 on small extracellular vesicles holds promise for achieving more precise and gentle immunosuppression and regulation at the wound site, avoiding secondary damage to tissues caused by long-term, excessive inflammation, and creating a favorable immune microenvironment for subsequent tissue regeneration and reconstruction. However, based on existing publicly available technologies, there is still a lack of systematic solutions for the remodeling and high-quality repair of the immune microenvironment of skin wounds using "dual-engineered PD-L1 and Siglec-15" small extracellular vesicles.

[0006] On the other hand, free-state small extracellular vesicles are easily lost with exudate and body fluids on the wound surface, resulting in a short local retention time and difficulty in maintaining a stable and sustained effective concentration at the wound site. Loading small extracellular vesicles into hydrogels with good biocompatibility and gelling properties, forming a three-dimensional cross-linked network structure on the wound surface, can not only provide a suitable moist healing environment but also significantly prolong the retention time of vesicles at the wound site, achieving sustained release and thus improving therapeutic efficacy. Existing exosome-hydrogel systems mostly focus on general repair and angiogenesis promotion functions. There is still a lack of reports on technologies that use engineering methods to enrich specific immunomodulatory molecules in vesicles and bind them to specific hydrogel systems to achieve precise regulation of the local immune microenvironment at the wound site.

[0007] In summary, current technologies cannot simultaneously meet the following requirements: Under safe and controllable conditions, to enrich immunomodulatory molecules such as PD-L1 and Siglec-15 in small extracellular vesicles through engineering methods, and to enhance the retention and sustained-release capacity of vesicles at the wound site using a suitable hydrogel carrier, thereby accelerating wound healing while significantly improving healing quality and promoting the regeneration of skin appendages. Therefore, it is necessary to develop a PD-L1 and Siglec-15 engineered small extracellular vesicle NHT hydrogel to regulate the local immune microenvironment of skin wounds and promote high-quality healing, thus overcoming the shortcomings of existing technologies. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an engineered small extracellular vesicle hydrogel enriched with PD-L1 and Siglec-15, its preparation method and application.

[0009] To achieve the above and other related objectives, the first aspect of this application provides an engineered small extracellular vesicle hydrogel suitable for promoting skin wound healing. The engineered small extracellular vesicle hydrogel is enriched with both PD-L1 and Siglec-15. The engineered small extracellular vesicle hydrogel includes a hydrogel matrix and PDL1-Siglec15-sEVs loaded in the hydrogel matrix. The PDL1-Siglec15-sEVs are small extracellular vesicles overexpressing PD-L1 and Siglec-15.

[0010] The second aspect of this application provides a method for preparing the engineered small extracellular vesicle hydrogel simultaneously enriched with PD-L1 and Siglec-15, comprising the following steps:

[0011] 1) Acrylamide monomers, N-hydroxysuccinimide and EDC are mixed and crosslinked under the action of a photoinitiator to obtain a hydrogel matrix;

[0012] 2) Construct a recombinant expression vector overexpressing PD-L1 and Siglec-15, transfect recipient cells, co-culture and collect the culture medium, and isolate and purify to obtain PDL1-Siglec15-sEVs;

[0013] 3) Mix the hydrogel matrix obtained in step 1) with the PDL1-Siglec15-sEVs obtained in step 2) and incubate to solidify and obtain PDL1-Siglec15-sEVs@NH hydrogel.

[0014] Optionally, step 4) involves mixing the PDL1-Siglec15-sEVs@NH hydrogel with a polyphenol compound to prepare an engineered small extracellular vesicle hydrogel.

[0015] The third aspect of this application provides the use of the above-described engineered small extracellular vesicle hydrogel in the preparation of products that promote skin wound healing.

[0016] In summary, this invention provides an engineered small extracellular vesicle hydrogel enriched with PD-L1 and Siglec-15, its preparation method, and its application, and achieves the following beneficial effects:

[0017] Compared to natural small extracellular vesicles and traditional wound treatment strategies, the PD-L1 and Siglec-15 engineered small extracellular vesicles and their NHT hydrogel formulation proposed in this invention can achieve more precise regulation of the local immune microenvironment at the wound site by enriching PD-L1 and Siglec-15 in the vesicles. This moderately inhibits excessive inflammatory responses, promotes the transformation of macrophages to phenotypes conducive to tissue regeneration, and helps to smoothly transition from the inflammatory phase to the proliferation and remodeling phase. Furthermore, animal experiments show that the engineered small extracellular vesicle NHT hydrogel can significantly accelerate wound closure and shorten the time to complete wound healing under the same medication conditions, which is significantly better than natural small extracellular vesicles and the blank gel control.

[0018] This invention not only emphasizes rapid wound closure but also significantly improves healing quality, manifested in reduced scar formation, collagen fiber arrangement closer to normal skin structure, and increased regeneration of skin appendages, making the repaired skin structurally and functionally closer to normal tissue. Furthermore, the NHT hydrogel, as a carrier, provides a moist healing environment and a three-dimensional scaffold structure, significantly prolonging the retention time of engineered small extracellular vesicles at the wound site, achieving sustained-release. Attached Figure Description

[0019] Figure 1 The following are the results of the NHT hydrogel performance testing: A: Microstructure of the hydrogel observed by scanning electron microscopy (SEM); B: Swelling test results of NHT hydrogel; C: Strain curve, tensile strength and Young's modulus results of NHT hydrogel; D: Fourier transform infrared (FTIR) structural identification of NHT hydrogel.

[0020] Figure 2 The image shows the fluorescence detection results after transfecting 293T cells with recombinant plasmids overexpressing PD-L1 and Siglec-15.

[0021] Figure 3 The image shows the results of Western blotting to verify successful transfection of 293T cells.

[0022] Figure 4 Figure 1 shows the performance test results of PDL1-Siglec15-sEVs; Figure 2 shows the morphology of PDL1-Siglec15-sEVs observed by transmission electron microscopy; Figure 3 shows the particle size results detected by a nanoparticle tracking analyzer; Figure 4 shows the potential test results of PDL1-Siglec15-sEVs; Figure 5 shows the results of Western blot detection of surface labeled proteins.

[0023] Figure 5Image A: PDL1-Siglec15-sEVs@NHT, showing projection electron microscopy and cell uptake. Image B: NIH-3T3 cell uptake. Image C: Validation results of in vitro hydrogel release.

[0024] Figure 6 The figure shows the effect of PDL1-Siglec15-sEVs-@NHT on wound healing.

[0025] Figure 7 Figure 1: Immunofluorescence staining, H&E staining, and Masson staining results of PDL1-Siglec15-sEVs-@NHT for wound healing; Figure 2: Immunofluorescence staining results; Figure 3: H&E staining and Masson staining results. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0028] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0029] In this application, the term "small extracellular vesicles (sEVs)" refers to an endoplasmic extracellular vesicle secreted by cells with a diameter of 30-150 nm. Its safety, stability, and ability to cross biological barriers make it an advantageous drug delivery carrier.

[0030] In this application, the term "hydrogel" refers to a traditional and classic biomaterial that plays an important role in the field of tissue repair and reconstruction, possesses good biocompatibility and loose porous structural characteristics, and can load drugs to achieve drug delivery.

[0031] This application first provides an engineered small extracellular vesicle hydrogel suitable for promoting skin wound healing. The engineered small extracellular vesicle hydrogel includes a hydrogel matrix and PDL1-Siglec15-sEVs loaded in the hydrogel matrix. The PDL1-Siglec15-sEVs are small extracellular vesicles overexpressing PD-L1 and Siglec-15.

[0032] In this invention, sEVs serve as an excellent drug delivery system. By engineering sEVs to enrich PD-L1 and Siglec-15, the reparative effects of PD-L1 and Siglec-15 in skin wounds are enhanced. Using a hydrogel matrix to load sEVs can compensate for the shortcomings of sEVs in terms of retention and sustained release at the wound site, enhance the stability of sEVs, and facilitate the delivery of sEVs to specific defect sites for sustained in-situ release. Loading polyphenolic compounds with hydrogels can endow the hydrogels with anti-inflammatory and antioxidant biological functions.

[0033] In some embodiments, the hydrogel matrix comprises an acrylamide monomer, N-hydroxysuccinimide (NHS), EDC, and sodium hyaluronate (Na-HA). In a preferred embodiment, the acrylamide monomer is N-acryloylglycine (NAGA).

[0034] Preferably, the mass ratio of the acrylamide monomer, N-hydroxysuccinimide (NHS), EDC, and sodium hyaluronate (Na-HA) is 18~22:0.8~1.2:0.8~1.2:1.8~2.2. Preferably, the mass ratio of N-acryloylglycineamide (NAGA), N-hydroxysuccinimide (NHS), EDC, and sodium hyaluronate (Na-HA) is 20:1:1:2.

[0035] Preferably, based on the hydrogel matrix, the addition amount of N-acryloylglycine amide is 10 w / v; the addition amount of N-hydroxysuccinimide is 0.5 w / v; the addition amount of EDC is 0.5 w / v; and the addition amount of sodium hyaluronate is 1 w / v.

[0036] It should be noted that, to maintain mechanical strength and low swelling rate, N-acryloylglycine was chosen as the main monomer to enhance photocrosslinking ability and form a hydrogel network structure. EDC / NHS chemical crosslinking was selected, with hyaluronic acid providing additional active sites to convert carboxyl groups into NHS-active esters and form HA chains. These chains then form hydrogen bonds with the amide side chains of NAGA. Visible light free radical polymerization was used to form a three-dimensional polyacryloylglycine network. HA macromolecular chains were grafted into this network, resulting in a "double network" structure that is both strong and tough, while retaining the biorecognition of HA.

[0037] Preferably, the mass ratio of the hydrogel matrix to PDL1-Siglec15-sEVs is 1200~1400:1; it can be, but is not limited to, 1200:1, 1300:1, 1400:1 or any ratio or range between the above two values.

[0038] In some embodiments, the particle size of PDL1-Siglec15-sEVs is 30-200 nm. Non-limitingly, the particle size of PDL1-Siglec15-sEVs can be, but is not limited to, 30 nm, 60 nm, 90 nm, 120 nm, 150 nm, 180 nm, 210 nm, 240 nm, 270 nm, 300 nm, or any value or range between two of the above.

[0039] In some embodiments, the small extracellular vesicles are derived from stem cells, T cells, HEK293T cells, CHO cells, Sf9 cells, Sf21 cells or Hi5 cells, preferably T cells, and more preferably HEK293T cells.

[0040] In some embodiments, the engineered small extracellular vesicle hydrogel is further loaded with polyphenolic compounds. Preferably, the number of phenolic hydroxyl groups in the polyphenolic compound is 5 to 25. Non-limitingly, the number of phenolic hydroxyl groups in the polyphenolic compound can be, but is not limited to, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or any value or range between two of the above.

[0041] In some embodiments, the molecular weight of the polyphenol compound is 200 Da to 2000 Da. Non-limitingly, the molecular weight of the polyphenol compound can be, but is not limited to, 200 Da, 400 Da, 600 Da, 800 Da, 1000 Da, 1200 Da, 1400 Da, 1600 Da, 1800 Da, 2000 Da, or any value or range between two of the above.

[0042] In some embodiments, the polyphenolic compound includes one or more of quercetin, catechins, tea polyphenols, epigallocatechin, and tannins. Further, the polyphenolic compound is tea polyphenol.

[0043] In some embodiments, the loading of the polyphenol compound is 10 w / w% to 20 w / w, based on engineered small extracellular vesicles.

[0044] In some embodiments, tea polyphenols are used as a post-treatment agent to induce shrinkage through hydrogen bonding and hydrophobic interactions, thereby improving the resolution and mechanical properties of the hydrogel.

[0045] This application also provides a method for preparing the above-mentioned engineered small extracellular vesicle hydrogel, including the following steps:

[0046] 1) Acrylamide monomers, N-hydroxysuccinimide, sodium hyaluronate and EDC are mixed and crosslinked under the action of a photoinitiator to obtain a hydrogel matrix;

[0047] 2) Construct a recombinant expression vector overexpressing PD-L1 and Siglec-15, transfect recipient cells, co-culture and collect the culture medium, and isolate and purify to obtain PDL1-Siglec15-sEVs;

[0048] 3) Mix the hydrogel matrix obtained in step 1) with the PDL1-Siglec15-sEVs obtained in step 2) and incubate to solidify and obtain PDL1-Siglec15-sEVs@NH hydrogel.

[0049] Optionally, step 4) involves mixing the PDL1-Siglec15-sEVs@NH hydrogel with a polyphenol compound to prepare an engineered small extracellular vesicle hydrogel.

[0050] In some embodiments, in step 1), the acrylamide monomer is N-acryloylglycine.

[0051] Preferably, the mass ratio of the acrylamide monomer, N-hydroxysuccinimide, EDC, and sodium hyaluronate is 18~22:0.8~1.2:0.8~1.2:1.8~2.2. More preferably, the mass ratio of the acrylamide monomer, N-hydroxysuccinimide, EDC, and sodium hyaluronate is 20:1:1:2.

[0052] It should be noted that under photoinitiator and UV irradiation, NAGA forms poly(N-acryloylglycamide) through free radical polymerization. EDC and NHS crosslink, providing additional active sites through hyaluronic acid, converting carboxyl groups into NHS-active esters and forming activated HA chains. The activated HA chains form an interpenetrating network by physically entangled with the NAGA network.

[0053] In some embodiments, the crosslinking time is 20 min to 30 min. Non-limitingly, the crosslinking time can be, but is not limited to, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, or any value or range between two of the above.

[0054] In some embodiments, in step 2), the recombinant expression vector is one or more of the following: recombinant lentiviral vector, recombinant adenovirus vector, recombinant retroviral vector, recombinant liposome and recombinant plasmid expression vector.

[0055] Preferably, the recipient cell is a stem cell, T cell, HEK293T cell, CHO cell, Sf9 cell, Sf21 cell, or Hi5 cell. Specifically, the recipient cell is a T cell, more preferably a HEK293T cell.

[0056] In some embodiments, the recombinant expression vector is a recombinant plasmid, and the promoter on the recombinant plasmid vector that initiates the expression of the PD-L1 and Siglec-15 encoding genes is EFla.

[0057] In a preferred embodiment, the method for transfecting the recombinant expression vector into recipient cells is selected from viral infection, liposome transfection, or electroporation transfection.

[0058] In some embodiments, the method for introducing the recombinant expression vector into recipient cells is liposome transfection.

[0059] In some embodiments, the PD-L1 protein comprises the amino acid sequence shown in SEQ ID NO.1; and the Siglec-15 protein comprises the amino acid sequence shown in SEQ ID NO.2.

[0060] In some embodiments, the gene encoding the PD-L1 protein comprises the nucleotide sequence shown in SEQ ID NO.3; and the Siglec-15 protein comprises the amino acid sequence shown in SEQ ID NO.4.

[0061] In some embodiments, the mass ratio of the hydrogel matrix to PDL1-Siglec15-sEVs is 1200 to 1400:1, which can be, but is not limited to, 1200:1, 1300:1, 1400:1, or any ratio or range between two of the above.

[0062] The hydrogel matrix is ​​incubated with PDL1-Siglec15-sEVs for 15-35 min. Preferably, the incubation time of the hydrogel matrix with PDL1-Siglec15-sEVs can be, but is not limited to, 15 min, 20 min, 25 min, 30 min, 35 min, or any value or range between two of the above values.

[0063] Preferably, the curing time is 10-30 min, and more preferably, it can be 10 min, 15 min, 20 min, 25 min, 30 min, or any value or range between the above two values.

[0064] In some embodiments, in step 4), the polyphenolic compound includes one or more of quercetin, catechins, tea polyphenols, epigallocatechin, and tannins. Further, the polyphenolic compound is tea polyphenol.

[0065] In a preferred embodiment, the concentration of tea polyphenols is 8-12 w / v%. Non-limitingly, the concentration of tea polyphenols can be, but is not limited to, 8 w / v%, 9 w / v%, 10 w / v%, 11 w / v%, 12 w / v%, or any value or range between the above two values.

[0066] Preferably, the mass ratio of PDL1-Siglec15-sEVs@NH hydrogel to tea polyphenols is (4~6):1. More preferably, the mass ratio of PDL1-Siglec15-sEVs@NH hydrogel to tea polyphenols can be, but is not limited to, 4:1, 5:1, 6:1, or any ratio or range between two of the above.

[0067] Preferably, the mixing time between PDL1-Siglec15-sEVs@NH hydrogel and tea polyphenols is 1h to 3h. More preferably, the mixing time between PDL1-Siglec15-sEVs@NH hydrogel and tea polyphenols can be, but is not limited to, 1h, 1.5h, 2.0h, 2.5h, 3.0h, or any value or range between two of the above.

[0068] This invention also provides the application of the above-described engineered small extracellular vesicle hydrogel in the preparation of products that promote skin wound healing.

[0069] In some embodiments, the skin wound includes one or more of the following: mechanical trauma, surgical incision, burns, diabetic foot ulcers, venous or arterial lower extremity ulcers, pressure sores, and radiation-induced skin injuries.

[0070] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0071] Example 1 – Preparation of PDL1-Siglec15-sEVs@NHT

[0072] 1. Preparation of NHT hydrogels

[0073] 1) Dissolve 1g of N-acryloylglycine in 10ml of deionized water and stir with a magnetic stirrer for 10min until completely dissolved. Under light-protected conditions, add 0.05g of 2,4,6-trimethylbenzoyl lithium phosphine sulfate to the solution and continue stirring. Then add 0.05g of N-hydroxysuccinimide and 0.05g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and continue stirring for 15min. Finally, add 0.1g of sodium hyaluronate and stir until no particles are visible.

[0074] 2) Transfer the above solution into a mold, irradiate with a UV lamp for 15 minutes and then solidify to obtain NH hydrogel.

[0075] 3) Dissolve 1g of tea polyphenols in 10ml of water to prepare a tea polyphenol solution, and soak the NH hydrogel in the tea polyphenol solution for 2h to obtain NHT hydrogel.

[0076] 4) Detection of NHT hydrogel

[0077] The microstructure of the hydrogel was observed using a scanning electron microscope (SEM). The morphology was imaged after gold was sputtered onto the NHT hydrogel for 30 seconds.

[0078] Swelling test of NHT hydrogel: Accurately weigh the initial weight (W0) of NH hydrogel and NHT hydrogel. Next, immerse the NH hydrogel and NHT hydrogel in deionized water, and take out the hydrogel samples at different time periods, absorb the surface moisture with filter paper and weigh them (Wt).

[0079] Uniaxial tensile tests were conducted on NH hydrogel and NHT hydrogel using a multi-functional testing machine, and stress-strain curves were obtained. The tensile strength and Young's modulus were calculated from the stress-strain values.

[0080] The chemical structures of NH hydrogel and NHT hydrogel were identified using Fourier transform infrared spectroscopy (FTIR). The scanning range was 4000 cm⁻¹. -1 -400 cm-1 The resolution is set to 4 cm. -1 The number of scans was 32.

[0081] The results are as follows Figure 1 As shown, the internal morphology of the NHT hydrogel observed by scanning electron microscopy (SEM) at an accelerating voltage of 5 kV is as follows. Figure 1 As shown in Figure A, swelling tests on NH hydrogel and NHT hydrogel revealed that the swelling capacity of NHT hydrogel was significantly higher than that of NH hydrogel. Figure 1 B), and its tensile strength is also significantly higher than that of NH hydrogel (B). Figure 1 C); Fourier transform infrared spectroscopy shows that at 1639 cm⁻¹ -1 The peak at that point is the C=O asymmetric stretching vibration peak of the amide. Figure 1 D).

[0082] 2. PD-L1 and Siglec-15 enrich engineered small extracellular vesicles

[0083] 1) Cell resuscitation and culture: Thaw frozen 293T cells rapidly (<2 min) in a 37℃ water bath; transfer all cell culture from the cryovial to a 125 ml shake flask containing 30 ml of pre-warmed HEK293F Hi-exp medium; place in a shaker at 37℃, 5% CO2, 130 rpm (amplitude 26 mm), and 80% humidity. Preheat the medium at 37℃ for 20-30 min; collect cells with a density ≥3×10⁶. 6 Cells with a cell density of ≥95% and in the mid-logarithmic growth phase were selected and cultured at a concentration of 0.5 × 10⁻⁶ cells / ml. 6 The cells / ml seeding density was transferred to preheated culture medium and cultured in a cell culture shaker at 37°C, 80% humidity, 130 rpm (26 mm amplitude), and 5% CO2.

[0084] 2) Based on the NCBI database search for human PD-L1 and Siglec-15 gene sequences, PD-L1 and Siglec-1 overexpressing cells were constructed using recombinant plasmids that overexpressed PD-L1 (amino acid sequence as shown in SEQ ID NO.1, encoding nucleotide sequence as shown in SEQ ID NO.3) and Siglec-15 (amino acid sequence as shown in SEQ ID NO.2, encoding nucleotide sequence as shown in SEQ ID NO.4). PD-L1 and Siglec-15 overexpressing plasmids were constructed by double digestion of GV658 plasmid with KpnI / PacI, and after verification, the cells were transfected. All overexpressing plasmids were constructed and produced by Shanghai Jikai Gene Medical Technology Co., Ltd. PD-L1-NC and Siglec15-NC control plasmids were also purchased. HEK293T cells were cultured in good condition, digested and resuspended in antibiotic-free complete medium. Cells were then cultured at 4-6 × 10⁶ cells per well. 5 Seed cells at a density of 100 cells / well in 6-well plates, with 2 mL of culture medium added to each well. Gently shake to mix thoroughly and incubate overnight (approximately 18-24 hours) in a 37°C, 5% CO2 incubator, allowing the cell density to reach 70%-90% (ideally 80%) at transfection. After 24 hours, prepare the transfection complex and proceed with transfection.

[0085] Prepare the DNA stock solution (in sterile centrifuge tube A): Take 125 µL of Opti-MEM. Add 2.5 µg each of the recombinant plasmids overexpressing PD-L1 and Siglec-15. Add 5 µL of P3000™ enhancer. Gently pipette to mix (do not vortex). Incubate at room temperature. Prepare the liposome stock solution (in sterile centrifuge tube B): Take another tube and add 125 µL of Opti-MEM. Add 5.0 µL of Lipofectamine 3000 reagent. Immediately pipette to mix gently (do not vortex). Incubate at room temperature for 5 minutes. Formation of the DNA-liposome complex: Add the entire diluted DNA stock solution (tube A) to the diluted Lipofectamine 3000 stock solution (tube B). Gently pipette to mix (usually 4-6 times). The solution may be slightly turbid at this point. Incubate at room temperature for 10-15 minutes to allow the complex to stabilize and form.

[0086] Adding the complex to the cells: Remove the 6-well plate from the incubator without changing the culture medium. Add 250 µL of the DNA-liposome complex dropwise to the corresponding well of the cell culture medium. After addition, gently shake the plate back and forth and side to side to distribute the complex evenly in the culture medium. Return the plate to the 37°C, 5% CO2 incubator. Observe the cells under a microscope 4-6 hours after transfection. If the cells are in good condition, aspirate the culture medium containing the complex, gently wash once with pre-warmed PBS, and then add 2 mL of fresh complete culture medium (antibiotics can be added again at this time). 48 hours after transfection: Begin expression detection. Observe the fluorescence expression directly under a fluorescence microscope for one part; extract cell proteins for another part and use Western blotting to detect the expression of PD-L1 and Siglec-15.

[0087] SEQ ID NO.1: MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILV VDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET;

[0088] SEQ ID NO.2: MEKSIWLLACLAWVLPTGSFVRTKIDTTENLLNTEVHSSPAQRWSMQVPPEVSEAAGDAAVLPCTFTPHRHYDGPLTAIWRAGEPYAGPQVFRCAAARGSELCQTALSLHGRFRLLGNPRRNDLSLRVERLALADDRRYFCRVEFAGDVHDRYESRHGVRL HVTAAPRIVNISVLPSPAHAFRALCTAEGEPPPALAWSGPALGNSLAAVRSPREGHGHLVTAELPALTHDGRYTCTAANSLGRSEASVYLFRFHGASGASTVALLLGALGFKALLLLGVLAARAARRRPEHLDTPDTPPSQAQESNYENLSQMNPRSPPATMCSP;

[0089] SEQ ID NO.3:ATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCTCTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGCACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCTTGGTGTAGCACTGACATTCATCTTCCGTTTAAGAAAAGGGAGAATGATGGATGTGAAAAAATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTAA;

[0090] SEQ ID NO.4:ATGGAAAAGTCCATCTGGCTGCTGGCCTGCTTGGCGTGGGTTCTCCCGACAGGCTCATTTGTGAGAACTAAAATAGATACTACGGAGAACTTGCTCAACACAGAGGTGCACAGCTCGCCAGCGCAGCGCTGGTCCATGCAGGTGCCACCCGAGGTGAGCGCGGAGGCAGGCGACGCGGCAGTGCTGCCCTGCACCTTCACGCACCCGCACCGCCACTACGACGGGCCGCTGACGGCCATCTGGCGCGCGGGCGAGCCCTATGCGGGCCCGCAGGTGTTCCGCTGCGCTGCGGCGCGGGGCAGCGAGCTCTGCCAGACGGCGCTGAGCCTGCACGGCCGCTTCCGGCTGCTGGGCAACCCGCGCCGCAACGACCTCTCGCTGCGCGTCGAGCGCCTCGCCCTGGCTGACGACCGCCGCTACTTCTGCCGCGTCGAGTTCGCCGGCGACGTCCATGACCGCTACGAGAGCCGCCACGGCGTCCGGCTGCACGTGACAGCCGCGCCGCGGATCGTCAACATCTCGGTGCTGCCCAGTCCGGCTCACGCCTTCCGCGCGCTCTGCACTGCCGAAGGGGAGCCGCCGCCCGCCCTCGCCTGGTCCGGCCCGGCCCTGGGCAACAGCTTGGCAGCCGTGCGGAGCCCGCGTGAGGGTCACGGCCACCTAGTGACCGCCGAACTGCCCGCACTGACCCATGACGGCCGCTACACGTGTACGGCCGCCAACAGCCTGGGCCGCTCCGAGGCCAGCGTCTACCTGTTCCGCTTCCATGGCGCCAGCGGGGCCTCGACGGTCGCCCTCCTGCTCGGCGCTCTCGGCTTCAAGGCGCTGCTGCTGCTCGGGGTCCTGGCCGCCCGCGCTGCCCGCCGCCGCCCAGAGCATCTGGACACCCCGGACACCCCACCACGGTCCCAGGCCCAGGAGTCCAATTATGAAAATTTGAGCCAGATGAACCCCCGGAGCCCACCAGCCACCATGTGCTCACCG。

[0091] 3) PD-L1 and Siglec-1 overexpressing cells were co-cultured with the 293T cells obtained in step 1) to obtain PDL1-Siglec15-293T cells overexpressing PD-L1 and Siglec-15. The 293T cells transfected with the plasmid were then cultured on a large scale. The culture supernatant was collected using serum-free medium over 48 hours, with a cell density exceeding 70%. The collected supernatant was centrifuged sequentially at 500g (4℃) and 2000g (4℃) for 10 min to remove dead cells and cell debris. The supernatant was then centrifuged at 4℃ and 10000g for 30 min to remove organelles. The supernatant was then transferred to a 100kDa MWCO ultrafiltration centrifuge tube and centrifuged at 2000g for 30 min. The concentrate was then collected and ultracentrifuged at 4℃ and 100000g for 70 min to collect the precipitate (i.e., small extracellular vesicles). Then, the small extracellular vesicles were resuspended in phosphate-buffered saline (PBS) and centrifuged again at 100,000 g for 70 min, and the precipitate was collected. Finally, the small extracellular vesicles were resuspended in phosphate-buffered saline (PBS), the purified small extracellular vesicles were collected, and filtered through a 0.22 μm pore filter to obtain a PDL1-Siglec15-sEVs small extracellular vesicle suspension, which was stored at -80°C for later use. PDL1-NC-sEVs, Siglec15-NC-sEVs, and PDL1-Siglec15-NC-sEVs were prepared using the same procedure.

[0092] 293T cells transfected with PD-L1 and Siglec-15 recombinant plasmids, fluorescence pattern as shown in the figure. Figure 2 As shown, transfection was successful, and Western blotting confirmed successful transfection of 293T cells. Figure 3 To expand the culture, 293T cells were cultured in serum-free medium. The culture supernatant was collected, centrifuged, filtered, and the precipitate was resuspended to obtain a suspension of small extracellular vesicles (sEVs). PDL1-sEVs and Siglec15-sEVs were obtained.

[0093] 4) Identification of PDL1-Siglec15-sEVs

[0094] The morphology of small extracellular vesicles was observed using transmission electron microscopy (TEM). Suspensions of PDL1-sEVs, Siglec15-sEVs, and PDL1-Siglec15-sEVs were respectively added to a 2 mm diameter copper grid. After standing at room temperature for 5 min, residual liquid at the edge of the grid was gently blotted away with filter paper. The grid was then inverted onto a droplet of 30 g / L phosphotungstic acid (pH 6.8) and negatively stained at room temperature for 5 min. Finally, the grid was dried under an incandescent lamp and observed and photographed under a TEM. The results are shown below. Figure 4 As shown in Figure A.

[0095] Nanoparticle tracking analysis (NTA) was used to detect particle size. First, the extracellular vesicle suspensions of PDL1-sEVs, Siglec15-sEVs, and PDL1-Siglec15-sEVs were diluted with PBS to the optimal concentration for instrument detection. After thorough mixing, 1 mL of sample was injected into the sample chamber using a disposable syringe. The particle size and concentration of the extracellular vesicles were displayed through the instrument's software. The results are shown below. Figure 4 As shown in Figure B, it can be seen that the particle size of PDL1-Siglec15-sEVs is not significantly different from that of PDL1-sEVs and Siglec15-sEVs. The particle size is mainly distributed in the range of 30nm~300nm, which are 110, 113, and 115nm respectively.

[0096] Potential measurement: The extracellular vesicle suspensions of PDL1-sEVs, Siglec15-sEVs, and PDL1-Siglec15-sEVs were measured using a Zeta potential analyzer. The results are as follows: Figure 4 As shown in Figure C, it can be seen that PDL1-Siglec15-sEVs are not significantly different from PDL1-sEVs and Siglec15-sEVs.

[0097] Western blot detection of surface-labeled proteins:

[0098] Cell protein extraction: PDL1-Siglec15-sEVs and PDL1-sEVs, Siglec15-sEVs were lysed with cell lysis buffer, and the supernatant was collected by centrifugation to obtain the sample. After protein concentration determination, loading buffer was added to the sample, and the mixture was vortexed and boiled in a water bath for 10 min, then cooled on ice. The prepared sample was stored frozen at -20°C.

[0099] Prepare SDS-PAGE separating gels of appropriate concentrations according to protein molecular weight and add the prepared protein samples. After sample addition, perform electrophoresis. Stop electrophoresis after all target bands have separated. Use a polyvinylidene fluoride (PVDF) membrane, activate the membrane with methanol, place the SDS-PAGE gel on the membrane, transfer it in a transfer clamp for 2 hours, and then block the PVDF membrane in 5% skim milk at room temperature for 2 hours. Wash with TBST, and cut the PVDF membrane into strips according to the target protein molecular weight. Incubate the strips with the corresponding primary antibody working solution overnight. The next day, remove the strips and wash with 1×TBST. Dilute the HRP-labeled secondary antibody with TBST at a ratio of 1:3000, incubate at room temperature for 1 hour, and wash with 1×TBST. Prepare ECL chemiluminescence working solution, add it evenly to the PVDF membrane, and use an exposure analyzer to develop the expression of PDL1 and Siglec15 proteins on the PVDF membrane.

[0100] The results are as follows Figure 3 As shown, the marker proteins PDL1 and Siglec15 of PDL1-Siglec15-sEVs are positively expressed.

[0101] 3. Preparation of PDL1-Siglec15-sEVs@NHT

[0102] 1) Dissolve 1 g of N-acryloylglycine amide in 10 ml of deionized water and stir with a magnetic stirrer for 10 min until completely dissolved. Add 0.05 g of 2,4,6-trimethylbenzoyl lithium phosphine sulfate to the solution under light-protected conditions and continue stirring. Then add 0.05 g of N-hydroxysuccinimide and 0.05 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride sequentially and continue stirring for 15 min. Finally, add 0.1 g of sodium hyaluronate and stir until no particles are visible.

[0103] 2) The above hydrogel solution was mixed with the prepared PDL1-Siglec15-sEVs small extracellular vesicle (1mg) suspension, and after standing for 15min, it was transferred to a mold, irradiated with ultraviolet light for 15min and cured to obtain PDL1-Siglec15-sEVs@NH hydrogel.

[0104] 3) Dissolve 1g of tea polyphenols in 10ml of water to prepare a tea polyphenol solution, and soak the NH hydrogel in the tea polyphenol solution for 2h to finally obtain PDL1-Siglec15-sEVs@NHT.

[0105] Based on the above steps, in the preparation of NHT hydrogel, before UV light curing, the prepared PDL1-sEVs, Siglec15-sEVs, and sEVs small extracellular vesicle suspensions are added to the mixed solution, and then UV light curing is performed to finally obtain PDL1-sEVs@NHT, Siglec15-sEVs@NHT, CON-sEVs@NHT, and PDL1-Siglec15-NC-sEVs@NHT.

[0106] Performance test results as follows Figure 5 As shown, PDL1-Siglec15-sEVs were labeled with Dil dye, and then the labeled PDL1-Siglec15-sEVs were encapsulated in an NHT hydrogel. Confocal microscopy was performed after adding Dil to the small extracellular vesicles, revealing that the sEVs were uniformly distributed within the hydrogel. Figure 5 A). After preparing Dil-labeled PDL1-Siglec15-sEVs@NHT and co-culturing them with NIH-3T3 cells, and after 24 hours of normal culture in an incubator, it was verified that PDL1-Siglec15-sEVs could be taken up by NIH-3T3 cells. Figure 5 B).

[0107] The in vitro hydrogel release assays have verified that NHT hydrogel can release sEVs for an extended period of time. Figure 5 C). The hydrogel was incubated in vitro in 1 mL of PBS solution. At one-day intervals, 500 μL of supernatant was collected and replaced with an equal volume of PBS solution. The collected samples were then analyzed using a bis(octanoic acid) assay to determine the amount of free sEVs in the supernatant. Daily and cumulative release curves over time were calculated and plotted, as shown in A and B of section 6. It can be seen that PDL1-Siglec15-sEVs@NHT can be released continuously for approximately one week, with over 80% of PDL1-Siglec15-sEVs released from the NHT hydrogel.

[0108] Example 2 – Effect of PDL1-Siglec15-sEVs@NH on wound healing

[0109] A skin injury model was established using SD rats. After acclimatization, a circular skin wound with a diameter of 1 cm was cut off on the back of the SD rats. They were divided into four groups: NHT group, CON-sEVs-@NHT group, PDL1-sEVs-@NHT group, Siglec15-sEVs-@NHT group, PDL1-Siglec15-sEVs-@NHT group, and PDL1-Siglec15-NC-sEVs-@NHT group. CON-sEVs-@NHT, PDL1-sEVs-@NHT, Siglec15-sEVs-@NHT, PDL1-Siglec15-sEVs-@NHT, or PDL1-Siglec15-NC-sEVs-@NHT were applied to the wound according to the different groups. The wound size was measured after 14 days, and the skin tissue at the injury site was extracted and prepared into sections for immunofluorescence staining, H&E staining, and Masson staining.

[0110] Immunofluorescence staining of tissue sections:

[0111] Paraffin sections were dried in a 65°C oven, transferred to a staining rack, and dewaxed in xylene. The sections were then hydrated by immersion in ethanol of varying concentrations, followed by washing with pure water and PBS to remove the ethanol. The sections were then immersed in 3% H2O2 to remove endogenous peroxidase. After washing, the sections were placed in heated sodium citrate buffer solution and autoclaved. The sections were then cooled to room temperature in sodium citrate buffer, washed, and the liquid around the tissue was wiped off. 5% BSA solution was added to the tissue, and the sections were incubated at 37°C for 30 min to block non-specific antigens. After removing the liquid around the tissue, 25 μL of diluted antibody solution was added to each tissue section, and the sections were incubated overnight at 4°C, then warmed for 30 min and washed. 25 μL of diluted fluorescent secondary antibody solution (1:1000; diluted with 1% BSA) was added, and the sections were incubated at 37°C for 1 minute. After h, the slides were cleaned; mounted with a mounting solution containing 4',6-diamidinyl-2-phenylindole (DAPI), an antifluorescence quencher; observed and imaged using a laser confocal microscope.

[0112] Hematoxylin-eosin (HE) staining procedure: After baking paraffin sections in an oven, dewax them to water. After washing, stain with hematoxylin for 5 min, then rinse with running water. Add 1% hydrochloric acid-ethanol for 20 s, then wash with ddH2O for 2 min. Blue with 1% ammonia for 20 s, observe under a microscope, and wash with ddH2O for 2 min. Stain with 0.5% eosin for 2 min, then wash with ddH2O for 30 s. Then dehydrate following the dewaxing-to-water reverse procedure, mount with neutral resin, scan with a slide scanner, and record the observation results.

[0113] Masson staining procedure: Dewax paraffin sections to water; stain cell nuclei with cycad hematoxylin: Immerse sections in cycad hematoxylin staining solution for 3-8 minutes, then rinse with running water; stain with Ponceau S-acid Fuchsin: Stain sections in Ponceau S-acid Fuchsin staining solution for 5 minutes, then rinse; phosphomolybdic acid differentiation: Immerse sections in phosphomolybdic acid for differentiation for a few seconds, then rinse with water; aniline blue staining: Immerse sections in aniline blue staining solution for 5 minutes, then rinse; rapid dehydration and clearing: Air dry sections and mount with neutral resin; microscopic examination, image acquisition and analysis.

[0114] The results are as follows Figures 6-7 As shown, compared with the CON-sEVs-@NHT group, PDL1-sEVs-@NHT group, Siglec15-sEVs-@NHT group, and PDL1-Siglec15-NC-sEVs-@NHT group, the use of the PDL1-Siglec15-sEVs-@NHT group can accelerate wound healing. Figure 6 Tissue samples were taken from the injured skin and prepared into sections for immunofluorescence staining, H&E staining, and Masson staining. Rats in the PDL1-Siglec15-sEVs@NHT group showed the fastest skin wound healing, significantly better angiogenesis and skin proliferation, and hair follicle recovery and fibrosis. Figure 7 ).

[0115] In summary, the above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An engineered small extracellular vesicle hydrogel suitable for promoting skin wound healing, wherein the engineered small extracellular vesicle hydrogel is enriched with both PD-L1 and Siglec-15, the engineered small extracellular vesicle hydrogel comprising a hydrogel matrix and PDL1-Siglec15-sEVs loaded in the hydrogel matrix, wherein the PDL1-Siglec15-sEVs are small extracellular vesicles overexpressing PD-L1 and Siglec-15.

2. The engineered small extracellular vesicle hydrogel as described in claim 1, characterized in that, The hydrogel matrix comprises acrylamide monomers, N-hydroxysuccinimide, EDC, and sodium hyaluronate; And / or, the mass ratio of the hydrogel matrix to PDL1-Siglec15-sEVs is 1200~1400:1; And / or, the small extracellular vesicles are derived from stem cells, T cells, HEK293T cells, CHO cells, Sf9 cells, Sf21 cells or Hi5 cells.

3. The engineered small extracellular vesicle hydrogel as described in claim 2, characterized in that, The mass ratio of the acrylamide monomer, N-hydroxysuccinimide, EDC, and sodium hyaluronate is 18~22:0.8~1.2:0.8~1.2:1.8~2.2; and / or, the small extracellular vesicles are derived from HEK293T cells.

4. The engineered small extracellular vesicle hydrogel as described in claim 2, characterized in that, The acrylamide monomer is N-acryloylglycine; and / or, the engineered small extracellular vesicle hydrogel is loaded with polyphenolic compounds.

5. The engineered small extracellular vesicle hydrogel as described in claim 4, characterized in that, The molecular weight of the polyphenolic compounds is 200 Da to 2000 Da; And / or, the number of phenolic hydroxyl groups in the polyphenolic compound is 5 to 25; And / or, the polyphenolic compounds include one or more of quercetin, catechins, tea polyphenols, epigallocatechin, and tannins; And / or, based on the engineered small extracellular vesicle hydrogel, the loading of the polyphenolic compound is 10w / w% to 20w / w.

6. A method for preparing engineered small extracellular vesicle hydrogels according to any one of claims 1-5, comprising the following steps: 1) Acrylamide monomers, N-hydroxysuccinimide, EDC and sodium hyaluronate are mixed and crosslinked under the action of a photoinitiator to obtain hydrogel particles; 2) Construct recombinant expression vectors that overexpress PD-L1 and Siglec-15, transfect recipient cells, co-culture and collect the culture medium, and isolate and purify to obtain PDL1-Siglec15-sEVs; 3) Mix the hydrogel particles obtained in step 1) with the PDL1-Siglec15-sEVs obtained in step 2) and incubate them to solidify and obtain PDL1-Siglec15-sEVs@NH hydrogel.

7. The preparation method according to claim 6, characterized in that, It also includes step 4) mixing PDL1-Siglec15-sEVs@NH hydrogel with polyphenolic compounds to prepare engineered small extracellular vesicle hydrogels.

8. The preparation method according to claim 6, characterized in that, Step 1) also includes one or more of the following: 11) The acrylamide monomer is N-acryloylglycine; 12) The mass ratio of the acrylamide monomer, N-hydroxysuccinimide, EDC, and sodium hyaluronate is 18~22:0.8~1.2:0.8~1.2:1.8~2.2; 13) The crosslinking time is 15 min to 25 min.

9. The preparation method according to claim 6, characterized in that, Step 2) also includes one or more of the following: 21) The recombinant expression vector is one or more of the following: recombinant lentiviral vector, recombinant adenovirus vector, recombinant retroviral vector, recombinant liposome and recombinant plasmid expression vector; 22) The recipient cells are stem cells, T cells, HEK293T cells, CHO cells, Sf9 cells, Sf21 cells or Hi5 cells; 23) The method for transfecting the recombinant expression vector into recipient cells is selected from viral infection, liposome transfection or electroporation transfection.

10. The preparation method according to claim 6, characterized in that, In step 3), the mass ratio of the hydrogel matrix to PDL1-Siglec15-sEVs is 1200~1400:1; the incubation time for mixing the hydrogel matrix and PDL1-Siglec15-sEVs is 22h~26h; and / or the curing time is 10-30min.

11. The preparation method according to claim 7, characterized in that, Step 4) also includes one or more of the following: 41) The polyphenolic compounds include one or more of quercetin, catechins, tea polyphenols, epigallocatechin, and tannic acid; 42) The concentration of polyphenol compound solutions is 8~12 w / v% 43) The mass ratio of the PDL1-Siglec15-sEVs@NH hydrogel to the polyphenolic compound is (4~6):1; 44) The PDL1-Siglec15-sEVs@NH hydrogel is mixed with polyphenolic compounds for 1 h to 3 h.

12. The use of the engineered microcellular extracellular vesicle hydrogel as described in any one of claims 1-5 in the preparation of products that promote skin wound healing.

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