Apelin and HBD3 delivery system based on hydrogel and engineering extracellular vesicles as well as preparation method and application of Apelin and HBD3 delivery system

Abstract

The invention provides an Apelin and HBD3 delivery system based on hydrogel and engineering extracellular vesicles as well as a preparation method and application of the Apelin and HBD3 delivery system, and belongs to the technical field of medicines. The preparation method comprises the following steps: transducing Wharton jelly-derived mesenchymal stem cells by utilizing a lentiviral vector, so that the Wharton jelly-derived mesenchymal stem cells are over-expressed with Apelin and HBD3 proteins, then extracting engineered EVs, and loading the engineered EVs in GelMA hydrogel, so as to obtain Apelin-HBD3-EVs-coated GelMA. According to the Apelin-HBD3-EVs (at) GelMA compound, the anti-inflammatory and antibacterial characteristics of the Apelin-HBD3-EVs (at) GelMA are verified through in-vitro experiments and in-vivo experiments, and the Apelin-HBD3-EVs (at) GelMA compound shows excellent anti-inflammatory, antibacterial and wound healing promoting effects in an old mouse infectious wound model, and shows huge application prospects for coping with special challenges of chronic wound treatment of old people.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a delivery system for Apelin and HBD3 based on hydrogels and engineered extracellular vesicles, its preparation method, and its application. Background Technology

[0002] Skin wound healing is a complex process involving multiple cell types, tissues, cytokines, and chemokines. In healthy individuals, the skin maintains a normal microenvironment, and wound healing begins with hemostasis and an inflammatory response, subsequently progressing through tissue regeneration and remodeling. However, aging skin is characterized by a persistent inflammatory response (i.e., inflammatory aging), which significantly impacts the wound healing process. In wounds of elderly patients, impaired microcirculation leads to a reduction in the number of inflammatory cells and chemical mediators reaching the wound surface, thereby altering the inflammatory response pattern. Relatively low perfusion capacity at the wound site results in insufficient supply of key nutrients and oxygen required for healing, leading to cell death through apoptosis or necrosis. Furthermore, the decline in skin barrier function and immune regulation in the elderly make them more susceptible to infection throughout the healing process. The observed decline in liver and kidney function in the elderly may affect antibiotic metabolism, thereby increasing their sensitivity to antibiotic treatment. Therefore, these adverse factors, combined with the impairment of skin barrier function, significantly increase the risk of wound infection in elderly patients. Global population aging exacerbates the challenges of chronic wound healing in the elderly. Considering these challenges, angiogenesis, inflammatory response regulation, and antimicrobial capacity are key elements for wound healing in the elderly. Therefore, there is an urgent need to find an integrated solution to accelerate wound healing and improve the quality of life for the elderly. Summary of the Invention

[0003] To address the problems existing in the prior art, the present invention aims to provide a delivery system for Apelin and HBD3 based on hydrogels and engineered extracellular vesicles, and a method for preparing the same.

[0004] The present invention also aims to provide an application of an Apelin and HBD3 delivery system based on hydrogels and engineered extracellular vesicles in the preparation of drugs that promote endothelial cell proliferation, angiogenesis, antibacterial activity, and reduce inflammatory response.

[0005] The present invention also aims to provide an application of an Apelin and HBD3 delivery system based on hydrogels and engineered extracellular vesicles in the preparation of drugs that promote the healing of wounds on aging skin.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a delivery system for Apelin and HBD3 based on hydrogel and engineered extracellular vesicles, wherein engineered extracellular vesicles containing Apelin and HBD3 are encapsulated in gelatin-methacrylamide hydrogel to obtain the delivery system.

[0007] Preferably, the engineered extracellular vesicles containing Apelin and HBD3 are secreted by engineered Wharton's collagen mesenchymal stem cells; the engineered Wharton's collagen mesenchymal stem cells contain an Apelin overexpression vector and an HBD3 overexpression vector.

[0008] The present invention also provides a method for preparing the above-mentioned delivery system, comprising the following steps: A 0.25% LAP solution was prepared and heated; then GelMA was added at a final concentration of 5%–15% to prepare a GelMA scaffold; engineered extracellular vesicles containing Apelin and HBD3 were added at a final concentration of 4 × 10⁻⁶. 10 Add cells / mL to a GelMA scaffold, incubate, and perform blue light chemical crosslinking to obtain the delivery system.

[0009] Preferably, the heat treatment conditions are 55°C for 15 minutes.

[0010] Preferably, the incubation conditions are 37°C for 10 minutes.

[0011] Preferably, the blue light chemical crosslinking condition is 10 mW / cm. 2 Blue light chemical crosslinking for 15 seconds.

[0012] Preferably, the engineered extracellular vesicles containing Apelin and HBD3 are obtained by isolating and purifying engineered Wharton's collagen mesenchymal stem cells; the engineered Wharton's collagen mesenchymal stem cells are transfected into Apelin overexpression vector and HBD3 overexpression vector via lentiviral transfection.

[0013] The present invention also provides the use of the above-described delivery system in the preparation of drugs that promote endothelial cell proliferation and / or angiogenesis.

[0014] The present invention also provides the use of the above-described delivery system in the preparation of antibacterial and / or anti-inflammatory drugs.

[0015] The present invention also provides the application of the above-described delivery system in the preparation of a medicament for promoting the healing of wounds on aging skin.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention develops a hydrogel-based sustained-release system that delivers humanized proteins via engineered extracellular vesicles (EVs), suitable for tissue repair in elderly patients. The Apelin-HBD3-EVs@GelMA complex provided by this invention has a porous structure, enabling controlled release of Apelin and HBD3. In vitro experiments show that this system promotes endothelial cell proliferation and angiogenesis, protects the body from inflammatory factors through the AMPK / KLF2 pathway, and restores mitochondrial membrane potential. The system exhibits antibacterial properties against the tested strains. In an aged mouse model, Apelin-HBD3-EVs@GelMA accelerates wound healing, increases epidermal thickness, improves collagen arrangement, reduces malondialdehyde (MDA) levels, and promotes microvascular formation. Furthermore, this complex inhibits neutrophil infiltration by reducing myeloperoxidase (MPO) levels, promotes M2 macrophage polarization, inhibits pro-inflammatory cytokine secretion, and enhances anti-inflammatory factor release, demonstrating significant repair effects. This invention demonstrates the efficacy of Apelin-HBD3-EVs@GelMA hydrogel in treating delayed healing caused by infection and inflammation by modulating the synergistic effects of endothelial cells, neutrophils, and macrophages. This Apelin-HBD3-EVs@GelMA system accelerates wound healing, reduces inflammatory responses, and possesses potent antibacterial properties. It promotes tissue regeneration while inhibiting inflammation and microbial growth, making it suitable for elderly patients with chronic wounds and demonstrating significant clinical translational potential for the comprehensive treatment of complex age-related diseases. Attached Figure Description

[0017] Figure 1 Engineering design and genetic modification validation of EVs; Figure 2 Loading strategies for engineered EVs and characterization of GelMA brackets; Figure 3 The effects of Apelin-HBD3-EVs@GelMA scaffold on aging HUVECs; Figure 4 The in vitro antibacterial properties of the Apelin-HBD3-EVs@GelMA scaffold; Figure 5 Apelin-HBD3-EVs@GelMA scaffolds accelerated the healing of MRSA-infected skin wounds in aged mice; Figure 6 The impact of Apelin-HBD3-EVs@GelMA scaffolds on healing quality and inflammatory response; Figure 7 Effects of Apelin-HBD3-EVs@GelMA on neutrophil infiltration and macrophage polarization. Detailed Implementation

[0018] This invention provides a delivery system for Apelin and HBD3 based on hydrogels and engineered extracellular vesicles. The engineered extracellular vesicles containing Apelin and HBD3 are encapsulated in a gelatin-methacrylamide hydrogel to obtain the delivery system, denoted as Apelin-HBD3-EVs@GelMA. Preferably, the engineered extracellular vesicles containing Apelin and HBD3 are secreted by engineered Wharton's collagen mesenchymal stem cells; the engineered Wharton's collagen mesenchymal stem cells contain an Apelin overexpression vector and an HBD3 overexpression vector. The Apelin-HBD3-EVs@GelMA complex provided by this invention has a porous structure, enabling controlled release of Apelin and HBD3.

[0019] This invention also provides a method for preparing the above-mentioned delivery system, comprising the following steps: preparing a LAP solution with a final concentration of 0.25% (w / v) and heating it; then adding GelMA (methacrylamide gelatin) at a final concentration of 5%~15% (w / v) to prepare a GelMA scaffold; and adding engineered extracellular vesicles containing Apelin and HBD3 at a final concentration of 4×10⁻⁶. 10 Add cells / mL to a GelMA scaffold, incubate, and perform blue light chemical crosslinking to obtain the delivery system.

[0020] Preferably, the heat treatment conditions of the present invention are: heat treatment at 55°C for 15 min; the incubation conditions are: incubation at 37°C for 10 min; and the blue light chemical crosslinking conditions are: 10 mW / cm 2 Blue light chemical crosslinking for 15 seconds. The solvent of the LAP solution in this invention is preferably PBS buffer; the GelMA is preferably added after the LAP solution has cooled; the final concentration of the GelMA is preferably 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, and 14%; the GelMA scaffold is preferably added after sterility testing through a 0.22 μm filter membrane.

[0021] Preferably, the engineered extracellular vesicles containing Apelin and HBD3 described in this invention are obtained by isolation and purification of engineered Wharton's collagen mesenchymal stem cells. As an optional embodiment, the isolation and purification steps include: collecting the culture supernatant when the cell confluence reaches 70%-90%, and separating the cells by centrifugation at 300g for 10 min; retaining the supernatant, and continuing centrifugation at 2000g for 10 min to remove dead cells; subsequently increasing the centrifugation force to 10000g, and centrifuging the mixture again for 30 min to remove cell debris; the treated supernatant is then ultracentrifuged (100000g, 70 min) to obtain crude EVs precipitate; the EVs precipitate is resuspended in PBS and then purified by centrifugation at 100000g for 70 min. All operations are performed at 4°C.

[0022] Preferably, the engineered Wharton's collagen mesenchymal stem cells of the present invention are introduced into the Apelin overexpression vector and the HBD3 overexpression vector via lentiviral transfection.

[0023] The present invention also provides the use of the above-described delivery system in the preparation of drugs that promote endothelial cell proliferation and / or angiogenesis, in the preparation of drugs that are antibacterial and / or reduce inflammatory responses, and in the preparation of drugs that promote the healing of wounds on aging skin.

[0024] The Apelin-HBD3-EVs@GelMA provided by this invention exhibits excellent performance in both in vitro and in vivo experiments, effectively addressing a key challenge in wound healing in the elderly. In vitro analysis shows that this delivery system significantly promotes the proliferation of human umbilical vein endothelial cells (HUVECs), reduces the level of pro-inflammatory cytokines, and stimulates angiogenesis in an aging environment, fully demonstrating its therapeutic potential. By activating the anti-aging-related AMPK / KLF2 signaling pathway and restoring mitochondrial function, the mechanism of action of Apelin has been thoroughly elucidated, indicating that its efficacy extends beyond anti-inflammatory applications. Apelin-HBD3-EVs@GelMA possesses antibacterial efficacy, effectively inhibiting both Gram-positive and Gram-negative bacteria, making it an important solution for wound care in the elderly. This human-derived antimicrobial peptide does not require good kidney or liver function, does not trigger allergic reactions, and does not introduce additional metal ions. In in vivo experiments, Apelin-HBD3-Evs@GelMA hydrogel demonstrated excellent wound healing effects in aged mice with MRSA-infected wounds. It not only accelerated the wound healing speed but also significantly improved the healing quality, fully demonstrating its precise targeted therapeutic effect on wound healing in elderly patients and showcasing its powerful anti-inflammatory properties. Through the combined action of Apelin and HBD3, it balances pro-inflammatory and anti-inflammatory cytokines, regulates neutrophil recruitment and macrophage polarization, and significantly increases the microvascular density in the newly formed skin, demonstrating the potential to promote angiogenesis and showing significant advantages for wound healing in elderly patients.

[0025] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0026] Unless otherwise specified, the following embodiments are all conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] In this specific embodiment of the invention, all experimental procedures were reviewed and approved by the Ethics Committee of the General Hospital of the Chinese People's Liberation Army. Animal experiments strictly followed the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals." Male C57BL / 6J mice aged 18-24 months were used in the experiments and purchased from Beijing SPF Biotechnology Co., Ltd. All mice were housed in a specific pathogen-free animal housing with a constant temperature (23°C) and humidity (60%), maintaining a 12-hour diurnal rhythm. To ensure experimental stability, mice underwent acclimatization feeding for one week prior to the experiment, during which they had free access to food and water. All human samples were obtained from specimens discarded during surgery. All subjects participated voluntarily after fully understanding the purpose of the experiment and signed written informed consent forms.

[0029] In a specific embodiment of this invention, Wharton colloid mesenchymal stem cells (WJ-MSCs) were cultured using a 1:1 mixture of high-glucose Durbeco modified Eagle medium (DMEM) and DMEM / F12 medium (Gibco, USA), supplemented with 10% exosome-free fetal bovine serum (SBI, USA) and 100 U / mL penicillin-streptomycin (Gibco, USA). The WJ-MSCs used in subsequent experiments were passaged 3 to 8 times. Human umbilical vein endothelial cells (HUVECs) and 293T cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin-streptomycin, and 2 mM L-glutamine (Gibco, USA). All cells were cultured in a humid environment at 37°C and 5% CO2.

[0030] In this specific embodiment of the invention, a blinded method was used. Excel and SPSS were used to process the data. The normality of continuous variables was tested using the Kolmogorov-Smirnov test, followed by a homogeneity of variance test. Normally distributed data were expressed as mean ± standard deviation and tested using a two-tailed unpaired Student's t-test or one-way ANOVA, followed by a Bonferroni test for multiple comparisons. A significance level of 0.05 was defined.

[0031] Example 1 A delivery system for Apelin and HBD3 based on hydrogels and engineered extracellular vesicles: 1. Plasmids and lentivirus-based gene transduction: The Apelin gene (Genbank ID NM_017413) was ligated into the pLVX-IRES-Puro plasmid. EcoR I and BamHBetween restriction endonuclease sites, a vector plasmid overexpressing apelin (pLVX-Apelin-IRES-Puro) was obtained. The HBD3 gene (Genbank ID NM_018661) was ligated into the pLVX-IRES-Puro plasmid. EcoR I and BamH Between the I restriction endonuclease sites, a vector plasmid overexpressing HBD3 (pLVX-HBD3-IRES-Puro) was obtained.

[0032] The necessary packaging plasmid (psPAX2) and envelope plasmid (pMD2.G) were co-transfected into human 293T cells with vectors overexpressing Apelin and HBD3. Forty-eight hours after transfection, the virus-containing supernatant was collected and filtered through a 0.45 μm filter to remove cell debris. The obtained lentiviral particles were used to transduce WJ-MSCs, and 8 μg / mL polybrene was added to improve transduction efficiency. Stable transduced cell lines were established after 14 days of selection using 2 μg / mL puromycin, and designated as engineered cells (Apelin-HBD3-WJ-MSCs). The expression levels of Apelin and HBD3 in the engineered cells were verified by Western blot and qRT-PCR.

[0033] 2. Isolation of extracellular vesicles (EVs): Extracellular vesicles were collected from engineered cells (Apelin-HBD3-WJ-MSCs) and cultured in serum-free medium (catalog number NC0106, China UKANG Company). When cell confluence reached 70%-90%, the culture supernatant was collected, and cells were separated by centrifugation at 300g for 10 min. After retaining the supernatant, the mixture was centrifuged again at 2000g for 10 min to remove dead cells. The centrifugation was then increased to 10000g, and the mixture was centrifuged again for 30 min to remove cell debris. The treated supernatant was then ultracentrifuged at 100000g for 70 min to obtain a crude EV pellet. The EV pellet was resuspended in PBS and centrifuged again at 100000g for 70 min to obtain purified EVs (Apelin-HBD3-EVs). All operations were performed at 4°C.

[0034] 3. Preparation of gel-encapsulated engineered EVs (Apelin-HBD3-EVs): Lithylphosphonate (LAP) was dissolved in PBS buffer to a final concentration of 0.25% (w / v), and then heated at 55°C for 15 min. After the LAP cooled, equal masses of GelMA were added to the solution at ratios of 5%, 10%, and 15% (w / v) to prepare hydrogels. The hydrogel solutions were then subjected to sterility testing by passing them through a 0.22 μm filter. EVs were then processed at a concentration of 4 × 10⁻⁶. 10 A final concentration of [number] cells / mL was added to the GelMA scaffold and incubated at 37°C for 10 min. [The remaining text appears to be incomplete and requires further context.] 2 After chemical cross-linking with blue light (405nm) for 15 seconds, the hydrogel was immersed in PBS or culture medium and placed in a 12-well plate for in vitro experiments.

[0035] Example 2 1. Plasmids and lentivirus-based gene transduction: The Apelin gene (Genbank ID NM_017413) was ligated into the pLVX-IRES-Puro plasmid. EcoR I and BamH Between restriction endonuclease sites, a vector plasmid overexpressing apelin (pLVX-Apelin-IRES-Puro) was obtained. The HBD3 gene (Genbank ID NM_018661) was ligated into the pLVX-IRES-Puro plasmid. EcoR I and BamH Between the I restriction endonuclease sites, a vector plasmid overexpressing HBD3 (pLVX-HBD3-IRES-Puro) was obtained.

[0036] The necessary packaging plasmid (psPAX2) and envelope plasmid (pMD2.G) were co-transfected into human 293T cells with vectors overexpressing Apelin and HBD3. Forty-eight hours after transfection, the virus-containing supernatant was collected and filtered through a 0.45 μm filter to remove cell debris. The obtained lentiviral particles were used to transduce WJ-MSCs, and 8 μg / mL polybrene was added to improve transduction efficiency. Stable transduced cell lines were established after 14 days of selection using 2 μg / mL puromycin, and designated as engineered cells (Apelin-HBD3-WJ-MSCs). The expression levels of Apelin and HBD3 in the engineered cells were verified by Western blot and qRT-PCR.

[0037] 2. Establish the HUVEC aging model:

[0038] To avoid replication-induced senescent cells, proliferative HUVECs with a low passage count (<6 times) were selected as a normal control. For the replication-induced senescence model, cells were passaged until senescence occurred. After approximately 18 passages or 24 population doublings, HUVECs reached the senescent state, which was verified by microscopic morphological observation and RT-qPCR detection of senescence biomarkers.

[0039] 3. Isolation and identification of extracellular vesicles (EVs): Extracellular vesicles were collected from engineered cells (Apelin-HBD3-WJ-MSCs) and cultured in serum-free medium (catalog number NC0106, China UKANG Company). When cell confluence reached 70%-90%, the culture supernatant was collected, and cells were separated by centrifugation at 300g for 10 min. After retaining the supernatant, the mixture was centrifuged again at 2000g for 10 min to remove dead cells. The centrifugation was then increased to 10000g, and the mixture was centrifuged again for 30 min to remove cell debris. The treated supernatant was then ultracentrifuged at 100000g for 70 min to obtain a crude EV pellet. The EV pellet was resuspended in PBS and centrifuged again at 100000g for 70 min to obtain purified EVs (Apelin-HBD3-EVs). All operations were performed at 4°C.

[0040] The ultrastructure and size distribution of extracted EVs were observed using a through-electron microscope (TEM, model JEM-1011, NEC Corporation), and nanoparticle tracking analysis was performed using a ZetaView nanoparticle analysis system (Particle Metrix GmbH, Germany). Western blotting was used to detect the expression levels of EV characteristic markers CD63, CD81, TSG101, and cadherin.

[0041] 4. RT-qPCR: Total RNA was extracted from engineered cells (Apelin-HBD3-WJ-MSCs). The extracted total RNA was reverse transcribed into complementary DNA using FastKing gDNA DispellingRT SuperMix (catalog number KR118, Tiangen Biotech). Quantification was then performed using SuperReal premixed SYBR Green fluorescent dye (catalog number FP215, Tiangen Biotech). Each sample was analyzed three times in duplicate on a CFXConnect real-time quantitative PCR system (Bio-Rad Laboratories, USA). -ΔΔct The relative expression levels of genes were assessed using a method. Primer sequences used for amplification are detailed in Table 1. In vitro experiments were conducted in groups N=3.

[0042] Table 1 Primer sequences

[0043] 5. Western blotting: Proteins were extracted using RIPA lysis buffer (R0010, Solarbio, China) containing 1% protease inhibitor (A8260, Solarbio, China) and a pre-chilled tissue homogenizer. After lysis on ice for 10 min, cell debris was removed by centrifugation at 14000g for 15 min, and the supernatant was transferred to new centrifuge tubes. Protein concentration was determined using a BCA protein assay kit (PC0020, Solarbio, China). The lysis buffer was boiled with 5×SDS loading buffer (P1040, Solarbio, China) for 5 min. Subsequently, 40 μg of protein samples were subjected to 8% and 12% Hepes-Tris precast gel electrophoresis (PG01010, PG01210, Solarbio, China) and transferred to polyvinylidene fluoride (PVDF) membranes (88518, Thermo Scientific, USA). After blocking with Tris buffer (TBST, pH 7.6) containing 3% skim milk for 1 h, the membrane was incubated overnight at 4°C with the corresponding primary antibody. After washing three times with TBST, it was incubated at room temperature with an appropriate amount of horseradish peroxidase (HRP)-labeled secondary antibody for 1.5 h. Protein bands were detected using a Bio-Rad ChemiDoc XRS gel imaging system (Bio-Rad, USA), and quantification was performed using Image Lab software (Bio-Rad, USA). Housekeeping protein levels were used as an internal control for standardization. In vitro experiments: N=3.

[0044] 6. Enzyme-linked immunosorbent assay (ELISA): Competitive enzyme-linked immunosorbent assay (ELISA) was used to detect apelin, HBD3, KLF2, and inflammation-related factors, strictly following the manufacturer's operating procedures. Samples were homogenized in a pre-chilled tissue homogenizer and then added to diluted microplates coated with the corresponding antigens (Sangon Biotech Co., Ltd., China) along with standard solutions. After incubation with biotin-labeled antibodies, HRP-labeled streptavidin was added to form immune complexes. Unbound enzyme was removed by washing, followed by the addition of the chromogenic substrate TMB. The absorbance was measured at 450 nm, and the concentrations were calculated based on a standard curve. The in vivo sample size was N=6, and the in vitro sample size was N=3.

[0045] 7. Preparation of encapsulated engineered EVs (Apelin-HBD3-EVs): Lithylphosphonate (LAP) was dissolved in PBS buffer to a final concentration of 0.25% (w / v), and then heated at 55°C for 15 min. After the LAP cooled, equal masses of GelMA were added to the solution at ratios of 5%, 10%, and 15% (w / v) to prepare hydrogels. The hydrogel solutions were then subjected to sterility testing by passing them through a 0.22 μm filter. EVs were then processed at a concentration of 4 × 10⁻⁶. 10 A final concentration of [number] cells / mL was added to the GelMA scaffold and incubated at 37°C for 10 min. [The remaining text appears to be incomplete and requires further context.] 2 After chemical cross-linking with blue light (405nm) for 15 seconds, the hydrogel was immersed in PBS or culture medium and placed in a 12-well plate for in vitro experiments.

[0046] The control group GelMA hydrogel was prepared under the same conditions, but with the addition of only PBS buffer and without EVs.

[0047] 8. Characterization of methylcellulose hydrogel (GelMA) scaffold: To characterize the morphology and chemical structure of the GelMA scaffold and its encapsulated extracellular vesicles (EVs), samples were fixed in 2.5% glutaraldehyde before analysis. The samples were placed on conductive tape fixed to the scanning electron microscope (SEM) stage and then subjected to a 120-second platinum coating process using a Leica EM ACE600 SEM. The structure of the GelMA scaffold and its encapsulated EVs was observed using a Hitachi S-4800 SEM at 10 kV. Simultaneously, the distribution characteristics of the EVs within the GelMA scaffold were imaged and analyzed using a Leica SP8 confocal microscope.

[0048] 9. Mechanical properties and viscosity analysis: Tensile tests were conducted using a general mechanical testing machine (MTS, USA). Cross-linked GelMA hydrogel samples were prepared into cylinders 30 mm in length and 10 mm in width and stretched horizontally at a rate of 2 mm / min. Stress-strain curves were continuously recorded until the specimen fractured. Rheological properties were tested using a rheometer (Anton Paar, Germany). Viscosity changes of the GelMA hydrogel samples were measured at a shear rate of 10 τs / s under a constant temperature of 25 °C, with viscosity values ​​recorded every 20 seconds for 20 min. Furthermore, under fixed strain and frequency conditions, the modulus of the hydrogel samples was examined as a function of temperature and time. The storage modulus (G') and loss modulus (G'') were measured and recorded.

[0049] 10. Release and Degradation Assays: In the release assay, the hydrogel was immersed in PBS solution in a 12-well plate, and the supernatant was collected for 15 consecutive days. The EV content in the supernatant was detected by an ELISA kit, and the quantification was based on the released CD63 level. In the degradation assay, the cross-linked GelMA hydrogel sample was immersed in PBS solution at 37°C. After removing moisture by vacuum drying at 50°C, it was weighed at predetermined time intervals. The degradation rate was calculated using the formula: Degradation rate = (Current weight - Initial weight) / Original weight × 100%.

[0050] 11. Biosafety evaluation of stents: To assess the in vitro cytotoxicity of the scaffold, human umbilical vein endothelial cells (HUVECs), epidermal stem cells (EpiSCs), and fibroblasts (FBs) were cultured. In the experiment, a 10% concentration of methylcellulose hydrogel (GelMA) scaffold containing EVs was added to the cell culture medium, and the cells were cultured for 48 hours. The control group received only PBS buffer. Cell viability changes were assessed using the Cell Counting Kit-8 (CCK-8): using a Thermo Fisher Scientific Synergy2 microplate reader, absorbance was measured at 450 nm 2 hours after supplementation with CCK-8 reagent (catalog number HY-K0301). Three replicates were set for each group, resulting in three experimental groups.

[0051] In vivo biosafety testing was conducted using aged mice as experimental subjects. A model was established by intraperitoneal injection of 100 μL of the immersion solution every two days for six consecutive weeks. The control group underwent the same method but used PBS as the solvent. The biotoxicity of Apelin-HBD3-EVs@GelMA was analyzed by collecting organ and blood samples from mice; six samples were collected from each group.

[0052] 12. Catheterization test: Human umbilical vein endothelial cells (10 per well) 4 (Numbers) were inoculated into matrix gel-coated 96-well plates and treated with different solutions for 10 h. Images of the tubular structures were taken using a microscope, and the branch lengths were calculated using ImageJ software.

[0053] 13. Mitochondrial detection: To assess changes in mitochondrial membrane potential (MMP), incubated human umbilical vein endothelial cells (HUVECs) were stained with TMRM dye (10 μM, catalog number HY-D0984, MCE Biosciences, USA) at 37°C for 15 min. Cells were then washed three times with PBS buffer to remove excess dye, and immediately subjected to flow cytometry for analysis. Mean fluorescence intensity was calculated to reflect MMP levels. Each experiment was repeated three times (N=3).

[0054] 14. In vitro antibacterial properties: Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus ( S.aureus The bacteria were cultured in tryptone soybean broth (TSB) medium. *E. coli* was cultured in LB medium. After removing the culture medium from the freshly amplified bacterial solution by centrifugation (3000 rpm, 5 min), the bacteria were resuspended in PBS to prepare a bacterial suspension. The absorbance (OD) of 300 μL of the bacterial suspension at 600 nm was measured using a turbidimeter. 600 For OD 600 The bacterial suspension at a concentration of 0.5 μL was quantified using the colony forming unit (CFU) method: MRSA and Staphylococcus aureus were calculated at 4.0 × 10^8 CFU / mL, and Escherichia coli at 2.5 × 10^8 CFU / mL. To evaluate the antibacterial effect of the GelMA scaffold, 100 μL of GelMA scaffold with or without extracellular vesicles (EVs) was added to a 300 μL bacterial suspension (OD) containing 0.5 μL of bacterial suspension. 600 The bacteria were cultured in 48-well plates with a concentration of 0.5 μL (0.5 μL / min). After incubation at 37°C and 220 rpm for 24 h with shaking, the remaining bacteria were quantified using the CFU method. A PBS control group was included as a reference.

[0055] 15. Animal models: Healthy aged male C57BL / 6J mice were randomly assigned to four groups: Ctrl group (treated with PBS), GelMA group (treated with GelMA hydrogel), Vector-EVs@GelMA group (empty vector control treatment, the only difference from Apelin-HBD3-EVs@GelMA group is the absence of the target functional molecules Apelin and HBD3), and Apelin-HBD3-EVs@GelMA group.

[0056] Mice were anesthetized with isoflurane, and a full-thickness skin defect with a diameter of 8 mm was created on their backs. MRSA bacteria (5 × 10⁻⁶) were then introduced. 8CFU / mL (20 μL) was inoculated into the wound. Immediately after model establishment, the wound was treated to prevent contracture, and a 10 mm diameter white perforated sheet was used as a reference. The residual wound area was measured at specified time points. One day after bacterial inoculation, wound pus was cultured in TSA medium to confirm MRSA infection. For mice with successfully established infection models, 100 μL of 10% hydrogel was injected into each wound, followed by cross-linking treatment with blue light (405 nm) for 15 s. The Ctrl group used 100 μL of PBS solution. On days 3, 7, and 10 after modeling, 6 mice from each group were randomly selected and sacrificed to assess the expression levels of Apelin, HBD3, and KLF2 in the 6 wounds. By day 10, all surviving mice were sacrificed, and tissue samples were collected to assess healing quality. Quantitative analysis was performed on photographs of 4 randomly selected mice (8 wounds in total) using ImageJ software. The healing rate was calculated as (1 - residual wound area / total wound area) × 100%. All measurements were performed blinded.

[0057] Surviving bacteria in the wound: The survival of MRSA in the post-implantation wound and MRSA after different treatments were evaluated on days 3, 7, and 10. Samples of wound and surrounding subcutaneous tissue were collected, homogenized with PBS, diluted, and then spread on TSA plates to determine bacterial load.

[0058] HE staining and MASSON staining: On day 10 of the experiment, three mice from each group were randomly selected and euthanized, and six tissue samples were collected from the area surrounding the wound for wound condition monitoring. After collection, the tissue samples were soaked overnight in 4% paraformaldehyde solution (4°C). After dehydration and embedding, sections were prepared, followed by dewaxing and hydration treatments, and then stained using an HE staining kit (catalog number G1120, CyberBio) and a MASSON trichrome staining kit (catalog number G1340, CyberBio). Six samples were used in each experimental group.

[0059] Immunofluorescence staining: Skin tissue samples were cut into thin sections. For antigen retrieval, the sections were placed at 37°C and incubated for 30 min with 20 μg / mL DNase-free proteinase K (product number P1120, Sartorius, China). Subsequently, they were treated with PBS containing 0.1% Triton X-100 (product number HFH10, Ingenic Semiconductor, USA) for 30 min. After blocking with 4% goat serum (product number SL038, Sartorius, China) for 30 min, the sections were incubated overnight at 4°C with the corresponding primary antibody. After incubation, the sections were washed three times with PBS (5 min each time) and then incubated with Alexa Fluor-labeled secondary antibody at room temperature for 1 h. After washing with PBS, the sections were stained with anti-quenching mounting medium containing 4,6-diamino-2-phenylindole (DAPI, product number P36962, Ingenic Semiconductor, USA). Images were acquired using a Caseviewer microscopic imaging system (3DHISTECH, Hungary). The average fluorescence intensity of DAPI was calculated using ImageJ software and used as a reference value for the relative expression level of the protein. The sample size for each experiment was N=6.

[0060] 16. Experimental Results and Statistics (1) Characterization of engineered cells (Apelin-HBD3-WJ-MSCs) and engineered EVs (Apelin-HBD3-EVs): See [link to EV engineering design and genetic modification validation] Figure 1 In the figure, A. is a schematic diagram illustrating the gene editing strategy used in the EV project; B. is an RT-PCR verification of gene modification in WJ-MSCs overexpressing Apelin and HBD3, with a sample size of N = 3. C, D, and E. The effect of apelin overexpression in engineered cells and engineered EVs was verified by Western blotting at the protein level, with a sample size of N = 3. F, G. The expression level of HBD3 in engineered cells and engineered EVs was quantitatively analyzed by ELISA, with a sample size of N = 3. H. Western blot analysis confirmed the expression of specific markers in engineered cells and engineered EVs, N = 3; I. Morphological characteristics of engineered EVs were assessed by transmission electron microscopy (TEM) and particle size distribution analysis, with a sample size of N = 3.

[0061] RT-PCR analysis confirmed that the mRNA expression levels of Apelin and HBD3 in Apelin-HBD3-WJ-MSCs were significantly increased by 6.24-fold and 13.25-fold, respectively. Figure 1 B in the sample, P < 0.01). Western blotting was used (…). Figure 1 C, D, E) and ELISA method ( Figure 1 Protein levels in stably transduced Apelin-HBD3-WJ-MSCs and their extracted engineered EVs were measured using F and G in the assay. Apelin-HBD3-WJ-MSCs showed a 3.56-fold increase in total Apelin concentration and a 5.26-fold increase in total HBD3 concentration (P<0.01). In contrast, Apelin-HBD3-EVs showed a 1.96-fold increase in Apelin concentration and a 2.77-fold increase in HBD3 concentration (P<0.01). This indicates that the engineered EVs exhibit strong payload encapsulation capabilities.

[0062] Western blot, transmission electron microscopy (TEM), and dynamic light scattering (DLS) analyses showed that the characteristics of Apelin-HBD3-EVs were essentially consistent with those of vector EVs. Figure 1 (H, I in the text). Western blot analysis showed that EV markers such as CD63, CD81, and TSG101 were present in both vector EVs and Apelin-HBD3-EVs. Calnexin, an endoplasmic reticulum marker and indicator of cellular contamination, was only detected in WJ-MSCs, with extremely low expression levels in both types of EVs. Both vector EVs and Apelin-HBD3-EVs exhibited a cup-shaped or circular structure with diameters of approximately 148.95 nm and 149.1 nm, respectively. Additional gene modifications did not significantly alter the diameter or morphological characteristics of the EVs.

[0063] (2) Characterization of GelMA and GelMA carrying engineered EVs (Apelin-HBD3-EVs@GelMA) Loading strategies for engineered EVs and characterization of GelMA brackets, as follows Figure 2 Figure A shows a schematic diagram of the engineered EVs strategy loaded onto a GelMA scaffold. Figure B shows a schematic diagram of the photocrosslinking properties of a GelMA scaffold loaded with engineered extracellular vesicles; the three-dimensional fluorescence image on the right shows the distribution of engineered extracellular vesicles within the GelMA scaffold. Figures C and D show scanning electron microscope images of GelMA loaded with engineered EVs at different concentrations, with the average pore size labeled. Scale bar = 100 μm, N = 6. E. Strain curves of Apelin-HBD3-EVs@GelMA at different concentrations. F. Viscosity curves of Apelin-HBD3-EVs@GelMA at different concentrations. G. Release rate of Apelin-HBD3-EVs@GelMA at different concentrations. H. Degradation rate of Apelin-HBD3-EVs@GelMA at different concentrations.

[0064] Hydrogels, as drug delivery carriers, significantly prolong drug retention in wounds by regulating drug release time. Hydrogels exhibit excellent adaptability to wounds of different shapes and effectively maintain a moist wound environment, thereby promoting the healing process. Figure 2 Figure A shows a schematic diagram of the structure of the Apelin-HBD3-EVs@GelMA formulation. This hydrogel is lightweight, and the extracellular vesicles (EVs) exhibit a three-dimensional uniform dispersion. Figure 2 (B in the text). Scanning electron microscopy (SEM) analysis showed that the Apelin-HBD3-EVs@GelMA hydrogel possesses a uniform, continuous, and interconnected porous three-dimensional network structure. At concentrations of 5%, 10%, and 15%, the pore sizes were 162.30 nm, 108.73 nm, and 67.72 nm, respectively. Figure 2 (C~D in the formula, P<0.01). Elongation at break tests showed that the 5%, 10%, and 15% concentration groups had elongation at break of 112.11%, 75.77%, and 102.93%, respectively, corresponding to tensile strengths of 8.47 kPa, 8.86 kPa, and 4.22 kPa. Figure 2 The field of rheology mainly studies the deformation and flow properties of materials. Self-healing hydrogels, due to their excellent properties, have become an ideal choice for treating uneven skin wounds. Rotational shear experiments were conducted on cross-linked Apelin-HBD3-EVs@GelMA materials at a shear rate of 10τ / s. Figure 2 (F in the text). Viscosity-time curves showed that the viscosity of the 10% and 5% hydrogels initially decreased and then stabilized rapidly upon application of shear force; while the viscosity of the 15% hydrogel increased initially and then stabilized. The 10% hydrogel exhibited the highest viscosity value, which is attributed to its optimal viscoelastic properties that meet the requirements of wound dressing formulations, making it a more ideal adjunct material for wound treatment applications. The synthesized Apelin-HBD3-EVs@GelMA hydrogel was immersed in phosphate-buffered saline (PBS), and the released extracellular vesicles (EVs) were quantitatively analyzed at specific time points. Figure 2 (G in the text). All three concentrations of Apelin-HBD3-EVs@GelMA hydrogels exhibited stable and sustained release rates in the early stages. The maximum release of EVs occurred between days 5 and 6, and this release state lasted for approximately 3 days. The 5% and 10% concentrations of hydrogels reached 80% release on days 7 and 9, respectively, while the 15% concentration of hydrogel only achieved 80% EV release on day 13. Figure 2As shown in H, the degradation rate of the hydrogel was assessed every two days after immersion in the culture medium. The results showed that the degradation rates of the 10% and 15% concentrations of Apelin-HBD3-EVs@GelMA hydrogel were significantly slower than those of the 10% concentration group. Based on the above data, the 10% concentration hydrogel formulation was determined to be the optimal solution.

[0065] (3) The modulus characteristics of the hydrogel after formation with temperature and time were investigated by analyzing the changes in storage modulus (G′) and loss modulus (G′′) to confirm its self-assembly stability. The cross-linked hydrogel remained solid in the temperature range of 4 to 40 °C and did not deform at 1% strain and 5 Hz frequency. For the 10% concentration Apelin-HBD3-EVs@GelMA hydrogel, when the temperature increased from 4 °C to 40 °C, G′ decreased from 6073.3 Pa to 5585.4 Pa, while G′′ increased from 31.037 Pa to 49.98 Pa. When the temperature decreased from 40 °C to 4 °C, G′ increased from 5405.27 Pa to 5949.1 Pa, while G′′ decreased from 41.48 Pa to 30.50 Pa. Time-dependent modulus analysis showed that the hydrogels of the 5% and 10% concentration groups exhibited better stability than the 15% concentration group.

[0066] All these combined data indicate that 10% Apelin-HBD3-EVs@GelMA hydrogel is an ideal candidate material for wound healing applications. The biocompatibility of the EV-loaded GelMA hydrogel was subsequently validated through in vitro cytotoxicity assessment, focusing on its compatibility with epidermal stem cells (EpiSCs), fibroblasts (FBs), and human umbilical vein endothelial cells (HUVECs). Furthermore, the researchers intravenously injected the engineered EV-loaded hydrogel into C57BL / 6J mice to evaluate its biocompatibility. After 6 weeks of intravenous administration, no significant abnormalities were observed in organ tissue sections of the 10% Apelin-HBD3-EVs@GelMA treatment group. Simultaneously, liver and kidney blood biochemical indicators and renal function test results were all within the normal reference range.

[0067] (4) Effects of Apelin-HBD3-EVs@GelMA on aging HUVECs The effects of Apelin-HBD3-EVs@GelMA on aging HUVECs are discussed in [link to relevant documentation]. Figure 3 The figure shows that the Apelin-HBD3-EVs@GelMA scaffold was evaluated using the CCK-8 assay to assess the proliferative capacity of aged human umbilical vein endothelial cells (HUVECs). The sample size N = 3, and statistical significance was marked as... B, C, and D. The effects of the Apelin-HBD3-EVs@GelMA scaffold on the release of inflammatory factors from aged human umbilical vein endothelial cells (HUVECs) were evaluated using ELISA. The experimental sample size was N = 3. E. The effect of the Apelin-HBD3-EVs@GelMA scaffold on the angiogenesis capacity of aged human umbilical vein endothelial cells (HUVECs) was evaluated using a tubular structure formation assay, and the relative total branch length was calculated. Sample size N = 3. , F. Western blotting was used to detect the effect of Apelin-HBD3-EVs@GelMA scaffolds on the AMPK / KLF2 signaling pathway in aged human umbilical vein endothelial cells (HUVECs). Three replicates were used in the experiment, and statistical significance was indicated by: .05, G. The effect of Apelin-HBD3-EVs@GelMA scaffold on mitochondrial membrane potential in aged human umbilical vein endothelial cells was detected by flow cytometry by measuring TMRM fluorescence intensity. N = 3. .

[0068] Immune dysregulation (i.e., persistent and prolonged inflammation) may lead to inflammatory aging, which has a significant impact on delayed wound healing. A persistent inflammatory environment and altered immune cell activity may disrupt angiogenesis, thereby hindering complete wound healing. Using young HUVECs as a control group, this study verified the effect of Apelin-HBD3-EVs@GelMA on aged human umbilical vein endothelial cells (HUVECs). The results showed that, as detected by CCK-8 assay (…),… Figure 3 In study A), it was found that the proliferation rate of senescent cells was significantly lower than that of young cells (P<0.01). The use of Vector-EVs@GelMA only slightly alleviated this phenomenon (P>0.05), while Apelin-HBD3-EVs@GelMA significantly enhanced the ability to promote cell proliferation (P<0.01). Furthermore, inflammatory marker analysis showed that compared with young cells, the levels of pro-inflammatory cytokines (such as IL-6, IL-1β, and TNF-α) in the supernatant of senescent cells were significantly increased (P<0.01). Figure 3 (B~D in the text). The application of Vector-EVs@GelMA partially reduced cytokine levels (P<0.05). In contrast, Apelin-HBD3-EVs@GelMA significantly reduced inflammatory cytokine levels (P<0.01). Apelin-HBD3-EVs@GelMA was more effective than Vector-EVs@GelMA in promoting vascular development in aged HUVECs. Figure 3The presence of E in this substance may be related to its anti-inflammatory properties. Western blot analysis of the AMPK / KLF2 signaling pathway revealed that, compared to Vector-EVs@GelMA, Apelin-HBD3-EVs@GelMA significantly induced AMPK phosphorylation and increased KLF2 expression (E). Figure 3 From a mitochondrial perspective, the Apelin-HBD3-EVs@GelMA treatment group showed significantly better results in restoring the mitochondrial membrane potential of aging human umbilical vein endothelial cells than the Vector-EVs@GelMA treatment group. Figure 3 (G in the text). In summary, the ability of Apelin-HBD3-EVs@GelMA to inhibit inflammatory responses and promote angiogenesis makes it a promising candidate for promoting wound healing under inflammatory aging conditions. Apelin-HBD3-EV@GelMA promotes angiogenesis through its anti-inflammatory properties.

[0069] (5) Validation of the antibacterial properties of Apelin-HBD3-EVs@GelMA The in vitro antibacterial properties of the Apelin-HBD3-EVs@GelMA scaffold are shown in [link to documentation]. Figure 4 In the figure, A and B show the quantitative analysis of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli by colony counting on agar plates. Sample size N = 3. C, D. Schematic diagram and results of the bacterial inhibition zone test. N = 3. .

[0070] The increased risk of microbial infections in elderly patients may be related to age-related decline in antimicrobial resistance and prolonged exposure of wounds to the external environment. For elderly patients with delayed wound healing, the antibacterial efficacy of wound dressings is crucial. This includes protection against Gram-positive Staphylococcus aureus (S. aureus). S. aureus ), methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Escherichia coli ( E. coli The broad-spectrum antibacterial efficacy of Apelin-HBD3-EVs@GelMA hydrogel was evaluated by culturing various bacterial suspensions, including those containing vector-exosomes@GelMA. Results showed that the bacterial populations in the control group, GelMA group, and Vector-exosomes@GelMA group all exhibited high viability. However, the bacterial populations co-cultured with Apelin-HBD3-EVs@GelMA hydrogel showed a significant decrease in viability, demonstrating a significant antibacterial effect. When the initial concentration was 4.0 × 10^8 CFU / mL (OD200), the antibacterial effect was significantly enhanced. 600At a concentration of 0.5, the hydrogel exhibited a mortality rate of up to 90.7% against Staphylococcus aureus and 90.2% against methicillin-resistant Staphylococcus aureus (MRSA). Against Escherichia coli, Apelin-HBD3-EVs@GelMA demonstrated a bacterial survival inhibition rate of 92.5%. Figure 4 (A~B in the diagram). Further inhibition zone experiments ( Figure 4 The results from C~D also confirmed the excellent antibacterial properties of Apelin-HBD3-EVs@GelMA, indicating its broad application prospects in inhibiting microbial proliferation during wound healing in elderly patients.

[0071] (6) Apelin-HBD3-EVs@GelMA accelerated the healing of MRSA-infected skin wounds in aged mice. The healing effect of Apelin-HBD3-EVs@GelMA on MRSA-infected skin wounds in aged mice is shown in the figure. Figure 5 In the diagram, A is a schematic diagram of the animal experiment process; B is a representative macroscopic schematic diagram of wound healing over a specified number of days; C is a schematic photograph of the wound area within 10 days, N=6; and D is a quantitative analysis of the wound healing rate. N = 6. E. Quantitative analysis of bacterial colony density in wound edge specimens. N = 6. F, G, and H. The concentrations of Apelin, HBD3, and KLF2 in wound edge samples were quantitatively detected using ELISA. Sample size N = 6. , .

[0072] More than half of wound infections are caused by Staphylococcus aureus, which can range from asymptomatic to severe and even fatal. Methicillin-resistant Staphylococcus aureus (MRSA), due to its antibiotic resistance, can cause persistent infection and lead to wounds that are difficult to heal. Micrographs were taken on days 3, 7, and 10 post-injury. The group treated with Apelin-HBD3-EVs@GelMA showed a significant improvement in wound healing, with a wound closure rate of 50.36% on day 3. Figure 5(B~D in the original text). In comparison, the control group had a wound closure rate of 12.71%, the GelMA group had a wound closure rate of 36.57%, and the Vector-EVs@GelMA group had a wound closure rate of 53.79%. As time progressed, the promoting effect of Apelin-HBD3-EVs@GelMA became increasingly apparent, reaching 87.43% by day 7. During the same period, the control group, GelMA group, and Vector-EVs@GelMA group reached 47.12%, 64.99%, and 78.33%, respectively. By day 10, the wound closure rate of the Apelin-HBD3-EVs@GelMA group reached as high as 99.67%, a stark contrast to the 72.52% of the control group, the 87.48% of the GelMA group, and the 90.52% of the Vector-EVs@GelMA group. This demonstrates the significant effect of Apelin-HBD3-EVs@GelMA in promoting wound healing, especially on days 7 and 10.

[0073] Apelin-HBD3-EVs@GelMA hydrogel demonstrated significant antibacterial effects during wound healing. By day three, the Apelin-HBD3-EVs@GelMA group showed no residual purulent exudate, a stark contrast to other groups. Figure 5 (B in the text). This difference may stem from the higher number of MRSA infection cases in each group ( Figure 5 (E in the text). By day 7, the bacterial load in the Apelin-HBD3-EVs@GelMA group decreased by approximately 41.49% compared to day 3, while the bacterial counts in the other three groups remained relatively stable. By day 10, the Apelin-HBD3-EVs@GelMA group had the fewest residual colonies, while the other three groups still had large bacterial communities. These results fully demonstrate that the Apelin-HBD3-EVs@GelMA hydrogel has a strong bactericidal ability in the in vivo environment and can effectively inhibit the proliferation of MRSA.

[0074] Wound edge samples were collected at different time points during the healing process, and the sustained release characteristics of the hydrogel were analyzed by ELISA. The results showed that the Apelin-HBD3-EVs@GelMA hydrogel group exhibited significantly increased Apelin concentrations at all stages of wound healing. Figure 5 The presence of F in the figure fully demonstrates its sustained release capability. HBD3 protein concentrations increased on days 3 and 7 after treatment with this hydrogel, but no statistically significant difference was observed by day 10. Figure 5(G in the text). Compared to Apelin's 9 kDa molecular weight, HBD3 is only 4.9 kDa, which may explain its faster release rate. Furthermore, KLF2 level detection showed that its expression level increased at all time points during the healing process, perfectly matching the increasing trend of Apelin concentration. Figure 5 (H in the text).

[0075] The results confirmed that Apelin-HBD3-EV S@Gel MA hydrogel accelerated the healing of MRSA-infected wounds in aged mice.

[0076] (7) Effects of Apelin-HBD3-EVs@GelMA scaffold on healing quality and inflammatory response The impact of Apelin-HBD3-EVs@GelMA scaffolds on healing quality and inflammatory response is discussed in [link to relevant documentation]. Figure 6 In the figure, A. HE staining and MASSON trichrome staining of each group on day 10. The boxed area next to each photograph shows a magnified version of the black box. B. Calculated neoepithelial thickness on day 10 to reflect the quality of neoepithelial tissue. N = 6. C. Percentage of collagen content in the dermis. N = 6. D. Immunofluorescence staining of CD34 and PCNA on day 10. N = 6. E, F, G, H, I, J. Tissue samples were taken from the wound edge, and the changes in the levels of IL-1β, IL-6, TNF-α, CXCL-1, IL-4, and IL-10 were analyzed at specified time points using ELISA. Sample size N = 6. , .

[0077] HE and MASSON staining were performed on day 10 to more comprehensively evaluate the therapeutic effect of Apelin-HBD3-EVs@GelMA hydrogel. Magnified observation of the skin tissue at the wound edge provided a clearer view of tissue morphology. Overall, the Apelin-HBD3-EVs@GelMA group showed a lower number of infiltrative inflammatory cells. Figure 6 (A) and the epidermal structure is intact, with significant thickening of the neoepithelial layer ( Figure 6 Group B in this group has a structure that is closer to that of a healthy epidermis than the other three groups. MASSON staining showed that the collagen fibers in this group exhibited higher orderliness and maturity. Figure 6 (A in the text). Meanwhile, dermal collagen content was significantly increased in the Apelin-HBD3-EVs@GelMA group ( Figure 6 C in the text). CD34 immunofluorescence staining confirmed that the number of vascular endothelial cells in the dermis increased after Apelin-HBD3-EVs@GelMA treatment. Figure 6The D-staining pattern indicates enhanced dermal vascularization. Furthermore, PCNA staining revealed an increased number of proliferating cells, particularly in the epidermal basal layer, further validating the proliferative effect of engineered EVs on EpiSCs.

[0078] In the wound healing process of elderly individuals, especially in the presence of infection, dysregulated inflammatory responses are often associated with persistent non-healing wounds. IL-1β, IL-6, and TNF-α are common pro-inflammatory factors. Cytokine-induced neutrophil chemokines belong to the CXC chemokine family, among which CXCL-1 is involved in neutrophil migration and activation, and its persistent accumulation in delayed-healing wounds can hinder the healing process. From day 3 to day 10, IL-1β at the wound edge in the EVs@GelMA group and the Apelin-HBD3-EVs@GelMA group ( Figure 6 E in IL-6 Figure 6 F in TNF-α Figure 6 G) and CXCL-1 ( Figure 6 The levels of IL-4 and IL-10 decreased. The Apelin-HBD3-EVs@GelMA group showed a significant downregulation trend compared to other groups, indicating that the hydrogel alleviated the inflammatory response. Conversely, IL-4 and IL-10 are regenerative cytokines that promote tissue repair, wound healing, and macrophage M2 polarization. The Apelin-HBD3-EVs@GelMA group showed the highest IL-4 levels. Figure 6 I) and IL-10 ( Figure 6 The results also confirmed that the Apelin-HBD3-EVs@GelMA group was the most effective in reducing inflammation, demonstrating that Apelin-HBD3-EVs@GelMA hydrogel improves healing quality and reduces inflammatory response.

[0079] (8) Effects of Apelin-HBD3-EVs@GelMA on neutrophil infiltration and macrophage polarization The effects of Apelin-HBD3-EVs@GelMA on neutrophil infiltration and macrophage polarization are shown in [reference needed]. Figure 7 Immunofluorescence staining was performed on day 10 post-injury to label MPO, CD80, and CD206, reflecting neutrophil infiltration and macrophage polarization. N = 6.

[0080] Following skin injury, neutrophils rapidly aggregate at the wound site, which is crucial for mitigating the risk of infection caused by a compromised skin barrier. However, in older individuals, chronic inflammation leads to neutrophil dysfunction, causing them to persistently migrate towards the wound margin and be activated via CXCL-1. Myeloperoxidase (MPO), a reliable biomarker of neutrophil activation, can be used to assess the extent of neutrophil recruitment and infiltration at the site of infection. Immunofluorescence staining results showed that the Apelin-HBD3-EVs@GelMA group had decreased neutrophil activation levels on day 10, consistent with the decreasing trend of CXCL-1 and pro-inflammatory cytokine levels.

[0081] Besides neutrophils, macrophages, as key immune cells, participate in inflammatory responses by mediating phagocytosis to clear pathogens and dead cells. However, in aged epidermis, macrophage dysfunction exacerbates inflammation and hinders wound healing. M1 macrophages secrete pro-inflammatory cytokines that are detrimental to wound healing, while M2 macrophages possess anti-inflammatory properties, promoting repair processes such as wound healing and tissue repair. Macrophage polarization from M1 to M2 helps accelerate the healing process. Immunohistochemical results of CD80 in M1 macrophages and CD206 in M2 macrophages were obtained 10 days after wound treatment in aged mice. The Apelin-HBD3-EVs@GelMA group had fewer CD80+ cells but more CD206+ cells. Furthermore, the frequency of CD80+ and CD206+ positive cells (total macrophages) was low in the Ctrl and GelMA groups, indicating that immunosuppression prevented sufficient macrophage accumulation. In conclusion, the Apelin-HBD3-EVs@GelMA group more effectively promoted M2 polarization in MRSA-infected wounds, which has a significant advantage for wound healing in elderly patients.

[0082] The above results prove that: This invention utilizes WJ-MSCs combined with gene editing technology to successfully prepare engineered EVs carrying apelin and HBD3 proteins. Experimental data show that gene modification did not significantly alter the appearance or size of the EVs, allowing them to retain the inherent characteristics of EVs while fully leveraging the advantages of the exogenous genes.

[0083] A 5% concentration hydrogel has a larger pore size and a faster release rate, which may be more suitable for the healing process of acute wounds; while a 15% concentration hydrogel, with its smaller pore size, not only has a longer-lasting sustained release rate but also improves the rigidity of the material. This characteristic is particularly beneficial for treating pressure ulcers in long-term pressure areas such as the sacrum and coccyx of the elderly, as its structural integrity can provide better protection for the wound by enhancing support. In this invention, considering that the wound in the mouse model is located on the back, a 10% concentration hydrogel was chosen to balance the release rate, viscosity, and rigidity, while improving adhesion and therapeutic effect at the wound site. When selecting other concentrations of hydrogel for different healing scenarios, a comprehensive consideration of the wound's nature and location is necessary.

[0084] In vitro experiments showed that this delivery system significantly promoted the proliferation of human umbilical vein endothelial cells (HUVECs), reduced the levels of pro-inflammatory cytokines, and stimulated angiogenesis in an aging environment, fully demonstrating its therapeutic potential. By activating the anti-aging-related AMPK / KLF2 signaling pathway and restoring mitochondrial function, the mechanism of action of Apelin was thoroughly elucidated, indicating that its efficacy extends beyond anti-inflammatory applications. Apelin-HBD3-EVs@GelMA possesses antibacterial properties, effectively inhibiting both Gram-positive and Gram-negative bacteria, making it an important solution for wound care in the elderly. This human-derived antimicrobial peptide does not require good kidney or liver function, does not trigger allergic reactions, and does not introduce additional metal ions. In in vivo experiments, Apelin-HBD3-Evs@GelMA hydrogel demonstrated excellent wound healing effects in aged mice with MRSA-infected wounds. It not only accelerated the wound healing speed but also significantly improved the healing quality, fully demonstrating its precise targeted therapeutic effect on wound healing in elderly patients and showcasing its powerful anti-inflammatory properties: through the combined action of Apelin and HBD3, it balances pro-inflammatory and anti-inflammatory cytokines, regulates neutrophil recruitment and macrophage polarization, and significantly increases the microvascular density in the new skin, demonstrating its potential to promote angiogenesis.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A delivery system for Apelin and HBD3 based on hydrogels and engineered extracellular vesicles, characterized in that, The delivery system was obtained by encapsulating engineered extracellular vesicles containing Apelin and HBD3 in a gelatin-methacrylamide hydrogel.

2. The delivery system according to claim 1, characterized in that, The engineered extracellular vesicles containing Apelin and HBD3 are secreted by engineered Wharton's collagen mesenchymal stem cells; the engineered Wharton's collagen mesenchymal stem cells contain Apelin overexpression vectors and HBD3 overexpression vectors.

3. A method for preparing the delivery system according to any one of claims 1 to 2, characterized in that, Includes the following steps: A 0.25% LAP solution was prepared and heated; then GelMA was added at a final concentration of 5%–15% to prepare a GelMA scaffold; engineered extracellular vesicles containing Apelin and HBD3 were added at a final concentration of 4 × 10⁻⁶. 10 Add cells / mL to a GelMA scaffold, incubate, and perform blue light chemical crosslinking to obtain the delivery system.

4. The preparation method according to claim 3, characterized in that, The heat treatment conditions are 55°C for 15 minutes.

5. The preparation method according to claim 3, characterized in that, The incubation conditions were 37°C for 10 minutes.

6. The preparation method according to claim 3, characterized in that, The conditions for the blue light chemical crosslinking are 10 mW / cm. 2 Blue light chemical crosslinking for 15 seconds.

7. The preparation method according to claim 3, characterized in that, The engineered extracellular vesicles containing Apelin and HBD3 were isolated and purified from engineered Wharton's collagen mesenchymal stem cells; the engineered Wharton's collagen mesenchymal stem cells were transfected into Apelin overexpression vector and HBD3 overexpression vector via lentiviral transfection.

8. The use of the delivery system according to any one of claims 1 to 2 in the preparation of a medicament that promotes endothelial cell proliferation and / or angiogenesis.

9. The use of the delivery system according to any one of claims 1 to 2 in the preparation of medicaments for antibacterial and / or anti-inflammatory purposes.

10. The use of the delivery system according to any one of claims 1 to 2 in the preparation of a medicament for promoting the healing of wounds on aging skin.

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