An engineered exosome-integrated pH-responsive hydrogel and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a preparation method of a pH-responsive hydrogel based on engineering exosome integration, which comprises the following steps: (1) mixing exosomes with a PEI solution, and then performing room temperature incubation treatment, and obtaining an exosome-PEI compound after the incubation is completed; (2) mixing the exosome-PEI compound with plasmid DNA, and then performing room temperature incubation treatment, and obtaining an exosome-PEI-plasmid DNA compound after the incubation is completed; (3) modifying gamma-PGA by using dopamine to obtain DA modified gamma-PGA; mixing a DA modified gamma-PGA solution with a 3-FPBA solution to obtain a PGA-DA / 3-FPBA solution; (4) adding the exosome-PEI-plasmid DNA compound prepared in the step (2) into the PGA-DA / 3-FPBA solution, mixing uniformly to obtain a mixed solution, and then mixing the mixed solution with a carboxymethyl chitosan solution to prepare a pH-responsive hydrogel through room temperature reaction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pH-responsive hydrogel based on engineered exosome integration, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a global chronic metabolic disease with an increasing prevalence. Chronic, slow-healing wounds caused by diabetes, particularly diabetic foot ulcers (DFU), are among its most serious and common complications. The healing process of these wounds is extremely complex and slow, characterized by persistent inflammation, impaired angiogenesis, decreased cell proliferation and migration, and severe oxidative stress. This complex pathological microenvironment leads to prolonged wound healing, a high risk of infection, and in severe cases, even amputation, causing immense physical and psychological suffering and imposing a heavy medical burden on patients.

[0003] To address this challenge, various wound management strategies have been developed in clinical and research fields. Traditional wound dressings, such as gauze and films, primarily serve as physical barriers and absorb exudate, offering limited functionality and failing to actively intervene in or regulate the complex wound microenvironment, thus having limited effectiveness in promoting substantial healing of diabetic wounds. In recent years, functional hydrogel dressings have attracted considerable attention due to their advantages, including providing a moist healing environment, good biocompatibility, and the ability to load active substances. However, most existing hydrogel dressings still have limitations. For example, some hydrogels loaded with growth factors (vascular endothelial growth factor VEGF, fibroblast growth factor FGF, etc.) can promote angiogenesis or cell proliferation to some extent, but these exogenous protein factors are expensive, unstable, and have short half-lives. Furthermore, they often exhibit uncontrolled "burst release" behavior in hydrogels, making it difficult to achieve precise and long-lasting release that matches different stages of wound healing, resulting in unsatisfactory therapeutic effects.

[0004] To overcome the limitations of single growth factors, cell-based therapies, particularly those utilizing cells and their secretions, have shown great potential. Exosomes, as key mediators of intercellular communication, are the core active components of cellular secretions. As vesicles with diameters ranging from 30 to 150 nanometers, exosomes can carry various bioactive molecules (such as proteins, miRNAs, and mRNAs), playing a crucial role in regulating inflammation, promoting angiogenesis, and tissue regeneration. Compared to directly using cells, exosomes, as a "cell-free" therapy, offer advantages such as low immunogenicity, good stability, and ease of storage and application.

[0005] Combining exosomes with hydrogels to construct a delivery system capable of in-situ, sustained release of therapeutic exosomes is currently a research hotspot. However, existing composite hydrogel systems still face several technical challenges. Firstly, the release mechanism lacks intelligent responsiveness. Most existing systems rely on the passive degradation or diffusion of the hydrogel to release exosomes, resulting in uncontrollable release behavior. The microenvironment of chronic diabetic wounds is typically weakly acidic, a key pathological characteristic, yet few delivery systems can utilize this characteristic to intelligently regulate the release of therapeutic factors. Traditional hydrogel networks primarily serve as passive scaffolds for carrying and releasing exosomes, typically lacking additional therapeutic functions themselves. However, the pathology of diabetic wounds is complex, with high levels of reactive oxygen species and the initial risk of bleeding requiring dressings to possess multiple functions. Therefore, hydrogel matrices lacking intrinsic antioxidant and hemostatic capabilities limit their comprehensive therapeutic effects. Furthermore, wound healing is a dynamic and phased process, including the inflammatory, proliferative, and remodeling phases. An ideal treatment strategy should be able to release appropriate amounts of therapeutic factors at the right time and place, according to the needs of different stages. Existing technologies often release active substances in a single or continuous manner, making it difficult to achieve "spatio-temporal control" synchronized with the healing process, resulting in low treatment efficiency. Therefore, there is a need to develop a novel smart wound dressing that can not only serve as a highly efficient delivery carrier for therapeutic factors but also intelligently respond to the pathological microcirculation of diabetic wounds, enabling the time-sequential, on-demand release of therapeutic factors. Summary of the Invention

[0006] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide a pH-responsive hydrogel based on engineered exosome integration, its preparation method and application.

[0007] To achieve the objectives of this invention, the technical solution adopted is as follows: The first aspect of this invention provides a method for preparing a pH-responsive hydrogel based on engineered exosome integration, comprising the following steps: (1) After mixing exosomes with PEI (polyethyleneimine) solution, the mixture was incubated at room temperature. After the incubation was completed, exosome-PEI complex was obtained. (2) The exosome-PEI complex was mixed with plasmid DNA and then incubated at room temperature. After the incubation was completed, the exosome-PEI-plasmid DNA complex was obtained. (3) γ-PGA (γ-polyglutamic acid) was modified with dopamine to obtain DA-modified γ-PGA; The DA-modified γ-PGA solution was mixed with a PBS solution of 3-FPBA (3-methylphenylboronic acid) to obtain a PGA-DA / 3-FPBA solution. (4) The exosome-PEI-plasmid DNA complex prepared in step (2) is added to the PGA-DA / 3-FPBA solution, mixed well, and then the mixture is mixed with carboxymethyl chitosan solution and reacted at room temperature to prepare pH-responsive hydrogel.

[0008] According to the above preparation method, preferably, the exosomes are exosomes secreted by mesenchymal stem cells; the plasmid DNA contains the VEGFA gene. More preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells; the plasmid DNA is the pECMV-Vegfa-m-FLAG plasmid.

[0009] According to the above preparation method, preferably, the mass ratio of exosomes to PEI is (1-5):1, and the mass ratio of PEI to plasmid DNA is (1-10):1. More preferably, the mass ratio of exosomes to PEI is 2:1, and the mass ratio of PEI to plasmid DNA is 5:1. According to the above preparation method, preferably, the concentration of PGA-DA in the PGA-DA / 3-FPBA solution is 8-12 wt%; and the mass concentration of 3-FPBA in the PGA-DA / 3-FPBA solution is 2.5 wt%. More preferably, the concentration of PGA-DA in the PGA-DA / 3-FPBA solution is 10 wt%.

[0010] According to the above preparation method, preferably, the concentration of the carboxymethyl chitosan solution is 5-10 wt%; more preferably, the concentration of the carboxymethyl chitosan solution is 7.5 wt%.

[0011] According to the above preparation method, preferably, in step (4), the concentration of plasmid DNA in the mixture is 1-100 µg / mL; the volume ratio of the mixture to the carboxymethyl chitosan solution is (1-5):(1-3). More preferably, the concentration of plasmid DNA in the mixture is 10-60 µg / mL; the volume ratio of the mixture to the carboxymethyl chitosan solution is 3:2. Most preferably, the concentration of plasmid DNA in the mixture is 30 µg / mL.

[0012] According to the above preparation method, preferably, the DA-modified γ-PGA is prepared by covalently linking the amino group of DA to the carboxyl group of γ-PGA. More preferably, the DA-modified γ-PGA is prepared by EDC / NHS coupling chemistry to link the amino group of DA to the carboxyl group of γ-PGA through an amide bond.

[0013] The second aspect of this patent provides a pH-responsive hydrogel prepared using the preparation method described in the first aspect above.

[0014] The third aspect of this patent provides the application of the pH-responsive hydrogel described in the second aspect above in the preparation of wound dressings for stopping bleeding and / or promoting wound healing.

[0015] According to the above application, the wound includes a skin ulcer.

[0016] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows: This invention prepares a novel EPD@Gel hydrogel by integrating exosomes and plasmids expressing the VEGFA gene into a hydrogel. This EPD@Gel hydrogel can effectively regulate the release of the EPD complex and pDNA under acidic conditions. Therefore, when used on diabetic infected wounds, the EPD@Gel hydrogel can trigger the precise release of the EPD complex and pDNA, significantly promoting cell proliferation, migration, and angiogenesis. Furthermore, the EPD@Gel hydrogel exhibits excellent antibacterial activity, self-healing ability, strong adhesion to moist tissues, and injectability. Moreover, the hydrogel network itself possesses inherent antioxidant properties and effective hemostatic function, enabling it to actively adapt to the complex environment and mechanical activity of the wound, providing long-term, stable, and complete protection. These superior functional properties collectively constitute the key basis for its ability to significantly promote the efficient repair and regeneration of skin ulcers (especially diabetic skin ulcers, such as diabetic foot ulcers), ultimately achieving rapid and effective treatment of chronic, difficult-to-heal diabetic wounds. Attached Figure Description

[0017] Figure 1Characterization of the Exos-PEI-pDNA (EPD) complex; (a) Schematic diagram of EPD complex preparation process; (b) Representative transmission electron microscopy (TEM) images of isolated Exos; (c) Nanoparticle tracking analysis (NTA) measurement of Exos particle size distribution and concentration; (d) Western blot verification of exosome markers (CD63, CD81, TSG101) and negative control (calcinin) expression in MSCs and their derived Exos; (e) Agarose gel electrophoresis showing the comparison between EPD complex formation (lane 4) and pDNA (lane 1), Exos (lane 2), and Exos / pDNA mixture (lane 3); (f) TEM images showing the morphological characteristics of PEI, EP, and EPD; (g) Hydrodynamic diameter determination of Exos, EP, and EPD by DLS; (h) Zeta potential measurement of Exos, PEI, EP, and EPD complexes, data expressed as mean ± standard deviation; (i) (j) Agarose gel retardation experiment of PEI / pDNA complexes formed with different PEI:pDNA mass ratios; Agarose gel electrophoresis showing the migration rate of EPD complexes with different Exos:PE mass ratios; Figure 2 Figure 1 shows the experimental results for evaluating the cytotoxicity of EPD complexes prepared with different Exos / PEI mass ratios for CCK-8. Data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined by one-way ANOVA combined with Tukey's multiple comparison test, p < 0.0001. Figure 3 (a) Fluorescence microscopy images of HaCaT cells treated with different Exos:PEI mass ratios, FITC-labeled PEI (green) and DiI-labeled Exos (red); (b) Pearson colocalization coefficient analysis of PEI (green) and Exos (red); (c) Quantitative flow cytometry analysis of EPD uptake efficiency in HaCaT cells; (d) Transfection efficiency of EPD complex in HEK293T cells as determined by flow cytometry. Figure 4Characterization of EPD@Gel; (a) Microscopic images of the hydrogel formation process; (b) FT-IR spectra of PGA, DA, PGA-DA, 3-FPBA, CMSC and hydrogel; (c) Results of hydrogel swelling performance test; (d) Results of hydrogel degradation performance test; (e) SEM images of representative cross-sections of hydrogel; (f) Release curve of EPD in EPD@Gel; (g) Release curve of pDNA in EPD@Gel; (h) Shape adaptability of EPD@Gel; (i) Self-healing ability of EPD@Gel; (j) Stability of EPD@Gel; (k) Adhesion of EPD@Gel to moist tissue; (l) Injection performance of EPD@Gel; (m) Amplitude scan; (n) Frequency scan; (o) Step strain recovery; (p) Complex viscosity; Figure 5 Three-dimensional confocal laser scanning microscopy (CLSM) image of a hydrogel loaded with Dil-labeled EPD (red), scale bar: 5 μm; Figure 6 To assess the in vitro biocompatibility and healing-promoting bioactivity of EPD@Gel; (a) Survival rate of HaCaT cells treated with different formulations on days 1 / 2 / 3 (CCK-8 assay); (b) Representative EdU staining of HaCaT cells: nuclei (DAPI, blue) and proliferating cells (EdU, red); (c) Scratch healing assay 24 hours after treatment; (d) Quantitative wound closure rate; (e) HaCaT cell Transwell migration assay; (f) Quantitative analysis of migrating cells; (g) Tube formation assay of HUVECs in Matrigel; (h) Quantitative analysis of total branch length; (i) qRT-PCR analysis of VEGFA mRNA expression; (j) Representative Western blot analysis of HaCaT cells after treatment; (k) Detection of intracellular ROS in HaCaT cells by DCFH-DA fluorescence (green); Figure 7 The results of blood compatibility testing are presented as mean ± standard deviation (n = 3); in the figure, DDW represents double-distilled water. Figure 8 The results of flow cytometry quantitative analysis of the intracellular reactive oxygen species scavenging ability of EPD@Gel hydrogel; Figure 9 The results are from the CCK-8 cell viability assay. Data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined using one-way ANOVA combined with Tukey's multiple comparison test. **p<0.01, ****p<0.0001; Figure 10(a) Antimicrobial properties of EPD@Gel; (b) Inhibition zones of *E. coli* and *S. aureus* around discs containing PBS, 3M gel extract, exos, EPD complex, or EPD@Gel extract; (c) Quantitative analysis of the diameter of the inhibition zones in (a); (d) Representative images of surviving *E. coli* and *S. aureus* colonies on LB agar plates; (e) Live / dead stained bacteria: live bacteria (green, SYTO 9), dead bacteria (red, propidium iodide); (f) Bacterial viability calculated from colony counts in (c); (g) Live / dead cell ratio quantified from fluorescence images in (d); (d) SEM images of *E. coli* and *S. aureus* after treatment (pre-fixation and dehydration). Figure 11 The in vivo therapeutic effects of EPD@Gel in a diabetic infected wound model are shown below: (a) Schematic timeline of wound creation and treatment; (b) Representative wound photographs on days 0 / 3 / 7 / 11 / 14 post-treatment; (c) Continuous images of wound closure from day 0 to day 14; (d) Staphylococcus aureus colonies cultured from wound homogenate on day 14; (e) Quantitative analysis of wound closure percentage; (f) Pseudocolor plot of wound area percentage at specified time points; (g) Representative H&E and Masson trichrome staining (histological analysis) of wound tissue on days 7 / 14; (h) Epithelial thickness and (i) collagen volume fraction on day 14. Figure 12 The results of immunofluorescence detection of CD31 and α-SMA expression in wound neoskin on days 7 and 14 are shown. (a) Representative immunofluorescence co-localization images of CD31 (red) and α-SMA (green) in neoskin at wound sites in each group on days 7 and 14, scale bar: 100 µm; (b) Quantitative analysis of CD31 expression levels. Data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined using two-way ANOVA combined with Dunnett's multiple comparison test. p > 0.05 indicated no significant difference (NS). P<0.05, **p<0.01, ****p<0.0001; Figure 13The results of HIF-1α expression in wound neodermal tissue on days 7 and 14 were obtained by immunofluorescence detection. (a) Representative immunofluorescence images of HIF-1α in newly formed skin tissue of each group on days 7 and 14. Scale bar: 100 µm; (b) Quantitative analysis of HIF-1α expression level; Data are expressed as mean ± standard deviation (n=3). Statistical significance was determined by two-way ANOVA combined with Dunnett's multiple comparison test. p>0.05 was considered as no significant difference (NS); *p<0.05, p<0.01, *p<0.001, p<0.0001; Figure 14 To investigate the angiogenesis and anti-inflammatory effects of EPD@Gel in diabetic mice; (a–d) Representative immunofluorescence images show the expression of (a) vascular endothelial growth factor (VEGF) A, (b) anti-inflammatory cytokine IL-10, (c) inflammatory pro-inflammatory cytokine TNF-α, and (d) cell proliferation marker Ki67 in wound-embryonic skin tissue; (e–h) Quantitative analysis of the relative expression levels of (e) VEGF A, (f) IL-10, (g) TNF-α, and (h) Ki67 in wound-embryonic skin tissue of different groups; Figure 15 The effects of EPD@Gel on biomechanical markers in diabetic mice; (a) Immunofluorescence image of collagen I; (c) Immunofluorescence image of integrin α3; (e) Immunofluorescence image of Vinculin protein; (b) Quantitative analysis of relative expression level of collagen I; (d) Quantitative analysis of relative expression level of integrin α3; (f) Quantitative analysis of relative expression level of Vinculin protein; Figure 16 Results of hemostatic performance testing of EPD@Gel hydrogel; (A) Representative bleeding images of each experimental group during liver hemostasis in mice; (B) Blood loss in liver tissue of each group of mice; (C) Representative bleeding images of each experimental group during tail hemostasis in mice; (D) Blood loss in tail region of each group of mice. Statistical significance: *P<0.05, ****P<0.0001, NS indicates no statistical difference. Detailed Implementation

[0018] To enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific embodiments. The following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0019] It should be noted that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the exemplary embodiments of the present invention. Experimental methods in the following embodiments, unless otherwise specified, employ conventional techniques in this art or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0020] Example 1: Isolation and Identification of Exosomes Exosomes were isolated from human umbilical cord mesenchymal stem cell (hUC-MSCs) conditioned medium using differential ultracentrifugation, and the isolated exosomes were identified.

[0021] The specific procedure for isolating exosomes from human umbilical cord mesenchymal stem cells (hUC-MSCs) conditioned medium using differential ultracentrifugation is as follows: hUC-MSCs cells are seeded into serum-free medium with clearly defined chemical composition (RegenExo series) and placed in a 37℃, 5% CO2 saturated humidity incubator for expansion culture. When the cell confluence reaches 70%–80%, fresh medium is added, and culture continues for 24–48 days. h, collect the supernatant to obtain the conditioned medium; perform differential ultracentrifugation on the collected conditioned medium. The specific steps of differential ultracentrifugation (all centrifugation steps are performed at 4℃ to maximize the protection of the biological activity of exosomes) are as follows: centrifuge at 300×g for 10 minutes, and collect the supernatant after centrifugation; centrifuge the supernatant at 2000×g for 20 minutes, and collect the supernatant after centrifugation; centrifuge the supernatant at 10000×g for 30 minutes, and collect the supernatant after centrifugation; centrifuge the supernatant at 120000×g for 70 minutes, discard the supernatant after centrifugation, then resuspend the precipitate with pre-cooled PBS buffer, and then ultracentrifuge at 120000×g for 70 minutes, discard the supernatant after centrifugation, collect the precipitate to obtain purified exosomes (denoted as Exos), resuspend the Exos in PBS buffer to obtain the Exos suspension, and store it at -80℃ for later use.

[0022] The morphological characteristics of Exos were observed using a transmission electron microscope (TEM; Hitachi HT-7800, Japan). The results are as follows: Figure 1 As shown in b. From Figure 1 As shown in b, Exos exhibits a typical cup-shaped morphology and a clear bilayer membrane structure with an average diameter of 110 ± 9 nm.

[0023] The particle concentration and size distribution of Exos were determined using a nanoparticle tracking and analysis system (NTA) (NanoSight NS300, Malvern Panalitick, UK). The results are as follows: Figure 1 As shown in c. From Figure 1As shown in section c, nanoparticle tracking analysis (NTA) confirmed a high particle concentration (6.6 × 10⁻⁶). 10 (particles / mL).

[0024] The presence of exosome marker proteins (CD63, catalog number 25682-1-AP, Proteintech; CD81, catalog number GB111073, Servicebio; TSG101, catalog number GB15618, Servicebio) and the absence of the negative marker protein calnetin (catalyst GB111369, Servicebio) were confirmed by Western blot. The results are as follows: Figure 1 As shown in d. From Figure 1 As shown in Figure d, Exos markers (CD63, CD81, TSG101) were significantly enriched, and the endoplasmic reticulum protein Calnexin was absent, confirming the successful extraction of Exos.

[0025] The hydrodynamic diameter and zeta potential of Exos were determined using a dynamic light scattering (DLS) instrument (Zetasizer Nano ZS90, Malvern Instruments, UK). The results are as follows: Figure 1 As shown in g and h. From Figure 1 As can be seen from g, the hydrodynamic diameter distribution range of Exos is relatively narrow, with a peak particle size of approximately 110 nm, indicating good particle size uniformity, consistent with the typical particle size range of exosomes. Figure 1 As can be seen from h, the zeta potential of Exos is negative, with an average potential of approximately -10.3 mV, indicating that its surface carries a negative charge, which is beneficial for forming stable complexes with cationic polymers (such as PEI) through electrostatic adsorption.

[0026] Example 2: Preparation of the Exos-PEI-pDNA complex 1. Exploration of the effect of the PEI to pDNA (plasmid DNA) mass ratio on the performance of the Exos-PEI-pDNA complex: To investigate the effect of the PEI to pDNA (plasmid DNA) mass ratio on the preparation of the Exos-PEI-pDNA complex, this invention sets different PEI to pDNA mass ratios to prepare PEI / pDNA complexes.

[0027] The specific steps for preparing the PEI / pDNA complex are as follows: Under continuous vortex conditions, 1 µg of pDNA is mixed with a PEI (polyethyleneimine) solution and incubated at room temperature for 20 min to obtain the PEI / pDNA complex; wherein, the pDNA is a plasmid for expressing the VEGFA gene (preferably pECMV-Vegfa-m-FLAG plasmid), and the mass ratio of PEI to pDNA is set to 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, and 40:1, respectively.

[0028] The pDNA preparation method is as follows: the dry powder of plasmid pECMV-Vegfa-m-FLAG (purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number: P6585) was transformed into Escherichia coli DH5α competent cells, plated on LB agar plates containing kanamycin (50 µg / mL), and single colonies were picked and inoculated into LB liquid medium containing the corresponding kanamycin for amplification culture. After the culture was completed, plasmid was extracted using the TIANprep Mini Plasmid Kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP103) according to the kit instructions. The plasmid was eluted with elution buffer EB and diluted with sterile deionized water to a working concentration of 500 ng / µL to obtain the pDNA solution.

[0029] The PEI / pDNA complexes prepared with different PEI / pDNA mass ratios were subjected to electrophoresis (TAE buffer) on a 1% (w / v) agarose gel containing GoldView nucleic acid staining reagent (catalog number NA0093, Leagene Biotechnology). The binding ability of PEI to pDNA was determined by observing the retention of the pDNA band. The results are as follows: Figure 1 As shown in i.

[0030] Depend on Figure 1 As shown in Figure i, when the mass ratio of PEI to pDNA is ≥ 1:1, the pDNA is completely retained. To balance the complete encapsulation efficiency of pDNA with cellular biosafety, the mass ratio of PEI to pDNA was fixed at 5:1 during the subsequent preparation of the EPD complex.

[0031] 2. Exploration of the effect of the mass ratio of Exos to PEI on the performance of the Exos-PEI-pDNA complex: To investigate the effect of the mass ratio of Exos to PEI on the performance of the Exos-PEI-pDNA complex, this invention sets different mass ratios of Exos to PEI to prepare the Exos-PEI-pDNA complex.

[0032] The specific steps of the Exos-PEI-pDNA complex preparation method (such as...) Figure 1 As shown in Figure a), under continuous vortex conditions, PEI (polyethyleneimine) solution was added dropwise to the Exos suspension prepared in Example 1, and incubated at room temperature for 30 min to obtain the Exos-PEI complex (denoted as the EP complex); the EP complex was mixed with 1 µg pDNA, and incubated at room temperature for another 20 min to obtain the Exos-PEI-pDNA complex (denoted as the EPD complex). The pDNA was a plasmid for expressing the VEGFA gene (preferably the pECMV-Vegfa-m-FLAG plasmid), the mass ratio of PEI to pDNA was 5:1, and the mass ratio of Exos to PEI was set sequentially to 0:1, 0.25:1, 0.5:1, 0.75:1, 1:1, 2:1, 3:1, 4:1, and 5:1.

[0033] The pDNA preparation method is as follows: the dry powder of plasmid pECMV-Vegfa-m-FLAG (purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number: P6585) was transformed into Escherichia coli DH5α competent cells, plated on LB agar plates containing kanamycin (50 µg / mL), and single colonies were picked and inoculated into LB liquid medium containing the corresponding kanamycin for amplification culture. After the culture was completed, plasmid was extracted using the TIANprep Mini Plasmid Kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP103) according to the kit instructions. The plasmid was eluted with elution buffer EB and diluted with sterile deionized water to a working concentration of 500 ng / µL to obtain the pDNA solution.

[0034] Furthermore, to facilitate subsequent detection of the uptake efficiency of EPD complexes prepared with different Exos / PEI mass ratios through cellular uptake experiments, Exos and PEI were fluorescently labeled during the preparation of the EPD complexes. The fluorescent labeling of Exos was performed as follows: Exos was incubated with 5 µg / mL DiI at room temperature for 30 minutes, followed by purification by ultracentrifugation (120000×g, 70 minutes, 4℃) to obtain fluorescently labeled Exos. The fluorescent labeling of PEI was performed as follows: PEI was reacted with FITC (1 mg / mL, dissolved in DMSO) overnight in the dark, and the resulting conjugate was dialyzed (MWCO: 1,000 Da) for 48 hours, followed by lyophilization.

[0035] (1) Detection of the effect of different Exos to PEI mass ratios on the migration rate of EPD complex: The EPD complexes prepared with different Exos to PEI mass ratios were subjected to electrophoresis (TAE buffer) on a 1% (w / v) agarose gel containing GoldView nucleic acid staining reagent (catalog number NA0093, Leagene Biotechnology). The effect of different Exos ratios on the binding ability of the EP complex (exosome-PEI) to pDNA was determined by observing the retention degree of pDNA bands in the sample wells and the presence or absence of free pDNA migration bands. The results are as follows. Figure 1 As shown in j.

[0036] Depend on Figure 1 As can be seen from the above, the pDNA bands of the EPD complexes prepared under different Exos to PEI mass ratios were all completely retained in the sample wells. Figure 1 (j) No obvious free pDNA migration bands were observed, indicating that the amount of exosomes added does not affect the encapsulation and complexation efficiency of PEI on pDNA (this efficiency is determined only by the mass ratio of PEI to pDNA), that is, the binding ability of the EP complex to pDNA is independent of the amount of Exos used.

[0037] (2) Detection of the effect of different Exos to PEI mass ratios on the cytotoxicity of EPD complex: The cytotoxicity of the EPD complexes prepared with different Exos to PEI mass ratios was evaluated using the Cell Counting Kit-8 (CCK-8, catalog number BS350A, Biosharp). The specific procedure was as follows: HaCaT cells were counted at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [insert density here] in 96-well plates and cultured for 24 hours to allow them to adhere. Subsequently, the cells were incubated with the EPD complex in serum-containing medium for 48 hours. After treatment, the cells were gently washed with PBS, and then incubated with CCK-8 reagent (diluted 1:10 in fresh medium) for 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability (%) = (Ah - Ab) / (Ac - Ab) × 100, where Ah, Ac, and Ab represent the optical density values ​​of the experimental group, cell control group, and blank control group, respectively. Results are as follows: Figure 2 As shown.

[0038] Depend on Figure 2The results showed that when the Exos to PEI mass ratio was 0:1, the exosome-free complex exhibited significant cytotoxicity, reducing HaCaT cell viability to 77.72 ± 3.5%. Conversely, biocompatibility significantly improved with increasing Exos incorporation. Notably, at exosome:PEI mass ratios of 1:1 and 5:1, cell viability was maintained at 99.74 ± 2.1% and 105.3 ± 4.2%, respectively, comparable to the untreated control group. These results indicate that Exos effectively mitigated the inherent cytotoxicity of PEI. Therefore, formulations with Exos to PEI mass ratios between 1:1 and 5:1 were selected for subsequent experiments, as these formulations minimized cytotoxicity while maintaining high pDNA binding rates.

[0039] (3) Detection of the effect of different Exos to PEI mass ratios on cellular uptake of EPD complex: The uptake efficiency of the EPD complexes prepared with different Exos to PEI mass ratios was evaluated using cellular uptake assays. The specific procedure was as follows: HaCaT cells were introduced at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density in 24-well plates and cultured for 24 hours to allow cell adhesion. After washing with PBS, cells were pre-incubated in serum-free medium for 1 hour. Subsequently, fluorescently labeled EPD complex was added to the wells, and the cells were co-incubated for 6 hours. After incubation, cells were fixed with 4% paraformaldehyde (PFA), and the nuclei were counterstained with Hoechst. Cell uptake was observed using fluorescence microscopy, and the Pearson colocalization coefficients of PEI (green) and Exos (red) were further analyzed. Additionally, for quantitative analysis, cells treated under the same conditions were collected and analyzed by flow cytometry.

[0040] Fluorescence microscopy images of HaCaT cells treated with EPD complexes prepared at different Exos to PEI mass ratios are shown below. Figure 3 As shown in Figure a. From Figure 3 As shown in Figure a, FITC-labeled PEI (green) and DiI-labeled Exos (red) exhibit significant co-localization (superimposed yellow signal), confirming successful complex assembly. Further analysis of the Pearson co-localization coefficients of PEI (green) and Exos (red) yielded the following results: Figure 3 As shown in b. From Figure 3 As shown in Figure b, the Pearson colocalization coefficient reaches its peak (0.86) when the Exos to PEI mass ratio is 2:1. Quantitative analysis results by flow cytometry are as follows: Figure 3 As shown in c. From Figure 3 As shown in Figure c, the cell uptake efficiency was highest (87.6%) when the mass ratio of Exos to PEI was 2:1.

[0041] (4) Detection of the effect of different Exos to PEI mass ratios on EPD complex cell transfection: The transfection efficiency of the EPD complexes prepared with different Exos to PEI mass ratios in cells was evaluated using in vitro gene transfection experiments. The specific procedure was as follows: HEK293T cells were transfected at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 24-well plates and cultured overnight until cell confluence reached 70%-80%. Before transfection, the medium was replaced with serum-free DMEM, and the EPD complex was added followed by incubation for 6 hours. After incubation, the medium was replaced with fresh complete medium and cultured for another 48 hours. Enhanced green fluorescent protein (EGFP) expression was observed using fluorescence microscopy, and transfection efficiency was quantified using flow cytometry. Results are as follows: Figure 3 As shown in d.

[0042] Depend on Figure 3 As shown in Figure d, the transfection efficiency is strictly dependent on the Exos to PEI mass ratio, and the transfection efficiency reaches its peak when the Exos to PEI mass ratio is 2:1. Therefore, an Exos:PEI mass ratio of 2:1 was selected as the optimal ratio for subsequent studies.

[0043] 3. Characterization of the EPD complex: (1) Preparation of EPD complex: The specific steps for preparing the EPD complex are as follows: Under continuous vortex conditions, PEI (polyethyleneimine) solution is added dropwise to the Exos suspension prepared in Example 1, and incubated at room temperature for 30 min to obtain the EP complex; the EP complex is then mixed with 1 µg pDNA and incubated at room temperature for another 20 min to obtain the EPD complex. The mass ratio of PEI to pDNA is 5:1, and the mass ratio of Exos to PEI is 2:1.

[0044] The pDNA is a plasmid used to express the VEGFA gene, preferably the pECMV-Vegfa-m-FLAG plasmid. The pDNA is prepared as follows: The pECMV-Vegfa-m-FLAG plasmid powder (purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number: P6585) is transformed into E. coli DH5α competent cells, plated on LB agar plates containing kanamycin (50 µg / mL), and single colonies are picked and inoculated into LB liquid medium containing the corresponding kanamycin for amplification and culture. After culture, plasmid is extracted using the TIANprep Mini Plasmid Kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP103) according to the kit instructions. The plasmid is eluted with elution buffer EB and diluted with sterile deionized water to a working concentration of 500 ng / µL to obtain the pDNA solution.

[0045] (2) Morphological characterization of the EPD complex: The morphology of the EPD complex, EP complex, and PEI was observed using transmission electron microscopy (TEM), and the results are as follows: Figure 1 As shown in f. From Figure 1 As shown in f, free PEI exhibits amorphous aggregation, while Exos presents a typical spherical vesicle structure (110±9 nm). After complexing with PEI, the EP complex maintains its spherical shape, with the particle size increasing to 142±12 nm and surface density increasing. Upon further loading with pDNA, the EPD complex particle size further increases to 175±15 nm, while still maintaining a well-dispersed spherical morphology, indicating that PEI and pDNA have been successfully assembled layer by layer onto the exosome surface.

[0046] (3) Detection of hydrodynamic diameter and zeta potential of EPD complex: Its hydrodynamic diameter and zeta potential were determined by dynamic light scattering (DLS), and the results are as follows: Figure 1 As shown in g and h. From Figure 1 As shown in Figure g, the hydrodynamic diameter gradually increases from Exos to the EP complex and then to the EPD complex, indicating that the introduction of PEI and pDNA gradually increases the hydrodynamic volume of the nanoparticles. Furthermore, the particle size distribution is relatively narrow, suggesting that the complexes have good dispersibility in the aqueous phase. Figure 1 As shown in the results, the surface charge of the particles reversed from negative (Exos) to positive (EP), and decreased after pDNA loading, indicating that the cation density was optimized, which helps to enhance cellular infiltration and reduce cytotoxicity. These results confirm that PEI and pDNA were sequentially deposited on the Exos surface.

[0047] Example 3: Preparation of hydrogel 1. Synthesis and characterization of dopamine-modified γ-PGA (denoted as PGA-DA) PGA-DA is synthesized by combining DA (dopamine) with the carboxyl group of γ-PGA (γ-polyglutamic acid) via EDC / NHS coupling chemistry. The specific synthetic steps are as follows: γ-PGA (1 g, containing 7.7 mmol carboxyl group) is dissolved in 100 mL of deionized water to obtain a γ-PGA solution; under continuous stirring in an ice bath, EDC (5 mmol) and NHS (10 mmol) are added to the γ-PGA solution, and after activation for 5 minutes, DA (10 mmol) is added, and the reaction is carried out at room temperature for 12 hours; after the reaction is complete, the obtained product is purified by dialysis (MWCO: 12-14 kDa) with deionized water for 72 hours, followed by lyophilization. 1 H nuclear magnetic resonance (NMR) 1Successful coupling was confirmed by H NMR (Bruker Advance 400, 400 MHz, Switzerland) and Fourier transform infrared spectroscopy (FT-IR, Nicolet iS20, Thermo Fisher Scientific, USA).

[0048] 2. Preparation and characterization of hydrogels (1) Preparation of hydrogels: The hydrogel was prepared via dynamic crosslinking mediated by borate esters and imine bonds. The specific preparation steps are as follows: 1) Dissolve 3-FPBA (3-methylphenylboronic acid) in PBS buffer (pH 7.4) to prepare a 7.5 wt% 3-FPBA solution; dissolve PGA-DA in PBS buffer (pH 7.4) to obtain a PGA-DA solution; 2) The 3-FPBA solution was mixed with PGA-DA solutions of different concentrations at a volume ratio of 1:2 to obtain three PGA-DA / 3-FPBA solutions (the concentrations of PGA-DA in the three PGA-DA / 3-FPBA solutions were 11.7wt%, 10.0wt%, and 8.3wt%, respectively). 3) Dissolve CMCS (carboxymethyl chitosan) in PBS buffer (pH 7.4) to prepare three CMCS solutions with concentrations of 5.0 wt%, 7.5 wt%, and 10.0 wt%. Mix the PGA-DA / 3-FPBA solution with the CMCS solution at a ratio of 3:2 (v / v) and react at room temperature to form a hydrogel (image of the hydrogel formation process is shown in the image). Figure 4 (As shown in Figure a). Specifically, the hydrogel formed by mixing an 11.7 wt% PGA-DA / 3-FPBA solution with a 5.0 wt% CMCS solution in a 3:2 (v / v) ratio was named P7F1.5C2; the hydrogel formed by mixing a 10.0 wt% PGA-DA / 3-FPBA solution with a 7.5 wt% CMCS solution in a 3:2 (v / v) ratio was named P6F1.5C3; and the hydrogel formed by mixing an 8.3 wt% PGA-DA / 3-FPBA solution with a 10.0 wt% CMCS solution in a 3:2 (v / v) ratio was named P5F1.5C4.

[0049] (2) Testing of hydrogel swelling and degradation properties: The swelling properties of hydrogels are crucial for absorbing wound exudate and maintaining a moist environment. Therefore, the swelling and degradation properties of the three hydrogels prepared in step (1) above were tested.

[0050] Swelling performance test of hydrogels: Three fully formed hydrogels (600 μL) prepared in step (1) above were immersed in PBS (pH 7.4, 37℃) to evaluate their swelling behavior. At predetermined time points, the samples were removed, the surface liquid was wiped off, and the weight was recorded. Swelling rate (%) = (W t / W0)×100, where W0 is the initial weight immediately after hydrogel formation (before immersion in PBS), W t The weight after drying at time t is the result of expansion.

[0051] In vitro degradation performance test of hydrogels: Three fully formed hydrogels (600 μL) prepared in step (1) above were incubated in PBS (pH 7.4, 37 °C). At specified time points, the samples were removed, gently rinsed with deionized water, lyophilized, and their dry weight (W) was measured. t Residual mass (%) = (W) t / W0)×100, where W0 represents the initial dry weight of the hydrogel after freeze-drying (before degradation) after molding.

[0052] The swelling performance test results of the hydrogel are as follows: Figure 4 As shown in c. From Figure 4 As shown in Figure c, the swelling ratio of the hydrogel decreases with increasing PGA-DA concentration in the solution. This may be because higher concentrations of PGA-DA introduce more catechol groups, thereby increasing the density of crosslinking points for forming borate ester bonds with 3-FPBA. The denser crosslinking network restricts polymer chain relaxation and water molecule penetration, leading to a decrease in swelling capacity. The degradation performance test results of the hydrogel are as follows... Figure 4 As shown in d. From Figure 4 As shown in Figure d, the in vitro degradation ability of the hydrogel exhibits an inverse trend compared to its swelling ability: formulations with higher PGA-DA content degrade more slowly. This is attributed to its higher crosslinking density, which enhances the structural stability of the network, thereby delaying the breakage of the polymer backbone or the hydrolysis of dynamic covalent bonds. Considering the antioxidant, swelling, and degradation properties, P6F1.5C3 hydrogel was ultimately selected for subsequent experiments.

[0053] (3) Fourier transform infrared spectroscopy (FT-IR) characterization of hydrogels: The P6F1.5C3 hydrogel prepared in step (1) above was characterized by FT-IR, and the results are as follows: Figure 4 As shown in b.

[0054] Gel formation depends on dual dynamic covalent crosslinking: the borate group of 3-FPBA forms a boronic acid ester bond with the catechol group of PGA-DA, and the amino group of CMCS forms an imine bond with the aldehyde group of 3-FPBA. Figure 4 As can be seen from b, the spectrum is at 3289 cm⁻¹ -1The hydroxyl stretching vibration peak at 1640 cm⁻¹ and the peak at 1640 cm⁻¹ -1 The presence of an amide I absorption peak at 1645 cm⁻¹ indicates the successful formation of amide bonds in PGA-DA; simultaneously, the hydrogel shows an absorption peak at 1645 cm⁻¹. -1 (C=N imine bond stretching vibration) and 1401 cm⁻¹ -1 The characteristic absorption peaks appearing at the (B–O stretching vibration) point jointly confirm the successful construction of its cross-linked network.

[0055] (4) SEM characterization of hydrogel: The P6F1.5C3 hydrogel prepared in step (1) above was characterized by SEM, and the results are as follows: Figure 4 As shown in e. From Figure 4 As can be seen from the image, the SEM image shows that the P6F1.5C3 hydrogel has a loose and porous three-dimensional structure, which is conducive to the exchange and transfer of substances.

[0056] (5) Hydrogel porosity detection: The porosity of the P6F1.5C3 hydrogel prepared in step (1) was determined using the ethanol displacement method. Specifically, the freeze-dried hydrogel sample was immersed in anhydrous ethanol for 24 hours. The weights before immersion (W1) and after immersion (W2) were recorded. The sample volume (V1) was measured before immersion. Porosity (%) = (W2 - W1) / (ρ × V1) × 10, where ρ is the density of anhydrous ethanol (0.789 g / mL). The porosity of the hydrogel was found to be 27.2 ± 0.5%.

[0057] Example 4: Preparation and characterization of pH-responsive hydrogels based on engineered exosome integration 1. Preparation of pH-responsive hydrogels based on engineered exosome integration (denoted as EPD@Gel hydrogels) The specific steps for preparing EPD@Gel hydrogel are as follows: 1) Dissolve 3-FPBA in PBS buffer (pH 7.4) to prepare a 7.5 wt% 3-FPBA solution; dissolve PGA-DA in PBS buffer (pH 7.4) to obtain a PGA-DA solution; 2) Mix 3-FPBA solution and PGA-DA solution at a volume ratio of 1:2 to obtain PGA-DA / 3-FPBA solution (the concentration of PGA-DA in PGA-DA / 3-FPBA solution is 10.0 wt%, and the concentration of 3-FPBA in PGA-DA / 3-FPBA solution is 2.5 wt%). 3) Add the EPD complex (the EPD complex prepared in step 3 of Example 2) to the PGA-DA / 3-FPBA solution obtained in step 2) above, mix well to obtain the EPD / PGA-DA / 3-FPBA mixture; the final concentration of pDNA in the EPD / PGA-DA / 3-FPBA mixture is 30 µg / mL; 4) Dissolve CMCS in PBS buffer (pH 7.4) to prepare a CMCS solution with a concentration of 7.5 wt%; mix the EPD / PGA-DA / 3-FPBA mixture obtained in step 3) with the CMCS solution at a ratio of 3:2 (v / v) and react at room temperature to generate a hydrogel, thus obtaining EPD@Gel hydrogel.

[0058] 2. Detection of EPD immobilization in EPD@Gel hydrogel To characterize the immobilization of EPD in the EPD@Gel hydrogel, Dil fluorescently labeled EPD was encapsulated within the hydrogel, and the prepared EPD@Gel hydrogel was examined using confocal laser scanning microscopy (CLSM). The results are as follows: Figure 5 As shown.

[0059] Depend on Figure 5 As can be seen, the CLSM image confirms that EPD has a uniform spatial distribution in the three-dimensional network, indicating that it is stably dispersed in the hydrogel system.

[0060] 3. Physical property characterization of EPD@Gel hydrogel The physical properties of the EPD@Gel hydrogel prepared in step 1 of this embodiment were characterized as follows: (1) Detection of EPD complex release performance of EPD@Gel hydrogel: The EPD release profile of the EPD@Gel hydrogel was evaluated using the BCA Protein Assay Kit (catalog number 143179, Biosharp) to quantify the cumulative release. Specifically, 100 μL of EPD@Gel was placed in a Transwell insert, immersed in 600 μL of PBS solution at pH 5.5 or 7.4, and incubated at 37°C. At specified time points within 6 days, 10 μL of release medium was collected and replaced with an equal volume of fresh PBS. The cumulative release percentage was calculated based on the initial EPD loading.

[0061] Test results as follows Figure 4 As shown in f. From Figure 4As shown in Figure f, EPD release is slow under neutral conditions, with less than half of the total amount released by day 6, which is consistent with the degradation rate of the hydrogel. However, under acidic conditions, EPD release is significantly accelerated due to the breaking of imine bonds, reaching a cumulative release of 96.66% by day 6. This indicates that acidic conditions can effectively regulate the release behavior of the EPD complex in the EPD@Gel hydrogel. Diabetic infected wounds, due to local hypoxia, anaerobic respiration of cells, and active bacterial (especially anaerobic) metabolism, lead to the accumulation of acidic metabolites such as lactic acid, forming a characteristic acidic microenvironment with a pH value typically between 5.5 and 6.5. Therefore, when the EPD@Gel hydrogel of this invention is used on diabetic infected wounds, it can trigger the precise release of the EPD complex.

[0062] (2) Detection of pDNA release performance of EPD@Gel hydrogel: The pDNA release profile of EPD@Gel hydrogels was evaluated using the 1×dsDNA HS Assay Kit (catalog number 12642ES60, YEASEN). The results are as follows: Figure 4 As shown in g. From Figure 4 As can be seen from the results, under simulated physiological conditions (pH 7.4), the release of pDNA from EPD@Gel hydrogel was relatively slow, with a cumulative release rate of only 15.0% after 48 hours; however, under simulated acidic microenvironment of diabetic infected wounds (pH 5.5), the release rate of pDNA was significantly accelerated, with a cumulative release rate of 46.3% after 48 hours, which is about 3.1 times that under neutral conditions.

[0063] (3) Plasticity test of EPD@Gel hydrogel: The experimental method for testing plasticity was as follows: EPD@Gel hydrogel was kneaded and shaped into different forms. The results are as follows: Figure 4 As shown in h. From Figure 4 As can be seen from h, EPD@Gel hydrogel exhibits excellent functional properties and can be molded into complex structures such as stars, hearts, triangles, and squares.

[0064] (4) Self-healing ability test of EPD@Gel hydrogel: The specific experimental method for testing self-healing ability was as follows: Two pieces of EPD@Gel hydrogel, one stained red and the other blue, were cut in half lengthwise. The cut surfaces of the different colored halves were then tightly joined together. After standing at room temperature for 10 minutes, the healing process of the joint was observed, and the mechanical stability after healing was evaluated through a manual tensile test. The results are as follows: Figure 4 As shown in i. From Figure 4 As can be seen from i, the cut hydrogel achieves interfacial fusion within 10 minutes and remains intact after stretching, which is attributed to the dynamic reversibility of imine bonds and borate ester bonds.

[0065] (5) Skin adhesion test of EPD@Gel hydrogel: The specific experimental method for testing the skin adhesion of EPD@Gel hydrogel was as follows: EPD@Gel hydrogel was applied to the skin surface on the dorsal side of the volunteer's finger joints. After it gelled in situ, the volunteer was instructed to perform repeated finger flexion movements (flexion angle range 60°–180°), and the adhesion state of the hydrogel during dynamic skin deformation was observed. The results are as follows: Figure 4 As shown in j. From Figure 4 As can be seen from the data, EPD@Gel hydrogel maintains adhesive integrity during finger joint flexion (60°-180°).

[0066] (6) Adhesion test of EPD@Gel hydrogel to moist tissue: The specific experimental method for detecting the adhesion of EPD@Gel hydrogel to moist tissues was as follows: EPD@Gel hydrogels were applied to the surfaces of fresh isolated mouse heart and liver tissues, and after gentle pressing to ensure close contact, the adhesion of the hydrogels to the moist tissue surfaces was observed. Results are as follows: Figure 4 As shown in k. From Figure 4 As can be seen from the data, EPD@Gel hydrogel has strong adhesion to wet tissues such as the heart and liver.

[0067] (7) Injection performance testing of EPD@Gel hydrogel: The specific experimental method for testing the injection performance of EPD@Gel hydrogel was as follows: EPD@Gel hydrogel was pre-filled into a sterile syringe equipped with a 26G needle, and manually injected at a constant rate. The needle blockage, continuity and morphology of the extruded material were observed, and the gel recovery of the hydrogel after extrusion was evaluated. Results are as follows: Figure 4 As shown in Figure 1. From Figure 4 As shown in Figure 1, the EPD@Gel hydrogel was not blocked when extruded through a 26G needle and quickly returned to its gel state after extrusion. This injectability is attributed to the shear-thinning behavior of the dynamic cross-linked network, which ensures its precise delivery in minimally invasive procedures.

[0068] (8) Rheological properties of EPD@Gel hydrogel: Rheological properties of EPD@Gel hydrogel were analyzed using a DHR-2 rheometer. The specific experimental method was as follows: Cylindrical hydrogel specimens with a diameter of 8 mm and a height of 2 mm were used, and the following tests were performed sequentially: First, strain amplitude scanning (0.1% to 1000% strain, frequency 1 Hz) was performed at 25℃ to determine the linear viscoelastic region (LVR); then, frequency scanning (0.1 to 100 Hz) was performed at a constant strain of 0.1% LVR at 25℃, and the relationship between frequency and storage modulus (G′) and loss modulus (G″) was recorded; finally, self-recovery characteristics were evaluated through three consecutive sets of step strain tests, which involved alternating small strain (1% maintaining structure) and large strain (300% destroying structure) at 1 Hz frequency and 25℃. The results are as follows: Figure 4 The values ​​m, n, o, and p are shown in the diagram.

[0069] Depend on Figure 4 From m, n, o, and p, it can be seen that the linear viscoelastic strain range of EPD@Gel hydrogel is as wide as 252% ( Figure 4 The critical failure strain is relatively high (m), indicating that it has good resistance to deformation; during the frequency scan, G' is always higher than G'' and is frequency independent (m). Figure 4 The invention demonstrates typical elastic gel behavior primarily characterized by covalent crosslinking, exhibiting a stable and uniform network structure. Furthermore, the modulus of the EPD@Gel hydrogel completely recovered after three strain cycles (1%→300%→1%). Figure 4 (The text appears to be incomplete and contains several errors. A more accurate translation would require the full context.) Figure 4 (p), confirming that the EPD@Gel hydrogel of the present invention has thixotropic properties.

[0070] The above results demonstrate that the EPD@Gel hydrogel prepared according to this invention exhibits pH-responsive drug release properties. In the acidic microenvironment (pH 5.5–6.5) of diabetic infected wounds, the imine bonds in the hydrogel network undergo acid-instability cleavage, resulting in a loosened or even dissociated cross-linked structure, thereby triggering the precise release of the EPD complex and pDNA. Under normal physiological conditions (pH 7.4), the network remains stable, effectively inhibiting non-specific drug release. Furthermore, the EPD@Gel hydrogel possesses excellent self-healing capabilities, high adhesion to moist tissues, and good injectability. The dynamic covalent bonds (imine and borate ester bonds) in its network endow the material with multiple functions, including shear thinning, self-repair, and interfacial adhesion. The synergistic integration of these multiple functions—pH-triggered precise drug release, self-repair to maintain structural integrity, and wet adhesion to achieve wound adhesion—constitutes the key basis for the EPD@Gel hydrogel's ability to significantly promote efficient repair and regeneration of diabetic infected wounds.

[0071] 4. Functional testing of EPD@Gel hydrogel Functional testing was performed on the EPD@Gel hydrogel prepared in step 1 of this embodiment, and simultaneously, Exos@Gel hydrogel, Gel hydrogel (i.e., the P6F1.5C3 hydrogel prepared in Example 3), and 3M gel (3M TM Tegaderm TM Hydrocolloid dressing (catalog number: 91110) and PBS buffer were used as controls.

[0072] The steps of the Exos@Gel hydrogel preparation method are as follows: 1) Dissolve 3-FPBA in PBS buffer (pH 7.4) to prepare a 7.5 wt% 3-FPBA solution; dissolve PGA-DA in PBS buffer (pH 7.4) to obtain a PGA-DA solution; 2) Mix 3-FPBA solution and PGA-DA solution at a volume ratio of 1:2 to obtain PGA-DA / 3-FPBA solution (the concentration of PGA-DA in PGA-DA / 3-FPBA solution is 10.0 wt%, and the concentration of 3-FPBA in PGA-DA / 3-FPBA solution is 2.5 wt%). 3) Add the Exos prepared in Example 1 to the PGA-DA / 3-FPBA solution obtained in step 2) above, and disperse evenly to obtain an Exos / PGA-DA / 3-FPBA mixture; the final concentration of pDNA in the Exos / PGA-DA / 3-FPBA mixture is 30 µg / mL. 4) Dissolve CMCS in PBS buffer (pH 7.4) to prepare a CMCS solution with a concentration of 7.5 wt%; mix the Exos / PGA-DA / 3-FPBA mixture obtained in step 3) with the CMCS solution at a ratio of 3:2 (v / v) and react at room temperature to form a hydrogel, thus obtaining the Exos@Gel hydrogel.

[0073] The specific functional tests of EPD@Gel hydrogel are as follows: (1) Preparation of EPD@Gel hydrogel extract: The EPD@Gel hydrogel was sterilized by irradiating both sides with a UV lamp (254 nm) for 30 minutes each in a clean bench. Then, it was immersed in complete cell culture medium and incubated at 37°C and 5% CO2 for 24 hours in the dark (the culture medium should completely submerge the hydrogel). After incubation, the supernatant was collected and centrifuged at 1000–1500 g for 5–10 minutes to remove hydrogel degradation particles. The supernatant was then filtered through a 0.22 μm filter membrane for sterilization to obtain the hydrogel extract, which was stored at 4°C for later use.

[0074] (2) Biocompatibility test of EPD@Gel hydrogel: The specific procedure for cell compatibility testing is as follows: HaCaT cells are prepared at a density of 2 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL in a 96-well plate. After overnight adhesion, the supernatant was aspirated, and 100 μL of the EPD@Gel hydrogel extract prepared in step (1) above was added to each well. The extract was then detected by the CCK-8 assay after 1, 2, and 3 days of culture.

[0075] The specific procedure for blood compatibility testing is as follows: EPD@Gel hydrogel extract is mixed with 4% mouse erythrocyte suspension at a 1:1 (volume ratio), incubated at 37°C for 2 hours, centrifuged after incubation, and the supernatant is collected. The absorbance of the supernatant is measured at a wavelength of 545 nm. The hemolysis rate is calculated using the formula: Hemolysis rate (%) = (At - An) / (Ap - An) × 100. Where At, An, and Ap represent the absorbance values ​​of the test sample, negative control (physiological saline), and positive control (deionized water), respectively.

[0076] For comparison, the same method was used to test the biocompatibility of Exos@Gel hydrogel, Gel hydrogel, 3M gel, and PBS buffer, respectively; double-distilled water (DDW) was used as a positive control. The preparation methods of Exos@Gel hydrogel extract, Gel hydrogel extract, and 3M gel extract are basically the same as the preparation method of EPD@Gel hydrogel extract described in step (1) above, except that EPD@Gel hydrogel is replaced with Exos@Gel hydrogel, Gel hydrogel, and 3M gel, respectively.

[0077] The results of the cell compatibility test are as follows Figure 6 As shown in Figure a. From Figure 6 As shown in Figure a, after 3 days of culture, the survival rate of HaCaT cells in the EPD@Gel group was significantly higher than that in the control group, indicating that the EPD@Gel hydrogel of the present invention not only has good biosafety, but also promotes cell proliferation.

[0078] The results of the blood compatibility test are as follows Figure 7 As shown. By Figure 7 It can be seen that the hemolysis rate of the EPD@Gel group and its component control groups is lower than the internationally recognized safety threshold of 5%, which meets the blood compatibility standard for medical materials. This indicates that it will not cause significant red blood cell rupture when in contact with blood components, and has reliable in vivo application safety.

[0079] (3) Detection of the effect of EPD@Gel hydrogel on cell proliferation: Cell proliferation was detected using EdU assay reagent. The specific procedure was as follows: HaCaT cells were cultured at a density of 1 × 10⁶ cells / well. 4 HaCaT cells were seeded at a density of [number] cells / well in 24-well plates. After overnight adhesion, the original culture medium was discarded, and 500 μL of EPD@Gel hydrogel extract was added to each well. The plates were then treated at 37°C and 5% CO2 for 24 hours. After 24 hours of treatment, HaCaT cells were co-incubated with 10 μM EdU for 2 hours. Cells were then fixed, permeabilized, and stained using an Alexa Fluor 555-labeled click reaction. Nuclei were counterstained with Hoechst. After counterstaining, cells were washed twice with PBS, and images were acquired under a fluorescence microscope. The proliferation index was calculated using ImageJ software using the formula: Proliferation Index (%) = (Number of EdU-positive cells / Number of Hoechst-positive cells) × 100%.

[0080] For comparison, the present invention also used the same method to detect the effects of Exos@Gel hydrogel, Gel hydrogel, 3M gel, and PBS buffer on cell proliferation. The preparation methods of Exos@Gel hydrogel extract, Gel hydrogel extract, and 3M gel extract are basically the same as the preparation method of EPD@Gel hydrogel extract described in step (1) above, except that EPD@Gel hydrogel is replaced with Exos@Gel hydrogel, Gel hydrogel, and 3M gel, respectively.

[0081] Test results as follows Figure 6 As shown in b. From Figure 6 As shown in Figure b, the EdU staining results showed that the proportion of EdU+ cells in the EPD@Gel group was significantly higher than that in other groups. This effect may be due to the good biological activity of CMCS and γ-PGA and the synergistic effect of the active factors carried by exosomes.

[0082] (4) Detection of the effect of EPD@Gel hydrogel on cell migration: The effect of EPD@Gel hydrogel on cell migration was investigated using cell scratch assays and Transwell migration assays.

[0083] The specific procedure for the scratch assay is as follows: HaCaT cells are placed at a density of 4 × 10⁶ cells per well. 4 Counts were inoculated at a density of 2 × 10⁶ cells per well in 24-well plates containing Ibidi silicone inserts (catalog number 80209, Ibidi) (2 × 10⁶ cells per well in each of the two chambers of the insert). 4Cells (70 μL / well) were cultured at 37°C and 5% CO2 until cell confluence >95% (approximately 24 hours). After the cells grew into a dense monolayer, the silicone insert was carefully removed with sterile forceps to form a uniformly wide scratch area. The well plate was gently washed twice with PBS to remove cell debris generated by the scratch. Subsequently, 500 μL of the hydrogel extract prepared in step (1) above was added to each well, and the plate was treated at 37°C and 5% CO2 for 24 hours. The scratch area was photographed using an inverted microscope at 0 hours after scratching and 24 hours after incubation. The scratch area was measured using ImageJ software, and the migration rate (%) was calculated as (A0 - A...). t ) / A0 × 100%, where A0 represents the initial scratch area, A t This represents the area of ​​the scratch at time t.

[0084] The specific procedure for the Transwell migration assay is as follows: HaCaT cells are digested and resuspended in serum-free culture medium at a concentration of 1 × 10⁶ cells per well. 5 Cells were seeded into the upper chamber of a Transwell plate (total volume 100 μL, placed in a 24-well plate). 500 μL of culture medium containing 30% FBS and hydrogel extract was added to the lower chamber as a chemotactic agent. The plates were incubated at 37°C and 5% CO2 for 24 hours. After incubation, unmigrated cells in the upper chamber were gently wiped with a cotton swab. Cells that migrated to the lower chamber were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 20 minutes, washed with PBS, and then photographed and counted under an inverted microscope.

[0085] For comparison, the present invention also used the same method to detect the effects of Exos@Gel hydrogel, Gel hydrogel, 3M gel, and PBS buffer on cell migration. The preparation methods of Exos@Gel hydrogel extract, Gel hydrogel extract, and 3M gel extract are basically the same as the preparation method of EPD@Gel hydrogel extract described in step (1) above, except that EPD@Gel hydrogel is replaced with Exos@Gel hydrogel, Gel hydrogel, and 3M gel, respectively.

[0086] The results of the scratch test are as follows Figure 6 As shown in C and D. The detection results of the Transwell migration experiment are as follows. Figure 6 As shown in e and f. From Figure 6As shown in c, d, e, and f, after co-culturing with the hydrogel extract for 24 hours, both the 3M group and the Gel group showed higher scratch closure rates than the PBS group. Among them, the Exos@Gel group showed significant enhanced migration, while the EPD@Gel group showed the best performance in terms of scratch closure and Transwell migration cell number, indicating that the EPD@Gel hydrogel of the present invention has a significant migration-promoting ability.

[0087] (5) Detection of the effect of EPD@Gel hydrogel on angiogenesis: The effect of EPD@Gel hydrogel on angiogenesis was investigated using a tubular formation assay. Specifically, human umbilical vein endothelial cells (HUVECs) were cultured at 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 500 μL each in a Matrigel® Basement Membrane Matrix (Catalog No. 356234, Corning) diluted with growth factors, and incubated at 37°C and 5% CO2 for 6 hours. The resulting tubular network structure was observed under a microscope. The total branch length of the tubular structure was quantitatively analyzed using the angiogenesis analysis plugin in ImageJ software. Results are as follows: Figure 6 As shown in g and h.

[0088] For comparison, the present invention also used the same method to detect the effects of Exos@Gel hydrogel, Gel hydrogel, 3M gel, and PBS buffer on angiogenesis. The preparation methods of Exos@Gel hydrogel extract, Gel hydrogel extract, and 3M gel extract are basically the same as the preparation method of EPD@Gel hydrogel extract described in step (1) above, except that EPD@Gel hydrogel is replaced with Exos@Gel hydrogel, Gel hydrogel, and 3M gel, respectively.

[0089] Depend on Figure 6 As shown in g and h, the EPD@Gel group induced a denser tubular network, and the total branch length was significantly better than other groups, suggesting that Exos and VEGFA have a synergistic pro-angiogenic effect. Angiogenesis provides oxygen and nutrient support to the wound site and is a key step in healing.

[0090] (6) Detection of the effect of EPD@Gel hydrogel on VEGFA mRNA and VEGFA protein expression: HaCaT cells were stored at a density of 2 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well plates and incubated overnight at 37°C with 5% CO2. After adhesion, the original culture medium was discarded, and 2 mL of hydrogel extract was added to each well. The treatment continued for 24 hours. After treatment, the supernatant was discarded, and the cell pellet was collected. The expression levels of VEGFA mRNA and VEGFA protein in the cell pellet were then detected.

[0091] The specific procedures for detecting VEGFA mRNA expression were as follows: the cell pellet was washed twice with pre-cooled PBS buffer, total RNA was extracted from the cell pellet using TRIzol reagent, and reverse transcribed into cDNA using HiScript III RT SuperMi. Using the cDNA as a template, quantitative PCR was performed on a real-time PCR system using SYBR Green qPCR MasterMi. The relative expression level of VEGFA was normalized using the internal control GAPDH as a benchmark and calculated using the 2-ΔΔCt method. The gene-specific primer sequences used for quantitative PCR are shown in Table 1.

[0092]

[0093] The specific procedure for EGFA protein expression detection was as follows: Cells were washed twice with pre-chilled PBS buffer, and 150–200 μL of RIPA lysis buffer (containing protease and phosphatase inhibitors) was added to each well. Lysis was performed on ice for 30 minutes, with gentle shaking every 10 minutes to ensure the lysis buffer evenly covered the bottom of the wells. After lysis, the lysate was collected using a cell scraper into pre-chilled centrifuge tubes and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected as the total protein sample. The sample was then separated by SDS-PAGE and transferred to a nitrocellulose (NC) membrane. The membrane was blocked with 5% skim milk and incubated overnight at 4°C with anti-VEGFA, followed by incubation with HRP-labeled secondary antibody. Protein bands were visualized using an enhanced chemiluminescence (ECL) reagent and imaged using a chemiluminescence detection system.

[0094] For comparison, the present invention also used the same method to detect the effects of Exos@Gel hydrogel, Gel hydrogel, 3M gel, and PBS buffer on VEGFA mRNA and VEGFA protein expression. The preparation methods of Exos@Gel hydrogel extract, Gel hydrogel extract, and 3M gel extract are basically the same as the preparation method of EPD@Gel hydrogel extract described in step (1) above, except that EPD@Gel hydrogel is replaced with Exos@Gel hydrogel, Gel hydrogel, and 3M gel, respectively.

[0095] The RT-qPCR results of VEGFA mRNA expression are as follows: Figure 6As shown in Figure i, the Western blotting results of VEGFA protein expression are as follows: Figure 6 As shown in j. From Figure 6 As shown in i and j, EPD@Gel treatment significantly upregulated the mRNA and protein expression levels of VEGFA in HaCaT cells, demonstrating that the EPD complex successfully mediated the overexpression of VEGFA.

[0096] (7) Detection of intracellular antioxidant capacity of EPD@Gel hydrogel: The hyperglycemic microenvironment in diabetic wounds leads to the accumulation of reactive oxygen species (ROS) and oxidative stress, severely hindering repair. Therefore, controlling ROS levels in wounds is essential for wound healing.

[0097] 1) The intracellular reactive oxygen species (ROS) scavenging capacity was detected using the DCFH-DA assay. The specific procedure was as follows: HaCaT cells were seeded in 24-well plates and cultured for 24 hours. Then, 500 μL of EPD@Gel hydrogel extract containing 1 mmol / L H2O2 was added to each well, and the treatment continued for another 24 hours. After washing with PBS, the cells were incubated with 10 μM DCFH-DA at 37°C in the dark for 30 minutes. Intracellular ROS levels were qualitatively assessed using fluorescence microscopy and quantitatively analyzed by flow cytometry.

[0098] For comparison, the same method was used to detect the intracellular antioxidant capacity of Exos@Gel hydrogel, Gel hydrogel, 3M gel, and PBS buffer, respectively, with cells treated only with H2O2 as a control. The preparation methods of Exos@Gel hydrogel extract, Gel hydrogel extract, and 3M gel extract are basically the same as the preparation method of EPD@Gel hydrogel extract described in step (1) above, except that EPD@Gel hydrogel is replaced with Exos@Gel hydrogel, Gel hydrogel, and 3M gel, respectively.

[0099] Results of qualitative assessment of intracellular ROS levels using fluorescence microscopy, such as Figure 6 As shown in k. From Figure 6 As shown in the data, the ROS level in the H2O2-stimulated group was significantly increased, while the fluorescence intensity of the EPD@Gel hydrogel-treated group was comparable to that of the PBS group. The antioxidant capacity of the EPD@Gel hydrogel is attributed to the abundant catechol groups in dopamine and the antioxidant properties exhibited by the borate ester bonds in EPD@Gel, thus enabling the hydrogel to significantly scavenge ROS. Quantitative analysis using flow cytometry to quantify fluorescence intensity is shown in the figure below. Figure 8 As shown. By Figure 8 It can be seen that the EPD@Gel group had the lowest average fluorescence intensity, and its ROS scavenging efficiency was significantly better than that of other control groups.

[0100] 2) To assess whether antioxidant capacity can be converted into substantial cell protection, cell viability was measured under oxidative stress using the CCK-8 assay. The specific procedure was as follows: HaCaT cells were seeded in 96-well plates and cultured for 24 hours. Then, 500 μL of EPD@Gel hydrogel extract containing 1 mmol / L H2O2 was added to each well, and the treatment continued for 5 hours. After washing, the cells were incubated with 10% (v / v) CCK-8 reagent for 2 hours, and the absorbance was measured at 450 nm. For comparison, the same method was used to detect Exos@Gel hydrogel, Gel hydrogel, 3M gel, and PBS buffer, with cells treated only with H2O2 serving as a control. The preparation methods of Exos@Gel hydrogel extract, Gel hydrogel extract, and 3M gel extract were basically the same as those described in step (1) above for EPD@Gel hydrogel extract, except that EPD@Gel hydrogel was replaced with Exos@Gel hydrogel, Gel hydrogel, and 3M gel, respectively.

[0101] The results of the CCK-8 cell viability assay are as follows: Figure 9 As shown. By Figure 9 It was found that H2O2 stimulation alone led to a significant decrease in cell viability, while the decrease in viability was significantly alleviated in cells pretreated with various hydrogel extracts. Among them, the EPD@Gel group showed the best cell protection effect, with cell viability restored to near normal levels. This excellent protective effect can be attributed to the triple synergistic effect of exosomes, the delivered VEGFA gene, and the hydrogel matrix: the ROS scavenging ability of the hydrogel itself alleviated the direct damage of oxidative stress; the natural active ingredients carried by exosomes helped maintain cell homeostasis; and the overexpression of VEGFA may further enhance the cell's resistance to and repair capacity against oxidative stress by activating downstream survival signaling pathways.

[0102] (8) Antibacterial performance test of EPD@Gel hydrogel: The antibacterial efficacy of EPD@Gel hydrogel against *Escherichia coli* and *Staphylococcus aureus* was assessed using the agar diffusion method and the surface contact method, while simultaneously evaluating bacterial membrane integrity and morphological characteristics. In the agar diffusion experiment, bacterial biofilms (10T) were... 9 CFU mL -1 The bacterial culture was incubated with filter discs containing hydrogel extract or PBS (control group) at 37°C for 12 hours, and the inhibition zone was then measured. In the surface contact experiment, bacterial flora (10... 6 CFU mL -1The bacteria were directly contacted with the hydrogel for 2 hours, and the survival rate was determined by serial dilution plate counting. Subsequently, the integrity of the bacterial membrane was analyzed using the LIVE / DEAD BacLight kit combined with fluorescence microscopy. After standard fixation, dehydration and critical point drying, morphological changes were observed using scanning electron microscopy (3kV).

[0103] For comparison, the same method was used to test the antibacterial properties of Exos (prepared in Example 1), EP complex (PEI solution was added dropwise to the Exos suspension prepared in Example 1, and incubated at room temperature for 30 min to obtain the EP complex, with a mass ratio of Exos to PEI of 2:1), 3M gel, and PBS buffer. The preparation method of the 3M gel extract was basically the same as the preparation method of the EPD@Gel hydrogel extract described in step (1) above, except that the EPD@Gel hydrogel was replaced with a 3M gel.

[0104] Test results as follows Figure 10 As shown. By Figure 10 It can be seen that the agar diffusion experiment shows (e.g.) Figure 10 As shown in Figure a), the PBS, 3M, and Exos groups only produced weak zones of inhibition (ZOI) or no inhibition areas, indicating limited antibacterial activity. In contrast, the EP complex, due to the inherent antibacterial properties of polyethyleneimine (PEI), showed significant ZOI against both strains. Notably, the EPD@Gel hydrogel exhibited the strongest antibacterial effect, significantly superior to other groups, and quantitative analysis of the inhibition zones further confirmed this conclusion (e.g., ...). Figure 10 (As shown in b). Antimicrobial activity was quantitatively assessed by colony counting. The bacterial-hydrogel co-culture suspension was serially diluted, plated, and incubated for 12 hours (e.g., ...). Figure 10 As shown in Figure c), the bacterial survival rates in the EP-treated group decreased to 55.4% (Escherichia coli) and 30.7% (Staphylococcus aureus); while EPD@Gel treatment caused the survival rates of Escherichia coli and Staphylococcus aureus to drop sharply to 11.1% and 3.6%, respectively (as shown in Figure c). Figure 10 As shown in Figure e), it demonstrates its superior antibacterial efficacy. Live / dead bacterial staining directly visualizes membrane integrity damage (e.g., ...). Figure 10 As shown in d and f), the control group mainly exhibited green fluorescence (SYTO9-labeled live bacteria), while the EPD@Gel group showed strong red fluorescence (propidium iodide-labeled membrane-damaged dead bacteria), indicating a large number of bacterial deaths. Scanning electron microscopy (SEM) was used to observe bacterial morphological changes (e.g., ...). Figure 10As shown in g), untreated bacteria maintained a smooth, intact morphology. Similar to the 3M, Exos, and PBS groups, EP treatment induced bacterial membrane shrinkage and perforation, while EPD@Gel treatment resulted in widespread bacterial lysis, leaving only cell debris in the field of view. Therefore, the EPD@Gel hydrogel prepared in this invention exhibits a certain potential to inhibit bacterial growth. This may be due to the hydrogel's excellent swelling properties, which enable it to effectively absorb excess wound exudate. By regulating the humidity of the wound microenvironment and diluting the local bacterial concentration, it creates a microenvironment unfavorable to excessive bacterial proliferation for wound healing.

[0105] Example 5: Study on the effect of EPD@Gel hydrogel in promoting the healing of diabetic wounds 1. Laboratory animals, experimental groups, and treatment methods for each group: All animal experiments were approved by the Animal Ethics Committee of Zhengzhou University (Program No. ZZU-LAC20241129). A type 1 diabetic mouse model was established by inducing 6-8 week old male BALB / c mice with streptozotocin (100 mg / kg / day, intraperitoneal injection, for 2 days). The diabetic state was confirmed by hyperglycemia (>16.7 mMol / L) 14 days later.

[0106] A 15 mm diameter full-thickness wound was created on the back of type 1 diabetic mice anesthetized with pentobarbital (50 mg / kg, intraperitoneal injection), and Staphylococcus aureus (20 μL, 10) was inoculated. 9 CFU mL -1 Then, type 1 diabetic mice with full-thickness wounds on their backs were randomly divided into five groups (n=3 per group): (1) PBS group, (2) 3M group, (3) Gel group, (4) Exos@Gel group, and (5) EPD@Gel group. The EPD@Gel group used EPD@Gel hydrogel prepared in Example 4 of this invention as a dressing applied to the wound; the Exos@Gel group used Exos@Gel hydrogel (prepared according to the preparation method described in step 4 of Example 4) as a dressing applied to the wound; the Gel group used P6F1.5C3 hydrogel prepared in Example 3 as a dressing applied to the wound; the 3M group used 3M gel (3M TM Tegaderm TM The hydrocolloid dressing (product number: 91110) was applied to the wound as a dressing; the PBS group used sterile PBS solution to be dripped onto the wound surface; the dressings of both groups were changed every 3 days.

[0107] Wound closure was monitored on days 0, 3, 7, 11, and 14, and the wound healing rate was calculated using the formula: Healing rate = (A0 - A) / (A0 - A) t ) / A0×100%, where A0 is the initial wound area, At The area represents the wound at time t. Additionally, the bacterial load in the wound homogenate of mice in each group was measured on day 14.

[0108] Wound tissue (including healing margin and new tissue) was collected on days 7 and 14 for histological (hematoxylin-eosin (H&E) staining and Masson trichrome staining) and immunofluorescence analysis. During immunofluorescence staining, the sections were stained with the following primary antibodies: anti-CD31 / α-SMA (catalog numbers GB120005 and GB111364, Servicebio), anti-HIF-1α (catalog number GB111339, Servicebio), anti-VEGFA (catalog number 19003-1-AP, Proteintech), anti-IL-10 (catalog number GB11534, Servicebio), anti-TNF-α (catalog number 60291-1-Ig, Proteintech), anti-Ki-67 (catalog number A16919, ABclonal), anti-type I collagen (catalog number GB124197, Servicebio), anti-integrin α3 (catalog number 66070-1-Ig, Proteintech), and anti-Vinculin (catalog number 26520-1-AP, Proteintech). After washing with PBS, sections were incubated with CoraLite 488 or 594-labeled secondary antibody for 1 hour, counterstained with DAPI, and observed under a fluorescence microscope. Image analysis was performed using ImageJ software.

[0109] Representative wound photographs of mice in each group on days 0 / 3 / 7 / 11 / 14 after treatment are shown below. Figure 11 As shown in Figure b, continuous images of wound closure from day 0 to day 14 are shown below. Figure 11 As shown in Figure 11c, on day 0, the wound sizes of all groups were consistent; on day 3, the wounds in the PBS and 3M groups showed weak contraction, while those in the hydrogel-treated groups shrank significantly; on day 7, all wounds continued to shrink, but only the EPD@Gel hydrogel group achieved near-complete closure by day 14. The Gel and Exos@Gel groups still had residual wounds, while the PBS and 3M groups showed obvious signs of infection throughout the healing process, resulting in the worst healing. A photograph of Staphylococcus aureus colonies cultured from the wound homogenate on day 14 is shown below. Figure 11 As shown in d. From Figure 11 As shown in the data, the PBS group and the 3M group had the highest bacterial density, the Gel group and the Exos@Gel group showed moderate colony growth, and the EPD@Gel group significantly inhibited bacterial proliferation, confirming the highly efficient antibacterial properties of EPD@Gel hydrogel in vivo.

[0110] Quantitative analysis of wound closure percentage and pseudo-color graph of wound area percentage at specified time points, as shown below. Figure 11As shown in e and f. From Figure 11 As shown in e and f, the closure rate of the EPD@Gel group on day 14 reached 97.10%, which was significantly better than that of Exos@Gel (92.7%), Gel (89.0%), 3M (83.5%) and PBS group (78.2%).

[0111] Representative H&E staining results of wound tissue on days 7 and 14 are as follows: Figure 11 As shown in Figure g. H&E staining revealed that on day 7, all groups exhibited predominantly granulation tissue without complete epithelial coverage; on day 14, the PBS and 3M groups showed excessively thickened newly formed epithelium accompanied by persistent inflammatory infiltration; furthermore, the epithelial thickness measurement results on day 14 showed that the Exos@Gel and EPD@Gel groups formed a thin layer of mature epithelium approaching the thickness of normal skin (as shown in Figure g). Figure 11 (h). This result demonstrates that the EPD@Gel hydrogel of this invention possesses both aesthetic and functional healing advantages.

[0112] Representative Masson trichrome staining results of wound tissue on days 7 and 14 are as follows: Figure 11 As shown in Figure g. Masson trichrome staining assessment of collagen deposition showed that on day 7, newly formed blue collagen fibers were visible in the hydrogel group, particularly prominent in the EPD@Gel group; on day 14, collagen arrangement was disordered in the control group, while dense and highly ordered collagen bundles formed in the EPD@Gel group. The epithelial collagen volume fraction quantitative analysis results on day 14 are shown in Figure g. Figure 11 As shown in i, by Figure 11 As can be seen from i, EPD@Gel hydrogel has the ability to promote collagen maturation.

[0113] Furthermore, biocompatibility was assessed 14 days after treatment by H&E staining of major organs (heart, liver, spleen, lungs, and kidneys). Results showed that no adverse reactions occurred in any group during treatment, and blood routine and biochemical parameters remained within normal ranges, demonstrating that the hydrogel system exhibits excellent biocompatibility during wound healing and does not cause systemic toxicity.

[0114] The results of immunofluorescence detection of the expression of CD31, α-SMA, HIF-1α, and VEGFA in wound neodermal tissue on days 7 and 14 are as follows: Figure 12 , Figure 13 , Figure 14 a, Figure 14 As shown in e. From Figure 12 , Figure 13 , Figure 14 a, Figure 14As shown in Figure e, the EPD@Gel group exhibited the strongest positive signals for CD31 and α-SMA on day 14, significantly higher than other groups. Furthermore, the expression of VEGFA and HIF-1α was also most pronounced in the EPD@Gel group. This is due to the local overexpression of VEGFA induced by the EPD@Gel hydrogel and the pro-angiogenic activity of Exos itself. In addition, the hydrogel scaffold provides a moist and protective microenvironment, supporting endothelial cell budding and lumen formation, ultimately promoting efficient wound angiogenesis.

[0115] The levels of IL-10 and TNF-α in the wound bed are key indicators for assessing inflammation and tissue healing. The results of immunofluorescence assays of IL-10 and TNF-α levels in the wound neodermis on days 7 and 14 are shown below. Figure 14 As shown in b, c, f, and g. Among them, by Figure 14 As shown in Figures b and f, on day 14 of treatment, the expression of the anti-inflammatory factor IL-10 in the EPD@Gel group was significantly higher than that in the control group, indicating that this treatment can enhance the anti-inflammatory microenvironment and is beneficial to the healing process. Figure 14 As shown in c and g, TNF-α expression was high in all groups during the peak of inflammation on day 7. By day 14, the PBS and 3M groups still maintained high expression, reflecting persistent inflammation and delayed healing. In contrast, the TNF-α levels in the Gel, Exos@Gel, and EPD@Gel groups decreased significantly, with the EPD@Gel group showing the most significant decrease, confirming its effective ability to alleviate the inflammatory response.

[0116] The results of immunofluorescence detection of the expression of the proliferation marker Ki67 in the wound neodermal tissue on days 7 and 14 are as follows: Figure 14 As shown in d and h. From Figure 14 As shown in d and h, on day 14, the expression of Ki67 in the EPD@Gel group cells was significantly enhanced, indicating that while the EPD@Gel hydrogel exerts anti-inflammatory and antioxidant effects, it also creates a favorable microenvironment for wound healing, thereby promoting cell proliferation and significantly accelerating the closure of diabetic wounds.

[0117] Collagen deposition is a crucial step in extracellular matrix (ECM) remodeling during wound healing, providing structural support and mechanical strength for newly formed tissue. Type I collagen, as a major structural protein of the ECM, not only maintains tissue integrity but also provides a scaffold for cell migration and angiogenesis, making it essential for functional regeneration. The results of immunofluorescence detection of collagen I levels in wound neodermal tissue on days 7 and 14 are shown below. Figure 15 As shown in a and b. From Figure 15As shown in Figures a and b, on day 14, compared with the PBS and 3M groups, all treatment groups (Gel, Exos@Gel, and EPD@Gel) exhibited higher collagen deposition, with the EPD@Gel group showing the most significant increase. This result is consistent with Masson's trichrome staining. Furthermore, the collagen fibers in the EPD@Gel group were more densely and orderly arranged, indicating that EPD@Gel hydrogel can effectively promote ECM functional reconstruction.

[0118] The composition and structure of the ECM directly affect cell adhesion, migration, proliferation, and differentiation, and its dynamic interaction with cells is a core mechanism of wound repair. Further analysis was conducted on key molecules related to this process. Integrin α3, as a major cell surface adhesion receptor, mediates the binding of cells to ECM components (such as laminin and collagen), regulating cell proliferation, migration, and survival. Vinculin, as a key downstream adaptor protein, is enriched in adhesion focals, stabilizing integrin-mediated cell-ECM connections and transmitting mechanical signals to the cytoskeleton, providing support for cell spread, migration, and vascular stability. Based on the crucial role of ECM-cell interactions in wound repair, the expression results of integrin α3 and Vinculin (vinculin) in wound neodermal tissue at days 7 and 14, detected by immunofluorescence, are as follows: Figure 15 As shown in points c, d, e, and f. (From...) Figure 15 As shown in C, D, E, and F, the expression of integrin α3 and Vinculin protein in the EPD@Gel treatment group showed an upregulated trend. This phenomenon may be due to EPD@Gel promoting collagen formation and ECM remodeling, providing a suitable environment for cell migration and proliferation. Furthermore, by activating integrin α3 and upregulating Vinculin, extracellular mechanical signals are transduced into the cell, thereby effectively regulating cell adhesion, spreading, proliferation, and migration behavior, ultimately accelerating the wound regeneration and closure process.

[0119] Example 6: Study on the hemostatic properties of EPD@Gel hydrogel BALB / c mice anesthetized with pentobarbital (50 mg / kg, intraperitoneal injection) were used to evaluate hemostasis using liver hemorrhage and tail amputation models. Simultaneously, Exos@Gel hydrogel, Gel hydrogel (i.e., the P6F1.5C3 hydrogel prepared in Example 3), and 3M gel (3M...) were used to assess hemostasis. TM Tegaderm TM Hydrocolloid dressing (catalog number: 91110) and PBS buffer were used as controls.

[0120] In a liver hemorrhage model, the liver was exposed via midline laparotomy, and a standard penetrating puncture wound was created in the mouse liver lobe using an 18-gauge needle. EPD@Gel hydrogel was immediately applied to seal the bleeding site, ensuring complete coverage. The wound was allowed to solidify in situ. Simultaneously, Exos@Gel hydrogel, gel hydrogel (i.e., the P6F1.5C3 hydrogel prepared in Example 3), and 3M gel (3M...) were used. TM Tegaderm TM Hydrocolloid dressing (catalog number: 91110) and PBS buffer were used as controls. Representative bleeding images of each experimental group were recorded during hemostasis of mouse liver, and the amount of blood loss in liver tissue of each group was counted. The results are as follows: Figure 16 As shown in Figures A and B. From Figure 16 As shown in Figures A and B, the EPD@Gel hydrogel group, Exos@Gel hydrogel group, and Gel hydrogel group rapidly formed a physical barrier after application, with only a very small amount of oozing observed, and the bleeding was controlled within a short time. In stark contrast, the PBS solution control group failed to provide any hemostasis, and the wound continued to bleed; while the commercially available 3M transparent dressing could cover the wound, it lacked active adhesion and sealing capabilities, and active bleeding was still visible underneath, resulting in significant blood loss. This preliminarily demonstrates that the EPD@Gel hydrogel prepared in this invention has excellent rapid sealing and hemostatic capabilities for visceral tissue wounds.

[0121] To further verify its performance in bleeding scenarios involving large-diameter blood vessels, a mouse tail truncation model was established. 30% of the distal tail of the mice was surgically removed, and the wound was immediately treated with hydrogel after truncation. Representative bleeding images of each experimental group were recorded during tail hemostasis, and the amount of blood loss in the tail region of each group was calculated. The results are as follows: Figure 16 As shown in C and D. From Figure 16 As shown in C and D, EPD@Gel hydrogel, Exos@Gel hydrogel, and Gel hydrogel can adhere tightly to the moist wound surface after transection, effectively sealing the severed ends of the caudal arteries and veins, and rapidly achieving hemostasis. In contrast, the 3M gel and PBS buffer-treated groups exhibited continuous active bleeding. These results further confirm the strong and reliable hemostatic properties of hydrogels in complex wounds involving muscle, skin, and larger blood vessels.

[0122] In summary, this hydrogel demonstrates excellent rapid hemostasis in both delicate visceral puncture wounds and large blood vessel bleeding caused by transection. Its efficacy likely stems from the synergistic effect of multiple mechanisms: 1) excellent wet tissue adhesion, enabling immediate and tight wound sealing; 2) rapid in-situ gelation properties, forming a physical barrier; and 3) its porous network structure, which may facilitate the accumulation of blood components and promote local coagulation. These properties make it a highly promising material for acute wound hemostasis.

[0123] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications. These are equivalent embodiments with variations. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical concept of the present invention still fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a pH-responsive hydrogel based on engineered exosome integration, characterized in that, Includes the following steps: (1) After mixing exosomes with PEI solution, the mixture was incubated at room temperature. After the incubation was completed, the exosome-PEI complex was obtained. (2) The exosome-PEI complex was mixed with plasmid DNA and then incubated at room temperature. After the incubation was completed, the exosome-PEI-plasmid DNA complex was obtained. (3) γ-PGA was modified with dopamine to obtain DA-modified γ-PGA; the DA-modified γ-PGA solution was mixed with 3-FPBA solution to obtain PGA-DA / 3-FPBA solution; (4) The exosome-PEI-plasmid DNA complex prepared in step (2) is added to the PGA-DA / 3-FPBA solution, mixed well, and then the mixture is mixed with carboxymethyl chitosan solution and reacted at room temperature to prepare pH-responsive hydrogel.

2. The preparation method according to claim 1, characterized in that, The exosomes are exosomes secreted by mesenchymal stem cells; the plasmid DNA contains the VEGFA gene.

3. The preparation method according to claim 2, characterized in that, The mesenchymal stem cells are human umbilical cord mesenchymal stem cells; the plasmid DNA is pECMV-Vegfa-m-FLAG plasmid.

4. The preparation method according to any one of claims 1-3, characterized in that, The mass ratio of exosomes to PEI is (1-5):1, and the mass ratio of PEI to plasmid DNA is (1-10):

1.

5. The preparation method according to claim 4, characterized in that, The concentration of PGA-DA in the PGA-DA / 3-FPBA solution is 8–12 wt%; the mass concentration of 3-FPBA in the PGA-DA / 3-FPBA solution is 2.5 wt%. The concentration of the carboxymethyl chitosan solution is 5–10 wt%.

6. The preparation method according to claim 5, characterized in that, In step (4), the concentration of plasmid DNA in the mixture is 1 to 100 µg / mL; the volume ratio of the mixture to the carboxymethyl chitosan solution is (1 to 5): (1 to 3).

7. The preparation method according to claim 6, characterized in that, The DA-modified γ-PGA is prepared by covalently linking the amino group of DA to the carboxyl group of γ-PGA.

8. A pH-responsive hydrogel prepared using any one of the preparation methods described in claims 1-7.

9. The use of the pH-responsive hydrogel of claim 8 in the preparation of wound dressings, the dressings being used for wound hemostasis and / or promoting wound healing.

10. The application according to claim 9, characterized in that, The wound includes skin ulcers.

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