Abstract The orderly regulation of immune inflammation and promotion of the regeneration of skin vessels and fibers are key to the treatment of diabetic skin injury (DSI). Although various traditional polypeptide biological dressings continue to be developed, their efficacy is not satisfactory. In recent years, plant-to-mammal regulation has provided an effective approach for chronic wound management, but the development of effective plant-based treatments remains challenging. The development of exosomes from Chinese herbs is promising for wound healing. In this study, plant exosomes derived from lemons (Citrus limon) were extracted, and their biological efficacy was verified. Lemon exosomes regulated the polarization reprogramming of macrophages, promoted the proliferation and migration of vascular endothelial cells and fibroblasts, and thus promoted the healing of diabetic wounds. To solve the problems of continuous drug delivery and penetration depth, Lemon Exosomes were loaded into a hydrogel constructed of Gelatin Methacryloyl (GelMA) and Dialdehyde Starch (DAS) that closely fits to the skin, absorbs water, swells, and is moist and breathable, effectively promoting the sustained and slow release of exosomes and resulting in excellent performance for diabetic wound healing. Our GelMA-DAS-Lemon Exosomes hydrogel (GelMA/DAS/Exo hydrogel) patch represents a potentially valuable option for repairing diabetic wounds in clinical applications. Graphical Abstracts [38]graphic file with name 12951_2025_3138_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03138-y. Keywords: Diabetic wound healing, Macrophage reprogramming, Lemon exosomes, Angiogenesis, Network pharmacology Introduction Diabetic skin wounds in the context of diabetes are often difficult to heal, which seriously affects the quality of life of patients and imposes an enormous burden on society and the economy [[39]1]. At present, the conventional treatment methods for diabetic skin injury (DSI) mainly include debridement, wound dressing, lesion decompression, anti-infection measures, peripheral vascular disease management and strict blood glucose control [[40]2]. However, these treatments have limited effects on skin healing. The incidence of poor prognoses, such as amputation, continues to increase annually (19.4% higher in 2023 than in 2013) [[41]3]. With the increasing number of diabetic patients worldwide and the lack of effective treatment methods [[42]4], there is an urgent need to develop new solutions for the treatment of diabetic wounds. To solve this issue, research has been conducted to develop drug delivery systems for diabetic skin lesions, such as polymer-based nanoparticles [[43]5], liposomes [[44]6], and micellar hydrogel dressings [[45]7], which are widely studied owing to their cost effectiveness and ease of modification. However, their disadvantages are that they generate certain hepatorenal toxicity, inflammation and immunogenicity. In recent years, biomimetic natural nanoparticles, such as mammalian cell-derived exosomes, have attracted attention for the treatment of various diseases because of their specific tissue targeting ability [[46]8], high biocompatibility, and limited cytotoxicity [[47]9]. As previously reported by Dae Hyun Ha et al., the use of mesenchymal stem cell/stromal cell-derived exosomes in the immunomodulatory treatment of skin regeneration has yielded satisfactory results [[48]10]. Yang An et al. used adipose-derived stem cell exogenesis to accelerate skin wound healing [[49]11]. However, exosomes of animal origin still face serious challenges, including (i) low production yield [[50]12], (ii) time-consuming and laborious production processes [[51]13], (iii) difficulty in producing many homogeneous exosomes [[52]14], and (iv) immunogenicity and toxicity between different animal cells [[53]15]. To solve these problems, exosomes from Chinese herbal plants have become an alternative treatment option [[54]16]. Exosomes of plant origin have the advantages of being easier to obtain, lower in cost, and not requiring extensive cell culture steps [[55]17]. Moreover, the lipids, proteins and nucleic acids carried by natural exosomes from plants have corresponding antioxidant, anti-inflammatory, antibacterial and antitumor properties and have a wide range of therapeutic value [[56]18, [57]19]. To date, plant exosomes derived from grapes, tea, grapefruit, ginseng, and turmeric have been reported to be remarkably effective in the treatment of colitis, liver damage, infections, and tumors [[58]20–[59]22]. However, the application of Lemon (Citrus limon) Exosomes with oxidative regulatory effects on wound healing in chronic diabetes has not been reported, and further exploration is needed. In addition, effective administration strategies, slowly release times, and material protection of the wound barrier are critical in the management of DSI. As a preferred alternative to betadine and silver ions, drug-encapsulated hydrogel dressings address the limitations observed in common wound dressings, such as medical cotton, bandages, and gauze, with a wide range of drug delivery efficiency and depth of penetration and good tissue adhesion [[60]23]. Hydrogels can absorb tissue exudate to maintain a dry environment; additionally, the barrier effect of hydrogels can result in good isolation of pathogenic microorganisms [[61]24–[62]26]. In addition, hydrogel-loaded dressings have the advantages of good air permeability, ultralight weight, and easy portability and storage [[63]26]. Therefore, hydrogels have important potential therapeutic value in the healing of DSI. In this work, inspired by natural adhesives, we designed and prepared a semisynthetic hydrogel that combines Lemon Exosomes with a Gelatin Methacryloyl (GelMA)-Dialdehyde starch (DAS) hydrogel that was identified as safe by the FDA [[64]27–[65]29] and further explored its wound healing effect and mechanism of action. Lemon exosomes (Exo) and GelMA are renewable compounds, and hydrogel preparation is convenient, environmentally friendly and inexpensive. In particular, GelMA-DAS-Lemon Exosomes hydrogel (GelMA/DAS/Exo hydrogel) can also be gelated in situ as adhesive dressings to better fill and seal irregular wounds. We systematically studied the biomaterial properties of GelMA/DAS/Exo hydrogels, including their stability, viscoelasticity, adhesion and biocompatibility. Transcriptomics and network pharmacology were combined to identify the pathogenic mechanism involved. Immunofluorescence staining, Western blotting and tissue staining were used to investigate the effects of Lemon Exosomes on immune regulation, vascular regeneration and collagen fiber generation. Moreover, we assessed the chronic wound healing effect of the GelMA/DAS/Exo hydrogel in a type 1 diabetic rat model. The results showed that the GelMA/DAS/Exo hydrogel may be a potential candidate material for the clinical treatment of DSI (Fig. [66]1). Fig. 1. [67]Fig. 1 [68]Open in a new tab GelMA/DAS/Exo hydrogel was used as a dressing for wound healing in DSI. (A) Preparation of the lemon exosome hydrogel. (B) The lemon exosome hydrogel promoted diabetic wound healing by regulating macrophage polarization and promoting fibroblast and vascular endothelial cell proliferation Materials and methods RNA sequencing (RNA-seq) and bioinformatic analysis Streptozotocin (STZ) was used to construct a DSI rat model, and RNA sequencing was conducted using samples from the experimental group and the blank control group. Total RNA was isolated and purified with Trizol (Thermo Fisher, 15596018) according to the manufacturer’s instructions. The quantity and purity of the total RNA were subsequently determined with a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and the integrity of the RNA was assessed with a Bioanalyzer 2100 (Agilent, CA, USA). The concentration was > 50 ng/µL, the RIN value was > 7.0, and the total RNA concentration was > 1 µg. Oligo (dT) magnetic beads (Dynabeads Oligo (dT), cat. 25-61005; Thermo Fisher, USA) were used to specifically capture mRNA containing polyadenylate (polyA) through two rounds of purification. cDNA was synthesized from the fragmented RNA via Invitrogen Super-Script™ II Reverse Transcriptase (cat. 1896649, CA, USA). cDNA fragments were connected to sequencing adapters to form a library. This process included terminal modifications, connection adapters, PCR amplification, etc. The constructed library contained DNA copies of the original RNA fragments. The sequencing data were analyzed using bioinformatics tools. Differential gene analysis was performed on the sequencing dataset using the Limma package in R. OV-related module genes were obtained via WGCNA using the WGCNA package. The cluster Profiler package was used to carry out GO and KEGG enrichment analyses of the DEGs and gene clusters, identify characteristic pathways, verify the relevant expression molecules in the pathways, and characterize the results through heatmaps. Network pharmacological screening of lemon-DSI-associated target pathways We searched the Bioinformatics Analysis Tool for Molecular Mechanisms of Traditional Chinese Medicine (BATMAN-TCM) ([69]http://bionet.ncpsb.org.cn/batman-tcm/)and relevant literature for the possible chemical composition of lemon by using the search term “Ingredient” OR “Herb name” OR “Compound” = “Lemon”. Material synthesis and characterization Gelatin (from porcine skin) and methacrylic anhydride were purchased from Sigma-Aldrich. DAS (with an aldehyde group content of 15.86 ± 0.07 mmol/g) was purchased from Jinshan Modified Starch Co., Ltd. (Shandong, China). 2-Hydroxy-1-(4- (hydroxyethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959) was purchased from BASF, Germany. Isolation and purification of lemon exosomes Fresh lemons were purchased from Squeeze to collect lemon juice. After the lemon juice was filtered with a 40 μm filter, large particles were removed via centrifugation at 1000 × g for 20 min, 3000 × g for 20 min, 10,000 × g for 30 min, and 70,000 × g for 1 h. The supernatant was subsequently centrifuged at 135,000 × g for 90 min, and the precipitate was suspended in PBS to obtain a crude lemon sample. The crude lemon sample was diluted with PBS. The mixture was centrifuged at 135,000 × g for 90 min. The collected lemon exosomes were stored at -80 °C for future use. Characterization of the lemon exosomes The morphology of the lemon exosomes was observed via transmission electron microscopy (TEM). The particle size and zeta potential of the lemon exosomes were measured using a Zetasizer Nano ZS (Malvern). Phagocytosis validated exosome targeting. Synthesis of GelMA To produce photocrosslinkable GelMA chains, gelatin was dissolved in phosphate-buffered saline (PBS) (10 % w/v) at 50 °C. Methacrylic anhydride (0.8 mL per gram of gelatin) was added, and the mixture was stirred for 2 h at 50 °C to modify the lysine groups on the gelatin chains. To terminate the methacryloyl modification reaction, the solution was diluted with PBS. The solution was dialyzed using dialysis membrane tubing with a 12–14 kDa molecular weight cutoff in distilled water for 7 days at 40 °C to remove unreacted methacrylic anhydride. Then, the solution was heated to 50 °C and filtered through a vacuum filter with a 0.22 μm pore size. Finally, the solution was lyophilized for 7 days to obtain dried GelMA foam. Nuclear magnetic resonance (1 H NMR) spectroscopy was employed to characterize the GelMA polymer. The samples were dissolved in deuterium oxide (D[2]O). Preparation of the GelMA/DAS/Exo hydrogel GelMA (6 g) was mixed in 30 mL of PBS containing 0.5% (w/v) Irgacure 2959 at 80 °C. A 10% (w/v) DAS solution was stirred in a boiling water bath until it became a paste. An equal volume of DAS solution was subsequently slowly added to the GelMA solution while stirring. The prepared hydrogels were designated GelMA-DAS hydrogels and it is cured by UV irradiation after preparation. GelMA/DAS/Exo hydrogel were prepared using the same procedure, and lemon exosomes were added at the concentration of 20 ug/mL. Characterization of the GelMA/DAS/Exo hydrogel To quantify the chemical constitution of the hydrogels, Fourier transform infrared (FTIR) spectroscopy was performed using a Nicolet-iS50R spectrophotometer (Thermo Fisher, US) in the range of 4000–400 cm^− 1. Scanning electron microscopy (SEM, Apreo S HiVac, FEI, US) was used to analyze the microstructure of the hydrogels. Rheology analysis of the GelMA/DAS/Exo hydrogel The mechanical properties of the GelMA/DAS/Exo hydrogel were assessed using a rotary rheometer (Physica MCR302, Anton Paar) in frequency sweep mode from 0.1 to 10 Hz with 5.0% strain. A strain amplitude sweep test (strain [γ] = 1–300%) was performed to obtain the critical strain point. The self-healing capacity of the hydrogels was assessed through an alternating step‒strain scanning test utilizing a constant frequency of 1 Hz. The oscillatory strain alternated between a small amplitude (γ = 1%) and a large amplitude (γ = 300%), each maintained for 10 s. The injectable properties of the gel samples were characterized by measuring the linear viscosity (η) in frequency sweep mode. Adhesion strength test of the GelMA/DAS/Exo hydrogel Lap shear tests were conducted using GelMA/DAS/Exo hydrogel samples attached to porcine skin tissue (20 mm long and 10 mm wide) via a dynamic mechanical analyzer (DMA Q800, USA) to assess the adhesive strength of the GelMA/DAS/Exo hydrogel to biological tissue. In vitro tests Live/dead cell staining A live/dead assay and cytoskeletal staining were applied to evaluate the in vitro biocompatibility of samples. To perform the live/dead staining, 5 × 10^5 L929 cells and HUVECs were seeded on each sample for 24 h. Next, live/dead staining solution containing Calcein-AM (Invitrogen, USA), propidium iodide (PI, Invitrogen, USA) and PBS was prepared and then added to each sample for 30 min at 37 °C, followed by observation under a laser confocal microscope (ZEISS LSM 980, Germany). Cell cytoskeletal staining Cytoskeletal staining of L929 cells and HUVECs was conducted as follows. In brief, 1 × 10^5 cells were cultured on each sample in 12-well plates for 3 days, fixed with 4% paraformaldehyde, stained with β-actin tracker green (Beyotime, China) and Hoechst (Sigma, USA), and observed under a laser confocal microscope (ZEISS LSM 980, Germany). Gene expression Total RNA was harvested using a total RNA kit (Omega, USA) and then reverse-transcribed into cDNA via a reverse transcription kit (Takara, Japan). Real-time qPCR (RT‒qPCR) was conducted using LightCycler 480 SYBR Green Master Mix (Takara, Japan). This experiment was repeated in triplicate, and expression was calculation using the 2–ΔΔCt method. Table [70]1 displays the primers used in the present study. Table 1. Displays the primers used in the present study Target Forward Reverse GAPDH CCTCGTCCCGTAGACAAAATG TGAGGTCAATGAAGGGGTCGT iNOS AGCTCGGGTTGAAGTGGTATG CACAGCCACATTGATCTCCG Arg-1 CTGGGGATTGGCAAGGTGAT CAGCCCGTCGACATCAAAG IL-10 GACTTTAAGGGTTACTTGGGTTGC CTTATTTTCACAGGGGAGAAATCG TNF-α AGCCGATGGGTTGTACCTTG ATAGCAAATCGGCTGACGGT [71]Open in a new tab Immunofluorescence (IF) Paraformaldehyde (4%) was used to fix the cells for 30 min. Next, the fixed samples were permeabilized with 0.2% Triton X-100 (Biofroxx, Germany) for 1 h at room temperature, followed by blocking with 3% BSA (Biofroxx, Germany) for 1 h. Then, the samples were incubated with the corresponding primary antibodies overnight at 4 °C (Table [72]2). After the samples were washed three times with PBS, secondary antibodies were added for another hour at room temperature. Finally, Hoechst 33342 (Sigma, USA) was used to stain the nuclei before fluorescence images were captured under a confocal reflection microscope (ZEISS LSM 980, Germany). Table 2. Related information about primary antibodies Antibodies Species Type Dilution(IF/WB/IHC) Source Anti-GAPDH Rabbit Monoclonal lgG 1:2000 ProteinTech, China Anti-Arg-1 Rabbit Polyclonal lgG 1:1000/1:200/1:100 ProteinTech, China Anti-iNOS Rabbit Recombinant lgG 1:1000/1:200/1:100 ProteinTech, China Anti-CD31 Rabbit Polyclonal lgG 1:1000/1:200/1:100 ProteinTech, China Anti-VEGFA Rabbit Polyclonal lgG 1:1000/1:200/1:100 ProteinTech, China Anti-αSMA Rabbit Polyclonal lgG 1:1000/1:200/1:100 ProteinTech, China [73]Open in a new tab Western blot (WB) assay Cells or tissues were washed with PBS and lysed in radioimmunoprecipitation assay (RIPA, CWBIO, China) buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher, USA). Next, the lysates were sonicated, followed by centrifugation at 4 °C. The obtained supernatant was transferred to a new centrifuge and stored at − 20 °C. The total protein concentration was measured with a BCA assay kit (Beyotime, China). After the addition of loading buffer (Beyotime, China) to the protein mixture and denaturation at 100 °C for 10 min, equal amounts (40 µg) of protein mixture were loaded on sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE, Beyotime, China) gels to separate the proteins. Then, the proteins were transferred onto polyvinylidene difluoride (PVDF, Thermo Fisher, USA) membranes. The PVDF membranes were then incubated in high-efficiency blocking solution (Genefist, China) for 10 min, followed by incubation with specific primary antibodies at 4 °C overnight (Table [74]2). After incubation with the corresponding secondary antibodies, the protein bands were visualized with an enhanced chemiluminescence (ECL, Thermo Fisher, USA) kit, and images were acquired using a GelView 6000 Pro (BLT, China). The density of the bands was quantitatively analyzed by ImageJ software. Flow cytometry A total of 1 × 10^5 ~ 1 × 10^7 cells were placed in a 1.5 mL centrifuge tube and centrifuged at 3000 r/min for 5 min, after which the supernatant was discarded. The cells were resuspended in 100 µL of FACS buffer. An anti-Fc receptor antibody (0.5 µL, 0.5 mg/mL) was added to each centrifuge tube, and the tubes were placed in a water bath for 3 min. Then, 1 µl (0.5 mg/mL) of fluorescent antibody was added, and the tubes were placed in a water bath for 30 min. Finally, 350 µL of FACS buffer was added, the samples were gently mixed, and then centrifuged for 5 min at 3000 rpm/min. The supernatant was discarded, and 100 µL of instrument buffer was added to each tube, followed by gentle mixing to resuspend the cells. The cell suspension was transferred into a special tube for FACS. The antibodies used for flow cytometry are listed below: FITC anti-mouse F4/80 (157310, BioLegend), PE anti-mouse CD86 (159204, BioLegend), APC anti-mouse CD206 (141708, BioLegend). Cell migration assay For the cell migration assay, a transverse scratch was created to simulate an artificial wound after HUVECs and L929 reached a confluence of approximately 90%. Next, PBS was used to wash the samples gently three times to remove floating cells. Then, the cells were stained with Calcein AM (Invitrogen, USA) for 0 h and 12 h. Micrographs were acquired with a confocal microscope (ZEISS LSM 980, Germany). Transwell assay L929 cells were seeded in 6-well plates and cultured overnight. The cells were washed and cultured in serum-free DMEM supplemented with TDNPs (10 µg mL^− 1) for 24 h. The cells were then collected and placed in the upper chamber of a 24-well plate. Transwell chambers (Corning) were used for transwell assays. DMEM supplemented with 10% FBS was added to the lower chamber. L929 cells were further cultured for 36 h, and non-migrated cells were removed using a cotton swab. The migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. In vivo analysis of diabetic wound healing Ethics statement All animal experiments were performed after receiving approval from the Animal Experimental Ethics Committee of Nanfang Hospital of Southern Medical University and complied with the requirements of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. DSI model in rats The in vivo wound healing efficacy of GelMA/DAS/Exo hydrogel was assessed utilizing a strip skin wound model. Forty SD rats were randomly divided into four groups. The rats were anesthetized via the intraperitoneal injection of 3% pentobarbital sodium and inhaled isoflurane (2%). The back surfaces of the rats were then shaved and disinfected. A strip wound (lengths = 10 mm) was formed on the back using a surgical scalpel. Then either a control, Lemon Exosomes, GelMA/DAS hydrogel or the GelMA/DAS/Exo hydrogel were applied to the wound surface and cured by UV (the hydrogel and exosomes were disinfected by irradiation or cell filter before mixing). The wound was then covered with Tegaderm (3 m Inc, USA) and bandages to keep the material from drying and dehydrating and to prevent the animals from scratching or biting the wounds. After recovery from anesthesia, the rats were placed back in their cages and monitored daily. A digital photo of the wound was obtained. After 14 days, the rats were anesthetized and killed, and the local skin tissues were sectioned and stained for analysis. Histology analysis Tissue samples were fixed in 10% formalin and embedded in paraffin. The tissue samples were cut into 5 μm thick sections, which were then deparaffinized and rehydrated, followed by hematoxylin and eosin (H & E) and Masson’s trichrome staining. The stained sections were imaged under a microscope. Immunohistochemical staining was performed according to standard protocols. The sections were incubated with the following antibodies: anti-Arginase-1 (16001-1-AP, ProteinTech), iNOS (80517-1-RR, ProteinTech), anti-VEGFA (19003-1-ProteinTech), anti-CD31 (11265-1-AP, ProteinTech), anti- alpha-SMA (14395-1-AP, ProteinTech). Statistical analysis The results are presented as means ± standard deviations (SDs), for which at least three samples were used. One-way analysis of variance (ANOVA) and Bonferroni-multiple comparisons were used to analyze the data. All analyses were performed using GraphPad Prism 9.0 and Origin 2019b. P values < 0.05 indicate statistical significance. ns in the graphs indicates no significance; * p < 0.05, ** p < 0.01, and *** p < 0.001. Results and discussion RNA sequencing reveals potential immune mechanisms of DSI To clarify the mechanism mediating the homeostasis of DSI, we predicted the molecular mechanism of the damaged diabetic cell model and the blank control group via RNA-seq. First, PCA and matrix normalization showed that the two data samples had good repeatability (Fig. [75]2A-B). With |log2 (FC) |>1 and FDR < 0.05 as criteria, DEGs were screened from GSVA and Limma data. Furthermore, 271 upregulated genes and 20 downregulated genes were identified via heatmaps and volcano maps (Fig. [76]2C-D). Through GO and KEGG enrichment analyses, differential expression was found to be closely related to the expression of TNF-17, NF-κB, IL-17 and other factors that play important roles in regulating the M1/M2 phenotype transformation, inflammation and immune function of macrophages (Fig. [77]2E-G). The above data suggest that diabetic skin damage may be caused by a poor wound microenvironment with an imbalance in macrophage polarization, leading to severe inflammation and cell dysfunction. Therefore, reducing inflammation and regulating dysregulated metabolic activity and communication between macrophages and fibroblasts in the diabetic wound microenvironment may promote the healing of chronic diabetic wounds (Fig. [78]2H). Fig. 2. [79]Fig. 2 [80]Open in a new tab RNA sequencing revealed potential immune mechanisms of diabetic skin damage. (A) Three samples were diabetic skin lesions, and 3 samples were controls. The whole gene expression of each sample was normalized. (B) There were obvious differences between the two groups of samples, and there are no confounding factors identified through PCA. (C) Heatmap showing the expression of all differential genes between the two groups of samples. (D) Differentially expressed genes were screened using the following criteria: |log2 (FC) |>1 and FDR < 0.05. (E-G) GO and KEGG pathway enrichment analyses revealed that DSI was closely related to inflammation, oxidative stress, cell proliferation and apoptosis. (G) Intercellular communication of pathway molecules Potential therapeutic relationship between lemon-related pharmaceutical active ingredients and DSI Related studies have reported that lemons are rich in a variety of antioxidant components, such as vitamin C and flavonoids [[81]30]. These ingredients neutralize free radicals and reduce cellular damage from oxidative stress. Through their antioxidant effects [[82]31], lemon exosomes can help protect cells from oxidative damage and help cells maintain their normal function and structure. In addition, lemon also has certain anti-inflammatory properties that can inhibit the production of inflammatory factors and reduce inflammation [[83]32]. In order to determine the active components of lemon exosomes, we first conducted non-targeted metabolomic sequencing on lemon juice and lemon exosomes, respectively, and found that lemon exosomes were rich in D-ribo-Phytosphingosine (47.68%), Citric acid (12.34%), alpha-Keto-gamma-(methylthio) butyric acid (8.1%), Diphenylphosphine oxide (5.46%), Dimethyl sulfoxide (5.31%) and other active components (Fig. [84]3A-B). Lemon juice mainly contains effective ingredients such as Citric acid (31.02%), (2R)-6-methylpiperidine-2-carboxylic acid (10.59%), Diphenylphosphine oxide (9.56%), alpha-Keto-gamma-(methylthio) butyric acid (8.62%), 5,7-dimethoxychromen-2-one (8.5%) and so on (Fig. [85]3C-D); In addition, compared with lemon juice, lemon exosomes also contain vitamin C and flavonoids and other antioxidants are basically similar (Fig. [86]3E-F). On this basis, we constructed pharmacological interaction network between lemon and DSI and identified 190 common targets in a Venn diagram (Fig. [87]4A). Further protein–protein interaction (PPI) network analysis revealed that these targets were closely related to inflammation, oxidative stress, cell proliferation and apoptosis (Fig. [88]4B-C). Moreover, KEGG pathway enrichment analysis further indicated that lemon was related to oxidative stress (Fig. [89]4D). Therefore, these results suggest that bioactive substances rich in lemons are associated with the regulation of diabetic skin cell pathways and may play a comprehensive therapeutic role in diabetic wound healing. Fig. 3. [90]Fig. 3 [91]Open in a new tab Non-targeted metabolomics of lemon juice and lemon exosomes. (A-B) Lemon juice non-targeted metabolomics pie and bar graphs; (C-D) Lemon exosomes non-targeted metabolomics pie and bar graphs; (E-F) Comparison of vitamin C and flavonoids in lemon juice and lemon exosomes Fig. 4. [92]Fig. 4 [93]Open in a new tab Potential therapeutic relationship between lemon-related pharmaceutical active ingredients and DSI. (A) Potential target gene crossover between lemon and diabetic skin lesions. (B-C) Lemon and potential target gene networks in diabetic skin lesions. (D) KEGG pathway enrichment map of potential target genes Characterization of GelMA/DAS/Exo hydrogel Plant exosomes are nanoscale vesicles that are extracts of plant active ingredients and contain a variety of parental molecules, such as proteins, nucleic acids, lipids and other bioactive substances, that act as messengers in cell communication [[94]33]. To improve the effectiveness of lemon treatment, lemon-derived nanoparticles were isolated from homogenized lemon juice via density gradient centrifugation. Lemon Exosomes were continuously enriched during this process (Fig. [95]5A). The physical picture of lemon exosomes is shown in Fig. [96]5B. The morphology and size of the lemon exosomes were characterized via transmission electron microscopy (TEM) and nanoparticle tracer analysis (NTA). The TEM results revealed a homogeneous membrane-coated vesicle structure with relatively low cell debris and lipoprotein contamination (Fig. [97]5C). In addition, the particle size distribution of the lemon exosomes ranged from 85.0 to 515.0 nm (Fig. [98]5D). Cytophagocytosis experiments revealed that lemon exosomes could be targeted by macrophages, fibroblasts and HUVECs and could be uniformly loaded in a hydrogel (Fig. [99]5E-H). Moreover, scanning electron microscopy revealed that the surface pore structure of the hydrogel was uniform, which could play an important role in cell adhesion, proliferation and intercellular communication. As shown in Fig. [100]5I, the alkenyl (CH2 = C-) hydrogen peak appeared at 5.5 and 5.8 ppm, and the methyl (CH3-) hydrogen peak appeared at 1.9 ppm, indicating the successful preparation of GelMA. As shown in Fig. [101]5J, in the FT-IR spectrum of the GelMA/DAS hydrogel, the characteristic peak of the aldehyde group (-C = O) of bialdehyde starch at 1726 cm − 1, the characteristic peak of the Schiff base bond (-C = N-) formed between GelMA and bialdehyde starch at 1633 cm − 1, and the peak of the N-H bending vibration of GelMA at 1540 cm − 1 proved the successful cross-linking of GelMA with DAS. To observe the micromorphology of the hydrogels after exosome doping, SEM characterization was conducted. As shown in Fig. [102]5K-L, all the hydrogels exhibited porous network structures, and doping with exosomes did not result in significant changes. Fig. 5. [103]Fig. 5 [104]Open in a new tab Preparation and characterization of GelMA/DAS/Exo hydrogel. (A) Diagram of the synthesis of hydrogels. (B) Physical picture of Lemon Exosomes. (C) Lemon Exosome TEM. (D) Lemon Exosome NTA. (E-H) Lemon Exosomes could be targeted by macrophages, fibroblasts and HUVECs and could be uniformly loaded in a hydrogel. (I) ^1H NMR spectra of gelatin and GelMA. (J) IR spectra of the GelMA, DAS, and GelMA/DAS hydrogels. (K-L) SEM images of the GelMA/DAS and GelMA/DAS/Exo hydrogels Rheological properties and shape adaptability of the GelMA/DAS/Exo hydrogel The mechanical properties of the hydrogel were studied via dynamic frequency scanning rheology. Figure [105]6A shows the storage modulus (G’) and loss modulus (G″) as a function of frequency before and after curing. G′ was always greater than G″ throughout the frequency range, indicating that the hydrogel was stable and acted as an elastic solid. UV curing of the hydrogel increased its mechanical strength, as shown in Fig. [106]6B. The incorporation of Lemon Exosomes did not affect the mechanical strength of the hydrogel. Therefore, subsequent strain amplitude scanning measurements were conducted exclusively on the GelMA/DAS hydrogel without Exos. As shown in Fig. [107]6C, the G′ and G″ values for the GelMA/DAS hydrogel converged at ≈ 75.8% strain, indicating that the hydrogel network broke down and became a sol-gel when the critical strain was exceeded; In contrast, the UV-cured GelMA/DAS hydrogels exhibited significantly enhanced mechanical properties, with a critical strain reaching ≈ 238%. In addition, the ability of the hydrogel to adapt to the shape of irregular surfaces was assessed. As shown in Fig. [108]6D, when the hydrogel was placed in glove and petal molds, the hydrogel conformed to the shape and filled the grooves, and the shape of the molds was maintained after UV curing. Without UV curing, there were only hydrogen bond cross-links and some weak Schiff-based bonds in the hydrogel, leading to poor mechanical properties and a certain degree of mobility. UV curing increased the degree of cross-linking in the hydrogel, enhancing its mechanical properties and allowing it to maintain a fixed shape. From the above results, it can be concluded that the hydrogel could adapt to irregular surfaces. The self-healing ability of hydrogels can significantly extend their service life. Therefore, to evaluate the self-healing performance of the GelMA/DAS hydrogel after UV curing, strain amplitude scanning measurements were conducted to investigate the rheological behavior of the hydrogel under external strain. The recoverability of the hydrogel was evaluated via continuous cyclic strain testing (from 1% strain to 300% strain to 1% strain) at a constant angular frequency of 10 rad/s. As shown in Fig. [109]6E, the hydrogel transformed into a sol-gel state (G’ < G″) when the critical strain exceeded 300%. When the strain returned to 5%, the gel reorganized, and the G′ and G″ values immediately returned to their initial values, without significant loss. To verify the self-healing properties of the hydrogel, we combined two triangular pieces of hydrogel, each cut into two halves, and after a certain period, the two pieces healed into a single hydrogel (Fig. [110]6F). The fast sol‒gel transition behavior of the hydrogel network was fully reversible and reproducible in the cyclic test. Viscosity measurements of the GelMA/DAS hydrogel were conducted. Increasing the shear rate resulted in a decrease in viscosity (Fig. [111]6G), demonstrating hydrogel injectability. In addition, owing to the shear-thinning property of hydrogels, aqueous colorant-stained GelMA/DAS hydrogel could be continuously injected via a 1 mL syringe into a syringe containing water, maintaining its gel state without dissolving (Fig. [112]6G). Fig. 6. [113]Fig. 6 [114]Open in a new tab Rheological properties and shape adaptability of the GelMA/DAS/Exo hydrogel. (A) Rheological analysis of the GelMA/DAS and GelMA/DAS/Exo hydrogels before and after UV curing in frequency scan mode. (B) Mean storage modulus of the GelMA/DAS and GelMA/DAS/Exo hydrogels before and after UV curing at 1 Hz, with error bars indicating the standard deviation (SD) (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3). (C) Scanning tests of strain amplitudes ranging from 0.001–300% at a fixed angular frequency (10 rad s-1). (D) Images showing shape changes in the GelMA/DAS hydrogel. (E) Sequential strain testing of the cured GelMA/DAS hydrogel ranging from a small strain (1%) to large strain (300%). (F) Image showing the self-healing ability of the cured GelMA/DAS hydrogel. (G) Shear-thinning experiment of the GelMA/DAS hydrogel. Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001) Adhesive properties of the prepared GelMA/DAS/Exo hydrogel To quantitatively evaluate the tissue adhesion properties of the prepared hydrogels and investigate the effect of UV curing on the tissue adhesion of the GelMA/DAS hydrogel and GelMA/DAS/Exo hydrogel, lap-shear adhesion tests were performed to measure the interfacial tissue adhesion strength. The hydrogels were placed at the junction between two pieces of pig skin (Fig. [115]7A). The adhesion mechanical curves and corresponding maximum adhesion strengths of the GelMA/DAS and GelMA/DAS/Exo hydrogels to fresh pig skin are shown in Fig. [116]7B and C, respectively. The adhesion strength of the hydrogels did not change significantly after the addition of Exos. The maximum adhesion strengths of the GelMA/DAS and GelMA/DAS/Exo hydrogels to pig skin were 1.63 ± 0.19 and 1.74 ± 0.25 kPa, respectively. As shown in Fig. [117]7D and E, UV curing significantly enhanced the adhesion properties of the UV-cured GelMA/DAS and GelMA/DAS/Exo hydrogels; the maximum adhesion strengths to pig skin were 23.24 ± 1.77 and 24.93 ± 2.37 kPa, respectively. This enhanced effect was attributed to the formation of a mechanical interlock between the UV-cured hydrogels and the substrate. The cohesive force of the UV-cured hydrogels significantly increased, changing the failure mode from adhesion failure to cohesion failure. Additionally, we investigated the adhesion of the UV-cured GelMA/DAS hydrogel to pig skin in vitro. After bending, twisting, and stretching, the hydrogel remained tightly adhered to the pig skin without any detachment, indicating that the UV-cured hydrogel possessed considerable tissue-binding strength (Fig. [118]7F). Fig. 7. [119]Fig. 7 [120]Open in a new tab Adhesive properties of the hydrogels. (A) Schematic diagram of lap-shear experiments involving hydrogels. (B) Lap-shear adhesion curves for the GelMA/DAS and GelMA/DAS/Exo hydrogels applied to pig skin. (C) Lap shear adhesion strengths of the GelMA/DAS and GelMA/DAS/Exo hydrogels applied to pig skin. (D) Lap-shear adhesion curves for UV-cured GelMA/DAS and GelMA/DAS/Exo hydrogels applied to pig skin. (E) Lap shear adhesion strengths of UV-cured GelMA/DAS and GelMA/DAS/Exo hydrogels applied to pig skin. (F) In situ bonding image of the hydrogel to pig skin. Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001) In vivo and in vitro biocompatibility of the GelMA/DAS/Exo hydrogel Skin tissue is composed mainly of epidermal and endothelial cells [[121]34]. Therefore, our study of the biocompatibility of the GelMA/DAS/Exo hydrogel is an important prerequisite for the treatment of diabetic skin damage. We cultured HUVECs and L929 cells on GelMA/DAS/Exo hydrogels (Fig. [122]8A). Live/dead staining revealed many live cells (green) and very few dead cells (red) in each group (Fig. [123]8B and I). Quantitative analysis revealed that there was no significant difference between dead and living cells (Fig. [124]8C and J). The CCK-8 assay results revealed that the proliferative activity of HUVECs and L929s was not significantly affected (Fig. [125]8D and K). In addition, cytoskeletal experiments demonstrated that HUVECs and L929 cells in the GelMA/DAS hydrogel, GelMA/DAS/Exo and Lemon Exosomes groups exhibited no significant growth effects compared with those observed in the control group, indicating that GelMA, DAS and Lemon Exosomes had prominent biological effects on cell growth (Fig. [126]8E-H). GelMA hydrogels can be used as scaffold materials to construct various tissues, such as cartilage, bone, and skin. GelMA hydrogels can provide support for cells and promote tissue regeneration; they can be adapted to the requirements of different tissues by adjusting their composition and degree of crosslinking. Studies have also shown that Lemon Exosomes can reduce oxidative stress damage to cells through antioxidant substances, promote cell migration and proliferation, and reduce cell apoptosis. In vitro hemolysis experiments revealed that the red blood cell rupture liquid in the Triton X-100 group was bright red, whereas the liquid in the PBS group and the GelMA/DAS/Exo hydrogel group was clear, indicating that the GelMA/DAS/Exo hydrogel had good blood compatibility (Fig. [127]8L-M). Fig. 8. [128]Fig. 8 [129]Open in a new tab Biocompatibility of the GelMA/DAS/Exo hydrogel. (A) Schematic diagram of the coculture of Lemon Exosome hydrogels with cells. (B) Live/dead staining of HUVECs cultured on each sample surface for 1 day. Live cells are stained green, and dead cells are stained red. The scale bar represents 200 μm. (C) Quantitative analysis of the live/dead staining results (n = 3). (D) The results of the CCK-8 assay demonstrated that the cell viability surpassed 80% for each sample for 48 h after seeding (n = 3). (E) Cytoskeleton images showing the adhesion of HUVECs cultured in each group after 3 days. The scale bar represents 100 μm. (F) Quantitative analysis of the cell spread area (n = 3). (G) Cytoskeleton images showing the adhesion of L929 cells cultured in each group after 3 days. The scale bar represents 100 μm. (H) Quantitative analysis of the cell spread area (n = 3). (I) Live/dead staining of L929 cells cultured on each sample surface for 1 day. Live cells are stained green, and dead cells are stained red. The scale bar represents 200 μm. (J) Quantitative analysis of the live/dead staining results (n = 3). (K) The results of the CCK-8 assay demonstrated that the cell viability surpassed 80% for each sample for 48 h after seeding (n = 3). (L) Hemolysis test of the materials. (M) Quantitative analysis of the hemolysis test results. Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001) In the field of tissue engineering, in vivo biocompatibility is the basis for building functional tissues and organs [[130]35]. Scaffold materials for tissue engineering need to provide a suitable environment for cells to grow while simultaneously integrating with human tissues. When new drug carriers such as nanoparticles and hydrogels are used to deliver drugs to the body, if the carrier material is not biocompatible, it may be quickly cleared by the human body and cannot effectively deliver drugs to the target site. In addition, poor biocompatibility may also lead to the accumulation of carrier materials in the body, causing hepatorenal toxicity [[131]36]. Therefore, we studied the biocompatibility of the GelMA/DAS/Exo hydrogel in vivo and found that there was no obvious toxic damage to the heart, liver, spleen, lung or kidney of rats (Fig. [132]9A). In addition, the biochemical indices of the GelMA/DAS/Exo hydrogel after routine blood tests, liver function analysis and kidney function analysis were not significantly different from those of the control group, demonstrating that the GelMA/DAS/Exo hydrogel had good biocompatibility in vivo (Fig. [133]9B). Fig. 9. [134]Fig. 9 [135]Open in a new tab In vivo biocompatibility of lemon exosome hydrogels. (A) Pathological examinations of the heart, liver, kidney and lung of rats in the control and GelMA/DAS/Exo groups. (B) Serum levels of biomarkers reflecting liver function and kidney function and blood cell parameters in rats treated with GelMA/DAS/Exo hydrogels (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001) GelMA/DAS/Exo hydrogel promotes M2 macrophage reprogramming In people with diabetes, a chronic high-glucose environment and metabolic disorders can affect immune system function [[136]37]. The overactivation of M1 macrophages aggravates tissue destruction and releases inflammatory factors, leading to local tissue redness, swelling and pain [[137]38]. Therefore, regulating macrophage polarization at the trauma site is essential for controlling tissue regeneration through the secretion of multiple cytokines that regulate the function of surrounding cells and promote the construction of the ECM. On the basis of the lemon network pharmacology results, lemon and its derivatives have a potential effect on M2 macrophage polarization. We obtained lemon extract and lemon exosomes to preliminarily verify their effects on the polarization of macrophages. The results showed that compared with lemon extract, lemon exosomes could effectively promote the polarization of M2 macrophages, and the fluorescence expression of Arg-1 was significantly increased (Fig. [138]S1), which may be due to the fact that lemon extract has no special protective structure after entering human body. It is easily broken down or inactivated by environmental factors (e.g. stomach acid, enzymes, etc.). Therefore, in further study, we loaded lemon exosomes on GelMA/DAS hydrogel for exploration. RAW264.7 cells were cocultured with the GelMA/DAS/Exo hydrogel (Fig. [139]10A). After incubation for 24 h, the expression levels of iNOS and TNF-α in M1 macrophages decreased in the Lemon Exosomes and GelMA/DAS/Exo groups, whereas the expression levels of Arg-1 and IL-10 in M2 macrophages increased (Fig. [140]10B-C). To simulate the inflammatory response of diabetic wound macrophages, RAW264.6 cells were subsequently stimulated with lipopolysaccharide (LPS), followed by intervention with the GelMA/DAS/Exo hydrogel. Immunofluorescence and WB color rendering revealed that the number of proinflammatory M1 macrophages (iNOS) decreased in the Lemon Exosomes and GelMA/DAS/Exo hydrogel groups, whereas the number of anti-inflammatory M2 macrophages (Arg-1) increased in the Lemon Exosomes and GelMA/DAS/Exo hydrogel groups (Fig. [141]10D-H). Moreover, flow cytometry revealed that CD86 + levels decreased from 13.3 to 2.05%, whereas CD206 + levels increased from 12.9 to 61.4% (Fig. [142]10I-J). These results indicate that the GelMA/DAS/Exo hydrogel can be used as an inflammatory modulator to regulate immune polarization homeostasis around DSI to promote wound recovery. Fig. 10. [143]Fig. 10 [144]Open in a new tab The GelMA/DAS/Exo hydrogel stimulated the polarization of RAW 264.7 cells from the M1 phenotype to the M2 phenotype through the NF-κB pathway. (A) An illustration of how GelMA/DAS/Exo modulates macrophage polarization from the M1 phenotype to the M2 phenotype. (B) Bar graph showing the RT‒qPCR results for the gene expression of anti-inflammatory cytokines (Arg-1 and IL-10) and proinflammatory cytokines (iNOS and TNF-α) in each group (n = 3). (C) Heatmap showing the RT‒qPCR results for the gene expression of anti-inflammatory cytokines (Arg-1 and IL-10) and proinflammatory cytokines (iNOS and TNF-α) in each group (n = 3). (D) IF images showing the number of Arg1 (red)-positive and iNOS (red)-positive RAW264.7 cells cultured on each hydrogel. The scale bar represents 50 μm. (E) Quantitative analysis of the fluorescence intensity of Arg-1 (n = 3). (F) Quantitative analysis of the fluorescence intensity of iNOS (n = 3). (G) WB analysis of Arg-1 and iNOS protein expression. (H) The protein band intensities of Arg-1 and iNOS were quantified using ImageJ (n = 3). (I) Flow cytometry analysis of the proportions of M1 macrophages (labeled with F4/80^+CD86^+) and M2 macrophages (labeled with F4/80 + CD206+); (F) percentages of F4/80^+CD86^+ and F4/80^+CD206^+ cells (n = 3); significant differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001) GelMA/DAS/Exo hydrogel promotes angiogenesis Angiogenesis is one of the critical processes in wound healing and directly affects wound healing outcomes [[145]39, [146]40]. Oxy-gen, nutrients and growth factors are delivered to injured sites through new blood vessels. Dys-angiogenesis is an important problem in DSI [[147]41]. The effects of lemon exosomes on angiogenesis have not been reported. To verify whether the Lemon Exosome hydrogel promoted angiogenesis, a tube formation assay was performed, and the results revealed that the number of closed tubular structures was greater in the GelMA/DAS/Exo group than in the GelMA/DAS hydrogel group and the control group (Fig. [148]11A-B). The expression levels of the vascular genes CD31 and VEGFA were significantly increased in the control group containing lemon exosomes (Fig. [149]11C-F). Similarly, WB also confirmed that Lemon Exosomes stimulated angiogenesis (Fig. [150]11G-H). In conclusion, GelMA/DAS/Exo hydrogel have potential therapeutic effects on the repair of endothelial cells damaged by diabetic skin. The effects may be related to the cell growth factors released by the hydrogel itself, or the antioxidant effect of lemon may alleviate the oxidative stimulation of the high-sugar environment in endothelial cells, thus promoting the repair of damaged endothelial cells. Fig. 11. [151]Fig. 11 [152]Open in a new tab Status of angiogenesis. (A) Vessel formation analysis of HUVECs cultured on different materials for 12 h. (B) Quantitative analysis of angiogenesis (n = 3). (C) IF images showing the number of VEGFA (red)-positive HUVECs cultured on each hydrogel. The scale bar represents 50 μm. (D) Quantitative analysis of the fluorescence intensity of VEGFA (n = 3). (E) IF images showing the number of CD31 (red)-positive HUVECs cultured on each hydrogel. The scale bar represents 50 μm. (F) Quantitative analysis of the fluorescence intensity of CD31 (n = 3). (G) WB analysis of CD31 and VEGFA protein expression. (H) The protein band intensities of CD31 and VEGFA were quantified using ImageJ (n = 3) Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001) GelMA/DAS/Exo hydrogel promotes the proliferation and migration of fibroblasts We investigated the proliferation and migration effects of lemon exosome hydrogel on L929 and HUVECs cells in response to the dysregulation of fibroblast metabolism and proliferation in DSI, which leads to the obstruction of tissue regeneration [[153]40, [154]42]. First, the results of cell scratch experiments revealed that, compared with those in the control group and the hydrogel group alone, fibroblasts migrated to the scratched area at an increased rate in the Lemon Exosome group (Fig. [155]12A and C); therefore, Lemon Exosomes promoted the sustained growth of fibroblasts from 0 h to 12 h. At 0 h and 12 h, the mobility of the Lemon Exosome-treated cells was approximately 1.78 and 1.89 times greater than that of the control cells, respectively (Fig. [156]12B and D). WB and immunofluorescence also proved that it has a good effect on promoting collagen fiber formation (Fig. [157]12E-H). The results of in vitro transwell experiments supported this conclusion, with more migrating cells observed in the Lemon Exosome-treated fibroblast group (Fig. [158]12I-J). Due to its nanoscale size and good skin penetration, lemon exosomes can penetrate deep into the bottom layer of the skin and deliver internal active ingredients (such as growth factors, etc.) to skin cells to promote skin cell repair and regeneration. In contrast, lemon extract has a smaller effect on the proliferation and migration of skin cells (Fig. [159]S2 and [160]S3), and the components in it may be limited by the size of the molecules and the skin barrier in penetrating the skin. These results confirmed that Lemon Exosomes positively regulate fibroblast proliferation and migration. Fig. 12. [161]Fig. 12 [162]Open in a new tab HUVECs and L929 cell migration and the fibrillogenesis were studied in vitro. (A) Wound-healing migration assay of HUVECs on each hydrogel at different time points. The scale bar represents 200 μm. (B) Quantitative analysis of the scratch area in the bare region at 0 and 12 h (n = 3). (C) Wound-healing migration assay of L929 cells on each hydrogel at different time points. The scale bar represents 200 μm. (D) Quantitative analysis of the scratch area in the bare region at 0 and 12 h (n = 3). (E) IF displaying the number of α-SMA (green)-positive L929 cells cultured in each group. The scale bar represents 50 μm. (F) Quantitative analysis of the fluorescence intensity of α-SMA (n = 3). (G) WB analysis of α-SMA protein expression. (H) The protein band intensity of α-SMA was quantified using ImageJ (n = 3). (I) Transwell experiment. The scale bar represents 200 μm. (J) Quantitative analysis of transwell experiment (n = 3). Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001) Hemostatic properties of the GelMA/DAS/Exo hydrogel The hemostatic properties of a hydrogel can create a relatively stable environment for a wound, which is conducive to the subsequent healing process [[163]43]. By stopping the bleeding quickly, blood immersion and interference in the wound can be reduced while also reducing the risk of infection [[164]44]. Moreover, hydrogels contain components that promote cell growth and tissue repair, which can further accelerate wound healing [[165]45, [166]46]. Therefore, evaluating the hemostatic properties of GelMA/DAS/Exo hydrogel is very important. Therefore, we first used a rat tail hemostasis experiment to evaluate the hemostatic performance of the GelMA/DAS/Exo hydrogel (Fig. [167]13A). We used a blank control (untreated) and compared it with a medical hemostatic sponge, and the results revealed that the gauze of the GelMA/DAS/Exo hydrogel group was stained with much less blood than that of the untreated and gelatin sponge groups (Fig. [168]13B). In addition, the quantitative results confirmed that, compared with other treatments, GelMA/DAS/Exo hydrogel therapy achieved sufficient hemostasis with lower blood loss (Fig. [169]13C). Moreover, to evaluate the hemostatic effect of the hydrogel on acute blood loss, we conducted a hemostatic experiment involving partial liver resection in rats (Fig. [170]13D). The degree of blood infiltration of gauze and quantitative analysis also revealed that the GelMA/DAS/Exo hydrogel treatment achieved faster hemostasis and less blood loss (Fig. [171]13E-F). Together, these results confirm that the GelMA/DAS/Exo hydrogel has significant hemostatic properties in vivo, indicating its potential as a treatment for skin injuries. Fig. 13. [172]Fig. 13 [173]Open in a new tab Hemostatic properties of the GelMA/DAS/Exo hydrogel. (A) Schematic diagram of rat tail hemostasis. (B-C) Gross hemostatic map of the rat tail and quantitative analysis. (D) Schematic diagram of liver hemostasis. (E-F) Liver hemostasis and quantitative analysis. Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001) In vivo diabetic wound healing by the GelMA/DAS/Exo hydrogel The wound-healing potential of the GelMA/DAS/Exo hydrogel was evaluated in vivo using a full-thickness skin defect model in diabetic rats (Fig. [174]14A). Diabetic rats were divided into four groups, namely, the phosphate-buffered saline (PBS) group, the Lemon Exosome group, the GelMA/DAS hydrogel group and the GelMA/DAS/Exo hydrogel treatment group, and observed for 14 days. Compared with that in the GelMA/DAS hydrogel group and the control group, the wound healing process in the Lemon Exosome group and the GelMA/DAS/Exo hydrogel group was significantly accelerated. In the control group and the GelMA/DAS hydrogel group, wound healing was delayed, and redness, swelling, exudation, and local scabbing clearly occurred. Although the Lemon exosome treatment group did not show significantly greater healing efficiency than the GelMA/DAS hydrogel group did, the therapeutic effects were relatively greater in the early stage of wound healing (day 7). These findings suggest that the hydrogel carrier facilitates the absorption of exudate and provides a balanced water‒gas microenvironment in the early stages of wound healing (Fig. [175]14B). Fig. 14. [176]Fig. 14 [177]Open in a new tab The GelMA/DAS/Exo hydrogel promote diabetic wound healing. (A) Diabetic skin damage modeling diagram. (B) Visual observation of DSI. (C) H&E images of the skin of diabetic model rats after different treatments. Scale bar, 2 mm at 2 x magnification and 400 μm at 10 x magnification. (D) Representative image of Masson’s trichrome staining. Scale bar, 2 mm at 2 x magnification and 400 μm at 10 x magnification In addition, we further assessed the therapeutic effect of the GelMA/DAS/Exo hydrogel on wounds via HE and Masson staining. After 14 days, in the Lemon Exosome and GelMA/DAS/Exo hydrogel groups, inflammatory infiltration in the wound tissue was reduced, the wound width was reduced, collagen deposition was increased, and the wound microenvironment structure was rearranged. In contrast, the control group and the GelMA/DAS hydrogel alone group Inflammation infiltration still exists, the wound width is relatively wide, and collagen formation is still in the early stage (Fig. [178]14C-D). Immunohistological staining revealed that, compared with those in the control group and the GelMA/DAS hydrogel group alone, the expression levels of the M2-type macrophage marker Arg-1 were greater in the Lemon exosome and GelMA/DAS/Exo hydrogel groups, and the expression level of the M1-type macrophage marker iNOS was lower. Inflammation was significantly decreased and tissue repair was significantly increased in the two treatment groups (Fig. [179]15A). In addition, wounds treated with preparations containing Lemon Exosomes presented increased angiogenesis (CD31 and VEGFA) (Fig. [180]15B) and collagen production (alpha-SMA) (Fig. [181]16). Notably, compared with the control group, the hydrogel alone group showed slight improvements in wound recovery and collagen formation, effects that were attributed to the exudate absorption properties provided by the hydrogel and the humid environment. These findings suggest that lemon exosome therapy provides an oxidation-balanced microenvironment that promotes diabetic wound healing through the regulation of multicellular networks mediated by plant-derived natural vesicles. In addition, our development strategy employed hydrogels as exosome carriers, ensuring a moist wound repair environment and continuous exosome release to effectively regulate the wound microenvironment without compromising the biological activity of lemon exosomes. Fig. 15. [182]Fig. 15 [183]Open in a new tab The GelMA/DAS/Exo hydrogel can reprogram macrophage polarization from the M1 to M2 phenotype and angiogenesis. (A) Immunohistochemistry of Arg-1 and iNOS in different groups. (B) Immunohistochemistry of CD31 and VEGFA in different groups. Scale bar, 2 mm at 2 x magnification, 100 μm at 40 x magnification, and 25 μm at the highest magnification Fig. 16. [184]Fig. 16 [185]Open in a new tab The GelMA/DAS/Exo hydrogel can collagen fiber formation. (A) Immunohistochemistry of α-SMA in different groups. Scale bar, 2 mm at 2 x magnification, 100 μm at 40 x magnification, and 25 μm at the highest magnification Conclusion In this study, a lemon-derived nanohydrogel dressing targeting the diabetic wound microenvironment was developed for damage repair in diabetic wounds. Through transcriptome sequencing analysis, we obtained a new understanding of the immune characteristics of diabetic wounds. Network pharmacological analysis and target gene prediction further increased our understanding of the therapeutic potential of lemon exosomes. In vitro experiments confirmed that the GelMA/DAS/Exo hydrogel participated in immune regulation, promoting the regeneration of blood vessels and fiber tissue on macrophages. Moreover, the hydrogel had good biocompatibility and hemostatic performance. Additionally, in vivo evaluations using diabetic rat models confirmed the effectiveness of GelMA/DAS/Exo hydrogel in promoting diabetic wound healing while addressing the specific associated challenges. Our study combined transcriptomics and network pharmacological analysis, thereby strengthening the relevance and applicability of our findings. These innovative approaches have the potential to significantly enhance the treatment of individuals with diabetic wounds by overcoming the limitations of traditional therapies and biology-based treatments. Electronic supplementary material Below is the link to the electronic supplementary material. [186]Supplementary Material 1^ (1.7MB, docx) Acknowledgements