Responsive Hydrogel Delivery of DFO-Programmed Apoptotic Bodies Drives Redox-Immuno-Angiogenic Remodeling in Diabetic Wounds

Responsive Hydrogel Delivery of DFO-Programmed Apoptotic Bodies Drives Redox-Immuno-Angiogenic Remodeling in Diabetic Wounds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Responsive Hydrogel Delivery of DFO-Programmed Apoptotic Bodies Drives Redox-Immuno-Angiogenic Remodeling in Diabetic Wounds Hao Guo, Jian Li, Chengcheng Gu, Ning Liu, Jie Sun, Danhui Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9522213/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract Mesenchymal stem cell (MSC)-derived exosomes show clear potential in cell-free regenerative medicine. However, their clinical translation is limited by low production yields and inconsistent therapeutic efficacy. To address this, we actively programmed stem cell apoptosis to generate high-yield, functional extracellular vesicles. By using deferoxamine (DFO) to stabilize HIF-1α prior to UV-induced apoptosis, we produced DFO-programmed apoptotic bodies (DABs). These programmed apoptotic bodies demonstrated a significantly higher production yield than conventional exosomes. Multi-omics analyses revealed that DABs inherit an enriched, pro-regenerative miRNA repertoire that functionally outperforms both native apoptotic bodies and exosomes. Beyond promoting classical angiogenic and migratory signaling, DABs support tissue repair by altering the mitochondrial bioenergetics of recipient cells. To prevent rapid in vivo clearance and address the highly oxidative diabetic microenvironment, we encapsulated DABs within a glucose- and ROS-responsive hydrogel, developing the DABs@PHA-PVA Gel . In vivo transcriptomics demonstrated that DABs@PHA-PVA Gel synchronized active ROS scavenging with the on-demand release of DABs. This localized delivery improved complete tissue reconstruction by simultaneously enhancing the Cxcr4/Nrp1/S1pr1 angiogenic axis, activating the Daglb/Cnr2 anti-inflammatory pathway, and engaging the Txnip/Foxo4/Nrf2 antioxidant cascade. Overall, this study establishes programmed apoptotic bodies as a scalable and effective alternative to standard exosomes therapies for tissue regeneration. Exosomes DFO-Programmed Apoptotic Bodies Transcriptomic Reprogramming Tissue Regeneration Redox-Immuno-Angiogenic Synergy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs), particularly exosomes, have attracted growing interest as cell-free therapeutics capable of delivering bioactive cargoes to injured tissues [ 1 – 3 ]. Despite these promising features, clinical translation of MSC-EVs remains limited. Exosomes biogenesis is inherently low yield, and even when sufficient quantities are obtained, their cargo composition is often inconsistent and insufficiently potent for use in diseased tissues [ 4 , 5 ]. These persistent problems have pushed researchers to look beyond exosomes for better-suited EVs sources. Apoptotic bodies (ABs) were long dismissed as inert cellular debris from programmed cell death, a view now challenged by their proven biological and therapeutic activities [ 6 , 7 ]. Apoptosis naturally generates these vesicles in massive quantities. They characteristically expose phosphatidylserine (PS) on their surface to act as an “eat-me” signal, which facilitates macrophage clearance and drives anti-inflammatory responses [ 8 ]. This combination of high yield and immune modulation makes them an attractive alternative to conventional exosomes [ 9 ]. However, a critical limitation is that native ABs are insufficient at promoting new blood vessel formation—a prerequisite for repair in ischemic or poorly vascularized tissues [ 10 , 11 ]. This gap in pro-angiogenic activity limits their utility in more demanding pathological settings, and to date, few studies have demonstrated a reliable strategy to overcome it. We reasoned that this limitation might be overcome not by modifying the vesicles themselves, but by reprogramming the cells that produce them. Specifically, we pretreated MSCs with deferoxamine (DFO), an FDA-approved iron chelator with a well-established clinical safety record [ 12 ], prior to apoptosis induction. DFO stabilizes HIF-1α by blocking its iron-dependent prolyl hydroxylase-mediated degradation [ 13 , 14 ], effectively placing the parent cells in a sustained hypoxia-mimicking state prior to undergoing apoptosis. The apoptotic bodies generated from these programmed cells, which we term DABs, are consequently loaded with a markedly enriched profile of pro-regenerative miRNAs compared to their native counterparts. The approach shifts the therapeutic intervention upstream to the cell itself rather than engineering the vesicles post-isolation. To test DABs under genuinely demanding conditions, we turned to the diabetic wound model. These wounds are notoriously difficult to treat, owing to the combined burden of dysregulated inflammation, poor perfusion, and oxidative stress [ 15 – 17 ]. From a vesicle delivery standpoint, this environment is particularly hostile: elevated proteolytic activity and ROS can structurally compromise EVs membranes, while wound exudate rapidly flushes away unprotected cargo before it can act [ 18 , 19 ]. To address this, we encapsulated DABs in a glucose- and ROS-responsive hydrogel (PHA-PVA Gel ) that doubles as a local ROS scavenger, enabling sustained on-demand release directly at the wound site [ 20 ]. In this study, we demonstrate that DABs carrying DFO-optimized miRNA cargo display both immunomodulatory and pro-angiogenic activity that clearly exceeds that of native ABs and conventional exosomes. Unexpectedly, this miRNA cargo also modulates mitochondrial function in recipient cells, enhancing ATP output. By integrating these miRNA optimized DABs with a ROS-scavenging hydrogel, we successfully translated these cellular advantages into tissue regeneration within a highly hostile diabetic in vivo model. Together, these findings position programmed apoptotic bodies as a scalable and mechanistically distinct alternative to exosomes, with broader implications for cell-free approaches to tissue repair (Fig. 1 a and 1 b). 2. Materials and methods 2.1. Materials Deferoxamine mesylate (DFO) was obtained from Abcam plc. Hyaluronic acid (HA) was purchased from Bloomage Biotech Co., Ltd. Human albumin (HSA) was purchased from ExCell Biotechnology, Sexton Biotechnology, and Grifols, respectively. High-glucose DMEM and DMEM/F12 basal media were purchased from Jiangsu KeyGEN BioTECH. For cell viability and apoptosis analyses, the AO/PI double staining kit was acquired from APExBIO Technology, while the Coomassie brilliant blue and cell apoptosis kits were from YEASEN Biotechnology. A comprehensive panel of biochemical reagents and antibodies was purchased from Beyotime Biotechnology; this included the BCA protein assay kit, BeyoExo™ Exosome Identification Kit (CD63, CD9, TSG101, Hsp70, and Calnexin), DiI/DiO lipophilic dyes, as well as all primary and secondary antibodies for immunofluorescence and immunoblotting (anti-HIF-1α, anti-CD86, anti-CD206, anti-CD31, anti-CD105, anti-VEGF, anti-TGF-β, anti-α-SMA, HRP-conjugated β-Actin, Anti-Rabbit IgG (H + L), and AF 488/647). Mouse ELISA kits for inflammatory and angiogenic cytokines were obtained from MULTI SCIENCES. Specific antibodies for flow cytometry were sourced from BD Pharmingen. 2.2. Cell culture Human umbilical cord blood tube skin cells (HUVEC), human keratinocytes cells (HaCaT), and mouse mononuclear macrophages cells (RAW264.7) were purchased from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). They were cultured in DMEM medium containing 10% FBS in a 37℃, 5% CO 2 incubator. Specifically, Human umbilical cord mesenchymal stem cells (MSCs) were kindly provided by Jiangsu Province Cell Therapy Manufacturing Center (Nanjing, China) and previously identified, were cultured in DMEM/F12 containing 5% human platelet lysate (Sexton) in a 37℃, 5% CO 2 incubator. 2.3. Animal Male C57BL/6 mice (6 weeks of age, 18–22 g) were sourced from Jiangsu Huachuang Sino Pharmaceutical Technology Co., Ltd. (Taizhou, China). Animal housing and care were managed by the Animal Experimental Center of China Pharmaceutical University. All in vivo procedures received ethical approval from the Ethics Committee of China Pharmaceutical University (Nanjing, China) and were executed in full compliance with institutional guidelines for animal care. 2.4. Apoptosis induction of MSCs MSCs at passages fewer than six were cultured in DMEM/F12 with 5% HPL containing DFO (250 µg/mL) or PBS for 24 h. The prime medium was discarded, cells were rinsed three times with PBS, and HPL-free medium was added. The confluent monolayers of DFO-MSCs and MSCs, maintained in uncovered cell culture dishes, were irradiated with ultraviolet light (UV) (30 W, 254 nm, Philip, China) at an intensity of 300 mJ/cm² for 10 min. After 24 h of apoptosis induction, apoptosis was evaluated by morphological observation and Annexin V-FITC/PI staining. In addition, apoptotic collapse in apoptotic DFO-MSCs (denoted as Apo-D-MSCs) and apoptotic MSCs (denoted as Apo-MSCs) was observed using inverted fluorescence microscopy. By contrast, confocal laser scanning microscopy (LSM800, Zeiss) was employed to monitor the generation of apoptotic vacuoles within DiI-stained MSCs. 2.5. Separation and purification of apoptotic bodies Apoptotic bodies were extracted from the culture supernatant of Apo-D-MSCs and Apo-MSCs using an extensively used differential centrifugation (denoted as DABs and ABs). Briefly, after 24 h UV irradiation induction, the HPL-free cell culture supernatant of Apo-D-MSCs and Apo-MSCs were collected and centrifuged at 400×g for 5 min to precipitate dead cells, and at 2000×g for 30 min to precipitate cell debris. Following the removal of dead cells and larger debris, the clarified supernatant was subjected to centrifugation at 40,000 × g for 40 min to sediment the apoptotic vesicles. To clear residual co-precipitating proteins, these crude pellets were washed by resuspension in ice-cold PBS and re-pelleted under identical centrifugation conditions. The final purified DABs and ABs were then dispersed in 100 µL of PBS and preserved at -80°C for downstream applications. To maintain vesicular integrity, the entire differential centrifugation protocol was strictly carried out at 4°C. 2.6. Characterization of DABs and ABs Total protein concentrations of DABs and ABs were measured by a bicinchoninic acid (BCA) assay. Particle size distribution and zeta potential were measured by a Zetasizer Nano (Nano-ZS90, Malvern, UK). Morphology was checked using transmission electron microscopy (TEM). Total protein from MSCs, Apo-D-MSCs, Apo-MSCs, ABs, and DABs was extracted with RIPA buffer. This protein was mixed with loading buffer and heated at 95°C for 5 minutes for SDS-PAGE. The protein bands were stained with Coomassie Brilliant Blue and photographed. Surface phosphatidylserine (PS) on DABs and ABs was measured by Annexin V-FITC labeling and flow cytometry. 2.7. Cellular uptake of DABs and ABs HUVEC, HaCaT, and RAW264.7 cells were seeded on 12-well plates at 1 × 10 5 cells/well and cultured overnight. These cells were incubated with DiI-labeled DABs or ABs for 4 hours. Cells were washed with cold PBS, fixed with 4% paraformaldehyde and stained with DAPI for confocal laser scanning microscopy. Cells in 12-well plates were collected using trypsin and analyzed by flow cytometry. 2.8. Preparation and Characterization of Exo MSCs were cultured in HPL-free DMEM/F12 for 24 h. The conditioned medium was collected and sequentially centrifuged (300×g, 2000×g, and 10000×g), filtered through a 0.22-µm membrane, and ultracentrifuged at 100000 ×g for 70 min at 4°C. The exosomes pellet was resuspended in PBS and stored at -80°C. The particle size distribution of exosomes was assessed by dynamic light scattering (DLS), vesicular morphology was visualized using transmission electron microscopy (TEM), and exosome-associated surface markers (CD9, CD63, TSG101, and Hsp70) were analyzed by western blotting. These analyses collectively confirmed the characteristic size distribution, canonical vesicular morphology, and expected marker expression of exosomes (Fig. S10 and S11). 2.9. miRNA sequencing and bioinformatic analysis Exosomes (Exo) and apoptotic bodies (ABs) were isolated from MSCs, and DFO-programmed apoptotic bodies (DABs) were isolated from DFO-preconditioned MSCs as described above. Small RNA sequencing was performed by Bioyi Biotechnology Co., Ltd. (Wuhan, China). Total RNA was extracted from Exo, ABs, and DABs. Small RNAs of 18 to 30 nucleotides were used to build libraries. These libraries were amplified by PCR and sequenced on the Illumina NovaSeq 6000 platform. Raw reads were filtered for clean data. Differentially expressed miRNAs (DEMs) were identified using thresholds of |fold change| > 2 and p-value < 0.05. Target transcripts were predicted using TargetScan and miRDB. GO and KEGG enrichment analyses were done on these targets. Chord diagrams were made using the Hiplot web tool ( http://hiplot.com.cn ) to show miRNA-function relationships. Pathway clustering was done using Metascape ( https://metascape.org/ ). Protein-protein interaction (PPI) networks and hub modules were built using Cytoscape with the MCODE plugin. 2.10. Analysis of HUVEC proliferation Cell proliferation was measured by CCK-8 assay. HUVEC were seeded in 96-well plates at 5 × 10 3 cells/well overnight and incubated with Exo, ABs, or DABs (40 µg/mL). Untreated cells served as the control. At 0, 12, 24, and 48 h, culture medium was replaced with fresh medium containing 10% (v/v) CCK-8 reagent and incubated for 1–2 h at 37°C. Absorbance at 450 nm was measured with a microplate reader (Multiskan MK3, Thermo). 2.11. Cell scratch assay Cell migration was measured by scratch assay. HUVEC and HaCaT cells were seeded in 6-well plates at 2 × 10 5 cells/well and cultured to confluency. A linear scratch was made with a sterile 200 µL pipette tip, and detached cells were removed by PBS. Cells were cultured in serum-free medium with 40 µg/mL Exo, ABs, or DABs. Images of the scratch area were taken at 0, 12, and 24 h using an inverted phase-contrast microscope, and gap areas were measured with ImageJ. 2.12. Transwell assay HUVEC suspensions (2 × 10 4 cells in 200 µL) were seeded in the upper chambers of transwell inserts (24-well, 8 µm pore size). The lower chambers contained 600 µL medium with 5% FBS and 40 µg/mL Exo, ABs, DABs, or PBS. Cells were cultured at 37°C for 24 and 48 h. The upper chamber membrane was fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Migrated cells were imaged at 24 and 48 h by an inverted optical microscope and counted with ImageJ. 2.13. Matrigel tube formation assay Angiogenesis was assessed by tube formation assay. HUVEC (5 × 10 4 cells/well) were seeded in 96-well plates coated with 100 µL growth factor-reduced Matrigel (Corning, USA) and incubated with 40 µg/mL Exo, ABs, or DABs for 8 h. Tube structures were imaged and quantified with ImageJ. 2.14. Macrophage polarization assay in vitro To evaluate the immunomodulatory capacity of the vesicles, RAW264.7 murine macrophages were plated in 12-well plates (1.5 × 10 5 cells/well) and primed with lipopolysaccharide (LPS, 1 µg/mL) for 24 h to induce an inflammatory phenotype. The LPS-challenged cells were then cultured for an additional 24 h in fresh complete medium containing PBS or 40 µg/mL of Exo, ABs, or DABs. For flow cytometric analysis, the treated macrophages were harvested, resuspended in 100 µL of PBS, and dual-stained with anti-mouse CD86 and anti-mouse CD206 antibodies for 30 min at 4°C in the dark. After standard washing procedures, the macrophage polarization states were quantified using a flow cytometer. 2.15. Macrophage immunoregulatory assay in vitro For an in vitro immunoregulatory study, RAW264.7 cells were treated with LPS (1 µg/ml) for 24 h and subsequently co-incubated with different formulations (40 µg/mL). Twenty-four hours later, the levels of immune factors (TNF-α, IL-6, IL-1β, IL-4, IL-10, and VEGF) in cell supernatants were measured using ELISA kits according to the manufacturer’s recommended protocols. 2.16. Macrophage ATP production assay in vitro To evaluate the total adenosine 5’-triphosphate (ATP) production capacity of macrophages in vivo , RAW264.7 cells were seeded in each well of twelve-well plates and treated with Exo, ABs, and DABs (40 µg/mL) for 24 h, and cells in medium containing PBS were prepared in parallel as the control group (1.5 × 10 5 cells). Afterwards, ATP in macrophages from different groups were extracted and detected using an Enhanced ATP Assay Kit. 2.17. Synthesis and characterization of phenylboronic acid-functionalized HA (PHA) To synthesize the PHA copolymer, 3-aminophenylboronic acid (3-APBA) was grafted onto the hyaluronic acid (HA) backbone via EDC/NHS-mediated amide coupling. First, the carboxyl groups of HA (80 mL, 7.5 mg/mL) were activated by stirring with EDC (540 mg) and NHS (330 mg) at room temperature for 6 h. Following activation, 3-APBA (450 mg) was introduced, and the conjugation reaction proceeded under vigorous stirring for an additional 24 h. To purify the synthesized PHA, the crude mixture was dialyzed (MWCO 3500 Da) against distilled water for 48 h, passed through a 0.8-µm filter, and lyophilized at -80°C. The chemical structure and successful grafting of the final PHA product were subsequently validated utilizing UV-vis spectrophotometry, 1 H NMR, and FTIR spectroscopy. 2.18. Preparation of the glucose/ROS dual-responsive PHA-PVA Gel hydrogel To fabricate the PHA-PVA Gel hydrogels, lyophilized PHA (at predetermined concentrations) and commercial PVA were individually dissolved in deionized water, with the pH of both precursor solutions subsequently neutralized to 7.4. The final hydrogel networks were rapidly formed at ambient temperature by co-extruding the PHA and PVA solutions at a 1:1 (v/v) ratio utilizing a double-barrel syringe. Five hydrogel compositions were fabricated with varying PHA solutions: Gel-1 (PHA (1.0%, w/v), PVA (8.0%, w/v)), Gel-2 (PHA (2.0%, w/v), PVA (8.0%, w/v)), Gel-3 (PHA (3.0%, w/v), PVA (8.0%, w/v)), Gel-4 (PHA (4.0%, w/v), PVA (8.0%, w/v)), and Gel-5 (PHA (5.0%, w/v), PVA (8.0%, w/v)). 2.19. Microstructure characterization The internal structure of PHA-PVA Gel specimens was examined by scanning electron microscopy (SEM). Specimens were snap-frozen in liquid nitrogen and freeze-dried for 24 h. Cross-sections were mounted on conductive carbon stubs, sputter-coated with gold, and imaged by SEM. 2.20. Rheological behavior Test Viscoelastic properties of the hydrogels were measured on a DHR-10 rheometer (TA Instruments) with a 10-mm parallel plate geometry at a 1-mm gap. Time sweeps at 1% strain were first performed to establish the linear viscoelastic region. Frequency sweeps (0.1–100 rad/s, 1% strain) and strain sweeps (0.1–1000%) were then performed at 37°C to measure storage modulus (G’) and loss modulus (G’’). 2.21. Self-healing properties evaluation Self-healing of PHA-PVA Gel was assessed visually and rheologically. Two hydrogel pieces dyed with rhodamine B and methylene blue were placed in contact for 2 h and then stretched to confirm bonding at the interface. Rheological recovery was measured by a step-strain test, where strain was alternated between 1000% and 5% to monitor recovery of G’ and G’’. 2.22. Adhesion strength test The in vitro adhesion of the PHA-PVA Gel was assessed on eight distinct organs (heart, liver, lung, spleen, kidney, bladder, colon, and stomach) that were excised from C57 mice. Specifically, 200 µL of freshly prepared PHA-PVA Gel was applied to stainless steel tweezers, which were subsequently used to attach the gel to the moist surface of the organs above. All these tests were conducted more than 5 times to ensure the results were consistent and reliable. 2.23. Intracellular ROS elimination and cytoprotective effect of PHA-PVA Gel To evaluate the intracellular ROS-scavenging capability of the hydrogels, HUVECs (2 × 10 5 cells/dish) were seeded into confocal imaging dishes and allowed to attach overnight. The cultures were pre-treated with medium containing either HA-PVA Gel or PHA-PVA Gel for 2 h, followed by a 4-h oxidative challenge using 5 mM H 2 O 2 . Intracellular ROS levels were subsequently probed by incubating the cells with 10 µM DCFH-DA (in serum-free medium) for 30 min prior to fluorescence microscopy. HUVEC (1 × 10 5 cells/well) were seeded in 12-well plates, pre-incubated with hydrogels for 2 h, and treated with 5 mM H 2 O 2 for 6 h. Cell viability was assessed by LIVE/DEAD® staining and fluorescence imaging. Apoptosis was measured by flow cytometry. 2.24. Evaluation of biocompatibility Cytotoxicity of PHA-PVA Gel was measured by CCK-8 assay in HUVEC and HaCaT cells. Hydrogel extracts were prepared following ISO 10993-5. To generate the hydrogel leachates for in vitro assays, the mixture was incubated at 37°C for 48 h. For biocompatibility evaluation, HUVECs and HaCaT cells were plated in 96-well plates at a density of 5 × 10 3 cells/well and allowed to adhere overnight. The standard culture medium was subsequently aspirated and substituted with 200 µL of fresh medium supplemented with the prepared hydrogel leachates. Cellular viability was continuously monitored over a 72-h period (evaluated at 24, 48, and 72 h) utilizing a CCK-8 assay. 2.25. Preparation and characterization of DABs@PHA-PVA Gel The DABs@PHA-PVA Gel hydrogel was prepared through the following sequential steps. Firstly, DABs were isolated as described previously and subsequently dispersed in a PVA solution at a concentration of 200 µg/mL. Afterwards, the DABs@PHA-PVA Gel hydrogel was formulated by combining a PVA solution containing DABs with a PHA solution using a dual-channel injector to achieve homogeneous mixing. To confirm the distribution of DABs in DABs@PHA-PVA Gel hydrogel, Dil-labelled DABs@PHA-PVA Gel was fabricated and observed through Z-stack model of CLSM. 2.26. In vitro responsive particle release assay To simulate the microenvironment of diabetic wounds, three release medium were prepared: (1) H 2 O 2 solution (200 µM), (2) glucose solution (16 mM), and (3) H 2 O 2 (200 µM) and glucose (16 mM) mixed solution. Then, the release profile of DABs from DABs@PHA-PVA Gel was examined when submerged in PBS and the medium at 37℃. Briefly, DABs@PHA-PVA Gel was positioned within 12-well plates, with each well containing 2 mL of the corresponding release medium, ensuring complete immersion of the hydrogel. The plates were subsequently incubated in a shaking incubator at 37°C with constant agitation. At predetermined time intervals (0, 2, 4, 6, 8, 12, 24, 36, 48, 60, and 72 h), 400 µL of the release medium was carefully collected from each well, and an equivalent volume of fresh medium was introduced to preserve a consistent volume of 2 mL. The number of DABs released at predetermined intervals was determined via BCA protein assay. In addition, the size distribution of DABs in the liquid medium at 72 h was measured using a laser particle size meter to assess shape integrity and changes in diameter after release. 2.27. In vitro responsive degradation test The degradation assay was conducted by immersing the DABs@PHA-PVA Gel in the three previously mentioned microenvironmental medium. In short, DABs@PHA-PVA Gel was freshly prepared, and the surface moisture was removed using filter paper. The initial weight (W 0 ) was then recorded. Subsequently, the weighed samples were incubated in the medium above and PBS at 37°C. At designated time points (3, 6, 9, 12, and 15 days), the medium was discarded, and the hydrogels were dried and weighed (W n ). The degradation rate of the hydrogels was calculated using the following formula: DR = (W 0 - W n ) / W 0 × 100%, where W 0 represents the initial weight of the hydrogel at day 0, W n denotes the weight of the hydrogel at day n, and DR indicates the degradation rate of the hydrogel. 2.28. Establishment of diabetic mouse cutaneous wound model All in vivo protocols were executed under the ethical approval of the Institutional Animal Care and Use Committee (IACUC) at China Pharmaceutical University (approval # 2024-09-017). Male C57BL/6 mice (6 weeks old, 18–22 g) were sourced from Jiangsu Huachuang Sino Pharmaceutical Technology Co., Ltd. and maintained on a 12-h light/dark cycle with ad libitum access to a high-fat diet and water. To induce the type 2 diabetes mellitus (T2DM) model, mice reaching a body weight of 30 g received consecutive daily intraperitoneal injections of streptozotocin (STZ, 50 mg/kg) for 5 days. Fasting blood glucose (FBG) was measured by a glucometer. Mice with FBG > 16.65 mM for two consecutive days were considered successfully modeled. Mice that did not reach this threshold received additional STZ (20 mg/kg, i.p.). Diabetic mice were anesthetized, dorsal hair was removed, and a 10-mm full-thickness excisional wound was made along the dorsal midline. 2.29. In vivo wound healing investigation The diabetic mice were randomly divided into 8 groups: 1) Control group (G1); 2) Exo group (G2); 3) ABs group (G3); 4) DABs group (G4); 5) PHA-PVA Gel group (G5); 6) ABs@PHA-PVA Gel group (G6); 7) DABs@usGel group (G7); and 8) DABs@PHA-PVA Gel group (G8). (Herein, ABs@PHA-PVA Gel denotes a responsive hydrogel system for ABs, whereas DABs@usGel represents an unresponsive hydrogel loaded with DABs, prepared through freeze-thaw crosslinking using HA and PVA as precursor solutions at the same concentration.) Each group contained at least six mice. Treatments were applied to wounds every three days. Exo, ABs, and DABs were dosed at 100 µg/mouse, and the control group received saline. Wounds were covered with 3M™ Tegaderm dressings. Mice were euthanized at day 7 and day 14, and full-thickness dorsal skin was collected for further analysis. Macroscopic wound closure was monitored by capturing digital photographs on days 0, 3, 7, and 14 post-surgeries. Additionally, The wound area ratio (%) can be calculated by the formula S n /S 0 × 100%, where S 0 is the wound area at day 0, and S n is the wound area at day n (n = 3, 7, 14). On day 7 and 14, the mice were anesthetized and euthanized, after which the wound tissues were harvested for histological analysis. 2.30. Histological, immunohistochemistry, and immunofluorescence analysis For histological evaluation, tissue sections were deparaffinized and rehydrated, then stained with H&E and Masson’s trichrome. To evaluate the expression of ROS in the wounds of diabetic mice, immunofluorescence staining was performed to examine ROS levels in the skin tissues of the wound areas during the intermediate treatment phase (day 7). To assess macrophage phenotypic transition in wounds, immunofluorescence staining for CD86 and CD206 was performed. To detect angiogenesis in the wound, wound tissue sections were stained with CD31, CD105, and α-SMA for immunofluorescence. In addition, IHC staining for VEGF and TGF-β was performed to detect changes in the wound microenvironment associated with neovascularization. To evaluate hypoxia-induced improvement in mouse wounds, immunofluorescence staining was performed to assess HIF-1α expression. Nuclei were stained with DAPI. Quantification of IHC and IF was performed using ImageJ software. 2.31. Evaluation of tissue inflammation At days 7 and 14, tissue sections were collected for the assessment of tissue inflammation. To quantify TNF-α, IL-6, IL-1β, IL-4, IL-10, and VEGF levels, cytokine data were standardized relative to the concentrations of these factors measured in normal skin using ELISA kits. Furthermore, flow cytometry was used to analyze macrophage polarization in skin wound tissues at the conclusion of the treatment period. 2.32. Transcriptome analysis Wound tissues from the control and DABs@PHA-PVA Gel groups (n = 3/group) were collected at day 14. Total RNA was extracted with TRIzol (Invitrogen) and quantified by a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). Libraries were constructed from high-quality RNA and sequenced on the BGISEQ-T7 platform (BGI, Shenzhen, China). Raw FASTQ reads were filtered with fastp (v0.21.0) and aligned to the mouse reference genome with HISAT2 (v2.1.0). Transcript expression was quantified as FPKM using StringTie (v2.1.5). DEGs were identified with DESeq2 using thresholds of |log2(fold change)| > 1 and p < 0.05. GO and KEGG enrichment analyses were performed with the clusterProfiler R package (p < 0.05). 2.33. Statistical analysis Statistical analysis was performed using GraphPad Prism 10.4.1. Data are presented as mean ± SD. Two-group comparisons were made by two-tailed unpaired Student’s t-test. Multi-group comparisons were made by one-way ANOVA with Tukey’s multiple comparisons. P < 0.05, p < 0.01, and p < 0.001 were considered statistically significant. 3. Results 3.1. Preparation and characterization of DABs The preparation of DABs is shown in Fig. 2 a. MSCs were preconditioned with DFO and then induced to undergo apoptosis by UV irradiation. To optimize DFO preconditioning, MSCs were treated with various concentrations of DFO. Based on AO/PI cell viability assays and Western blot analysis, 250 µg/mL was selected as the optimal concentration, as it induced the maximum upregulation of HIF-1α while maintaining excellent cell viability (Fig. S1 and Fig. S2). Subsequently, the optimal UV-induced apoptosis condition was determined to be 10 minutes of irradiation, based on analysis of cell morphology and apoptosis rates (Fig. S3 and Fig. S4). Following UV irradiation, the morphology of the apoptotic cells was examined. At six hours post-treatment, both apoptotic MSCs (Apo-MSCs) and apoptotic DFO-MSCs (Apo-D-MSCs) exhibited clear morphological signs of apoptosis, including the formation of prominent vacuole-like structures and membrane blebbing, which were absent in the non-irradiated control group (Fig. 2 b and 2 c). Following 24 h of induction, apoptotic bodies (ABs and DABs) were isolated through differential sequential centrifugation (Fig. S5). Transmission electron microscopy (TEM) revealed that both vesicle types displayed characteristic spherical or cup-shaped forms with distinct membrane structures (Fig. 2 d). Dynamic light scattering (DLS) analysis revealed a size distribution ranging from 50 nm to 800 nm, characteristic of apoptotic bodies (Fig. 2 e). Both ABs and DABs displayed a negative surface charge of approximately − 20 mV, consistent with their origin from the negatively charged cell membrane (Fig. 2 f). We then examined the key surface properties of the vesicles. Coomassie brilliant blue staining verified that both ABs and DABs contained a complex protein from their parent cells (Fig. 2 h). Both ABs and DABs were found to be highly enriched with the “eat-me” signal, phosphatidylserine (PS), with flow cytometry showing exposure rates of 84.60% and 83.40%, respectively (Fig. 2 i). BCA assay showed that DAB yield was higher than ABs yield and Exo yield from the same number of parent cells (Fig. 2 g). This indicated that DFO preconditioning acted as a dual-functional strategy, simultaneously programming vesicle cargo and amplifying production efficiency. Afterwards, HUVEC, RAW264.7, and HaCaT cells were incubated with DiI-labeled ABs and DABs. Confocal microscopy showed that all three cell types internalized ABs and DABs with perinuclear localization. Flow cytometry showed that RAW264.7 had higher fluorescence intensity than HUVEC and HaCaT cells, indicating greater uptake efficiency (Fig. 2 j, 2 k; Fig. S6 and Fig. S7). This may be due to PS on the surface of ABs and DABs, which promotes recognition and uptake by macrophages via efferocytosis. 3.2. Transcriptomics analysis of DABs miRNAs play key roles in post-transcriptional gene regulation and cell modulation [ 21 , 22 ]. The miRNA content of vesicles reflects the physiological state of the parent cell [ 23 ]. To investigate the impact of DFO programming on vesicular cargo, we compared miRNA profiles of ABs and DABs by high-throughput sequencing. PCA showed clear separation between the two groups, with DABs showing tighter clustering and greater sample homogeneity (Fig. 3 a). Differential expression analysis identified 214 altered miRNAs in DABs, including 108 upregulated and 106 downregulated (Fig. 3 b). To elucidate the functional implications of the differentially expressed miRNAs, we performed GO and KEGG enrichment analyses. Then GO and KEGG enrichment analyses were performed on predicted target genes of the differentially expressed miRNAs (DEMs). GO analysis showed enrichment in protein kinase activity and chemokine receptor signaling (Fig. 3 c). KEGG analysis showed enrichment in immune-related pathways such as Th1/Th2 differentiation, as well as focal adhesion and axon regeneration pathways (Fig. 3 d). Analysis of the top 50 DEMs showed that 34 were upregulated in DABs (Fig. 3 e). miRNAs linked to angiogenesis and cell migration, including hsa-miR-4674, hsa-miR-1246, hsa-miR-3976, and hsa-miR-320e, were enriched in DABs (Fig. S8a and S8b). To understand how DABs drive regeneration, we looked at the target networks of the top ten upregulated miRNAs in DABs. Pathway enrichment showed these targets are linked to key repair processes, including angiogenesis, cell migration, and VEGF signaling (Fig. 3 f and 3 g). We also found that these miRNAs appear to regulate mitochondrial bioenergetics and cellular respiration (Fig. 3 h), which was somewhat unexpected. This suggests DABs may promote tissue repair by activating angiogenic pathways while also supporting the metabolic demands of the repair process, though the exact interplay between these two mechanisms needs further investigation. Beyond metabolic reprogramming, enrichment analysis of the top 10 upregulated miRNA targets in DABs revealed a significant clustering in pathways essential for cellular dynamics, such as Rho GTPase signaling and cytoskeleton organization (Fig. 3 i) [ 24 ]. Combined with enrichments in the mitotic cell cycle and chromatin remodeling, these findings underscore a shift toward a more active, migratory phenotype. How DFO programming integrates these diverse structural and bioenergetic processes remains an open question. Mapping the predicted miRNA targets onto a protein-protein interaction network revealed a rather heterogeneous regulatory landscape, with MCODE identifying two functionally distinct hubs (Fig. 3 j, Fig. S9) [ 24 ]. A prominent energy-metabolism module (MCODE 2) captures much of the mitochondrial ATP synthesis and electron transport machinery—specifically components like NDUFB5, NDUFA9, COX3, and ND1, which appear tightly governed by the miR-320 family and miR-3976 [ 25 ]. The second was a cytoskeletal dynamics module (MCODE 4) centered on RAC1 signaling, where CRK, NCKAP1, and ABI2 formed a closely connected motility network. These results suggest that DABs may work by boosting mitochondrial energy production while also activating RAC1-driven cytoskeletal changes, which together could support cell migration and tissue repair though the relative contribution of each arm warrants further study [ 26 , 27 ]. Finally, we extended the transcriptomic profiling to include parent cell-derived exosomes (Exo) for comparison. The data showed that both ABs and DABs possessed a more complex and abundant miRNA repertoire compared to the Exo group (Fig. S12-S16). Chord diagram analysis of the top upregulated miRNAs in DABs relative to Exo unveiled a coordinated bipartite regulatory network that simultaneously orchestrates the resolution of inflammation and the activation of structural regeneration pathways. This data established a robust mechanistic basis for the superior therapeutic efficacy of DABs over conventional Exo-based therapies (Fig. S17 and S18). 3.3. In vitro biological evaluation of DABs Determining the functional impact of this molecular became our primary focus, particularly given the distinct pro-regenerative miRNA signature we found packaged within the DABs. To provide the therapeutic benchmark, we selected stem cell-derived exosomes (Exo), currently under investigation in clinical trials, as the positive control [ 28 , 29 ]. Given that effective wound healing necessitates the coordinated proliferation and migration of endothelial and epidermal cells [ 30 ], we systematically evaluated the efficacy of DABs against native ABs and Exo in driving these cellular behaviors. DABs visibly forced a rapid G1-to-S transition in HUVECs, pooling the population in the active S and G2/M phases (Fig. 4 a and 4 b). Far from just dividing faster, these cells became highly mobile. In fact, 24 hours was all it took for DAB-treated HUVEC and HaCaT cultures to completely seal scratch wounds, leaving ABs and Exo lagging far behind (Fig. 4 c-e; Fig. S19a and 19b). CCK-8 assays also showed increased cell proliferation after DABs treatment (Fig. S20). Transwell assays showed DABs increased HUVEC migration by 1.95-fold, compared to 1.25-fold with Exo and 1.16-fold with native ABs (Fig. 4 f and 4 g). In Matrigel tube formation assays, DABs also produced the greatest increases in total tube length and network area across all groups (Fig. 4 h and 4 i). To evaluate immunomodulatory effects in vitro , RAW264.7 were stimulated with LPS to mimic an inflammatory environment (Fig. 4 j) [ 31 ]. Flow cytometry showed that the control and Exo groups maintained a high proportion of CD86 + (M1) macrophages, while DABs treatment shifted the balance toward CD206 + (M2) macrophages (Fig. 4 k). The CD206/CD86 ratio was highest in the DABs group compared to both Exo and native ABs (Fig. 4 l). ELISA results further showed that DABs treatment lowered pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) (Fig. 4 n) and raised anti-inflammatory and angiogenic cytokines, including IL-4, IL-10, and VEGF (Fig. 4 o). Polarization into an M2 state fundamentally relies on oxidative phosphorylation. Tracking this specific energy requirement showed a distinct metabolic difference, with DABs-treated macrophages producing substantially more ATP (Fig. 4 m). This suggests the metabolic boost provided by DABs may help support the energy demands of phenotypic switching and sustained anti-inflammatory activity. 3.4. Preparation and characterization of DABs@PHA-PVA Gel To enable controlled delivery of DABs, we developed a responsive hydrogel using a phenylboronic acid-modified hyaluronic acid copolymer (PHA). PHA was synthesized by conjugating 3-aminophenylboronic acid onto the hyaluronic acid backbone, and successful grafting was confirmed by UV-vis, 1 H-NMR, and FT-IR spectroscopy (Fig. S21-S23). The copolymer was then crosslinked with polyvinyl alcohol (PVA) to form the hydrogel network (Fig. 5 a). Different PHA/PVA ratios were tested to optimize gelation and stability, and vial inversion tests identified Gel-2, Gel-3, and Gel-4 as suitable candidates based on their rapid sol-gel transition and stable formation (Fig. S24). Rheological characterizations showed that all three formulations were elastic-dominant (G’ > G’’) (Fig. 5 b and 5 c). However, Gel-3 displayed fluctuations in its time-sweep profile, suggesting network inhomogeneity (Fig. 5 d). Gel-4 maintained a consistent rheological profile and was selected for subsequent experiments (designated as PHA-PVA Gel ). Scanning electron microscopy (SEM) of PHA-PVA Gel revealed a porous, interconnected microstructure (Fig. S25 and Fig. 5 e). Macroscopic evaluation of self-healing properties showed that two PHA-PVA Gel pieces (stained red and blue) fused within minutes and withstood subsequent stretching (Fig. 5 f). Rheological step-strain tests indicated that the hydrogel recovered over 95% of its initial storage modulus immediately following a 1000% high-strain cycle (Fig. 5 g). The hydrogel also adhered to various murine organs ex vivo , including the heart, liver, spleen, and lungs (Fig. 5 h). We evaluated the intrinsic ROS-scavenging capacity of the hydrogel in vitro using an H 2 O 2 -induced oxidative stress model in HUVEC. DCFH-DA staining indicated high intracellular ROS levels in the H 2 O 2 -treated group (G2), which were effectively neutralized in the presence of PHA-PVA Gel (G4) (Fig. 5 i and 5 l). Flow cytometry showed that H 2 O 2 induced apoptosis in 76.80% of cells (G2). Treatment with PHA-PVA Gel reduced the apoptotic rate to 18.65% (G4), compared to 64.50% with the control hydrogel (G3) (Fig. 5 j and 5 k). Live/Dead staining confirmed higher cell viability in the PHA-PVA Gel group (Fig. S26). Quantitative assays demonstrated that cells cultured in hydrogel leachates-maintained proliferation rates comparable to untreated controls (Fig. S27). DABs were then loaded into the hydrogel (DABs@PHA-PVA Gel ) for further characterization. Confocal Z-stack imaging showed that DiI-labeled DABs were distributed evenly throughout the porous hydrogel network (Fig. 5 m). We evaluated the in vitro release of DABs under conditions simulating the high glucose and ROS levels of a diabetic wound [ 32 ]. Under dual-stimulus conditions (glucose + H 2 O 2 ), DABs release reached 95.33% at 72 h, which was significantly higher than either single-stimulus (glucose or H 2 O 2 alone) or PBS groups (Fig. 5 n). This matched the hydrogel degradation pattern, where the greatest mass loss also occurred under dual-stimulus conditions (Fig. 5 o). DLS confirmed that the released DABs maintained their original size distribution (Fig. 5 p). 3.5. Synergistic in vivo wound healing in a diabetic mouse model Following the in vitro results, we next evaluated the therapeutic performance of DABs@PHA-PVA Gel in a full-thickness diabetic wound model. The experimental setup and STZ-induced diabetic model are described in Fig. 6 a and Fig. S28. Mice were randomized into eight groups: untreated (G1), Exo (G2), native ABs (G3), DABs (G4), PHA-PVA Gel (G5), ABs@PHA-PVA Gel (G6), DABs@usGel (unresponsive hydrogel loaded with DABs, G7), and DABs@PHA-PVA Gel (G8). Wound closure was tracked over 14 days. Representative images (Fig. 6 b and 6 c) and wound area heatmaps (Fig. 6 e) showed faster healing in the DABs@PHA-PVA Gel group (G8), which reached a closure rate of 80.69% by day 7 (Fig. 6 f). DABs alone (G4, 69.87%) also outperformed the Exo group (G2), and the difference between G8 and G4 likely reflects the benefit of sustained therapeutic retention provided by the hydrogel. G8 treated wounds reached complete re-epithelialization by day 14, with hair follicle regeneration already visible at the macroscopic level. This physical closure was anchored by a robust architectural recovery; H&E sections from days 7 and 14 detailed a continuous, well-consolidated epidermis packed with newly formed sweat glands and follicles (Fig. 6 d, Fig. S29). This highlighted the failure of the untreated G1 group, which stalled at under 61% closure and remained structurally deficient, burdened by unresolved inflammatory infiltrates and a persistently incomplete epidermis. Collagen accumulation reached high of 74.57% in the G8 tissues. This matrix accumulation showed moderate outcomes across the G7, G6, and G4 groups, with values ranging from 56.59% to 62.85%. Zooming in on the vesicle-only treatments isolates a fundamental functional gap. The DABs (G4) alone drove deposition up to 56.59% while the Exo (G2) alone stalled at 40.98% (Fig. 6 h). DFO programming evidently equips DABs with an intrinsic tissue-remodeling strength that Exo lack entirely. Scar index results followed the same trend, with G8 achieving the lowest score (3.05%) compared to G2 (42.11%), G4 (23.62%), G5 (45.98%), and G1 (68.59%) (Fig. 6 g). 3.6. In situ mechanistic evaluation The regenerative success seen at day 14 is fundamentally rooted in earlier microenvironmental shifts. We examined wound tissues at day 7 to capture this initial remodeling phase. Excessive ROS notoriously drives persistent inflammation and impairs diabetic healing [ 33 ]. DHE staining of these early samples provided a direct readout of the underlying oxidative stress burden. Tissues from the untreated group (G1) showed intense red fluorescence, indicating high levels of oxidative stress (Fig. 7 a). In stark contrast, the DABs@PHA-PVA Gel group (G8) demonstrated near-complete ROS elimination (0.32% positive area), as quantified in Fig. 7 f. This ROS-scavenging efficacy was significantly more potent than that of the hydrogel alone (G5: PHA-PVA Gel ), suggesting a crucial synergistic effect between the active PHA-PVA Gel carrier and the sustained, inflammation-modulating release of DABs. This quick reduction in oxidative stress is essential for breaking the feedback cycle that sustains chronic inflammation. We therefore analyzed macrophage phenotype and distribution by immunofluorescence. As hypothesized, the G8 group showed a dramatic reduction in M1 macrophage (CD86 + ) infiltration (Fig. 7 b and 7 f). At the same time, it showed the highest levels of anti-inflammatory M2 macrophages (CD206 + ), suggesting an early phenotypic switch. This beneficial immune environment persisted until the end, as indicated by consistent immunofluorescence results at day 14 (Fig. S31 and Fig. S32). To further validate this long-term effect, we analyzed the tissue cytokine profile by ELISA at day 14. As shown in Fig. 7 g and Fig. S33, the G8 group maintained the most profound reduction in pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and the highest levels of anti-inflammatory cytokines (IL-4, IL-10, VEGF). Proper vascularization supports the proliferative healing phase [ 34 ]. The G8 treatment initiated this response effectively, with day 7 immunofluorescence capturing a dense CD31 + and α-SMA + vessel network that continued expanding through day 14 (Fig. 7 c and 7 f; Fig. S34 and S35). Local protein expression aligned directly with this structural growth. Tissues receiving the G8 formulation yielded the highest concentrations of TGF-β and VEGF (Fig. 7 e and 7 f) and maintained these elevated pro-angiogenic signals into the later stages of repair (Fig. S36 and S37). HIF-1α expression offered a functional readout for local tissue hypoxia. Unresolved oxygen deficiency characterized the untreated G1 group since these defects maintained consistently high HIF-1α levels across the entire observation period. In contrast, the G8 group exhibited a rapid and significant decrease in HIF-1α expression (Fig. 7 d and 7 f; Fig. S38), consistent with the restoration of tissue perfusion. Finally, the biosafety profile of DABs@PHA-PVA Gel was tested to ensure clinical applicability. Histological examination of major organs and local muscle tissue, combined with serum biochemical analysis, revealed no systemic toxicity or inflammatory response (Fig. S39-S41), confirming the excellent biocompatibility of DABs@PHA-PVA Gel for regenerative medicine. 3.7. Transcriptomic analysis of the DABs@PHA-PVA Gel promoting the diabetic wound healing While histological evaluations confirmed the superior tissue quality in the DABs@PHA-PVA Gel group, diabetic wound regeneration involves a complex symphony of signaling events that extends beyond structural observations. Therefore, to gain an unbiased, global understanding of the molecular reprogramming driving the regenerative effects of our system, we performed RNA-seq on wound tissues harvested on day 14 (G1: Untreated vs. G8: DABs@PHA-PVA Gel ), with sequencing quality control and alignment statistics summarized in Supporting Tab. S1 and S2. First, Principal Component Analysis (PCA) demonstrated that the untreated (G1) and DABs@PHA-PVA Gel -treated (G8) groups segregated into distinct, non-overlapping clusters (Fig. 8 a). Differential expression analysis identified 4779 differentially expressed genes (DEGs) between the two groups (FDR 1), comprising 2493 upregulated and 2286 downregulated genes in the G8 group, as visualized in the volcano plot (Fig. 8 b). GO and KEGG enrichment analyses were performed to evaluate the biological pathways associated with these DEGs. GO analysis indicated that the downregulated genes in the G8 group were primarily enriched in pro-inflammatory biological processes, including ‘response to interferon-gamma’, ‘inflammatory response’, and ‘cytokine response’ (Fig. 8 c and Fig. S42). Conversely, the upregulated genes were primarily associated with regenerative and metabolic processes, such as ‘cell projection’, ‘cytoskeleton organization’, and ‘ATP binding’. KEGG pathway analysis confirmed a suppression of classical inflammatory networks in the treated tissues. Specific cascades like TNF, NF-κB, and general cytokine signaling were distinctly underrepresented. This dampened inflammatory state allowed tissue repair and metabolic programs to dominate the enrichment profiles, with Foxo and Rap1 signaling emerging as the primary driven pathways (Fig. 8 d and Fig. S43). To further examine transcriptional changes corresponding to the wound healing, we analyzed targeted gene sets associated with oxidative stress, inflammation, and angiogenesis [ 35 – 37 ]. Heatmaps demonstrated a coordinated upregulation of antioxidant genes (e.g., Trp53inp1, Foxo4, Pink1), immunomodulatory genes (e.g., Cebpa, Cd300a, Cnr2), and pro-angiogenic genes (e.g., Cxcr4, Nrp1, S1pr1) in the G8 group relative to G1 (Fig. 8 e, 8 g, and 8 i). The expression levels (FPKM) of representative hub genes were plotted to quantify these cluster trends (Fig. 8 f, 8 h, and 8 j). Compared to the untreated control, the DABs@PHA-PVA Gel treatment resulted in significantly higher expression levels of specific antioxidant (Cat, Txnip, Foxo4), anti-inflammatory (Cebpa, Cd300a, Cnr2), and pro-angiogenic (Cxcr4, Nrp1, Rhob) genes. Together, these targeted transcriptional analyses provide direct validation of the upstream miRNA-driven and pathway-level findings, demonstrating that DABs@PHA-PVA Gel coordinately reprogram antioxidant defense, inflammatory regulation, and angiogenic responses at the gene expression level. Based on these transcriptomic findings, we tentatively interpret a molecular mechanism (Fig. 8 k). These mechanisms suggest that DABs@PHA-PVA Gel may influence multiple biological processes rather than a single dominant pathway. Alterations in Txnip, Foxo4, and Pink1 expression are consistent with modulation of Pink1-mediated mitochondrial quality control, which may contribute to improved redox balance [ 38 , 39 ]. In parallel, increased expression of genes within the Daglb/Cnr2 axis may reflect engagement of endogenous pathways linked to inflammation resolution, which have recently been implicated in skin repair processes [ 40 , 41 ]. The activation of the Cxcr4/Nrp1/S1pr1 axis points to a coordinated vascular repair mechanism, where Cxcr4 recruits endothelial progenitor cells and Nrp1 and S1pr1 support Vegfr2 signaling and Rhob GTPase activation to drive endothelial migration and vessel maturation [ 42 , 43 ]. 4. Discussion Clinical translation of MSC-derived exosomes remains limited by their relatively low natural secretion yield and the instability of their therapeutic performance under pathological conditions [ 4 , 5 , 44 , 45 ]. Apoptotic bodies (ABs) offer a high-yield alternative and possess intrinsic immunomodulatory properties largely attributed to phosphatidylserine (PS) exposure on their surface [ 6 , 7 , 46 ]. However, their native pro-angiogenic activity is generally insufficient to meet the substantial vascularization demands of severely ischemic environments such as diabetic wounds [ 10 , 11 , 47 , 48 ]. In the present study, we demonstrate that pre-programming MSCs with DFO prior to apoptosis enables the generation of programmed vesicles (DABs) that effectively address both of these limitations. DABs possess an enhanced biological activity that is likely driven by their abundant miRNA cargo. Although conventional preconditioning often relies on DFO to stabilize HIF-1α [ 49 , 50 ], our work reveals an additional effect that applying DFO prior to apoptosis induction fundamentally alters the vesicle’s cargo composition. For instance, DABs were found to be heavily loaded with specific miRNAs known to influence angiogenesis and cell migration such as hsa-miR-4674, hsa-miR-3976, and hsa-miR-320e [ 51 ]. The levels of these molecules far exceeded what we observed in typical MSC-derived exosomes or native apoptotic bodies. These altered miRNAs likely contribute to the superior pro-angiogenic and immunomodulatory effects observed in our study. Surface exposure of PS is known to facilitate macrophage efferocytosis [ 52 , 53 ], a property that is retained in DABs. Analysis of recipient macrophages suggests the involvement of additional regulatory mechanisms beyond canonical pathways. The miRNA repertoire delivered by DABs appears to influence mitochondrial energy metabolism, as evidenced by increased cellular ATP production. To date, most EV-based therapeutic strategies have focused on modulating inflammatory signaling [ 54 , 55 ], whereas modulation of mitochondrial metabolism in recipient immune cells by programmed apoptotic bodies has not been reported. Whether this metabolic shift directly drives the M1-to-M2 polarization observed in our study remains unclear. However, given the well-established dependence of M2 macrophages on oxidative phosphorylation [ 56 ], this association merits further investigation. To improve vesicle stability within the hostile microenvironment of diabetic wounds, DABs were further encapsulated in a glucose- and ROS-responsive PHA-PVA Gel . The in vivo results showed this approach was effective, with DABs@PHA-PVA Gel promoting early wound closure faster than either exosome therapies or no treatment at all. We then ran RNA sequencing on the treated wound tissues to get a clearer picture of the biological changes driving this accelerated healing. The transcriptomic profiles showed a few distinct but coordinated trends. We noted a drop in the Txnip/Foxo4 cascade alongside the restoration of Nrf2-driven redox homeostasis [ 38 , 39 ]. At the same time, the Daglb/Cnr2 signaling axis lit up that makes sense given the emerging role of cannabinoid receptor 2 in clearing up skin inflammation [ 40 , 41 ]. Furthermore, there was an upregulation across the Cxcr4/Nrp1/S1pr1 receptor network, which is known to recruit endothelial progenitor cells for vascular repair [ 42 , 43 ]. The simultaneous modulation of these pathways by a single vesicle-based intervention is noteworthy and likely reflects the diverse regulatory capacity of the miRNAs carried by DABs. Despite these encouraging findings, our current study does have a few constraints. Although the STZ-induced diabetic mouse model reproduces certain aspects of diabetic pathology, it does not fully capture the complex systemic complications present in human type 2 diabetes [ 57 , 58 ]. In addition, murine wound healing relies heavily on contraction, whereas re-epithelialization plays a more dominant role in human chronic wounds [ 59 , 60 ]. The 14-day observation period used in this study is also insufficient to fully characterize the long-term degradation behavior and systemic clearance profile of PHA-PVA Gel . From a translational perspective, further work will also be required to ensure batch-to-batch consistency of DFO-programmed ABs and to establish scalable purification strategies suitable for large-scale production. Future studies using large-animal wound models, such as porcine skin defects that more closely resemble human dermal architecture, will therefore be important. In conclusion, our findings demonstrate the potential of upstream cellular programming as a practical strategy to generate functionally enhanced apoptotic vesicles. Notably, a single preconditioning step using an approved small molecule was sufficient to substantially reshape apoptotic vesicle cargo, conferring both immunoregulatory and pro-angiogenic properties. Moreover, the fact that these programmed vesicles might actively alter recipient cell metabolism opens up a new therapeutic angle worth exploring. This strategy could extend well past diabetic wound repair. It holds promise for other conditions where conventional exosome therapies fall short, such as peripheral nerve injuries, inflammatory skin disorders, and ischemic vascular diseases. Declarations Ethics declarations All in vivo protocols were executed under the ethical approval of the Institutional Animal Care and Use Committee (IACUC) at China Pharmaceutical University (approval # 2024-09-017). Competing interests The authors declare no conflict of interest. Supplementary Information Supplementary data to this article can be found in Supporting Information file. Funding This work was supported by the National Natural Science Foundation of China (82272801, 82073175); Shandong Provincial Natural Science Foundation (ZR2025MS1272); Taishan Scholars Program for Young Expert of Shandong Province (tsqn202306383); Science and Technology Co-construction Project of National Administration of Traditional Chinese Medicine (GZY-KJS-SD-2024-103); Jinan Municipal Science and Technology Plan Project (202512040); China Postdoctoral Science Foundation (2025M772160); Basic Research Program of Jiangsu (BK20251569). Author Contribution H. Guo drafted the manuscript, reviewed and edited the paper, administered the project, developed the methodology, validated the results, curated the data, and contributed to the study conception. J. Li drafted the manuscript, developed the methodology, validated the results, curated the data, and contributed to the study conception. C.C. Gu, N. Liu, J. Sun, and D.H. Wang contributed to methodology development, result validation, and data curation. S.Y. Meng and D.S. Geng contributed to methodology development and data curation. Y.F. Lian reviewed and edited the manuscript, supervised the study, administered the project, developed the methodology, validated the results, curated the data, and contributed to the study conception. F.N. Lv reviewed and edited the manuscript, acquired funding, developed the methodology, validated the results, curated the data, and contributed to the study conception. M.R. Huo drafted the manuscript, reviewed and edited the paper, supervised and administered the project, acquired funding, developed the methodology, validated the results, curated the data, and contributed to the study conception. All authors reviewed and approved the final version of the manuscript. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. 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Supplementary Files SupportingInformation.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 16 May, 2026 Reviews received at journal 15 May, 2026 Reviews received at journal 12 May, 2026 Reviews received at journal 09 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers agreed at journal 01 May, 2026 Reviewers agreed at journal 01 May, 2026 Reviewers agreed at journal 01 May, 2026 Reviewers invited by journal 01 May, 2026 Editor assigned by journal 30 Apr, 2026 Submission checks completed at journal 30 Apr, 2026 First submitted to journal 25 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9522213","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":637196867,"identity":"f578d5c2-cf94-404c-8a4a-9e8375e82554","order_by":0,"name":"Hao Guo","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Guo","suffix":""},{"id":637196868,"identity":"66324014-abbf-4be2-b298-71607ad69afa","order_by":1,"name":"Jian Li","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Li","suffix":""},{"id":637196869,"identity":"e524bd4b-001b-4d01-8962-c1184926ac6d","order_by":2,"name":"Chengcheng Gu","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Chengcheng","middleName":"","lastName":"Gu","suffix":""},{"id":637196870,"identity":"f77e2ab3-ddf0-40c0-8760-7990a63bca0e","order_by":3,"name":"Ning Liu","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Liu","suffix":""},{"id":637196871,"identity":"05752fc1-8da0-4c8c-9339-bd768b171ad7","order_by":4,"name":"Jie Sun","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Sun","suffix":""},{"id":637196872,"identity":"1b2a8187-8819-4b7c-9b9c-456981718e2c","order_by":5,"name":"Danhui Wang","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Danhui","middleName":"","lastName":"Wang","suffix":""},{"id":637196873,"identity":"9fac833b-ac4a-4241-a3f2-d2c635f4c7a7","order_by":6,"name":"Siyuan Meng","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Siyuan","middleName":"","lastName":"Meng","suffix":""},{"id":637196874,"identity":"2b33e5b0-72bf-4366-8829-f757d5e78b41","order_by":7,"name":"Dongshu Geng","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Dongshu","middleName":"","lastName":"Geng","suffix":""},{"id":637196875,"identity":"32a1d5a8-793b-47f3-a3d5-3ad167fc2d9c","order_by":8,"name":"Yunfei Lian","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yunfei","middleName":"","lastName":"Lian","suffix":""},{"id":637196878,"identity":"d7f6fa50-73d9-473a-abe3-cc4dc34858f3","order_by":9,"name":"Fangnan Lv","email":"","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Fangnan","middleName":"","lastName":"Lv","suffix":""},{"id":637196879,"identity":"fe532d70-2ffa-4b3c-b7b6-5f1923597acd","order_by":10,"name":"Meirong Huo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYBACAyQSCCok5ORJ1HLGwtiwgSgtMMDYVpHIcICAFnOJ5KMbPhTckzPnX/zsMe88iQTGBuaHj27g0WI5Iy3t5gyDYmPLGc/MjXm3SeSxM7AZG+fgc9iNHLPbPAYJiRtunGGTBmopZmzgYZMmqOUPXMscicSGA8RoYQBpOd8D1NJAjJYzz9Ju9hgkGBvcYDM3nHNMwtiwmZBfjicfu/HjT4KcwfnDzx68qamTk2dvfvgYnxYEkEhggzCYiVIOAvwH2IhWOwpGwSgYBSMLAADhIExwOSvaagAAAABJRU5ErkJggg==","orcid":"","institution":"China Pharmaceutical University","correspondingAuthor":true,"prefix":"","firstName":"Meirong","middleName":"","lastName":"Huo","suffix":""}],"badges":[],"createdAt":"2026-04-25 04:53:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9522213/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9522213/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108970492,"identity":"5be1cc21-fee7-42ea-bdd2-8d8757144269","added_by":"auto","created_at":"2026-05-11 10:16:28","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6981389,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e for promoting diabetic wound healing. (a) Preparation of DFO-programmed apoptotic bodies (DABs) and their encapsulation in the glucose/ROS-responsive PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (b) Proposed mechanism of DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e in diabetic wound healing, highlighting ROS scavenging with DABs on-demand release, macrophage polarization, and angiogenesis.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/13595284661c5e55166ba1cc.jpeg"},{"id":108970450,"identity":"417bb227-296d-42b9-8547-052fabbad79d","added_by":"auto","created_at":"2026-05-11 10:16:01","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1333056,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation and characterization of ABs and DABs. (a) Schematic illustration of the isolation and purification of DABs from DFO-treated MSCs. (b) Morphological changes of MSCs 6 h after apoptosis. (Scale bars = 200 μm). (c) Formation of apoptotic vacuoles in MSCs 6 h after apoptosis. (Scale bars = 20 μm). (d) TEM of ABs and DABs (Scale bars = 100 nm). (e) Particle size distribution of ABs and DABs. (f) Zeta potential of ABs and DABs. (g) Relative protein content in Exo, ABs, and DABs (n = 3). (h) Protein composition of MSCs, Apo-MSCs, Apo-D-MSCs, ABs, and DABs was analyzed by Coomassie blue staining. (i) Flow cytometric analysis of Annexin V staining in ABs and DABs. (j) \u0026amp; (k) Cellular uptake of HUVEC and RAW264.7 cells after being treated with ABs and DABs for 4 h by CLSM and flow cytometry. (Scale bar = 10 μm). **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/65fdf50bec442de1100699b4.jpeg"},{"id":108970461,"identity":"e5722198-e9e8-4a28-b069-b82403fede5b","added_by":"auto","created_at":"2026-05-11 10:16:07","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1464408,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatic comparison between DABs and ABs. (a) Principal component analysis (PCA) of the transcriptomic profiles between ABs and DABs. Each data point represents a single sample. (b) Volcano plots to exhibit the upregulated (108) and downregulated (106) miRNAs (ABs vs DABs). (c) Gene ontology (GO) enrichment and (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis upon 214 differential miRNAs. (e) Heatmap showing the significantly differential miRNA (top 50) expression between ABs and DABs (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, FDR \u0026lt; 0.05). (f) Chord plot illustrating the functional associations between the top 10 upregulated miRNAs in DABs and key regenerative biological processes. (g) Chord plot showing the relationships between the top 10 upregulated miRNAs in DABs and representative angiogenesis-related signaling pathways, including Wnt signaling, VEGF signaling, FGF signaling, and vascular maturation. (h) Chord plot showing the relationships between the top 10 upregulated miRNAs in DABs and representative mitochondrial processes, including mitochondrial organization, energy derivation, cellular respiration, and mitochondrial transport. (i) Metascape functional visualization analysis showing the process and pathway enrichment network (colored by cluster ID) from the target genes of the top 10 upregulated miRNAs in DABs. (j) PPI network showing the interaction relationship among the predicted target genes of the top 10 significantly upregulated miRNAs in DABs, with the hub gene networks identified by the MCODE algorithm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/584f360ae14df06e86743e3e.jpeg"},{"id":108970491,"identity":"a59289f7-524b-489b-85b0-6c2029262936","added_by":"auto","created_at":"2026-05-11 10:16:28","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1866428,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of DABs on cell proliferation, migration, angiogenesis, and anti-inflammation. (a) Flow cytometry analysis of HUVEC cells’ cycle distribution and (b) corresponding quantitative analysis (n = 3). (c) Schematic illustration showing the design of the scratch assay. (d) Representative images of scratch assay of HUVEC at 24 h. (e) Quantification of cell migration rate at 24 h (n = 3). (f) Representative images of the transwell assay in HUVEC cells treated with Exo, ABs, and DABs, and (g) Quantitative analysis of the migrated cells (n = 3). (h) Representative images of tube formation assays in HUVEC treated with Exo, ABs, and DABs. (Scale bar = 100 μm) (i) Quantitative analysis of the tube formation assays in HUVEC treated with Exo, ABs, and DABs (n = 3). (j) Schematic illustration of the \u003cem\u003ein vitro\u003c/em\u003e immunomodulation assay using LPS-stimulated RAW264.7. (k) Representative flow cytometry plots showing the population shift from pro-inflammatory M1 to anti-inflammatory M2 phenotypes following different treatments. (l) Quantitative analysis of the M2/M1 polarization ratio (CD206\u003csup\u003e+\u003c/sup\u003e/CD86\u003csup\u003e+\u003c/sup\u003e) (n = 3). (m) Effects of Exo, ABs, and DABs on the total adenosine 5’-triphosphate (ATP) production in RAW264.7 \u003cem\u003ein vitro \u003c/em\u003e(n = 3). (n) Detection of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and (o) anti-inflammatory cytokines (IL-4, IL-10, and VEGF) in the supernatant of LPS-stimulated RAW264.7 treated with Exo, ABs, and DABs (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/cda7262101faf93dabaa5cab.jpeg"},{"id":108970487,"identity":"39cb4a5a-ebe2-4987-b3a6-34d8a73a8241","added_by":"auto","created_at":"2026-05-11 10:16:26","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1329094,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation and characterization of DABs-loaded dual-responsive hydrogel System (DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e). (a) Schematic illustration showing the fabrication process of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (b) Frequency sweeps tests of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (c) Amplitude sweeps tests of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (d) Time sweeps tests of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (e) SEM characterizes the morphology of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (Scale bar = 100 μm). (f) Schematic diagram of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e self-healing function and Macroscopic self-healing property of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e over time. (g) Microscopic self-healing performance of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e with stress variation. (h) The adhesive effect of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e on major organs of C57 mice. (i) Representative ROS staining (green fluorescence) of HUVEC with DCFH-DA probe. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 1 mM. Scale bar, 20 μm. (j) Quantification of ROS fluorescence (n = 3). (k) Flow cytometry analysis of apoptosis in HUVEC cells co-cultured with PHA-PVA\u003csup\u003eGel\u003c/sup\u003e under high oxidative stress conditions. (l) Quantitative analysis of HUVEC apoptosis cells after different treatments (n = 3). (m) Representative 3D CLSM image of DiD-labeled DABs@ PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (n) DABs release features in different medium (n = 3). (o) \u003cem\u003eIn vitro\u003c/em\u003e degradation feature of DABs@PHA-PVA\u003csup\u003eGel \u003c/sup\u003e(n = 3). (p) Particle size of DABs distribution after releasing from DABs@ PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.)\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/2baa883d906e6f223e179125.jpeg"},{"id":108977483,"identity":"c6731da4-1d02-4f27-9198-751365de7687","added_by":"auto","created_at":"2026-05-11 11:31:53","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2241612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e therapeutic efficacy of DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e in diabetic wound healing. (a) Schematic illustration of the treatment schedule for the diabetic full-thickness skin wound in C57 mice. (b) Photographs of wounds and (c) traces of wound closure in different treatment groups from day 0 to day 14. (d) Histological analysis of the wounds stained with H\u0026amp;E and Masson on day 14. Scale bar = 400 and 100 (enlarged) μm. (e) wound area (cm\u003csup\u003e2\u003c/sup\u003e) and (f) wound closure rate (n = 6 biologically independent wounds) over time. (g) Scar index and (h) Collagen volume fraction (n = 3). (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.)\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/acbc917fab1ccd4b969bf075.jpeg"},{"id":108970462,"identity":"7348f509-090f-4ee5-aa59-bcb8a80ddf7b","added_by":"auto","created_at":"2026-05-11 10:16:07","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2589641,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of oxidative stress, regulation of inflammation, and promotion of vascular network reconstruction by DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e in diabetic wound healing. (a) Immunofluorescence staining for ROS (red) in tissue sections obtained from different treatment groups on day 7. (b) Immunofluorescence staining of the M1 macrophage marker CD86 (red) and the M2 macrophage marker CD206 (green). (c) Immunofluorescence staining for CD31(green)/CD105(red) and α-SMA (green) in tissue sections obtained from different treatment groups on day 7. (d) Immunofluorescence staining for the hypoxia-related marker HIF-1α (green) in tissue sections obtained from different treatment groups on day 7. (e) Immunohistochemical staining of TGF-β and VEGF in tissue sections obtained from different treatment groups on day 7. (Scale bar = 50 μm) (f) Quantification of ROS; CD86, CD206, CD31, α-SMA, HIF-1α, and TGF-β from immunofluorescence staining images and Immunohistochemical staining images (n = 3). (g) ELISA detection of IL-6, IL-1β, and TNF-α in wound areas after being treated with different treatment groups on day 14 (n=3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/11efe85708d485a8333c6abe.jpeg"},{"id":108970454,"identity":"fd0fe8a3-eaba-4680-a717-5c041d9e6de9","added_by":"auto","created_at":"2026-05-11 10:16:03","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1141057,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq analysis of the diabetic wound tissue treated with DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. (a) Principal component analysis (PCA) of the transcriptomic profiles between the untreated and DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e treated groups. Each data point represents a single sample. (b) Volcano plots showing the upregulated (2493) and downregulated (2286) genes. (c) Gene ontology (GO) enrichment and (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis upon 2493 differential genes and down-regulated (2286) genes. Heatmaps of significantly upregulated genes related to (e) antioxidant stress, (g) inflammation regulation, and (i) angiogenesis in the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e treated groups as compared to the untreated group. Fragments per kilobase million (FPKM) of (f) Cat, Txnip, Pink1, and Foxo4, (h) Cebpa, Cd300a, Daglb, and Cnr2, (j) Cxcr4, Nrp1, S1pr1, and Rhob (n=3). (k) Schematic illustration showing the potential mechanism underlying the enhancement of diabetic wound healing by the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/a493da6e2162cc998ec9753d.jpeg"},{"id":108979734,"identity":"3f8d55f1-1c01-4c71-b7f0-0211fc7c1000","added_by":"auto","created_at":"2026-05-11 12:00:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19445992,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/592e5e94-05f2-4eaa-bd84-97173138e179.pdf"},{"id":108970490,"identity":"e654d415-8c38-4ba4-ab35-e4cc85fcb6c5","added_by":"auto","created_at":"2026-05-11 10:16:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":25380461,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9522213/v1/0e0fea3b83eeb1740d5677c9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Responsive Hydrogel Delivery of DFO-Programmed Apoptotic Bodies Drives Redox-Immuno-Angiogenic Remodeling in Diabetic Wounds","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMesenchymal stem cell-derived extracellular vesicles (MSC-EVs), particularly exosomes, have attracted growing interest as cell-free therapeutics capable of delivering bioactive cargoes to injured tissues [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite these promising features, clinical translation of MSC-EVs remains limited. Exosomes biogenesis is inherently low yield, and even when sufficient quantities are obtained, their cargo composition is often inconsistent and insufficiently potent for use in diseased tissues [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These persistent problems have pushed researchers to look beyond exosomes for better-suited EVs sources.\u003c/p\u003e \u003cp\u003eApoptotic bodies (ABs) were long dismissed as inert cellular debris from programmed cell death, a view now challenged by their proven biological and therapeutic activities [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Apoptosis naturally generates these vesicles in massive quantities. They characteristically expose phosphatidylserine (PS) on their surface to act as an \u0026ldquo;eat-me\u0026rdquo; signal, which facilitates macrophage clearance and drives anti-inflammatory responses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This combination of high yield and immune modulation makes them an attractive alternative to conventional exosomes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, a critical limitation is that native ABs are insufficient at promoting new blood vessel formation\u0026mdash;a prerequisite for repair in ischemic or poorly vascularized tissues [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This gap in pro-angiogenic activity limits their utility in more demanding pathological settings, and to date, few studies have demonstrated a reliable strategy to overcome it.\u003c/p\u003e \u003cp\u003eWe reasoned that this limitation might be overcome not by modifying the vesicles themselves, but by reprogramming the cells that produce them. Specifically, we pretreated MSCs with deferoxamine (DFO), an FDA-approved iron chelator with a well-established clinical safety record [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], prior to apoptosis induction. DFO stabilizes HIF-1α by blocking its iron-dependent prolyl hydroxylase-mediated degradation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], effectively placing the parent cells in a sustained hypoxia-mimicking state prior to undergoing apoptosis. The apoptotic bodies generated from these programmed cells, which we term DABs, are consequently loaded with a markedly enriched profile of pro-regenerative miRNAs compared to their native counterparts. The approach shifts the therapeutic intervention upstream to the cell itself rather than engineering the vesicles post-isolation.\u003c/p\u003e \u003cp\u003eTo test DABs under genuinely demanding conditions, we turned to the diabetic wound model. These wounds are notoriously difficult to treat, owing to the combined burden of dysregulated inflammation, poor perfusion, and oxidative stress [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. From a vesicle delivery standpoint, this environment is particularly hostile: elevated proteolytic activity and ROS can structurally compromise EVs membranes, while wound exudate rapidly flushes away unprotected cargo before it can act [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To address this, we encapsulated DABs in a glucose- and ROS-responsive hydrogel (PHA-PVA\u003csup\u003eGel\u003c/sup\u003e) that doubles as a local ROS scavenger, enabling sustained on-demand release directly at the wound site [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that DABs carrying DFO-optimized miRNA cargo display both immunomodulatory and pro-angiogenic activity that clearly exceeds that of native ABs and conventional exosomes. Unexpectedly, this miRNA cargo also modulates mitochondrial function in recipient cells, enhancing ATP output. By integrating these miRNA optimized DABs with a ROS-scavenging hydrogel, we successfully translated these cellular advantages into tissue regeneration within a highly hostile diabetic \u003cem\u003ein vivo\u003c/em\u003e model. Together, these findings position programmed apoptotic bodies as a scalable and mechanistically distinct alternative to exosomes, with broader implications for cell-free approaches to tissue repair (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eDeferoxamine mesylate (DFO) was obtained from Abcam plc. Hyaluronic acid (HA) was purchased from Bloomage Biotech Co., Ltd. Human albumin (HSA) was purchased from ExCell Biotechnology, Sexton Biotechnology, and Grifols, respectively. High-glucose DMEM and DMEM/F12 basal media were purchased from Jiangsu KeyGEN BioTECH. For cell viability and apoptosis analyses, the AO/PI double staining kit was acquired from APExBIO Technology, while the Coomassie brilliant blue and cell apoptosis kits were from YEASEN Biotechnology. A comprehensive panel of biochemical reagents and antibodies was purchased from Beyotime Biotechnology; this included the BCA protein assay kit, BeyoExo\u0026trade; Exosome Identification Kit (CD63, CD9, TSG101, Hsp70, and Calnexin), DiI/DiO lipophilic dyes, as well as all primary and secondary antibodies for immunofluorescence and immunoblotting (anti-HIF-1α, anti-CD86, anti-CD206, anti-CD31, anti-CD105, anti-VEGF, anti-TGF-β, anti-α-SMA, HRP-conjugated β-Actin, Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L), and AF 488/647). Mouse ELISA kits for inflammatory and angiogenic cytokines were obtained from MULTI SCIENCES. Specific antibodies for flow cytometry were sourced from BD Pharmingen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell culture\u003c/h2\u003e \u003cp\u003eHuman umbilical cord blood tube skin cells (HUVEC), human keratinocytes cells (HaCaT), and mouse mononuclear macrophages cells (RAW264.7) were purchased from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). They were cultured in DMEM medium containing 10% FBS in a 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Specifically, Human umbilical cord mesenchymal stem cells (MSCs) were kindly provided by Jiangsu Province Cell Therapy Manufacturing Center (Nanjing, China) and previously identified, were cultured in DMEM/F12 containing 5% human platelet lysate (Sexton) in a 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Animal\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (6 weeks of age, 18\u0026ndash;22 g) were sourced from Jiangsu Huachuang Sino Pharmaceutical Technology Co., Ltd. (Taizhou, China). Animal housing and care were managed by the Animal Experimental Center of China Pharmaceutical University. All \u003cem\u003ein vivo\u003c/em\u003e procedures received ethical approval from the Ethics Committee of China Pharmaceutical University (Nanjing, China) and were executed in full compliance with institutional guidelines for animal care.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Apoptosis induction of MSCs\u003c/h2\u003e \u003cp\u003eMSCs at passages fewer than six were cultured in DMEM/F12 with 5% HPL containing DFO (250 \u0026micro;g/mL) or PBS for 24 h. The prime medium was discarded, cells were rinsed three times with PBS, and HPL-free medium was added. The confluent monolayers of DFO-MSCs and MSCs, maintained in uncovered cell culture dishes, were irradiated with ultraviolet light (UV) (30 W, 254 nm, Philip, China) at an intensity of 300 mJ/cm\u0026sup2; for 10 min. After 24 h of apoptosis induction, apoptosis was evaluated by morphological observation and Annexin V-FITC/PI staining. In addition, apoptotic collapse in apoptotic DFO-MSCs (denoted as Apo-D-MSCs) and apoptotic MSCs (denoted as Apo-MSCs) was observed using inverted fluorescence microscopy. By contrast, confocal laser scanning microscopy (LSM800, Zeiss) was employed to monitor the generation of apoptotic vacuoles within DiI-stained MSCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Separation and purification of apoptotic bodies\u003c/h2\u003e \u003cp\u003eApoptotic bodies were extracted from the culture supernatant of Apo-D-MSCs and Apo-MSCs using an extensively used differential centrifugation (denoted as DABs and ABs). Briefly, after 24 h UV irradiation induction, the HPL-free cell culture supernatant of Apo-D-MSCs and Apo-MSCs were collected and centrifuged at 400\u0026times;g for 5 min to precipitate dead cells, and at 2000\u0026times;g for 30 min to precipitate cell debris. Following the removal of dead cells and larger debris, the clarified supernatant was subjected to centrifugation at 40,000 \u0026times; g for 40 min to sediment the apoptotic vesicles. To clear residual co-precipitating proteins, these crude pellets were washed by resuspension in ice-cold PBS and re-pelleted under identical centrifugation conditions. The final purified DABs and ABs were then dispersed in 100 \u0026micro;L of PBS and preserved at -80\u0026deg;C for downstream applications. To maintain vesicular integrity, the entire differential centrifugation protocol was strictly carried out at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Characterization of DABs and ABs\u003c/h2\u003e \u003cp\u003eTotal protein concentrations of DABs and ABs were measured by a bicinchoninic acid (BCA) assay. Particle size distribution and zeta potential were measured by a Zetasizer Nano (Nano-ZS90, Malvern, UK). Morphology was checked using transmission electron microscopy (TEM). Total protein from MSCs, Apo-D-MSCs, Apo-MSCs, ABs, and DABs was extracted with RIPA buffer. This protein was mixed with loading buffer and heated at 95\u0026deg;C for 5 minutes for SDS-PAGE. The protein bands were stained with Coomassie Brilliant Blue and photographed. Surface phosphatidylserine (PS) on DABs and ABs was measured by Annexin V-FITC labeling and flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cellular uptake of DABs and ABs\u003c/h2\u003e \u003cp\u003eHUVEC, HaCaT, and RAW264.7 cells were seeded on 12-well plates at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and cultured overnight. These cells were incubated with DiI-labeled DABs or ABs for 4 hours. Cells were washed with cold PBS, fixed with 4% paraformaldehyde and stained with DAPI for confocal laser scanning microscopy. Cells in 12-well plates were collected using trypsin and analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Preparation and Characterization of Exo\u003c/h2\u003e \u003cp\u003eMSCs were cultured in HPL-free DMEM/F12 for 24 h. The conditioned medium was collected and sequentially centrifuged (300\u0026times;g, 2000\u0026times;g, and 10000\u0026times;g), filtered through a 0.22-\u0026micro;m membrane, and ultracentrifuged at 100000 \u0026times;g for 70 min at 4\u0026deg;C. The exosomes pellet was resuspended in PBS and stored at -80\u0026deg;C. The particle size distribution of exosomes was assessed by dynamic light scattering (DLS), vesicular morphology was visualized using transmission electron microscopy (TEM), and exosome-associated surface markers (CD9, CD63, TSG101, and Hsp70) were analyzed by western blotting. These analyses collectively confirmed the characteristic size distribution, canonical vesicular morphology, and expected marker expression of exosomes (Fig. S10 and S11).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. miRNA sequencing and bioinformatic analysis\u003c/h2\u003e \u003cp\u003eExosomes (Exo) and apoptotic bodies (ABs) were isolated from MSCs, and DFO-programmed apoptotic bodies (DABs) were isolated from DFO-preconditioned MSCs as described above. Small RNA sequencing was performed by Bioyi Biotechnology Co., Ltd. (Wuhan, China). Total RNA was extracted from Exo, ABs, and DABs. Small RNAs of 18 to 30 nucleotides were used to build libraries. These libraries were amplified by PCR and sequenced on the Illumina NovaSeq 6000 platform. Raw reads were filtered for clean data. Differentially expressed miRNAs (DEMs) were identified using thresholds of |fold change| \u0026gt; 2 and p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Target transcripts were predicted using TargetScan and miRDB. GO and KEGG enrichment analyses were done on these targets. Chord diagrams were made using the Hiplot web tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://hiplot.com.cn\u003c/span\u003e\u003cspan address=\"http://hiplot.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to show miRNA-function relationships. Pathway clustering was done using Metascape (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/\u003c/span\u003e\u003cspan address=\"https://metascape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein-protein interaction (PPI) networks and hub modules were built using Cytoscape with the MCODE plugin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Analysis of HUVEC proliferation\u003c/h2\u003e \u003cp\u003eCell proliferation was measured by CCK-8 assay. HUVEC were seeded in 96-well plates at 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well overnight and incubated with Exo, ABs, or DABs (40 \u0026micro;g/mL). Untreated cells served as the control. At 0, 12, 24, and 48 h, culture medium was replaced with fresh medium containing 10% (v/v) CCK-8 reagent and incubated for 1\u0026ndash;2 h at 37\u0026deg;C. Absorbance at 450 nm was measured with a microplate reader (Multiskan MK3, Thermo).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Cell scratch assay\u003c/h2\u003e \u003cp\u003eCell migration was measured by scratch assay. HUVEC and HaCaT cells were seeded in 6-well plates at 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and cultured to confluency. A linear scratch was made with a sterile 200 \u0026micro;L pipette tip, and detached cells were removed by PBS. Cells were cultured in serum-free medium with 40 \u0026micro;g/mL Exo, ABs, or DABs. Images of the scratch area were taken at 0, 12, and 24 h using an inverted phase-contrast microscope, and gap areas were measured with ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Transwell assay\u003c/h2\u003e \u003cp\u003eHUVEC suspensions (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells in 200 \u0026micro;L) were seeded in the upper chambers of transwell inserts (24-well, 8 \u0026micro;m pore size). The lower chambers contained 600 \u0026micro;L medium with 5% FBS and 40 \u0026micro;g/mL Exo, ABs, DABs, or PBS. Cells were cultured at 37\u0026deg;C for 24 and 48 h. The upper chamber membrane was fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Migrated cells were imaged at 24 and 48 h by an inverted optical microscope and counted with ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Matrigel tube formation assay\u003c/h2\u003e \u003cp\u003eAngiogenesis was assessed by tube formation assay. HUVEC (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in 96-well plates coated with 100 \u0026micro;L growth factor-reduced Matrigel (Corning, USA) and incubated with 40 \u0026micro;g/mL Exo, ABs, or DABs for 8 h. Tube structures were imaged and quantified with ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Macrophage polarization assay in vitro\u003c/h2\u003e \u003cp\u003eTo evaluate the immunomodulatory capacity of the vesicles, RAW264.7 murine macrophages were plated in 12-well plates (1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) and primed with lipopolysaccharide (LPS, 1 \u0026micro;g/mL) for 24 h to induce an inflammatory phenotype. The LPS-challenged cells were then cultured for an additional 24 h in fresh complete medium containing PBS or 40 \u0026micro;g/mL of Exo, ABs, or DABs. For flow cytometric analysis, the treated macrophages were harvested, resuspended in 100 \u0026micro;L of PBS, and dual-stained with anti-mouse CD86 and anti-mouse CD206 antibodies for 30 min at 4\u0026deg;C in the dark. After standard washing procedures, the macrophage polarization states were quantified using a flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Macrophage immunoregulatory assay in vitro\u003c/h2\u003e \u003cp\u003eFor an \u003cem\u003ein vitro\u003c/em\u003e immunoregulatory study, RAW264.7 cells were treated with LPS (1 \u0026micro;g/ml) for 24 h and subsequently co-incubated with different formulations (40 \u0026micro;g/mL). Twenty-four hours later, the levels of immune factors (TNF-α, IL-6, IL-1β, IL-4, IL-10, and VEGF) in cell supernatants were measured using ELISA kits according to the manufacturer\u0026rsquo;s recommended protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Macrophage ATP production assay in vitro\u003c/h2\u003e \u003cp\u003eTo evaluate the total adenosine 5\u0026rsquo;-triphosphate (ATP) production capacity of macrophages \u003cem\u003ein vivo\u003c/em\u003e, RAW264.7 cells were seeded in each well of twelve-well plates and treated with Exo, ABs, and DABs (40 \u0026micro;g/mL) for 24 h, and cells in medium containing PBS were prepared in parallel as the control group (1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells). Afterwards, ATP in macrophages from different groups were extracted and detected using an Enhanced ATP Assay Kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. Synthesis and characterization of phenylboronic acid-functionalized HA (PHA)\u003c/h2\u003e \u003cp\u003eTo synthesize the PHA copolymer, 3-aminophenylboronic acid (3-APBA) was grafted onto the hyaluronic acid (HA) backbone via EDC/NHS-mediated amide coupling. First, the carboxyl groups of HA (80 mL, 7.5 mg/mL) were activated by stirring with EDC (540 mg) and NHS (330 mg) at room temperature for 6 h. Following activation, 3-APBA (450 mg) was introduced, and the conjugation reaction proceeded under vigorous stirring for an additional 24 h. To purify the synthesized PHA, the crude mixture was dialyzed (MWCO 3500 Da) against distilled water for 48 h, passed through a 0.8-\u0026micro;m filter, and lyophilized at -80\u0026deg;C. The chemical structure and successful grafting of the final PHA product were subsequently validated utilizing UV-vis spectrophotometry, \u003csup\u003e1\u003c/sup\u003eH NMR, and FTIR spectroscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18. Preparation of the glucose/ROS dual-responsive PHA-PVA\u003csup\u003eGel\u003c/sup\u003e hydrogel\u003c/h2\u003e \u003cp\u003eTo fabricate the PHA-PVA\u003csup\u003eGel\u003c/sup\u003e hydrogels, lyophilized PHA (at predetermined concentrations) and commercial PVA were individually dissolved in deionized water, with the pH of both precursor solutions subsequently neutralized to 7.4. The final hydrogel networks were rapidly formed at ambient temperature by co-extruding the PHA and PVA solutions at a 1:1 (v/v) ratio utilizing a double-barrel syringe. Five hydrogel compositions were fabricated with varying PHA solutions: Gel-1 (PHA (1.0%, w/v), PVA (8.0%, w/v)), Gel-2 (PHA (2.0%, w/v), PVA (8.0%, w/v)), Gel-3 (PHA (3.0%, w/v), PVA (8.0%, w/v)), Gel-4 (PHA (4.0%, w/v), PVA (8.0%, w/v)), and Gel-5 (PHA (5.0%, w/v), PVA (8.0%, w/v)).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.19. Microstructure characterization\u003c/h2\u003e \u003cp\u003eThe internal structure of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e specimens was examined by scanning electron microscopy (SEM). Specimens were snap-frozen in liquid nitrogen and freeze-dried for 24 h. Cross-sections were mounted on conductive carbon stubs, sputter-coated with gold, and imaged by SEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.20. Rheological behavior Test\u003c/h2\u003e \u003cp\u003eViscoelastic properties of the hydrogels were measured on a DHR-10 rheometer (TA Instruments) with a 10-mm parallel plate geometry at a 1-mm gap. Time sweeps at 1% strain were first performed to establish the linear viscoelastic region. Frequency sweeps (0.1\u0026ndash;100 rad/s, 1% strain) and strain sweeps (0.1\u0026ndash;1000%) were then performed at 37\u0026deg;C to measure storage modulus (G\u0026rsquo;) and loss modulus (G\u0026rsquo;\u0026rsquo;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.21. Self-healing properties evaluation\u003c/h2\u003e \u003cp\u003eSelf-healing of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was assessed visually and rheologically. Two hydrogel pieces dyed with rhodamine B and methylene blue were placed in contact for 2 h and then stretched to confirm bonding at the interface. Rheological recovery was measured by a step-strain test, where strain was alternated between 1000% and 5% to monitor recovery of G\u0026rsquo; and G\u0026rsquo;\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e2.22. Adhesion strength test\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e adhesion of the PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was assessed on eight distinct organs (heart, liver, lung, spleen, kidney, bladder, colon, and stomach) that were excised from C57 mice. Specifically, 200 \u0026micro;L of freshly prepared PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was applied to stainless steel tweezers, which were subsequently used to attach the gel to the moist surface of the organs above. All these tests were conducted more than 5 times to ensure the results were consistent and reliable.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e2.23. Intracellular ROS elimination and cytoprotective effect of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eTo evaluate the intracellular ROS-scavenging capability of the hydrogels, HUVECs (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/dish) were seeded into confocal imaging dishes and allowed to attach overnight. The cultures were pre-treated with medium containing either HA-PVA\u003csup\u003eGel\u003c/sup\u003e or PHA-PVA\u003csup\u003eGel\u003c/sup\u003e for 2 h, followed by a 4-h oxidative challenge using 5 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Intracellular ROS levels were subsequently probed by incubating the cells with 10 \u0026micro;M DCFH-DA (in serum-free medium) for 30 min prior to fluorescence microscopy. HUVEC (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded in 12-well plates, pre-incubated with hydrogels for 2 h, and treated with 5 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 6 h. Cell viability was assessed by LIVE/DEAD\u0026reg; staining and fluorescence imaging. Apoptosis was measured by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e2.24. Evaluation of biocompatibility\u003c/h2\u003e \u003cp\u003eCytotoxicity of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was measured by CCK-8 assay in HUVEC and HaCaT cells. Hydrogel extracts were prepared following ISO 10993-5. To generate the hydrogel leachates for \u003cem\u003ein vitro\u003c/em\u003e assays, the mixture was incubated at 37\u0026deg;C for 48 h. For biocompatibility evaluation, HUVECs and HaCaT cells were plated in 96-well plates at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well and allowed to adhere overnight. The standard culture medium was subsequently aspirated and substituted with 200 \u0026micro;L of fresh medium supplemented with the prepared hydrogel leachates. Cellular viability was continuously monitored over a 72-h period (evaluated at 24, 48, and 72 h) utilizing a CCK-8 assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e2.25. Preparation and characterization of DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eThe DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e hydrogel was prepared through the following sequential steps. Firstly, DABs were isolated as described previously and subsequently dispersed in a PVA solution at a concentration of 200 \u0026micro;g/mL. Afterwards, the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e hydrogel was formulated by combining a PVA solution containing DABs with a PHA solution using a dual-channel injector to achieve homogeneous mixing. To confirm the distribution of DABs in DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e hydrogel, Dil-labelled DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was fabricated and observed through Z-stack model of CLSM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e2.26. In vitro responsive particle release assay\u003c/h2\u003e \u003cp\u003eTo simulate the microenvironment of diabetic wounds, three release medium were prepared: (1) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution (200 \u0026micro;M), (2) glucose solution (16 mM), and (3) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (200 \u0026micro;M) and glucose (16 mM) mixed solution. Then, the release profile of DABs from DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was examined when submerged in PBS and the medium at 37℃. Briefly, DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was positioned within 12-well plates, with each well containing 2 mL of the corresponding release medium, ensuring complete immersion of the hydrogel. The plates were subsequently incubated in a shaking incubator at 37\u0026deg;C with constant agitation. At predetermined time intervals (0, 2, 4, 6, 8, 12, 24, 36, 48, 60, and 72 h), 400 \u0026micro;L of the release medium was carefully collected from each well, and an equivalent volume of fresh medium was introduced to preserve a consistent volume of 2 mL. The number of DABs released at predetermined intervals was determined via BCA protein assay. In addition, the size distribution of DABs in the liquid medium at 72 h was measured using a laser particle size meter to assess shape integrity and changes in diameter after release.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e2.27. In vitro responsive degradation test\u003c/h2\u003e \u003cp\u003eThe degradation assay was conducted by immersing the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e in the three previously mentioned microenvironmental medium. In short, DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was freshly prepared, and the surface moisture was removed using filter paper. The initial weight (W\u003csub\u003e0\u003c/sub\u003e) was then recorded. Subsequently, the weighed samples were incubated in the medium above and PBS at 37\u0026deg;C. At designated time points (3, 6, 9, 12, and 15 days), the medium was discarded, and the hydrogels were dried and weighed (W\u003csub\u003en\u003c/sub\u003e). The degradation rate of the hydrogels was calculated using the following formula: DR = (W\u003csub\u003e0\u003c/sub\u003e - W\u003csub\u003en\u003c/sub\u003e) / W\u003csub\u003e0\u003c/sub\u003e \u0026times; 100%, where W\u003csub\u003e0\u003c/sub\u003e represents the initial weight of the hydrogel at day 0, W\u003csub\u003en\u003c/sub\u003e denotes the weight of the hydrogel at day n, and DR indicates the degradation rate of the hydrogel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e2.28. Establishment of diabetic mouse cutaneous wound model\u003c/h2\u003e \u003cp\u003eAll \u003cem\u003ein vivo\u003c/em\u003e protocols were executed under the ethical approval of the Institutional Animal Care and Use Committee (IACUC) at China Pharmaceutical University (approval # 2024-09-017). Male C57BL/6 mice (6 weeks old, 18\u0026ndash;22 g) were sourced from Jiangsu Huachuang Sino Pharmaceutical Technology Co., Ltd. and maintained on a 12-h light/dark cycle with ad libitum access to a high-fat diet and water. To induce the type 2 diabetes mellitus (T2DM) model, mice reaching a body weight of 30 g received consecutive daily intraperitoneal injections of streptozotocin (STZ, 50 mg/kg) for 5 days. Fasting blood glucose (FBG) was measured by a glucometer. Mice with FBG\u0026thinsp;\u0026gt;\u0026thinsp;16.65 mM for two consecutive days were considered successfully modeled. Mice that did not reach this threshold received additional STZ (20 mg/kg, i.p.). Diabetic mice were anesthetized, dorsal hair was removed, and a 10-mm full-thickness excisional wound was made along the dorsal midline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e2.29. In vivo wound healing investigation\u003c/h2\u003e \u003cp\u003eThe diabetic mice were randomly divided into 8 groups: 1) Control group (G1); 2) Exo group (G2); 3) ABs group (G3); 4) DABs group (G4); 5) PHA-PVA\u003csup\u003eGel\u003c/sup\u003e group (G5); 6) ABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e group (G6); 7) DABs@usGel group (G7); and 8) DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e group (G8). (Herein, ABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e denotes a responsive hydrogel system for ABs, whereas DABs@usGel represents an unresponsive hydrogel loaded with DABs, prepared through freeze-thaw crosslinking using HA and PVA as precursor solutions at the same concentration.) Each group contained at least six mice. Treatments were applied to wounds every three days. Exo, ABs, and DABs were dosed at 100 \u0026micro;g/mouse, and the control group received saline. Wounds were covered with 3M\u0026trade; Tegaderm dressings. Mice were euthanized at day 7 and day 14, and full-thickness dorsal skin was collected for further analysis.\u003c/p\u003e \u003cp\u003eMacroscopic wound closure was monitored by capturing digital photographs on days 0, 3, 7, and 14 post-surgeries. Additionally, The wound area ratio (%) can be calculated by the formula S\u003csub\u003en\u003c/sub\u003e/S\u003csub\u003e0\u003c/sub\u003e \u0026times; 100%, where S\u003csub\u003e0\u003c/sub\u003e is the wound area at day 0, and S\u003csub\u003en\u003c/sub\u003e is the wound area at day n (n\u0026thinsp;=\u0026thinsp;3, 7, 14). On day 7 and 14, the mice were anesthetized and euthanized, after which the wound tissues were harvested for histological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e2.30. Histological, immunohistochemistry, and immunofluorescence analysis\u003c/h2\u003e \u003cp\u003eFor histological evaluation, tissue sections were deparaffinized and rehydrated, then stained with H\u0026amp;E and Masson\u0026rsquo;s trichrome. To evaluate the expression of ROS in the wounds of diabetic mice, immunofluorescence staining was performed to examine ROS levels in the skin tissues of the wound areas during the intermediate treatment phase (day 7). To assess macrophage phenotypic transition in wounds, immunofluorescence staining for CD86 and CD206 was performed. To detect angiogenesis in the wound, wound tissue sections were stained with CD31, CD105, and α-SMA for immunofluorescence. In addition, IHC staining for VEGF and TGF-β was performed to detect changes in the wound microenvironment associated with neovascularization. To evaluate hypoxia-induced improvement in mouse wounds, immunofluorescence staining was performed to assess HIF-1α expression. Nuclei were stained with DAPI. Quantification of IHC and IF was performed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e2.31. Evaluation of tissue inflammation\u003c/h2\u003e \u003cp\u003eAt days 7 and 14, tissue sections were collected for the assessment of tissue inflammation. To quantify TNF-α, IL-6, IL-1β, IL-4, IL-10, and VEGF levels, cytokine data were standardized relative to the concentrations of these factors measured in normal skin using ELISA kits. Furthermore, flow cytometry was used to analyze macrophage polarization in skin wound tissues at the conclusion of the treatment period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e2.32. Transcriptome analysis\u003c/h2\u003e \u003cp\u003eWound tissues from the control and DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e groups (n\u0026thinsp;=\u0026thinsp;3/group) were collected at day 14. Total RNA was extracted with TRIzol (Invitrogen) and quantified by a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). Libraries were constructed from high-quality RNA and sequenced on the BGISEQ-T7 platform (BGI, Shenzhen, China). Raw FASTQ reads were filtered with fastp (v0.21.0) and aligned to the mouse reference genome with HISAT2 (v2.1.0). Transcript expression was quantified as FPKM using StringTie (v2.1.5). DEGs were identified with DESeq2 using thresholds of |log2(fold change)| \u0026gt; 1 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. GO and KEGG enrichment analyses were performed with the clusterProfiler R package (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e2.33. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 10.4.1. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Two-group comparisons were made by two-tailed unpaired Student\u0026rsquo;s t-test. Multi-group comparisons were made by one-way ANOVA with Tukey\u0026rsquo;s multiple comparisons. \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Preparation and characterization of DABs\u003c/h2\u003e \u003cp\u003eThe preparation of DABs is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. MSCs were preconditioned with DFO and then induced to undergo apoptosis by UV irradiation. To optimize DFO preconditioning, MSCs were treated with various concentrations of DFO. Based on AO/PI cell viability assays and Western blot analysis, 250 \u0026micro;g/mL was selected as the optimal concentration, as it induced the maximum upregulation of HIF-1α while maintaining excellent cell viability (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig. S2). Subsequently, the optimal UV-induced apoptosis condition was determined to be 10 minutes of irradiation, based on analysis of cell morphology and apoptosis rates (Fig. S3 and Fig. S4).\u003c/p\u003e \u003cp\u003eFollowing UV irradiation, the morphology of the apoptotic cells was examined. At six hours post-treatment, both apoptotic MSCs (Apo-MSCs) and apoptotic DFO-MSCs (Apo-D-MSCs) exhibited clear morphological signs of apoptosis, including the formation of prominent vacuole-like structures and membrane blebbing, which were absent in the non-irradiated control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Following 24 h of induction, apoptotic bodies (ABs and DABs) were isolated through differential sequential centrifugation (Fig. S5). Transmission electron microscopy (TEM) revealed that both vesicle types displayed characteristic spherical or cup-shaped forms with distinct membrane structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Dynamic light scattering (DLS) analysis revealed a size distribution ranging from 50 nm to 800 nm, characteristic of apoptotic bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Both ABs and DABs displayed a negative surface charge of approximately\u0026thinsp;\u0026minus;\u0026thinsp;20 mV, consistent with their origin from the negatively charged cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eWe then examined the key surface properties of the vesicles. Coomassie brilliant blue staining verified that both ABs and DABs contained a complex protein from their parent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Both ABs and DABs were found to be highly enriched with the \u0026ldquo;eat-me\u0026rdquo; signal, phosphatidylserine (PS), with flow cytometry showing exposure rates of 84.60% and 83.40%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). BCA assay showed that DAB yield was higher than ABs yield and Exo yield from the same number of parent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). This indicated that DFO preconditioning acted as a dual-functional strategy, simultaneously programming vesicle cargo and amplifying production efficiency.\u003c/p\u003e \u003cp\u003eAfterwards, HUVEC, RAW264.7, and HaCaT cells were incubated with DiI-labeled ABs and DABs. Confocal microscopy showed that all three cell types internalized ABs and DABs with perinuclear localization. Flow cytometry showed that RAW264.7 had higher fluorescence intensity than HUVEC and HaCaT cells, indicating greater uptake efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek; Fig. S6 and Fig. S7). This may be due to PS on the surface of ABs and DABs, which promotes recognition and uptake by macrophages via efferocytosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Transcriptomics analysis of DABs\u003c/h2\u003e \u003cp\u003emiRNAs play key roles in post-transcriptional gene regulation and cell modulation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The miRNA content of vesicles reflects the physiological state of the parent cell [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To investigate the impact of DFO programming on vesicular cargo, we compared miRNA profiles of ABs and DABs by high-throughput sequencing. PCA showed clear separation between the two groups, with DABs showing tighter clustering and greater sample homogeneity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Differential expression analysis identified 214 altered miRNAs in DABs, including 108 upregulated and 106 downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eTo elucidate the functional implications of the differentially expressed miRNAs, we performed GO and KEGG enrichment analyses. Then GO and KEGG enrichment analyses were performed on predicted target genes of the differentially expressed miRNAs (DEMs). GO analysis showed enrichment in protein kinase activity and chemokine receptor signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). KEGG analysis showed enrichment in immune-related pathways such as Th1/Th2 differentiation, as well as focal adhesion and axon regeneration pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Analysis of the top 50 DEMs showed that 34 were upregulated in DABs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). miRNAs linked to angiogenesis and cell migration, including hsa-miR-4674, hsa-miR-1246, hsa-miR-3976, and hsa-miR-320e, were enriched in DABs (Fig. S8a and S8b).\u003c/p\u003e \u003cp\u003eTo understand how DABs drive regeneration, we looked at the target networks of the top ten upregulated miRNAs in DABs. Pathway enrichment showed these targets are linked to key repair processes, including angiogenesis, cell migration, and VEGF signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). We also found that these miRNAs appear to regulate mitochondrial bioenergetics and cellular respiration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), which was somewhat unexpected. This suggests DABs may promote tissue repair by activating angiogenic pathways while also supporting the metabolic demands of the repair process, though the exact interplay between these two mechanisms needs further investigation. Beyond metabolic reprogramming, enrichment analysis of the top 10 upregulated miRNA targets in DABs revealed a significant clustering in pathways essential for cellular dynamics, such as Rho GTPase signaling and cytoskeleton organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Combined with enrichments in the mitotic cell cycle and chromatin remodeling, these findings underscore a shift toward a more active, migratory phenotype. How DFO programming integrates these diverse structural and bioenergetic processes remains an open question.\u003c/p\u003e \u003cp\u003eMapping the predicted miRNA targets onto a protein-protein interaction network revealed a rather heterogeneous regulatory landscape, with MCODE identifying two functionally distinct hubs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej, Fig. S9) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A prominent energy-metabolism module (MCODE 2) captures much of the mitochondrial ATP synthesis and electron transport machinery\u0026mdash;specifically components like NDUFB5, NDUFA9, COX3, and ND1, which appear tightly governed by the miR-320 family and miR-3976 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The second was a cytoskeletal dynamics module (MCODE 4) centered on RAC1 signaling, where CRK, NCKAP1, and ABI2 formed a closely connected motility network. These results suggest that DABs may work by boosting mitochondrial energy production while also activating RAC1-driven cytoskeletal changes, which together could support cell migration and tissue repair though the relative contribution of each arm warrants further study [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, we extended the transcriptomic profiling to include parent cell-derived exosomes (Exo) for comparison. The data showed that both ABs and DABs possessed a more complex and abundant miRNA repertoire compared to the Exo group (Fig. S12-S16). Chord diagram analysis of the top upregulated miRNAs in DABs relative to Exo unveiled a coordinated bipartite regulatory network that simultaneously orchestrates the resolution of inflammation and the activation of structural regeneration pathways. This data established a robust mechanistic basis for the superior therapeutic efficacy of DABs over conventional Exo-based therapies (Fig. S17 and S18).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003e3.3. In vitro biological evaluation of DABs\u003c/h2\u003e \u003cp\u003eDetermining the functional impact of this molecular became our primary focus, particularly given the distinct pro-regenerative miRNA signature we found packaged within the DABs. To provide the therapeutic benchmark, we selected stem cell-derived exosomes (Exo), currently under investigation in clinical trials, as the positive control [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Given that effective wound healing necessitates the coordinated proliferation and migration of endothelial and epidermal cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], we systematically evaluated the efficacy of DABs against native ABs and Exo in driving these cellular behaviors. DABs visibly forced a rapid G1-to-S transition in HUVECs, pooling the population in the active S and G2/M phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Far from just dividing faster, these cells became highly mobile. In fact, 24 hours was all it took for DAB-treated HUVEC and HaCaT cultures to completely seal scratch wounds, leaving ABs and Exo lagging far behind (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-e; Fig. S19a and 19b). CCK-8 assays also showed increased cell proliferation after DABs treatment (Fig. S20). Transwell assays showed DABs increased HUVEC migration by 1.95-fold, compared to 1.25-fold with Exo and 1.16-fold with native ABs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). In Matrigel tube formation assays, DABs also produced the greatest increases in total tube length and network area across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eTo evaluate immunomodulatory effects \u003cem\u003ein vitro\u003c/em\u003e, RAW264.7 were stimulated with LPS to mimic an inflammatory environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Flow cytometry showed that the control and Exo groups maintained a high proportion of CD86\u003csup\u003e+\u003c/sup\u003e (M1) macrophages, while DABs treatment shifted the balance toward CD206\u003csup\u003e+\u003c/sup\u003e (M2) macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). The CD206/CD86 ratio was highest in the DABs group compared to both Exo and native ABs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el). ELISA results further showed that DABs treatment lowered pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en) and raised anti-inflammatory and angiogenic cytokines, including IL-4, IL-10, and VEGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eo). Polarization into an M2 state fundamentally relies on oxidative phosphorylation. Tracking this specific energy requirement showed a distinct metabolic difference, with DABs-treated macrophages producing substantially more ATP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em). This suggests the metabolic boost provided by DABs may help support the energy demands of phenotypic switching and sustained anti-inflammatory activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec40\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Preparation and characterization of DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eTo enable controlled delivery of DABs, we developed a responsive hydrogel using a phenylboronic acid-modified hyaluronic acid copolymer (PHA). PHA was synthesized by conjugating 3-aminophenylboronic acid onto the hyaluronic acid backbone, and successful grafting was confirmed by UV-vis, \u003csup\u003e1\u003c/sup\u003eH-NMR, and FT-IR spectroscopy (Fig. S21-S23). The copolymer was then crosslinked with polyvinyl alcohol (PVA) to form the hydrogel network (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Different PHA/PVA ratios were tested to optimize gelation and stability, and vial inversion tests identified Gel-2, Gel-3, and Gel-4 as suitable candidates based on their rapid sol-gel transition and stable formation (Fig. S24).\u003c/p\u003e \u003cp\u003eRheological characterizations showed that all three formulations were elastic-dominant (G\u0026rsquo; \u0026gt; G\u0026rsquo;\u0026rsquo;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). However, Gel-3 displayed fluctuations in its time-sweep profile, suggesting network inhomogeneity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Gel-4 maintained a consistent rheological profile and was selected for subsequent experiments (designated as PHA-PVA\u003csup\u003eGel\u003c/sup\u003e). Scanning electron microscopy (SEM) of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e revealed a porous, interconnected microstructure (Fig. S25 and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eMacroscopic evaluation of self-healing properties showed that two PHA-PVA\u003csup\u003eGel\u003c/sup\u003e pieces (stained red and blue) fused within minutes and withstood subsequent stretching (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Rheological step-strain tests indicated that the hydrogel recovered over 95% of its initial storage modulus immediately following a 1000% high-strain cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). The hydrogel also adhered to various murine organs \u003cem\u003eex vivo\u003c/em\u003e, including the heart, liver, spleen, and lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eWe evaluated the intrinsic ROS-scavenging capacity of the hydrogel \u003cem\u003ein vitro\u003c/em\u003e using an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress model in HUVEC. DCFH-DA staining indicated high intracellular ROS levels in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated group (G2), which were effectively neutralized in the presence of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e (G4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el). Flow cytometry showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e induced apoptosis in 76.80% of cells (G2). Treatment with PHA-PVA\u003csup\u003eGel\u003c/sup\u003e reduced the apoptotic rate to 18.65% (G4), compared to 64.50% with the control hydrogel (G3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek). Live/Dead staining confirmed higher cell viability in the PHA-PVA\u003csup\u003eGel\u003c/sup\u003e group (Fig. S26). Quantitative assays demonstrated that cells cultured in hydrogel leachates-maintained proliferation rates comparable to untreated controls (Fig. S27).\u003c/p\u003e \u003cp\u003eDABs were then loaded into the hydrogel (DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e) for further characterization. Confocal Z-stack imaging showed that DiI-labeled DABs were distributed evenly throughout the porous hydrogel network (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em). We evaluated the \u003cem\u003ein vitro\u003c/em\u003e release of DABs under conditions simulating the high glucose and ROS levels of a diabetic wound [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Under dual-stimulus conditions (glucose\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), DABs release reached 95.33% at 72 h, which was significantly higher than either single-stimulus (glucose or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e alone) or PBS groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003en). This matched the hydrogel degradation pattern, where the greatest mass loss also occurred under dual-stimulus conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eo). DLS confirmed that the released DABs maintained their original size distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ep).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec41\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Synergistic in vivo wound healing in a diabetic mouse model\u003c/h2\u003e \u003cp\u003eFollowing the \u003cem\u003ein vitro\u003c/em\u003e results, we next evaluated the therapeutic performance of DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e in a full-thickness diabetic wound model. The experimental setup and STZ-induced diabetic model are described in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and Fig. S28. Mice were randomized into eight groups: untreated (G1), Exo (G2), native ABs (G3), DABs (G4), PHA-PVA\u003csup\u003eGel\u003c/sup\u003e (G5), ABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e (G6), DABs@usGel (unresponsive hydrogel loaded with DABs, G7), and DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e (G8).\u003c/p\u003e \u003cp\u003eWound closure was tracked over 14 days. Representative images (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) and wound area heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee) showed faster healing in the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e group (G8), which reached a closure rate of 80.69% by day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). DABs alone (G4, 69.87%) also outperformed the Exo group (G2), and the difference between G8 and G4 likely reflects the benefit of sustained therapeutic retention provided by the hydrogel. G8 treated wounds reached complete re-epithelialization by day 14, with hair follicle regeneration already visible at the macroscopic level. This physical closure was anchored by a robust architectural recovery; H\u0026amp;E sections from days 7 and 14 detailed a continuous, well-consolidated epidermis packed with newly formed sweat glands and follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, Fig. S29). This highlighted the failure of the untreated G1 group, which stalled at under 61% closure and remained structurally deficient, burdened by unresolved inflammatory infiltrates and a persistently incomplete epidermis.\u003c/p\u003e \u003cp\u003eCollagen accumulation reached high of 74.57% in the G8 tissues. This matrix accumulation showed moderate outcomes across the G7, G6, and G4 groups, with values ranging from 56.59% to 62.85%. Zooming in on the vesicle-only treatments isolates a fundamental functional gap. The DABs (G4) alone drove deposition up to 56.59% while the Exo (G2) alone stalled at 40.98% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). DFO programming evidently equips DABs with an intrinsic tissue-remodeling strength that Exo lack entirely. Scar index results followed the same trend, with G8 achieving the lowest score (3.05%) compared to G2 (42.11%), G4 (23.62%), G5 (45.98%), and G1 (68.59%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec42\" class=\"Section2\"\u003e \u003ch2\u003e3.6. In situ mechanistic evaluation\u003c/h2\u003e \u003cp\u003eThe regenerative success seen at day 14 is fundamentally rooted in earlier microenvironmental shifts. We examined wound tissues at day 7 to capture this initial remodeling phase. Excessive ROS notoriously drives persistent inflammation and impairs diabetic healing [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. DHE staining of these early samples provided a direct readout of the underlying oxidative stress burden. Tissues from the untreated group (G1) showed intense red fluorescence, indicating high levels of oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). In stark contrast, the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e group (G8) demonstrated near-complete ROS elimination (0.32% positive area), as quantified in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef. This ROS-scavenging efficacy was significantly more potent than that of the hydrogel alone (G5: PHA-PVA\u003csup\u003eGel\u003c/sup\u003e), suggesting a crucial synergistic effect between the active PHA-PVA\u003csup\u003eGel\u003c/sup\u003e carrier and the sustained, inflammation-modulating release of DABs.\u003c/p\u003e \u003cp\u003eThis quick reduction in oxidative stress is essential for breaking the feedback cycle that sustains chronic inflammation. We therefore analyzed macrophage phenotype and distribution by immunofluorescence. As hypothesized, the G8 group showed a dramatic reduction in M1 macrophage (CD86\u003csup\u003e+\u003c/sup\u003e) infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). At the same time, it showed the highest levels of anti-inflammatory M2 macrophages (CD206\u003csup\u003e+\u003c/sup\u003e), suggesting an early phenotypic switch. This beneficial immune environment persisted until the end, as indicated by consistent immunofluorescence results at day 14 (Fig. S31 and Fig. S32). To further validate this long-term effect, we analyzed the tissue cytokine profile by ELISA at day 14. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg and Fig. S33, the G8 group maintained the most profound reduction in pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and the highest levels of anti-inflammatory cytokines (IL-4, IL-10, VEGF).\u003c/p\u003e \u003cp\u003eProper vascularization supports the proliferative healing phase [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The G8 treatment initiated this response effectively, with day 7 immunofluorescence capturing a dense CD31\u003csup\u003e+\u003c/sup\u003e and α-SMA\u003csup\u003e+\u003c/sup\u003e vessel network that continued expanding through day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef; Fig. S34 and S35). Local protein expression aligned directly with this structural growth. Tissues receiving the G8 formulation yielded the highest concentrations of TGF-β and VEGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef) and maintained these elevated pro-angiogenic signals into the later stages of repair (Fig. S36 and S37). HIF-1α expression offered a functional readout for local tissue hypoxia. Unresolved oxygen deficiency characterized the untreated G1 group since these defects maintained consistently high HIF-1α levels across the entire observation period. In contrast, the G8 group exhibited a rapid and significant decrease in HIF-1α expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef; Fig. S38), consistent with the restoration of tissue perfusion.\u003c/p\u003e \u003cp\u003eFinally, the biosafety profile of DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e was tested to ensure clinical applicability. Histological examination of major organs and local muscle tissue, combined with serum biochemical analysis, revealed no systemic toxicity or inflammatory response (Fig. S39-S41), confirming the excellent biocompatibility of DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e for regenerative medicine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec43\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Transcriptomic analysis of the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e promoting the diabetic wound healing\u003c/h2\u003e \u003cp\u003eWhile histological evaluations confirmed the superior tissue quality in the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e group, diabetic wound regeneration involves a complex symphony of signaling events that extends beyond structural observations. Therefore, to gain an unbiased, global understanding of the molecular reprogramming driving the regenerative effects of our system, we performed RNA-seq on wound tissues harvested on day 14 (G1: Untreated vs. G8: DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e), with sequencing quality control and alignment statistics summarized in Supporting Tab. S1 and S2. First, Principal Component Analysis (PCA) demonstrated that the untreated (G1) and DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e-treated (G8) groups segregated into distinct, non-overlapping clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Differential expression analysis identified 4779 differentially expressed genes (DEGs) between the two groups (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |log2FC| \u0026gt; 1), comprising 2493 upregulated and 2286 downregulated genes in the G8 group, as visualized in the volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eGO and KEGG enrichment analyses were performed to evaluate the biological pathways associated with these DEGs. GO analysis indicated that the downregulated genes in the G8 group were primarily enriched in pro-inflammatory biological processes, including \u0026lsquo;response to interferon-gamma\u0026rsquo;, \u0026lsquo;inflammatory response\u0026rsquo;, and \u0026lsquo;cytokine response\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and Fig. S42). Conversely, the upregulated genes were primarily associated with regenerative and metabolic processes, such as \u0026lsquo;cell projection\u0026rsquo;, \u0026lsquo;cytoskeleton organization\u0026rsquo;, and \u0026lsquo;ATP binding\u0026rsquo;. KEGG pathway analysis confirmed a suppression of classical inflammatory networks in the treated tissues. Specific cascades like TNF, NF-κB, and general cytokine signaling were distinctly underrepresented. This dampened inflammatory state allowed tissue repair and metabolic programs to dominate the enrichment profiles, with Foxo and Rap1 signaling emerging as the primary driven pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed and Fig. S43).\u003c/p\u003e \u003cp\u003eTo further examine transcriptional changes corresponding to the wound healing, we analyzed targeted gene sets associated with oxidative stress, inflammation, and angiogenesis [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Heatmaps demonstrated a coordinated upregulation of antioxidant genes (e.g., Trp53inp1, Foxo4, Pink1), immunomodulatory genes (e.g., Cebpa, Cd300a, Cnr2), and pro-angiogenic genes (e.g., Cxcr4, Nrp1, S1pr1) in the G8 group relative to G1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg, and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ei). The expression levels (FPKM) of representative hub genes were plotted to quantify these cluster trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eh, and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ej). Compared to the untreated control, the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e treatment resulted in significantly higher expression levels of specific antioxidant (Cat, Txnip, Foxo4), anti-inflammatory (Cebpa, Cd300a, Cnr2), and pro-angiogenic (Cxcr4, Nrp1, Rhob) genes. Together, these targeted transcriptional analyses provide direct validation of the upstream miRNA-driven and pathway-level findings, demonstrating that DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e coordinately reprogram antioxidant defense, inflammatory regulation, and angiogenic responses at the gene expression level.\u003c/p\u003e \u003cp\u003eBased on these transcriptomic findings, we tentatively interpret a molecular mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ek). These mechanisms suggest that DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e may influence multiple biological processes rather than a single dominant pathway. Alterations in Txnip, Foxo4, and Pink1 expression are consistent with modulation of Pink1-mediated mitochondrial quality control, which may contribute to improved redox balance [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In parallel, increased expression of genes within the Daglb/Cnr2 axis may reflect engagement of endogenous pathways linked to inflammation resolution, which have recently been implicated in skin repair processes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The activation of the Cxcr4/Nrp1/S1pr1 axis points to a coordinated vascular repair mechanism, where Cxcr4 recruits endothelial progenitor cells and Nrp1 and S1pr1 support Vegfr2 signaling and Rhob GTPase activation to drive endothelial migration and vessel maturation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eClinical translation of MSC-derived exosomes remains limited by their relatively low natural secretion yield and the instability of their therapeutic performance under pathological conditions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Apoptotic bodies (ABs) offer a high-yield alternative and possess intrinsic immunomodulatory properties largely attributed to phosphatidylserine (PS) exposure on their surface [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, their native pro-angiogenic activity is generally insufficient to meet the substantial vascularization demands of severely ischemic environments such as diabetic wounds [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In the present study, we demonstrate that pre-programming MSCs with DFO prior to apoptosis enables the generation of programmed vesicles (DABs) that effectively address both of these limitations.\u003c/p\u003e \u003cp\u003eDABs possess an enhanced biological activity that is likely driven by their abundant miRNA cargo. Although conventional preconditioning often relies on DFO to stabilize HIF-1α [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], our work reveals an additional effect that applying DFO prior to apoptosis induction fundamentally alters the vesicle\u0026rsquo;s cargo composition. For instance, DABs were found to be heavily loaded with specific miRNAs known to influence angiogenesis and cell migration such as hsa-miR-4674, hsa-miR-3976, and hsa-miR-320e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The levels of these molecules far exceeded what we observed in typical MSC-derived exosomes or native apoptotic bodies. These altered miRNAs likely contribute to the superior pro-angiogenic and immunomodulatory effects observed in our study.\u003c/p\u003e \u003cp\u003eSurface exposure of PS is known to facilitate macrophage efferocytosis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], a property that is retained in DABs. Analysis of recipient macrophages suggests the involvement of additional regulatory mechanisms beyond canonical pathways. The miRNA repertoire delivered by DABs appears to influence mitochondrial energy metabolism, as evidenced by increased cellular ATP production. To date, most EV-based therapeutic strategies have focused on modulating inflammatory signaling [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], whereas modulation of mitochondrial metabolism in recipient immune cells by programmed apoptotic bodies has not been reported. Whether this metabolic shift directly drives the M1-to-M2 polarization observed in our study remains unclear. However, given the well-established dependence of M2 macrophages on oxidative phosphorylation [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], this association merits further investigation.\u003c/p\u003e \u003cp\u003eTo improve vesicle stability within the hostile microenvironment of diabetic wounds, DABs were further encapsulated in a glucose- and ROS-responsive PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. The \u003cem\u003ein vivo\u003c/em\u003e results showed this approach was effective, with DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e promoting early wound closure faster than either exosome therapies or no treatment at all. We then ran RNA sequencing on the treated wound tissues to get a clearer picture of the biological changes driving this accelerated healing. The transcriptomic profiles showed a few distinct but coordinated trends. We noted a drop in the Txnip/Foxo4 cascade alongside the restoration of Nrf2-driven redox homeostasis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. At the same time, the Daglb/Cnr2 signaling axis lit up that makes sense given the emerging role of cannabinoid receptor 2 in clearing up skin inflammation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Furthermore, there was an upregulation across the Cxcr4/Nrp1/S1pr1 receptor network, which is known to recruit endothelial progenitor cells for vascular repair [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The simultaneous modulation of these pathways by a single vesicle-based intervention is noteworthy and likely reflects the diverse regulatory capacity of the miRNAs carried by DABs.\u003c/p\u003e \u003cp\u003eDespite these encouraging findings, our current study does have a few constraints. Although the STZ-induced diabetic mouse model reproduces certain aspects of diabetic pathology, it does not fully capture the complex systemic complications present in human type 2 diabetes [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In addition, murine wound healing relies heavily on contraction, whereas re-epithelialization plays a more dominant role in human chronic wounds [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The 14-day observation period used in this study is also insufficient to fully characterize the long-term degradation behavior and systemic clearance profile of PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. From a translational perspective, further work will also be required to ensure batch-to-batch consistency of DFO-programmed ABs and to establish scalable purification strategies suitable for large-scale production. Future studies using large-animal wound models, such as porcine skin defects that more closely resemble human dermal architecture, will therefore be important.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings demonstrate the potential of upstream cellular programming as a practical strategy to generate functionally enhanced apoptotic vesicles. Notably, a single preconditioning step using an approved small molecule was sufficient to substantially reshape apoptotic vesicle cargo, conferring both immunoregulatory and pro-angiogenic properties. Moreover, the fact that these programmed vesicles might actively alter recipient cell metabolism opens up a new therapeutic angle worth exploring. This strategy could extend well past diabetic wound repair. It holds promise for other conditions where conventional exosome therapies fall short, such as peripheral nerve injuries, inflammatory skin disorders, and ischemic vascular diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics declarations\u003c/strong\u003e \u003cp\u003eAll \u003cem\u003ein vivo\u003c/em\u003e protocols were executed under the ethical approval of the Institutional Animal Care and Use Committee (IACUC) at China Pharmaceutical University (approval # 2024-09-017).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupplementary Information\u003c/h2\u003e \u003cp\u003eSupplementary data to this article can be found in \u003cem\u003eSupporting Information\u003c/em\u003e file.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82272801, 82073175); Shandong Provincial Natural Science Foundation (ZR2025MS1272); Taishan Scholars Program for Young Expert of Shandong Province (tsqn202306383); Science and Technology Co-construction Project of National Administration of Traditional Chinese Medicine (GZY-KJS-SD-2024-103); Jinan Municipal Science and Technology Plan Project (202512040); China Postdoctoral Science Foundation (2025M772160); Basic Research Program of Jiangsu (BK20251569).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH. Guo drafted the manuscript, reviewed and edited the paper, administered the project, developed the methodology, validated the results, curated the data, and contributed to the study conception. J. Li drafted the manuscript, developed the methodology, validated the results, curated the data, and contributed to the study conception. C.C. Gu, N. Liu, J. Sun, and D.H. Wang contributed to methodology development, result validation, and data curation. S.Y. Meng and D.S. Geng contributed to methodology development and data curation. Y.F. Lian reviewed and edited the manuscript, supervised the study, administered the project, developed the methodology, validated the results, curated the data, and contributed to the study conception. F.N. Lv reviewed and edited the manuscript, acquired funding, developed the methodology, validated the results, curated the data, and contributed to the study conception. M.R. Huo drafted the manuscript, reviewed and edited the paper, supervised and administered the project, acquired funding, developed the methodology, validated the results, curated the data, and contributed to the study conception. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePhinney DG, Di Giuseppe M, Njah J, et al. Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. 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Bioeng Translational Med. 2022;7(3):e10344. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/btm2.10344\u003c/span\u003e\u003cspan address=\"10.1002/btm2.10344\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Exosomes, DFO-Programmed Apoptotic Bodies, Transcriptomic Reprogramming, Tissue Regeneration, Redox-Immuno-Angiogenic Synergy","lastPublishedDoi":"10.21203/rs.3.rs-9522213/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9522213/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMesenchymal stem cell (MSC)-derived exosomes show clear potential in cell-free regenerative medicine. However, their clinical translation is limited by low production yields and inconsistent therapeutic efficacy. To address this, we actively programmed stem cell apoptosis to generate high-yield, functional extracellular vesicles. By using deferoxamine (DFO) to stabilize HIF-1α prior to UV-induced apoptosis, we produced DFO-programmed apoptotic bodies (DABs). These programmed apoptotic bodies demonstrated a significantly higher production yield than conventional exosomes. Multi-omics analyses revealed that DABs inherit an enriched, pro-regenerative miRNA repertoire that functionally outperforms both native apoptotic bodies and exosomes. Beyond promoting classical angiogenic and migratory signaling, DABs support tissue repair by altering the mitochondrial bioenergetics of recipient cells. To prevent rapid \u003cem\u003ein vivo\u003c/em\u003e clearance and address the highly oxidative diabetic microenvironment, we encapsulated DABs within a glucose- and ROS-responsive hydrogel, developing the DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e transcriptomics demonstrated that DABs@PHA-PVA\u003csup\u003eGel\u003c/sup\u003e synchronized active ROS scavenging with the on-demand release of DABs. This localized delivery improved complete tissue reconstruction by simultaneously enhancing the Cxcr4/Nrp1/S1pr1 angiogenic axis, activating the Daglb/Cnr2 anti-inflammatory pathway, and engaging the Txnip/Foxo4/Nrf2 antioxidant cascade. Overall, this study establishes programmed apoptotic bodies as a scalable and effective alternative to standard exosomes therapies for tissue regeneration.\u003c/p\u003e","manuscriptTitle":"Responsive Hydrogel Delivery of DFO-Programmed Apoptotic Bodies Drives Redox-Immuno-Angiogenic Remodeling in Diabetic Wounds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 10:13:32","doi":"10.21203/rs.3.rs-9522213/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-16T10:27:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-16T00:57:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T12:45:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T04:14:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247766352425857350547551946607880359832","date":"2026-05-06T06:37:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162383411523029355829244622343884796434","date":"2026-05-06T05:02:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134312326287855125723736178307666088685","date":"2026-05-01T18:08:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"140702200962077668343039460281491461710","date":"2026-05-01T14:44:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300439846614160017355133625293498504538","date":"2026-05-01T06:45:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-01T06:20:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T14:35:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T14:34:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2026-04-25T04:49:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"676f74a8-0808-4413-bccf-d712beb9eba1","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-16T10:27:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-16T00:57:08+00:00","index":20,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T12:45:02+00:00","index":19,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T04:14:39+00:00","index":18,"fulltext":""},{"type":"reviewerAgreed","content":"247766352425857350547551946607880359832","date":"2026-05-06T06:37:42+00:00","index":17,"fulltext":""},{"type":"reviewerAgreed","content":"162383411523029355829244622343884796434","date":"2026-05-06T05:02:47+00:00","index":16,"fulltext":""},{"type":"reviewerAgreed","content":"134312326287855125723736178307666088685","date":"2026-05-01T18:08:36+00:00","index":14,"fulltext":""},{"type":"reviewerAgreed","content":"140702200962077668343039460281491461710","date":"2026-05-01T14:44:55+00:00","index":13,"fulltext":""},{"type":"reviewerAgreed","content":"300439846614160017355133625293498504538","date":"2026-05-01T06:45:45+00:00","index":12,"fulltext":""},{"type":"reviewersInvited","content":"6","date":"2026-05-01T06:20:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T14:35:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T14:34:57+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-16T10:39:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 10:13:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9522213","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9522213","identity":"rs-9522213","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}