Endothelial cell-targeting aptamer-empowered exosomes accelerate wound healing by promoting specialized angiogenesis in type 1 diabetic mice

Endothelial cell-targeting aptamer-empowered exosomes accelerate wound healing by promoting specialized angiogenesis in type 1 diabetic mice | 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 Endothelial cell-targeting aptamer-empowered exosomes accelerate wound healing by promoting specialized angiogenesis in type 1 diabetic mice Na Zhao, Zi-Zhen Guo, Sheng-Feng Bai, Ying-Feng Gao, Jing Lei, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7518048/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Dec, 2025 Read the published version in Stem Cell Research & Therapy → Version 1 posted 7 You are reading this latest preprint version Abstract Background The reduced angiogenesis in diabetes mellitus (DM) represents a critical barrier to effective skin wound healing. Therapeutic strategies involving mesenchymal stem cells (MSCs) and MSC-derived exosomes (EXOs) have demonstrated potential in promoting wound healing in diabetic contexts. However, each approach presents specific limitations. Methods Apt-PEG-DSPE was synthesized via amide condensation between DSPE-PEG-COOH and NH₂-Apt, followed by incubation with EXOs to yield Apt-EXOs, then mix with HA to form Apt-EXOs-HA. C57BL/6 mice were injected intraperitoneally with 50 mg/kg STZ daily for 5 days to induce type 1 diabetes (T1D). Under anesthesia, dorsal fur was shaved and full-thickness skin defects (1.0 cm diameter) were created. The therapeutic effect of Apt-EXOs-HA on T1D skin injury was evaluated by wound healing, vascularization and collagen deposition. The Student’s t -test (two-tailed) was used to assess statistical significance. Results In this study, we identified that vascular structures, specifically CD31⁺EMCN⁺ vessels, are impaired in T1D, which contributes to delayed wound healing and aberrant collagen deposition. Following proteomic analysis and related vascular endothelial cell experiments (including cell migration and tube formation) demonstrating the superior angiogenic potential of EXOs compared to MSCs, we engineered endothelial-targeting EXOs by conjugating them with aptamers (Apt). The application of these Apt-conjugated EXOs in combination with a hyaluronic acid scaffold significantly enhanced angiogenesis under both physiological and DM conditions, thereby accelerating wound healing. Conclusions Collectively, our findings emphasize the essential role of specialized angiogenesis in wound repair and propose a novel, advanced EXOs modification-based therapeutic approach to enhance wound healing in both normal and diabetes-related pathophysiological conditions. diabetic wound healing aptamer exosomes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Skin lesions are a common occurrence throughout life and are generally associated with a robust self-healing capacity. However, the rising prevalence of aging, obesity, and diabetes within the global population has imposed a substantial financial burden on healthcare systems worldwide [ 1 ]. Diabetes mellitus (DM), a chronic metabolic disorder, is projected to affect approximately 700 million adults by 2045 [ 2 ]. Type 1 diabetes (T1D), in particular, is distinguished by an absolute deficiency in insulin production resulting from the autoimmune destruction of pancreatic β-cells, which are erroneously attacked by a dysregulated immune system [ 3 ]. This condition is often considered to be associated with more severe skin damage compared to type 2 diabetes (T2D), primarily due to reduced angiogenesis and impaired vascular repair mechanisms, both of which are consequences of autoimmune-mediated damage and the complete loss of endogenous insulin production [ 4 – 6 ]. Patients with T1D experience earlier and more severe damage to vascular endothelial cells due to hyperglycemia, leading to impaired skin microvascular formation and structural abnormalities, which easily cause problems such as dry skin and ulcers. Patients with T2D may develop vascular endothelial dysfunction due to factors such as insulin resistance, but abnormal skin angiogenesis usually chronic and occurs later. As a consequence, individuals with T1D often experience delayed wound healing, suboptimal healing outcomes, or chronic wounds that fail to heal entirely [ 7 ]. Clinically, hydrogels are often employed to maintain wound moisture, thereby reducing inflammation and infection. However, their efficacy in fully restoring compromised wound healing remains limited [ 8 ]. Furthermore, numerous strategies to promote angiogenesis have been developed over the past decades to address this critical challenge with unsatisfied outcomes [ 7 , 9 ]. Mesenchymal stem cell (MSC)-based therapy has been developed and offers a promising therapeutic approach for T2D skin wound repair by promoting specialized CD31 + Endomucin (EMCN) + vessels formation, this therapeutic outcome is achieved through the secretion of vesicles enriched with angiogenic proteins [ 10 ]. Besides, current research indicates that the regenerative capabilities of MSCs are predominantly influenced by their paracrine actions rather than direct transplantation and differentiation. Exosomes (EXOs) secreted by MSCs are considered primary mediators of these paracrine effects [ 11 ]. These EXOs, a category of extracellular vesicles with diameters ranging from 30 to 150 nm, are known to transport a wide array of proteins, mRNA, and miRNAs [ 12 – 14 ], have been reported to enhance vascular endothelial cells (ECs) proliferation and migration and to upregulate the expression of molecules intricately associated with angiogenesis [ 15 , 16 ], suggesting the potential application of MSC-derived EXOs in reconstructing vasculature in diabetic skin. Nonetheless, the non-specific biodistribution of unmodified EXOs in vivo diminishes their efficacy, necessitating repeated administrations [ 17 – 19 ]. To address the limited availability of MSC-derived EXOs, several EXOs modification technologies have been developed to enhance their bioavailability. Aptamers (Apt), synthetic single-stranded oligonucleotides, can be screened, amplified, and enriched in vitro using systematic evolution of ligands by exponential enrichment (SELEX) [ 20 , 21 ]. These Apt often adopt secondary structures such as hairpins and stem-loops, as well as three-dimensional configurations, allowing them to bind target molecules with high affinity and specificity [ 22 , 23 ]. Apt-functionalized drug loaded liposomes have been reported to enhance drug stability, targeting and delivery efficiency [ 24 , 25 ]. To further address the weak mechanical properties of EXOs, the combination of hydrogels and hyaluronic acid (HA) are used as scaffold to strength the biocompatibility, biodegradability, water absorption and retention properties of EXOs [ 26 – 28 ]. The resulting Apt-EXOs-HA complex prompts investigation into whether the sustained binding of EXOs to ECs can preferentially promote the formation of CD31⁺EMCN⁺ vessels, a vascular subtype associated with regeneration that is reduced in T1D skin [ 10 , 29 ]. In the current study, we seek to elucidate the role of angiogenesis in T1D in mice and to develop bioengineered EXOs to enhance angiogenesis in diabetic skin. Utilizing liquid chromatography-mass spectrometry (LC-MS) and related vascular endothelial cell experiments (including cell migration and tube formation), we identified a superior angiogenic function of EXOs compared to MSCs. We synthesized an Apt-EXOs-HA complex, employing EC-specific Apt as targeting agents and HA as a scaffold to optimize the mechanical properties of EXOs. We examined the angiogenic effects of Apt-EXOs-HA, with particular emphasis on CD31⁺EMCN⁺ vessel formation, both in vitro and in vivo , and analyzed its role in promoting wound healing in T1D skin. Overall, our research aims to clarify the critical role of specialized angiogenesis in T1D wound healing and to advance the application of engineered EXOs in addressing unmet clinical needs in diabetic skin treatment. To our knowledge, this study is the first to demonstrate the therapeutic potential of Apt-equipped EXOs for T1D wound healing. Methods Animals Eight-week-old female C57BL/6 mice were provided by Laboratory Animal Center of Fourth Military Medical University. All experimental protocols were approved by the Fourth Military Medical University. All animal experiments conducted in this research were performed in accordance with the guidelines of the Fourth Military Medical University Intramural Animal Use and Care Committee and met the NIH guidelines for the care and use of laboratory animals. Animals were randomly assigned to different experimental groups, maintained with good ventilation and a 12 h light / dark cycle, and were kept feeding and drinking before being sacrificed. The work has been reported in line with the ARRIVE guidelines 2.0. T1D full-layer cutaneous wound modeling C57BL/6 mice were injected intraperitoneally with 50 mg/kg STZ daily for 5 days to induct DM, mice were defined as T1D when glucose levels were >11.1 mmol/L under fasting conditions. After anesthesia, the dorsal fur of T1D mice was shaved, and full-layer cutaneous wounds with a diameter of 1.0 cm were carefully made using ophthalmic scissors under sterile surgical conditions, then the wound was covered with transparent film dressing. Following diabetic wound induction, all experimental mice were housed in standard individually ventilated cages (IVC). Each cage contained autoclaved bedding that was refreshed every 48 hours to maintain optimal hygiene. The housing environment was strictly maintained at 22 ± 2°C with 50 ± 10% relative humidity under a controlled 12 h light / dark cycle. Postoperative monitoring included daily visual inspection of wounds with photographic documentation at predetermined time points. Any displaced dressings were promptly replaced with sterile alternatives to ensure proper wound protection. Throughout the study period, all animals had continuous access to autoclaved feed and purified drinking water ad libitum. Two distinct anesthetic protocols were employed for different procedures. For in vivo wound imaging: mice were anesthetized using isoflurane inhalation to ensure immobility for consistent and high-quality photography. Anesthesia was induced by placing mice in an induction chamber with a flow of 2% isoflurane in oxygen. Once anesthetized, the mice were transferred to a nose cone for the duration of the imaging procedure, where anesthesia was maintained with 1.5% isoflurane. For terminal procedures and tissue collection: at the experimental endpoint, mice were deeply anesthetized via intraperitoneal injection of 100 mg/kg sodium pentobarbital. This protocol ensures a surgical plane of anesthesia, leading to euthanasia. Cutaneous wound healing assessment Wound bed sizes during approximately 2-week experimental periods were observed daily and imaged at indicated time points by digital camera and data were calculated as follows: (actual wound area/original wound area) ×100%. Following euthanasia by intraperitoneal injection of a barbiturate drug, the wound surface and the surrounding skin within a 0.5 cm radius were collected for further analysis. H&E and Masson’s trichrome staining Biopsies of the original wound area and the remaining wound beds were sampled on day 7 or 14 after administration and fixed with 4% paraformaldehyde for 24 h, followed by dehydration and embedded in paraffin. The samples were sectioned perpendicularly to the wound surface in 4 µm consecutive sections by a microtome and were prepared for H&E and Masson’s trichrome staining. H&E staining was conducted as previously described [ 30 ]. For analysis of collagen deposition, Masson’s trichrome staining was performed with a commercial kit (BaSO, China) according to manufacturer’s instructions. Vascularization assessment Biopsy samples of the original and remaining wounds were placed in culture dishes, inwardly facing down. Vascularization states of each specimen were assessed under incandescent illumination and photographed with a digital camera (Leica, Germany). Immunofluorescent assay The original and remaining wounds on day 7 after administration were biopsied, fixed with 4% paraformaldehyde for 4 ~ 6 h and dehydrated with 30% sucrose for 24 ~ 48 h. 20 µm cryosections were permeabilized by Triton X-100 (Sigma-Aldrich, USA) for 20 min at room temperature, blocked with goat serum (Boster, China) for 1 h at 4°C and then stained with primary antibodies overnight at 4°C as follows: anti-mouse Alexa Fluor™ 488-conjugated anti-CD31 (FAB3628G, R&D Systems, USA; diluted 1:100) co-stained with anti-mouse Endomucin (sc-65495, Santa Cruz Biotechnology, USA; diluted 1:100), anti-mouse PCNA (ab92552, Abcam, USA; diluted 1:200). Whereafter incubated with the following fluorescence-conjugated secondary antibodies at room temperature for 1 h: Cy3-conjugated goat anti-rat IgG (33308ES60, YEASEN, China; diluted 1:200) or Fluor™ 488 Goat Anti-Rabbit lgG (33106ES60, YEASEN, China; diluted 1:200). The sections subsequently mounted with antifade mounting medium containing DAPI. Quantification of CD31 + EMCN + vessels and PCNA + cells was carried out with ImageJ software (NIH, USA). Cell culture MSCs were isolated from umbilical cords of women who gave birth in Xi'an Fourth Military Medical University Xijing Hospital. All subjects provided informed consent. It was approved by the Ethics Committee of Xi'an Fourth Military Medical University. MSCs were isolated by tissue block culture attachment method [ 31 ]. Simply put, the umbilical veins and arteries and their surrounding Wharton jelly are separated from the stroma, the Wharton jelly mesenchymal tissue is cut into 2–3 mm 3 chunks and transferred to a petri dish. The MSCs were cultured with alpha-minimum essential medium (𝛼-MEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Sijiqing, China) and 1% penicillin/streptomycin (Invitrogen, USA) in a humidified atmosphere of 5% CO 2 at room temperature. The C166 and C2C12 cells were cultured with dulbecco's modified eagle medium (DMEM, Invitrogen, USA) with 10% FBS and 1% penicillin-streptomycin. The medium was refreshed every 3 days, and the cells were passaged once they were 80%-90% confluence. EXOs isolation For EXOs isolation, we initiated each batch with 1×10 7 cells (from a T75 culture flask). When the cells reached 80% confluence, the medium was substituted with fresh inducing medium (α-MEM basal medium) without FBS. After 48 h, the supernatant was collected. Initial clarification at 300 g for 10 min to remove cells, followed by 2,000 g for 20 min and 16,000 g for 30 min to eliminate cellular debris and larger extracellular vesicles. Finally, we used a Beckman Coulter Optima XPN-100 ultracentrifuge equipped with a 32 Ti fixed-angle rotor, and centrifuged at 150,000 g for 90 minutes to achieve the final EXO precipitation. We maintained rigorous temperature control (4℃) throughout all centrifugation steps to preserve vesicle integrity. Following ultracentrifugation, the pelleted EXOs were resuspended in 200 µL of 0.22 µm-filtered phosphate-buffered saline (PBS, pH 7.4), a volume optimized through preliminary experiments to ensure appropriate EXO concentrations for downstream analyses. To achieve the standardization of EXOs, the BCA protein assay method (TianGen, China) was employed. The morphology of EXOs was assessed through transmission electron microscopy (TEM, Thermo fisher, USA). The size distribution of EXOs was quantitated using dynamic light scattering (DLS) with a Zetasizer (Malvern, UK). To ensure measurement accuracy, the EXO samples were diluted in 0.22 µm-filtered PBS buffer (pH 7.4). The expression of marker proteins was examined by western blot analysis. Specifically, EXOs lysates were prepared by cell lysis buffer (Beyotime, China) containing protease inhibitor (Roche, Switzerland), the extracted proteins were separated by SDS-PAGE electrophoresis, transferred onto polyvinylidene fluoride membranes (Roche, Switzerland) and blocked in 5% BSA (MP Biomedicals, USA) at room temperature for 2 h. The membranes were incubated with the following primary antibodies: CD9 (ab236630, Abcam, USA, 1:1000), CD63 (ab271286, Abcam, USA, 1:1000), CD81 (sc-166029, Santa Cruz Biotechnology, USA, diluted 1:500), COX Ⅳ (4844, CST, USA, 1:1000) and LAMP 1 (ab24170, Abcam, USA, 1:1000) and then incubated with peroxidase-conjugated secondary antibody: Goat Anti Mouse IgG (RS0001, Immunoway, USA, 1:10000) or Goat Anti Rabbit IgG (RS0002, Immunoway, USA, 1:10000). Proteomic Analysis The protein lysates of MSCs and EXOs were prepared and analyzed by LC-MS/MS on Orbitrap Exploris™ 480 mass spectrometer with a NanoSpray III ion source. The original data was analyzed using the Proteome Discoverer system (v2.4.1.15). Proteins were identified by comparison with the Uniport database, and the error finding rate was set to 0.01 for both peptides and proteins. Proteins are quantified using MaxQuant's default parameters. Among the identified proteins, differential expression proteins (DEPs) were included (fold change > 1.5, p < 0.05), and further functional analysis was conducted based on GO and KEGG databases. Validation of Apt affinity Apt that target ECs (5'-CCC ACG TCT GCG CTT AGC TCC TGG GCC TGG ATG GGC-3') was synthesized by Sangon Biotech Co., Ltd. (China). For the Apt affinity assay, MSCs, C166 and C2C12 cells were seeded at a density of 3×10 4 cells/well onto a 24-well plate with cell climbing slides. Then, carboxyfluorescein-labeled Apt (Apt-FAM) with a concentration of 10 µM were incubated with cells for 3 h. After rinsed with PBS, cells were successively fixed, mounted with antifade mounting medium containing DAPI (Beyotime, China) and imaged by a fluorescence microscope (Olympus, Japan). Synthesis of Apt-EXOs-HA Apt-PEG-DSPE was first prepared by an amide condensation reaction between DSPE-PEG-COOH and 5’-NH 2 -modified Apt as follows: DSPE-PEG-COOH (Avanti, USA), NHS (Macklin, China) and EDC (Macklin, China) were separately dissolved in ddH 2 O, adjusted the solvent pH to 5 ~ 6 and stirring at room temperature. After dialysed several times to remove unreacted NHS and EDC, 5’-NH 2 -modified Apt was added and stirred for another 12 h. Then Apt-PEG-DSPE and EXOs were stirred at room temperature for 1 ~ 2 h in pyrocarbonate-treated PBS, unconjugated Apt-PEG-DSPE was removed by ultrafiltered several times to prepare Apt-modified EXOs suspension, namely Apt-EXOs. Prior to use, the HA (Macklin, China) was fully dissolved in PBS (pH adjusted to ~ 7.0) and subsequently mixed with either EXOs or Apt-EXOs to form the composite gel. The final concentrations in the prepared gel were 50 mg/mL for HA and 1.25 mg/mL for EXOs or Apt-EXOs. Apt-EXOs Characterization TEM, DLS and western blot analysis of Apt-EXOs were performed. Moreover, the preparation of Apt-EXOs was evaluated by gel retardation assay. In short, Apt, DSPE-PEG-Apt and Apt-EXOs were mixed with the sample buffer respectively and adjust the voltage of agar-electrophoresis at 30–40 V/cm for 90 min, followed by imaging using the gel imaging system. Cellular internalization of Apt-EXOs The PKH26 labeling of purified EXOs was performed by resuspending the EXOs in Diluent C buffer, followed by mixing with freshly prepared PKH26 dye solution (dilute the PKH26 dye in diluent C). After a 5-minute incubation at room temperature in the dark, the reaction was terminated with 1/2 volume of EXO-depleted FBS, and unbound dye was removed through PBS washing and ultracentrifugation [ 32 , 33 ]. For the EXOs internalization experiments, C166 cells were seeded at a density of 3×10 4 cells/well onto a 24-well plate with cell climbing slides. Then, 100 µg/mL PKH26 labeled Apt-EXOs was incubated with C166 cells for 3 h at room temperature. Subsequently, the cells were fixed with 4% paraformaldehyde and mounted with antifade mounting medium containing DAPI. The signals of Apt-EXOs uptake were observed using a fluorescence microscope. PKH26 labeled EXOs treated C166 cells were used as a negative control. In vitro scratch Assay C166 cells were seeded at a density of 3×10 5 cells/well onto a 6-well plate and the scratch assay was performed to evaluate their migration ability, as previously reported. In brief, when the cells reached 90% confluence, sterile 1 ml pipette tips were used to create scratches on the plates by scraping cells away. After washing with PBS, the medium was changed to one without FBS. MSC conditioned medium, 100 µg/mL EXOs or Apt-EXOs and added severally, and three locations were randomly selected for imaging 24 h later with an inverted microscope (Leica, Germany). In vitro tube formation assay MSC conditioned medium, 100 µg/mL EXOs or Apt-EXOs were premixed with C166 cells and seeded at a density of 1×10 4 cells/well onto the 96-well plate coated with Matrigel (Corning, USA) and incubated for 6 h. The enclosed networks of complete tubes were digitally imaged. For quantification of the Matrigel tube formation assay, we utilized ImageJ software (NIH, USA) with the Angiogenesis Analyzer plugin to measure tube length. Statistical analysis All data are expressed as mean ± standard deviation (SD). The Student’s t -test (two-tailed) was used to assess statistical significance. Values of P < 0.05 were considered statistically significant. Statistical and graphical analysis using GraphPad Prism 9.0 (GraphPad Software, USA). Results Impaired angiogenesis is associated with delayed skin wound healing in mice with T1D T1D is associated with severe microvascular complications attributed to absolute insulin deficiency, a dysregulated immune system, and abnormal tissue metabolism, which together contribute to impaired wound healing [ 34 ]. At the beginning of this study, we examined changes in angiogenesis, with a particular focus on CD31⁺EMCN⁺ vessels, during wound healing in a T1D context. For this purpose, we employed a high-dose streptozotocin (STZ)-induced T1D mouse model and established a wound healing framework, systematically monitoring wound area progression at defined time points. The results indicated a marked inhibition of wound closure rates in T1D mice (Fig. 1 a-b), which correlated with decreased collagen deposition (Fig. 1 c). Parameters such as wound bed size, neo-epithelialization gap, and collagen index were quantified and statistically analyzed (Fig. 1 d-f). Furthermore, the distribution of CD31⁺EMCN⁺ vessels and capillaries was significantly compromised (Fig. 1 g), and statistical analysis of the CD31⁺EMCN⁺ area and capillary area percentages was conducted (Fig. 1 h-i). These findings indicate that impaired angiogenesis, along with the presence of CD31⁺EMCN⁺ vessels, contributes to delayed wound healing and diminished collagen deposition. Further suppression of skin angiogenesis in T1D mice exacerbates impaired wound healing The impairment of angiogenesis in T1D skin wound healing has been established. To further confirm the relationship between angiogenesis and wound healing in T1D mice, we administered intraperitoneal injections of DAPT, a γ-secretase inhibitor known to suppress Notch signaling [ 35 ], to inhibit angiogenesis and observed the wound healing process. On day 7, the distribution of CD31⁺EMCN⁺ vessels and capillaries was reduced, though not entirely abolished under our experimental conditions (SI. 1a-c). Following significant inhibition of angiogenesis, the wound healing area was markedly diminished, particularly after day 7 (SI. 1d). Histological analysis using H&E and Masson staining revealed an increased epithelial gap in the DAPT-treated group and decreased collagen deposition (SI. 1e, f). Wound bed size, neo-epithelialization gap, and collagen index were quantified and statistically analyzed (SI. 1g-i). Collectively, these findings demonstrate that vascular structures are diminished in T1D skin and highlight the possibility of CD31⁺EMCN⁺ vessels in promoting the healing of T1D skin wounds. EXOs exhibit superior angiogenesis-stimulating potential compared to MSCs After establishing the critical role of angiogenesis in wound healing, we explored the use of MSC-based therapy to stimulate angiogenesis. Previous studies have reported that MSCs, particularly MSC-derived EXOs, are among the most effective agents for promoting angiogenesis and enhancing skin wound healing [ 36 ]. To identify more effective strategies for promoting angiogenesis, we conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to evaluate angiogenesis-associated proteins present in EXOs and MSCs (Table. S1). EXOs and MSCs differ in protein signature, showing 4568 overlapping proteins and 55 proteins specifically expressed in EXOs in the Venn diagram (Fig. 2 a). Among co-expressed proteins, 1188 proteins were upregulated in EXOs compared to the MSCs counterparts (Fig. 2 b). As for the potential functions of differentially expressed proteins, the enrichment analysis in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed a number of related terms, among which "cell adhesion molecules" stood out (Fig. 2 c). It's worth noting that gene ontology (GO) enrichment analysis of significantly upregulated proteins revealed an abundance of several terms related to angiogenesis in EXOs, including "sprouting angiogenesis", "angiogenesis", "regulation of sprouting angiogenesis", "regulation of angiogenesis" and "positive regulation of angiogenesis" (Fig. 2 d). According to functional analysis, multiple individual proteins involved in angiogenesis were upregulated in EXOs. For example, we found significant upregulation of several proteins in EXOs, namely Epidermal growth factor receptor (EGFR), Notch 2, Caveolin-1 (CAV1), Transforming growth factor (TGF)-β1, Janus kinase (JAK), Annexin A1 (ANXA1) and ANXA3 (Fig. 2 e), which have been reported to be involved in germination, dilation and formation of blood vessels [ 37 – 43 ]. Notch activation directs tip-derived ECs into developing arteries, and further, Dll4-Notch signaling combines budding angiogenesis with arterial formation [ 40 ]. Endothelial CAV1 plays an important role in endothelium-dependent hyperpolarization mediated vasodilation and ischemic angiogenesis by modifying post-translational proteins through nitro oxidative stress [ 41 ]. TGF-β1 induces the production of vascular endothelial growth factor by TGF-β receptor, Smad2/3, PI3K/AKT and JNK1/2 related signaling pathways and further promoted in vitro angiogenesis of ECs and in vitro vessel germination of the aortic ring [ 42 ]. Consistent with the results of LC-MS/MS analysis, experiments related to vascular endothelial cells also demonstrated that the EXOs exhibit stronger pro-angiogenic activity than MSC-conditioned medium, manifested by enhanced cell migration and tube formation (Fig. 2 f-i). Overall, these data suggest that EXOs exhibit superior functionality in promoting angiogenesis compared to MSCs. Characterization of EC-targeting EXOs The superior angiogenic potency of EXOs has been demonstrated. However, EXOs alone still present several limitations, including restricted availability and suboptimal bioavailability. To address these challenges, Apt exhibiting strong affinity, high specificity, low immunogenicity, and ease of modification were selected to specifically target ECs [ 44 ]. We first identified the synthesized Apt by mass spectrometry, and observed that the molecular weight was consistent with the expectation (SI. 2a). Particularly, the results of specific targeting ability showed that the fluorescence area of FAM-labeled Apt in C166 ECs was tens of times that of UC-MSCs and C2C12 myoblast cells in the control group, confirming its high selectivity in binding to ECs (Fig. 3 a, b). In addition, we performed migration and tube formation assay on C166 cells to evaluate the pro-angiogenic ability of Apt. The results demonstrated that Apt had no effect on migration and tube formation of C166 cells (SI. 2b-e). Meanwhile, we applied Apt to a mouse model of wound healing and found that the formation of CD31 + EMCN + vessels and capillary in the Apt group did not change compared to the control (SI. 2f-h). Based on the above characteristics of Apt, we modify the EXOs surface with the Apt targeting ECs. To be specific, Apt-PEG-DSPE was prepared by coupling the 5'-NH 2 -modified Apt with DSPE-PEG-COOH through an amide condensation reaction (SI. 3a), and then co-incubated with EXOs to obtain Apt-functionalized EXOs Apt-EXOs (Fig. 3 c). The successful conjugation of the DSPE-PEG-Apt and Apt-EXOs were evaluated by gel retardation assay, the results illustrated that DSPE-PEG-Apt and Apt-EXOs were successfully synthesized since the molecular weight of DSPE-PEG-Apt and complex increased in different degrees, showing electrophoretic bands at diverse locations (Fig. 3 d). Furthermore, the cup-shaped EXOs with clear membrane structure about 100 nm in diameter, and the particle size of Apt-EXOs is mainly distributed around 300 nm, which is a homogeneous bilayer spherical nanoparticles surrounded by a layer of black nuclea-like material (Fig. 3 e-f). The scanning electron microscope (SEM) of plain HA and HA containing Apt-EXOs demonstrated similar morphology, although slight alterations in the morphology of the adhered exosomes occurred during sample preparation (Fig. 3 g). Western blot analysis revealed the enrichment of typical EXOs markers, such as CD9, CD63 and CD81 in Apt-EXOs, but lacked the mitochondrial marker COV Ⅳ and the lysosomal marker LAMP1, indicating that EXOs were successfully collected with a high purity, and were not damaged during the reaction (Fig. 3 h). Taken together, Apt functionalized EXOs targeting ECs were successfully produced. Apt-conjugated EXOs exhibit preferred internalization efficiency and enhanced pro-angiogenic activity in vitro After the successful synthesis of Apt-EXOs, we proceeded to analyze their bioavailability, focusing on ECs internalization and pro-angiogenic activity. We first labeled EXOs with PKH26 to examine whether the conjugated Apt enable EXOs to specifically target ECs. As expected, massive PKH26 fluorescence signals were detected in C166 cells incubated with Apt-EXOs, while slight were detected in UC-MSCs and C2C12 cells (Fig. 4 a). Moreover, PKH26-labeled Apt-EXOs was incubated with C166 cells, and PKH26-labeled EXOs served as a negative control, we observed that the PKH26 fluorescence signal in Apt-EXOs treated C166 cells was much greater than that in the EXOs group (Fig. 4 b). In terms of the cellular mechanism through which Apt-EXOs is taken up by C166 cells, we pretreat C166 cells with EIPA (a micropinocytosis inhibitor), nystatin (a caveolae-mediated endocytosis inhibitor) and chlorpromazine (a clathrin-mediated endocytosis inhibitor) respectively, then they are processed with PKH26-labeled Apt-EXOs. Confocal images displayed that Apt-EXOs can be taken up through all of the three uptake pathways, especially by micropinocytosis (Fig. 4 c). To confirm the cellular internalization of EXOs, the fluorescence intensity of PKH26 was measured and subjected to statistical analysis (Fig. 4 d-f). The property of Apt-EXOs in promoting angiogenic capacity was also assessed. Compared with the control group, C166 cells exposed to EXOs and Apt-EXOs exhibited greater migration and tube formation ability, especially in the Apt-EXOs group, suggesting that Apt functionalization promotes the pro-angiogenic capacity of EXOs (Fig. 4 g), further quantification confirmed the above observation (Fig. 4 h). Additionally, migration and tube formation assays were quantified and statistically analyzed to evaluate their pro-angiogenic potential (Fig. 4 i-j). Overall, these findings demonstrate the enhanced internalization of Apt-EXOs by ECs and are anticipated to promote angiogenesis and accelerate wound healing. Apt-EXOs promote vascular regeneration and accelerate wound healing in normal skin Following the evaluation of the angiogenic capacity of Apt-EXOs in vitro , we examined their vascular regeneration potential in vivo . In this process, HA was employed as a carrier for EXOs and Apt-EXOs to enable controlled release, thereby preventing sudden release and minimizing liquid leakage. Mice were administered EXOs or Apt-EXOs in a standardized volume of 200 µL HA at a dose of 150 µg. Compared with the control, Apt-EXOs-HA treatment significantly accelerated the wound closure rate (Fig. 5 a). Histological analysis of the dissected skin tissues via H&E and Masson’s staining further showed that Apt-EXOs-HA treatment remarkably enhanced skin healing, as shown by reduced epithelial distance (Fig. 5 b) and increased collagen deposition (Fig. 5 c). The wound closure rate, neo-epithelialization, and collagen deposition were quantified and subjected to statistical analysis (Fig. 5 d-f). Notably, after 7 days of treatment, the immunofluorescence co-staining of CD31 and EMCN showed that Apt-EXOs-HA significantly increased CD31 + EMCN + vessels as well as the capillary growth in injury sites (Fig. 5 g), the number of PCNA + proliferative cells were also remarkably increased of the Apt-EXOs-HA group (Fig. 5 h). The percentage of CD31 + EMCN + area, capillary area, and PCNA + area was measured and subjected to statistical analysis (Fig. 5 i-k). Collectively, these findings indicate that Apt-EXOs-HA effectively enhances skin wound healing by promoting angiogenesis. Apt-EXOs preserve their biological functionality in T1D skin lesions Microvascular disease is a common complication of diabetes, where angiogenesis inhibitors, hyperglycemia, and various abnormal factors impede vascular regrowth. Consequently, promoting vascular regeneration in T1D skin wound healing remains a significant challenge [ 45 ]. Given the excellent performance of Apt-EXOs in the healing of normal mouse skin wounds, we further investigated its potential therapeutic effects of Apt-EXOs by establishing T1D full-layer cutaneous wounds. Each diabetic mouse was also administered a dose of 150 µg of EXOs or Apt-EXOs. Similarly, Apt-EXOs-HA treatment substantially accelerated cutaneous wound healing, while EXOs-HA treatment had a relatively slight effect and HA had almost no effect on day 7 (Fig. 6 a). For characteristics of the wound lesion, H&E staining of the wound bed samples at day 14 demonstrated that faster epithelialization in Apt-EXOs-HA group while slower epithelialization in EXOs-HA, HA and control groups, indicating that Apt-EXOs-HA had excellent ability to promote granulation tissue maturation and epidermal regeneration (Fig. 6 b). Masson’s trichrome staining showed a superior collagen deposition with certain direction and thicker bundle for Apt-EXOs-HA group versus that of EXOs-HA, HA and control groups with short collagen deposition (Fig. 6 c). The wound closure rate, neo-epithelialization, and collagen deposition were quantified and subjected to statistical analysis (Fig. 6 d-f). In terms of promoting angiogenesis, immunofluorescence co-staining of CD31 and EMCN, as well as the changes of capillaries strongly demonstrated the excellent angiogenesis promoting ability of Apt-EXOs-HA (Fig. 6 g). The number of proliferative cells in Apt-EXOs-HA group was also significantly increased by immunofluorescence assay of PCNA (Fig. 6 h). The percentage of CD31 + EMCN + area, capillary area, and PCNA + area was measured and subjected to statistical analysis (Fig. 6 i-k). Collectively, these findings suggest that Apt-EXOs-HA effectively contribute to the improvement of a pro-regenerative microenvironment, thereby promoting skin wound healing through the enhancement of angiogenesis in the context of T1D. Discussion Diabetic wounds are a prevalent complication of diabetes, characterized by impaired angiogenesis. Among the vascular subtypes essential for regeneration and wound healing, CD31⁺EMCN⁺ vessels play a pivotal role. However, in diabetes, vascular regrowth is hindered by various factors, including hyperglycemia, neuropathy, and abnormal extracellular matrix (ECM) deposition [ 46 , 47 ]. Hyperglycemia exacerbates oxidative stress and inflammation, while neuropathy disrupts angiogenic processes by impairing sensory perception and motor control, ultimately reducing oxygen supply and further impeding angiogenesis [ 48 , 49 ]. Additionally, the altered ECM composition in diabetes interferes with the critical signaling and molecular interactions required for proper angiogenic progression [ 50 ]. Under normal physiological conditions, angiogenesis is initiated by ECs forming immature microvessels, which subsequently mature through pericyte-mediated signaling to ensure vascular bed stability and integrity [ 51 ]. In diabetes, this process is disrupted, with delayed recruitment of pericytes leading to extravascular leakage and vascular edema [ 52 ]. Even after wound closure is achieved, diabetic wounds often fail to restore adequate pericyte coverage, compromising the establishment of a well-perfused vascular network, a fundamental requirement for effective wound healing. In this study, our analysis of vascular distribution in T1D wounds revealed a marked reduction in CD31⁺EMCN⁺ vessels, a critical factor contributing to delayed wound healing in diabetes. These findings underscore the importance of elucidating the biological mechanisms underlying CD31⁺EMCN⁺ vessel formation and assessing strategies to promote their regeneration, which could provide valuable insights into enhancing tissue repair in diabetic wounds. To the best of our knowledge, no existing medications specifically promote angiogenesis or the formation of CD31⁺EMCN⁺ vessels for the treatment of diabetic wound healing. The application of MSC aggregates has been reported to stimulate wound healing by promoting angiogenesis in T2D, but its clinical application is limited by challenges such as low survival rates, questionable cell retention, and the complexity of implantation strategies [ 53 ]. Recent studies suggest that the therapeutic effects of MSC transplantation are mediated primarily through cell-cell interactions rather than direct angiogenic differentiation of MSCs [ 54 ]. EXOs, which serve as key mediators of intercellular communication, deliver signaling molecules such as proteins, microRNAs, and nucleic acids, and have been reported to play a critical role in wound repair by promoting blood vessel formation [ 55 ]. It is reported that EXOs derived from stem cells can participate in the regulation of cell function in the process of diabetic wound healing, promote the formation of new blood vessels, and thus accelerate wound healing [ 56 ]. Notably, present study demonstrated that EXOs contain a higher abundance of pro-angiogenic proteins compared to MSCs, providing compelling evidence that EXOs may be more effective than MSCs in promoting angiogenesis. Nevertheless, the clinical application of EXOs remains challenging, primarily due to the limited yield of EXOs extraction and their suboptimal bioavailability. Specifically, less than 1 µg of exosomal protein can be isolated from 1 mL of culture medium [ 57 ]. For in vivo applications, the effective dosage of EXOs ranges from 10 to 500 µg for mice, while clinical trials require approximately 0.5–1.4 × 10¹¹ µg of EXOs [ 58 ]. Furthermore, in situ experiments have shown that EXOs can be taken up by nearly all cell types. Considering the diverse cell populations in subcutaneous tissue, macrophages are identified as the primary cells absorbing EXOs, followed by MSCs. To enhance the efficiency of EXOs utilization, modification techniques have been developed to direct EXOs to specific cellular membrane sites, thereby improving their targeted delivery. The surface of EXOs can be directly engineered through various chemical or physical modifications to induce targeted therapy [ 59 ], and EXOs modification technology was developed in past decades. In recent years, research has found that nucleic acid Apt have the characteristics of high affinity, strong specificity, low immunogenicity, and easy synthesis and modification in vitro . They are oligonucleotide fragments obtained by screening, amplifying and enriching from nucleic acid molecular libraries through SELEX, frequently adopt secondary structures such as hairpins and stem-loops, as well as three-dimensional spatial configurations, binding to their targets via precise molecular conformational matching [ 60 , 61 ]. In the context of skin lesions, Apt-mediated nanozymes have the ability to recognize and bind specifically to bacterial surfaces, facilitating in situ generation of hydroxyl radicals (-OH) for chemokinetic sterilization [ 25 ]. This provides evidence that the nucleic acid structure of the Apt remains intact and undamaged by bacteria present in skin lesions. In this study, an Apt specifically targeting ECs was identified through the SELEX method and applied to modify EXOs. The Apt-functionalized EXOs exhibited targeted delivery to ECs, resulting in enhanced endocytosis by ECs. This increased uptake significantly improved angiogenic capacity and facilitated the formation of CD31⁺EMCN⁺ blood vessels. Moreover, this modification approach was found to be more straightforward and efficient compared to previously reported methods, with a reduced risk of introducing organic compounds, thereby offering a greener and safer strategy for targeted angiogenesis [ 62 ]. Future research will focus on further advancing endothelial targeting strategies by incorporating highly specific targeting molecules, such as P8RI peptides [ 63 ]. Another challenge in the application of EXOs in wound healing is their rapid clearance and short survival time. The hydrogel based on natural polysaccharide is considered to be an ideal dressing for diabetes wounds. Meanwhile, quite a lot studies have shown that hydrogels can encapsulate EXOs, form scaffolds and serve as drug carriers to reduce the degradation rate [ 64 ]. HA is a non-sulfated glycosaminoglycan that is a major component of the ECM in skin cells. Due to its excellent biocompatibility, biodegradability, and hydrophilicity, HA has been used in the production of various wound dressings [ 65 ]. In this study, we encapsulate EXOs with HA to prevent their sudden release in large quantities. Consistent with findings from other studies, Apt-EXOs-HA demonstrated the capability to promote wound healing in normal skin. Remarkably, Apt-EXOs-HA also successfully stimulated vascular regrowth within the vasculature-inhibiting microenvironment associated with type 1 diabetes, indicating that the spontaneous release of EXOs may contribute to the amelioration of the T1D microenvironment. The relationship between insulin and angiogenesis is well-established [ 66 , 67 ], Given the insulin-deficient and more hostile microenvironment in T1D compared to the reduced, yet insulin-present, environment in T2D, our study focuses on addressing this distinct and harsher condition. We successfully enhance wound healing in this challenging context by optimizing EXOs to augment their angiogenic capacity and facilitate the formation of CD31⁺EMCN⁺ vessels. To further elucidate the mechanisms underlying EXO-mediated improvement of the T1D microenvironment, a detailed analysis of the protein composition of EXOs should be conducted. In the present study, we established a well-characterized T1D full-thickness cutaneous wound model by precisely controlling wound creation using ophthalmic scissors, with particular attention to maintaining consistent wound size and depth across all experimental animals. While our model recapitulates several key pathophysiological features of impaired diabetic wound healing, including persistent hyperglycemia-induced tissue damage, dysregulated inflammatory responses, impaired angiogenesis, abnormal extracellular matrix deposition, and delayed epithelialization, all of which are mediated through conserved molecular pathways involving oxidative stress, growth factor dysregulation, and cellular apoptosis. These shared pathological mechanisms, combined with the model's reproducibility and experimental tractability, make it a valuable and widely accepted tool for preliminary investigations of diabetic wound healing mechanisms and therapeutic interventions. While the STZ-induced diabetic mouse model offers advantages including controlled genetic background, standardized wound characteristics, and well-defined experimental parameters that facilitate mechanistic and functional studies, it cannot fully replicate the complex multifactorial nature of human diabetic wounds, which involve variable etiologies (such as neuropathy, microangiopathy, and recurrent infections), diverse wound characteristics, and significant interindividual variability influenced by factors including disease duration, comorbidities, and lifestyle factors [ 68 ]. These findings will ultimately require validation in more complex systems before clinical translation. Conclusions Our study provides evidence suggesting that impaired angiogenesis, including a reduction in CD31⁺EMCN⁺ vessels, may contribute to delayed wound healing in T1D. These findings highlight CD31⁺EMCN⁺ vessels as a potential therapeutic target worthy of further investigation. Apt-EXOs were prepared by conjugating the aptamer to the surface of exosomes via amide condensation. Our in vitro experiments demonstrated that Apt-EXOs significantly enhanced the intake, migration and tube formation capabilities of vascular endothelial cells. For in vivo evaluation, we established models of normal mice and STZ-induced diabetic mice, and the results revealed that Apt-EXOs markedly promoted angiogenesis, wound healing and collagen deposition. The application of modified EXOs demonstrated the capacity to improve the T1D skin microenvironment and promote wound healing by enhancing angiogenesis. These findings offer a robust experimental foundation and a promising therapeutic strategy for addressing T1D skin wound healing. Abbreviations ANX annexin Apt aptamers CAV1 caveolin-1 DM diabetes mellitus DLS dynamic light scattering ECs endothelial cells ECM extracellular matrix EGFR epidermal growth factor receptor EMCN endomucin EXOs exosomes GO gene ontology HA hyaluronic acid JAK Janus kinase KEGG Kyoto Encyclopedia of Genes and Genomes LC-MS liquid chromatography-mass spectrometry LC-MS/MS liquid chromatography-tandem mass spectrometry MSCs mesenchymal stem cells SELEX evolution of ligands by exponential enrichment SEM scanning electron microscope STZ streptozotocin T1D type 1 diabetes T2D type 2 diabetes TEM transmission electron microscopy TGF transforming growth factor UC-MSCs umbilical cord-MSCs Declarations Ethics approval and consent to participate MSCs were isolated from human umbilical cords, which were collected immediately after delivery from donors at Xi'an No. 4 Hospital following the acquisition of written informed consent. Ethics approval was obtained from the Ethics Committee of Xi’an Fourth Hospital (Reference No. 20190012; Approval Project Title: "Study on the Correlation of Umbilical Cord Mesenchymal Stem Cells in Gestational Diabetes and Infant Metabolic Abnormalities"; Date of Approval: Aug 7, 2019). The laboratory animals were handled in accordance with the "Guidelines for the Care and Use of Laboratory Animals" and the "Animal Welfare Act in China". The animal experiments were approved by the Medical and Laboratory Animal Ethics Committee of Northwestern Polytechnical University (Reference No. 202501153; Approval Project Title: "Application of Nucleic Acid-Based Novel Vesicular Biomaterials in Skin Regeneration"; Date of Approval: May 6, 2025). Consent for publication Not applicable. Availability of data and materials Information required to reanalyze the data reported in this work is available from the corresponding authors upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work is supported by grants from the National Key Research and Development Program of China (2022YFA1104400), the National Natural Science Foundation of China (82301028, 82401201, 82371020, 82100985, 32101096 and 82170988), the Project of State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration (2024MS04, 2024KA01), the Shaanxi Provincial Health Research and Innovation Platform Construction Plan (2024PT-04), the Young Science and Technology Rising Star Project of Shaanxi Province (2024ZC-KJXX-122), the Project of General Hospital of Eastern Theater Command (22LCZLXJS12), the China Postdoctoral Science Foundation (BX20230485 and 2021M693954) and the "Rapid Response" Research projects (2023KXKT017 and 2023KXKT090). Authors' contributions N.Z., Z.-Z.G. and S.-F.B. contributed equally to this work. Conceptualization: B.-D.S., C.-H.H., and C.-X.Z.; Methodology: N.Z., Z.-Z.G., S.-F.B., Y.-F.G., P.L., Y-H.J., X-Y. Q., Y.S., and M.Y.; Investigation: N.Z., Z.-Z.G., S.-F.B., Y.-F.G., J.L., J. C., and H.-K.X.; Visualization: N.Z., Z.-Z.G., S.-F.B., L.-H.B., H.N., and Q.-N. W.; Supervision: B.-D.S., C.-H.H., and C.-X.Z.; Writing—original draft: N.Z., Z.-Z.G. and S.-F.B.; Writing—review & editing: B.-D.S., C.-H.H., and C.-X.Z. Acknowledgements The authors declare that they have not use AI-generated work in this manuscript. References H.S. Kim, X. Sun, J.-H. Lee, H.-W. Kim, X. Fu, K.W. Leong, Advanced drug delivery systems and artificial skin grafts for skin wound healing, Advanced Drug Delivery Reviews 146 (2019) 209-239. J.B. Cole, J.C. Florez, Genetics of diabetes mellitus and diabetes complications, Nature Reviews Nephrology 16(7) (2020) 377-390. J. Ozougwu, K. Obimba, C. Belonwu, C. 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1","display":"","copyAsset":false,"role":"figure","size":1475107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpairment of wound healing and angiogenesis in T1D skin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a)\u003cstrong\u003e \u003c/strong\u003eRepresentative photographs and schematic images of full-thickness cutaneous wounds in control and T1D mice. Scale bar: 1 cm. (b) H\u0026amp;E staining of wound bed samples of control and diabetic mice on day 14 post-wound modeling showing neo-epithelization gap. Scale bar: 1 mm. (c) Masson’s trichrome staining of wound bed samples of control and T1D mice on day 14 post-wound modeling showing collagen deposition. Scale bar: 100 μm. (d) Quantification of wound area of control and T1D mice.\u003cstrong\u003e \u003c/strong\u003e(e-f) Quantification of neo-epithelization gap and collagen index of wounded skin of control and T1D mice.\u003cstrong\u003e \u003c/strong\u003e(g) Immunofluorescent staining of CD31 (green) and Emdomucin (EMCN; red) co-immunostaining and vascularization states of the wounded skin of control and T1D mice on day 7 post-wound modeling. White scale bar: 100 μm, black scale bar: 1 cm. (h-i) Quantification of CD31\u003csup\u003e+\u003c/sup\u003eEMCN\u003csup\u003e+\u003c/sup\u003e vessel and capillary area at the wounded skin of control and T1D mice on day 7 post-wound modeling.\u003cstrong\u003e \u003c/strong\u003e(n=3 per group, data represent mean ± SD. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. Two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/16126e56b6ea72d96e318798.png"},{"id":93759913,"identity":"7e8248f8-ac8a-49a4-ad96-73599ebc4994","added_by":"auto","created_at":"2025-10-17 09:28:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1632852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic analysis of EXOs and its promotion of ECsmigration and angiogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Venn diagram depicting gene distribution detected in RNA-seq. (b) Volcano plot showing significantly upregulated (orange dots) and downregulated (green dots) proteins in EXOs, compared to mesenchymal stem cells (MSCs). (c) Kyoto Encyclopedia of Genes and Genomes enrichment analysis of up-regulated proteins of EXOs over MSCs. (d) Gene ontology (GO) enrichment analysis of significantly upregulated proteins in EXOs. (e) Hierarchical clustering of the proteins involved in the GO terms. (f) Representative images of scratch assay of ECs treated by MSC conditioned medium and EXOs at indicated timepoints. Scale bar: 300 μm. (g) Quantification of migration rate of ECs treated by MSC conditioned medium and EXOs. (h) Representative images of tube formation assay of ECs treated by MSC conditioned medium and EXOs at indicated timepoints. Scale bar: 100 μm. (i) Quantification of formed tube length of ECs treated by MSC conditioned medium and EXOs. (n=3 per group, data represent mean ± SD. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/46829459c87af1b98c0be45b.png"},{"id":93758952,"identity":"ed15bc23-5074-43eb-9437-1c0ccf2245e2","added_by":"auto","created_at":"2025-10-17 09:20:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1463676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of Aptamer (Apt)-EXOs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative fluorescence microscopic image of FAM-labeled Apt (green) internalization by C166, UC-MSCs and C2C12. Scale bar: 100 μm. (b) Quantification of FAM\u003csup\u003e +\u003c/sup\u003e area. (c) Diagram demonstrates how Apt-EXOs to prepare. (d) Agarose gel electrophoresis image of Apt, Apt-PEG-DSPE and Apt-EXOs. (e) Representative image of EXOs and Apt-EXOs morphology observed by transmission electron microscopy. Scale bar: 200 nm. (f) The diameter distribution of EXOs (black) and Apt-EXOs (red) detected by DLS. (g) Representative image of plain HA and HA containing Apt-EXOs monitored by SEM. Scale bar: 1 μm. (h) Western blot analysis of EXOs surface markers CD9, CD63, CD81, COX Ⅳ and LAPM1 in EXOs and Apt-EXOs. (n=3 per group, data represent mean ± SD. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. Two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/d8a2dc8d0d215be1e734ed06.png"},{"id":93758948,"identity":"a9c5da93-d06a-493a-85fb-eb89d08e1d86","added_by":"auto","created_at":"2025-10-17 09:20:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2225872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecific aptamers (Apt) promote the internalization of EXOs into ECs and promote its migration and angiogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative fluorescence microscopic image of PKH26-labeled Apt-EXOs (red) internalization by C166 ECs, UC-MSCs and C2C12 myoblast cells. Scale bar: 100 μm. (b) Representative fluorescence microscopic image of PKH26-labeled EXOs and Apt-EXOs (red) internalization by C166. Scale bar: 100 μm. (c) Representative fluorescence microscopic image of PKH26-labeled Apt-EXOs (red) internalization by C166 pretreated with EIPA, nystatin and chlorpromazine. Scale bar: 100 μm. (d-f) Quantification of PKH26\u003csup\u003e+\u003c/sup\u003e area in Figure 5a-c. (g) Representative images of scratch assay of C166 treated by EXOs and Apt-EXOs at indicated timepoints. Scale bar: 300 μm. (h) Quantification of migration rate of C166 treated by EXOs and Apt-EXOs. (i) Representative images of tube formation assay of C166 treated by EXOs and Apt-EXOs at indicated timepoints. Scale bar: 100 μm. (j) Quantification of formed tube length of C166 treated by EXOs and Apt-EXOs. (n=3 per group, data represent mean ± SD. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/e016599a6e0aff19662cff5d.png"},{"id":93758950,"identity":"15d16125-eaa6-46ae-8209-e7f16b4bcb22","added_by":"auto","created_at":"2025-10-17 09:20:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3516808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhancement of revascularization and alleviation of wound healing in normal skin by Apt-EXOs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative photographs and schematic images of the wounded skin in control, Hyaluronic acid (HA), EXOs and Apt-EXOs groups of normal mice. Scale bar: 1 cm.\u003cstrong\u003e \u003c/strong\u003e(b) H\u0026amp;E staining of wound bed samples in control, HA, EXOs and Apt-EXOs groups on day 7 post-wound modeling showing neo-epithelization gap. Scale bar: 1 mm.\u003cstrong\u003e \u003c/strong\u003e(c) Masson’s trichrome staining of wound bed samples in control, HA, EXOs and Apt-EXOs groups on day 7 post-wound modeling showing collagen deposition. Scale bar: 100 μm.\u003cstrong\u003e \u003c/strong\u003e(d) Quantification of the wound area in control, HA, EXOs and Apt-EXOs groups.\u003cstrong\u003e \u003c/strong\u003e(e-f) Quantification of neo-epithelization gap and collagen index of wounded skin in control, HA, EXOs and Apt-EXOs groups.\u003cstrong\u003e \u003c/strong\u003e(g) Immunofluorescent staining of CD31 (green) and EMCN (red) co-immunostaining and vascularization states of the wounded skin in control, HA, EXOs and Apt-EXOs groups on day 7 post-wound modeling. White scale bar: 100 μm, black scale bar: 1 cm.\u003cstrong\u003e \u003c/strong\u003e(h) Immunofluorescent staining of PCNA (green) of the wounded skin in control, HA, EXOs and Apt-EXOs groups on day 7 post-wound modeling. Scale bar: 100 μm.\u003cstrong\u003e \u003c/strong\u003e(i-k) Quantification of CD31\u003csup\u003e+\u003c/sup\u003eEMCN\u003csup\u003e+\u003c/sup\u003e vessel area, number of capillary area and PCNA\u003csup\u003e+\u003c/sup\u003e area of the wounded skin in control, HA, EXOs and Apt-EXOs groups.\u003cstrong\u003e \u003c/strong\u003e(n=3 per group, data represent mean ± SD. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/e87df0f58f3c212d09950fca.png"},{"id":93759919,"identity":"fc771533-012a-4dc1-a99e-0842a6c8e480","added_by":"auto","created_at":"2025-10-17 09:28:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3365215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApt-EXOs maintains its efficacy in T1D skin lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative photographs and schematic images of the wounded skin in control, HA, EXOs and Apt-EXOs groups of T1D mice. Scale bar: 1 cm.\u003cstrong\u003e \u003c/strong\u003e(b) H\u0026amp;E staining of wound bed samples in control, HA, EXOs and Apt-EXOs groups on day 7 post-wound modeling showing neo-epithelization gap. Scale bar: 1 mm.\u003cstrong\u003e \u003c/strong\u003e(c) Masson’s trichrome staining of wound bed samples in control, HA, EXOs and Apt-EXOs groups on day 7 post-wound modeling showing collagen deposition. Scale bar: 100 μm.\u003cstrong\u003e \u003c/strong\u003e(d) Quantification of the wound area in control, HA, EXOs and Apt-EXOs groups.\u003cstrong\u003e \u003c/strong\u003e(e-f) Quantification of neo-epithelization gap and collagen index of wounded skin in control, HA, EXOs and Apt-EXOs groups.\u003cstrong\u003e \u003c/strong\u003e(g) Immunofluorescent staining of CD31 (green) and EMCN (red) co-immunostaining and vascularization states of the wounded skin in control, HA, EXOs and Apt-EXOs groups on day 7 post-wound modeling. White scale bar: 100 μm, black scale bar: 1 cm.\u003cstrong\u003e \u003c/strong\u003e(h) Immunofluorescent staining of PCNA (green) of the wounded skin in control, HA, EXOs and Apt-EXOs groups on day 7 post-wound modeling. Scale bar: 100 μm.\u003cstrong\u003e \u003c/strong\u003e(i-k) Quantification of CD31\u003csup\u003e+\u003c/sup\u003eEMCN\u003csup\u003e+\u003c/sup\u003e vessel area, number of capillary area and PCNA\u003csup\u003e+\u003c/sup\u003e area of the wounded skin in control, HA, EXOs and Apt-EXOs groups.\u003cstrong\u003e \u003c/strong\u003e(n=3 per group, data represent mean ± SD. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/7d722ed0c77b1e5681967925.png"},{"id":97723904,"identity":"6243707b-b061-4011-a380-930f920fbd3f","added_by":"auto","created_at":"2025-12-08 16:09:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13827374,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/0e2156cf-8a46-4eb5-a525-8d92b0a6506d.pdf"},{"id":93759916,"identity":"fabc5a19-ff12-4cb2-989c-7a6850fa0208","added_by":"auto","created_at":"2025-10-17 09:28:58","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1434857,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/99b3cc90f193f135fc82ef91.xlsx"},{"id":93758960,"identity":"e15e1577-cbfe-4d5a-a6af-5866399290a9","added_by":"auto","created_at":"2025-10-17 09:20:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1377420,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7518048/v1/fa72583d275dcfb62707e306.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Endothelial cell-targeting aptamer-empowered exosomes accelerate wound healing by promoting specialized angiogenesis in type 1 diabetic mice","fulltext":[{"header":"Background","content":"\u003cp\u003eSkin lesions are a common occurrence throughout life and are generally associated with a robust self-healing capacity. However, the rising prevalence of aging, obesity, and diabetes within the global population has imposed a substantial financial burden on healthcare systems worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Diabetes mellitus (DM), a chronic metabolic disorder, is projected to affect approximately 700\u0026nbsp;million adults by 2045 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Type 1 diabetes (T1D), in particular, is distinguished by an absolute deficiency in insulin production resulting from the autoimmune destruction of pancreatic β-cells, which are erroneously attacked by a dysregulated immune system [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This condition is often considered to be associated with more severe skin damage compared to type 2 diabetes (T2D), primarily due to reduced angiogenesis and impaired vascular repair mechanisms, both of which are consequences of autoimmune-mediated damage and the complete loss of endogenous insulin production [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Patients with T1D experience earlier and more severe damage to vascular endothelial cells due to hyperglycemia, leading to impaired skin microvascular formation and structural abnormalities, which easily cause problems such as dry skin and ulcers. Patients with T2D may develop vascular endothelial dysfunction due to factors such as insulin resistance, but abnormal skin angiogenesis usually chronic and occurs later. As a consequence, individuals with T1D often experience delayed wound healing, suboptimal healing outcomes, or chronic wounds that fail to heal entirely [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Clinically, hydrogels are often employed to maintain wound moisture, thereby reducing inflammation and infection. However, their efficacy in fully restoring compromised wound healing remains limited [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, numerous strategies to promote angiogenesis have been developed over the past decades to address this critical challenge with unsatisfied outcomes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMesenchymal stem cell (MSC)-based therapy has been developed and offers a promising therapeutic approach for T2D skin wound repair by promoting specialized CD31\u003csup\u003e+\u003c/sup\u003eEndomucin (EMCN)\u003csup\u003e+\u003c/sup\u003e vessels formation, this therapeutic outcome is achieved through the secretion of vesicles enriched with angiogenic proteins [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Besides, current research indicates that the regenerative capabilities of MSCs are predominantly influenced by their paracrine actions rather than direct transplantation and differentiation. Exosomes (EXOs) secreted by MSCs are considered primary mediators of these paracrine effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These EXOs, a category of extracellular vesicles with diameters ranging from 30 to 150 nm, are known to transport a wide array of proteins, mRNA, and miRNAs [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], have been reported to enhance vascular endothelial cells (ECs) proliferation and migration and to upregulate the expression of molecules intricately associated with angiogenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], suggesting the potential application of MSC-derived EXOs in reconstructing vasculature in diabetic skin. Nonetheless, the non-specific biodistribution of unmodified EXOs \u003cem\u003ein vivo\u003c/em\u003e diminishes their efficacy, necessitating repeated administrations [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo address the limited availability of MSC-derived EXOs, several EXOs modification technologies have been developed to enhance their bioavailability. Aptamers (Apt), synthetic single-stranded oligonucleotides, can be screened, amplified, and enriched \u003cem\u003ein vitro\u003c/em\u003e using systematic evolution of ligands by exponential enrichment (SELEX) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These Apt often adopt secondary structures such as hairpins and stem-loops, as well as three-dimensional configurations, allowing them to bind target molecules with high affinity and specificity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Apt-functionalized drug loaded liposomes have been reported to enhance drug stability, targeting and delivery efficiency [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To further address the weak mechanical properties of EXOs, the combination of hydrogels and hyaluronic acid (HA) are used as scaffold to strength the biocompatibility, biodegradability, water absorption and retention properties of EXOs [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The resulting Apt-EXOs-HA complex prompts investigation into whether the sustained binding of EXOs to ECs can preferentially promote the formation of CD31⁺EMCN⁺ vessels, a vascular subtype associated with regeneration that is reduced in T1D skin [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the current study, we seek to elucidate the role of angiogenesis in T1D in mice and to develop bioengineered EXOs to enhance angiogenesis in diabetic skin. Utilizing liquid chromatography-mass spectrometry (LC-MS) and related vascular endothelial cell experiments (including cell migration and tube formation), we identified a superior angiogenic function of EXOs compared to MSCs. We synthesized an Apt-EXOs-HA complex, employing EC-specific Apt as targeting agents and HA as a scaffold to optimize the mechanical properties of EXOs. We examined the angiogenic effects of Apt-EXOs-HA, with particular emphasis on CD31⁺EMCN⁺ vessel formation, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and analyzed its role in promoting wound healing in T1D skin. Overall, our research aims to clarify the critical role of specialized angiogenesis in T1D wound healing and to advance the application of engineered EXOs in addressing unmet clinical needs in diabetic skin treatment. To our knowledge, this study is the first to demonstrate the therapeutic potential of Apt-equipped EXOs for T1D wound healing.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eEight-week-old female C57BL/6 mice were provided by Laboratory Animal Center of Fourth Military Medical University. All experimental protocols were approved by the Fourth Military Medical University. All animal experiments conducted in this research were performed in accordance with the guidelines of the Fourth Military Medical University Intramural Animal Use and Care Committee and met the NIH guidelines for the care and use of laboratory animals. Animals were randomly assigned to different experimental groups, maintained with good ventilation and a 12 h light / dark cycle, and were kept feeding and drinking before being sacrificed. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eT1D full-layer cutaneous wound modeling\u003c/h3\u003e\n\u003cp\u003eC57BL/6 mice were injected intraperitoneally with 50 mg/kg STZ daily for 5 days to induct DM, mice were defined as T1D when glucose levels were \u0026gt;11.1 mmol/L under fasting conditions. After anesthesia, the dorsal fur of T1D mice was shaved, and full-layer cutaneous wounds with a diameter of 1.0 cm were carefully made using ophthalmic scissors under sterile surgical conditions, then the wound was covered with transparent film dressing.\u003c/p\u003e\u003cp\u003eFollowing diabetic wound induction, all experimental mice were housed in standard individually ventilated cages (IVC). Each cage contained autoclaved bedding that was refreshed every 48 hours to maintain optimal hygiene. The housing environment was strictly maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with 50\u0026thinsp;\u0026plusmn;\u0026thinsp;10% relative humidity under a controlled 12 h light / dark cycle. Postoperative monitoring included daily visual inspection of wounds with photographic documentation at predetermined time points. Any displaced dressings were promptly replaced with sterile alternatives to ensure proper wound protection. Throughout the study period, all animals had continuous access to autoclaved feed and purified drinking water ad libitum. Two distinct anesthetic protocols were employed for different procedures. For in vivo wound imaging: mice were anesthetized using isoflurane inhalation to ensure immobility for consistent and high-quality photography. Anesthesia was induced by placing mice in an induction chamber with a flow of 2% isoflurane in oxygen. Once anesthetized, the mice were transferred to a nose cone for the duration of the imaging procedure, where anesthesia was maintained with 1.5% isoflurane. For terminal procedures and tissue collection: at the experimental endpoint, mice were deeply anesthetized via intraperitoneal injection of 100 mg/kg sodium pentobarbital. This protocol ensures a surgical plane of anesthesia, leading to euthanasia.\u003c/p\u003e\n\u003ch3\u003eCutaneous wound healing assessment\u003c/h3\u003e\n\u003cp\u003eWound bed sizes during approximately 2-week experimental periods were observed daily and imaged at indicated time points by digital camera and data were calculated as follows: (actual wound area/original wound area) \u0026times;100%. Following euthanasia by intraperitoneal injection of a barbiturate drug, the wound surface and the surrounding skin within a 0.5 cm radius were collected for further analysis.\u003c/p\u003e\n\u003ch3\u003eH\u0026E and Masson’s trichrome staining\u003c/h3\u003e\n\u003cp\u003eBiopsies of the original wound area and the remaining wound beds were sampled on day 7 or 14 after administration and fixed with 4% paraformaldehyde for 24 h, followed by dehydration and embedded in paraffin. The samples were sectioned perpendicularly to the wound surface in 4 \u0026micro;m consecutive sections by a microtome and were prepared for H\u0026amp;E and Masson\u0026rsquo;s trichrome staining. H\u0026amp;E staining was conducted as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For analysis of collagen deposition, Masson\u0026rsquo;s trichrome staining was performed with a commercial kit (BaSO, China) according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eVascularization assessment\u003c/h3\u003e\n\u003cp\u003eBiopsy samples of the original and remaining wounds were placed in culture dishes, inwardly facing down. Vascularization states of each specimen were assessed under incandescent illumination and photographed with a digital camera (Leica, Germany).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescent assay\u003c/h2\u003e\u003cp\u003eThe original and remaining wounds on day 7 after administration were biopsied, fixed with 4% paraformaldehyde for 4\u0026thinsp;~\u0026thinsp;6 h and dehydrated with 30% sucrose for 24\u0026thinsp;~\u0026thinsp;48 h. 20 \u0026micro;m cryosections were permeabilized by Triton X-100 (Sigma-Aldrich, USA) for 20 min at room temperature, blocked with goat serum (Boster, China) for 1 h at 4\u0026deg;C and then stained with primary antibodies overnight at 4\u0026deg;C as follows: anti-mouse Alexa Fluor\u0026trade; 488-conjugated anti-CD31 (FAB3628G, R\u0026amp;D Systems, USA; diluted 1:100) co-stained with anti-mouse Endomucin (sc-65495, Santa Cruz Biotechnology, USA; diluted 1:100), anti-mouse PCNA (ab92552, Abcam, USA; diluted 1:200). Whereafter incubated with the following fluorescence-conjugated secondary antibodies at room temperature for 1 h: Cy3-conjugated goat anti-rat IgG (33308ES60, YEASEN, China; diluted 1:200) or Fluor\u0026trade; 488 Goat Anti-Rabbit lgG (33106ES60, YEASEN, China; diluted 1:200). The sections subsequently mounted with antifade mounting medium containing DAPI. Quantification of CD31\u003csup\u003e+\u003c/sup\u003eEMCN\u003csup\u003e+\u003c/sup\u003e vessels and PCNA\u003csup\u003e+\u003c/sup\u003e cells was carried out with ImageJ software (NIH, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eMSCs were isolated from umbilical cords of women who gave birth in Xi'an Fourth Military Medical University Xijing Hospital. All subjects provided informed consent. It was approved by the Ethics Committee of Xi'an Fourth Military Medical University. MSCs were isolated by tissue block culture attachment method [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Simply put, the umbilical veins and arteries and their surrounding Wharton jelly are separated from the stroma, the Wharton jelly mesenchymal tissue is cut into 2\u0026ndash;3 mm\u003csup\u003e3\u003c/sup\u003e chunks and transferred to a petri dish. The MSCs were cultured with alpha-minimum essential medium (\u0026#120572;-MEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Sijiqing, China) and 1% penicillin/streptomycin (Invitrogen, USA) in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at room temperature. The C166 and C2C12 cells were cultured with dulbecco's modified eagle medium (DMEM, Invitrogen, USA) with 10% FBS and 1% penicillin-streptomycin. The medium was refreshed every 3 days, and the cells were passaged once they were 80%-90% confluence.\u003c/p\u003e\n\u003ch3\u003eEXOs isolation\u003c/h3\u003e\n\u003cp\u003eFor EXOs isolation, we initiated each batch with 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells (from a T75 culture flask). When the cells reached 80% confluence, the medium was substituted with fresh inducing medium (α-MEM basal medium) without FBS. After 48 h, the supernatant was collected. Initial clarification at 300 g for 10 min to remove cells, followed by 2,000 g for 20 min and 16,000 g for 30 min to eliminate cellular debris and larger extracellular vesicles. Finally, we used a Beckman Coulter Optima XPN-100 ultracentrifuge equipped with a 32 Ti fixed-angle rotor, and centrifuged at 150,000 g for 90 minutes to achieve the final EXO precipitation. We maintained rigorous temperature control (4℃) throughout all centrifugation steps to preserve vesicle integrity. Following ultracentrifugation, the pelleted EXOs were resuspended in 200 \u0026micro;L of 0.22 \u0026micro;m-filtered phosphate-buffered saline (PBS, pH 7.4), a volume optimized through preliminary experiments to ensure appropriate EXO concentrations for downstream analyses. To achieve the standardization of EXOs, the BCA protein assay method (TianGen, China) was employed. The morphology of EXOs was assessed through transmission electron microscopy (TEM, Thermo fisher, USA). The size distribution of EXOs was quantitated using dynamic light scattering (DLS) with a Zetasizer (Malvern, UK). To ensure measurement accuracy, the EXO samples were diluted in 0.22 \u0026micro;m-filtered PBS buffer (pH 7.4). The expression of marker proteins was examined by western blot analysis. Specifically, EXOs lysates were prepared by cell lysis buffer (Beyotime, China) containing protease inhibitor (Roche, Switzerland), the extracted proteins were separated by SDS-PAGE electrophoresis, transferred onto polyvinylidene fluoride membranes (Roche, Switzerland) and blocked in 5% BSA (MP Biomedicals, USA) at room temperature for 2 h. The membranes were incubated with the following primary antibodies: CD9 (ab236630, Abcam, USA, 1:1000), CD63 (ab271286, Abcam, USA, 1:1000), CD81 (sc-166029, Santa Cruz Biotechnology, USA, diluted 1:500), COX Ⅳ (4844, CST, USA, 1:1000) and LAMP 1 (ab24170, Abcam, USA, 1:1000) and then incubated with peroxidase-conjugated secondary antibody: Goat Anti Mouse IgG (RS0001, Immunoway, USA, 1:10000) or Goat Anti Rabbit IgG (RS0002, Immunoway, USA, 1:10000).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eProteomic Analysis\u003c/h2\u003e\u003cp\u003eThe protein lysates of MSCs and EXOs were prepared and analyzed by LC-MS/MS on Orbitrap Exploris\u0026trade; 480 mass spectrometer with a NanoSpray III ion source. The original data was analyzed using the Proteome Discoverer system (v2.4.1.15). Proteins were identified by comparison with the Uniport database, and the error finding rate was set to 0.01 for both peptides and proteins. Proteins are quantified using MaxQuant's default parameters. Among the identified proteins, differential expression proteins (DEPs) were included (fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and further functional analysis was conducted based on GO and KEGG databases.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eValidation of Apt affinity\u003c/h2\u003e\u003cp\u003eApt that target ECs (5'-CCC ACG TCT GCG CTT AGC TCC TGG GCC TGG ATG GGC-3') was synthesized by Sangon Biotech Co., Ltd. (China). For the Apt affinity assay, MSCs, C166 and C2C12 cells were seeded at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well onto a 24-well plate with cell climbing slides. Then, carboxyfluorescein-labeled Apt (Apt-FAM) with a concentration of 10 \u0026micro;M were incubated with cells for 3 h. After rinsed with PBS, cells were successively fixed, mounted with antifade mounting medium containing DAPI (Beyotime, China) and imaged by a fluorescence microscope (Olympus, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of Apt-EXOs-HA\u003c/h2\u003e\u003cp\u003eApt-PEG-DSPE was first prepared by an amide condensation reaction between DSPE-PEG-COOH and 5\u0026rsquo;-NH\u003csub\u003e2\u003c/sub\u003e-modified Apt as follows: DSPE-PEG-COOH (Avanti, USA), NHS (Macklin, China) and EDC (Macklin, China) were separately dissolved in ddH\u003csub\u003e2\u003c/sub\u003eO, adjusted the solvent pH to 5\u0026thinsp;~\u0026thinsp;6 and stirring at room temperature. After dialysed several times to remove unreacted NHS and EDC, 5\u0026rsquo;-NH\u003csub\u003e2\u003c/sub\u003e-modified Apt was added and stirred for another 12 h. Then Apt-PEG-DSPE and EXOs were stirred at room temperature for 1\u0026thinsp;~\u0026thinsp;2 h in pyrocarbonate-treated PBS, unconjugated Apt-PEG-DSPE was removed by ultrafiltered several times to prepare Apt-modified EXOs suspension, namely Apt-EXOs. Prior to use, the HA (Macklin, China) was fully dissolved in PBS (pH adjusted to ~\u0026thinsp;7.0) and subsequently mixed with either EXOs or Apt-EXOs to form the composite gel. The final concentrations in the prepared gel were 50 mg/mL for HA and 1.25 mg/mL for EXOs or Apt-EXOs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eApt-EXOs Characterization\u003c/h2\u003e\u003cp\u003eTEM, DLS and western blot analysis of Apt-EXOs were performed. Moreover, the preparation of Apt-EXOs was evaluated by gel retardation assay. In short, Apt, DSPE-PEG-Apt and Apt-EXOs were mixed with the sample buffer respectively and adjust the voltage of agar-electrophoresis at 30\u0026ndash;40 V/cm for 90 min, followed by imaging using the gel imaging system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCellular internalization of Apt-EXOs\u003c/h2\u003e\u003cp\u003eThe PKH26 labeling of purified EXOs was performed by resuspending the EXOs in Diluent C buffer, followed by mixing with freshly prepared PKH26 dye solution (dilute the PKH26 dye in diluent C). After a 5-minute incubation at room temperature in the dark, the reaction was terminated with 1/2 volume of EXO-depleted FBS, and unbound dye was removed through PBS washing and ultracentrifugation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. For the EXOs internalization experiments, C166 cells were seeded at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well onto a 24-well plate with cell climbing slides. Then, 100 \u0026micro;g/mL PKH26 labeled Apt-EXOs was incubated with C166 cells for 3 h at room temperature. Subsequently, the cells were fixed with 4% paraformaldehyde and mounted with antifade mounting medium containing DAPI. The signals of Apt-EXOs uptake were observed using a fluorescence microscope. PKH26 labeled EXOs treated C166 cells were used as a negative control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eIn vitro scratch Assay\u003c/h2\u003e\u003cp\u003eC166 cells were seeded at a density of 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well onto a 6-well plate and the scratch assay was performed to evaluate their migration ability, as previously reported. In brief, when the cells reached 90% confluence, sterile 1 ml pipette tips were used to create scratches on the plates by scraping cells away. After washing with PBS, the medium was changed to one without FBS. MSC conditioned medium, 100 \u0026micro;g/mL EXOs or Apt-EXOs and added severally, and three locations were randomly selected for imaging 24 h later with an inverted microscope (Leica, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eIn vitro tube formation assay\u003c/h2\u003e\u003cp\u003eMSC conditioned medium, 100 \u0026micro;g/mL EXOs or Apt-EXOs were premixed with C166 cells and seeded at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well onto the 96-well plate coated with Matrigel (Corning, USA) and incubated for 6 h. The enclosed networks of complete tubes were digitally imaged. For quantification of the Matrigel tube formation assay, we utilized ImageJ software (NIH, USA) with the Angiogenesis Analyzer plugin to measure tube length.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (two-tailed) was used to assess statistical significance. Values of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Statistical and graphical analysis using GraphPad Prism 9.0 (GraphPad Software, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eImpaired angiogenesis is associated with delayed skin wound healing in mice with T1D\u003c/h2\u003e\u003cp\u003eT1D is associated with severe microvascular complications attributed to absolute insulin deficiency, a dysregulated immune system, and abnormal tissue metabolism, which together contribute to impaired wound healing [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. At the beginning of this study, we examined changes in angiogenesis, with a particular focus on CD31⁺EMCN⁺ vessels, during wound healing in a T1D context. For this purpose, we employed a high-dose streptozotocin (STZ)-induced T1D mouse model and established a wound healing framework, systematically monitoring wound area progression at defined time points. The results indicated a marked inhibition of wound closure rates in T1D mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b), which correlated with decreased collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Parameters such as wound bed size, neo-epithelialization gap, and collagen index were quantified and statistically analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f). Furthermore, the distribution of CD31⁺EMCN⁺ vessels and capillaries was significantly compromised (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), and statistical analysis of the CD31⁺EMCN⁺ area and capillary area percentages was conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-i). These findings indicate that impaired angiogenesis, along with the presence of CD31⁺EMCN⁺ vessels, contributes to delayed wound healing and diminished collagen deposition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eFurther suppression of skin angiogenesis in T1D mice exacerbates impaired wound healing\u003c/h2\u003e\u003cp\u003eThe impairment of angiogenesis in T1D skin wound healing has been established. To further confirm the relationship between angiogenesis and wound healing in T1D mice, we administered intraperitoneal injections of DAPT, a γ-secretase inhibitor known to suppress Notch signaling [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], to inhibit angiogenesis and observed the wound healing process. On day 7, the distribution of CD31⁺EMCN⁺ vessels and capillaries was reduced, though not entirely abolished under our experimental conditions (SI. 1a-c). Following significant inhibition of angiogenesis, the wound healing area was markedly diminished, particularly after day 7 (SI. 1d). Histological analysis using H\u0026amp;E and Masson staining revealed an increased epithelial gap in the DAPT-treated group and decreased collagen deposition (SI. 1e, f). Wound bed size, neo-epithelialization gap, and collagen index were quantified and statistically analyzed (SI. 1g-i). Collectively, these findings demonstrate that vascular structures are diminished in T1D skin and highlight the possibility of CD31⁺EMCN⁺ vessels in promoting the healing of T1D skin wounds.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eEXOs exhibit superior angiogenesis-stimulating potential compared to MSCs\u003c/h2\u003e\u003cp\u003eAfter establishing the critical role of angiogenesis in wound healing, we explored the use of MSC-based therapy to stimulate angiogenesis. Previous studies have reported that MSCs, particularly MSC-derived EXOs, are among the most effective agents for promoting angiogenesis and enhancing skin wound healing [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To identify more effective strategies for promoting angiogenesis, we conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to evaluate angiogenesis-associated proteins present in EXOs and MSCs (Table. S1). EXOs and MSCs differ in protein signature, showing 4568 overlapping proteins and 55 proteins specifically expressed in EXOs in the Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Among co-expressed proteins, 1188 proteins were upregulated in EXOs compared to the MSCs counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). As for the potential functions of differentially expressed proteins, the enrichment analysis in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed a number of related terms, among which \"cell adhesion molecules\" stood out (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). It's worth noting that gene ontology (GO) enrichment analysis of significantly upregulated proteins revealed an abundance of several terms related to angiogenesis in EXOs, including \"sprouting angiogenesis\", \"angiogenesis\", \"regulation of sprouting angiogenesis\", \"regulation of angiogenesis\" and \"positive regulation of angiogenesis\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). According to functional analysis, multiple individual proteins involved in angiogenesis were upregulated in EXOs. For example, we found significant upregulation of several proteins in EXOs, namely Epidermal growth factor receptor (EGFR), Notch 2, Caveolin-1 (CAV1), Transforming growth factor (TGF)-β1, Janus kinase (JAK), Annexin A1 (ANXA1) and ANXA3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), which have been reported to be involved in germination, dilation and formation of blood vessels [\u003cspan additionalcitationids=\"CR38 CR39 CR40 CR41 CR42\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Notch activation directs tip-derived ECs into developing arteries, and further, Dll4-Notch signaling combines budding angiogenesis with arterial formation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Endothelial CAV1 plays an important role in endothelium-dependent hyperpolarization mediated vasodilation and ischemic angiogenesis by modifying post-translational proteins through nitro oxidative stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. TGF-β1 induces the production of vascular endothelial growth factor by TGF-β receptor, Smad2/3, PI3K/AKT and JNK1/2 related signaling pathways and further promoted \u003cem\u003ein vitro\u003c/em\u003e angiogenesis of ECs and \u003cem\u003ein vitro\u003c/em\u003e vessel germination of the aortic ring [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Consistent with the results of LC-MS/MS analysis, experiments related to vascular endothelial cells also demonstrated that the EXOs exhibit stronger pro-angiogenic activity than MSC-conditioned medium, manifested by enhanced cell migration and tube formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-i). Overall, these data suggest that EXOs exhibit superior functionality in promoting angiogenesis compared to MSCs.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eCharacterization of EC-targeting EXOs\u003c/h2\u003e\u003cp\u003eThe superior angiogenic potency of EXOs has been demonstrated. However, EXOs alone still present several limitations, including restricted availability and suboptimal bioavailability. To address these challenges, Apt exhibiting strong affinity, high specificity, low immunogenicity, and ease of modification were selected to specifically target ECs [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. We first identified the synthesized Apt by mass spectrometry, and observed that the molecular weight was consistent with the expectation (SI. 2a). Particularly, the results of specific targeting ability showed that the fluorescence area of FAM-labeled Apt in C166 ECs was tens of times that of UC-MSCs and C2C12 myoblast cells in the control group, confirming its high selectivity in binding to ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). In addition, we performed migration and tube formation assay on C166 cells to evaluate the pro-angiogenic ability of Apt. The results demonstrated that Apt had no effect on migration and tube formation of C166 cells (SI. 2b-e). Meanwhile, we applied Apt to a mouse model of wound healing and found that the formation of CD31\u003csup\u003e+\u003c/sup\u003eEMCN\u003csup\u003e+\u003c/sup\u003e vessels and capillary in the Apt group did not change compared to the control (SI. 2f-h). Based on the above characteristics of Apt, we modify the EXOs surface with the Apt targeting ECs. To be specific, Apt-PEG-DSPE was prepared by coupling the 5'-NH\u003csub\u003e2\u003c/sub\u003e-modified Apt with DSPE-PEG-COOH through an amide condensation reaction (SI. 3a), and then co-incubated with EXOs to obtain Apt-functionalized EXOs Apt-EXOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The successful conjugation of the DSPE-PEG-Apt and Apt-EXOs were evaluated by gel retardation assay, the results illustrated that DSPE-PEG-Apt and Apt-EXOs were successfully synthesized since the molecular weight of DSPE-PEG-Apt and complex increased in different degrees, showing electrophoretic bands at diverse locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Furthermore, the cup-shaped EXOs with clear membrane structure about 100 nm in diameter, and the particle size of Apt-EXOs is mainly distributed around 300 nm, which is a homogeneous bilayer spherical nanoparticles surrounded by a layer of black nuclea-like material (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f). The scanning electron microscope (SEM) of plain HA and HA containing Apt-EXOs demonstrated similar morphology, although slight alterations in the morphology of the adhered exosomes occurred during sample preparation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Western blot analysis revealed the enrichment of typical EXOs markers, such as CD9, CD63 and CD81 in Apt-EXOs, but lacked the mitochondrial marker COV Ⅳ and the lysosomal marker LAMP1, indicating that EXOs were successfully collected with a high purity, and were not damaged during the reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Taken together, Apt functionalized EXOs targeting ECs were successfully produced.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eApt-conjugated EXOs exhibit preferred internalization efficiency and enhanced pro-angiogenic activity in vitro\u003c/h2\u003e\u003cp\u003eAfter the successful synthesis of Apt-EXOs, we proceeded to analyze their bioavailability, focusing on ECs internalization and pro-angiogenic activity. We first labeled EXOs with PKH26 to examine whether the conjugated Apt enable EXOs to specifically target ECs. As expected, massive PKH26 fluorescence signals were detected in C166 cells incubated with Apt-EXOs, while slight were detected in UC-MSCs and C2C12 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Moreover, PKH26-labeled Apt-EXOs was incubated with C166 cells, and PKH26-labeled EXOs served as a negative control, we observed that the PKH26 fluorescence signal in Apt-EXOs treated C166 cells was much greater than that in the EXOs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In terms of the cellular mechanism through which Apt-EXOs is taken up by C166 cells, we pretreat C166 cells with EIPA (a micropinocytosis inhibitor), nystatin (a caveolae-mediated endocytosis inhibitor) and chlorpromazine (a clathrin-mediated endocytosis inhibitor) respectively, then they are processed with PKH26-labeled Apt-EXOs. Confocal images displayed that Apt-EXOs can be taken up through all of the three uptake pathways, especially by micropinocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). To confirm the cellular internalization of EXOs, the fluorescence intensity of PKH26 was measured and subjected to statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f). The property of Apt-EXOs in promoting angiogenic capacity was also assessed. Compared with the control group, C166 cells exposed to EXOs and Apt-EXOs exhibited greater migration and tube formation ability, especially in the Apt-EXOs group, suggesting that Apt functionalization promotes the pro-angiogenic capacity of EXOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), further quantification confirmed the above observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Additionally, migration and tube formation assays were quantified and statistically analyzed to evaluate their pro-angiogenic potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei-j). Overall, these findings demonstrate the enhanced internalization of Apt-EXOs by ECs and are anticipated to promote angiogenesis and accelerate wound healing.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eApt-EXOs promote vascular regeneration and accelerate wound healing in normal skin\u003c/h2\u003e\u003cp\u003eFollowing the evaluation of the angiogenic capacity of Apt-EXOs \u003cem\u003ein vitro\u003c/em\u003e, we examined their vascular regeneration potential \u003cem\u003ein vivo\u003c/em\u003e. In this process, HA was employed as a carrier for EXOs and Apt-EXOs to enable controlled release, thereby preventing sudden release and minimizing liquid leakage. Mice were administered EXOs or Apt-EXOs in a standardized volume of 200 \u0026micro;L HA at a dose of 150 \u0026micro;g. Compared with the control, Apt-EXOs-HA treatment significantly accelerated the wound closure rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Histological analysis of the dissected skin tissues \u003cem\u003evia\u003c/em\u003e H\u0026amp;E and Masson\u0026rsquo;s staining further showed that Apt-EXOs-HA treatment remarkably enhanced skin healing, as shown by reduced epithelial distance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and increased collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The wound closure rate, neo-epithelialization, and collagen deposition were quantified and subjected to statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-f). Notably, after 7 days of treatment, the immunofluorescence co-staining of CD31 and EMCN showed that Apt-EXOs-HA significantly increased CD31\u003csup\u003e+\u003c/sup\u003eEMCN\u003csup\u003e+\u003c/sup\u003e vessels as well as the capillary growth in injury sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg), the number of PCNA\u003csup\u003e+\u003c/sup\u003e proliferative cells were also remarkably increased of the Apt-EXOs-HA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). The percentage of CD31\u003csup\u003e+\u003c/sup\u003eEMCN\u003csup\u003e+\u003c/sup\u003e area, capillary area, and PCNA\u003csup\u003e+\u003c/sup\u003e area was measured and subjected to statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei-k). Collectively, these findings indicate that Apt-EXOs-HA effectively enhances skin wound healing by promoting angiogenesis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eApt-EXOs preserve their biological functionality in T1D skin lesions\u003c/h2\u003e\u003cp\u003eMicrovascular disease is a common complication of diabetes, where angiogenesis inhibitors, hyperglycemia, and various abnormal factors impede vascular regrowth. Consequently, promoting vascular regeneration in T1D skin wound healing remains a significant challenge [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Given the excellent performance of Apt-EXOs in the healing of normal mouse skin wounds, we further investigated its potential therapeutic effects of Apt-EXOs by establishing T1D full-layer cutaneous wounds. Each diabetic mouse was also administered a dose of 150 \u0026micro;g of EXOs or Apt-EXOs. Similarly, Apt-EXOs-HA treatment substantially accelerated cutaneous wound healing, while EXOs-HA treatment had a relatively slight effect and HA had almost no effect on day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). For characteristics of the wound lesion, H\u0026amp;E staining of the wound bed samples at day 14 demonstrated that faster epithelialization in Apt-EXOs-HA group while slower epithelialization in EXOs-HA, HA and control groups, indicating that Apt-EXOs-HA had excellent ability to promote granulation tissue maturation and epidermal regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Masson\u0026rsquo;s trichrome staining showed a superior collagen deposition with certain direction and thicker bundle for Apt-EXOs-HA group versus that of EXOs-HA, HA and control groups with short collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The wound closure rate, neo-epithelialization, and collagen deposition were quantified and subjected to statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-f). In terms of promoting angiogenesis, immunofluorescence co-staining of CD31 and EMCN, as well as the changes of capillaries strongly demonstrated the excellent angiogenesis promoting ability of Apt-EXOs-HA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). The number of proliferative cells in Apt-EXOs-HA group was also significantly increased by immunofluorescence assay of PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). The percentage of CD31\u003csup\u003e+\u003c/sup\u003eEMCN\u003csup\u003e+\u003c/sup\u003e area, capillary area, and PCNA\u003csup\u003e+\u003c/sup\u003e area was measured and subjected to statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei-k). Collectively, these findings suggest that Apt-EXOs-HA effectively contribute to the improvement of a pro-regenerative microenvironment, thereby promoting skin wound healing through the enhancement of angiogenesis in the context of T1D.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDiabetic wounds are a prevalent complication of diabetes, characterized by impaired angiogenesis. Among the vascular subtypes essential for regeneration and wound healing, CD31⁺EMCN⁺ vessels play a pivotal role. However, in diabetes, vascular regrowth is hindered by various factors, including hyperglycemia, neuropathy, and abnormal extracellular matrix (ECM) deposition [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Hyperglycemia exacerbates oxidative stress and inflammation, while neuropathy disrupts angiogenic processes by impairing sensory perception and motor control, ultimately reducing oxygen supply and further impeding angiogenesis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Additionally, the altered ECM composition in diabetes interferes with the critical signaling and molecular interactions required for proper angiogenic progression [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Under normal physiological conditions, angiogenesis is initiated by ECs forming immature microvessels, which subsequently mature through pericyte-mediated signaling to ensure vascular bed stability and integrity [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In diabetes, this process is disrupted, with delayed recruitment of pericytes leading to extravascular leakage and vascular edema [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Even after wound closure is achieved, diabetic wounds often fail to restore adequate pericyte coverage, compromising the establishment of a well-perfused vascular network, a fundamental requirement for effective wound healing. In this study, our analysis of vascular distribution in T1D wounds revealed a marked reduction in CD31⁺EMCN⁺ vessels, a critical factor contributing to delayed wound healing in diabetes. These findings underscore the importance of elucidating the biological mechanisms underlying CD31⁺EMCN⁺ vessel formation and assessing strategies to promote their regeneration, which could provide valuable insights into enhancing tissue repair in diabetic wounds.\u003c/p\u003e\u003cp\u003eTo the best of our knowledge, no existing medications specifically promote angiogenesis or the formation of CD31⁺EMCN⁺ vessels for the treatment of diabetic wound healing. The application of MSC aggregates has been reported to stimulate wound healing by promoting angiogenesis in T2D, but its clinical application is limited by challenges such as low survival rates, questionable cell retention, and the complexity of implantation strategies [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Recent studies suggest that the therapeutic effects of MSC transplantation are mediated primarily through cell-cell interactions rather than direct angiogenic differentiation of MSCs [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. EXOs, which serve as key mediators of intercellular communication, deliver signaling molecules such as proteins, microRNAs, and nucleic acids, and have been reported to play a critical role in wound repair by promoting blood vessel formation [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. It is reported that EXOs derived from stem cells can participate in the regulation of cell function in the process of diabetic wound healing, promote the formation of new blood vessels, and thus accelerate wound healing [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Notably, present study demonstrated that EXOs contain a higher abundance of pro-angiogenic proteins compared to MSCs, providing compelling evidence that EXOs may be more effective than MSCs in promoting angiogenesis. Nevertheless, the clinical application of EXOs remains challenging, primarily due to the limited yield of EXOs extraction and their suboptimal bioavailability. Specifically, less than 1 \u0026micro;g of exosomal protein can be isolated from 1 mL of culture medium [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. For \u003cem\u003ein vivo\u003c/em\u003e applications, the effective dosage of EXOs ranges from 10 to 500 \u0026micro;g for mice, while clinical trials require approximately 0.5\u0026ndash;1.4 \u0026times; 10\u0026sup1;\u0026sup1; \u0026micro;g of EXOs [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Furthermore, in situ experiments have shown that EXOs can be taken up by nearly all cell types. Considering the diverse cell populations in subcutaneous tissue, macrophages are identified as the primary cells absorbing EXOs, followed by MSCs. To enhance the efficiency of EXOs utilization, modification techniques have been developed to direct EXOs to specific cellular membrane sites, thereby improving their targeted delivery.\u003c/p\u003e\u003cp\u003eThe surface of EXOs can be directly engineered through various chemical or physical modifications to induce targeted therapy [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and EXOs modification technology was developed in past decades. In recent years, research has found that nucleic acid Apt have the characteristics of high affinity, strong specificity, low immunogenicity, and easy synthesis and modification \u003cem\u003ein vitro\u003c/em\u003e. They are oligonucleotide fragments obtained by screening, amplifying and enriching from nucleic acid molecular libraries through SELEX, frequently adopt secondary structures such as hairpins and stem-loops, as well as three-dimensional spatial configurations, binding to their targets via precise molecular conformational matching [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In the context of skin lesions, Apt-mediated nanozymes have the ability to recognize and bind specifically to bacterial surfaces, facilitating in situ generation of hydroxyl radicals (-OH) for chemokinetic sterilization [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This provides evidence that the nucleic acid structure of the Apt remains intact and undamaged by bacteria present in skin lesions. In this study, an Apt specifically targeting ECs was identified through the SELEX method and applied to modify EXOs. The Apt-functionalized EXOs exhibited targeted delivery to ECs, resulting in enhanced endocytosis by ECs. This increased uptake significantly improved angiogenic capacity and facilitated the formation of CD31⁺EMCN⁺ blood vessels. Moreover, this modification approach was found to be more straightforward and efficient compared to previously reported methods, with a reduced risk of introducing organic compounds, thereby offering a greener and safer strategy for targeted angiogenesis [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Future research will focus on further advancing endothelial targeting strategies by incorporating highly specific targeting molecules, such as P8RI peptides [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAnother challenge in the application of EXOs in wound healing is their rapid clearance and short survival time. The hydrogel based on natural polysaccharide is considered to be an ideal dressing for diabetes wounds. Meanwhile, quite a lot studies have shown that hydrogels can encapsulate EXOs, form scaffolds and serve as drug carriers to reduce the degradation rate [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. HA is a non-sulfated glycosaminoglycan that is a major component of the ECM in skin cells. Due to its excellent biocompatibility, biodegradability, and hydrophilicity, HA has been used in the production of various wound dressings [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In this study, we encapsulate EXOs with HA to prevent their sudden release in large quantities. Consistent with findings from other studies, Apt-EXOs-HA demonstrated the capability to promote wound healing in normal skin. Remarkably, Apt-EXOs-HA also successfully stimulated vascular regrowth within the vasculature-inhibiting microenvironment associated with type 1 diabetes, indicating that the spontaneous release of EXOs may contribute to the amelioration of the T1D microenvironment. The relationship between insulin and angiogenesis is well-established [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], Given the insulin-deficient and more hostile microenvironment in T1D compared to the reduced, yet insulin-present, environment in T2D, our study focuses on addressing this distinct and harsher condition. We successfully enhance wound healing in this challenging context by optimizing EXOs to augment their angiogenic capacity and facilitate the formation of CD31⁺EMCN⁺ vessels. To further elucidate the mechanisms underlying EXO-mediated improvement of the T1D microenvironment, a detailed analysis of the protein composition of EXOs should be conducted.\u003c/p\u003e\u003cp\u003eIn the present study, we established a well-characterized T1D full-thickness cutaneous wound model by precisely controlling wound creation using ophthalmic scissors, with particular attention to maintaining consistent wound size and depth across all experimental animals. While our model recapitulates several key pathophysiological features of impaired diabetic wound healing, including persistent hyperglycemia-induced tissue damage, dysregulated inflammatory responses, impaired angiogenesis, abnormal extracellular matrix deposition, and delayed epithelialization, all of which are mediated through conserved molecular pathways involving oxidative stress, growth factor dysregulation, and cellular apoptosis. These shared pathological mechanisms, combined with the model's reproducibility and experimental tractability, make it a valuable and widely accepted tool for preliminary investigations of diabetic wound healing mechanisms and therapeutic interventions. While the STZ-induced diabetic mouse model offers advantages including controlled genetic background, standardized wound characteristics, and well-defined experimental parameters that facilitate mechanistic and functional studies, it cannot fully replicate the complex multifactorial nature of human diabetic wounds, which involve variable etiologies (such as neuropathy, microangiopathy, and recurrent infections), diverse wound characteristics, and significant interindividual variability influenced by factors including disease duration, comorbidities, and lifestyle factors [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. These findings will ultimately require validation in more complex systems before clinical translation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study provides evidence suggesting that impaired angiogenesis, including a reduction in CD31⁺EMCN⁺ vessels, may contribute to delayed wound healing in T1D. These findings highlight CD31⁺EMCN⁺ vessels as a potential therapeutic target worthy of further investigation. Apt-EXOs were prepared by conjugating the aptamer to the surface of exosomes via amide condensation. Our in vitro experiments demonstrated that Apt-EXOs significantly enhanced the intake, migration and tube formation capabilities of vascular endothelial cells. For in vivo evaluation, we established models of normal mice and STZ-induced diabetic mice, and the results revealed that Apt-EXOs markedly promoted angiogenesis, wound healing and collagen deposition. The application of modified EXOs demonstrated the capacity to improve the T1D skin microenvironment and promote wound healing by enhancing angiogenesis. These findings offer a robust experimental foundation and a promising therapeutic strategy for addressing T1D skin wound healing.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANX\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;annexin\u003c/p\u003e\n\u003cp\u003eApt\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;aptamers\u003c/p\u003e\n\u003cp\u003eCAV1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;caveolin-1\u003c/p\u003e\n\u003cp\u003eDM diabetes mellitus\u003c/p\u003e\n\u003cp\u003eDLS dynamic light scattering\u003c/p\u003e\n\u003cp\u003eECs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;endothelial cells\u003c/p\u003e\n\u003cp\u003eECM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;extracellular matrix\u003c/p\u003e\n\u003cp\u003eEGFR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;epidermal growth factor receptor\u003c/p\u003e\n\u003cp\u003eEMCN\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;endomucin\u003c/p\u003e\n\u003cp\u003eEXOs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;exosomes\u003c/p\u003e\n\u003cp\u003eGO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;gene ontology\u003c/p\u003e\n\u003cp\u003eHA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;hyaluronic acid\u003c/p\u003e\n\u003cp\u003eJAK\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Janus kinase\u003c/p\u003e\n\u003cp\u003eKEGG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003eLC-MS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;liquid chromatography-mass spectrometry\u003c/p\u003e\n\u003cp\u003eLC-MS/MS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;liquid chromatography-tandem mass spectrometry\u003c/p\u003e\n\u003cp\u003eMSCs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;mesenchymal stem cells\u003c/p\u003e\n\u003cp\u003eSELEX\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;evolution of ligands by exponential enrichment\u003c/p\u003e\n\u003cp\u003eSEM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;scanning electron microscope\u003c/p\u003e\n\u003cp\u003eSTZ\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;streptozotocin\u003c/p\u003e\n\u003cp\u003eT1D\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;type 1 diabetes\u003c/p\u003e\n\u003cp\u003eT2D\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;type 2 diabetes\u003c/p\u003e\n\u003cp\u003eTEM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;transmission electron microscopy\u003c/p\u003e\n\u003cp\u003eTGF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;transforming growth factor\u003c/p\u003e\n\u003cp\u003eUC-MSCs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;umbilical cord-MSCs\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSCs were isolated from human umbilical cords, which were collected immediately after delivery from donors at Xi\u0026apos;an No. 4 Hospital following the acquisition of written informed consent. Ethics approval was obtained from the Ethics Committee of Xi\u0026rsquo;an Fourth Hospital (Reference No. 20190012; Approval Project Title: \u0026quot;Study on the Correlation of Umbilical Cord Mesenchymal Stem Cells in Gestational Diabetes and Infant Metabolic Abnormalities\u0026quot;; Date of Approval: Aug 7, 2019). The laboratory animals were handled in accordance with the \u0026quot;Guidelines for the Care and Use of Laboratory Animals\u0026quot; and the \u0026quot;Animal Welfare Act in China\u0026quot;. The animal experiments were approved by the Medical and Laboratory Animal Ethics Committee of Northwestern Polytechnical University (Reference No. 202501153; Approval Project Title: \u0026quot;Application of Nucleic Acid-Based Novel Vesicular Biomaterials in Skin Regeneration\u0026quot;; Date of Approval: May 6, 2025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformation required to reanalyze the data reported in this work is available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by grants from the National Key Research and Development Program of China (2022YFA1104400), the National Natural Science Foundation of China (82301028, 82401201, 82371020, 82100985, 32101096 and 82170988), the Project of State Key Laboratory of Oral \u0026amp; Maxillofacial Reconstruction and Regeneration (2024MS04, 2024KA01), the Shaanxi Provincial Health Research and Innovation Platform Construction Plan (2024PT-04), the Young Science and Technology Rising Star Project of Shaanxi Province (2024ZC-KJXX-122), the Project of General Hospital of Eastern Theater Command (22LCZLXJS12), the China Postdoctoral Science Foundation (BX20230485 and 2021M693954) and the \u0026quot;Rapid Response\u0026quot; Research projects (2023KXKT017 and 2023KXKT090).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.Z., Z.-Z.G. and S.-F.B. contributed equally to this work. Conceptualization: B.-D.S., C.-H.H., and C.-X.Z.; Methodology: N.Z., Z.-Z.G., S.-F.B., Y.-F.G., P.L., Y-H.J., X-Y. Q., Y.S., and M.Y.; Investigation: N.Z., Z.-Z.G., S.-F.B., Y.-F.G., J.L., J. C., and H.-K.X.; Visualization: N.Z., Z.-Z.G., S.-F.B., L.-H.B., H.N., and Q.-N. W.; Supervision: B.-D.S., C.-H.H., and C.-X.Z.; Writing\u0026mdash;original draft: N.Z., Z.-Z.G. and S.-F.B.; Writing\u0026mdash;review \u0026amp; editing: B.-D.S., C.-H.H., and C.-X.Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eH.S. Kim, X. Sun, J.-H. Lee, H.-W. Kim, X. Fu, K.W. Leong, Advanced drug delivery systems and artificial skin grafts for skin wound healing, Advanced Drug Delivery Reviews 146 (2019) 209-239.\u003c/li\u003e\n\u003cli\u003eJ.B. Cole, J.C. 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King, The use of animal models in diabetes research, British Journal of Pharmacology 166(3) (2012) 877-894.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"diabetic wound healing, aptamer, exosomes","lastPublishedDoi":"10.21203/rs.3.rs-7518048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7518048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe reduced angiogenesis in diabetes mellitus (DM) represents a critical barrier to effective skin wound healing. Therapeutic strategies involving mesenchymal stem cells (MSCs) and MSC-derived exosomes (EXOs) have demonstrated potential in promoting wound healing in diabetic contexts. However, each approach presents specific limitations.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eApt-PEG-DSPE was synthesized via amide condensation between DSPE-PEG-COOH and NH₂-Apt, followed by incubation with EXOs to yield Apt-EXOs, then mix with HA to form Apt-EXOs-HA. C57BL/6 mice were injected intraperitoneally with 50 mg/kg STZ daily for 5 days to induce type 1 diabetes (T1D). Under anesthesia, dorsal fur was shaved and full-thickness skin defects (1.0 cm diameter) were created. The therapeutic effect of Apt-EXOs-HA on T1D skin injury was evaluated by wound healing, vascularization and collagen deposition. The Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (two-tailed) was used to assess statistical significance.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn this study, we identified that vascular structures, specifically CD31⁺EMCN⁺ vessels, are impaired in T1D, which contributes to delayed wound healing and aberrant collagen deposition. Following proteomic analysis and related vascular endothelial cell experiments (including cell migration and tube formation) demonstrating the superior angiogenic potential of EXOs compared to MSCs, we engineered endothelial-targeting EXOs by conjugating them with aptamers (Apt). The application of these Apt-conjugated EXOs in combination with a hyaluronic acid scaffold significantly enhanced angiogenesis under both physiological and DM conditions, thereby accelerating wound healing.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eCollectively, our findings emphasize the essential role of specialized angiogenesis in wound repair and propose a novel, advanced EXOs modification-based therapeutic approach to enhance wound healing in both normal and diabetes-related pathophysiological conditions.\u003c/p\u003e","manuscriptTitle":"Endothelial cell-targeting aptamer-empowered exosomes accelerate wound healing by promoting specialized angiogenesis in type 1 diabetic mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 09:20:53","doi":"10.21203/rs.3.rs-7518048/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-28T17:52:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T15:02:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237128287593270128743400600487546312318","date":"2025-10-08T15:25:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-06T13:19:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-23T09:31:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-17T10:46:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2025-09-16T13:17:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62379a2b-04b2-4f95-848c-8192581d4b6f","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:02:34+00:00","versionOfRecord":{"articleIdentity":"rs-7518048","link":"https://doi.org/10.1186/s13287-025-04846-w","journal":{"identity":"stem-cell-research-and-therapy","isVorOnly":false,"title":"Stem Cell Research \u0026 Therapy"},"publishedOn":"2025-12-07 15:58:00","publishedOnDateReadable":"December 7th, 2025"},"versionCreatedAt":"2025-10-17 09:20:53","video":"","vorDoi":"10.1186/s13287-025-04846-w","vorDoiUrl":"https://doi.org/10.1186/s13287-025-04846-w","workflowStages":[]},"version":"v1","identity":"rs-7518048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7518048","identity":"rs-7518048","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}