Anti-inflammatory and anti-oxidative electrospun nanofiber membrane promotes diabetic wound healing via macrophage modulation

Anti-inflammatory and anti-oxidative electrospun nanofiber membrane promotes diabetic wound healing via macrophage modulation | 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 Anti-inflammatory and anti-oxidative electrospun nanofiber membrane promotes diabetic wound healing via macrophage modulation Jibing He, Shasha Zhou, Jiaxing Wang, Binbin Sun, Dalong Ni, Jinglei Wu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3853738/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Mar, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 7 You are reading this latest preprint version Abstract Background In the inflammatory milieu of diabetic chronic wounds, macrophages undergo substantial metabolic reprogramming and play a pivotal role in orchestrating the immune response. Itaconic acid, primarily synthesized by inflammatory macrophages as a byproduct in the tricarboxylic acid cycle, has recently gained increasing attention as an immunomodulator. This study aims to assess the immunomodulatory capacity of an itaconic acid derivative, 4-Octyl itaconate (OI), which was covalently conjugated to electrospun nanofibers and investigated through in vitro studies and a full-thickness wound model of diabetic mice. Results OI was feasibly conjugated onto chitosan (CS), which was then grafted to electrospun PCL/gelatin (PG) nanofibers to obtain P/G-CS-OI membranes. The P/G-CS-OI membrane exhibited good mechanical strength, compliance, and biocompatibility. In addition, the sustained OI release endowed the nanofiber membrane with great antioxidative and anti-inflammatory activity both in vitro and in vivo . Specifically, the P/G-CS-OI membrane activated nuclear factor-erythroid-2-related factor 2 (NRF2) by alkylating Kelch-like ECH-associated protein 1 (KEAP1). This antioxidative response led to macrophage modulation of mitigated inflammatory responses, enhanced phagocytic activity, and recovered angiogenesis of endothelial cells, finally contributing to improved healing of diabetic wounds. Conclusions The P/G-CS-OI nanofiber membrane shows good capacity in macrophage modulation and might be promising for diabetic chronic wound treatment. electrospinning nanofiber membrane itaconic acid anti-inflammatory diabetic wound Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Diabetes mellitus (DM), a prevalent chronic metabolic disorder, affects an estimated 536 million people globally [ 1 ]. Approximately 25% of these patients eventually develop diabetic foot ulcers (DFUs), with an amputation rate of 43.8% and a mortality rate of 51.7% within 6 years post-hospitalization for the ulcer, resulting in an estimated global economic burden of $ 8.5 billion [ 2 ]. Conservative medical interventions, including antibiotics, magnetic thermal therapy, and hyperbaric oxygen chamber therapy, are often associated with adverse reactions and incomplete treatment outcomes [ 3 , 4 ]. Therefore, there is an urgent need for the development of new therapies to address chronic diabetic wounds. The process of wound healing includes three overlapping stages: inflammation, proliferation, and remodeling [ 5 ]. Macrophages, as the most active non-specific immune cells throughout the entire process, play a double-edged sword role [ 6 ]. On the one hand, the activation of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) biases macrophage polarization toward M1 phenotype in chronic inflammatory milieu, leading to the secretion of pro-inflammatory mediators [ 7 ]. On the other hand, an increased transformation of M2 phenotype macrophages in the latter stages secretes pro-healing chemokines and growth factors, which foster the recruitment of endothelial progenitor cells that are essential for neovascularization and ensure sufficient blood flow to the wound site [ 8 ]. However, in the typical hyperglycemic microenvironment of diabetic conditions, both the phenotypic shift of macrophages and their debris-clearing efficiency are compromised, resulting in chronic inflammation [ 9 , 10 ]. Thus, strategies that modulate the physiological roles of macrophages pose significant implications for diabetic wounds. Intermediates in macrophage glycolysis and the tricarboxylic acid cycle (TAC), such as lactic acid and citric acid, are increasingly recognized as key elements affecting macrophage function and phenotype [ 11 , 12 ]. Previous studies have demonstrated that activated M1 macrophages exhibit elevated expression of immune-responsive gene 1 (IRG1), which encodes aconitate decarboxylase and further catalyzes the conversion of the tricarboxylic acid cycle intermediate cis-aconitate into itaconic acid [ 13 ]. Structurally akin to methylene, itaconic acid modulates immune responses through the competitive inhibition and alkylation of target proteins [ 14 ]. Specifically, itaconic acid impedes the activity of succinate dehydrogenase in macrophages, and reverses the mitochondrial electron transport chain, thereby reducing inflammation by diminishing reactive oxygen species (ROS) production and altering macrophage metabolism [ 15 ]. 4-Octyl itaconate (OI), a derivative of itaconic acid, is more permeable to cell membranes than its parent compound [ 16 ]. Previous studies have demonstrated that OI can enter macrophages and be converted into itaconic acid to regulate mitochondrial metabolism [ 17 ]. Therefore, in this study, we selected OI as a targeted therapeutic agent for modulating macrophage activity in the inflammatory context of diabetic wounds. Nowadays, many biomaterials with multifaceted functionalities, including anti-inflammatory, reparative, and antibacterial properties, have been developed for resolving chronic inflammation and facilitating diabetic wound healing [ 18 , 19 ]. However, despite these promising functionalities, how to balance the biological and mechanical properties of these dressing materials to achieve optimal in vivo effectiveness remains a critical challenge. Chitosan (CS), a linear, semi-crystalline natural polysaccharide derived from chitin, can act as a biocompatible, biodegradable biological scaffold with antioxidant and antibacterial properties [ 20 ]. Electrospun nanofiber membranes are featured by their biomimetic structure, high porosity, extensive surface area, softness, and compliance and have been extensively investigated for wound dressing applications [ 21 ]. Especially, CS-coated electrospun nanofiber membranes show great antibacterial and antioxidative activities and are attractive for wound treatment [ 22 , 23 ]. In this study, we reported a CS-OI grafted nanofiber membrane by electrospinning and assessed its excellent capability for dressing diabetic wounds in a mouse model (Scheme 1 ). First, OI was conjugated to CS to obtain CS-OI conjugate, which was then covalently grafted to electrospun PCL/gelatin nanofibers to obtain PCL/gelatin-CS-IO (P/G-CS-OI) membrane. Physicochemical properties of the P/G-CS-OI membrane were characterized and its anti-inflammatory, antibacterial, and antioxidative activities, as well as biocompatibility and bioactivity, were assessed. Finally, the P/G-CS-OI membrane was used as dressings in a diabetic mouse model to assess its biological performance for curing chronic wounds. Materials and Methods Materials Polycaprolactone (PCL, Mn = 80 kDa) and type A gelatin (300 g Bloom) were obtained from Sigma Aldrich (St. Louis, Missouri, USA). Chitosan (CS, 90% deacetylated powder, Mw = 200 kDa), 4-octenyl Itaconate (OI), and 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Glacial acetic acid and 2,2,2-trifluoroethanol (TFE) were obtained from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Sterile gauzes were obtained from Hainuo Medical Technologies Co., Ltd. (Qingdao, China). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). N-hydroxysuccinimide (NHS) was obtained from Adamas-beta (Shanghai, China). LB Broth and LB Broth Agar were purchased from Sangon Biotech (Shanghai, China). Electrospinning of P/G membranes PCL and type A gelatin were dissolved in TFE to achieve a total polymer concentration of 10% (w/v). The ratio of PCL to gelatin was 8:2, and the mixture was stirred at room temperature to ensure homogeneity. To enhance the miscibility between PCL and gelatin, a small quantity (0.2%, v/v) of glacial acetic acid was added to the solution. The electrospinning process was conducted by feeding this solution through a 20 G needle at a consistent rate of 1.5 mL/h, with an applied voltage of 15 kV to facilitate the formation of nanofibers. The as-spun nanofibers were collected on a slowly rotating mandrel (60 mm diameter, rotating at 120 rpm) to obtain PCL/gelatin (P/G) membranes. Preparation of P/G-CS-OI membranes The synthesis of the CS-OI conjugate was achieved using EDC/NHS carbodiimide chemistry, which facilitates the formation of amide bonds between the amino groups of CS and the carboxyl groups of OI. Initially, chitosan was dissolved in 0.1M dilute hydrochloric acid to a concentration of 1% (w/v). Subsequently, OI, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and NHS (N-hydroxysuccinimide) were added in succession, ensuring a final molar ratio of CS:OI:EDC:NHS at 50:1:10:5. The reaction was maintained at room temperature in a dark environment for 24 h. Then the reaction was air-dried, washed with ethanol to remove any unreacted residues, and air-dried again to obtain the CS-OI conjugate. For the functionalization of the P/G membranes, the membranes were immersed in a CS-OI aqueous solution (1 mg/mL ) at 37°C and agitated for 24 h. Post-infusion with the CS-OI conjugate, the membranes were soaked in MES (2-(N-morpholino) ethanesulfonic acid) buffer to facilitate the crosslinking process, which was carried out at room temperature over 24 h using an EDC:NHS molar ratio of 2:1. To remove any unreacted EDC and NHS, membranes were washed extensively with deionized water, undergoing six cycles of 30 min each. Finally, electrospun nanofiber membranes were vacuum-dried in an oven to remove residual solvent for subsequent studies. Characterization of P/G-CS-OI membranes The morphology of electrospun membranes was observed by scanning electron microscope (SEM, Phenom, XL, Netherlands). Fiber diameters of P/G-CS-OI membranes were measured from SEM micrographs by using Image J. An FTIR spectrometer (Nicolet iS 10, Thermo Fisher Scientific, USA) was used to identify the chemical structures of electrospun nanofiber membranes. The FTIR spectra were obtained by recording 32 scans between 4000 and 400 cm − 1 with a resolution of 0.5 cm − 1 . The XRD patterns of CS-OI were determined by X-ray diffractometer (D8 Advance, Bruker, Germany), detecting the crystalline phase of samples at 2θ between 5° and 60°. We employed the SL200A contact angle analyzer (Solon Tech., Shanghai, China) to obtain the water contact angle (WCA) of nanofiber membranes. A water droplet (5 µL) was placed on the surface of nanofiber membranes and the dynamic water contact angle was recorded. The contact angle was measured by using Image J (n = 3). The mechanical properties of electrospun membranes were evaluated using an uniaxial tensile test as described in our prior publication [ 24 ]. The rectangular samples (10 × 40 mm) underwent incubation in phosphate-buffered saline (PBS) at a temperature of 37 ℃ for 24 h. Afterward, samples were positioned within the grips of a uniaxial testing machine (Instron 5567, Norwood, MA) equipped with a 50 N load cell and tested until failure at a crosshead speed of 10 mm/min. The ultimate tensile strength (UTS) was defined as the maximum load before failure and Young's modulus was calculated as the slope of the stress-strain curve's initial 5% linear section (n = 3). The water absorption and moisturizing rate of electrospun membranes were measured by weighing method. To determine the water absorption rate, electrospun nanofiber membranes were tailored into 1 × 1 cm squares and weighed (W 0 ), then immersed in deionized water at room temperature, wiping away the surface water carefully using filter paper and weighing again (W t ) (n = 3). The water absorption rate of membranes was calculated by the following equation: \(water absorption rate \left(\text{%}\right)= ({W}_{t}-{W}_{0})/{W}_{0}\times 100\text{%}\) . To determine the moisturizing rate, electrospun nanofiber membranes were tailored into 1 cm ×1 cm squares and weighed (W 0 ) and incubated in deionized water for 20 min at room temperature, and weighed (W 1 ). Membranes were placed in a room temperature environment and weighed (W 2 ) at predesignated time points (n = 3). The moisturizing rate of membranes was calculated by the following equation: \(\text{m}\text{o}\text{i}\text{s}\text{t}\text{u}\text{r}\text{i}\text{z}\text{i}\text{n}\text{g} \text{r}\text{a}\text{t}\text{e} \left(\text{%}\right)= ({W}_{2}-{W}_{0})/({W}_{1}-{W}_{0})\times 100\text{%}\) In vitro degradation and drug release were conducted by immersing electrospun membranes in PBS under a shaker incubator at 37°C. To determine the in vitro degradation, electrospun nanofiber membranes were tailored into 1 cm ×1 cm squares and weighed (W 0 ). Incubated samples were retrieved, rinsed with deionized water, lyophilized, and weighed (W w ) at predesignated time points (n = 3). The remaining mass was determined according to the following equation: \(Percentage of remained mass \left(\text{%}\right)=\frac{{W}_{w}}{{W}_{0}}\times 100\text{%}\) . The drug release of OI from the P/G-CS-OI membrane was assessed through UV-vis spectrophotometry. UV analysis was conducted on the incubated solution (1 mL) at specified time intervals, and an equivalent volume of fresh PBS was substituted. The concentration of OI released from P/G-CS-OI membranes was determined by comparing it to the standard curve based on known OI concentrations (n = 6). Antibacterial test The antibacterial activity of the P/G-CS-OI membranes was evaluated against two commonly found bacterial species. More specifically, E. coli (ATCC 25922) and S. aureus (ATCC 25923) were cultured in Luria broth under shaking conditions for 24 h at a temperature of 37 ℃. Subsequently, each sample was inoculated with 200 µL of bacterial inoculum solution (10 8 CFU/mL ) and then incubated at 37 ℃ for 24 h. The bacterial solution was then diluted and cultured with 100 µL of diluent on LB Agar for 12 h, allowing for the observation of colony counts and the subsequent calculation of the Normalized survival rate. Cell culture The RAW 264.7 macrophages and human immortalized keratinocytes (HaCaT) were maintained in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 , with the culture medium being refreshed every two days to maintain optimal cell growth and viability. Before cell seeding, electrospun nanofiber membranes were prepared for biological interaction studies. Each membrane was carefully punched using a sterile hole punch to match the dimensions of the wells in standard cell culture plates. Following this, the membranes were subjected to UV sterilization to ensure aseptic conditions. Cytocompatibility assessments Initially, the nanofiber membranes were cut to match the size of the wells in a 48-well plate and subjected to 24 h of UV sterilization, with a midway turnover to ensure complete exposure. Following sterilization, RAW 264.7 macrophages and HaCaT keratinocytes were revived and cultured. These cells were then seeded into the 48-well plates containing the sterilized nanofiber membranes at a density of 20,000 cells per well. The cultures were maintained at 37°C, with the culture medium changing every other day. Cell viability and proliferation on the electrospun nanofiber membranes were assessed at 1, 4, and 7 days post-seeding. Before the test, the culture medium was aspirated, and the cells were washed thrice with PBS. The viability and proliferation rates were quantitatively measured using live/dead staining reagents and a Cell Counting Kit-8 assay (Biosharp), following the manufacturer's instructions (n = 9). The cell survival rate was calculated using the formula: cell survival rate (%) = (Viable cells/Total cells) × 100%. In addition to these quantitative assays, cell adhesion and morphology on the nanofiber membranes were visually analyzed. At day 7, the cells cultured on the membranes were fixed with 4% paraformaldehyde, sequentially dehydrated in graded ethanol solutions, and air-dried at room temperature. The samples were then sputter-coated with gold to enhance conductivity and imaged using a scanning electron microscope (SEM). Hemocompatibility assessments Fresh blood was collected from healthy mice and centrifuged at 1000 rpm for 10 min to isolate pure red blood cells (RBCs). The RBCs were washed three times and subsequently diluted in PBS to achieve a final concentration of 2%. For the assay, electrospun nanofiber membranes with a diameter of 8 mm were introduced into 250 µL of the RBC suspension and incubated at 37°C for 1 h. As controls, PBS and Triton X-100 were employed as the negative and positive controls, respectively, and mixed with the RBC suspension under identical conditions. Following the incubation period, all samples underwent centrifugation at 1000 rpm for 5 min. The supernatant of each sample was then analyzed spectrophotometrically to measure the absorbance of released hemoglobin at 540 nm. The hemolysis ratio, indicative of the degree of damage to the RBCs, was calculated using the following formula: Hemolysis ratio (%) = [(Abs - Absn)/(Absp - Absn)] × 100%, where Abs represents the absorbance of the sample, Absp the absorbance of the positive control, and Absn the absorbance of the negative control (n = 3). Subcutaneous implantation Electrospun membranes were punched into 8 mm discs and sterilized by UV (12 h for each side) and were subcutaneously implanted in seven-week-old female rats (weighing approximately 280–320 g, purchased from Shanghai Slack Laboratory Animal Limited Liability Company, Shanghai, China). The rats were anesthetized by intraperitoneal injection of chloral hydrate (10%), and pockets were created by blunt dissection of the fascia between the skin and muscle. Electrospun membranes were embedded in the four pockets of the same rat and closed with sutures (n = 3). After 7 and 28 days, the entire subcutaneous tissue containing electrospun nanofiber membranes was excised, and fixed with 4% paraformaldehyde overnight. Paraffin-embedded tissues and sectioned into 5 µm sections for staining. Flow cytometry The electrospun nanofiber membranes were first placed in 6-well plates, and RAW264.7 cells were seeded on their surfaces at a density of 2 × 10 5 cells per well. Following the incubation, the culture medium was discarded, and the cells were washed three times with PBS to remove debris and non-adherent cells. Lipopolysaccharide (LPS, TargetMol) was then added to the culture to induce an inflammatory response, simulating diabetic wound conditions. After another 24 h culture period, cells were collected. For immunophenotyping, the cell pellets were resuspended in 100 µL of PBS containing 1.25 µg of PE-conjugated anti-mouse CD86 antibody and 0.25 µg of FITC-conjugated anti-mouse CD206 antibody (Biolegend). This step was performed on ice for 30 min to preserve cell viability and prevent nonspecific antibody binding. The expression of CD86 and CD206 was subsequently analyzed using the CytoFLEX flow cytometry system (Beckman Coulter). Additionally, the intracellular ROS level was detected using DCFH-DA (Yeasen), following the same procedure as described above. Real‑time polymerase chain reaction (RT‑PCR) Total RNA was extracted from the samples using the EZ-press RNA Purification Kit (EZBioscience, Roseville, MN, USA). Following extraction, the RNA was reverse transcribed into complementary DNA (cDNA) utilizing the Colour Reverse Transcription Kit (EZBioscience). Quantitative RT-PCR was then performed using the 2 × Colour SYBR Green qPCR Master Mix (EZBioscience). The relative expression levels of the target genes were calculated employing the 2 −ΔΔCt method (n = 3). Primer sequences are shown in Table S1 (Additional file 1). Scratch assay HaCaT cells were seeded into 6-well plates at a density of 1 × 10 5 cells per well. Upon reaching approximately 90% confluence, a scratch was carefully made through the cell monolayer to simulate a wound. Following that, macrophage-conditioned medium was added to the wells. To monitor and quantify the process of wound closure, three random fields of view along the scratch were selected in each well. These areas were photographed using an optical microscope at 0, 24, and 48 h post-scratch. Streptozotocin (STZ)‑induced diabetic mice The animal study protocols were rigorously designed and executed in compliance with the ethical guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Sixth People's Hospital Affiliated with the Shanghai Jiao Tong University School of Medicine. A cohort of 36 male ICR mice, aged between 6 and 8 weeks, was utilized for the establishment of a diabetic model. The mice were housed under controlled environmental conditions, maintained at 22 ± 2℃, with three animals per cage to ensure adequate space and social interaction. The diabetic state in the mice was induced by 150 mg/kg of STZ (≥ 98% purity; Beyotime). To enhance the effectiveness of STZ, mice were subjected to a 24 h fasting before the administration of STZ. Subsequently, the blood glucose levels of mice were monitored regularly by sampling from the tail vein 3 days post-injection. A continuous blood glucose level greater than or equal to 16.7 mM was considered the successful establishment of diabetes. Wound healing evaluation After anesthesia, the dorsal fur of the mice was carefully removed using an electric animal razor, and the surgical area was disinfected with 75% alcohol. Mice were subsequently divided into three distinct groups: Control, P/G-CS, and P/G-CS-OI, with each group comprising 12 animals. In each mouse, a full-thickness skin wound of 8 mm diameter was created on the back using a sterile biopsy punch. The wounds were then covered with the respective electrospun nanofiber membranes, wrapped by sterile gauzes and secured with sutures. The progression of wound healing was documented at 0, 3, 7, 10, and 14 days post-wounding by capturing digital photographs of the wounds. Histological analysis Healing tissues of animals 3, 7, 10, and 14 days post-surgery were harvested and subjected to a series of histological preparations. Samples were then stained using hematoxylin and eosin (H&E; Solarbio) and Masson’s trichrome (Solarbio). Immunofluorescence staining was performed to evaluate the macrophage phenotype and extent of tissue repair in the injured skin tissues. Specific antibodies including CD31 (1:4000; Proteintech), CD86 (1:200; Abways), CD206 (1:300; Proteintech), α-SMA (1:300; Proteintech), K10 (1:100; Abways), K14 (1:100; Abways).And Goat Anti-Mouse IgG (H + L) Cy3 (1:200; Abways) and Goat Anti-Rabbit IgG (H + L) Alexa Fluor 488 (1:200; Abways), were used to label the target cells and proteins. The stained sections were examined under a Ci-S microscope (Nikon) for morphological and quantitative analysis. ELISA assay The tissue samples were homogenized and sonicated in pre-cooled PBS. The homogenates were then centrifuged at 12,000 g for 5 min, and the supernatants were collected. The expression levels of various cytokines were quantitatively assessed using an ELISA kit (Servicebio, Wuhan, China), following the manufacturer's instructions. Statistical analysis Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism. One-way or two-way analysis of variance (ANOVA) with post hoc multiple comparisons and Student's t-test were used to analyze significance, where appropriate. A p -value of less than 0.05 was considered statistically significant. Results and Discussion Characterization of electrospun membranes The fabrication and structural characterization of surface-modified nanofiber membranes P/G-CS-OI are detailed in Fig. 1 A. SEM images reveal a dense arrangement of porous, randomly oriented nanofibers (Fig. 1 B). The average diameters of P/G, P/G-CS, and P/G-CS-OI membranes were 299 ± 54 nm, 352 ± 63 nm, and 371 ± 91 nm, respectively. Covalent grafting of CS and CS-OI to P/G nanofibers led to increased fiber diameter (Fig. 1 C) but did not alter the randomly oriented nanofiber structure. FTIR was employed to analyze the chemical composition of the CS-OI conjugate and electrospun nanofiber membranes (Fig. 1 D). CS exhibited characteristic absorption bands of functional groups: amide I (C = O stretching) at 1654 cm − 1 , amide II (N-H bending) at 1555 cm − 1 , and amide III (C-N stretching) at 1318 cm − 1 [ 25 ]. In the OI spectra, the prominent band at 1721 cm − 1 corresponded to the C = O stretching of carboxyl groups. For CS-OI conjugate, the band intensity increased at 1643 cm − 1 and 1521 cm − 1 , indicating the formation of amide I and amide II bonds between the carboxyl group of OI and the amino group of the CS chain through amide bonds. In addition, the characteristic absorption band of the OI monomer at 1727 cm − 1 was also observed in the CS-OI conjugate, further proving the existence of the OI component in the CS-OI conjugate. Therefore, these results demonstrate that the grafting reaction occurs in the carboxyl group of OI and the amino group of CS through the amide bond. For P/G electrospun membranes, the characteristic peaks of amide I and amide II at 1643 cm − 1 and 1543 cm − 1 , respectively, confirmed the presence of gelatin within the electrospun membranes. For P/G-CS and P/G-CS-OI membranes, the characteristic peak of the amide Ⅰ band shifts toward a higher wavenumber (from 1643 to 1650 cm − 1 ), and such shift in peak positions implies weakening or lengthening of bonds, confirming the existence of an interaction of the OI with the CS [ 26 , 27 ]. XRD patterns (Fig. 1 E) demonstrate that CS had a broad peak at 2θ = 19.9 showing its high crystallinity, aligning with previous reports [ 28 , 29 ]. The high crystallinity of chitosan is generally caused by inter- and intra-molecular hydrogen bonds [ 30 ]. OI displayed multiple narrow and sharp diffraction peaks that indicate its characteristic of crystalline materials. The CS-OI conjugate exhibited a smooth diffraction pattern, indicating reduced crystallinity and the consequent increase of the amorphous phase, confirming the successful conjugation of OI onto CS. Lower crystallinity would lead to the increase of solubility in water, CS can only be soluble in acid conditions (pKa ∼ 5.5) before reaction and CS-OI were easily soluble in water. This was probably due to that OI hinders the inter- and intra-molecular hydrogen bonds network of chitosan molecules [ 31 , 32 ]. CS-OI showed water-solubility facilitating subsequent P/G-CS-OI membrane preparation in our study. Hydrophilicity is a critical property influencing material-tissue contact of wound dressings. The electrospun P/G nanofiber membrane had a water contact angle of 53 ± 7°, indicating its hydrophilic nature (Fig. 1 F). This is due to the presence of gelatin that greatly reduces the hydrophobicity of PCL. The introduction of After grafting with CS and CS-OI, the obtained nanofiber membranes had contact angles of 57 ± 5° and 47 ± 1°, respectively, which were not significant from the P/G nanofiber membrane ( p > 0.05). This result suggests that the overall hydrophilicity is not significantly impacted by the addition of CS or CS-OI. To guarantee a close and comfortable interaction with the wound, as well as to regulate the tension exerted on it, the mechanical characteristics of the wound dressing should resemble those of human skin. As depicted in Fig. 1 G, the UTS for P/G, P/G-CS, and P/G-CS-OI membranes were measured at 12 ± 3 MPa, 19 ± 2 MPa, and 20 ± 2 MPa, respectively, highlighting a marked increase in strength with the conjugation of CS and CS-OI. Moreover, P/G-CS had the highest Young's modulus (36 ± 4 MPa), surpassing both P/G and P/G-CS-OI (18 ± 2 MPa and 25 ± 1 MPa, respectively). The strains at failure were 153 ± 7% for P/G, 112 ± 13% for P/G-CS, and 192 ± 13% for P/G-CS-OI, P/G-CS-OI membranes exhibited the greatest strains at failure. Human skin typically exhibits a tensile modulus ranging from 15 to 150 MPa, an ultimate tensile stress between 1 and 32 MPa, and an ultimate tensile strain from 35 to 115% [ 33 ]. These results indicate that our nanofiber membranes could provide suitable tensile properties for wound dressing. The water absorption and retention capabilities of nanofiber membranes are critical for their efficacy as wound dressings, particularly in absorbing tissue exudates. The water absorption rates of the nanofiber membranes were evaluated over 60 min, as shown in Fig. 1 J. The rates were determined to be 743 ± 42%, 670 ± 40%, and 584 ± 62% for P/G, P/G-CS, and P/G-CS-OI membranes, respectively, with all three types reaching maximum absorption within the first 15 min. In terms of moisture retention, as indicated in Fig. 1 H, the rates were 19 ± 2%, 15 ± 3%, and 21 ± 3% for P/G, P/G-CS, and P/G-CS-OI membranes, respectively. These results show that our nanofiber membrane can absorb the solution quickly, reach the maximum absorption ratio in a short time, and remain stable. As a wound dressing, it can maintain a moist environment around the wound, which is favorable for wound healing [ 34 ]. In vitro degradation of nanofiber membranes was evaluated in PBS solution at 37°C for up to 42 days (Fig. 1 J). The remaining mass of P/G-CS and P/G-CS-OI samples was around 95% after 2 weeks, decreasing to 82% after 6 weeks. For P/G samples, the remaining mass was 93% in the second week, decreasing to 80%, suggesting a slightly faster degradation rate compared to P/G-CS and P/G-CS-OI membranes. The nanofiber membrane exhibits slow degradation characteristics, which fulfill the requirements of wound dressings. This feature effectively prevents the negative impact of degradation products on the wound. Additionally, it aids in extending the lifespan of the dressing during the wound-healing process, reducing the need for frequent dressing replacements. As a result, it minimizes the secondary trauma caused by dressing changes [ 35 ]. The slow degradation of the membrane favors the long-term release of the active ingredient, and we evaluated the cumulative release of OI from the P/G-CS-OI membranes for up to 28 days (Fig. 1 K). Within the first 2 weeks, P/G-CS-OI membranes released 75% of OI and exhibited a relatively slow release of OI for a period of up to 28 days. In our study, conjugating small molecule OI to polymeric carries CS has been applied to increase the bioavailability of OI, and the drug delivery system can be further stabilized by crosslinking CS-OI conjugates to nanofibers. In the drug release experiments, P/G-CS-OI membranes achieved long-term release effects, which help to play the biological role of OI for a long time to help skin repair. The impaired immune defense system induced by diabetes can easily lead to recurrent bacterial infections in chronic hard-to-heal wounds, and the increasing exudate provides a breeding ground for bacterial growth [ 36 ]. The antibacterial efficacy of the nanofiber membranes was identified using common wound pathogens, E.coli (Gram-negative) and S.aureus (Gram-positive) (Fig. S2, Additional file 1). The control and P/G groups showed more bacterial colonies, while the P/G-CS and P/G-CS-OI groups had significantly fewer bacterial colonies. The normalized survival of E. coli co-cultured with P/G-CS and P/G-CS-OI membranes was 60 ± 7% and 71 ± 2%, respectively, and S. aureus was 74 ± 9% and 63 ± 3%, respectively. Despite the antimicrobial effect of OI [ 37 ], it is mainly CS that exerts the antimicrobial function in the P/G-CS-OI membranes. In the acidic microenvironment of wounds, its amino group is positively charged, and it can bind with the negative charge on the surface of the bacterial cell membrane, which alters the permeability of the cell membrane and leads to metabolic disorders of the bacterium, to achieve the antibacterial effect [ 38 ]. In the CS-OI conjugate, part of the amino group of CS was covalently coupled with the carboxyl group of OI, and for the P/G-CS-OI membranes, the amino group of CS was further substituted, which reduced the bacteriostatic effect of the P/G-CS-OI membranes. However, it is still suitable for the prevention of bacterial infection in wound care. Biocompatibility of electrospun membranes The cytocompatibility of electrospun membranes was evaluated with RAW 264.7 and HaCaT cells. Live/dead staining shows that both RAW 264.7 (Fig. 2 A) and HaCaT (Fig. 2 B) cells experience robust proliferation on electrospun membranes. Most cells were live (green stain) with very few dead cells (red stain) from day 1 to 7. Both cells showed a great increase in populations over time. SEM images reveal that cells are closely attached to the nanofibers. CCK-8 assays demonstrated sustained increases in cellular metabolic activity, with no discernible cytotoxic effects attributable to the electrospun nanofiber membranes (Figs. 2 C and E). Moreover, both cells had viability rates greater than 90% after 24 h on electrospun membranes (Figs. 2 D and 2 F). These results suggest that the P/G-CS-OI membrane has good cytocompatibility. Given that wound dressings are destined for direct contact with blood in vivo, assessing the potential for hemolysis is a crucial aspect of hemocompatibility testing (Fig. S1 , Additional file 1). Results from these assays yielded supernatants that were light yellow in color post-contact with the different membranes. This outcome paralleled the negative control and stood in stark contrast to the bright red of the positive control. The P/G, P/G-CS, and P/G-CS-OI membranes were associated with hemolysis rates of less than 2%, a figure well below the international safety benchmark of 5%. The aforementioned in vitro biocompatibility findings were validated through in vivo experiments that involved subcutaneous implantation of the electrospun nanofiber membranes in a mouse model. Subsequent histological analyses at days 7 and 28 post-implantation indicated only minimal immune reactions to the P/G, P/G-CS, and P/G-CS-OI membranes, as evidenced by H&E staining (Fig. S2A, Additional file 1). Although encapsulated by fibroblast layers, significant infiltration by neutrophils or monocytes was notably absent. Masson's trichrome staining illustrated tissue-related fiber proliferation and collagen deposition, indicative of integration following membrane implantation (Fig. S2B, Additional file 1). Measurement of the fibrous capsule thickness, one marker of immune response, revealed a decrease in thickness for samples P/G-CS and P/G-CS-OI, from 408 ± 33 µm and 237 ± 37 µm at day 7, to 233 ± 23 µm and 161 ± 14 µm on day 28, respectively (Fig. S2C, Additional file 1). In contrast, P/G samples exhibited an increase in capsule thickness over the same period. Furthermore, analysis of cell infiltration depth at the tissue-membrane interface revealed lower figures for the P/G-CS and P/G-CS-OI membranes compared to P/G alone at both 7 and 28 days post-implantation (Fig. S2D, Additional file 1). Collectively, these findings reinforce the conclusion that the fabricated electrospun nanofiber membranes exhibit substantial biocompatibility, advocating for their application in wound healing interventions. Macrophage anti-inflammatory modulation of P/G-CS-OI membrane Macrophages are indispensable in mounting a local immune response within injured tissues, an imbalance characterized by the predominance of M1 macrophages and impaired conversion to M2 macrophages can exacerbate tissue damage and impede wound healing processes [ 39 ]. Therefore, we investigated the impact of the P/G-CS-OI membranes on macrophage polarization under LPS challenge (Fig. 3 ). Figure 3 A shows a notable upregulation of the levels of CC Chemokine Receptor 7 (CCR7) upon LPS exposure. Remarkably, the application of P/G-CS-OI membranes led to significant attenuation in the fluorescence signal of CCR7, concomitantly with an upsurge in the Arginase 1 (ARG1) signal, indicative of a shift towards M2 macrophage polarization. Flow cytometry analysis reveals a substantial decrease (7.26%) in the percentage of M1 macrophages (CD86+), alongside a marked increase (20.84%) in M2 macrophages (CD206+), after P/G-CS-OI treatment (Fig. 3 B-D). To further elucidate the role of P/G-CS-OI in macrophage polarization and the regulation of inflammatory mediators, RT-PCR was employed (Figs. 3 E-L). The RT-PCR results demonstrated a significant increase in the gene expression of CCR7, tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, and IL-6 in LPS-activated RAW264.7 cells, coupled with a marked decrease in IL-4 expression. Interestingly, the level of IL-10 did not exhibit the anticipated decrease, suggesting an inherent cellular self-regulation mechanism aimed at preventing excessive inflammation. In the P/G-CS-OI group, RT-PCR results indicated a substantial downregulation in the expression of pro-inflammatory genes (CCR7, TNF-α, IL-1β, and IL-6) and upregulation in ARG1, Macrophage Scavenger Receptor 1 (MSR1), IL-4, and IL-10, underscoring the potent immunomodulatory and pro-repair capabilities of the P/G-CS-OI membranes. It is noteworthy that MSR1, as one of the markers of M2 macrophages, can enhance the phagocytosis ability of macrophages. This facilitates the clearance of bacterial fragments and apoptotic cells, thereby contributing to the improvement of the chronic wound inflammatory microenvironment [ 40 ]. These findings collectively suggest that the use of P/G-CS-OI membranes, not only promotes the transformation of macrophages from the M1 to the M2 phenotype but also enhances their phagocytic function and anti-inflammatory activity. This dual action considerably augments the potential for improved skin repair in chronic wound scenarios, highlighting the therapeutic value of P/G-CS-OI membranes in modulating the immune response and facilitating tissue regeneration. Antioxidant activity of P/G-CS-OI membrane The excessive ROS accumulation in diabetic wounds can not only oxidize surrounding lipids and proteins, causing cellular damage but also promote the chemotaxis of inflammatory factors to the wound site, thus hindering skin regeneration [ 41 ]. Therefore, the development of wound dressings with antioxidant capabilities has become the focus of current research [ 42 ]. To this end, RAW 264.7 cells were cultured in a pathological oxidative microenvironment, induced by 100 ng/mL LPS, and intracellular ROS levels were assessed using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA). The resulting fluorescence signal was quantified and characterized using both flow cytometry and fluorescence microscopy. The results, as illustrated in Fig. 4 A, indicated that in the LPS-treated groups (Control and P/G-CS), DCFH-DA exhibited significantly higher green fluorescence compared to the control group. Notably, the use of P/G-CS-OI membranes effectively reduced intracellular oxidative stress. Flow cytometry quantitative data, presented in Fig. 4 B, showed that the percentage of FITC-positive channels in the P/G-CS-OI group was significantly lower compared to other experimental groups. This finding highlights the potent ROS scavenging ability of P/G-CS-OI membranes, underscoring its potential as an effective antioxidant in wound dressing applications. Nitric Oxide (NO) is a versatile signaling molecule produced by various cell types and plays a pivotal role in inflammation and oxidative stress in skin tissue [ 43 ]. Being a free radical, NO can react with superoxide anions to form peroxynitrite, a potent oxidizing agent that inflicts damage on surrounding cells and tissues [ 44 ]. Superoxide Dismutase (SOD) is a metalloenzyme that catalyzes the disproportionation of superoxide anions, thereby maintaining cellular REDOX balance [ 45 ]. Our study demonstrates that the application of P/G-CS and P/G-CS-OI membranes significantly reduces NO levels, increases SOD activity, and consequently mitigates cellular oxidative stress damage, as shown in Figs. 4 C and 4 D. In chronic diabetic wounds, persistent hyperglycemia induces an accumulation of damaged mitochondria, leading to an overproduction of coenzyme Q and subsequent impairment of the normal electron transport within the respiratory chain [ 46 ]. This mitochondrial dysfunction redirects a substantial number of electrons back to mitochondrial complex I, creating a significant imbalance between ROS production and elimination [ 47 ]. Such an imbalance can result in lipid peroxidation within macrophages, leading to stromal swelling and rupture of the outer membrane [ 48 ]. Nuclear Factor Erythroid 2–Related Factor 2 (NRF2) is a key transcription factor that coordinates cellular defense mechanisms against oxidative and electrophilic stress [ 49 ]. It regulates the transcription of antioxidant genes dependent on the Antioxidant Response Element (ARE). This regulation includes the expression of genes such as NAD(P)H Quinone Dehydrogenase 1 (NQO1), Glutamate Cysteine Ligase Catalytic Subunit (GCLC), Glutamate Cysteine Ligase Modifier Subunit (GCLM), and Heme Oxygenase 1 (HO-1) [ 50 ]. Kelch-like ECH-associated protein 1 (KEAP1), a dimeric protein, normally binds to NRF2 in the cytoplasm and facilitates its degradation via the proteasome system. Hence, KEAP1 is also recognized as an inhibitor of NRF2. Our experiment had confirmed that P/G-CS-OI membranes could significantly up-regulate the expression of NRF2 and its downstream antioxidant components NQO1, GCLC, GCLM, and HO-1 (Figs. 4 E-J), thereby clearing excess ROS and maintaining REDOX homeostasis in the cellular environment. This result is mainly attributable to OI, which plays a crucial role in this regulatory mechanism by alkylating cysteine residues (151, 257, 288, 273, and 297) on KEAP1. This modification prompts the dissociation of NRF2 from KEAP1, allowing NRF2 to translocate to the nucleus where it interacts with ARE, thereby enhancing the expression of downstream genes with antioxidant properties and creating suitable conditions for the healing of chronic wounds. However, since the main function of OI is to alkalize KEAP1 and promote its dissociation, the influence on the expression level of KEAP1 is relatively small (Fig. 4 F) [ 51 ]. Bioactivity of P/G-CS-OI for proliferation of keratinocytes To evaluate the effect of macrophage immune response on the formation and differentiation of blood vessels and epidermis, RAW 264.7 cells were planted in different groups of electrospun nanofiber membranes and activated with lipopolysaccharide (LPS) to simulate the inflammatory microenvironment. The conditioned medium obtained was used to grow HaCaTs and evaluate the effects of cytokines and growth factors produced by macrophages under different conditions on the differentiation and maturation of HaCaTs (Fig. 5 A). We investigated the effects of different medium conditions on HaCaT cell migration through scratch experiments. As shown in Fig. 5 B, neither P/G nor P/G-CS showed enhanced cell migration, while P/G-CS-OI showed a higher ability to promote cell migration. This trend was consistent with the polarization change of macrophages, indicating that the conditioned medium obtained by macrophages after remodeling had obvious chemotactic effect on epidermal repair cells. However, the mechanism underlying accelerated HaCaT cell migration remains unknown in the current study. We further measured gene expression in different groups of RAW 264.7 and HaCaT cells. We found that RAW 264.7 cells up-regulated vascular endothelial growth factor (VEGF) under the influence of P/G-CS-OI membranes (Fig. 5 D), VEGF can promote endothelial cell migration, proliferation, and differentiation. It also helps to form new blood vessel structures, providing enough oxygen and nutrients to promote growth, differentiation, and recovery of damaged tissues. Based on good vascularization, the proliferation and differentiation of keratin can provide an important structural role in wound healing. Keratin 10 (K10) belongs to the type II keratin family and is closely associated with terminal differentiation in the epidermis of the skin. Keratin 14 (K14), a type I keratin family, is a marker of the undifferentiated state of epidermal cells, and an increased ratio of K14/ K10 often indicates that epidermal cells are in a period of rapid proliferation. Involucrin (IVI) is an amorphous protein that is one of the early markers of epidermal differentiation, and the level of IVI expression in HaCaT cells can be used to assess the degree of cell differentiation. Our detection results showed that HaCaT cells under the influence of P/G-CS-OI condition medium showed lower K10 expression and higher K14 and IVI expression than P/G and P/G-CS groups (Figs. 5 E-G). These results may be attributed to the remodeling of macrophages by electrospun nanofiber membranes. Macrophages are key players in the innate immune system and are closely involved in tissue repair and regeneration. They are not only an important source of pro-fibrotic mediators and various growth factors, but also a balancer of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. The remodeling of macrophages can drive cell activation and extracellular matrix turnover in the inflammatory microenvironment after injury [ 52 ]. Diabetic wound healing In vivo studies were conducted to evaluate the role of the P/G-CS-OI membranes in promoting wound healing in a diabetic full-thickness skin defect model, and the wound repair process in each treatment group was observed at days 0, 3, 7, 10, and 14 (Fig. 6 A). As shown in Fig. 6 B and Fig. 6 C, on day 3, the P/G-CS-OI membranes already showed a higher wound closure rate (50 ± 4%) compared with P/G (7 ± 5%) and P/G-Cs (20 ± 13%). After 14 days, wounds in P/G-CS-OI were almost completely healed, while marked scarring in the other groups indicated delayed wound repair. To further elucidate the role of electrospun nanofiber membranes in promoting chronic wound healing, histological analyses were performed. H&E staining images showed that compared with P/G and P/G-CS groups, there was more neovasculation and hair follicle formation in the regenerated skin of P/G-CS-OI groups, and there was less infiltration of inflammatory cells such as monocytes and neutrophils. After 14 days of treatment, complete re-epithelialization was observed in the wounds treated with P/G-CS-OI, characterized by typical cortical structures and morphological features (Fig. 6 D). Additionally, the thickness of the granulation tissue, a vital indicator of the wound healing process, was assessed using Masson tri-color staining to determine collagen deposition and scar length in the tissue. It is not difficult to see that compared with P/G and P/G-CS, the granulation tissue thickness of P/G-CS-OI is thicker, scar length and wound margin are significantly reduced, and there is a lot of dense and orderly collagen accumulation under the epidermis (Fig. 6 E). Chronic wounds are hallmarked by prolonged inflammation, which is characterized by a dominant M1 macrophage polarization, elevated levels of inflammatory cytokines, and compromised processes of angiogenesis and epithelial regeneration [ 53 ]. In addition to in vitro results of P/G-CS-OI membranes facilitating macrophage phenotypic remodeling, our study further investigated the impact of these electrospun nanofiber membranes on inflammatory infiltration and tissue repair in vivo . Immunofluorescence images showed a widespread distribution of M2 macrophages (CD206+) within the subcutaneous layer of treated wounds, while concurrently, the presence of M1 macrophages (CD86+) was notably diminished (Fig. 7 A). This observation indicates that P/G-CS-OI membranes effectively induce the polarization shift from the M1 to M2 phenotype in vivo . The subsidence of inflammation is followed by hastened angiogenesis. To quantitatively analyze neovascularization within the granulation tissue, immunofluorescence staining for CD31 and α-Smooth Muscle Actin (α-SMA) was employed, which showed that the P/G-CS-OI membranes significantly upregulated the expression of both CD31 and α-SMA in the regenerated skin on days 7 and 14. CD31 can label endothelial cells responsible for the formation of vascular lumen, and α-SMA can label pericytes/smooth muscle cells involved in structural stability and regulation of vascular permeability. Vessels displaying both CD31 + and α-SMA + were characterized as mature, whereas vessels labeled only with CD31 + were considered immature [ 54 ]. Our findings suggest that electrospun nanofiber membranes are effective in promoting the formation of mature blood vessels. Keratin 10 (K10) and keratin 14 (K14) serve as critical markers for epidermal differentiation and epidermal basal cells, respectively [ 55 ]. Fluorescence results showed that P/G and P/G-CS groups had weak K10 + epidermis and scattered K14 + epidermal basal layer, indicating that the wound tissue had not regenerated a mature epidermal structure. In contrast, wounds treated with P/G-CS-OI membranes displayed a well-formed epidermis, characterized by a completely covered K10 + epidermal basal layer and a K14 + epidermal lining. This finding demonstrates that P/G-CS-OI membranes promote rapid and effective epithelialization of wounds. After 7 days of treatment under the same conditions, immunohistochemical staining showed the expression of TNF-𝛼 in the P/G-CS-OI group was significantly decreased, while the expression of IL-10, VEGF, and NRF2 was significantly increased (Figs. 7 B-F), which was aligned with the in vitro results. ELISA was also used to quantitatively analyze the expressions of TNF-𝛼, IL-1β, Interferon (IFN)-γ, and VEGF, and corresponding kits were used to detect the expressions of oxidase SOD and oxidative stress product malondialdehyde (MDA) (Figs. 7 G-L). The inflammatory cytokines TNF-𝛼, IL-1β, and IFN-γ inhibit wound healing, while VEGF acts on endothelial cells to aid endothelial chemotactic migration, which is essential for angiogenesis during cell proliferation. These outcomes are consistent with observations from immunohistochemistry and immunofluorescence staining, as anticipated. The expressions of TNF-𝛼, IL-1β, IFN-γ, and MDA were significantly decreased, while the expressions of VEGF and SOD were increased. These results suggest that the P/G-CS-OI membrane not only inhibits tissue inflammation but also induces regeneration of blood vessels and skin appendages and promotes wound healing. Overall, electrospun nanofiber membranes show significant potential in enhancing the healing of chronic wounds by simultaneously alleviating oxidative stress, inhibiting inflammation, and promoting new blood vessel formation. Conclusions In summary, our study focused on the development and characterization of a bioactive electrospun nanofiber membrane, composed of PCL, Gelatin, CS, and OI, envisioned as an innovative wound dressing material. The P/G-CS-OI membranes demonstrate a remarkable ability to inhibit the activation of specific inflammatory mediators, effectively contributing to macrophage modulation, and thereby promoting the healing of chronic wounds. A noteworthy aspect of the P/G-CS-OI membrane is its capacity for the sustained release of OI. This feature is instrumental in activating ARE through the NRF2/KEAP1 pathway, which relieves oxidative stress and promotes wound healing. In vivo studies in a diabetic mouse skin wound model further validated the significant therapeutic benefits of the P/G-CS-OI membrane. These benefits include the modulation of inflammation, promotion of neovascularization, and enhancement of collagen deposition. Therefore, this inherently multifunctional electrospun nanofiber membrane not only reconstructs the tissue microenvironment but also accelerates tissue regeneration. Its diverse and potent capabilities position it as a promising biological dressing, meriting further exploration and potential applications in tissue engineering and regenerative medicine. Abbreviations OI 4-octyl itaconate DAPI 4',6-diamidino-2-phenylindole DCFH-DA 2,7-dichlorodihydrofluorescein FITC Fluorescein isothiocyanate HO-1 Heme Oxygenase-1 NRF2 Nuclear factor (erythroid derived 2)-related factor 2 KEAP1 Kelch-like ECH-associated protein 1 NQO1 NAD(P)H:quinone oxidoreductase 1 PBS Phosphate Buffer Saline SOD Superoxide Dismutase MDA malondialdehyde STZ Streptozocin TAC tricarboxylic acid cycle α-SMA α-Smooth muscle actin ELISA Enzyme-linked immunosorbent assay IHC Immunohistochemical IL Interleukin TNF Tumor necrosis factor IFN Interferon LPS Lipopolysaccharide ROS Reactive oxygen species SEM Scanning electron microscopy Declarations Acknowledgments None. Author contributions XP, JW, and ND designed the study. JH, SZ, and JW carried out experiments and drafted the manuscript. JW and SB contributed to sample preparation. ND, JW, and XP provided guidance and assistance on relevant experiments. All authors reviewed and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 81974326, 82372427) and the Excellent Youth Training Program of Shanghai Sixth People's Hospital (Grant No. ynyq202202). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Sixth People's Hospital Affiliated with the Shanghai Jiao Tong University School of Medicine. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, Stein C, Basit A, Chan JCN, Mbanya JC, et al: IDF Diabetes Atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. 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Wu Y, Wang Y, Zheng C, Hu C, Yang L, Kong Q, Zhang H, Wang Y: A Versatile Glycopeptide Hydrogel Promotes Chronic Refractory Wound Healing Through Bacterial Elimination, Sustained Oxygenation, Immunoregulation, and Neovascularization. Advanced Functional Materials. 2023;33:2305992. Kirkton RD, Santiago-Maysonet M, Lawson JH, Tente WE, Dahl SLM, Niklason LE, Prichard HL: Bioengineered human acellular vessels recellularize and evolve into living blood vessels after human implantation. Science Translational Medicine. 2019;11:eaau6934. Qiang L, Yang S, Cui Y-H, He Y-Y: Keratinocyte autophagy enables the activation of keratinocytes and fibroblastsand facilitates wound healing. Autophagy. 2021;17:2128-2143. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Additionalfile.docx scheme1.jpg Scheme 1. Schematic illustration of a Chitosan-4-Octyl itaconate grafted nanofiber membrane (P/G-CS-OI), which can promote diabetic wound healing via macrophage modulation. Cite Share Download PDF Status: Published Journal Publication published 16 Mar, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 27 Jan, 2024 Reviews received at journal 23 Jan, 2024 Reviewers agreed at journal 16 Jan, 2024 Reviewers invited by journal 16 Jan, 2024 Editor assigned by journal 12 Jan, 2024 Submission checks completed at journal 12 Jan, 2024 First submitted to journal 11 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3853738","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266770964,"identity":"3c87de5c-39b6-42e3-9ff8-3cee8b55e8f8","order_by":0,"name":"Jibing He","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jibing","middleName":"","lastName":"He","suffix":""},{"id":266770965,"identity":"e9033130-f4f2-40df-8efd-ae069294f39e","order_by":1,"name":"Shasha Zhou","email":"","orcid":"","institution":"Donghua University","correspondingAuthor":false,"prefix":"","firstName":"Shasha","middleName":"","lastName":"Zhou","suffix":""},{"id":266770966,"identity":"961c0efa-09d1-4c66-b3ed-747a30f7214a","order_by":2,"name":"Jiaxing Wang","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiaxing","middleName":"","lastName":"Wang","suffix":""},{"id":266770967,"identity":"efa7bb0a-dc0e-4235-9865-1a3c7863be99","order_by":3,"name":"Binbin Sun","email":"","orcid":"","institution":"Donghua University","correspondingAuthor":false,"prefix":"","firstName":"Binbin","middleName":"","lastName":"Sun","suffix":""},{"id":266770968,"identity":"03f794ab-fe24-4905-b4fe-aa37d91e65ee","order_by":4,"name":"Dalong Ni","email":"","orcid":"","institution":"Ruijin Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Dalong","middleName":"","lastName":"Ni","suffix":""},{"id":266770969,"identity":"93524354-045c-4e33-9bd2-64dddd9cd185","order_by":5,"name":"Jinglei Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoElEQVRIiWNgGAWjYBACxgbmA9Jg1gHitbAlkKiFgYHHgEQtzDNyPt4ubGOQ47uRwPi5gCiHzcjdbD2zjcFY8kYCs/QMorTMzt0mzdvGkLjhRgIbMw9xWnKegbTUk6SFDaQlwYB4LfOfGVvznJMwnHnmYbM0UVoMew4/vM1TZiPPdzz54GfitDSAKQmQhQ3EaGBgkCdO2SgYBaNgFIxoAAAdMizVidrCywAAAABJRU5ErkJggg==","orcid":"","institution":"Donghua University","correspondingAuthor":true,"prefix":"","firstName":"Jinglei","middleName":"","lastName":"Wu","suffix":""},{"id":266770970,"identity":"c62c8e74-a3f8-4b95-9daf-a020d3a0cd42","order_by":6,"name":"Xiaochun Peng","email":"","orcid":"","institution":"Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaochun","middleName":"","lastName":"Peng","suffix":""}],"badges":[],"createdAt":"2024-01-11 14:19:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3853738/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3853738/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-02385-9","type":"published","date":"2024-03-16T15:01:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49711242,"identity":"024ff868-e189-435f-9a8f-77076fa919b1","added_by":"auto","created_at":"2024-01-16 19:56:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2101986,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation and physiochemical properties of electrospun nanofiber membranes. Schematic of electrospun nanofiber membrane preparation (A). SEM images of electrospun nanofiber membranes (B) with nanofiber diameter distribution (C). FTIR spectra, (D) and XRD patterns (E), water contact angle (F), mechanical properties (G), water absorption rate (H), moisturizing rate (I), \u003cem\u003ein vitro\u003c/em\u003e degradation (J), and drug release profile (K) of electrospun nanofiber membranes. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and n.s., not significant.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/88c5286c273ae43a9ce95100.jpg"},{"id":49711246,"identity":"4be15a57-7d01-4f93-84cd-ea99b21f1560","added_by":"auto","created_at":"2024-01-16 19:56:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2386441,"visible":true,"origin":"","legend":"\u003cp\u003eCytocompatibility assessments. Live/dead staining and SEM images of RAW 264.7 macrophages (A) and HaCaT cells (B) on electrospun nanofiber membranes. Cell proliferation rates (C and E) were assessed by CCK-8 assay. Cell survival rates (D and F) were determined by live/dead staining at day 1. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and n.s., not significant.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/f87058b14434647e24d1a71a.jpg"},{"id":49711244,"identity":"cbd5930e-243e-4de0-9d8a-a2509ec74432","added_by":"auto","created_at":"2024-01-16 19:56:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2196852,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003eanti-inflammatory assessments. Both IF imaging (A) and flow cytometry (B-D) demonstrate the P/G-CS-OI membrane is capable of downregulating M1 and upregulating M2 phenotypes of macrophages. RT-PCR analysis also confirms that the P/G-CS-OI membrane downregulates M1 (E) and upregulates M2 (F and G) phenotypes of macrophages, as well as downregulates pro-inflammatory genes of TNF-α(H), IL-1β (I), and IL-6(J) and upregulates anti-inflammatory genes of IL-4 (K) and IL-10 (L). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and n.s., not significant.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/159b7c4b4d9a1f4a653d2423.jpg"},{"id":49711245,"identity":"540d1d21-8753-4b5a-afa1-9fc1d9199196","added_by":"auto","created_at":"2024-01-16 19:56:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1522141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003eantioxidative assessments. DCFH staining (A) and flow cytometry (B) analysis reveal that the P/G-CS-OI membrane shows the greatest ROS clearance of LPS-challenged macrophages. It also significantly lowers the NO production (C) and enhances SOD activity (D) of LPS-challenged macrophages. RT-PCR analysis demonstrates that the P/G-CS-OI membrane upregulates NRF2 (E) and downregulates KEAP1 (F), as well as upregulates NRF2 downstream genes of HO-1 (G), NQO1 (H), GCLC (I), and GCLM (J). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, and n.s., not significant.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/c5276baed28ef1595291042b.jpg"},{"id":49711504,"identity":"3ceb45e0-ac76-472e-a3c3-0f369ddeb479","added_by":"auto","created_at":"2024-01-16 20:04:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1691702,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of P/G-CS-OI membrane treated macrophage conditioned medium on HaCaT cell migration and gene expression. Schematic of experimental design (A). Conditioned medium significantly promotes HaCaT cell migration (B) as evidenced by accelerated healing rate of scratch (C). It significantly upregulates VEGF expression (D) and downregulates K10 (E) expression and upregulates K14 (F) and IVI (G) expressions. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and n.s., not significant.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/c4725b77efcda5abf6a921d1.jpg"},{"id":49711248,"identity":"dc48961c-705e-4fc3-b22f-a37e8af8ad4d","added_by":"auto","created_at":"2024-01-16 19:56:53","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2951507,"visible":true,"origin":"","legend":"\u003cp\u003eGross observation and histological analyses of wound healing quality. Schematic of diabetic mouse preparation and wound dressing treatment with subsequent analysis endpoint (A). Gross images of wounds (B) with plotted wound closure curves (C). Histological images of H\u0026amp;E (D) and Masson’s trichrome (E) staining from day 3 to 14.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/406d277d28938099a9aac575.jpg"},{"id":49711249,"identity":"2d0da7ff-af8e-419a-852b-4071dfc49999","added_by":"auto","created_at":"2024-01-16 19:56:53","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2968955,"visible":true,"origin":"","legend":"\u003cp\u003eIF and IHC staining and ELISA analyses of wound healing quality. IF staining shows that the P/G-CS-OI membrane modulates macrophage polarization by down-regulating CD86+ cells and up-regulating CD206+ cells (A). The P/G-CS-OI membrane improves neovascularization as evidenced by increased numbers CD31+/α-SMA+ blood vessels and promotes epithelialization by intense K10+/K14+ layers (A). IHC staining (B) shows that the P/G-CS-OI membrane significantly alleviates inflammation by down-regulating TNF-α expression (C) and upregulating the expressions of IL-10 (D), VEGF (E), and NRF2 (F). ELISA analysis of healing tissues also confirms the P/G-CS-OI membrane significantly reduces the pro-inflammatory cytokine accumulation of TNF-α (G), IL-1β (H), and IFN-γ(I). It shows powerful \u003cem\u003ein vivo\u003c/em\u003e antioxidative capacity by stimulating SOD (J) and reducing MDA (K) production and also upregulating VEGF secretion (L). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and n.s., not significant.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/a468650b72ebb3eae19f668a.jpg"},{"id":52907288,"identity":"ee75701e-7907-45e5-9575-1549da3bcf88","added_by":"auto","created_at":"2024-03-18 15:11:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2115499,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/076a87b4-5852-4a61-bba3-1af6d6f6186f.pdf"},{"id":49711505,"identity":"c5f87e80-97ca-40b3-8b3b-18c910cef9ca","added_by":"auto","created_at":"2024-01-16 20:04:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5669475,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/b39e02fcbcd774830b8cdfaa.docx"},{"id":49711250,"identity":"8dc5b862-ea15-4a4a-abd4-46d8a94e25a4","added_by":"auto","created_at":"2024-01-16 19:56:53","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":709150,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Schematic illustration of a Chitosan-4-Octyl itaconate grafted nanofiber membrane (P/G-CS-OI), which can promote diabetic wound healing via macrophage modulation.\u003c/p\u003e","description":"","filename":"scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3853738/v1/8b057498c95fc25ed43bf551.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-inflammatory and anti-oxidative electrospun nanofiber membrane promotes diabetic wound healing via macrophage modulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003e Diabetes mellitus (DM), a prevalent chronic metabolic disorder, affects an estimated 536\u0026nbsp;million people globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Approximately 25% of these patients eventually develop diabetic foot ulcers (DFUs), with an amputation rate of 43.8% and a mortality rate of 51.7% within 6 years post-hospitalization for the ulcer, resulting in an estimated global economic burden of \u003cspan\u003e$\u003c/span\u003e8.5\u0026nbsp;billion [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Conservative medical interventions, including antibiotics, magnetic thermal therapy, and hyperbaric oxygen chamber therapy, are often associated with adverse reactions and incomplete treatment outcomes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, there is an urgent need for the development of new therapies to address chronic diabetic wounds.\u003c/p\u003e \u003cp\u003eThe process of wound healing includes three overlapping stages: inflammation, proliferation, and remodeling [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Macrophages, as the most active non-specific immune cells throughout the entire process, play a double-edged sword role [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. On the one hand, the activation of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) biases macrophage polarization toward M1 phenotype in chronic inflammatory milieu, leading to the secretion of pro-inflammatory mediators [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. On the other hand, an increased transformation of M2 phenotype macrophages in the latter stages secretes pro-healing chemokines and growth factors, which foster the recruitment of endothelial progenitor cells that are essential for neovascularization and ensure sufficient blood flow to the wound site [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, in the typical hyperglycemic microenvironment of diabetic conditions, both the phenotypic shift of macrophages and their debris-clearing efficiency are compromised, resulting in chronic inflammation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, strategies that modulate the physiological roles of macrophages pose significant implications for diabetic wounds.\u003c/p\u003e \u003cp\u003eIntermediates in macrophage glycolysis and the tricarboxylic acid cycle (TAC), such as lactic acid and citric acid, are increasingly recognized as key elements affecting macrophage function and phenotype [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Previous studies have demonstrated that activated M1 macrophages exhibit elevated expression of immune-responsive gene 1 (IRG1), which encodes aconitate decarboxylase and further catalyzes the conversion of the tricarboxylic acid cycle intermediate cis-aconitate into itaconic acid [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Structurally akin to methylene, itaconic acid modulates immune responses through the competitive inhibition and alkylation of target proteins [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Specifically, itaconic acid impedes the activity of succinate dehydrogenase in macrophages, and reverses the mitochondrial electron transport chain, thereby reducing inflammation by diminishing reactive oxygen species (ROS) production and altering macrophage metabolism [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. 4-Octyl itaconate (OI), a derivative of itaconic acid, is more permeable to cell membranes than its parent compound [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previous studies have demonstrated that OI can enter macrophages and be converted into itaconic acid to regulate mitochondrial metabolism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, in this study, we selected OI as a targeted therapeutic agent for modulating macrophage activity in the inflammatory context of diabetic wounds.\u003c/p\u003e \u003cp\u003eNowadays, many biomaterials with multifaceted functionalities, including anti-inflammatory, reparative, and antibacterial properties, have been developed for resolving chronic inflammation and facilitating diabetic wound healing [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, despite these promising functionalities, how to balance the biological and mechanical properties of these dressing materials to achieve optimal \u003cem\u003ein vivo\u003c/em\u003e effectiveness remains a critical challenge. Chitosan (CS), a linear, semi-crystalline natural polysaccharide derived from chitin, can act as a biocompatible, biodegradable biological scaffold with antioxidant and antibacterial properties [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Electrospun nanofiber membranes are featured by their biomimetic structure, high porosity, extensive surface area, softness, and compliance and have been extensively investigated for wound dressing applications [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Especially, CS-coated electrospun nanofiber membranes show great antibacterial and antioxidative activities and are attractive for wound treatment [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we reported a CS-OI grafted nanofiber membrane by electrospinning and assessed its excellent capability for dressing diabetic wounds in a mouse model (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). First, OI was conjugated to CS to obtain CS-OI conjugate, which was then covalently grafted to electrospun PCL/gelatin nanofibers to obtain PCL/gelatin-CS-IO (P/G-CS-OI) membrane. Physicochemical properties of the P/G-CS-OI membrane were characterized and its anti-inflammatory, antibacterial, and antioxidative activities, as well as biocompatibility and bioactivity, were assessed. Finally, the P/G-CS-OI membrane was used as dressings in a diabetic mouse model to assess its biological performance for curing chronic wounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003ePolycaprolactone (PCL, Mn\u0026thinsp;=\u0026thinsp;80 kDa) and type A gelatin (300 g Bloom) were obtained from Sigma Aldrich (St. Louis, Missouri, USA). Chitosan (CS, 90% deacetylated powder, Mw\u0026thinsp;=\u0026thinsp;200 kDa), 4-octenyl Itaconate (OI), and 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Glacial acetic acid and 2,2,2-trifluoroethanol (TFE) were obtained from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Sterile gauzes were obtained from Hainuo Medical Technologies Co., Ltd. (Qingdao, China). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). N-hydroxysuccinimide (NHS) was obtained from Adamas-beta (Shanghai, China). LB Broth and LB Broth Agar were purchased from Sangon Biotech (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eElectrospinning of P/G membranes\u003c/h2\u003e \u003cp\u003ePCL and type A gelatin were dissolved in TFE to achieve a total polymer concentration of 10% (w/v). The ratio of PCL to gelatin was 8:2, and the mixture was stirred at room temperature to ensure homogeneity. To enhance the miscibility between PCL and gelatin, a small quantity (0.2%, v/v) of glacial acetic acid was added to the solution. The electrospinning process was conducted by feeding this solution through a 20 G needle at a consistent rate of 1.5 mL/h, with an applied voltage of 15 kV to facilitate the formation of nanofibers. The as-spun nanofibers were collected on a slowly rotating mandrel (60 mm diameter, rotating at 120 rpm) to obtain PCL/gelatin (P/G) membranes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of P/G-CS-OI membranes\u003c/h2\u003e \u003cp\u003eThe synthesis of the CS-OI conjugate was achieved using EDC/NHS carbodiimide chemistry, which facilitates the formation of amide bonds between the amino groups of CS and the carboxyl groups of OI. Initially, chitosan was dissolved in 0.1M dilute hydrochloric acid to a concentration of 1% (w/v). Subsequently, OI, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), and NHS (N-hydroxysuccinimide) were added in succession, ensuring a final molar ratio of CS:OI:EDC:NHS at 50:1:10:5. The reaction was maintained at room temperature in a dark environment for 24 h. Then the reaction was air-dried, washed with ethanol to remove any unreacted residues, and air-dried again to obtain the CS-OI conjugate.\u003c/p\u003e \u003cp\u003eFor the functionalization of the P/G membranes, the membranes were immersed in a CS-OI aqueous solution (1 mg/mL ) at 37\u0026deg;C and agitated for 24 h. Post-infusion with the CS-OI conjugate, the membranes were soaked in MES (2-(N-morpholino) ethanesulfonic acid) buffer to facilitate the crosslinking process, which was carried out at room temperature over 24 h using an EDC:NHS molar ratio of 2:1. To remove any unreacted EDC and NHS, membranes were washed extensively with deionized water, undergoing six cycles of 30 min each. Finally, electrospun nanofiber membranes were vacuum-dried in an oven to remove residual solvent for subsequent studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of P/G-CS-OI membranes\u003c/h2\u003e \u003cp\u003eThe morphology of electrospun membranes was observed by scanning electron microscope (SEM, Phenom, XL, Netherlands). Fiber diameters of P/G-CS-OI membranes were measured from SEM micrographs by using Image J. An FTIR spectrometer (Nicolet iS 10, Thermo Fisher Scientific, USA) was used to identify the chemical structures of electrospun nanofiber membranes. The FTIR spectra were obtained by recording 32 scans between 4000 and 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a resolution of 0.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The XRD patterns of CS-OI were determined by X-ray diffractometer (D8 Advance, Bruker, Germany), detecting the crystalline phase of samples at 2θ between 5\u0026deg; and 60\u0026deg;.\u003c/p\u003e \u003cp\u003eWe employed the SL200A contact angle analyzer (Solon Tech., Shanghai, China) to obtain the water contact angle (WCA) of nanofiber membranes. A water droplet (5 \u0026micro;L) was placed on the surface of nanofiber membranes and the dynamic water contact angle was recorded. The contact angle was measured by using Image J (n\u0026thinsp;=\u0026thinsp;3). The mechanical properties of electrospun membranes were evaluated using an uniaxial tensile test as described in our prior publication [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The rectangular samples (10 \u0026times; 40 mm) underwent incubation in phosphate-buffered saline (PBS) at a temperature of 37 ℃ for 24 h. Afterward, samples were positioned within the grips of a uniaxial testing machine (Instron 5567, Norwood, MA) equipped with a 50 N load cell and tested until failure at a crosshead speed of 10 mm/min. The ultimate tensile strength (UTS) was defined as the maximum load before failure and Young's modulus was calculated as the slope of the stress-strain curve's initial 5% linear section (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003eThe water absorption and moisturizing rate of electrospun membranes were measured by weighing method. To determine the water absorption rate, electrospun nanofiber membranes were tailored into 1 \u0026times; 1 cm squares and weighed (W\u003csub\u003e0\u003c/sub\u003e), then immersed in deionized water at room temperature, wiping away the surface water carefully using filter paper and weighing again (W\u003csub\u003et\u003c/sub\u003e) (n\u0026thinsp;=\u0026thinsp;3). The water absorption rate of membranes was calculated by the following equation: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(water absorption rate \\left(\\text{%}\\right)= ({W}_{t}-{W}_{0})/{W}_{0}\\times 100\\text{%}\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTo determine the moisturizing rate, electrospun nanofiber membranes were tailored into 1 cm \u0026times;1 cm squares and weighed (W\u003csub\u003e0\u003c/sub\u003e) and incubated in deionized water for 20 min at room temperature, and weighed (W\u003csub\u003e1\u003c/sub\u003e). Membranes were placed in a room temperature environment and weighed (W\u003csub\u003e2\u003c/sub\u003e) at predesignated time points (n\u0026thinsp;=\u0026thinsp;3). The moisturizing rate of membranes was calculated by the following equation:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\text{m}\\text{o}\\text{i}\\text{s}\\text{t}\\text{u}\\text{r}\\text{i}\\text{z}\\text{i}\\text{n}\\text{g} \\text{r}\\text{a}\\text{t}\\text{e} \\left(\\text{%}\\right)= ({W}_{2}-{W}_{0})/({W}_{1}-{W}_{0})\\times 100\\text{%}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e degradation and drug release were conducted by immersing electrospun membranes in PBS under a shaker incubator at 37\u0026deg;C. To determine the \u003cem\u003ein vitro\u003c/em\u003e degradation, electrospun nanofiber membranes were tailored into 1 cm \u0026times;1 cm squares and weighed (W\u003csub\u003e0\u003c/sub\u003e). Incubated samples were retrieved, rinsed with deionized water, lyophilized, and weighed (W\u003csub\u003ew\u003c/sub\u003e) at predesignated time points (n\u0026thinsp;=\u0026thinsp;3). The remaining mass was determined according to the following equation:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(Percentage of remained mass \\left(\\text{%}\\right)=\\frac{{W}_{w}}{{W}_{0}}\\times 100\\text{%}\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe drug release of OI from the P/G-CS-OI membrane was assessed through UV-vis spectrophotometry. UV analysis was conducted on the incubated solution (1 mL) at specified time intervals, and an equivalent volume of fresh PBS was substituted. The concentration of OI released from P/G-CS-OI membranes was determined by comparing it to the standard curve based on known OI concentrations (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial test\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the P/G-CS-OI membranes was evaluated against two commonly found bacterial species. More specifically, \u003cem\u003eE. coli\u003c/em\u003e (ATCC 25922) and \u003cem\u003eS. aureus\u003c/em\u003e (ATCC 25923) were cultured in Luria broth under shaking conditions for 24 h at a temperature of 37 ℃. Subsequently, each sample was inoculated with 200 \u0026micro;L of bacterial inoculum solution (10\u003csup\u003e8\u003c/sup\u003e CFU/mL ) and then incubated at 37 ℃ for 24 h. The bacterial solution was then diluted and cultured with 100 \u0026micro;L of diluent on LB Agar for 12 h, allowing for the observation of colony counts and the subsequent calculation of the Normalized survival rate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe RAW 264.7 macrophages and human immortalized keratinocytes (HaCaT) were maintained in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Cells were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e, with the culture medium being refreshed every two days to maintain optimal cell growth and viability. Before cell seeding, electrospun nanofiber membranes were prepared for biological interaction studies. Each membrane was carefully punched using a sterile hole punch to match the dimensions of the wells in standard cell culture plates. Following this, the membranes were subjected to UV sterilization to ensure aseptic conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCytocompatibility assessments\u003c/h2\u003e \u003cp\u003eInitially, the nanofiber membranes were cut to match the size of the wells in a 48-well plate and subjected to 24 h of UV sterilization, with a midway turnover to ensure complete exposure. Following sterilization, RAW 264.7 macrophages and HaCaT keratinocytes were revived and cultured. These cells were then seeded into the 48-well plates containing the sterilized nanofiber membranes at a density of 20,000 cells per well. The cultures were maintained at 37\u0026deg;C, with the culture medium changing every other day.\u003c/p\u003e \u003cp\u003eCell viability and proliferation on the electrospun nanofiber membranes were assessed at 1, 4, and 7 days post-seeding. Before the test, the culture medium was aspirated, and the cells were washed thrice with PBS. The viability and proliferation rates were quantitatively measured using live/dead staining reagents and a Cell Counting Kit-8 assay (Biosharp), following the manufacturer's instructions (n\u0026thinsp;=\u0026thinsp;9). The cell survival rate was calculated using the formula: cell survival rate (%) = (Viable cells/Total cells) \u0026times; 100%.\u003c/p\u003e \u003cp\u003eIn addition to these quantitative assays, cell adhesion and morphology on the nanofiber membranes were visually analyzed. At day 7, the cells cultured on the membranes were fixed with 4% paraformaldehyde, sequentially dehydrated in graded ethanol solutions, and air-dried at room temperature. The samples were then sputter-coated with gold to enhance conductivity and imaged using a scanning electron microscope (SEM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHemocompatibility assessments\u003c/h2\u003e \u003cp\u003eFresh blood was collected from healthy mice and centrifuged at 1000 rpm for 10 min to isolate pure red blood cells (RBCs). The RBCs were washed three times and subsequently diluted in PBS to achieve a final concentration of 2%. For the assay, electrospun nanofiber membranes with a diameter of 8 mm were introduced into 250 \u0026micro;L of the RBC suspension and incubated at 37\u0026deg;C for 1 h. As controls, PBS and Triton X-100 were employed as the negative and positive controls, respectively, and mixed with the RBC suspension under identical conditions. Following the incubation period, all samples underwent centrifugation at 1000 rpm for 5 min.\u003c/p\u003e \u003cp\u003eThe supernatant of each sample was then analyzed spectrophotometrically to measure the absorbance of released hemoglobin at 540 nm. The hemolysis ratio, indicative of the degree of damage to the RBCs, was calculated using the following formula: Hemolysis ratio (%) = [(Abs - Absn)/(Absp - Absn)] \u0026times; 100%, where Abs represents the absorbance of the sample, Absp the absorbance of the positive control, and Absn the absorbance of the negative control (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSubcutaneous implantation\u003c/h2\u003e \u003cp\u003eElectrospun membranes were punched into 8 mm discs and sterilized by UV (12 h for each side) and were subcutaneously implanted in seven-week-old female rats (weighing approximately 280\u0026ndash;320 g, purchased from Shanghai Slack Laboratory Animal Limited Liability Company, Shanghai, China). The rats were anesthetized by intraperitoneal injection of chloral hydrate (10%), and pockets were created by blunt dissection of the fascia between the skin and muscle. Electrospun membranes were embedded in the four pockets of the same rat and closed with sutures (n\u0026thinsp;=\u0026thinsp;3). After 7 and 28 days, the entire subcutaneous tissue containing electrospun nanofiber membranes was excised, and fixed with 4% paraformaldehyde overnight. Paraffin-embedded tissues and sectioned into 5 \u0026micro;m sections for staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eThe electrospun nanofiber membranes were first placed in 6-well plates, and RAW264.7 cells were seeded on their surfaces at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. Following the incubation, the culture medium was discarded, and the cells were washed three times with PBS to remove debris and non-adherent cells. Lipopolysaccharide (LPS, TargetMol) was then added to the culture to induce an inflammatory response, simulating diabetic wound conditions. After another 24 h culture period, cells were collected.\u003c/p\u003e \u003cp\u003eFor immunophenotyping, the cell pellets were resuspended in 100 \u0026micro;L of PBS containing 1.25 \u0026micro;g of PE-conjugated anti-mouse CD86 antibody and 0.25 \u0026micro;g of FITC-conjugated anti-mouse CD206 antibody (Biolegend). This step was performed on ice for 30 min to preserve cell viability and prevent nonspecific antibody binding. The expression of CD86 and CD206 was subsequently analyzed using the CytoFLEX flow cytometry system (Beckman Coulter). Additionally, the intracellular ROS level was detected using DCFH-DA (Yeasen), following the same procedure as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eReal‑time polymerase chain reaction (RT‑PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the samples using the EZ-press RNA Purification Kit (EZBioscience, Roseville, MN, USA). Following extraction, the RNA was reverse transcribed into complementary DNA (cDNA) utilizing the Colour Reverse Transcription Kit (EZBioscience). Quantitative RT-PCR was then performed using the 2 \u0026times; Colour SYBR Green qPCR Master Mix (EZBioscience). The relative expression levels of the target genes were calculated employing the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method (n\u0026thinsp;=\u0026thinsp;3). Primer sequences are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (Additional file 1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eScratch assay\u003c/h2\u003e \u003cp\u003eHaCaT cells were seeded into 6-well plates at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. Upon reaching approximately 90% confluence, a scratch was carefully made through the cell monolayer to simulate a wound. Following that, macrophage-conditioned medium was added to the wells. To monitor and quantify the process of wound closure, three random fields of view along the scratch were selected in each well. These areas were photographed using an optical microscope at 0, 24, and 48 h post-scratch.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStreptozotocin (STZ)‑induced diabetic mice\u003c/h2\u003e \u003cp\u003e The animal study protocols were rigorously designed and executed in compliance with the ethical guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Sixth People's Hospital Affiliated with the Shanghai Jiao Tong University School of Medicine. A cohort of 36 male ICR mice, aged between 6 and 8 weeks, was utilized for the establishment of a diabetic model. The mice were housed under controlled environmental conditions, maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃, with three animals per cage to ensure adequate space and social interaction. The diabetic state in the mice was induced by 150 mg/kg of STZ (\u0026ge;\u0026thinsp;98% purity; Beyotime). To enhance the effectiveness of STZ, mice were subjected to a 24 h fasting before the administration of STZ. Subsequently, the blood glucose levels of mice were monitored regularly by sampling from the tail vein 3 days post-injection. A continuous blood glucose level greater than or equal to 16.7 mM was considered the successful establishment of diabetes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWound healing evaluation\u003c/h2\u003e \u003cp\u003eAfter anesthesia, the dorsal fur of the mice was carefully removed using an electric animal razor, and the surgical area was disinfected with 75% alcohol. Mice were subsequently divided into three distinct groups: Control, P/G-CS, and P/G-CS-OI, with each group comprising 12 animals. In each mouse, a full-thickness skin wound of 8 mm diameter was created on the back using a sterile biopsy punch. The wounds were then covered with the respective electrospun nanofiber membranes, wrapped by sterile gauzes and secured with sutures. The progression of wound healing was documented at 0, 3, 7, 10, and 14 days post-wounding by capturing digital photographs of the wounds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eHealing tissues of animals 3, 7, 10, and 14 days post-surgery were harvested and subjected to a series of histological preparations. Samples were then stained using hematoxylin and eosin (H\u0026amp;E; Solarbio) and Masson\u0026rsquo;s trichrome (Solarbio). Immunofluorescence staining was performed to evaluate the macrophage phenotype and extent of tissue repair in the injured skin tissues. Specific antibodies including CD31 (1:4000; Proteintech), CD86 (1:200; Abways), CD206 (1:300; Proteintech), α-SMA (1:300; Proteintech), K10 (1:100; Abways), K14 (1:100; Abways).And Goat Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Cy3 (1:200; Abways) and Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Alexa Fluor 488 (1:200; Abways), were used to label the target cells and proteins. The stained sections were examined under a Ci-S microscope (Nikon) for morphological and quantitative analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eELISA assay\u003c/h2\u003e \u003cp\u003eThe tissue samples were homogenized and sonicated in pre-cooled PBS. The homogenates were then centrifuged at 12,000 g for 5 min, and the supernatants were collected. The expression levels of various cytokines were quantitatively assessed using an ELISA kit (Servicebio, Wuhan, China), following the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using GraphPad Prism. One-way or two-way analysis of variance (ANOVA) with post hoc multiple comparisons and Student's t-test were used to analyze significance, where appropriate. A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of electrospun membranes\u003c/h2\u003e \u003cp\u003eThe fabrication and structural characterization of surface-modified nanofiber membranes P/G-CS-OI are detailed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. SEM images reveal a dense arrangement of porous, randomly oriented nanofibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The average diameters of P/G, P/G-CS, and P/G-CS-OI membranes were 299\u0026thinsp;\u0026plusmn;\u0026thinsp;54 nm, 352\u0026thinsp;\u0026plusmn;\u0026thinsp;63 nm, and 371\u0026thinsp;\u0026plusmn;\u0026thinsp;91 nm, respectively. Covalent grafting of CS and CS-OI to P/G nanofibers led to increased fiber diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) but did not alter the randomly oriented nanofiber structure.\u003c/p\u003e \u003cp\u003eFTIR was employed to analyze the chemical composition of the CS-OI conjugate and electrospun nanofiber membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). CS exhibited characteristic absorption bands of functional groups: amide I (C\u0026thinsp;=\u0026thinsp;O stretching) at 1654 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, amide II (N-H bending) at 1555 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and amide III (C-N stretching) at 1318 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the OI spectra, the prominent band at 1721 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponded to the C\u0026thinsp;=\u0026thinsp;O stretching of carboxyl groups. For CS-OI conjugate, the band intensity increased at 1643 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1521 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating the formation of amide I and amide II bonds between the carboxyl group of OI and the amino group of the CS chain through amide bonds. In addition, the characteristic absorption band of the OI monomer at 1727 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was also observed in the CS-OI conjugate, further proving the existence of the OI component in the CS-OI conjugate. Therefore, these results demonstrate that the grafting reaction occurs in the carboxyl group of OI and the amino group of CS through the amide bond. For P/G electrospun membranes, the characteristic peaks of amide I and amide II at 1643 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1543 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, confirmed the presence of gelatin within the electrospun membranes. For P/G-CS and P/G-CS-OI membranes, the characteristic peak of the amide Ⅰ band shifts toward a higher wavenumber (from 1643 to 1650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and such shift in peak positions implies weakening or lengthening of bonds, confirming the existence of an interaction of the OI with the CS [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eXRD patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) demonstrate that CS had a broad peak at 2θ\u0026thinsp;=\u0026thinsp;19.9 showing its high crystallinity, aligning with previous reports [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The high crystallinity of chitosan is generally caused by inter- and intra-molecular hydrogen bonds [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. OI displayed multiple narrow and sharp diffraction peaks that indicate its characteristic of crystalline materials. The CS-OI conjugate exhibited a smooth diffraction pattern, indicating reduced crystallinity and the consequent increase of the amorphous phase, confirming the successful conjugation of OI onto CS. Lower crystallinity would lead to the increase of solubility in water, CS can only be soluble in acid conditions (pKa \u0026sim; 5.5) before reaction and CS-OI were easily soluble in water. This was probably due to that OI hinders the inter- and intra-molecular hydrogen bonds network of chitosan molecules [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. CS-OI showed water-solubility facilitating subsequent P/G-CS-OI membrane preparation in our study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHydrophilicity is a critical property influencing material-tissue contact of wound dressings. The electrospun P/G nanofiber membrane had a water contact angle of 53\u0026thinsp;\u0026plusmn;\u0026thinsp;7\u0026deg;, indicating its hydrophilic nature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). This is due to the presence of gelatin that greatly reduces the hydrophobicity of PCL. The introduction of After grafting with CS and CS-OI, the obtained nanofiber membranes had contact angles of 57\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg; and 47\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;, respectively, which were not significant from the P/G nanofiber membrane (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This result suggests that the overall hydrophilicity is not significantly impacted by the addition of CS or CS-OI.\u003c/p\u003e \u003cp\u003eTo guarantee a close and comfortable interaction with the wound, as well as to regulate the tension exerted on it, the mechanical characteristics of the wound dressing should resemble those of human skin. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, the UTS for P/G, P/G-CS, and P/G-CS-OI membranes were measured at 12\u0026thinsp;\u0026plusmn;\u0026thinsp;3 MPa, 19\u0026thinsp;\u0026plusmn;\u0026thinsp;2 MPa, and 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 MPa, respectively, highlighting a marked increase in strength with the conjugation of CS and CS-OI. Moreover, P/G-CS had the highest Young's modulus (36\u0026thinsp;\u0026plusmn;\u0026thinsp;4 MPa), surpassing both P/G and P/G-CS-OI (18\u0026thinsp;\u0026plusmn;\u0026thinsp;2 MPa and 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 MPa, respectively). The strains at failure were 153\u0026thinsp;\u0026plusmn;\u0026thinsp;7% for P/G, 112\u0026thinsp;\u0026plusmn;\u0026thinsp;13% for P/G-CS, and 192\u0026thinsp;\u0026plusmn;\u0026thinsp;13% for P/G-CS-OI, P/G-CS-OI membranes exhibited the greatest strains at failure. Human skin typically exhibits a tensile modulus ranging from 15 to 150 MPa, an ultimate tensile stress between 1 and 32 MPa, and an ultimate tensile strain from 35 to 115% [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These results indicate that our nanofiber membranes could provide suitable tensile properties for wound dressing.\u003c/p\u003e \u003cp\u003eThe water absorption and retention capabilities of nanofiber membranes are critical for their efficacy as wound dressings, particularly in absorbing tissue exudates. The water absorption rates of the nanofiber membranes were evaluated over 60 min, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ. The rates were determined to be 743\u0026thinsp;\u0026plusmn;\u0026thinsp;42%, 670\u0026thinsp;\u0026plusmn;\u0026thinsp;40%, and 584\u0026thinsp;\u0026plusmn;\u0026thinsp;62% for P/G, P/G-CS, and P/G-CS-OI membranes, respectively, with all three types reaching maximum absorption within the first 15 min. In terms of moisture retention, as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, the rates were 19\u0026thinsp;\u0026plusmn;\u0026thinsp;2%, 15\u0026thinsp;\u0026plusmn;\u0026thinsp;3%, and 21\u0026thinsp;\u0026plusmn;\u0026thinsp;3% for P/G, P/G-CS, and P/G-CS-OI membranes, respectively. These results show that our nanofiber membrane can absorb the solution quickly, reach the maximum absorption ratio in a short time, and remain stable. As a wound dressing, it can maintain a moist environment around the wound, which is favorable for wound healing [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e degradation of nanofiber membranes was evaluated in PBS solution at 37\u0026deg;C for up to 42 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). The remaining mass of P/G-CS and P/G-CS-OI samples was around 95% after 2 weeks, decreasing to 82% after 6 weeks. For P/G samples, the remaining mass was 93% in the second week, decreasing to 80%, suggesting a slightly faster degradation rate compared to P/G-CS and P/G-CS-OI membranes. The nanofiber membrane exhibits slow degradation characteristics, which fulfill the requirements of wound dressings. This feature effectively prevents the negative impact of degradation products on the wound. Additionally, it aids in extending the lifespan of the dressing during the wound-healing process, reducing the need for frequent dressing replacements. As a result, it minimizes the secondary trauma caused by dressing changes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe slow degradation of the membrane favors the long-term release of the active ingredient, and we evaluated the cumulative release of OI from the P/G-CS-OI membranes for up to 28 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Within the first 2 weeks, P/G-CS-OI membranes released 75% of OI and exhibited a relatively slow release of OI for a period of up to 28 days. In our study, conjugating small molecule OI to polymeric carries CS has been applied to increase the bioavailability of OI, and the drug delivery system can be further stabilized by crosslinking CS-OI conjugates to nanofibers. In the drug release experiments, P/G-CS-OI membranes achieved long-term release effects, which help to play the biological role of OI for a long time to help skin repair.\u003c/p\u003e \u003cp\u003eThe impaired immune defense system induced by diabetes can easily lead to recurrent bacterial infections in chronic hard-to-heal wounds, and the increasing exudate provides a breeding ground for bacterial growth [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The antibacterial efficacy of the nanofiber membranes was identified using common wound pathogens, \u003cem\u003eE.coli\u003c/em\u003e (Gram-negative) and \u003cem\u003eS.aureus\u003c/em\u003e (Gram-positive) (Fig. S2, Additional file 1). The control and P/G groups showed more bacterial colonies, while the P/G-CS and P/G-CS-OI groups had significantly fewer bacterial colonies. The normalized survival of \u003cem\u003eE. coli\u003c/em\u003e co-cultured with P/G-CS and P/G-CS-OI membranes was 60\u0026thinsp;\u0026plusmn;\u0026thinsp;7% and 71\u0026thinsp;\u0026plusmn;\u0026thinsp;2%, respectively, and \u003cem\u003eS. aureus\u003c/em\u003e was 74\u0026thinsp;\u0026plusmn;\u0026thinsp;9% and 63\u0026thinsp;\u0026plusmn;\u0026thinsp;3%, respectively. Despite the antimicrobial effect of OI [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], it is mainly CS that exerts the antimicrobial function in the P/G-CS-OI membranes. In the acidic microenvironment of wounds, its amino group is positively charged, and it can bind with the negative charge on the surface of the bacterial cell membrane, which alters the permeability of the cell membrane and leads to metabolic disorders of the bacterium, to achieve the antibacterial effect [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the CS-OI conjugate, part of the amino group of CS was covalently coupled with the carboxyl group of OI, and for the P/G-CS-OI membranes, the amino group of CS was further substituted, which reduced the bacteriostatic effect of the P/G-CS-OI membranes. However, it is still suitable for the prevention of bacterial infection in wound care.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eBiocompatibility of electrospun membranes\u003c/h2\u003e \u003cp\u003eThe cytocompatibility of electrospun membranes was evaluated with RAW 264.7 and HaCaT cells. Live/dead staining shows that both RAW 264.7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and HaCaT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) cells experience robust proliferation on electrospun membranes. Most cells were live (green stain) with very few dead cells (red stain) from day 1 to 7. Both cells showed a great increase in populations over time. SEM images reveal that cells are closely attached to the nanofibers. CCK-8 assays demonstrated sustained increases in cellular metabolic activity, with no discernible cytotoxic effects attributable to the electrospun nanofiber membranes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and E). Moreover, both cells had viability rates greater than 90% after 24 h on electrospun membranes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results suggest that the P/G-CS-OI membrane has good cytocompatibility.\u003c/p\u003e \u003cp\u003eGiven that wound dressings are destined for direct contact with blood in vivo, assessing the potential for hemolysis is a crucial aspect of hemocompatibility testing (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Additional file 1). Results from these assays yielded supernatants that were light yellow in color post-contact with the different membranes. This outcome paralleled the negative control and stood in stark contrast to the bright red of the positive control. The P/G, P/G-CS, and P/G-CS-OI membranes were associated with hemolysis rates of less than 2%, a figure well below the international safety benchmark of 5%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe aforementioned in vitro biocompatibility findings were validated through in vivo experiments that involved subcutaneous implantation of the electrospun nanofiber membranes in a mouse model. Subsequent histological analyses at days 7 and 28 post-implantation indicated only minimal immune reactions to the P/G, P/G-CS, and P/G-CS-OI membranes, as evidenced by H\u0026amp;E staining (Fig. S2A, Additional file 1). Although encapsulated by fibroblast layers, significant infiltration by neutrophils or monocytes was notably absent. Masson's trichrome staining illustrated tissue-related fiber proliferation and collagen deposition, indicative of integration following membrane implantation (Fig. S2B, Additional file 1). Measurement of the fibrous capsule thickness, one marker of immune response, revealed a decrease in thickness for samples P/G-CS and P/G-CS-OI, from 408\u0026thinsp;\u0026plusmn;\u0026thinsp;33 \u0026micro;m and 237\u0026thinsp;\u0026plusmn;\u0026thinsp;37 \u0026micro;m at day 7, to 233\u0026thinsp;\u0026plusmn;\u0026thinsp;23 \u0026micro;m and 161\u0026thinsp;\u0026plusmn;\u0026thinsp;14 \u0026micro;m on day 28, respectively (Fig. S2C, Additional file 1). In contrast, P/G samples exhibited an increase in capsule thickness over the same period.\u003c/p\u003e \u003cp\u003eFurthermore, analysis of cell infiltration depth at the tissue-membrane interface revealed lower figures for the P/G-CS and P/G-CS-OI membranes compared to P/G alone at both 7 and 28 days post-implantation (Fig. S2D, Additional file 1). Collectively, these findings reinforce the conclusion that the fabricated electrospun nanofiber membranes exhibit substantial biocompatibility, advocating for their application in wound healing interventions.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMacrophage anti-inflammatory modulation of P/G-CS-OI membrane\u003c/h2\u003e \u003cp\u003eMacrophages are indispensable in mounting a local immune response within injured tissues, an imbalance characterized by the predominance of M1 macrophages and impaired conversion to M2 macrophages can exacerbate tissue damage and impede wound healing processes [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, we investigated the impact of the P/G-CS-OI membranes on macrophage polarization under LPS challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows a notable upregulation of the levels of CC Chemokine Receptor 7 (CCR7) upon LPS exposure. Remarkably, the application of P/G-CS-OI membranes led to significant attenuation in the fluorescence signal of CCR7, concomitantly with an upsurge in the Arginase 1 (ARG1) signal, indicative of a shift towards M2 macrophage polarization. Flow cytometry analysis reveals a substantial decrease (7.26%) in the percentage of M1 macrophages (CD86+), alongside a marked increase (20.84%) in M2 macrophages (CD206+), after P/G-CS-OI treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eTo further elucidate the role of P/G-CS-OI in macrophage polarization and the regulation of inflammatory mediators, RT-PCR was employed (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-L). The RT-PCR results demonstrated a significant increase in the gene expression of CCR7, tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, and IL-6 in LPS-activated RAW264.7 cells, coupled with a marked decrease in IL-4 expression. Interestingly, the level of IL-10 did not exhibit the anticipated decrease, suggesting an inherent cellular self-regulation mechanism aimed at preventing excessive inflammation. In the P/G-CS-OI group, RT-PCR results indicated a substantial downregulation in the expression of pro-inflammatory genes (CCR7, TNF-α, IL-1β, and IL-6) and upregulation in ARG1, Macrophage Scavenger Receptor 1 (MSR1), IL-4, and IL-10, underscoring the potent immunomodulatory and pro-repair capabilities of the P/G-CS-OI membranes. It is noteworthy that MSR1, as one of the markers of M2 macrophages, can enhance the phagocytosis ability of macrophages. This facilitates the clearance of bacterial fragments and apoptotic cells, thereby contributing to the improvement of the chronic wound inflammatory microenvironment [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese findings collectively suggest that the use of P/G-CS-OI membranes, not only promotes the transformation of macrophages from the M1 to the M2 phenotype but also enhances their phagocytic function and anti-inflammatory activity. This dual action considerably augments the potential for improved skin repair in chronic wound scenarios, highlighting the therapeutic value of P/G-CS-OI membranes in modulating the immune response and facilitating tissue regeneration.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant activity of P/G-CS-OI membrane\u003c/h2\u003e \u003cp\u003eThe excessive ROS accumulation in diabetic wounds can not only oxidize surrounding lipids and proteins, causing cellular damage but also promote the chemotaxis of inflammatory factors to the wound site, thus hindering skin regeneration [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, the development of wound dressings with antioxidant capabilities has become the focus of current research [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To this end, RAW 264.7 cells were cultured in a pathological oxidative microenvironment, induced by 100 ng/mL LPS, and intracellular ROS levels were assessed using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA). The resulting fluorescence signal was quantified and characterized using both flow cytometry and fluorescence microscopy. The results, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, indicated that in the LPS-treated groups (Control and P/G-CS), DCFH-DA exhibited significantly higher green fluorescence compared to the control group. Notably, the use of P/G-CS-OI membranes effectively reduced intracellular oxidative stress. Flow cytometry quantitative data, presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, showed that the percentage of FITC-positive channels in the P/G-CS-OI group was significantly lower compared to other experimental groups. This finding highlights the potent ROS scavenging ability of P/G-CS-OI membranes, underscoring its potential as an effective antioxidant in wound dressing applications.\u003c/p\u003e \u003cp\u003eNitric Oxide (NO) is a versatile signaling molecule produced by various cell types and plays a pivotal role in inflammation and oxidative stress in skin tissue [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Being a free radical, NO can react with superoxide anions to form peroxynitrite, a potent oxidizing agent that inflicts damage on surrounding cells and tissues [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Superoxide Dismutase (SOD) is a metalloenzyme that catalyzes the disproportionation of superoxide anions, thereby maintaining cellular REDOX balance [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our study demonstrates that the application of P/G-CS and P/G-CS-OI membranes significantly reduces NO levels, increases SOD activity, and consequently mitigates cellular oxidative stress damage, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD.\u003c/p\u003e \u003cp\u003eIn chronic diabetic wounds, persistent hyperglycemia induces an accumulation of damaged mitochondria, leading to an overproduction of coenzyme Q and subsequent impairment of the normal electron transport within the respiratory chain [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This mitochondrial dysfunction redirects a substantial number of electrons back to mitochondrial complex I, creating a significant imbalance between ROS production and elimination [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Such an imbalance can result in lipid peroxidation within macrophages, leading to stromal swelling and rupture of the outer membrane [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Nuclear Factor Erythroid 2\u0026ndash;Related Factor 2 (NRF2) is a key transcription factor that coordinates cellular defense mechanisms against oxidative and electrophilic stress [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. It regulates the transcription of antioxidant genes dependent on the Antioxidant Response Element (ARE). This regulation includes the expression of genes such as NAD(P)H Quinone Dehydrogenase 1 (NQO1), Glutamate Cysteine Ligase Catalytic Subunit (GCLC), Glutamate Cysteine Ligase Modifier Subunit (GCLM), and Heme Oxygenase 1 (HO-1) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Kelch-like ECH-associated protein 1 (KEAP1), a dimeric protein, normally binds to NRF2 in the cytoplasm and facilitates its degradation via the proteasome system. Hence, KEAP1 is also recognized as an inhibitor of NRF2. Our experiment had confirmed that P/G-CS-OI membranes could significantly up-regulate the expression of NRF2 and its downstream antioxidant components NQO1, GCLC, GCLM, and HO-1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-J), thereby clearing excess ROS and maintaining REDOX homeostasis in the cellular environment. This result is mainly attributable to OI, which plays a crucial role in this regulatory mechanism by alkylating cysteine residues (151, 257, 288, 273, and 297) on KEAP1. This modification prompts the dissociation of NRF2 from KEAP1, allowing NRF2 to translocate to the nucleus where it interacts with ARE, thereby enhancing the expression of downstream genes with antioxidant properties and creating suitable conditions for the healing of chronic wounds. However, since the main function of OI is to alkalize KEAP1 and promote its dissociation, the influence on the expression level of KEAP1 is relatively small (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eBioactivity of P/G-CS-OI for proliferation of keratinocytes\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of macrophage immune response on the formation and differentiation of blood vessels and epidermis, RAW 264.7 cells were planted in different groups of electrospun nanofiber membranes and activated with lipopolysaccharide (LPS) to simulate the inflammatory microenvironment. The conditioned medium obtained was used to grow HaCaTs and evaluate the effects of cytokines and growth factors produced by macrophages under different conditions on the differentiation and maturation of HaCaTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe investigated the effects of different medium conditions on HaCaT cell migration through scratch experiments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, neither P/G nor P/G-CS showed enhanced cell migration, while P/G-CS-OI showed a higher ability to promote cell migration. This trend was consistent with the polarization change of macrophages, indicating that the conditioned medium obtained by macrophages after remodeling had obvious chemotactic effect on epidermal repair cells. However, the mechanism underlying accelerated HaCaT cell migration remains unknown in the current study.\u003c/p\u003e \u003cp\u003eWe further measured gene expression in different groups of RAW 264.7 and HaCaT cells. We found that RAW 264.7 cells up-regulated vascular endothelial growth factor (VEGF) under the influence of P/G-CS-OI membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), VEGF can promote endothelial cell migration, proliferation, and differentiation. It also helps to form new blood vessel structures, providing enough oxygen and nutrients to promote growth, differentiation, and recovery of damaged tissues. Based on good vascularization, the proliferation and differentiation of keratin can provide an important structural role in wound healing. Keratin 10 (K10) belongs to the type II keratin family and is closely associated with terminal differentiation in the epidermis of the skin. Keratin 14 (K14), a type I keratin family, is a marker of the undifferentiated state of epidermal cells, and an increased ratio of K14/ K10 often indicates that epidermal cells are in a period of rapid proliferation. Involucrin (IVI) is an amorphous protein that is one of the early markers of epidermal differentiation, and the level of IVI expression in HaCaT cells can be used to assess the degree of cell differentiation. Our detection results showed that HaCaT cells under the influence of P/G-CS-OI condition medium showed lower K10 expression and higher K14 and IVI expression than P/G and P/G-CS groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-G). These results may be attributed to the remodeling of macrophages by electrospun nanofiber membranes. Macrophages are key players in the innate immune system and are closely involved in tissue repair and regeneration. They are not only an important source of pro-fibrotic mediators and various growth factors, but also a balancer of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases. The remodeling of macrophages can drive cell activation and extracellular matrix turnover in the inflammatory microenvironment after injury [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eDiabetic wound healing\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e studies were conducted to evaluate the role of the P/G-CS-OI membranes in promoting wound healing in a diabetic full-thickness skin defect model, and the wound repair process in each treatment group was observed at days 0, 3, 7, 10, and 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, on day 3, the P/G-CS-OI membranes already showed a higher wound closure rate (50\u0026thinsp;\u0026plusmn;\u0026thinsp;4%) compared with P/G (7\u0026thinsp;\u0026plusmn;\u0026thinsp;5%) and P/G-Cs (20\u0026thinsp;\u0026plusmn;\u0026thinsp;13%). After 14 days, wounds in P/G-CS-OI were almost completely healed, while marked scarring in the other groups indicated delayed wound repair.\u003c/p\u003e \u003cp\u003eTo further elucidate the role of electrospun nanofiber membranes in promoting chronic wound healing, histological analyses were performed. H\u0026amp;E staining images showed that compared with P/G and P/G-CS groups, there was more neovasculation and hair follicle formation in the regenerated skin of P/G-CS-OI groups, and there was less infiltration of inflammatory cells such as monocytes and neutrophils. After 14 days of treatment, complete re-epithelialization was observed in the wounds treated with P/G-CS-OI, characterized by typical cortical structures and morphological features (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Additionally, the thickness of the granulation tissue, a vital indicator of the wound healing process, was assessed using Masson tri-color staining to determine collagen deposition and scar length in the tissue. It is not difficult to see that compared with P/G and P/G-CS, the granulation tissue thickness of P/G-CS-OI is thicker, scar length and wound margin are significantly reduced, and there is a lot of dense and orderly collagen accumulation under the epidermis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eChronic wounds are hallmarked by prolonged inflammation, which is characterized by a dominant M1 macrophage polarization, elevated levels of inflammatory cytokines, and compromised processes of angiogenesis and epithelial regeneration [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In addition to \u003cem\u003ein vitro\u003c/em\u003e results of P/G-CS-OI membranes facilitating macrophage phenotypic remodeling, our study further investigated the impact of these electrospun nanofiber membranes on inflammatory infiltration and tissue repair \u003cem\u003ein vivo\u003c/em\u003e. Immunofluorescence images showed a widespread distribution of M2 macrophages (CD206+) within the subcutaneous layer of treated wounds, while concurrently, the presence of M1 macrophages (CD86+) was notably diminished (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). This observation indicates that P/G-CS-OI membranes effectively induce the polarization shift from the M1 to M2 phenotype \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe subsidence of inflammation is followed by hastened angiogenesis. To quantitatively analyze neovascularization within the granulation tissue, immunofluorescence staining for CD31 and α-Smooth Muscle Actin (α-SMA) was employed, which showed that the P/G-CS-OI membranes significantly upregulated the expression of both CD31 and α-SMA in the regenerated skin on days 7 and 14. CD31 can label endothelial cells responsible for the formation of vascular lumen, and α-SMA can label pericytes/smooth muscle cells involved in structural stability and regulation of vascular permeability. Vessels displaying both CD31\u0026thinsp;+\u0026thinsp;and α-SMA\u0026thinsp;+\u0026thinsp;were characterized as mature, whereas vessels labeled only with CD31\u0026thinsp;+\u0026thinsp;were considered immature [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Our findings suggest that electrospun nanofiber membranes are effective in promoting the formation of mature blood vessels.\u003c/p\u003e \u003cp\u003eKeratin 10 (K10) and keratin 14 (K14) serve as critical markers for epidermal differentiation and epidermal basal cells, respectively [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Fluorescence results showed that P/G and P/G-CS groups had weak K10\u0026thinsp;+\u0026thinsp;epidermis and scattered K14\u0026thinsp;+\u0026thinsp;epidermal basal layer, indicating that the wound tissue had not regenerated a mature epidermal structure. In contrast, wounds treated with P/G-CS-OI membranes displayed a well-formed epidermis, characterized by a completely covered K10\u0026thinsp;+\u0026thinsp;epidermal basal layer and a K14\u0026thinsp;+\u0026thinsp;epidermal lining. This finding demonstrates that P/G-CS-OI membranes promote rapid and effective epithelialization of wounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 7 days of treatment under the same conditions, immunohistochemical staining showed the expression of TNF-\u0026#120572; in the P/G-CS-OI group was significantly decreased, while the expression of IL-10, VEGF, and NRF2 was significantly increased (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-F), which was aligned with the \u003cem\u003ein vitro\u003c/em\u003e results. ELISA was also used to quantitatively analyze the expressions of TNF-\u0026#120572;, IL-1β, Interferon (IFN)-γ, and VEGF, and corresponding kits were used to detect the expressions of oxidase SOD and oxidative stress product malondialdehyde (MDA) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-L). The inflammatory cytokines TNF-\u0026#120572;, IL-1β, and IFN-γ inhibit wound healing, while VEGF acts on endothelial cells to aid endothelial chemotactic migration, which is essential for angiogenesis during cell proliferation. These outcomes are consistent with observations from immunohistochemistry and immunofluorescence staining, as anticipated. The expressions of TNF-\u0026#120572;, IL-1β, IFN-γ, and MDA were significantly decreased, while the expressions of VEGF and SOD were increased. These results suggest that the P/G-CS-OI membrane not only inhibits tissue inflammation but also induces regeneration of blood vessels and skin appendages and promotes wound healing.\u003c/p\u003e \u003cp\u003eOverall, electrospun nanofiber membranes show significant potential in enhancing the healing of chronic wounds by simultaneously alleviating oxidative stress, inhibiting inflammation, and promoting new blood vessel formation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our study focused on the development and characterization of a bioactive electrospun nanofiber membrane, composed of PCL, Gelatin, CS, and OI, envisioned as an innovative wound dressing material. The P/G-CS-OI membranes demonstrate a remarkable ability to inhibit the activation of specific inflammatory mediators, effectively contributing to macrophage modulation, and thereby promoting the healing of chronic wounds. A noteworthy aspect of the P/G-CS-OI membrane is its capacity for the sustained release of OI. This feature is instrumental in activating ARE through the NRF2/KEAP1 pathway, which relieves oxidative stress and promotes wound healing. \u003cem\u003eIn vivo\u003c/em\u003e studies in a diabetic mouse skin wound model further validated the significant therapeutic benefits of the P/G-CS-OI membrane. These benefits include the modulation of inflammation, promotion of neovascularization, and enhancement of collagen deposition. Therefore, this inherently multifunctional electrospun nanofiber membrane not only reconstructs the tissue microenvironment but also accelerates tissue regeneration. Its diverse and potent capabilities position it as a promising biological dressing, meriting further exploration and potential applications in tissue engineering and regenerative medicine.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4-octyl itaconate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4',6-diamidino-2-phenylindole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCFH-DA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,7-dichlorodihydrofluorescein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFITC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescein isothiocyanate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHO-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeme Oxygenase-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNRF2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear factor (erythroid derived 2)-related factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEAP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKelch-like ECH-associated protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNQO1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNAD(P)H:quinone oxidoreductase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate Buffer Saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSuperoxide Dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTZ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStreptozocin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etricarboxylic acid cycle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eα-SMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eα-Smooth muscle actin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemical\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIFN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterferon\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eScanning electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXP, JW, and ND designed the study. JH, SZ, and JW carried out experiments and drafted the manuscript. JW and SB contributed to sample preparation. ND, JW, and XP provided guidance and assistance on relevant experiments. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 81974326, 82372427) and the Excellent Youth Training Program of Shanghai Sixth People\u0026apos;s Hospital (Grant No. ynyq202202).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Sixth People\u0026apos;s Hospital Affiliated with the Shanghai Jiao Tong University School of Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, Stein C, Basit A, Chan JCN, Mbanya JC, et al: IDF Diabetes Atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. 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Autophagy. 2021;17:2128-2143.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\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":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"electrospinning, nanofiber membrane, itaconic acid, anti-inflammatory, diabetic wound","lastPublishedDoi":"10.21203/rs.3.rs-3853738/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3853738/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn the inflammatory milieu of diabetic chronic wounds, macrophages undergo substantial metabolic reprogramming and play a pivotal role in orchestrating the immune response. Itaconic acid, primarily synthesized by inflammatory macrophages as a byproduct in the tricarboxylic acid cycle, has recently gained increasing attention as an immunomodulator. This study aims to assess the immunomodulatory capacity of an itaconic acid derivative, 4-Octyl itaconate (OI), which was covalently conjugated to electrospun nanofibers and investigated through \u003cem\u003ein vitro\u003c/em\u003e studies and a full-thickness wound model of diabetic mice.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOI was feasibly conjugated onto chitosan (CS), which was then grafted to electrospun PCL/gelatin (PG) nanofibers to obtain P/G-CS-OI membranes. The P/G-CS-OI membrane exhibited good mechanical strength, compliance, and biocompatibility. In addition, the sustained OI release endowed the nanofiber membrane with great antioxidative and anti-inflammatory activity both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Specifically, the P/G-CS-OI membrane activated nuclear factor-erythroid-2-related factor 2 (NRF2) by alkylating Kelch-like ECH-associated protein 1 (KEAP1). This antioxidative response led to macrophage modulation of mitigated inflammatory responses, enhanced phagocytic activity, and recovered angiogenesis of endothelial cells, finally contributing to improved healing of diabetic wounds.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe P/G-CS-OI nanofiber membrane shows good capacity in macrophage modulation and might be promising for diabetic chronic wound treatment.\u003c/p\u003e","manuscriptTitle":"Anti-inflammatory and anti-oxidative electrospun nanofiber membrane promotes diabetic wound healing via macrophage modulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-16 19:56:48","doi":"10.21203/rs.3.rs-3853738/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-27T23:17:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-23T21:20:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2504902b-5457-4e28-9e4f-ee3084efcd6c","date":"2024-01-17T02:00:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-16T22:11:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-12T12:04:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-12T12:04:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-01-11T14:03:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ca673c2-d849-4a36-9a72-15c55c576715","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-18T15:06:20+00:00","versionOfRecord":{"articleIdentity":"rs-3853738","link":"https://doi.org/10.1186/s12951-024-02385-9","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-03-16 15:01:21","publishedOnDateReadable":"March 16th, 2024"},"versionCreatedAt":"2024-01-16 19:56:48","video":"","vorDoi":"10.1186/s12951-024-02385-9","vorDoiUrl":"https://doi.org/10.1186/s12951-024-02385-9","workflowStages":[]},"version":"v1","identity":"rs-3853738","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3853738","identity":"rs-3853738","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}