An immune reprogramming electrospun membrane of EGCG and cerium-doped mesoporous bioactive glass composite, and a preparation method and application thereof

Abstract

This invention discloses an immune reprogramming electrospun membrane (PA-Ce@EG) composed of EGCG and cerium-doped mesoporous bioactive glass, its preparation method, and applications. The PA-Ce@EG membrane uses polylactic acid (PLLA) as a substrate, and an extracellular matrix-mimicking nanofiber structure is constructed using electrospinning technology. Epigallocatechin gallate (EGCG) is loaded into cerium-doped mesoporous bioactive glass (Ce-MBG) and uniformly embedded in the PLLA nanofiber matrix. Rapid release of EGCG achieves early broad-spectrum antibacterial and anti-inflammatory effects; Ce... 3+ / Ce 4+ The redox cycle-mediated continuous scavenging of reactive oxygen species enables long-term antioxidant and immunomodulatory effects. This membrane possesses an extracellular matrix (ECM)-like structure, synergistically promoting antibacterial activity, immune regulation, and angiogenesis, accelerating high-quality healing of diabetic infected wounds, exhibiting good biocompatibility, and is suitable for the repair and treatment of chronic, refractory wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an immune reprogramming electrospun membrane for the repair of infectious diabetic wounds, its preparation method, and its application.

[0002] More specifically, this invention relates to a composite membrane material that integrates epigallocatechin gallate (EGCG) and cerium-doped mesoporous bioactive glass (Ce-MBG) into a polylactic acid (PLLA) nanofiber matrix. Through time-programmed antibacterial, antioxidant, and immunomodulatory effects, it achieves high-quality healing of infectious diabetic wounds. This invention belongs to the cross-technical field of wound dressings, tissue engineering scaffolds, and drug sustained-release materials. Background Technology

[0003] Diabetes-related chronic wounds constitute a significant global health burden. Statistics show that over 25% of people with diabetes will experience chronic wounds such as diabetic foot ulcers at some point in their lives, with infected diabetic wounds being one of the most serious clinical challenges. These wounds have complex pathophysiological characteristics, primarily including persistent bacterial colonization (often in the form of biofilms), chronic inflammation, excessive oxidative stress, and impaired angiogenesis. These factors interact to create a vicious cycle: the hyperglycemic environment weakens the bactericidal function of neutrophils and macrophages, making it difficult to clear bacteria; the bacterial biofilm continuously stimulates the release of pro-inflammatory cytokines, hindering antibiotic penetration; simultaneously, the chronic accumulation of reactive oxygen species (ROS) damages vascular endothelial cells and disrupts the integrity of the extracellular matrix (ECM), ultimately leading to delayed granulation tissue formation. Therefore, infected diabetic wounds often fall into a vicious cycle of "persistent infection-chronic inflammation-regeneration failure," significantly increasing the risk of amputation and death.

[0004] To break through this pathological impasse, researchers have developed a variety of bioactive dressings that attempt to actively modulate the wound microenvironment by integrating antibacterial, anti-inflammatory, or pro-angiogenic signals.

[0005] In the field of hydrogel dressings, various hydrogel systems have been reported for the repair of diabetic wounds. For example, Lin et al. developed a GelMA hydrogel prepared using 3D printing technology. This hydrogel contains photosynthetic microalgae and probiotics, which can enhance the oxygenation capacity and antibacterial effect of diabetic wounds (Lin et al., J Control Release, 2026:114653). Fu et al. successfully prepared a double-crosslinked collagen bio-ink functionalized with QHREDGS peptides using DLP bioprinting technology to promote angiogenesis (Fu et al., Int J Biol Macromol, 2026, 343:150408). However, hydrogel-based systems still have limitations such as insufficient mechanical strength, inaccurate release kinetics, and poor extracellular matrix simulation (Xu et al., Mater Des, 2025, 260:115207; Han et al., Mater Des, 2025, 260:115171). These defects often result in explosive drug release, a time mismatch with the healing phase, and a lack of effective structural guidance for cell migration and tissue remodeling.

[0006] In the field of electrospun nanofiber membranes, electrospun nanofiber membranes have become a highly promising alternative material for wound repair due to their extracellular matrix-like structure, high specific surface area, and tunable porosity. Current technologies have integrated various bioactive agents into electrospun fibers, including plant-derived polysaccharides with antibacterial and anti-inflammatory functions, and polyphenols such as curcumin that exert antioxidant effects and promote tissue regeneration (Tahamtan et al., Int J Biol Macromol, 2026, 338:149681; Rajora et al., Int J Biol Macromol, 2026:150462). However, most existing electrospun systems still rely on simple physical encapsulation techniques, mainly releasing single-function signals, with limited attention to the dynamic microenvironment requirements during the healing process, especially the stage-specific needs from infection control and inflammation resolution to cell proliferation and tissue remodeling. The main current technological limitation lies in the lack of a programmable fiber platform, which prevents the time-sequential synergistic release of multiple agents to regulate stage-specific biological responses.

[0007] In the application of bioactive molecules, EGCG has attracted much attention due to its broad-spectrum antibacterial activity through membrane disruption, its anti-inflammatory effect through NF-κB inhibition, and its potential to regulate macrophage polarization (Sahadevan et al., Crit Rev Food Sci Nutr, 2023, 63(30):10382-10411). However, its clinical application is limited by chemical instability and the problem of rapid release from traditional carriers, resulting in difficulty in sustaining efficacy in the treatment of chronic wounds (Li et al., Mater Des, 2025, 253:113876; Hu et al., Mater Today Bio, 2025, 32:101907; Alamet al., Mol Biomed, 2024, 5(1):73). In contrast, bioactive glasses (BGs) have gained widespread recognition due to their biocompatibility and angiogenesis potential. Ion doping strategies further endow BGs with antibacterial, antioxidant, and immunomodulatory functions (Liu et al., Biomaterials, 2025, 322:123376; Hu et al., Adv Sci, 2024, 11(5):2302674). Notably, cerium-doped mesoporous bioactive glasses (Ce-MBGs) utilize Ce... 3+ / Ce 4+ The redox cycle enables catalytic scavenging of reactive oxygen species and has been reported to promote M2 macrophage polarization and angiogenesis (Wang et al., Adv Sci, 2025:e01567; Jiang et al., J Nanobiotechnology, 2024, 22(1):639).

[0008] In summary, current technologies face three core challenges. First, insufficient multifunctional integration capabilities mean that existing dressings cannot simultaneously achieve synergistic effects of antibacterial, anti-inflammatory, antioxidant, and angiogenesis-promoting properties. Second, poor spatiotemporal control over therapeutic delivery, lacking a time-programmed release mechanism, fails to meet the stage-specific needs of wound healing. Third, poor synergy between structure and function, making it difficult to simultaneously regulate the immune microenvironment while providing structural support. Although EGCG and Ce-MBG have complementary mechanisms of action, there is currently no systematic research on the rational integration of both into a time-coordinated electrospinning platform to achieve staged regulation of infected wound healing. Summary of the Invention

[0009] This invention addresses the technical deficiencies of existing infectious diabetic wound dressings by providing an electrospun membrane and its preparation method that achieve synergistic effects of antibacterial, antioxidant, immunomodulatory, and angiogenesis. Specifically, this invention aims to solve the following technical problems:

[0010] First, it addresses the problem that existing dressings have limited functionality and are unable to simultaneously address the imbalance of the microenvironment caused by multiple factors in infectious diabetic wounds, such as bacterial colonization, chronic inflammation, oxidative stress, and impaired angiogenesis.

[0011] Second, to address the issues of inaccurate drug delivery kinetics and time mismatch with the wound healing stage in existing drug delivery systems, a fiber platform capable of time-programmed treatment delivery is provided.

[0012] Third, to address the problem that existing dressings cannot simultaneously regulate the immune microenvironment while providing structural support, a composite scaffold material with both extracellular matrix-like structure and immune reprogramming function is provided.

[0013] To address the aforementioned technical problems, this invention provides an immune-programmed electrospun membrane PLLA / Ce-MBG / EGCG (abbreviated as PA-Ce@EG), which integrates EGCG and cerium-doped mesoporous bioactive glass (Ce-MBG) into a polylactic acid (PLLA) nanofiber matrix. By loading EGCG into the Ce-MBG mesopores and embedding it into PLLA fibers, staged microenvironment regulation is achieved: rapid EGCG release enables early antibacterial and anti-inflammatory intervention, and Ce ions (Ce 3+ / Ce 4+ The continuous release of these substances drives subsequent redox regulation and macrophage reprogramming. Simultaneously, the ECM-like fibrous structure provides physical guidance for tissue remodeling. This invention, through a time-programmed immune reprogramming mechanism, achieves the synergistic integration of antibacterial, antioxidant, immunomodulatory, and angiogenesis mechanisms, providing a novel technical solution for the treatment of infectious diabetic chronic wounds.

[0014] To achieve the above objectives, the present invention adopts the following specific technical solution:

[0015] In a first aspect, the present invention provides an immune reprogramming electrospun membrane for the repair of infectious diabetic wounds, wherein the electrospun membrane is prepared by loading EGCG onto cerium-doped mesoporous bioactive glass and then integrating it into a polylactic acid nanofiber matrix.

[0016] Furthermore, the cerium doping content in the cerium-doped mesoporous bioactive glass (Ce-MBG) is 5 mol%, Ce 3+ With Ce 4+ coexist.

[0017] Furthermore, the mass ratio of EGCG to cerium-doped mesoporous bioactive glass (Ce-MBG) is 1:10.

[0018] Furthermore, the mass ratio of polylactic acid (PLLA) to cerium-doped mesoporous bioactive glass (Ce-MBG) loaded with EGCG is 10:1.

[0019] Furthermore, the electrospun membrane possesses time-programmed release characteristics: EGCG is rapidly and cumulatively released at a rate exceeding 70% within 7 days, achieving early antibacterial and anti-inflammatory effects; Ce... 3+ / Ce 4+ Ions are continuously released for more than 14 days through the gradual degradation of cerium-doped mesoporous bioactive glass, achieving late-stage antioxidant and immunomodulatory effects.

[0020] Furthermore, the electrospun membrane has a nanofiber structure that mimics the extracellular matrix, with a fiber diameter of 200 nm and a water contact angle of 78.6°.

[0021] In a second aspect, the present invention provides a method for preparing the above-mentioned electrospun film, the method comprising the following steps:

[0022] (1) Synthesis of cerium-doped mesoporous bioactive glass: Cerium-doped mesoporous bioactive glass was prepared by microemulsion-assisted sol-gel method, using tetraethoxysilane as silicon source, calcium nitrate tetrahydrate as calcium source, and cerium nitrate as cerium source, through sol-gel reaction, centrifugation washing, drying and calcination at 750℃.

[0023] (2) Loading of EGCG: The cerium-doped mesoporous bioactive glass obtained in step (1) is dispersed in an EGCG aqueous solution, stirred in the dark for 12 hours, and then centrifuged, washed and freeze-dried to obtain EGCG-loaded cerium-doped mesoporous bioactive glass.

[0024] (3) Electrospinning to form a film: L-polylactic acid is dissolved in hexafluoroisopropanol, and cerium-doped mesoporous bioactive glass loaded with EGCG obtained in step (2) is added. After being uniformly dispersed, the film is formed by electrospinning and then dried under vacuum to obtain the immune reprogramming electrospinned film.

[0025] Furthermore, the final concentration of the cerium-doped mesoporous bioactive glass loaded with EGCG in step (3) in the spinning solution is 0.5 mg / mL.

[0026] Furthermore, the electrospinning parameters in step (3) are: voltage 16kV, flow rate 1mL / h, receiving distance 15cm, and receiving drum speed 2800rpm.

[0027] Furthermore, the electrospun membrane possesses time-programmed release characteristics: EGCG accumulates to approximately 70% release within 7 days, achieving early antibacterial and anti-inflammatory effects; Ce... 3+ / Ce 4+ Ions are continuously released for more than 14 days, achieving long-lasting antioxidant and immunomodulatory effects.

[0028] Furthermore, the electrospun membrane has the following characteristics: the nanofibers are randomly oriented and have a uniform diameter; cerium-doped mesoporous bioactive glass is uniformly embedded in the fiber matrix; the membrane material has a hydrophilic surface with a water contact angle of 78.6°; and it has a biomimetic structure similar to the extracellular matrix.

[0029] In a third aspect, the present invention provides the use of the above-mentioned electrospun membrane in the preparation of a medical dressing for treating infectious diabetic wounds.

[0030] In a fourth aspect, the present invention also provides a medical dressing comprising the above-described electrospun membrane.

[0031] Furthermore, the medical dressing is in the form of a patch, gel, sponge, or foam.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) This invention integrates EGCG and Ce-MBG into PLLA electrospun membrane for the first time, constructing a ternary composite system, realizing the synergistic integration of four functions: antibacterial, antioxidant, immunomodulatory and angiogenesis-promoting, overcoming the technical defects of existing dressings with single functions.

[0034] (2) This invention achieves time-programmed therapeutic delivery through the rapid release of EGCG and the continuous release of Ce ions, perfectly matching the staged needs of wound healing.

[0035] (3) The present invention adopts a non-antibiotic antibacterial mechanism. Through the synergistic effect of Ce ion-induced oxidative stress and EGCG-mediated bacterial membrane destruction, it effectively kills drug-resistant bacteria and avoids the problem of antibiotic resistance.

[0036] (4) This invention reprograms the immune microenvironment by inducing macrophages to polarize to the M2 phenotype, and then enhances endothelial cell migration and lumen formation through paracrine signals, thus establishing an immune-vascular coupling enhancement mechanism.

[0037] (5) The nanofiber membrane constructed by electrospinning technology in this invention has an extracellular matrix-like structure, providing an ideal physical microenvironment for cell adhesion, migration and proliferation, while also having good mechanical strength and flexibility.

[0038] (6) The material of the present invention has good biocompatibility and blood compatibility, significantly accelerates wound healing in a diabetic mouse model of infected wounds, and no systemic toxicity was observed, indicating good biocompatibility.

[0039] (7) The preparation process of this invention is simple and controllable, the raw materials are widely available, it is easy to scale up production, and it has good prospects for clinical application transformation. Attached Figure Description

[0040] Figure 1 This is a characterization analysis diagram of the PA-Ce@EG electrospun film of the present invention. Among them, Figure 1 In the image, 'a' represents a Ce-MBG scanning electron microscope image; Figure 1 In the image, b represents the transmission electron microscope image of Ce-MBG and Ce-MBG@EG; Figure 1 In the diagram, 'c' represents the particle size distribution of Ce-MBG and Ce-MBG@EG. Figure 1 In the diagram, d represents the zeta potential of Ce-MBG and Ce-MBG@EG; Figure 1 In the image, 'e' represents the XPS Ce 3d spectrum of Ce-MBG. Figure 1 f in the figure represents the transmission electron microscope image of PA-Ce@EG and the corresponding EDS elemental distribution map; Figure 1 In the figure, g represents the scanning electron microscope and EDS elemental distribution map of PLLA and PA-Ce@EG; Figure 1 The hi in the text is based on Figure 1 The semi-quantitative elemental composition analysis results of the EDS spectrum shown in g are as follows; Figure 1 In the figure, j represents the Fourier transform infrared spectrum of PLLA and PA-Ce@EG, where the upper line represents PLLA and the lower line represents PA-Ce@EG. Figure 1 In the diagram, k represents the X-ray diffraction patterns of PLLA and PA-Ce@EG, with the upper line representing PLLA and the lower line representing PA-Ce@EG.

[0041] Figure 2 This diagram illustrates the surface properties, release behavior, antioxidant activity, and biocompatibility of PA-Ce@EG according to the present invention. Figure 2 In the figure, 'a' represents the PA-Ce@EG 3D surface reconstruction model obtained through 3D CLSM technology. Figure 2 In the figure, b is a quantitative analysis diagram of the surface roughness parameter; Figure 2 cd in the image is the contact angle of the electrospun membrane measured using deionized water and diiodomethane; Figure 2 In this context, 'e' represents the water contact angle value for different samples. Figure 2 In this context, f represents the calculated surface energy. Figure 2 In this context, g represents the water vapor permeability of different membranes; Figure 2h Figure 2 In this context, h represents the swelling rate in PBS buffer over 24 hours. Figure 2 In this context, ij represents the tensile strength and Young's modulus of the membrane. Figure 2 In this context, k represents the cumulative release curve of EGCG in the PA-Ce@EG vector over 14 days. Figure 2 ln in the figure represents the effects of DPPH·, ·OH, and O2· on DPPH·, ·OH, and O2·, respectively. - Free radical scavenging activity.

[0042] Figure 3 This is a graph illustrating the intracellular reactive oxygen species scavenging capacity and biocompatibility assessment of PA-Ce@EG according to the present invention. Figure 3 In the image, 'a' represents a representative fluorescence image stained with DCFH-DA, showing the level of intracellular reactive oxygen species under H2O2 stimulation. Figure 3 In this context, b represents the quantitative analysis of the average fluorescence intensity of DCFH-DA; Figure 3 In the figure, c represents the flow cytometry histogram of ROS-positive cells under different treatment conditions; Figure 3 In the diagram, d represents the in vitro cell compatibility assessment protocol. Figure 3 The ef values ​​represent the Calcein-AM / PI fluorescence staining results of NIH-3T3 cells and human umbilical vein endothelial cells cultured on different membranes, with green fluorescence (Calcein-AM) marking live cells and red fluorescence (PI) marking dead cells. Figure 3 The gh in the figure represents the CCK8 assay, which shows the proliferation of NIH-3T3 cells and HUVECs on days 1, 3, and 5. Figure 3 In this context, 'i' represents the hemolysis test result that assesses the blood compatibility of the membrane material.

[0043] Figure 4 This is a graph illustrating the in vitro antibacterial performance evaluation of PA-Ce@EG according to the present invention. Wherein, Figure 4 In the figures, ab represents the results of live / dead cell fluorescence staining for MRSA and E. coli, respectively. Figure 4 The cd image in the image represents a representative agar plate image showing the colony formation of MRSA and Escherichia coli. Figure 4 In this context, 'e' represents the quantitative analysis of the green / red fluorescence intensity ratio in MRSA and E. coli live / dead cell staining. Figure 4 f in the figure represents the quantitative analysis of the bacterial survival rate of MRSA and Escherichia coli based on colony-forming units; Figure 4 In the figure, gh represents the flow cytometry analysis results of PI-stained MRSA and Escherichia coli, respectively. Figure 4 In this context, 'i' represents the statistical quantitative analysis of PI-positive bacteria detected by flow cytometry. Figure 4 In the figure, j represents the quantitative analysis of the fluorescence intensity of MRSA and Escherichia coli PI.

[0044] Figure 5 This diagram illustrates the immunomodulatory effect of PA-Ce@EG on macrophage polarization in vitro. (The diagram shows the effect of this invention.) Figure 5 In the diagram, 'a' represents a schematic diagram of PA-Ce@EG-mediated macrophage reprogramming under LPS stimulation. Figure 5 In the image, b represents a representative immunofluorescence image of RAW264.7 macrophages after different treatments; Figure 5 In this context, 'c' represents the quantitative analysis of the fluorescence intensity of iNOS and CD206. Figure 5 In the diagram, d represents a flow cytometry dot matrix image showing the expression of CD86 and CD206 in RAW264.7 macrophages; Figure 5 In this context, 'e' represents CD86 obtained via flow cytometry. + With CD206 + Quantitative analysis of macrophage populations; Figure 5 In the figure, f represents the relative mRNA expression level.

[0045] Figure 6 This is a diagram illustrating the immune-mediated endothelial angiogenesis response induced by PA-Ce@EG according to the present invention. Among them, Figure 6 In the diagram, 'a' represents a schematic diagram of the scratch migration experiment. Figure 6 In the image, b represents a representative image from the HUVECs scratch migration experiment. Figure 6 In this context, 'c' represents the quantitative analysis of the remaining wound area in the scratch test. Figure 6 In the image, d represents a fluorescence image of the HUVEC angiogenesis process on the matrix gel; Figure 6 In this context, eh represents the quantitative analysis of angiogenesis parameters, including the number of nodules, branches, segments, and total length. Figure 6 In the figure, i represents the result of HUVECs CD31 immunofluorescence staining.

[0046] Figure 7 This is an in vivo evaluation diagram of wound healing in an infectious diabetic mouse model of the present invention. Among them, Figure 7 In this diagram, 'a' represents the experimental design. Figure 7 In the image, b represents a macroscopic image of an infected wound after different treatments; Figure 7 In the diagram, 'c' represents the corresponding wound area contour map and merged map. Figure 7 In the figure, d represents a heat map visualization of the evolution of the wound area; Figure 7 In this context, 'e' represents the quantitative analysis of the wound area. Figure 7 f in the diagram represents the bacterial colony map recovered from the wound tissue; Figure 7 In this context, 'g' represents the quantitative analysis of bacterial load in the incision tissue.

[0047] Figure 8 This is an in vivo pathological evaluation diagram of an infected diabetic wound treated with this invention. Among them, Figure 8In this text, 'a' represents the H&E staining result of the wound tissue. Figure 8 In this context, 'b' represents the quantitative analysis of granulation tissue width. Figure 8 In the figure, c represents the Masson trichrome staining result of the wound tissue; Figure 8 In this context, d represents the quantitative analysis of collagen volume fraction; Figure 8 In the figure, 'e' represents the immunofluorescence results of iNOS and CD206 staining in wound tissue; Figure 8 f in the figure represents the quantitative analysis of iNOS-positive cells; Figure 8 In this context, 'g' represents the quantitative analysis of CD206-positive cells. Figure 8 In the figure, h represents the immunofluorescence staining results of wound sections stained with α-SMA and CD31; Figure 8 In this context, 'i' represents the quantitative analysis of microvessel density calculated using CD31 or α-SMA positive structures.

[0048] Figure 9 This is a transcriptomic analysis diagram of wound tissue after PA-Ce@EG treatment according to the present invention. Among them, Figure 9 In the diagram, 'a' represents a volcano plot showing the differentially expressed genes between the PA-Ce@EG group and the control group. Figure 9 b in the figure is a Pearson correlation heatmap, showing the correlation coefficients of biological replicates between the Ctrl group and the PA-Ce@EG group; Figure 9 In the diagram, 'c' represents a hierarchical clustering heatmap of differentially expressed genes. Figure 9 In the diagram, d represents the GO enrichment bubble chart of differentially expressed genes.

[0049] Figure 10 This is a diagram showing the KEGG pathway enrichment analysis of wound tissue transcriptomics after PA-Ce@EG treatment according to the present invention. Detailed Implementation

[0050] The technical solution of the present invention will be described in more detail with reference to specific embodiments and accompanying drawings. In implementing the present invention, except for the experimental steps, conditions, and methods described in detail below, all other techniques and common knowledge in the art are employed, and no additional special limitations are imposed.

[0051] It should be noted that the embodiments described herein are only some typical examples of the present invention, and not all implementation methods. The following description of exemplary embodiments is for illustrative purposes only and does not constitute any limitation on the present invention or its application scenarios.

[0052] In the demonstration and discussion of the various embodiments, the specific numerical values ​​and parameters involved are for illustrative purposes only and are not intended to be limiting. Therefore, other embodiments based on the concept of this invention can have their relevant numerical values ​​adjusted according to actual needs.

[0053] In addition, unless otherwise specified, the experimental materials used in the examples are all commercially available biochemical reagents, and the experimental operations also follow the standard procedures in this field.

[0054] To more clearly illustrate the technical solution of the present invention, the present invention will be further described in detail below with reference to specific embodiments. Those skilled in the art should understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of protection of the present invention. All equivalent substitutions or modifications made based on the technical concept of the present invention fall within the scope of protection of the present invention.

[0055] Example 1: Synthesis of Cerium-Doped Mesoporous Bioactive Glass (Ce-MBG)

[0056] This embodiment provides a method for preparing cerium-doped mesoporous bioactive glass, the specific steps of which are as follows:

[0057] 0.6 g of hexadecyltrimethylammonium bromide (CTAB, AR, ≥99%) was dissolved in 30 mL of deionized water and vigorously stirred magnetically at 60 °C to form a homogeneous surfactant solution. After complete dissolution, 8 mL of ethyl acetate (AR, ≥99%) was added to form a microemulsion system, and stirring was continued for 20 minutes. Subsequently, 6 mL of 1M ammonia solution (pre-diluted with ammonia water (AR, 25–28 wt%)) was added dropwise to adjust the alkaline environment and promote the silica condensation reaction. After stirring for another 20 minutes, 2.88 mL of tetraethoxysilane (TEOS, 98%) was slowly added as a silica precursor. Next, 0.571 g of calcium nitrate tetrahydrate (AR, ≥99%) was added, and finally 0.35 g of cerium nitrate (AR, ≥99%) was added as a dopant precursor. The sol-gel reaction was carried out under continuous stirring for 4 hours. The resulting colloidal suspension was collected by centrifugation at 8000×g for 10 minutes and washed successively with deionized water and anhydrous ethanol (AR, ≥99.7%). The precipitate was dried at 60°C overnight and then calcined in air at 750°C for 3 hours to obtain Ce-MBG.

[0058] Example 2: Loading of EGCG

[0059] The Ce-MBG nanoparticles prepared in Example 1 were dispersed in an aqueous EGCG solution, wherein the mass ratio of EGCG to Ce-MBG nanoparticles was 1:10. The mixture was stirred in the dark for 12 hours to promote the diffusion of EGCG into the mesopores. The resulting Ce-MBG@EG was collected by centrifugation, washed, freeze-dried, and stored at 4°C.

[0060] Example 3: Preparation of PA-Ce@EG electrospun film

[0061] Polylactic acid (PLLA) was dissolved in hexafluoroisopropanol (HFIP) at a concentration of 10 wt%, and magnetically stirred overnight at room temperature until completely dissolved. Separately, Ce-MBG@EG prepared in Example 2 was uniformly dispersed in HFIP and added to the PLLA solution, wherein the mass ratio of PLLA to Ce-MBG@EG was 10:1, and the final concentration of Ce-MBG@EG in the mixture was 0.5 mg / mL. The resulting homogeneous mixture was transferred to a 5 mL plastic syringe equipped with a blunt-tipped stainless steel needle, and electrospinned using a high-voltage power supply. The spinning parameters were: 16 kV DC voltage, flow rate 1 mL / h, receiving distance 15 cm, and rotating drum speed 2800 rpm. The nanofiber mat was collected on aluminum foil and vacuum dried at room temperature for 6 hours to remove residual solvent, yielding the PA-Ce@EG membrane.

[0062] Ce-MBG composite membranes without EGCG were prepared using the same method and labeled as PA-Ce.

[0063] Example 4: Material Characterization

[0064] The morphology and microstructure of Ce-MBG and electrospun films were observed using scanning electron microscopy (SU8600, Hitachi) and transmission electron microscopy (HT7800, Hitachi). Chemical composition and structural characteristics were analyzed using Fourier transform infrared spectroscopy (Nicolet iS50, Thermo Fisher Scientific) and X-ray diffraction (X'Pert3 Powder, Malvern Panaco). Surface elemental valence states were characterized using X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Fisher Scientific). Surface wettability was assessed using a static water contact angle meter (DSA25S, Krüger). Mechanical properties were tested using a universal testing machine (AGS-X, Shimadzu). The films were cut into 30×10mm rectangular strips for uniaxial tensile testing, and tensile strength and Young's modulus were calculated.

[0065] Water vapor permeability was measured using a flexible bottle permeation system (gas permeation tester, 2 / 61, Memcon). The membrane was sealed in a bottle containing deionized water and incubated at 37°C for 12 hours. The mass loss due to water vapor permeation was recorded and calculated using WVP=w / (A×T), where w is the amount of water loss, A is the membrane surface area, and T is the time.

[0066] Swelling behavior was assessed using the gravimetric method. A 2×2cm... 2 After weighing, the samples were immersed in PBS buffer at 37°C for 1-24 hours, removed, dried, and weighed again to calculate the swelling rate.

[0067] The release profiles of EGCG and Ce ions were evaluated in PBS (pH 7.4, 37°C) for 14 days. EGCG concentration was determined by UV-Vis spectrophotometry (PerkinElmer Lambda 35, PerkinElmer, USA) at 274 nm, and Ce ion concentration was determined by inductively coupled plasma optical emission spectrometry (ICP-2060T, Tianrui Instruments).

[0068] The characterization results are as follows:

[0069] Scanning electron microscopy and transmission electron microscopy revealed that Ce-MBG consisted of uniform spherical particles with a diameter of approximately 100 nm and a clearly visible mesoporous structure. Figure 1 (ab in the text). After EGCG loading, dynamic light scattering analysis showed a moderate increase in hydrodynamic dimensions (ab in the text). Figure 1 In c), the zeta potential increases from -4.6 mV to -13.3 mV. Figure 1 (d) In the figure. XPS analysis verified Ce 3+ With Ce 4+ coexist( Figure 1 (e in the text).

[0070] Transmission electron microscopy and scanning electron microscopy combined with elemental analysis confirmed that Ce-MBG was successfully incorporated into the electrospun membrane, with particulate nanoparticles embedded in the fiber matrix. Figure 1 f in the text). Energy dispersive spectroscopy (EDS) analysis detected signals of C, O, Si, and Ce. Figure 1 f in the text). Both PLLA and PA-Ce@EG films exhibit a randomly oriented nanofiber network structure. Figure 1 No nanoparticle aggregation was observed. Semi-quantitative energy dispersive spectroscopy (EDS) analysis showed that Si and Ce elements were not detected in the PLLA film, while Si and Ce signals were detected in the PA-Ce@EG film. Figure 1 hi in the middle.

[0071] FTIR spectra show characteristic PLLA absorption bands, including C=O stretching vibrations (approximately 1750 cm⁻¹). -1 ) and COC vibration (approximately 1180-1080cm) -1 () Figure 1 (j) The XRD pattern shows that PLLA exhibits a typical amorphous halo structure, without obvious crystalline peaks of Ce-MBG or EGCG. Figure 1 k in the middle.

[0072] Surface wettability assessment showed that the water contact angle of the PLLA membrane was 106.4°, while that of the PA-Ce membrane decreased to 73.8°, and that of the PA-Ce@EG membrane was 78.6°. Figure 2 (c, e in the text). Surface energy calculations show that PLLA is 31.63 mJ / m. 2 PA-Ce is 37.91 mJ / m 2 PA-Ce@EG has a strength of 40.58 mJ / m 2 ( Figure 2 f in the middle.

[0073] Water vapor permeability measurements showed that all groups exhibited approximately 2.76 mg / cm³. 2 Similar transmission rate per h ( Figure 2 The swelling behavior assessment showed that all groups exhibited rapid water absorption in the initial stage, which gradually increased until an equilibrium state was reached, with similar swelling ratios among the groups (g). Figure 2 (h in the middle).

[0074] Mechanical property tests showed that, compared to PLLA, the tensile strength was significantly improved after incorporating Ce-MBG, with PA-Ce@EG exhibiting the highest tensile strength. Figure 2 The i in the text). Young's modulus also shows a similar trend ( Figure 2 j in the middle.

[0075] The release curve shows that EGCG is released at a cumulative rate of approximately 70% within 7 days, reaching a plateau on day 14. Figure 2 The release of Ce ions is more gradual, increasing from 4.2 ppm on day 1 to 19.6 ppm on day 14. This differentiated release kinetics indicates that EGCG is mainly released from the fibrous matrix through diffusion, while the release of Ce ions is regulated by the gradual degradation of Ce-MBG and ion exchange processes. This staged release mechanism allows EGCG to exert its antibacterial and antioxidant effects early on, followed by synergistic support through Ce ion-mediated sustained redox regulation and pro-angiogenic effects.

[0076] In summary, this embodiment successfully synthesized Ce-MBG with a mesoporous structure and a particle size of approximately 100 nm. 3+ and Ce 4+ Two valence states coexist. A PA-Ce@EG composite membrane was successfully prepared by embedding Ce-MBG@EG into a PLLA nanofiber matrix using electrospinning technology. This membrane exhibits a randomly oriented nanofiber structure, with Ce-MBG@EG nanoparticles uniformly distributed within the fiber matrix without significant aggregation. Compared to the pure PLLA membrane, the water contact angle of the PA-Ce@EG membrane decreased from 106.4° to 78.6°, and the surface energy increased from 31.63 mJ / m². 2 Increased to 40.58 mJ / m 2The tensile strength and Young's modulus of the PA-Ce@EG membrane are significantly higher than those of the pure PLLA membrane. The water vapor permeability of the PA-Ce@EG membrane is comparable to that of the PLLA membrane (approximately 2.76 mg / cm³). 2 The swelling ratio is similar to that of PLLA membranes, meeting the basic requirements for wound dressings. In vitro release experiments showed that EGCG exhibited a continuous release characteristic, accumulating approximately 70% release over 7 days, reaching a plateau on day 14; Ce ions showed a gradual release characteristic, increasing from 4.2 ppm on day 1 to 19.6 ppm on day 14. These results indicate that the PA-Ce@EG membrane prepared in this invention possesses excellent physicochemical properties and antioxidant activity, providing a material basis for subsequent biological evaluation.

[0077] Example 5: Detection of antioxidant activity and intracellular reactive oxygen species scavenging

[0078] I. Antioxidant Activity Assessment

[0079] DPPH radicals, hydroxyl radicals (·OH), and superoxide anions (O2·) are produced. - Scavenging assay. Membrane extracts were co-incubated with the corresponding free radical solutions, and absorbance was measured using a microplate reader (BioTek Synergy H1, BioTek Biotech, USA) to calculate scavenging efficiency. All experiments were performed with three replicates.

[0080] PA-Ce@EG membranes exhibited excellent scavenging activity against all tested free radical species. The DPPH free radical scavenging rate exceeded 85% within 30 minutes of incubation. Figure 2 The levels of hydroxyl radicals (·OH) and superoxide anions (O2·) were significantly higher than those in the PLLA and PA-Ce groups. - The clearance capabilities reached 68% and 74% respectively. Figure 2 The mn content in the PA-Ce group was significantly better than that in the control group and the PLLA group. The PA-Ce group showed moderate free radical scavenging activity, but it was significantly lower than that in the PA-Ce@EG group.

[0081] The introduction of EGCG significantly enhances the antioxidant properties of the composite membrane. PA-Ce@EG interacts with Ce through the phenolic hydroxyl structure of EGCG. 3+ / Ce 4+ The synergistic effect of the redox cycle enables highly efficient free radical scavenging capabilities.

[0082] II. Detection of Intracellular Reactive Oxygen Species Scavenging

[0083] Intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe. Cells were seeded in 24-well plates and co-cultured with different membrane extracts for 24 hours. Oxidative stress was induced by treatment with 200 μM H2O2 for 2 hours. Subsequently, cells were incubated with 10 μM DCFH-DA at 37°C in the dark for 30 minutes. After washing with PBS, the cells were imaged using a fluorescence microscope (Olympus IX83, Olympus Corporation, Japan), and the mean fluorescence intensity was measured using ImageJ image analysis software (National Institutes of Health, USA). ROS levels were simultaneously detected by flow cytometry (BD Accuri C6 Plus, BD Biosciences, USA).

[0084] DCFH-DA fluorescence staining results showed that, compared with the control group, H2O2 treatment significantly increased the fluorescence intensity of DCFH-DA, indicating a large accumulation of reactive oxygen species in cells. Figure 3 (a) The PLLA group did not significantly reduce fluorescence intensity, and there was no statistically significant difference compared with the H2O2 group. Both the PA-Ce group and the PA-Ce@EG group could attenuate intracellular reactive oxygen species signaling, with the PA-Ce@EG group showing the most significant inhibitory effect. Figure 3 (b) Flow cytometry analysis further validated the above findings. Figure 3 (c in the text)

[0085] PA-Ce@EG via Ce 3+ / Ce 4+ The synergistic effect of cyclic redox activity and the phenolic hydroxyl structure of EGCG effectively neutralizes excess reactive oxygen species in cells.

[0086] The above results demonstrate that the PA-Ce@EG membrane prepared in this invention exhibits excellent antioxidant properties in both chemical free radical scavenging and intracellular reactive oxygen species neutralization, providing an experimental basis for subsequent cell biology evaluation.

[0087] Example 6: In vitro biocompatibility assessment

[0088] Cell compatibility was assessed using mouse embryonic fibroblasts (NIH-3T3, ATCC) and human umbilical vein endothelial cells (HUVECs, ATCC). Cells were cultured at 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 cells / well on membrane samples in 24-well plates. After 24 hours, live / dead cells were stained with calcein AM and propidium iodide. The cells were observed and photographed using a fluorescence microscope (Olympus IX83). Cell proliferation was quantified on days 1, 3, and 5 using the CCK-8 assay, and absorbance at 450 nm was measured using a microplate reader (BioTek Synergy H1).

[0089] Blood compatibility was assessed by a hemolysis test: Anticoagulated rat blood was diluted with PBS and incubated with the membrane sample at 37°C for 1 hour. The sample was then centrifuged at 3000 rpm for 10 minutes. The supernatant was collected and the absorbance at 545 nm was measured using a UV-Vis spectrophotometer (PerkinElmer Lambda 35). The hemolysis rate was then calculated.

[0090] Live / dead cell staining results showed that NIH-3T3 cells and HUVECs cultured on PLLA, PA-Ce, and PA-Ce@EG scaffolds maintained high viability after 24 hours, with no obvious cell death observed. Figure 3 Both cell types exhibited normal morphological characteristics and typical spindle-shaped spreading morphology, indicating a good interaction between the cells and the material.

[0091] CCK-8 assay results showed that all groups exhibited cell proliferation progression within 5 days, and no significant cytotoxicity was detected in PA-Ce or PA-Ce@EG compared to PLLA. Figure 3 (gh in the middle).

[0092] The hemolysis test results showed that the hemolysis rate of all membrane materials was below the safety threshold of 5%. Figure 3 (i) confirms that it has good blood compatibility.

[0093] The results of this embodiment demonstrate that the incorporation of Ce-MBG and EGCG does not affect cell viability or growth. NIH-3T3 cells and HUVECs cultured on PLLA, PA-Ce, and PA-Ce@EG scaffolds maintained high viability, normal cell morphology, and a typical spindle-shaped spreading pattern. CCK-8 proliferation assays showed that cells on all membranes exhibited good proliferation trends within 5 days, and no significant cytotoxicity was detected in PA-Ce or PA-Ce@EG compared to PLLA. Hemolysis assays showed that the hemolysis rate of all membrane materials was below the safety threshold of 5%. These results confirm that the electrospun membranes prepared in this invention possess good biocompatibility and blood compatibility.

[0094] Example 7: In vitro antibacterial evaluation

[0095] Antimicrobial activity was evaluated using methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300) and Escherichia coli (ATCC, 8739). The bacteria were cultured overnight in Luria-Bertani (LB) broth at 37°C and adjusted to a concentration of 1×10⁻⁶. 6 CFU / mL concentration. Membrane sample (1×1 cm⁻¹) 2 The bacterial suspension (1 mL) was incubated with the bacterial suspension in a 24-well plate at 37°C for 12 hours.

[0096] Bacterial viability was detected by the LIVE / DEAD BacLight™ staining method and analyzed by confocal laser scanning microscopy (CLSM, Leica TCS SP8, Leica GmbH, Germany). Colony forming units (CFUs) were quantified by serial dilution and LB agar plate culture. Membrane integrity was assessed by propidium iodide (PI) staining (5 μg / mL, 15 min, protected from light). PI-positive bacteria were quantified by flow cytometry (BD Accuri C6 Plus, BD Biosciences, USA), and fluorescence intensity (excitation / emission wavelengths: 535 / 617 nm) was measured using a microplate reader (BioTek Synergy H1, BioTek Biosciences, USA). To assess intracellular component leakage, the treated bacterial suspension was centrifuged (8000×g, 10 min), and the absorbance of the supernatant was recorded at 260 nm (nucleic acid) and 280 nm (protein) wavelengths using a UV-Vis spectrophotometer (PerkinElmer Lambda 35, PerkinElmer, USA). All experiments were performed in triplicate.

[0097] The results of live / dead bacterial fluorescence staining showed significant differences in bacterial survival rates among the test groups. Figure 4 (ab in the text). Compared with the control group and the PLLA group, the PA-Ce@EG treatment group showed a significant increase in the number of red fluorescent (dead bacteria) bacteria against both MRSA and Escherichia coli, while the PA-Ce group only showed a moderate antibacterial effect.

[0098] The results of the agar plate colony assay further support this conclusion. Figure 4 (cd in the text): Samples treated with PA-Ce@EG showed a significantly reduced colony formation compared to all other groups. Quantitative analysis of colony forming units confirmed that the viable cell count of both strains decreased significantly after PA-Ce@EG treatment. Figure 4 f in the middle.

[0099] Calculate the green / red fluorescence intensity ratio from live / dead bacteria staining images. Figure 4 In the example of PA-Ce@EG, the lowest green / red ratio was observed, indicating severe damage to its membrane structure and loss of cell viability.

[0100] Flow cytometry analysis showed that the proportion of MRSA and Escherichia coli PI-positive cells in the PA-Ce@EG group was significantly increased in PI-stained bacteria. Figure 4 (gi in the text). Fluorescence intensity analysis further confirmed that PA-Ce@EG can enhance membrane permeability ( Figure 4 j in the middle.

[0101] The extravasation of intracellular nucleic acids and proteins in MRSA and Escherichia coli under different treatment conditions was evaluated by absorbance measurements at 260 nm and 280 nm. Compared with the control group, PLLA group, and PA-Ce group, the absorbance of both strains in the PA-Ce@EG treatment group was significantly increased at both wavelengths. Figure 4 The j in the figure indicates that a large amount of intracellular components are expelled.

[0102] The results of this embodiment demonstrate that PA-Ce@EG can severely disrupt the membrane integrity of Gram-positive bacteria (MRSA) and Gram-negative bacteria (Escherichia coli), leading to extravasation of cytoplasmic components and achieving effective bacterial inactivation. PA-Ce@EG exhibits excellent antibacterial properties through the synergistic effect of cerium ion-induced oxidative stress and EGCG-mediated membrane damage, providing a reliable non-antibiotic strategy for reducing bacterial load in diabetic wound infections.

[0103] Example 8: Evaluation of Macrophage Immunomodulatory Effects

[0104] Evaluation was performed using mouse RAW 264.7 macrophages (ATCC® TIB-71™). Cells were seeded onto sterile membranes in 24-well plates (2 × 10⁶ cells per well). 5 (Number of cells), with lipopolysaccharide (LPS, 100 ng / mL) as the M1 polarization control. After 24 hours of incubation, iNOS and CD206 were detected by immunofluorescence staining, imaged using a confocal laser scanning microscope (FV3000, Olympus), and semi-quantitatively analyzed using ImageJ software. For flow cytometry, cells were stained with FITC-labeled CD86 antibody and PE-labeled CD206 antibody, and analysis was performed on a flow cytometer (BD Accuri C6 Plus, BD Laboratories, USA). Total RNA was extracted using the TRIzol method and reverse transcribed to generate cDNA. The expression levels of IL-1β, TNF-α, TGF-β, and Cd206 were quantitatively detected using SYBR Green qPCR, with Gapdh as an internal control for standardization. Cytokine levels (IL-1β, TNF-α, TGF-β, and IL-10) in the supernatant were measured using ELISA kits according to the manufacturer's instructions.

[0105] Immunofluorescence staining showed that, compared with the control group and the PLLA group, cells cultured with PA-Ce@EG exhibited decreased expression of the M1 marker iNOS and increased expression of the M2 marker CD206. Figure 5 (bc in the text). PA-Ce@EG is significantly superior to PA-Ce in promoting M2 polarization, indicating that Ce ions and EGCG have a synergistic effect in regulating macrophage phenotype.

[0106] Flow cytometry analysis showed that LPS stimulation induced CD86 + Macrophages (M1 phenotype) were significantly enriched. PA-Ce and PA-Ce@EG treatments significantly altered the distribution of macrophage subsets: CD206 was significantly enriched in the PA-Ce@EG group. + The proportion of cells increased significantly, while CD86 + The proportion of macrophages decreased accordingly. Figure 5 (de in the text). Compared with the control group and the PLLA group, PA-Ce@EG significantly increased the proportion of anti-inflammatory M2 macrophages.

[0107] RT-qPCR results showed that in macrophages treated with PA-Ce@EG, the expression of IL-1β and TNF-α was significantly decreased, while the expression of TGF-β and Cd206 was increased. Figure 5 The f in the figure is consistent with the M2 type polarization phenotype.

[0108] ELISA further confirmed the secretory phenotype: IL-1β and TNF-α levels were significantly downregulated in the PA-Ce@EG group, while IL-10 and TGF-β levels were upregulated.

[0109] The results of this embodiment demonstrate that PA-Ce@EG can effectively reprogram macrophage responses by downregulating the pro-inflammatory cytokine axis and promoting a regenerative anti-inflammatory phenotype. The immunomodulatory capacity of this membrane originates from Ce. 4+ / Ce 3+ Synergistic effect of redox activity and anti-inflammatory properties of EGCG. PA-Ce@EG demonstrates the potential to alleviate chronic inflammatory responses and promote efficient wound healing by restoring immune balance and enhancing the M2 immune-mediated repair pathway.

[0110] Example 9: Evaluation of the angiogenesis effect of macrophage conditioned medium induced by HUVECs

[0111] To evaluate in vitro angiogenesis activity, this study employed a macrophage conditioned medium strategy. RAW264.7 macrophages (1×10⁶ cells / year) were cultured in this medium. 6 Human umbilical vein endothelial cells (cells / well, 6-well plate) were co-cultured with material extracts for 48 hours. The supernatant was collected, centrifuged, and then mixed with endothelial growth medium at a ratio of 1:3 to prepare macrophage conditioned medium. Human umbilical vein endothelial cells were cultured in macrophage conditioned medium for angiogenesis experiments.

[0112] In the tube formation experiment, cells (2 × 10⁻⁶) were used. 4 (10 cells / well) were seeded into a matrix gel-coated 96-well plate, and after 6 hours of incubation, the capillary-like structures were imaged and quantitatively analyzed using ImageJ software with an angiogenesis analysis plugin.

[0113] The migration experiment used the scratch method to assess wound closure at 0 and 12 hours.

[0114] In gene expression analysis, RNA was extracted from endothelial cells after culturing them in macrophage conditioned medium for 24 hours using the TRIzol method, and the activation status of endothelial cells was detected by CD31 immunofluorescence assay. Endothelial cells cultured on coverslips were fixed, permeabilized, and blocked, then incubated with anti-CD31 primary antibody and Alexa Fluor 488 secondary antibody. Cell nuclei were stained with DAPI and imaged using a fluorescence microscope. CD31 expression levels were semi-quantitatively analyzed using ImageJ software.

[0115] The scratch migration assay results showed that endothelial cells treated with PA-Ce@EG conditioned medium experienced a significantly faster wound closure rate. Figure 6 (b) Quantitative analysis confirmed that the migration area of ​​the PA-Ce@EG group was significantly larger than that of the control group, PLLA group, and PA-Ce group ( Figure 6 (c in the text)

[0116] In the tube formation experiment, endothelial cells exposed to PA-Ce@EG conditioned medium formed a dense and highly interconnected capillary-like network. Figure 6 In the d section, the quantitative indicators (including the number of nodes, branches, segments, and total tubular structure length) of the PA-Ce@EG group were significantly increased compared to other treatment groups. Figure 6 (eh in the middle).

[0117] CD31 immunofluorescence staining showed that CD31 expression was significantly enhanced in endothelial cells treated with PA-Ce@EG conditioned medium, exhibiting a more continuous and extended morphology, suggesting endothelial cell junction stabilization and maturation of angiogenic structures. In contrast, CD31 expression in endothelial cells of the control and PLLA groups was sparse and network formation ability was limited. Figure 6 (i in the text).

[0118] The results of this embodiment demonstrate that PA-Ce@EG indirectly enhances endothelial cell angiogenesis by remodeling macrophage-derived paracrine signaling pathways. This enhanced angiogenesis response is closely related to PA-Ce@EG-induced macrophage phenotype reprogramming: PA-Ce@EG significantly promotes the transformation to an M2-like macrophage phenotype, accompanied by increased levels of IL-10 and TGF-β secretion, two key paracrine mediators known to stimulate endothelial cell migration, survival, and angiogenesis. Simultaneously, inhibition of pro-inflammatory cytokines alleviates the inflammation-mediated angiogenesis inhibition. This immuno-angiogenic coupling mechanism is particularly advantageous in diabetic wounds, where persistent inflammation and vascular dysfunction are tightly intertwined, highlighting the therapeutic potential of PA-Ce@EG as an immunomodulatory pro-regenerative platform.

[0119] Example 10: In vivo assessment of wound healing in diabetic infections

[0120] All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji University. Male C57BL / 6J mice (6-8 weeks old) were induced to develop a diabetic model by intraperitoneal injection of streptozotocin (STZ, 30 mg / kg) for 7 consecutive days. Mice with blood glucose ≥16.7 mmol / L 4 weeks after induction were included in subsequent experiments. To establish an infected wound model, 8 mm full-thickness dorsal skin defects were created in mice after anesthesia, and MRSA (5 × 10⁻⁶ mcg / kg) was inserted. 7 CFU / mL (50 μL) was applied to the wound to induce local infection. Experimental animals were randomly divided into four groups (n=12): PBS group, PLLA group, PLLA@Ce group, and PA-Ce@EG group. The membrane was attached to the wound and fixed with a sterile transparent film. Wound closure was recorded every 2 days and quantitatively analyzed using ImageJ software. After 14 days, mice were euthanized, and wound tissue was collected for histological and immunohistochemical analysis, including hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and staining for CD31, inducible nitric oxide synthase (iNOS), and CD206. For transcriptomics analysis, total RNA was extracted from the wound tissue, and high-throughput RNA sequencing was used to assess differential gene expression and pathway enrichment. Major organs (heart, liver, spleen, lung, and kidney) were collected for systemic biosafety histopathological assessment.

[0121] Representative gross images of the wounds showed that both the control group and the PLLA treatment group exhibited persistent inflammation and delayed healing, while the PA-Ce and PA-Ce@EG treatment groups showed significantly accelerated wound contraction. The PA-Ce@EG treatment group showed earlier eschar shedding and faster wound area reduction during the healing process. Figure 7 (b) The corresponding wound area contour map and fusion map further revealed the dynamic changes during the wound closure process, clearly showing that the PA-Ce@EG group had a continuously faster healing trajectory ( Figure 7 (c) Quantitative thermographic analysis of wound area evolution showed that the residual wound area in PA-Ce@EG treated mice was significantly reduced, especially during the critical proliferative phase from day 7 to day 14. Figure 7 (d) Statistical analysis confirmed that, compared with other groups, PA-Ce@EG achieved the smallest wound area on day 14 ( Figure 7 The presence of EGCG (e) demonstrates its superior wound-healing effect. Although PA-Ce alone already showed some improvement compared to the PLLA group, the addition of EGCG further enhanced wound closure.

[0122] To assess whether accelerated healing was associated with improved infection control, the study quantified bacterial load in the early stages. Colony imaging and CFU analysis showed that the bacterial load was significantly lower in the PA-Ce@EG group compared to the control and PLLA groups. Figure 7 (fg in the text). This reduction occurs in the early stages of inflammation, indicating that effective infection control precedes tissue regeneration. Compared to PA-Ce alone, PA-Ce@EG further reduced bacterial counts.

[0123] Histological evaluation results showed that the PA-Ce@EG group had continuous epidermis, good dermal structure, enhanced collagen deposition, decreased iNOS-positive cells, increased CD206-positive cells, and significantly increased microvessel density in the wound tissue. Figure 8 The transcriptomic analysis showed that differentially expressed genes were enriched in cell adhesion, cytokine-cytokine receptor interactions, and the PI3K-Akt signaling pathway, etc. Figure 9 ad and Figure 10 ).

[0124] The results of this study demonstrate that PA-Ce@EG significantly reduced bacterial load and accelerated wound closure in a diabetic mouse model of infected wounds. The PA-Ce@EG treatment group exhibited faster wound contraction, earlier eschar shedding, and smaller residual wound area, showing superior healing effects compared to the PA-Ce and PLLA groups. Effective infection control precedes tissue regeneration, creating favorable conditions for subsequent immune regulation and tissue remodeling.

[0125] Example 11: Dressing Formulation Example

[0126] This embodiment provides a medical dressing containing a PA-Ce@EG membrane.

[0127] The PA-Ce@EG film prepared in Example 3 was cut to the required size (e.g., 2cm×2cm, 5cm×5cm, or 10cm×10cm), sterilized with ethylene oxide, and then sealed in a sterile aluminum foil bag. Each dressing was individually packaged and accompanied by instructions for use. This dressing is used for the covering and repair of infected diabetic wounds.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An immune reprogramming electrospun membrane composed of EGCG and cerium-doped mesoporous bioactive glass, characterized in that, The electrospun membrane is prepared by loading epigallocatechin gallate onto cerium-doped mesoporous bioactive glass and then integrating it into a L-type polylactic acid nanofiber matrix.

2. The electrospun film according to claim 1, characterized in that, The cerium doping concentration in the cerium-doped mesoporous bioactive glass is 5 mol%, Ce. 3+ With Ce 4+ coexist.

3. The electrospun film according to claim 1, characterized in that, The mass ratio of epigallocatechin gallate to cerium-doped mesoporous bioactive glass is 1:

10.

4. The electrospun film according to claim 1, characterized in that, The mass ratio of the L-polylactic acid to the cerium-doped mesoporous bioactive glass loaded with epigallocatechin gallate is 10:

1.

5. The electrospun film according to claim 1, characterized in that, The electrospun membrane exhibits time-programmed release characteristics: epigallocatechin gallate rapidly accumulates and releases over 70% within 7 days, achieving early antibacterial and anti-inflammatory effects; Ce 3+ / Ce 4+ Ions are continuously released for more than 14 days through the gradual degradation of cerium-doped mesoporous bioactive glass, achieving late-stage antioxidant and immunomodulatory effects.

6. The electrospun film according to claim 1, characterized in that, The electrospun membrane has a nanofiber structure that mimics the extracellular matrix, with a fiber diameter of 200 nm and a water contact angle of 78.6°.

7. A method for preparing the electrospun film according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Synthesis of cerium-doped mesoporous bioactive glass: Cerium-doped mesoporous bioactive glass was prepared by microemulsion-assisted sol-gel method, using tetraethoxysilane as silicon source, calcium nitrate tetrahydrate as calcium source, and cerium nitrate as cerium source, through sol-gel reaction, centrifugation washing, drying and calcination at 750℃. (2) Loading of epigallocatechin gallate: The cerium-doped mesoporous bioactive glass obtained in step (1) was dispersed in an aqueous solution of epigallocatechin gallate, stirred in the dark for 12 hours, centrifuged, washed and freeze-dried to obtain cerium-doped mesoporous bioactive glass loaded with epigallocatechin gallate. (3) Electrospinning to form a film: L-polylactic acid is dissolved in hexafluoroisopropanol, and cerium-doped mesoporous bioactive glass loaded with epigallocatechin gallate obtained in step (2) is added. After being uniformly dispersed, the film is formed by electrospinning and then dried under vacuum.

8. The preparation method according to claim 7, characterized in that, In step (3), the electrospinning parameters are: voltage 16kV, flow rate 1mL / h, receiving distance 15cm, and receiving drum speed 2800rpm.

9. The preparation method according to claim 7, characterized in that, The final concentration of the cerium-doped mesoporous bioactive glass loaded with epigallocatechin gallate in step (3) in the spinning solution is 0.5 mg / mL.

10. A dressing or gel for treating infectious diabetic wounds, characterized in that, It includes the electrospun film according to any one of claims 1-6.

11. A medical dressing, characterized in that, It includes the electrospun film according to any one of claims 1-6.

12. The medical dressing according to claim 11, characterized in that, The medical dressing is in the form of a patch, gel, sponge, or foam.

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