A composite hydrogel for promoting healing of diabetic wounds, its preparation method and application

Abstract

The present application relates to the technical field of biomedical materials, in particular to a composite hydrogel for promoting healing of diabetic wounds, a preparation method and application thereof. In the prior art, diabetic wounds are difficult to heal under the conditions of high glucose and high oxidative stress for a long time. The present application discloses a composite hydrogel for promoting healing of diabetic wounds, a preparation method and application thereof. The composite hydrogel is NSC / Met@HAMA, and the preparation method comprises the following steps: preparing an acrylate-metformin conjugate solution; under sterile conditions, mixing a neural stem cell suspension, a methylacrylated hyaluronic acid precursor solution and the acrylate-metformin conjugate solution at a volume ratio of 1:5:4, and performing a photo-crosslinking reaction in the presence of a photo initiator to obtain the composite hydrogel. The composite hydrogel synergistically regulates immunity and promotes vascular regeneration, and accelerates healing of diabetic wounds.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a composite hydrogel for promoting the healing of diabetic wounds, its preparation method, and its applications. Background Technology

[0002] Diabetic patients are in a state of chronic hyperglycemia, with coexisting microvascular and neuropathy, which significantly delays the skin wound healing process, making them prone to recurrent infections and developing into chronic ulcers, and in severe cases, even requiring amputation. Compared with normal wounds, diabetic wounds commonly exhibit pathological features such as persistent inflammation, elevated oxidative stress levels, insufficient local blood supply, and impaired extracellular matrix remodeling.

[0003] During the inflammatory phase, neutrophils and macrophages are crucial in clearing pathogens and tissue debris. However, in the diabetic microenvironment, immune regulation is often imbalanced: neutrophils are prone to overactivation and the formation of numerous extracellular traps, causing secondary tissue damage and hindering cell migration; macrophages are slow to transition from a pro-inflammatory to a pro-repair phenotype and have reduced efficiency in clearing apoptotic cells, thus creating a vicious cycle of persistent inflammation. Simultaneously, vascular endothelial cells are susceptible to mitochondrial dysfunction and apoptosis in a high-oxidative-stress environment. Insufficient angiogenesis prevents adequate oxygen and nutrient supply to meet regeneration needs, further limiting granulation tissue formation, collagen deposition, and re-epithelialization. Therefore, how to simultaneously regulate the immune microenvironment and promote angiogenesis is a key scientific and clinical challenge in the treatment of diabetic wounds.

[0004] Current treatment methods mainly include debridement, anti-infection, decompression, negative pressure drainage, and topical growth factors or dressings, but their comprehensive improvement on the complex pathological microenvironment is limited. In recent years, functional dressings and delivery systems based on biomaterials have attracted attention. Hydrogels, due to their advantages such as high water content three-dimensional network structure, good tissue compatibility, and ability to load drugs or cells, have been used for wound coverage and microenvironment regulation.

[0005] However, single-function hydrogels often struggle to simultaneously address multiple needs such as anti-oxidation, anti-inflammation, angiogenesis, and tissue remodeling. Furthermore, physical drug encapsulation can lead to burst release and insufficient maintenance of effective concentrations, impacting efficacy and safety. Stem cell therapy shows potential in promoting wound repair, but stem cells often face challenges such as low survival rates, short dwell times, and functional decline due to inflammation and oxidative stress at the wound site. Moreover, the regulation of immune-vascular remodeling by cell therapy alone remains unstable. In addition, most studies elucidating the synergistic repair mechanisms of materials, cells, and drugs remain at the histological and limited marker levels, failing to reveal the dynamic changes of different cell subpopulations and intercellular communication networks, thus limiting the targetedness and reproducibility of material design. Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide a composite hydrogel for promoting the healing of diabetic wounds, its preparation method, and its applications. By encapsulating neural stem cells in a three-dimensional network through HAMA photocrosslinking and covalently immobilizing an acrylate-metformin conjugate through crosslinking, in-situ gelation and sustained drug release are achieved, synergistically regulating immunity and promoting angiogenesis, thereby accelerating high-quality healing.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, a method for preparing a composite hydrogel for promoting the healing of diabetic wounds includes the following steps: Preparation of acrylate-metformin conjugate solution; Under sterile conditions, a neural stem cell suspension, a methacrylamide hyaluronic acid precursor solution, and an acrylate-metformin conjugate solution were mixed at a volume ratio of 1:5:4, and a photocrosslinking reaction was carried out in the presence of a photoinitiator to prepare a composite hydrogel.

[0008] Secondly, the methacrylamide hyaluronic acid precursor solution is a mixture of methacrylamide hyaluronic acid and a photoinitiator.

[0009] Thirdly, the photoinitiator is lithium acyl phosphate.

[0010] Fourthly, the concentration of methacrylamide in the hyaluronic acid precursor solution is 10%, and the concentration of lithium acylphosphate is 5%.

[0011] Fifthly, the density of neural stem cells is 2×10⁻⁶. 7 / mL, the final concentration of methacrylated hyaluronic acid is 5%, the final concentration of lithium acyl phosphate is 2.5%, and the final concentration of metformin is 200mM.

[0012] Sixthly, the conditions for the photocrosslinking reaction are: a wavelength range of 380~420nm, an illumination time of 5~20s, and a light source irradiance of 10~50mW / cm². 2 .

[0013] The seventh aspect involves the preparation of the acrylate-metformin conjugate solution, which includes the following steps: Metformin and AC-PEG-NHS were dissolved in phosphate buffer at a mass ratio of 1:25 and reacted at 4°C in the dark for 24 hours to introduce acrylate groups, thus obtaining an acrylate-metformin conjugate solution. The metformin concentration was 500 mM.

[0014] Eighthly, a composite hydrogel for promoting the healing of diabetic wounds, the composite hydrogel being prepared by the above method.

[0015] Ninthly, the use of the aforementioned composite hydrogel in the preparation of drugs that promote wound healing.

[0016] Compared with existing technologies, the present invention provides a composite hydrogel for promoting the healing of diabetic wounds, its preparation method, and its application, which bring the following significant effects: 1. This invention encapsulates neural stem cells in a HAMA light-crosslinked three-dimensional network and covalently fixes metformin in the form of acrylate conjugated crosslinking, thereby achieving an integrated dressing combining neural stem cells and drugs: the hydrogel three-dimensional high-water-content network can simulate the native ECM-like microenvironment and provide mechanical support for cells, while reducing the risk of rapid drug loss through drug covalent fixation, enabling metformin to achieve more stable and continuous release, thereby creating a more favorable local microenvironment for wound regeneration and helping to maintain ECM homeostasis; 2. This invention improves the quality of diabetic wound repair by synergistically reducing oxidative stress and regulating immune vascular remodeling: Under high oxidative stress conditions, it can reduce intracellular ROS / mtROS, inhibit endothelial cell apoptosis, improve mitochondrial membrane potential and energy metabolism, enhance key biological functions of endothelial cells, and promote angiogenesis, thereby helping to restore blood supply; at the same time, it promotes the transformation of macrophages to a repair phenotype and enhances the ability to clear apoptotic cells, reduces the deposition of extracellular traps (NETs) of neutrophils, and promotes the regression of inflammation into the regeneration stage; in vivo, it further manifests as accelerated wound closure, more complete and orderly collagen deposition, and increased levels of microvascular and mature vascular markers, resulting in higher quality tissue regeneration; 3. This invention introduces single-cell RNA sequencing to comprehensively map and quantify the cellular atlas of the wound microenvironment: By performing 10× single-cell sequencing on wound tissues of the control group and the NSC / Met@HAMA treatment group, the cellular heterogeneity of the wound microenvironment was systematically analyzed and a cellular atlas was established, which finely depicted the subpopulation division, differentiation trajectory, and RNA velocity characteristics of macrophages and neutrophils; and based on methods such as CellChat, intercellular communication was quantified, confirming that the overall intercellular crosstalk was enhanced after treatment, especially the number and intensity of interactions between immune cells and endothelial cells, thus providing direct support for the mechanism of action of this invention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1In the diagram, A shows the hydrogel encapsulation of neural stem cells and metformin hydrochloride; B shows the carbon-carbon double bond modification of metformin hydrochloride; C shows the gelation of HAMA and NSC / Met@HAMA hydrogels; D shows the nuclear magnetic resonance (NMR) analysis of HAMA; E shows the Fourier transform infrared (FT-IR) spectroscopy of HAMA; F shows the rheological test of HAMA under blue light irradiation; G shows the metformin release data with and without AC-PEG-NHS modification; H shows the scanning electron microscope (SEM) image of HAMA (scale bar = 500 μm); I shows the identification of neural stem cells using specific markers such as Nestin, GFAP, SOX2, and Tuj1 in bright field and immunofluorescence (scale bar = 100 μm for bright field and 500 μm for immunofluorescence images). Figure 2 In the table, A shows the live / dead staining of HUVECs treated with hydrogel at 24 and 48 hours (scale bar = 500 μm); B shows images of lumen formation in HUVECs treated with different hydrogels (scale bar = 500 μm); C shows the number of nodes in the lumen formation assay (n = 3); D shows the total branch length in the lumen formation assay (n = 3); E shows the fluorescence images of ROS in HUVECs after various treatments (scale bar = 500 μm); F shows the flow cytometry analysis of ROS in HUVECs treated with different hydrogels; G shows the average fluorescence intensity of HUVECs quantified by flow cytometry (n = 3); H shows the average fluorescence intensity of HUVECs quantified by fluorescence images using ImageJ (n = 3); I shows the quantitative flow cytometry analysis of the apoptosis ratio in HUVECs by calculating the cumulative ratio of B2+B4 (n = 3); and J shows the flow cytometry analysis of the anti-apoptotic ability of different hydrogels. Statistical analysis was performed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 3 In the table, A is a schematic diagram of the STZ-induced diabetic mouse animal experiment process; B is a representative image and a simulated image of the wound area in each group; C is the quantification of the remaining wound area (n=5); D is the quantification of the wound length on day 7 after treatment (n=4); E is the quantification of collagen deposition on day 14 after treatment (n=4); F is a representative image of HE staining of wound tissue on day 7 after treatment (scale bar = 500 μm); G is a representative image of Masson staining of wound tissue on day 14 after treatment (scale bar = 500 μm in normal view, scale bar = 20 μm in magnified view). Statistical analysis was performed using one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 4In the diagram, A shows the workflow of single-cell RNA sequencing for each group; B shows the unassessed cellular heterogeneity of wound tissue in the control and NSC / Met@HAMA groups plotted using UMAP; C shows the proportion of different cell types; D shows the UMAP plot of candidate marker genes in different cell types; E compares the differences in the number of interactions between all cell types in the wound region (NSC / Met@HAMA group and control group); F compares the strength of interactions between cells in the two groups, with red curves for enhancement and blue curves for weakness; G shows a dot plot displaying candidate marker genes for 10 different cell types. Figure 5 In the table, A represents the re-clustering analysis of macrophage subsets in the control group and the NSC / Met@HAMA group; B represents the RNA velocity analysis of macrophages; C represents the heatmap of macrophage subset-specific marker genes; D represents the re-clustering analysis of neutrophil subsets in the control group and the NSC / Met@HAMA group; E represents the RNA velocity analysis of neutrophils; F represents the heatmap of neutrophil subset-specific marker genes; G represents the comparison of the differences in interaction numbers among macrophages, neutrophils, and endothelial cells; H represents the comparison of interaction strength (NSC / Met@HAMA group and control group), where red curves represent enhanced interactions and blue curves represent weakened interactions; I represents the analysis of input and output interaction strengths in different groups; J and K represent pseudo-time analysis of macrophage subsets; L and M represent pseudo-time analysis of neutrophil subsets. Figure 6In the table, A shows the OCR analysis of HUVECs monitored by the hippocampal system; B shows the quantitative analysis of basal respiration, ATP production, reserve respiration capacity, and maximum respiration of HUVECs after hydrogel treatment (n=5); C shows the analysis of extracellular acidification rate (ECAR) of HUVECs detected by the Seahorse system; D shows the quantitative analysis of glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification of HUVECs after hydrogel treatment (n=5); E shows a schematic diagram of hippocampal and flow cytometry (MitoSOXRed and JC-1) mitochondrial function assessment; F shows the quantitative analysis of MitoSOXRed levels in HUVECs by flow cytometry (n=3); G shows the quantitative analysis of JC-1 staining in HUVECs after different treatments by calculating the JC-1 aggregate / JC-1 monomer ratio (n=3); H shows the MitoSOX levels in HUVECs under different treatments. Flow cytometry analysis of Red staining; I represents flow cytometry analysis of JC-1 staining in HUVECs under different treatments, ECAR: extracellular acidification rate; OCR: oxygen consumption rate; ATP: adenosine triphosphate; Student's t-test was used for statistical analysis of B and D. One-way ANOVA was used for statistical analysis of F and G, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=no significance; Figure 7 In the table, A shows representative images of CD206 and iNOS in RAW264.7 cells treated with LPS and different hydrogels, scale bar = 20 μm; B is a schematic diagram of cell effect measurement in RAW264.7 cells; C shows Annexin V-FITC / PI staining of Jurkat cells before and after UV irradiation; D shows flow cytometry analysis of each hydrogel group (n=3); E shows the quantification of fluorescence intensity of CD206 and (F) iNOS (n=4); G shows the quantification of cell burial rate of RAW264.7 cells after different treatments (n=3). Statistical analysis was performed using one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Figure 8In the image, A represents the representative immunofluorescence images of H3Cit (green) and MPO (red) labeled NETs in the wound sample on day 3 post-treatment (scale bar = 200 μm); B represents the representative immunochemical images of the inflammation-related markers CD206 and iNOS in the wound tissue on day 7 post-treatment; C represents the representative immunochemical image of GAS6 in the wound tissue on day 7 post-treatment; D represents the representative immunochemical images of CD31 and α-SMA on day 14 post-treatment; E and F represent the quantitative analysis of the relative fluorescence intensity of H3Cit and MPO (n=4); G-I represent the quantitative analysis of the positive area ratio of CD206, iNOS, and GAS6 (n=4); J represents the quantitative analysis of the number of blood vessels (obtained from CD31 expression statistics) (n=4); K represents the quantitative analysis of the positive area ratio of α-SMA (n=4). The immunohistochemical image scale bar is 40 μm. Statistical analysis was performed using one-way ANOVA. ANOVA), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Detailed Implementation

[0019] This application proposes a composite hydrogel for promoting the healing of diabetic wounds, its preparation method, and its application. It primarily addresses existing technologies for treating diabetic wounds under high glucose and oxidative stress conditions, including persistent inflammation, imbalance between immune regulation and angiogenesis, endothelial cell apoptosis and mitochondrial dysfunction, the difficulty of combining immune regulation and angiogenesis promotion with single treatments, rapid drug dissolution leading to insufficient effective concentration maintenance, and limited survival and function of stem cells in adverse microenvironments. To address these issues, this application constructs an NSC / Met@HAMA composite hydrogel system: using methacryloyl hyaluronic acid (HAMA) as the backbone material, a three-dimensional high-water-content network is formed through photocrosslinking under photoinitiator. Neural stem cells (NSCs) are encapsulated within this network, and metformin is introduced into the network via acrylate groups to form an acrylate-metformin conjugate, which is covalently fixed to the network during crosslinking, thereby achieving stable drug loading and sustained release. Therefore, this application can form a composite dressing in situ on the wound surface that has both cell protection and microenvironment regulation capabilities, synergistically reducing oxidative stress and cell apoptosis, improving mitochondrial function, promoting the immune microenvironment to shift towards a repair-promoting direction, and promoting angiogenesis and tissue reconstruction, thereby improving the healing speed and healing quality of diabetic wounds.

[0020] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be obtained by purchasing them on the market or by existing methods.

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0022] Example 1 1. Brief Introduction Advances in stem cell therapy have garnered significant attention due to their applications in tissue repair and regeneration. With self-renewal capacity and pluripotent differentiation potential, stem cells, including bone marrow-derived mesenchymal stem cells, adipose-derived stem cells, and umbilical cord mesenchymal stem cells, have shown promising effects in promoting wound healing. However, research in the field of neural stem cell therapy for diabetic wound healing is limited. Previous studies have typically focused on using neural stem cells to treat traumatic brain injury, spinal cord injury, and multiple sclerosis. The underlying mechanisms may involve modulating the inflammatory microenvironment and promoting neural regeneration. Therefore, exploring the potential of neural stem cells in diabetic wound healing aims to broaden their application scope.

[0023] Given the challenges associated with NSCs, such as poor viability, instability, and short lifespan in the external environment, further exploration has been undertaken to design suitable scaffolds or combine them with synergistic therapeutic agents to create a niche conducive to regeneration. Hydrogels, due to their highly hydrated three-dimensional network structure, can closely mimic the physiological environment of the native extracellular matrix. When loaded with stem cells, they not only provide suitable mechanical support but also partially protect cells from direct immune attack by the host. Utilizing the mild sol-gel transition and low cellular damage associated with photocrosslinked hydrogels, this invention selected hyaluronic acid methacrylate as the scaffold material for the hydrogel. Considering the complexity of wound regeneration, a combined strategy beyond standalone stem cell therapies is needed. Therefore, this invention designs a system that couples a cell-compatible hydrogel with the sustained release of metformin, a drug known for its ability to improve the wound microenvironment through antioxidant, anti-inflammatory, and AMPK-mediated bioenergetic effects. Preventing rapid drug dissolution constitutes a primary design challenge. The strategy of this invention addresses this problem by covalently linking metformin to the HAMA scaffold. This was achieved through the synthesis of an acrylate-metformin conjugate, in which acrylate-PEG-NHS (AC-PEG-NHS) reacts with the amino groups in metformin. Subsequently, photoinitiated crosslinking using lithium acyl phosphate (LAP) as an initiator permanently bridged this drug-polymer adduct to the hydrogel matrix, creating a unified platform for controlled release and enhanced regeneration outcomes. More discriminative sequencing methods are needed to more accurately characterize the dynamic shifts in intercellular communication between different cell types during wound regeneration. Single-cell RNA sequencing, as a powerful tool, allows for whole-genome analysis of individual cells within tissues. Integrating bioinformatics analyses can provide a more reliable interpretation of biological events. However, the application of NSC-based therapies, particularly in diabetic wound repair, remains unexplored at the single-cell level. Therefore, the goal of this invention is to utilize this technology to reveal changes in cell subpopulations and potential differentiation trajectories mediated by the combined delivery of NSCs and metformin, thereby providing a more robust foundation for treatment evaluation.

[0024] In this study, the invention comprehensively evaluated the biocompatibility of the system in vitro and in vivo, establishing fundamental prerequisites for its therapeutic applications. Benefiting from the potent secretomic activity of NSCs and the antioxidant and mitochondrial regulatory capabilities of metformin, the system effectively reduced oxidative stress, enhanced resistance to apoptosis, and improved mitochondrial membrane potential and energy metabolism. These effects collectively create a favorable microenvironment for the formation of mature and highly ordered blood vessels. Furthermore, the heterogeneous hydrogel promoted M2 polarization while inhibiting the M1 polarization phenotype of macrophages. More importantly, it exhibited a remarkable ability to scavenge apoptosis. In vivo experiments further confirmed its ability to reduce NET deposition. All these observed effects contribute to maintaining an immune microenvironment conducive to resolving inflammation. Through single-cell RNA sequencing, the invention delineated cellular heterogeneity in the regenerated wound on day 7. Specifically, the invention established a fine sub-clustering and differentiation trajectory for two major cell subsets—macrophages and neutrophils. The invention also quantified intercellular communication between immune cells and endothelial cells. In conclusion, engineered hydrogels containing NSC and metformin may be a powerful weapon against non-healing wounds.

[0025] 2. Materials and Methods 2.1. Cell Culture Mouse monocytes / macrophages (RAW264.7) and human umbilical vein endothelial cells (HUVECs) (from Shanghai Zhongqiaoxinzhou Biotech) were cultured in DMEM (Basal Media) containing 10% fetal bovine serum (FBS, VivaCell) and 1% penicillin / streptomycin (Pen / Strep, C100C5, NCM Biotech). Human T lymphocytes (Jurkat) were purchased from Procell (CL-0129) and cultured in RPMI 1640 medium (Gibco) containing 10% FBS and 1% penicillin / streptomycin. Primary neural stem cells were cultured in Neurobasal medium (21103049, Gibco) supplemented with 2% B27 (12587010, Gibco), 1% N2 (17502048, Gibco), 20 ng / mL recombinant human basic fibroblast growth factor (AF-100-18B, Peprotech), and 20 ng / mL recombinant human epidermal growth factor (AF-100-15, Peprotech). All cells were cultured at 37°C in a 5% CO2 incubator.

[0026] 2.2. Isolation and Characterization of NSC To isolate primary neural stem cells (NSCs) from mice, pregnant C57BL / 6 mice at 14.5 days of gestation were purchased from the Animal Experiment Center of Air Force Medical University. The ganglionic eminence (GE) of the fetal mouse brain was microscopically dissected. The dissected tissue was mechanically dissociated, and the resulting cell suspension was filtered through a 200 μm mesh to remove undissociated tissue debris. Cell debris was then removed by low-speed centrifugation (800 rpm, 5 minutes, 4°C). To purify the NSCs, adherent cells were removed after 24 hours of culture to eliminate non-NSCs.

[0027] 2.3. Characterization of NSCs by immunofluorescence Cells were seeded at an appropriate density on poly-L-lysine (PLL, Beyotime, C0313)-coated culture plates. After cell adhesion, they were fixed with 4% paraformaldehyde (Beyotime, P0099), infiltrated with 0.2% Triton X-100 (Beyotime, P0096), and blocked with 3% BSA. The samples were then incubated overnight at 4°C with primary antibodies against SOX2, Nestin, GFAP, and Tuj-1. The following day, after culturing with the corresponding fluorescent secondary antibodies, the immunostained cells were visualized and imaged under a fluorescence microscope.

[0028] 2.4. Preparation of hydrogels The hydrogel scaffold is methacrylamide hyaluronic acid (HAMA, EngineeringForLife, China). To functionalize the drug, metformin (catalog number: A122997, Ambeed; solid, purity ≥99%) was first dissolved in PBS at a mass ratio of 1:25 with AC-PEG-NHS (EngineeringForLife, China; solid, white powder, molecular weight 2k; purity ≥99%) and incubated statically at 4°C in the dark for 24 hours to introduce acrylate groups. For cell encapsulation, neural stem cell suspension, methacrylamide hyaluronic acid precursor solution (HAMA concentration 10%, LAP concentration 5%), and acrylate-metformin conjugate solution (metformin concentration 500mM) were mixed at a volume ratio of 1:5:4 under sterile conditions at room temperature. The final concentrations of HAMA, photoinitiator lithium acyl phosphate (LAP), and metformin were 200 mM and 2 × 10⁻⁶ mM respectively. 7 / mL; Conditions for the photocrosslinking reaction of the mixture: wavelength range of light irradiation 380~420nm, irradiation time 5~20s, and irradiance of the light source (mW / cm²) 10~50mW / cm². 2The hydrogel sample can be gelled by directly irradiating one side at a distance of 5cm.

[0029] 2.5. Characterization of hydrogels To confirm the successful grafting of methacrylic acid groups, HAMA was dissolved in deuterated water (D113904, Aladdin) and analyzed by ¹H NMR spectroscopy (Bruker AVANCE NEO 600, Germany). Fourier transform infrared spectroscopy (FT-IR) (Thermo Fisher Nicolet IS50) further confirmed the presence of the characteristic functional groups. The rheological properties of the HAMA hydrogel were evaluated using a rheometer (HaakeMars 40, Germany) to compare the storage modulus (G') and loss modulus (G) before and after photocrosslinking.

[0030] 2.6. Degradation and Swelling The in vitro degradation profile of the hydrogel was evaluated by incubating a pre-weighed sample (W0) in DPBS at 37°C. At predetermined time points, the hydrogel was recovered, surface moisture was carefully removed, and the wet mass (W0) was recorded. t The degradation process was monitored for 20 days, and the remaining mass percentage was calculated as (W). t / W0)×100%.

[0031] Swelling capacity was determined by measuring the mass change of hydrogels cultured in DPBS at 37°C. The initial mass (W0) of newly formed hydrogels was recorded. At specified intervals, samples were removed, excess liquid on the surface was blotted with filter paper, and the samples were weighed (W0). t The expansion rate is calculated as [(W)]. t –W0) / W0]×100%.

[0032] 2.7. Drug Release Analysis To evaluate the release kinetics of metformin in HAMA hydrogels, 500 μL of metformin hydrogel was immersed in 4 mL of DPBS. At each sampling time point, 100 μL of the release medium was collected and stored at -80°C for subsequent analysis, with an equal volume of fresh DPBS added to the system. The concentration of metformin in all samples was determined by measuring absorbance at 233 nm, and a standard curve was established using solutions with known metformin concentrations. The corresponding cumulative release percentage was calculated. All release experiments were performed three times (n=3).

[0033] 2.8. Biocompatibility and safety assessment 1) In vitro biocompatibility Two complementary assays were used to assess cell compatibility. For the live / dead assay, HUVECs were seeded in 96-well plates and co-cultured with various hydrogels at 37°C for 24 hours. Cells were then stained with a calcitonin-AM / PI double staining kit (LK-1516, LinGkeBio) and visualized under an inverted fluorescence microscope. For quantitative analysis, cell viability was determined using CCK-8. After 24 hours of co-culture, the medium was replaced with fresh medium containing CCK-8 reagent (C6005, NCM Biotechnology), and the cells were then cultured at 37°C for 2 hours. Absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as follows: (Osample - Oblank) / (Ocontrol - Oblank) × 100%, where Osample, Oblank, and Ocontrol represent the absorbance values ​​of the treatment group (co-cultured with various hydrogels), blank group (cell-free), and control group (untreated, normally cultured cells), respectively.

[0034] 2) Biocompatibility in vivo To assess the system's biocompatibility, adult wild-type C57BL / 6 mice were purchased from the Animal Experiment Center of Air Force Medical University, and NSC / Met@HAMA hydrogel was subcutaneously implanted into the mice for 14 days. After the implantation period, major organs (heart, liver, spleen, lungs, and kidneys) were collected for hematoxylin-eosin (H&E) staining to observe potential morphological changes. In addition, blood was collected for whole blood panel and biochemical analysis to assess any blood toxicity.

[0035] 3) Hemostatic ability The hemostatic efficacy of the hydrogel was tested in a wild-type C57 mouse liver hemorrhage model. Anesthetized mice were fixed to an operating table, and an abdominal incision was performed to expose the liver. After placing a sheet of filter paper under the liver, a 5 mm incision was made in the liver lobe. The hydrogel was immediately applied to cover the wound. The amount of blood loss was quantified by measuring the difference in weight of the filter paper before and after blood absorption.

[0036] Hemolytic activity was assessed using erythrocytes (RBCs) isolated from wild-type C57 mouse blood. Whole blood was centrifuged at 1000 rpm for 10 min, and the collected RBCs were washed three times with PBS. Subsequently, 500 μL of hydrogel was incubated with 500 μL of RBC suspension at 37°C for 1 h. After incubation, the mixture was centrifuged, and 100 μL of supernatant was transferred to a 96-well plate. Absorbance at 540 nm was measured using a microplate reader. The hemolysis ratio was calculated as follows: (A_Sample - A_PBS) / (A_Triton-X100 - A_PBS) × 100%, where A_sample, A_PBS, and A_Triton-X100 represent the absorbance values ​​of the hydrogel sample, negative control (PBS), and positive control (1% Triton-X100), respectively.

[0037] 2.9. Lumen Formation Experiment 24-well plates were pre-coated with Matrigel (356234, Corning) and incubated at 37°C for 1 hour. HUVECs were then seeded onto the plates and co-incubated with different hydrogels for 6 hours. After staining with Calcein-AM (LK-1516, LinGkeBio), cells were observed using an inverted fluorescence microscope (Nikon, DS-Ri2, Japan). The number of nodes and total branch length were measured using Angiogenesis_Analyzer in ImageJ.

[0038] 2.10. Apoptosis assay To evaluate the anti-apoptotic effect of the hydrogel, flow cytometry was used to perform an Annexin V-FITC / PI apoptosis assay. HUVECs were co-cultured with the hydrogel for 24 hours while being exposed to 300 mM H2O2. After treatment, cells were collected and stained with Annexin V-FITC and propidium iodide (PI) (556547, BD Pharmingen) at 4°C in the dark for 30 minutes, followed by washing to remove unbound dye. The samples were then analyzed on a flow cytometer (Beckman Coulter, XL), and the data were processed using EXPO32ADC analysis software.

[0039] 2.11. Cell cycle assay To assess the effect of hydrogels on the cell cycle of HUVECs, this invention analyzed the distribution of cell cycle phases using flow cytometry. Cells were first cultured in hydrogels or untreated for 24 hours. After collection, cells were fixed overnight in 70% ethanol and then stained with PI / RNase (550825, BD Pharmingen) at 4°C for 30 minutes. After a washing step to remove unbound dye, the samples were analyzed on a Beckman Coulter XL flow cytometer. The generated data were processed using ModFit LT 3.0 software to determine the percentage of cell cycle phases.

[0040] 2.12. Determination of ROS and mt-ROS To detect intracellular reactive oxygen species (ROS) and mitochondrial superoxide levels, this invention established an oxidative stress model in H2O2-induced HUVECs. Cells were co-cultured with different hydrogels and simultaneously exposed to 400 μM H2O2 for 24 hours. After treatment, cells were collected and stained using DCFH-DA (S0033S, Beyotime) and MitoSOXRed (S0061S, Beyotime) fluorescent probes. After two washes to remove excess dye, cells were analyzed by flow cytometry. Data were processed using Novo Express software.

[0041] 2.13. Mitochondrial membrane potential detection Mitochondrial membrane potential (ΔΨm) in this study was assessed by flow cytometry. HUVECs were co-cultured with various hydrogels and exposed to 400 μM H₂O₂ for 24 h. Cells were then collected by centrifugation and stained with JC-1 dye (C2003S, Beyotime) at 37°C for 20 min. After washing twice with the provided staining buffer, samples were analyzed on a Beckman Coulter XL flow cytometer. Data were processed using EXPO32 ADC analysis software.

[0042] 2.14. Macrophage polarization assay A model of LPS-induced macrophage polarization was established to demonstrate the inflammatory modulatory properties of the hydrogel. In short, RAW264.7 cells were treated with various methods for 48 hours in the presence of 200 ng / ml LPS. Samples were fixed with 4% PFA for 30 min, infiltrated with 0.2% Triton X-100, and blocked with 5% BSA for 2 h, followed by overnight incubation with primary antibodies at 4°C. The next day, cells were incubated with fluorescent secondary antibodies for 1 h and observed using a confocal microscope (FV3000, Olymbus). The primary antibodies used in this work were CD206 (1 μg / ml, ab64693, abcam) and iNOS (1:400, 18985-1-AP, ProteinTech).

[0043] 2.15. Cell proliferation experiment Cell proliferation capacity was assessed by flow cytometry. Jurkat cells were irradiated with UV light for 25 minutes to induce apoptosis, then cultured for 3 hours and labeled with Cell Tracker Deep Red (630 / 660nm, C34565, Thermo Fisher Scientific). These apoptotic cells were then co-incubated with RAW264.7 macrophages at a 5:1 ratio (apoptotic cells to macrophages) for 90 minutes. After washing to remove non-phagocytic cells, macrophages were labeled with F4 / 80 staining. Analysis was performed on an ACEA Biosciences flow cytometer, where efferocyty macrophages were identified as F4 / 80. + / Cell Tracker Deep Red + Population. Efficiency is calculated as F4 / 80. + Percentage of double-positive cells in the total population. Data processing was performed using NovoExpress software.

[0044] 2.16. Hippocampal Analysis Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using an Agilent Hipmaster XFe96 analyzer.

[0045] For the mitochondrial stress assay (OCR), HUVECs were seeded into TC-treated XF96 cell culture plates (103794-100, Agilent) and treated with different hydrogels for 24 hours in a 37°C, 5% CO2 incubator. On the day of the assay, the sensor cartridge was hydrated overnight in XF calibration solution in a non-CO2 incubator. Hippocampal XF bases supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine were filtered, sterilized, and adjusted to pH 7.4. The assay medium was preheated to 37°C before use. The cell culture medium was replaced with the preheated assay medium, and the cell plates were incubated at 37°C in a non-CO2 incubator for 60 minutes to maintain temperature and pH equilibrium. Mitochondrial regulators, 1.5 mM oligomycin, 1.0 mM FCCP, and 0.5 mM roxithone / antimycin A were loaded into the instrument according to the manufacturer's instructions. The plate was then loaded into the HippoXFe96 analyzer and mitochondrial stress tests were performed according to the standard programming protocol.

[0046] For the extracellular acidification rate (ECAR) assay to assess glycolytic function, HUVECs underwent the same pretreatment as described above. The assay medium for ECAR measurement was also similar. Prior to the assay, cells were equilibrated for 60 minutes in a glucose-free, carbon dioxide-free incubator to deplete endogenous glucose. Compounds including 10 mM glucose, 1.0 μM oligomycin, and 50 mM 2-deoxy-D-glucose (2-DG) were pre-loaded into the instrument. Real-time changes in ECAR were recorded after successive injections of these compounds. All data were normalized to total protein content and analyzed using Wave software (version 2.6, Agilent).

[0047] 2.17. Animal Research All animal experiments were approved by the Ethics Committee of the Air Force Military Medical University (Approval No.: 20250121). As previously described, a diabetic mouse model was established. Briefly, 5-6 week old male C57BL / 6 mice were fed a high-fat diet for 4 weeks, followed by intraperitoneal injections of streptococcus (STZ, 50 mg / kg body weight daily) for 5 consecutive days. Blood glucose levels were measured one week after the last injection. Mice with blood glucose concentrations exceeding 16.8 mmol / L in two consecutive measurements were identified as diabetic and used for subsequent wound healing experiments. For the wound healing experiment, the back hair of the mice was removed using an electric razor, followed by the application of depilatory cream to ensure complete hair removal. Mice were anesthetized with isoflurane of appropriate oxygen concentration, and two full-thickness skin wounds (8 mm in diameter) were formed on the back of each mouse using a punch. The wounds were filled with uniform doses (60 μL) of the following treatments: untreated group, HAMA group, NSC@HAMA group, Met@HAMA group, and NSC / Met@HAMA group. Following treatment, all wounds were covered and secured with a transparent film dressing (3MTegaderm™, 9534HP). Mice were then returned to their cages and closely monitored. Wound areas were photographed on days 0, 3, 7, 10, and 14 post-surgery to monitor healing progress. For histological analysis, mice were euthanized, and wound tissue and surrounding skin were harvested on days 3, 7, and 14. Wound areas measured from the photographs were analyzed using ImageJ software. Wound healing rate was calculated as: (S0 - St) / S0 × 100%, where S0 is the initial wound area (day 0) and St is the wound area on day t.

[0048] 2.18. Histological and Immunofluorescence Staining Tissue samples were fixed in 4% formaldehyde for at least 24 hours, followed by dehydration via a series of graded ethanol fractions. The dehydrated tissues were embedded in paraffin using an embedding machine. The embedded paraffin blocks were trimmed and cut into 4 μm thick sections. The sections were baked at 60°C and then stored at room temperature for subsequent staining.

[0049] For hematoxylin-eosin (H&E) staining, after dewaxing tissue sections to water, stain with hematoxylin for 4 minutes, differentiate with hydrochloric acid-ethanol for 1-50 seconds, return to blue with a weak alkaline solution, and then stain with eosin for 2-5 minutes. After dehydration and clearing, mount with neutral resin and examine under a microscope. For Masson staining, after dewaxing tissue sections to water, immerse them in Bouin solution. Then stain according to the manufacturer's instructions (G1340, Solarbio), dehydrate and clear, and mount with neutral resin. Observe the collagen distribution characteristics under a microscope.

[0050] For immunohistochemistry (IHC), after dewaxing tissue sections to water, antigen retrieval was performed using EDTA buffer at pH 9.0. After cooling to room temperature, slides were incubated in 3% H2O2 for 30 minutes to inhibit endogenous peroxidase activity. After drawing circles around the tissue sections with a hydrophobic pen, they were blocked with 10% goat serum at room temperature for 30 minutes, followed by incubation with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-CD206 (1:2000, ab300621, Abcam), anti-iNOS (1:2000, ab283655, Abcam), anti-Gas6 (1:100, A8545, ABclonal), anti-α-SMA (1:2000, 14395-1-AP, ProteinTech), and anti-CD31 (0.5 μg / mL, ab9498, Abcam). After incubation, the slides were washed three times with TBST and incubated with secondary antibody containing HRP for 1 hour at room temperature. Color development was performed using freshly prepared DAB solution. Finally, the cell nuclei were stained with DAPI, mounted with neutral resin, and examined under a microscope.

[0051] For immunofluorescence staining, after deparaffinizing the sections, circles were drawn around the tissue sections with a hydrophobic pen. The sections were blocked with 10% donkey serum at 37°C for 30 minutes, followed by incubation with primary antibody at 4°C overnight. The following major antibodies were used for immunofluorescence: anti-MPO (10 μg / mL, AF3667, R&D Systems), anti-H3Cit (1:200, ab5103, Abcam), anti-SOX2 (1:200, 11064-1-AP, ProteinTech), anti-Nestin (1:200, 19483-1-AP, ProteinTech), anti-GFAP (1:200, 16825-1-AP, ProteinTech), and Tuj1 (1:500, 10094-1-AP, ProteinTech). The next day, the sections were washed three times with TBST and incubated at 37°C with a secondary antibody containing an appropriate fluorescent group for 60 minutes. Cell nuclei were stained with DAPI and mounted with an antifluorescence quencher. All stained slides were stored in the dark to prevent fluorescence quenching. Slides were imaged using an automated slide scanner (OLYMPUS VS200) or a confocal microscope (FV3000, Olympus) for subsequent analysis.

[0052] 2.19. Single-cell RNA sequencing and quality control Tissue samples were collected from the control group and the NSC / Met@HAMA treatment group on day 7 after wound formation and preserved in tissue preservation solution. Subsequently, the tissue was dissociated using digestion buffer to obtain single-cell suspensions, and cell viability was determined using CountsStar. The single-cell suspensions, premixed solutions, gel beads containing unique sequence barcodes, and oil were then co-encapsulated in microfluidic channels to form oil-coated single-cell microreactors, termed gel beads in emulsion (GEMs). The GEMs were collected and reverse transcribed to achieve barcoding of cellular mRNA. The barcoded cDNA was then amplified, and a sequencing library was constructed for next-generation sequencing. Finally, the library was sequenced on the high-throughput platforms Illumina Xplus or DNBSEQT7.

[0053] For the raw sequencing data, initial quality control was performed using Cell Ranger (v7.1.0) to obtain basic metrics, including cell count, gene count, and sequencing data quality. Subsequently, the Seurat (v4) package was used for further rigorous quality control and filtering to remove low-quality cells and other suboptimal data, ensuring the reliability of downstream analyses.

[0054] 2.20. Statistical Analysis This invention presents data as mean ± standard deviation (SD) and performs statistical analysis using GraphPad Prism 10.5. Technically, this invention uses Student's t-test for comparisons between two groups and one-way ANOVA and Tukey's test for comparisons among multiple groups. The p-values ​​are expressed as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: no significance.

[0055] 3. Results 3.1. Fabrication and characterization of HAMA hydrogels encapsulated with neural stem cells and metformin To synthesize a hydrogel containing neural stem cells (NSCs) and modified metformin, this invention first selected methacryloyl hyaluronic acid (HAMA) as the scaffold. NSCs were isolated from fetal rat brain tissue and cultured and expanded in vitro. Considering that NSC therapy alone may not be sufficient to promote diabetic wound healing, this invention developed a synergistic drug delivery system by combining acrylate-modified metformin, which provides anti-apoptotic, intracellular ROS scavenging, polarization regulation, and metabolic reprogramming functions. To address the problem of rapid leaching of metformin from the scaffold and utilizing the presence of its amino group (NH2), this invention used AC-PEG-NHS to modify metformin, enabling its sustained release from the hydrogel scaffold via covalent bonds. Figure 1A). The hydrogel precursor solution (containing the photoinitiator LAP), cell suspension, and modified metformin solution were mixed in a specified ratio and thoroughly mixed, then irradiated with 405nm blue light for approximately 10 seconds to form the hydrogel NSC / Met@HAMA. Figure 1 B, 1C).

[0056] Next, the hydrogel was characterized multiple times. ¹H NMR spectroscopy confirmed the successful methacrylate esterification of hyaluronic acid (HA) to form HAMA. Characteristic signals of the methacrylate group were clearly observed: two vinyl protons at 6.08 ppm and 5.65 ppm, and an allyl methyl proton at 1.85 ppm. These findings, along with the characteristic signals of the HA backbone, including sugar ring protons (3.00–4.00 ppm and 4.20–4.60 ppm) and an N-acetylmethyl proton at 1.92 ppm, collectively demonstrate the successful synthesis of HAMA. It can also be concluded that the grafting ratio of the MA group is approximately 35% ( Figure 1 D). Fourier transform infrared (FTIR) spectroscopy reveals the characteristic transmittance of HAMA at specific wavenumbers. Figure 1 E). As a photopolymerizable hydrogel system, rheological tests were performed to examine the relationship between the storage modulus (G') and the loss modulus (G'') before and after UV irradiation. After exposure to 405 nm light, G' exceeded G'', indicating successful gelation. Figure 1 F). Furthermore, comparisons of the release profiles showed that the release rate of metformin in the AC-PEG-NHS modified group was significantly slower. Specifically, by day 4, the cumulative release of metformin in the unmodified group reached 62.8±2.51%, while the cumulative release in the modified group was only 36.58±2.36%. Moreover, this release-triggered effect persisted throughout the study period. By day 12, the modified group had released only 55.12±2.42% of the drug, significantly lower than the 80.89±3.15% released in the control group. Figure 1 G).

[0057] The microstructure of the HAMA hydrogel was characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). SEM images of the lyophilized sample revealed a tightly packed and interconnected network of pores, which is thought to facilitate cell penetration. Supplemental EDS analysis confirmed the expected elemental composition of the hydrogel, identifying carbon (C), nitrogen (N), and oxygen (O) as the major components of the polymer backbone. Furthermore, the detection of phosphorus (P) indicated the successful incorporation of phosphate-containing groups from the photoinitiation system during crosslinking. Figure 1 H).

[0058] To ensure the hydrogel system meets the requirements for wound healing applications, this invention evaluated its degradation and swelling capabilities. During 14 days of continuous monitoring, the hydrogel retained approximately 77.55 ± 0.02% of its initial mass. These findings confirm the strong water absorption capacity of the hydrogel of this invention, which is beneficial in absorbing wound exudate in healing scenarios. The selection criteria for neural stem cells (NSCs) as the loading component focused on their defined biocompatibility: proven self-renewal and proliferative capacity, broad pluripotent differentiation potential, and the generation of potent secretions that actively promote the repair process. Immunofluorescence staining confirmed the expression of NSC-specific markers, including Sox2, Nestin, Tuj1, and GFAP (…). Figure 1 I).

[0059] Here, the present invention develops a therapeutic system based on HAMA hydrogel co-packaged with NSC and modified metformin, and conducts a comprehensive evaluation to confirm its desired properties.

[0060] 3.2. Biocompatibility and in vitro biological effects of hydrogels Since excellent biocompatibility is a fundamental prerequisite for the effective application of hydrogel-based systems, their cellular compatibility must be evaluated before functional studies. Therefore, this invention incubated endothelial cells (HUVECs) with various hydrogel components for 24 and 48 hours, subsequently analyzing cell viability and proliferation to determine the safety profile of each formulation. Live / dead staining showed strong green fluorescence and minimal red fluorescence in all groups (control, HAMA, NSC@HAMA, Met@HAMA, and NSC / Met@HAMA), indicating good cell viability and confirming the excellent biocompatibility of this hydrogel system. Figure 2 A).

[0061] Angiogenesis plays a crucial role in restoring blood supply and remodeling the wound microenvironment. In the lumen formation assay, both the Met@HAMA and NSC@HAMA groups showed increased numbers of vascular branches and luminal structures during the same treatment period compared to the control group, indicating that both metformin and NSC promote angiogenesis. Furthermore, a synergistic effect was evident in the NSC / Met@HAMA group, which showed enhanced connectivity and branch length (…). Figure 2 (B-2D). In summary, this hydrogel system significantly enhances key biological functions of endothelial cells, particularly in cell cycle progression and angiogenesis.

[0062] Furthermore, diabetic wound healing is often affected by high oxidative stress, leading to cellular dysfunction. To simulate this, this invention established an oxidative damage model in HUVECs using H2O2 (500 mM, 12 h). H2O2 treatment significantly increased the intensity of green fluorescence, indicating a marked increase in intracellular ROS levels. Treatment with Met@HAMA or NSC@HAMA greatly reduced the fluorescence signal, although some green fluorescence remained. In contrast, the co-delivery system almost completely eliminated intracellular ROS (…). Figure 2 E). Flow cytometry analysis supported the following findings: H2O2 treatment caused a significant rightward shift of the fluorescence peak, while hydrogel loading shifted the peak to the left, indicating a decrease in ROS levels. Combination therapy showed the greatest efficacy ( Figure 2 F-2H). This invention also evaluated the ability of the hydrogel to protect HUVECs from apoptosis. Annexin V / PI staining showed that H2O2 treatment increased the total apoptosis rate (B2+B4) from 0.53±0.71% to 20.57±1.33%. Although HAMA itself had no significant effect, the addition of NSC or metformin significantly reduced the apoptosis rate to 12.83±0.70% and 11.23±1.23%, respectively. The co-delivery system further reduced the apoptosis rate to 7.53±0.87%, demonstrating the strong anti-apoptotic ability of the composite hydrogel. Figure 2 (I, 2J). In summary, hydrogels exhibit significant cytoprotective effects on endothelial cells by mitigating oxidative stress and inhibiting apoptosis. This dual capability is crucial for enhancing the resilience of endothelial cells in the hostile microenvironment of diabetic wounds.

[0063] 3.3. Bio-inspired hydrogels accelerated wound healing in diabetic mice. The in vivo therapeutic performance of the developed hydrogel system was systematically evaluated in a mature type 2 diabetic mouse model. This model was induced by a combination of a 4-week high-fat diet followed by a 5-day STZ injection regimen to achieve sustained hyperglycemia. Full-thickness excision wounds formed on the backs of these mice were topically treated with their respective hydrogel dressings. To quantitatively monitor the healing process, detailed photographs and digital planar mapping of the wound area were performed at predetermined intervals (days 0, 3, 7, and 14) after wound treatment. Figure 3 A).

[0064] Quantitative analysis of wound closure dynamics revealed the treatment outcomes in different treatment groups. Wound healing was severely impaired in the control group and the HAMA hydrogel-only group, with 58.35±7.69% and 48.24±4.22% of the initial wound area remaining unhealed by day 7, respectively. By day 14, these groups still exhibited considerable open wounds, with 30.49±5.91% and 28.17±7.82% of the area remaining unhealed, ultimately indicating that the HAMA hydrogel matrix itself did not provide a significant healing-promoting benefit in diabetic patients. In contrast, intervention with NSC or metformin significantly accelerated healing, achieving substantial wound closure by day 14 in 90.22±0.95% and 87.72±4.04%, respectively. The most significant therapeutic effect was observed in the co-delivery group (NSC / Met@HAMA), which promoted almost complete regenerative healing, with only 4.29±2.68% of the wound area remaining at the endpoint. Statistical analysis of the time-dependent healing curves further confirmed that the co-delivery group exhibited a more pronounced healing trajectory at all observed time points, highlighting a synergistic enhancement of the repair process. Figure 3 B, 3C).

[0065] In addition to gross wound closure, this invention also included a comprehensive histological evaluation to assess the quality of newly regenerated tissue. H&E staining was performed on the wound portion on day 7 to assess wound length and the structural integrity of the newly formed epidermis and dermis. Both NSC@HAMA and Met@HAMA monotherapy groups demonstrated significant efficacy in reducing wound gaps and promoting robust regeneration of the epidermal and dermal septa. However, the NSC / Met@HAMA co-delivery group exhibited the most pronounced recovery phenotype, characterized by the shortest residual wound length and the best tissue regeneration. Figure 3 D, 3F).

[0066] Furthermore, the maturation and organization of the extracellular matrix (ECM) were assessed using Masson staining of tissue sections on day 14, a key determinant of treatment quality. Histological examination revealed that the wounds in the control group and the HAMA-only group were characterized by sparse and fragmented collagen fibers, a typical feature of poor-quality scar tissue in diabetic wounds. In contrast, both NSC@HAMA and Met@HAMA resulted in a significant improvement in collagen deposition, evidenced by increased fiber density and better alignment. Most notably, wounds treated with NSC / Met@HAMA exhibited strong and uniform blue staining, indicating abundant total collagen content and the formation of a mature and robust ECM, supporting a morphological structure similar to intact physiological wound closure. Figure 3 E, 3G).

[0067] Overall, these combined findings provide compelling evidence that hydrogel-based co-delivery systems not only accelerate wound coverage by simultaneously delivering NSC and metformin, but also fundamentally coordinate superior regenerative quality.

[0068] 3.4. Single-cell analysis resolved the cellular heterogeneity and intercellular crosstalk in wound tissues between the untreated group and the NSC / Met@HAMA group. To further elucidate the biological events by which NSC / Met@HAMA promotes diabetic wound healing, this invention constructed a cDNA library and performed 10× single-cell RNA sequencing on skin tissue samples collected on day 7 of wound healing in both the control group and the NSC / Met@HAMA treatment group. Figure 4 A). First, this invention analyzes the cellular heterogeneity of the wound site through cluster analysis, dividing cells into 10 different subpopulations, including macrophages, neutrophils, dendritic cells, T cells, B cells, NK cells, Schwann cells, fibroblasts, endothelial cells, and smooth muscle cells, and describes the correlations, RNA velocity, and latent differentiation time (A). Figure 4 B). The cell population percentages in the hydrogel-treated group were 35.92% (macrophages), 45.13% (neutrophils), and 2.97% (endothelial cells), respectively, while those in the control group were 35.53%, 51.46%, and 1.98%, respectively. Figure 4 C). Subsequently, the present invention projects the marker genes of each cell subpopulation onto the UMAP map, which is highly consistent with cell type annotation; for example, S100a8 is used as a neutrophil marker, Lyz2 as a macrophage marker, and PECAM1 as an endothelial cell marker. Figure 4 D). The dot plot also vividly depicts candidate marker genes from 10 different cell populations ( Figure 4 E). Further CellChat analysis (NSC / Met@HAMAvs.Control) showed that hydrogel treatment generally enhanced the intensity and extent of intercellular crosstalk across all cell subtypes. Figure 4 F-4G).

[0069] Then, the present invention re-clustered the macrophages, subdividing them into eight sub-clusters ( Figure 5 Similarly, neutrophils were subdivided into six sub-clusters at a resolution of 0.5 (A). Figure 5 D). This invention performed RNA velocity and potential differentiation time analyses to describe the transitional relationships between cell states. Figure 5 B, 5E). Characteristic markers of macrophage and neutrophil subsets were visualized using heatmaps. Figure 5(C, 5F). At the set threshold, compared with the control group, macrophages in the treatment group showed 233 upregulated genes and 118 downregulated genes, while neutrophils showed 317 upregulated genes and 216 downregulated genes.

[0070] Furthermore, this invention aims to investigate, from a tissue-level perspective, whether hydrogel treatment promotes intercellular communication, particularly between immune cells and endothelial cells, using single-cell data. Figure 5 G, 5H). Using CellChat analysis, this invention found that, compared with the control group, the NSC / Met@HAMA group significantly increased the number and intensity of interactions between macrophages and endothelial cells, while the interaction between neutrophils and endothelial cells showed a decreasing trend. Figure 5 I). This invention utilizes smooth curves with arrows to visualize the differentiation trajectories of macrophages and neutrophils. Figure 5 Based on these trajectories, this invention designates macrophage subsets 0 and 5 as the major subsets enriched during the differentiation initiation phase (J, 5L). Figure 5 K). Notably, subset 0 exhibited significantly high Arg1 expression, indicating a potent anti-inflammatory effect. For neutrophils, subsets 0 and 3 were assigned to early developmental stages (K). Figure 5 M).

[0071] In summary, by combining hydrogel processing with high-resolution characterization provided by single-cell sequencing, this invention comprehensively maps and quantifies the cellular atlas of the wound microenvironment. Furthermore, this invention has demonstrated that the hydrogel system plays a crucial role in immune vascular remodeling, thus providing compelling evidence to support its potent therapeutic effect in promoting the healing of diabetic wounds.

[0072] 3.5. Bio-inspired hydrogels enhance mitochondrial function in endothelial cells. To gain a deeper understanding of the effects of treatment on the energy metabolism of HUVECs, this invention conducted glycolytic stress tests (by measuring extracellular acidification rate, ECAR) and mitochondrial stress tests (by measuring oxygen consumption rate, OCR) to assess glycolytic flux and oxidative phosphorylation levels.

[0073] In the OCR test, compared with the control group, the NSC / Met@HAMA treatment group significantly enhanced several key parameters of mitochondrial function in HUVECs ( Figure 6 A). These include increased basal respiration, ATP production, reserve respiratory capacity, and maximum respiratory capacity (A). Figure 6B). These promoting effects ensure better energy supply for HUVECs, enabling them to better adapt to the microenvironment of diabetic wounds and enhance their ability to proliferate, differentiate, and form tubes. In the ECAR assay, characteristic changes occurred in extracellular acidification levels after sequential administration of glucose, oligomycin, and 2-deoxyglucose (2-DG): an initial increase, a further rise, and a subsequent decrease. Figure 6 C). Hydrogel treatment consistently reduced the glycolytic level, glycolytic capacity, non-glycolytic acidification, and glycolytic reserves of HUVECs to varying degrees. Figure 6 D). In summary, the hydrogel-based therapy of this invention promotes the shift of cellular energy metabolism to mitochondrial aerobic oxidation, resulting in highly efficient oxidative phosphorylation, accompanied by a reduction in glycolysis.

[0074] Furthermore, timely clearance of mitochondrial superoxide is closely related to the stability of the mitochondrial electron transport chain. Flow cytometry is used to quantify the types of mitochondrial reactive oxygen species (mtROS). Figure 6 E). This invention observed a significant increase after H2O2 treatment, indicating a rightward shift in the fluorescence curve. The positive percentage of mtROS increased dramatically from 12.34±3.032% to 87.55±5.053%. Treatment with standard HAMA hydrogel did not significantly improve mtROS levels, which remained high at 85.08±4.306%. However, loading metformin or NSC hydrogels alone effectively reduced mtROS levels, decreasing the positive percentages to 67.66±4.486% and 68.60±3.026%, respectively. The synergistic combination therapy in the NSC / Met@HAMA group achieved the most substantial reduction, lowering mtROS levels to 56.93% (…). Figure 6 F, 6H).

[0075] Given the crucial role of mitochondrial membrane potential (ΔΨm) in driving ATP synthesis, this invention evaluated the ability of treatments to restore ΔΨm under oxidative stress. JC-1 staining combined with flow cytometry was used to determine changes in ΔΨm. This invention observed that H2O2 treatment significantly reduced the red / green fluorescence ratio compared to the control group, indicating ΔΨm dissipation and a corresponding increase in the population of the B4 region. All treatments reduced the proportion of cells in the B4 region to varying degrees. The Met@HAMA group and the NSC@HAMA group showed similar recovery effects. Notably, the NSC / Met@HAMA treatment produced the best results, restoring the B4 region to levels comparable to the control group and demonstrating a strong ability to restore mitochondrial membrane integrity. Figure 6 G, 6I).

[0076] 3.6. Therapeutic hydrogels enhance the reparative polarization and extracellular accumulation of macrophages. The regulation of the inflammatory microenvironment is a key determinant of successful wound healing, and macrophages are key regulators in this process. This invention first uses immunofluorescence analysis to analyze macrophage phenotypic transformation. RAW264.7 cells were stimulated with LPS to simulate an inflammatory stress microenvironment, followed by treatment with different hydrogels. Using CD206 as a marker for M2 macrophages, this invention observed that LPS stimulation significantly attenuated green fluorescence, indicating a downregulation of CD206 expression. NSC@HAMA treatment significantly increased CD206 expression, while Met@HAMA also showed a promoting effect. The combined treatment (NSC / Met@HAMA) showed the strongest green fluorescence intensity, indicating that this hydrogel system effectively promoted polarization towards the pro-repair M2 phenotype. Figure 7 A, 7E).

[0077] For M1 pro-inflammatory assessment, this invention selected iNOS as a marker. LPS stimulation strongly enhanced red fluorescence, confirming a significant inflammatory response. NSC@HAMA and Met@HAMA significantly attenuated this upregulation, although some residual red fluorescence remained. In contrast, NSC / Met@HAMA treatment almost eliminated iNOS expression, as indicated by the disappearance of red fluorescence, restoring it to a state close to that observed in the control group. Overall, these findings confirm that the composite hydrogel effectively modulates macrophage polarization to an anti-inflammatory phenotype and inhibits inflammatory activation. Figure 7 A, 7F).

[0078] Macrophages effectively and promptly clear excess apoptosis at diabetic wound sites, a process known as defollicular cytotoxicity, which is crucial for preventing secondary necrosis and persistent inflammation. This invention uses flow cytometry to assess whether hydrogels can enhance this process. UV-induced apoptosis Jurkat cells were labeled with CellTrackerDeepRed and co-cultured with macrophages at a ratio of 1:5 (macrophages:apoptotic cells). Macrophages were subsequently stained with F4 / 80 (…). Figure 7 B, 7C). Data showed that, compared with the untreated group and the standard HAMA group, both Met@HAMA and NSC@HAMA treatments increased the proportion of double-positive cells (representing hemorrhage cells and macrophages). The synergistic combination further amplified this effect, significantly increasing phagocytosis rate. Figure 7 (D, 7G). These results demonstrate that the hydrogel system also possesses a strong and efficient ability to eliminate apoptotic cells, which is crucial for preventing secondary damage and promoting a regenerative microenvironment.

[0079] 3.7. NSC / Met@HAMA promotes diabetic wound healing by facilitating regenerative immune vascular remodeling.

[0080] To further elucidate the molecular mechanisms of wound healing, this invention performed immunofluorescence and immunohistochemical analyses on skin wound tissue. Neutrophils undergo NETosis in the early stages of skin injury, releasing extracellular neutrophil traps (NETs). Excessive NETs can lead to persistent wound inflammation. For immunofluorescence detection, this invention used MPO (red) and H3Cit (green) to characterize the NET regions. Figure 8 A). The significant decrease in fluorescence intensity in the red and green channels confirms that encapsulation of NSC or metformin significantly reduces NETs levels. The co-delivery system exhibits the most efficient NETs gap, with virtually undetectable red and green fluorescence signals across the entire field of view. Figure 8 E, 8F).

[0081] To evaluate the in vivo regulation of inflammation by hydrogels, immunohistochemical staining was performed on the anti-inflammatory marker CD206 and the inflammatory marker iNOS. The results showed that delivery of NSC or metformin promoted an increasing trend in CD206 expression and a decreasing trend in iNOS expression. Figure 8 B). The NSC / Met@HAMA group showed the highest CD206 expression density and the lowest iNOS expression level, indicating that the hydrogel promotes macrophage polarization to a repair-prone phenotype and regulates the in vivo inflammatory immune microenvironment. Figure 8 G, 8H). Furthermore, given that Gas6 is a protein associated with phagocytic cell proliferation and can enhance macrophage recognition of apoptotic cells, thereby promoting the clearance of apoptotic cells, this invention performed immunohistochemical staining of Gas6 in wound tissue on day 7. Figure 8 C). This invention found that NSC / Met@HAMA significantly enhanced Gas6 expression, with a more pronounced effect than either component alone. Figure 8 I).

[0082] To assess whether and to what extent the hydrogel improved angiogenesis in diabetic wounds, CD31 staining was performed to assess microvessel density and α-SMA staining was performed to assess vascular smooth muscle wall formation in tissue collected at late healing stage (day 14). Figure 8 D). In the control and HAMA groups, few vascular structures were observed. However, delivery of NSC@HAMA significantly improved vascular density and integrity. The addition of metformin further amplified this effect, with the NSC / Met@HAMA group exhibiting denser vascular structures and lumen formation. Figure 8 In summary, this hydrogel system accelerates diabetic wound healing through multiple mechanisms: reducing NET formation, promoting repair macrophage polarization, enhancing cellular effects, and supporting angiogenesis.

[0083] 4. Summary This invention develops a combined delivery system for neural stem cells (NSCs) and metformin based on HAMA hydrogel. This invention provides the first comprehensive evaluation of the therapeutic efficacy of NSCs and their scaffold-based delivery strategy in wound healing, further enhancing therapeutic efficiency by incorporating metformin as a drug component. The system exhibits excellent biocompatibility, enhancing the antioxidant and anti-apoptotic capabilities of vascular endothelial cells in vitro, while improving oxidative phosphorylation and inhibiting glycolysis. It also reduces mitochondrial superoxide levels and increases mitochondrial membrane potential, thereby supporting efficient energy synthesis, transfer, and utilization. Furthermore, this therapeutic agent promotes anti-inflammatory polarization of macrophages, increases their sensitivity to apoptotic cells, and accelerates the clearance of apoptotic debris. In vivo wound healing experiments in diabetic mice demonstrate the system's significant pro-regenerative effect: it modulates neutrophil fate early in the process, reduces NETosis, and simultaneously alleviates inflammation and enhances cellular effects through macrophages. In later stages, it promotes collagen deposition and angiogenesis. Furthermore, through single-cell RNA sequencing, this invention decoded the distribution of cell types in regenerating wounds and revealed key biological events associated with NSC / Met@HAMA promoting healing, particularly active immune-endothelial cell interactions and their numbers.

[0084] Example 2: Preparation and application of hydrogel-nonwoven composite dressing The hydrogel obtained in Example 1 was bonded to a medical nonwoven fabric substrate to obtain a hydrogel-nonwoven composite dressing. The hydrogel side of the hydrogel-nonwoven composite dressing was applied to the diabetic wound and secured with medical tape; the dressing was changed periodically according to the amount of exudation. The composite dressing can create a moist microenvironment at the wound site and achieve a continuous effect on the local wound, thereby promoting the healing of diabetic wounds.

[0085] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a composite hydrogel for promoting the healing of diabetic wounds, characterized in that, The preparation method includes the following steps: Preparation of acrylate-metformin conjugate solution; Under sterile conditions, a neural stem cell suspension, a methacrylamide hyaluronic acid precursor solution, and an acrylate-metformin conjugate solution were mixed at a volume ratio of 1:5:4, and a photocrosslinking reaction was carried out in the presence of a photoinitiator to prepare a composite hydrogel.

2. The preparation method according to claim 1, characterized in that, The methacrylamide hyaluronic acid precursor solution is a mixture of methacrylamide hyaluronic acid and a photoinitiator.

3. The preparation method according to claim 2, characterized in that, The photoinitiator is lithium acyl phosphate.

4. The preparation method according to claim 3, characterized in that, The methacrylated hyaluronic acid precursor solution contained 10% methacrylated hyaluronic acid and 5% lithium acyl phosphate.

5. The preparation method according to claim 4, characterized in that, The density of neural stem cells is 2×10 7 / mL, the final concentration of methacrylated hyaluronic acid is 5%, the final concentration of lithium acyl phosphate is 2.5%, and the final concentration of metformin is 200mM.

6. The preparation method according to claim 1, characterized in that, The conditions for the photocrosslinking reaction are: a wavelength range of 380–420 nm, an illumination time of 5–20 s, and an irradiance of 10–50 mW / cm². 2 .

7. The preparation method according to claim 1, characterized in that, The preparation of the acrylate-metformin conjugate solution includes the following steps: Metformin and AC-PEG-NHS were dissolved in phosphate buffer at a mass ratio of 1:25 and reacted at 4°C in the dark for 24 hours to introduce acrylate groups, thus obtaining an acrylate-metformin conjugate solution. The metformin concentration was 500 mM.

8. A composite hydrogel for promoting the healing of diabetic wounds, characterized in that, The composite hydrogel is prepared by the method described in any one of claims 1 to 7.

9. Use of the composite hydrogel of claim 8 in the preparation of a wound-healing drug.

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