A magnesium-based nanofiber with healing-promoting function and its application in diabetic wound healing.

Abstract

This invention provides a magnesium-based nanofiber dressing with healing-promoting function, its preparation method, and its application, belonging to the field of medical dressing technology. Aminated magnesium boron nanosheets, chitosan, curcumin, and a fiber-forming polymer are dispersed in a mixed solvent to form a spinning solution, which is then electrospun to obtain a composite nanofiber membrane. This dressing uses aminated magnesium boron nanosheets as a controllable hydrogen source, enabling the continuous and stable release of therapeutic hydrogen gas, effectively neutralizing excessive reactive oxygen species generated in diabetic wounds and alleviating oxidative stress. Simultaneously, it integrates the antibacterial properties of chitosan with the anti-inflammatory and antioxidant activities of curcumin, synergistically promoting vascular endothelial cell migration and tubular structure formation, accelerating granulation tissue growth and epithelialization. The dressing prepared by this invention has a three-dimensional porous network structure, good biocompatibility, and strong functional synergy, enabling a full-chain, intelligent treatment of refractory diabetic wounds, from microenvironment regulation to active tissue regeneration. The preparation process is simple and suitable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to a magnesium-based nanofiber with healing-promoting function, its preparation method, and its application in the healing of diabetic wounds. Background Technology

[0002] Chronic, non-healing wounds in diabetes, especially diabetic foot ulcers, are among the most serious and costly complications of diabetes. Their pathological mechanisms are extremely complex, involving a vicious cycle of multiple factors, including oxidative stress storms caused by persistent hyperglycemia, chronic inflammation, susceptibility to secondary infections, impaired angiogenesis, and neuropathy. This abnormal wound microenvironment limits the effectiveness of traditional dressings and single-therapies; wound healing often stalls in the inflammatory or early proliferative phase, leading to prolonged illness and a heavy physical and psychological burden on patients. Globally, diabetic foot ulcers not only have a high prevalence but are also the leading cause of non-traumatic lower limb amputations, placing a significant strain on healthcare systems.

[0003] To address these challenges, current research focuses on several key areas: Antioxidant therapy strategies: Given that oxidative stress is a core obstacle to wound healing in diabetic patients, current research focuses on delivering exogenous antioxidants (such as vitamin C / E and superoxide dismutase mimics) and using materials with intrinsic antioxidant activity (such as certain natural polymers and selenium nanoparticles). However, these strategies often face problems such as poor stability of active substances, low bioavailability, short duration of action, or lack of continuous supply, making it difficult to meet the long-term antioxidant needs of chronic wounds.

[0004] Gas therapy strategies: Gas therapy molecules, represented by molecular hydrogen (H2), have attracted much attention due to their high selectivity in neutralizing highly toxic ROS, excellent anti-inflammatory properties, and good tissue penetration. Magnesium-based materials (such as elemental magnesium, magnesium hydride, and magnesium boride) are considered potential local portable hydrogen sources because they can react with water to generate hydrogen gas. However, key technical bottlenecks lie in the fact that most magnesium-based materials react violently in water, leading to an "explosive" release of hydrogen gas, making it difficult to maintain a long-term, stable therapeutic concentration; the violent reaction is accompanied by a sharp increase in local pH, which may cause secondary damage to newly formed tissue; at the same time, the rapid consumption of materials also limits their long-term hydrogen supply capacity. How to achieve "controllable, continuous, and gentle" release of hydrogen gas is the key to the application of gas therapy dressings.

[0005] Natural polymeric drug delivery systems: These systems utilize natural polymeric materials such as chitosan, hyaluronic acid, and collagen to construct dressing matrices. These materials typically possess good biocompatibility, biodegradability, inherent antibacterial or healing-promoting activities (e.g., chitosan), and can serve as carriers for various therapeutic drugs. However, their mechanical properties, loading efficiency for hydrophobic drugs, and ability to precisely control release behavior are sometimes insufficient, often requiring combination with other materials or technologies to improve overall performance.

[0006] However, current approaches still suffer from several significant shortcomings, including: limited and unsustainable functionality; antioxidant effects rely on the chemical clearance of active ingredients, a passive consumption mechanism that is easily depleted in a sustained high-ROS environment, failing to provide long-term protection; insufficient synergy; primarily consisting of simple physical mixtures of multiple active ingredients, with each function operating independently in time and space, resulting in a mere "addition" rather than "synergy"; unintelligent release behavior; drug release mainly relies on passive diffusion, unable to respond to dynamic changes in the wound microenvironment (such as pH and ROS levels), leading to a mismatch between release and treatment needs and low precision; incomplete treatment chain; focus on antibacterial and basic antioxidant effects, with insufficient intervention in the crucial angiogenesis repair process, and a lack of effective strategies to regulate macrophage polarization towards repair and break the chronic inflammatory cycle; and stability and bioavailability issues; some active ingredients (such as curcumin) are easily degraded during processing and storage, and physical encapsulation methods may affect their complete release and bioavailability, posing challenges to the reliability and consistency of therapeutic efficacy. Therefore, developing a novel dressing that achieves orderly synergy of functions and intelligent release response has become an important direction for overcoming existing technological bottlenecks.

[0007] Therefore, this invention is proposed to solve the above-mentioned technical problems. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a magnesium-based nanofiber dressing with healing-promoting function, its preparation method, and its application. This dressing can intelligently and continuously release therapeutic hydrogen to neutralize oxidative stress, and simultaneously integrates highly effective antibacterial, potent anti-inflammatory / antioxidant, and angiogenesis-promoting functions, achieving a full-chain, synergistic treatment of diabetic refractory wounds, from basic microenvironment improvement to active tissue regeneration promotion.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a magnesium-based nanofiber dressing with a healing-promoting function. The dressing is a composite nanofiber membrane comprising 2-7 amino boron nanosheets, chitosan, curcumin and a fiber-forming polymer, wherein the amino boron nanosheets are uniformly dispersed in the fibers as a controllable hydrogen source, and the composite nanofiber membrane has a porous interconnected three-dimensional network structure.

[0010] Furthermore, the preferred weight ratio of the aminated magnesium boron nanosheets, chitosan, curcumin, and fiber-forming polymer is (2~7):(2~7):(2~7):(80~100), more preferably 5:5:5:90. Secondly, the present invention also provides a method for preparing the above-mentioned magnesium-based nanofiber dressing with healing-promoting function, comprising the following steps: S1. Aminated magnesium boron nanosheets, chitosan, curcumin and fiber-forming polymer are dissolved or dispersed in a mixed solvent to form a spinning solution; S2. Electrospinning the spinning solution to obtain the magnesium-based nanofiber dressing.

[0011] Furthermore, the aminated magnesium boron nanosheets are obtained by controlling the etching and stripping of the MgB2 precursor under the action of a weak acid and an oxidant, followed by surface modification with an amino-containing silane coupling agent.

[0012] Furthermore, the weak acid is selected from at least one of acetic acid, citric acid, formic acid, and lactic acid.

[0013] Furthermore, the amino-containing silane coupling agent is selected from at least one of 3-aminopropyltrimethoxysilane, N-2-aminoethyl-3-aminopropyltrimethoxysilane, and 3-2-aminoethylaminopropyltrimethoxysilane.

[0014] Furthermore, the hydrated particle size distribution of the aminated magnesium boron nanosheets has a main peak in the range of 40-80 nm.

[0015] Furthermore, the mixed solvent in step S2 comprises hexafluoroisopropanol, deionized water, and glacial acetic acid.

[0016] Furthermore, the electrospinning process parameters are: voltage 10-20kV, spinning solution propulsion flow rate 0.3-0.8mL / h, and receiving distance 10-20cm.

[0017] Thirdly, the present invention provides the application of the magnesium-based nanofiber dressing described in the first aspect and the magnesium-based nanofiber dressing prepared by the preparation method described in the second aspect in the healing of diabetic wounds.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to integrate aminated magnesium boron nanosheets as a controllable hydrogen source into nanofiber dressings. Unlike the "burst" hydrogen release of conventional magnesium-based materials, through nanosheet structural design and surface amino modification, combined with the physical barrier and slow-release effect of the fiber matrix, a stable and continuous release of hydrogen (H2) is achieved in the microenvironment of diabetic wounds. This can selectively neutralize excessive hydroxyl radicals and other highly toxic reactive oxygen species generated in the wound over a long period of time, effectively alleviating oxidative stress from the source and creating a favorable "low-oxidation" microenvironment for subsequent repair.

[0019] This invention constructs a multifunctional synergistic system that integrates hydrogen release for antioxidant effects, curcumin for anti-inflammatory effects, chitosan for antibacterial effects, and synergistic promotion of angiogenesis. Aminated magnesium boron nanosheets first respond to the weakly acidic environment of the wound by releasing hydrogen, initially alleviating oxidative stress and inflammation; subsequently, curcumin is released, enhancing anti-inflammatory and antioxidant effects; chitosan provides immediate antibacterial protection. The three components work synergistically to effectively break the vicious cycle of "high oxidation-high inflammation-susceptibility to infection" in diabetic wounds and significantly promote endothelial cell migration and angiogenesis, thereby achieving a full-chain treatment from microenvironment regulation to active tissue regeneration.

[0020] This invention utilizes electrospinning technology to construct a three-dimensional porous nanofiber network, which not only possesses high specific surface area, excellent air permeability, and exudate management capabilities, but its structure can also intelligently respond to changes in the wound microenvironment, such as pH value, and regulate the reactivity of aminated magnesium boron nanosheets and the hydrogen release rate. Simultaneously, hydrophobic drugs such as curcumin can achieve stable encapsulation and controlled release within the nanofibers, solving the problems of poor stability and low bioavailability.

[0021] The preparation method of this invention uses readily available raw materials and has mature process steps (controllable etching and stripping combined with electrospinning), making it easy to achieve large-scale production. Cell experiments (MTT, live-dead staining) and animal experiments show that the obtained composite nanofiber dressing is non-toxic to various key repair cells such as macrophages and vascular endothelial cells, and can significantly promote cell proliferation, migration and angiogenesis. Its biocompatibility and healing-promoting effect are significantly better than those of single materials.

[0022] This invention provides a novel nanofiber dressing that integrates intelligent response, continuous hydrogen supply, strong antioxidant / anti-inflammatory / antibacterial properties, and efficient angiogenesis promotion. Targeting the core pathological obstacles to wound healing in diabetic patients, it achieves multifunctional orderly synergy and precise intervention, providing a promising new material solution for the clinical treatment of chronic, refractory wounds such as diabetic foot ulcers. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the structure of the magnesium-based nanofiber dressing (MPCC) of Example 3 of the present invention; Figure 2 The image shows the microstructure and structural characterization of the aminated magnesium boron nanosheets (MBNS) prepared in Example 1 of this invention. Figure 3 This is a SEM image of the MPCC nanofiber membrane prepared in Example 3 of the present invention; Figure 4 The hydrogen quantitative release curves based on the methylene blue method are for Aminated Magnesium Boron Nanosheets (MBNS) in Example 1 and Magnesium-based Nanofiber Dressings (MPCC) in Example 3 of this invention. Figure 5 This is a graph showing the in vitro antioxidant capacity evaluation of the MPCC nanofiber dressing in this embodiment of the invention. Figure 6 This is an in vitro biocompatibility evaluation diagram of amino-modified magnesium boron nanosheets (MBNS), PCC, and MPCC on RAW264.7 macrophages in the embodiments of the present invention; Figure 7 This is a diagram showing the in vitro biocompatibility evaluation of amino-modified magnesium boron nanosheets (MBNS), PCC, and MPCC on human umbilical vein endothelial cells (HUVEC) in the embodiments of the present invention. Figure 8 This is a graph showing the protective effect of aminated magnesium boron nanosheets (MBNS), PCC, and MPCC on RAW264.7 macrophages under oxidative stress and their ability to scavenge intracellular ROS. Figure 9 This is a diagram evaluating the protective effect of aminated magnesium boron nanosheets (MBNS), PCC, and MPCC on HUVEC cells under oxidative stress and their intracellular ROS scavenging ability in the embodiments of the present invention. Figure 10 This is an evaluation diagram of the promoting effect of aminated magnesium boron nanosheets (MBNS), PCC, and MPCC nanofiber dressings on the migration ability of human umbilical vein endothelial cells (HUVEC) in the embodiments of the present invention. Figure 11 This is a comprehensive evaluation chart showing the in vitro angiogenesis-promoting ability and in vivo diabetic wound healing effect of the MPCC nanofiber dressing in this embodiment of the invention. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] This invention provides a magnesium-based nanofiber dressing with healing-promoting function. The dressing is a composite nanofiber membrane comprising aminated magnesium boron nanosheets, chitosan, curcumin and fiber-forming polymer. The aminated magnesium boron nanosheets are uniformly dispersed in the fibers as a controllable hydrogen source, and the composite nanofiber membrane has a porous interconnected three-dimensional network structure.

[0026] In this invention, the preferred weight ratio of the aminated magnesium boron nanosheets, chitosan, curcumin and fiber-forming polymer is (2~7):(2~7):(2~7):(80~100), and more preferably 5:5:5:90.

[0027] The present invention does not impose any special limitations on the fiber-forming polymers used. Those skilled in the art can select them according to actual needs. For example, the fiber-forming polymers are selected from at least one of synthetic biodegradable polymers and natural polymers and their derivatives. Among them, synthetic biodegradable polymers can be polylactic acid, polycaprolactone, polylactic-co-glycolic acid copolymers, etc., which have good biocompatibility and biodegradability, can be gradually decomposed and metabolized and excreted in the body, and will not cause long-term effects on the body; natural polymers and their derivatives can be collagen, gelatin, cellulose derivatives, etc., which generally have better bioactivity and cell affinity.

[0028] The present invention also provides a method for preparing the above-mentioned magnesium-based nanofiber dressing with healing-promoting function, comprising the following steps: S1. Aminated magnesium boron nanosheets, chitosan, curcumin and fiber-forming polymer are dissolved or dispersed in a mixed solvent to form a spinning solution; S2. Electrospinning the spinning solution to obtain the magnesium-based nanofiber dressing.

[0029] In this invention, the aminated magnesium boron nanosheets are obtained by controllingly etching and stripping the MgB2 precursor under the action of a weak acid and an oxidant, and then modifying the surface with an amino-containing silane coupling agent.

[0030] In this invention, the preparation of aminated magnesium boron nanoparticles includes four steps: dispersion and protection, controlled etching and exfoliation, surface amino functionalization, purification and preservation. Dispersion and protection: Disperse 100-1000 mg of MgB2 powder with 0.5-5 g of a polymeric dispersant (such as polyethylene glycol 4000) in 100-200 mL of deionized water to form a mixed suspension. Then place the system in an ice bath to cool for 5-15 minutes while continuously purging with an inert gas (such as nitrogen). This operation aims to inhibit premature and violent hydrolysis of MgB2.

[0031] Controlled Etching and Stripping: Under continuous inert gas protection and stirring, 50-500 µL of a weak acid (acetic acid) was slowly and batch-wise added to the pre-cooled system. Subsequently, 1-10 mL of a 5-15% oxidant solution (hydrogen peroxide, H₂O₂) was added. This step is crucial; the acid and H₂O₂ work synergistically to gently and controllably etch the MgB₂ interlayer structure, simultaneously stripping it into boron nanosheets (MBNS), with the generation of a small amount of hydrogen gas. The entire reaction was carried out in an ice bath and nitrogen atmosphere to ensure a gentle and controllable reaction.

[0032] In this step, there are no special restrictions on the weak acid, and those skilled in the art can select it according to actual needs. The weak acid is preferably at least one of acetic acid, citric acid, formic acid, and lactic acid, and more preferably acetic acid.

[0033] Surface amino functionalization: Immediately after etching and stripping, 20-100 µL of an amino-containing silane coupling agent is added to the reaction system. Subsequently, the reaction system is kept at a constant temperature of 40-60°C and stirred for 3-6 hours under continuous inert gas protection. During this process, APTES undergoes hydrolysis, and its silanol groups bind to hydroxyl groups or defect sites on the MBNS surface. Simultaneously, the silanol groups formed by the hydrolysis of ethoxy groups can undergo self-condensation, ultimately resulting in the stable grafting of amino (-NH2) functional groups onto the MBNS surface.

[0034] In this step, surface amination improves the hydrophilicity and dispersion stability of MBNS in the spinning solution. When it is encapsulated in fibers and comes into contact with wound exudate, the MgB2 on the surface reacts slowly with water, continuously generating hydrogen gas (H2). The nanosheet structure increases the reaction area, making hydrogen release more stable and sustained, and also provides the possibility for subsequent loading or attraction of biomolecules through electrostatic interactions. This invention does not impose any particular limitation on the amino-containing silane coupling agent, and those skilled in the art can select according to actual needs. The amino-containing silane coupling agent is preferably at least one of 3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and 3-(2-aminoethyl)aminopropyltrimethoxysilane, more preferably 3-aminopropyltriethoxysilane (APTES).

[0035] Purification and preservation: After the reaction, the solid product was collected by centrifugation and washed alternately with water and ethanol to remove impurities. Finally, it was freeze-dried to obtain fluffy aminated MBNS powder. The hydrated particle size distribution of the aminated magnesium boron nanosheets was mainly in the range of 40-80 nm. It was stored in an inert gas for later use.

[0036] After completing the preparation of aminated magnesium boron nanosheets, the present invention prepares a composite spinning solution to obtain a uniform and stable spinning solution.

[0037] In this invention, the mixed solvent comprises hexafluoroisopropanol, deionized water, and glacial acetic acid. The ratio of these components can be adjusted according to the actual spinning effect, and the preferred volume ratio is 8:1:1. Generally, hexafluoroisopropanol is used as the main solvent to dissolve chitosan and polyvinyl alcohol (PVA). The addition of HAc can further promote the protonation and dissolution of chitosan. Deionized water is used to adjust the evaporation rate and conductivity of the solution to adapt to the subsequent electrospinning process.

[0038] Aminated magnesium boron nanosheets, chitosan, curcumin, and fiber-forming polymer are added to the mixed solvent in a predetermined weight ratio and uniformly dispersed. The method of uniform dispersion is not particularly limited and can be fully dissolved or dispersed by magnetic stirring, ultrasonic treatment, etc., to form a uniform spinning solution.

[0039] The electrospinning process parameters are: voltage 10-20kV, spinning solution propulsion flow rate 0.3-0.8mL / h, and receiving distance 10-20cm.

[0040] In this invention, the electrospinning process involves transferring the prepared spinning solution into the syringe of the electrospinning device. The electrospinning process parameters are set, with the voltage controlled at 10-20 kV. This voltage range allows the spinning solution to form a stable Taylor cone and generate a suitable jet. The spinning solution propulsion rate is set to 0.3-0.8 mL / h; too high a rate will result in uneven fiber thickness, while too slow a rate will affect spinning efficiency. The receiving distance is selected as 10-20 cm, as a suitable receiving distance facilitates sufficient fiber stretching and solidification. Under these parameters, the spinning solution is electrospinned. Under the action of a high-voltage electric field, the spinning solution forms a jet. After processes such as stretching and solvent evaporation, a magnesium-based nanofiber dressing with a porous interconnected three-dimensional network structure is finally obtained on the receiving device. MBNS nanosheets, CS, and Cur molecules are "frozen" in the rapidly solidified PVA fiber matrix, thereby forming a functional composite nanofiber membrane.

[0041] The magnesium-based nanofiber dressing of the present invention has a continuous phase fiber matrix consisting of a PVA / CS blend at the microscale, wherein curcumin (Cur) molecules and embedded MBNS nanosheets are uniformly dispersed therein.

[0042] The membrane has high porosity (>80%) and interconnected channels, which facilitates exudate absorption, gas exchange and cell migration.

[0043] When the dressing is applied to a diabetic wound, the weakly acidic (pH 4.5-6.5) exudate from the wound permeates into the porous structure of the fibrous membrane.

[0044] Intelligent activation and continuous hydrogen release: H⁺ ions in the exudate activate MBNS inside and on the surface of the fiber, causing it to react (MgB₂ + 4H₂O → 2H₂ + Mg²⁺ + B(OH)₃), continuously producing hydrogen (H₂) molecules. The small H₂ molecules can freely diffuse into the surrounding tissue, selectively neutralizing highly toxic ROS such as •OH and ONOO⁻, reducing oxidative stress at its source.

[0045] Synergistic anti-inflammatory and antioxidant effects: While oxidative stress is initially relieved by H2, ① curcumin (Cur) in the fiber begins to be slowly released, further clearing various free radicals and downregulating the expression of key pro-inflammatory factors (such as TNF-α, IL-6), thus enhancing the anti-inflammatory effect; ② chitosan (CS) itself is positively charged, which can destroy bacterial cell membranes, provide basic antibacterial protection, and prevent infection from aggravating inflammation.

[0046] Promoting Repair and Angiogenesis: Through the above steps, the wound microenvironment transforms from a state of "high oxidation and high inflammation" to a "repair-friendly" state. In this optimized environment: ① H2 and Cur have been shown to synergistically promote the migration and tubule formation of endothelial cells (HUVECs); ② MBNS and its products may guide macrophages to polarize towards the repair-oriented M2 phenotype, secreting pro-repair factors; ③ CS degradation products can further stimulate cell proliferation. Ultimately, on the three-dimensional scaffold provided by the dressing, new granulation tissue and blood vessels grow rapidly.

[0047] The entire process constitutes a complete positive cycle: responding to the microenvironment by releasing hydrogen → neutralizing ROS and alleviating oxidative stress → synergistic anti-inflammatory and antibacterial effects → improving local microcirculation and immune environment → creating optimal conditions for cell migration, proliferation and vascularization → accelerating wound closure and tissue remodeling.

[0048] The application of the magnesium-based nanofiber dressing described in the above technical solution in the healing of diabetic wounds.

[0049] To further illustrate the present invention, the following detailed description, in conjunction with examples, of the pirfenidone nanofiber film agent, its preparation method, and its application, is provided but should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1: Preparation of Aminated Magnesium Boron Nanosheets (MBNS) Weigh 200 mg of MgB2 powder and 2 g of PEG4000, and disperse them together in 150 mL of deionized water to form a mixed suspension. Cool the mixed suspension in an ice bath for 10 minutes while continuously purging with nitrogen. Under continuous nitrogen purging and magnetic stirring, slowly add 200 µL of acetic acid (1 M) using a syringe pump. Subsequently, slowly add 5 mL of 10% (w / w) H2O2 solution. After the addition is complete, continue the reaction in an ice bath for 1 hour. Immediately add 50 µL of LAPTES to the reaction system, remove the ice bath, and react under nitrogen protection at 50°C with stirring for 4 hours. After the reaction is complete, centrifuge the product (10000 rpm, 10 min), wash three times alternately with water and ethanol, and finally freeze-dry for 24 hours to obtain a fluffy aminated MBNS powder, which is stored in a nitrogen-purged desiccator for later use.

[0051] Example 2: Preparation of composite spinning solution 8 mL of hexafluoroisopropanol (HFIP), 1 mL of deionized water, and 1 mL of glacial acetic acid (HAc) were measured into a 20 mL glass bottle and magnetically stirred at 500 rpm for 5 minutes to form a homogeneous and transparent solvent system. Then, 0.05 g of MBNS powder prepared in Example 1, 0.05 g of chitosan (CS), 0.9 g of polyvinyl alcohol (PVA) powder, and 0.05 g of curcumin (Cur) powder were added sequentially to the above mixed solvent. All components were magnetically stirred continuously at 500 rpm for 12 hours at room temperature (25±2℃) in the dark until a homogeneous, viscous, and clear spinnable solution was obtained.

[0052] Example 3: Preparation of magnesium-based nanofiber dressing (MPCC) In Example 2, the prepared composite spinning solution was injected into a syringe and installed on the propulsion pump of the electrospinning device. A flat-headed metal needle was used as the spinning nozzle. A 15kV high-voltage electrostatic field was applied between the needle and the grounded roller collector. The propulsion pump flow rate was set to 0.5mL / h, allowing the spinning solution to form a stable "Taylor cone" at the needle. Under the action of the electrostatic field, the droplets were stretched, split, and whipped, while the solvent evaporated rapidly. Finally, the solution was deposited on the collector 15cm away from the needle, solidified into continuous nanofibers, and accumulated to form a nonwoven membrane material (MPCC). A schematic diagram of the dressing structure is shown below. Figure 1 . Example

[0053] Except for the absence of MBNS powder, the remaining steps were exactly the same as in Examples 2 and 3, and PCC nanofiber membranes were obtained.

[0054] Material characterization and performance testing MBNS morphology and structural characterization The morphology and structure of the MBNS prepared in Example 1 were characterized using scanning electron microscopy (SEM), dynamic light scattering (DLS), Zeta potentiometer, and Fourier transform infrared spectroscopy (FTIR). The results are shown in the figure. Figure 2 .

[0055] Figure 2 a is a scanning electron microscope image of MBNS: The image shows that the synthesized MBNS has a lamellar structure with a lateral size in the range of hundreds of nanometers and the lamellars are relatively thin, indicating that the nano-exfoliation of the MgB2 precursor has been successfully achieved.

[0056] Figure 2 b is the dynamic light scattering (DLS) particle size distribution of MBNS: This figure, with intensity as the weight, shows the hydrated particle size distribution of MBNS in water. The main peak is located in the 40-80 nm range and is relatively concentrated, further confirming the nanoscale size and good dispersibility of the product.

[0057] Figure 2 Figure c shows the Zeta potential test results of MBNS: The data shows that after APTES modification, the Zeta potential of MBNS in water is positive (e.g., in the range of +20mV to +40mV), which confirms that positively charged amino groups have been successfully introduced into its surface, which is beneficial to its stable dispersion in aqueous phase and spinning solution.

[0058] Figure 2 Figure d shows the Fourier transform infrared (FTIR) spectrum of MBNS: the figure compares the infrared spectra of the raw material MgB2 and the product MBNS. In the MBNS spectrum, vibrations attributed to NH bending vibrations (3300 cm⁻¹) can be observed. -1 1550cm -1 ) and Si-O-Si stretching vibration (1100cm) -1 The characteristic absorption peaks of APTES are direct evidence of the successful grafting of APTES onto the surface of nanosheets.

[0059] comprehensive Figure 2 (a) to (d) fully verify that the oxidative exfoliation and amination process can successfully prepare nanoscale hydrogen-releasing materials with uniform morphology, charged surface, and expected chemical structure.

[0060] 2. Morphology and structural characterization of MPCC The morphology and structure of the MBNS prepared in Example 3 were characterized using scanning electron microscopy (SEM), and the results are shown in the figure. Figure 3 .

[0061] Figure 3 The image on the left is a low-magnification panoramic image of the MPCC nanofiber membrane obtained by scanning electron microscopy (SEM): The image shows that a large-area, continuous, nonwoven fabric-like fiber membrane was successfully prepared using electrospinning technology. The fibers are randomly interlocked and stacked, forming a porous, interconnected three-dimensional network structure.

[0062] Figure 3 The image on the right is a high-magnification detailed image of the MPCC nanofiber membrane obtained by scanning electron microscopy (SEM). At high magnification, it can be observed that the surface of each individual fiber is smooth, continuous, and free of beads, with a uniform diameter distribution, mainly between 100 nm and 500 nm. No obvious particle aggregation or phase separation was observed in the image, indicating that MBNS, CS, Cur, and the PVA matrix have achieved effective nanoscale composite formation.

[0063] In vitro sustained hydrogen release performance test To further quantitatively evaluate the hydrogen release behavior of different material systems, we systematically monitored the hydrogen release of MBNS and its coated system MPCC (containing an equal mass of MBNS) in PBS at pH 7.4 using the methylene blue (MB) colorimetric method. The results are shown below. Figure 4 As shown, the horizontal axis of the graph represents time (hours, h), and the vertical axis represents the cumulative hydrogen release (unit: μmolH2 / mgMBNS).

[0064] Depend on Figure 4 It can be seen that the MBNS system exhibits a significant burst release phenomenon within 10 hours, with a significantly increased hydrogen release rate, indicating its rapid response and efficient release characteristics. The MPCC system, on the other hand, shows no significant burst release phenomenon in its hydrogen release curve throughout the entire monitoring period (0–24 hours), exhibiting a smooth, continuous, and controllable release behavior. Compared to MBNS, MPCC demonstrates superior sustained-release performance, with a more stable release rate and longer release time, meeting the fundamental technical requirements for "controlled hydrogen release" materials in the long-term treatment of diabetic chronic wounds.

[0065] Antioxidant capacity test The antioxidant activity of the materials was evaluated using the 1,1-diphenyl-2-picrylhydrazine (DPPH) method. The specific detection steps are as follows: First, a 100 μM DPPH ethanol solution was prepared as the reaction solution. A certain amount of the test samples (MBNS, PCC, MPCC) were weighed and added to 3 mL of the above DPPH solution, and the mixture was shaken to ensure sufficient contact between the sample and DPPH. Then, the mixture was incubated at room temperature in the dark for 30 minutes to complete the free radical scavenging reaction. After the reaction, 100 μL of the reaction solution was transferred to a 96-well plate, and the absorbance (OD) value was measured at 517 nm using a microplate reader. The antioxidant capacity of each sample was quantitatively evaluated by calculating the DPPH free radical scavenging rate. The results are shown in the appendix. Figure 5 a.

[0066] The antioxidant activity of the materials was evaluated using the 2,2'-adiazono-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method. The specific detection steps are as follows: First, prepare 7 mM ABTS solution and 4.95 mM potassium persulfate solution using PBS buffer. Mix 2 mL of ABTS solution with 2 mL of potassium persulfate solution and oxidize in the dark for 12 hours to generate an ABTS cationic free radical stock solution. Then, dilute the stock solution to 5% of its original concentration using PBS buffer (pH=7.4) to prepare the ABTS working solution. Weigh a certain amount of the test samples (MBNS, PCC, MPCC) and add them to 1 mL of the above ABTS working solution. Mix well and react in the dark for 30 minutes. After the reaction, transfer 100 μL of the reaction solution to a 96-well plate and measure the absorbance (OD) at 734 nm using a microplate reader. Calculate and compare the ABTS free radical scavenging ability of each sample. The results are shown in the appendix. Figure 5 b.

[0067] In the above experiments, the MBNS group was provided with a certain concentration of MBNS; the MPCC group was a composite dressing containing the same concentration of MBNS; and the amount of base dressing used in the PCC group and the MPCC group was the same.

[0068] Appendix Figure 5 The data are presented in bar chart form, demonstrating the comprehensive antioxidant capacity measured using the ABTS cationic radical scavenging method and the DPPH radical scavenging method. The MPCC nanofiber dressing prepared in this invention exhibited the strongest free radical scavenging capacity in both test systems, indicating that the MPCC dressing possesses broad-spectrum and highly efficient antioxidant activity. It can effectively neutralize various reactive oxygen species (ROS) generated excessively in diabetic wounds, precisely matching the core pathological requirement of "high oxidative stress" in diabetic wounds, and providing a key materials science solution for breaking the vicious cycle of oxidative stress that hinders healing.

[0069] Biocompatibility testing PCC composite fiber membrane extract and MPCC composite fiber membrane extract: PCC composite fiber membranes and MPCC composite fiber membranes were cut into samples of a specified area (1cm × 1cm) and placed aseptically into the wells of a tissue culture plate. An appropriate amount (2mL) of complete culture medium (e.g., DMEM containing 10% fetal bovine serum) was added to each well, ensuring complete immersion of the membrane. The culture plate was then placed in a cell culture incubator at 37°C and 5% CO2 for 6 hours. After incubation, the fiber membrane was carefully removed using sterile forceps, and the culture medium containing the extract was filtered through a sterile microporous membrane with a pore size of 0.22μm to remove any small particles that might detach. The resulting clear filtrate was the extract.

[0070] The in vitro biocompatibility test results of RAW264.7 macrophages are shown in the figure. Figure 6 .

[0071] The cell safety of the core material and the composite dressing extract was comprehensively evaluated using two methods: cell proliferation and viability staining.

[0072] Appendix Figure 6 a, 6b, and 6c are bar charts showing the relative cell viability detected by the Cell Counting Kit-8 (CCK-8) method. The figures show the results of RAW264.7 cells after 24 hours of treatment with different or fixed concentrations of MBNS raw material, PCC composite fiber membrane extract, and MPCC composite fiber membrane extract.

[0073] Depend on Figure 6 As can be seen from this, compared with the untreated blank control group (survival rate set at 100%), the cell survival rate of MBNS-treated cells remained at a high level (e.g., >90%), indicating that MBNS after oxidative stripping and amination treatment had no obvious toxicity to cells.

[0074] Depend on Figure 6 b shows that the cell survival rate of the PCC extract group was not significantly different from that of the control group or was slightly promoted, indicating that the composite matrix composed of chitosan (CS), polyvinyl alcohol (PVA) and curcumin (Cur) has excellent biocompatibility.

[0075] Depend on Figure 6 As shown in c, the cell survival rate of the group treated with MPCC extract containing the core hydrogen-releasing component MBNS was comparable to that of the other groups. This data strongly demonstrates that the final product of this invention, MPCC nanofiber dressing, is not only safe and non-toxic, but its released components may also have a certain positive impact on cell metabolism.

[0076] Appendix Figure 6 Images d, 6e, and 6f are confocal microscopy images obtained by dual fluorescence staining of live and dead cells. All images in the group are accompanied by a uniform 100µm scale bar. Each sub-image is composed of three fluorescence images merged together and separately shows live cells stained with Calcein-AM (green fluorescence) and dead cells stained with propidium iodide (PI) (red fluorescence).

[0077] Depend on Figure 6 As can be seen from d, in the Merge image, the field of view is filled with dense green fluorescent cells with a relaxed shape, and only a few scattered or very few red fluorescent spots, which intuitively shows that the vast majority of cells are in good condition.

[0078] Depend on Figure 6As can be seen from the image, the cells spread out well in the field of view, showing large areas of green fluorescence, and almost no dead cells with red fluorescence can be observed, further confirming the safety of the PCC substrate.

[0079] Depend on Figure 6 As shown in f, the green fluorescent cells in the image have a high density and healthy morphology, similar to groups (d) and (e), and no obvious red fluorescent dead cell areas are observed.

[0080] comprehensive Figure 6 The quantitative data (ac) and visualization images (df) fully demonstrate that all key materials in the entire preparation chain, from raw materials (MBNS) to intermediate products (PCC) and then to the final product (MPCC), exhibit excellent in vitro biocompatibility, laying a solid biological foundation for their safe application as wound dressings.

[0081] The in vitro biocompatibility test results of umbilical vein endothelial cells (HUVECs) are shown in the figure. Figure 7 .

[0082] Appendix Figure 7 The safety and potential promoting effect of the material on endothelial cells, a key cell type in angiogenesis, were evaluated through cell activity and migration tests.

[0083] Appendix Figure 7 Figures a, 7b, and 7c are bar charts showing the relative cell viability as detected by the Cell Counting Kit-8 (CCK-8) method. The figures show the results after treating HUVEC cells with MBNS, PCC composite fiber membrane extract, and MPCC composite fiber membrane extract for 24 hours, respectively.

[0084] Depend on Figure 7 As can be seen from a, compared with the blank control group (survival rate set at 100%), the survival rate of the treated cells remained at a similarly high level, indicating that the material is non-toxic to HUVEC cells.

[0085] Depend on Figure 7 As shown in b, the cell survival rate of the PCC extract group was not significantly different from that of the control group, indicating that the basic composite material has good cell compatibility.

[0086] Depend on Figure 7 As shown in c, the cell survival rate of the MPCC extract group was not significantly different from that of the control group, indicating that the basic composite material has good cell compatibility.

[0087] Appendix Figure 7Images d, 7e, and 7f are confocal microscopy images of HUVEC cells with dual fluorescence staining of live and dead cells. All images are accompanied by a uniform 100µm scale bar. Each sub-image contains three columns: the left side shows the nuclei of dead cells stained with propidium iodide (PI) (red fluorescence), the middle side shows the cytoplasm of live cells stained with calcein-AM (calcein-AM) (green fluorescence), and the right side shows the superimposed image of the two (Merge).

[0088] Depend on Figure 7 As can be seen from image d, a large number of well-formed, adherent green fluorescent live cells are visible in the Merge image, while red fluorescent dead cells are extremely rare in the field of view. Depend on Figure 7 As can be seen from the image, the cells are well spread out, and the field of view is filled with green fluorescence, with almost no red fluorescence, indicating that the material is safe.

[0089] As shown in 7f, the green fluorescent cells in the image are tightly connected, morphologically healthy, and show almost no red fluorescent signal. This corroborates the CCK-8 data in Figure (c), intuitively demonstrating that MPCC can significantly promote the growth and survival of HUVEC cells.

[0090] comprehensive Figure 7 The quantitative data (ac) and morphological images (df) fully demonstrate that the MPCC nanofiber dressing prepared in this invention has excellent biocompatibility and a positive proliferative effect on vascular endothelial cells. This provides important cellular evidence for achieving the key therapeutic goal of "promoting angiogenesis" in diabetic wound repair.

[0091] Test on the protective effect of RAW264.7 macrophages against oxidative stress damage To evaluate the ability of the material of this invention to resist oxidative stress damage at the cellular level, an in vitro oxidative stress model was established using RAW264.7 macrophages induced by hydrogen peroxide (H2O2). The ability of the material to resist oxidative damage at the cellular level was comprehensively evaluated using two methods: cell viability recovery assay and intracellular reactive oxygen species (ROS) level detection. The results are shown in [Figure number missing]. Figure 8 .

[0092] Appendix Figure 8Figures a, 8b, and 8c are bar charts showing the relative cell viability as detected by the Cell Counting Kit-8 (CCK-8) method. The figures illustrate how RAW264.7 cells were first treated with hydrogen peroxide (H2O2) to induce oxidative damage, then replaced with complete culture medium containing different concentrations of MBNS (5 μg / ml, 10 μg / ml, 20 μg / ml), PCC, or MPCC, and cultured for 24 hours. After culture, CCK-8 reagent was added to each well, and after incubation for an appropriate time, the absorbance (OD) value of each well was measured at 450 nm using a microplate reader. The relative cell viability of each experimental group was calculated and compared based on this. The substrate material for MPCC (MBNS content 20 μg / mL), PCC, and MPCC was consistent and did not contain MBNS.

[0093] Depend on Figure 8 As can be seen from this, compared with the H2O2-damaged group (model group) without material treatment, the cell survival rate of MBNS-treated cells was significantly improved, indicating that the components released by MBNS help to reduce oxidative damage and protect cells.

[0094] Depend on Figure 8 b shows that the cell survival rate of the PCC extract treatment group was also significantly higher than that of the model group, which proves the antioxidant protective effect of the curcumin (Cur) and chitosan (CS) complex system.

[0095] Depend on Figure 8 As shown in c, the MPCC extract treatment group showed the highest degree of cell survival recovery, significantly better than the MBNS and PCC groups alone. This strongly demonstrates that the synergistic effect of "MBNS hydrogen release" and "chitosan-curcumin" has the strongest antioxidant protective effect at the cellular level, and can most effectively reverse cell damage caused by oxidative stress.

[0096] The procedure for the ROS detection kit using the fluorescent probe DCFH-DA is as follows: After grouping and oxidative stress intervention with the experimental CCK-8, the culture medium is discarded. The DCFH-DA probe is loaded onto serum-free culture medium prepared according to the instructions and incubated at 37°C in the dark for a certain period. To facilitate cell localization, Hoechst 33342 can be used to stain the cell nuclei in parallel.

[0097] Appendix Figure 8Images d, 8e, and 8f are confocal fluorescence images used for detecting intracellular reactive oxygen species (ROS) levels. All images are accompanied by a uniform 100µm scale bar. Intracellular ROS were labeled with the DCFH-DA fluorescent probe (green fluorescence), and the cell nucleus was labeled with Hoechst33342 (blue fluorescence). Each sub-image is composed of three merged images, and the DCFH fluorescence channel and Hoechst fluorescence channel are displayed separately.

[0098] Depend on Figure 8 As can be seen from d, after being stimulated by H2O2, the intensity of green fluorescence (ROS signal) in the Merge image of cells treated with MBNS is at a moderate level, indicating that it can effectively clear some intracellular ROS.

[0099] Depend on Figure 8 As can be seen from the results, the green fluorescence intensity of cells treated with PCC was significantly reduced compared to the model group, indicating that the antioxidant effect of Cur effectively reduced the intracellular ROS level.

[0100] Depend on Figure 8 As shown in f, the green fluorescence intensity in the image is the weakest, close to the level of the normal control group that has not been damaged by H2O2. Meanwhile, the blue fluorescence shows a large number of cell nuclei with intact morphology. This directly demonstrates that MPCC treatment is the most effective way to remove excess intracellular ROS induced by H2O2, protecting cells from oxidative damage.

[0101] comprehensive Figure 8 The cell viability recovery data (ac) and the intuitive images (df) of intracellular ROS clearance fully demonstrate, from both functional recovery and mechanism verification perspectives, the superior antioxidant stress protection capabilities of the MPCC nanofiber dressing of this invention at the cellular level. This provides crucial experimental evidence for its ability to protect and repair cells and promote healing in the microenvironment of diabetic high oxidative stress wounds.

[0102] Test on the protective effect of human umbilical vein endothelial cells (HUVECs) against oxidative stress injury To further verify the universality of the material of this invention and its antioxidant protective effect at the vascular-related cell level, an in vitro oxidative stress model was established using hydrogen peroxide (H2O2)-induced human umbilical vein endothelial cells (HUVECs). The material's ability to resist oxidative damage in HUVECs was comprehensively evaluated using both cell viability recovery experiments and intracellular reactive oxygen species (ROS) level detection methods. The results are shown in [Figure number missing]. Figure 9 .

[0103] Appendix Figure 9Figures a, 9b, and 9c are bar charts showing the relative cell viability as detected by the Cell Counting Kit-8 (CCK-8) method. The figures illustrate the initial treatment of RAW264.7 cells with hydrogen peroxide (H2O2) to induce oxidative damage, followed by culture in media containing different materials. The specific procedure for the CCK-8 method involved seeding cells in 96-well plates and allowing them to adhere overnight. First, oxidative stress was induced by treating with 500 μM H2O2 solution for 30 minutes. Then, the medium was replaced with complete medium containing different concentrations of MBNS (5 μg / ml, 10 μg / ml, 20 μg / ml), PCC, or MPCC, and cultured for another 24 hours. After culture, CCK-8 reagent was added to each well, and after incubation for an appropriate time, the absorbance (OD) of each well was measured at 450 nm using a microplate reader. The relative cell viability of each experimental group was then calculated and compared. Among them, MPCC (MBNS content is 20μg / mL) and PCC have the same base material and do not contain MBNS.

[0104] Depend on Figure 9 As can be seen from this, compared with the model group that was only damaged by H2O2, the cell survival rate of MBNS treatment was significantly improved, indicating that it has the ability to protect endothelial cells from oxidative damage.

[0105] Depend on Figure 9 b shows that the cell survival rate of the PCC extract treatment group was also significantly higher than that of the model group, confirming the cell protective effect of the basic composite material (CS / PVA / Cur).

[0106] Depend on Figure 9 As shown in c, the MPCC extract treatment group exhibited the most significant recovery in cell viability, far exceeding not only the model group but also the MBNS and PCC groups alone. This quantitatively demonstrates that the MPCC complex system possesses the strongest antioxidant protection and repair capabilities for endothelial cells, which is crucial for maintaining the integrity of fragile vascular endothelium at diabetic wound sites.

[0107] The procedure for the ROS detection kit using the fluorescent probe DCFH-DA is as follows: After grouping and oxidative stress intervention with the experimental CCK-8, the culture medium is discarded. The DCFH-DA probe is loaded onto serum-free culture medium prepared according to the instructions and incubated at 37°C in the dark for a certain period. To facilitate cell localization, Hoechst 33342 can be used to stain the cell nuclei in parallel.

[0108] Appendix Figure 9Images d, 9e, and 9f show fluorescence detection images of reactive oxygen species (ROS) levels in HUVEC cells. ROS were labeled with the DCFH-DA probe (green fluorescence), and cell nuclei were labeled with Hoechst33342 (blue fluorescence). Each set of images includes a Merge plot, a separate DCFH channel plot, and a Hoechst channel plot.

[0109] Depend on Figure 9 As can be seen from d, after stimulation with H2O2, the MBNS-treated group showed moderate green fluorescence in the Merge image, indicating that it could partially clear intracellular ROS.

[0110] Depend on Figure 9 As can be seen, the green fluorescence intensity of the PCC-treated group was significantly lower than that of the model group, indicating that the intracellular ROS level was effectively controlled.

[0111] Depend on Figure 9 As shown in f, the green fluorescence signal is the weakest in the image, with an intensity similar to that of the undamaged normal control group. Simultaneously, the Hoechst channel reveals a large number of cell nuclei with regular morphology. This directly and qualitatively demonstrates that MPCC can most effectively remove excess ROS induced by oxidative stress within HUVEC cells, creating a favorable low-oxidative microenvironment for endothelial cell survival and function.

[0112] comprehensive Figure 9 Quantitative data on cell viability (ac) and fluorescence images (df) of intracellular ROS clearance confirm, from both functional recovery and molecular mechanism perspectives, that the MPCC nanofiber dressing of this invention can effectively protect vascular endothelial cells—key repair cells in diabetic wounds—from damage by the high-oxidative-stress microenvironment. This provides crucial cellular evidence for its core therapeutic goals of promoting angiogenesis and accelerating tissue repair.

[0113] Tests on the promoting effect on the migration ability of human umbilical vein endothelial cells (HUVEC).

[0114] The effects of materials on vascular endothelial cell migration behavior were qualitatively evaluated using two classic models: the cell scratch healing assay and the Transwell chamber migration assay. The results are shown in [Figure number missing]. Figure 10 .

[0115] The impact of assessment materials on the horizontal migration ability of HUVECs Logarithmic growth phase HUVECs were digested, centrifuged, resuspended, and counted. They were then seeded evenly in 6-well plates at a density of 350,000 cells per well. After complete cell adhesion, a straight line was drawn perpendicular to the bottom of the plate on the cell monolayer in each well using a 200 μL sterile pipette tip. The old culture medium was discarded, and the cells were gently washed twice with PBS to remove floating cells. Subsequently, the cells were treated according to the experimental design: the blank control group received fresh complete culture medium; the nanomaterial treatment group received complete culture medium containing the corresponding test material (MBNS, PCC, or MPCC); the H2O2 intervention group received culture medium containing 500 μM H2O2; and the co-treatment group received culture medium containing both 500 μM H2O2 and the corresponding test material (MBNS, PCC, or MPCC). The culture plates were incubated at 37°C in a 5% CO2 incubator. The effect of the materials on the transmembrane chemotactic migration ability of HUVECs was evaluated. HUVECs underwent the same pretreatment grouping as in previous experiments: treatment with complete medium, medium containing nanomaterials (MBNS, PCC, or MPCC), medium containing 500 μM H2O2, and medium containing both (H2O2 and nanomaterials) for 24 hours. After pretreatment, all groups of cells were replaced with serum-free medium and starved for another 24 hours. Cells were then digested, collected, resuspended in serum-free medium, and accurately counted. The cell suspension density was adjusted to 5000 cells per 200 μL, and 200 μL was added to the upper chamber of a Transwell plate (8 μm pore size). 700 μL of DMEM medium containing 20% ​​fetal bovine serum was added to the lower chamber (24-well plate wells) as a chemokine. The plate was incubated for 48 hours. After incubation, the chambers were removed, and unmigrated cells were gently wiped away from the inner surface of the upper chamber membrane with a cotton swab. The chambers were fixed in 4% paraformaldehyde for 15 minutes, followed by staining with 0.1% crystal violet solution for 20 minutes. After gentle rinsing with PBS, multiple fields of view were randomly selected under an optical microscope to photograph the cells that had migrated to the submembrane surface.

[0116] In the above experiments, the amounts of MBNS (20 μg / ml), PCC, and MPCC (containing 20 μg / ml MBNS) were the same for both PCC and MPCC substrates.

[0117] Depend on Figure 10 As can be seen from this, after 24 hours, the cells migrated from the edge of the scratch to the center, and the width of the scratch decreased significantly.

[0118] Depend on Figure 10 b shows that the scratched area was also partially covered by the migrated cells.

[0119] Depend on Figure 10 As shown in c, after 24 hours, the scratch was almost completely closed by the migrated cells, and the healing speed and area were significantly better than those of other treatments. Figure 10 Groups a and 10b.

[0120] Figure 10 Image d is a representative image from the HUVEC cell Transwell chamber migration experiment.

[0121] Image section: Shows cells that have migrated to the lower surface of the Transwell chamber after being stained with crystal violet. The image clearly shows: Blank control group: The number of cells on the lower surface is relatively small.

[0122] MBNS treatment group and PCC treatment group: The number of migrating cells increased compared with the control group.

[0123] MPCC treatment group: The number and density of cells stained on the lower surface were the highest, indicating that the number of cells passing through the membrane pores was significantly greater than in other groups.

[0124] comprehensive Figure 10 The results of scratch and Transwell migration experiments, from both two-dimensional planar migration and three-dimensional directional migration perspectives, jointly confirm that the MPCC nanofiber dressing of this invention can strongly promote the migration of human umbilical vein endothelial cells (HUVECs). This function is an indispensable cell biological basis for initiating angiogenesis and forming new capillary networks, providing direct and strong experimental evidence for the core therapeutic advantage of MPCC dressing in diabetic wounds: "pro-angiogenesis".

[0125] In vitro angiogenesis-promoting ability and efficacy of MPCC nanofiber dressing in diabetic wound healing tests The effect of the materials on the tube-forming ability of HUVECs, i.e., their angiogenic activity, was assessed. The Matrigel was thawed overnight at 4°C the day before the experiment. 20 μL (16.57 mg / mL) of Matrigel was added to each well of a pre-chilled 24-well plate, gently shaken to spread the gel evenly at the bottom of the well, and then the plate was placed in a 4°C refrigerator for 30 minutes to allow further spreading. During the experiment, the 24-well plate was transferred to a 37°C incubator and incubated for 40 minutes to allow the Matrigel to fully polymerize and solidify. Simultaneously, HUVECs underwent 24 hours of pretreatment and subsequent 24 hours of serum-free starvation, following the same grouping and procedure as the Transwell migration assay (blank control, nanomaterial treatment (MBNS, PCC, MPCC), H2O2 intervention, and co-treatment with H2O2 and nanomaterials). Cells were then digested and counted, resuspended in ECM medium containing 2% fetal bovine serum, and the cell density was adjusted to 150,000 cells per 500 μL suspension. Remove the solidified Matrigel 24-well plate from the incubator and carefully add 500 μL of cell suspension to each well. Return the plate to the incubator and continue culturing for 12 hours. After culturing, observe the tubular network structure under an optical microscope and take pictures of randomly selected fields of view. Figure 11 Image a is a representative image from an in vitro angiogenesis (tubule formation) experiment using HUVEC cells.

[0126] In the above experiments, the amounts of MBNS (20 μg / ml), PCC, and MPCC (containing 20 μg / ml MBNS) were the same for both PCC and MPCC substrates.

[0127] Image section: Showing the morphology of HUVEC cells under a light microscope after culturing on Matrigel at different time points (12 hours). The images show: Blank control group: Cells can connect to form simple network structures.

[0128] PCC treatment group: The network structure formed by cells was relatively complex, and the number of nodes and the total length of tubules increased compared with the blank control group, indicating that PCC has a certain promoting effect on angiogenesis.

[0129] MPCC treatment group: cells are more tightly connected, forming a more complex and complete tubular network structure with a significant increase in branching points and closed loops, simulating a more mature neovascularization-like structure.

[0130] Figure 11 b is the flowchart for establishing and experimenting with a full-thickness skin defect model in diabetic mice.

[0131] This flowchart, presented as a block diagram with connected arrows or a timeline, clearly illustrates the key steps and timelines in animal experiments. The process typically includes: inducing diabetes → model stabilization → creating a full-thickness skin defect on the back → randomization to groups (e.g., blank control group, PCC group, MPCC group) → dressing application and fixation → periodic observation, photography, and dressing changes → euthanizing animals at predetermined time points (days 3, 7, 10, and 14) and collecting tissue samples → performing histological and molecular biological analysis. This diagram illustrates the systematic and scientific nature of in vivo efficacy evaluation.

[0132] Figure 11 c represents the dynamic change curve of wound healing rate in diabetic mice. The graph shows time (days) on the x-axis and wound healing rate (%) on the y-axis. It plots the healing rate over time for different treatment groups (including the control group, PCC group, and MPCC group). The curves clearly show that from the early stages of treatment, the wound healing rate of the MPCC treatment group was consistently higher than that of the PCC group and the control group, and its healing speed was the fastest. At the experimental endpoint (day 14), the final healing rate of the MPCC group was significantly the highest. This quantitative data directly demonstrates that MPCC dressings have a significant effect on accelerating the closure of diabetic wounds in vivo.

[0133] Figure 11 Image d shows representative macroscopic healing images of wounds in diabetic mice at different time points. All images are accompanied by a uniform scale (1 cm is marked on the scale).

[0134] This series of photographs visually demonstrates how wounds change over time. Each group (PCC and MPCC) includes wound photographs taken on day 0 (immediately after modeling), day 3, day 7, day 10, and day 14 (or other key time points).

[0135] The comparison clearly shows that: PCC group: The wound gradually shrinks, but the healing speed is slow, and there may be exudation or scab formation.

[0136] MPCC group: At the same time point, the wound area shrank more significantly, the granulation tissue grew more rosy, the epithelialization process was faster, and the inflammatory response (such as redness and swelling) was milder. In the later stages of the experiment, the wound in the MPCC group was almost completely covered by new skin.

[0137] comprehensive Figure 11 From a to 11 days, a complete chain of evidence was provided, from the in vitro angiogenesis-promoting function to the in vivo healing dynamics and final effect, fully demonstrating that the MPCC nanofiber dressing of the present invention not only has the core cellular function of promoting angiogenesis, but also can safely and effectively accelerate the healing process of refractory wounds in the complex diabetic in vivo environment.

[0138] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A magnesium-based nanofiber dressing with healing-promoting function, characterized in that, The dressing is a composite nanofiber membrane comprising aminated magnesium boron nanosheets, chitosan, curcumin and fiber-forming polymer, wherein the aminated magnesium boron nanosheets are uniformly dispersed in the fibers as a controllable hydrogen source, and the composite nanofiber membrane has a porous interconnected three-dimensional network structure.

2. The magnesium-based nanofiber dressing according to claim 1, characterized in that, The preferred weight ratio of the amino-modified magnesium boron nanosheets, chitosan, curcumin, and fiber-forming polymer is (2~7):(2~7):(2~7):(80~100).

3. A method for preparing a magnesium-based nanofiber dressing according to claim 1 or 2, characterized in that, Includes the following steps: S1. Aminated magnesium boron nanosheets, chitosan, curcumin and fiber-forming polymer are uniformly dispersed in a mixed solvent to form a spinning solution; S2. Electrospinning is performed on the spinning solution to obtain the magnesium-based nanofibers.

4. The preparation method according to claim 3, characterized in that, The aminated magnesium boron nanosheets are obtained by controlling the etching and stripping of the MgB2 precursor under the action of weak acid and oxidant, followed by surface modification with an amino-containing silane coupling agent.

5. The preparation method according to claim 4, characterized in that, The weak acid is selected from at least one of acetic acid, citric acid, formic acid, and lactic acid.

6. The preparation method according to claim 4, characterized in that, The amino-containing silane coupling agent is selected from at least one of 3-aminopropyltrimethoxysilane, N-2-aminoethyl-3-aminopropyltrimethoxysilane, and 3-2-aminoethylaminopropyltrimethoxysilane.

7. The preparation method according to claim 4, characterized in that, The hydrated particle size distribution of the aminated magnesium boron nanosheets has a main peak in the range of 40-80 nm.

8. The preparation method according to claim 3, characterized in that, The mixed solvent in step S2 comprises hexafluoroisopropanol, deionized water, and glacial acetic acid.

9. The preparation method according to claim 3, characterized in that, The electrospinning process parameters are: voltage 10-20kV, spinning solution propulsion flow rate 0.3-0.8mL / h, and receiving distance 10-20cm.

10. The application of the magnesium-based nanofiber dressing according to any one of claims 1-2, and the magnesium-based nanofiber dressing prepared by the preparation method according to any one of claims 3-9, in the healing of diabetic wounds.

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