Degradable electrospinning dressing

Abstract

The invention discloses a degradable electrospinning dressing, and belongs to the technical field of biological materials.The degradable electrospinning dressing is prepared by dispersing aminosilane modified FeO in a chitosan solution, adding a gelatin solution, stirring and mixing evenly, conducting gradient freezing under the action of a rotating magnetic field, conducting permeation with a PLGA solution, then conducting electrospinning, conducting crosslinking with a genipin solution, conducting washing and conducting drying. And sterilizing to obtain the degradable electrospinning dressing. According to the degradable electrospinning dressing prepared by the preparation method disclosed by the invention, an electrospinning auxiliary material is obtained through three-layer gradient electrospinning by modifying a Fe3O4 enhanced chitosan-gelatin composite base material with amino silane and combining magnetic field assistance and gradient freezing forming, a bionic fiber structure is constructed, wound exudation dynamic balance and long-acting antibiosis are realized, the optimal moisture balance of a wound part is kept, and the antibacterial property of the wound part is improved. And wound healing is effectively promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a degradable electrospun dressing. Background Art

[0002] Modern wound dressings have evolved from traditional gauze to two major categories: interactive and bioactive. Interactive dressings such as nanofiber membranes and hydrogels accelerate healing by dynamically regulating wound moisture, antibacterial properties, and breathability, but they have functional limitations. Foam dressings are opaque and not suitable for dry wounds, and hydrogels have high water content and are difficult to handle with highly exudative wounds. Although bioactive dressings can monitor and actively intervene in the healing process in real time, their high cost due to the integration of electronic components limits their clinical popularity. The current technical difficulty lies in balancing exudate management: maintaining a moist environment containing growth factors while avoiding infection caused by excessive exudate. This places higher demands on the dressing's wetting gradient design and antibacterial / healing-promoting synergistic mechanism.

[0003] Electrospinning technology offers a new approach to overcoming these bottlenecks. The nanofiber membranes produced using this technology precisely mimic the fibrous structure of the natural extracellular matrix. By manipulating the ratio of gelatin to chitosan, biomimetic scaffolds with both biological activity and mechanical strength can be constructed. However, existing electrospun membranes still face challenges with their limited wettability and inadequate interface control. Deep wounds experience increased capillary permeability, leading to the coexistence of nutrients and pathogens in exudate. Traditional homogenized dressings struggle to achieve targeted absorption of exudate and release of antimicrobial factors. Summary of the Invention

[0004] The present invention discloses a degradable electrospun dressing. A chitosan-gelatin composite substrate is reinforced by aminosilane modification of Fe3O4, combined with magnetic field assistance and gradient freezing molding, and an electrospun auxiliary material is obtained by three-layer gradient electrospinning. A biomimetic fiber structure is constructed to achieve dynamic balance of wound exudation and long-term antibacterial effect, maintain optimal moisture balance in the wound area, and effectively promote wound healing.

[0005] The technical solution of the present invention is achieved as follows: The present invention provides a preparation method of a degradable electrospun dressing, which comprises the following steps: dispersing aminosilane-modified Fe3O4 in a chitosan solution, adding a gelatin solution, stirring and mixing the mixture uniformly, performing gradient freezing under the action of a rotating magnetic field, infiltrating the mixture with a PLGA solution, and then electrospinning the mixture. The mixture is cross-linked with a genipin solution, washed, dried, and sterilized to obtain the degradable electrospun dressing.

[0006] As a further improvement of the present invention, the following steps are included: Step 1, preparing a chitosan-Fe3O4 dispersion: dissolving chitosan in an acetic acid solution to prepare a chitosan solution, and adding aminosilane-modified Fe3O4 to obtain a chitosan-Fe3O4 dispersion; Step 2: Preparation of substrate pre-frozen solution: dissolving gelatin in deionized water to prepare a gelatin solution, and mixing the chitosan-Fe3O4 dispersion with the gelatin solution to obtain a substrate pre-frozen solution; Step 3: Preparation of frozen substrate: injecting the substrate pre-frozen solution into a polytetrafluoroethylene mold, performing gradient freezing, and applying a rotating magnetic field during the gradient freezing to obtain a frozen-molded substrate; Step 4: Electrospinning: Immerse the freeze-formed substrate in a PLGA solution, drain the immersed substrate, filter and stir the immersed PLGA solution to obtain an electrospinning solution, and use the drained substrate as a receiving plate for electrospinning to obtain an electrospun membrane. Step 5. Preparation of degradable electrospun dressing: After vacuum drying the electrospun membrane, place it in a genipin solution for cross-linking. After the cross-linking is completed, wash, dry, and sterilize to obtain a degradable electrospun dressing.

[0007] As a further improvement of the present invention, in step 1, the concentration of the acetic acid solution is 2%, the mass ratio of chitosan, acetic acid solution and aminosilane-modified Fe3O4 is 3-5:100:1-5, the ultrasonic dispersion power is 300-450W, the time is 20-30min, and the frequency is 20-40kHz. The relative molecular weight of chitosan is ≥100,000 and the degree of deacetylation is ≥90%. The preparation method of aminosilane-modified Fe3O4 comprises the following steps: (1) Fe3O4 nanoparticles were dispersed in Tris buffer at pH 8.5 and ultrasonicated for 10 min. Dopamine hydrochloride was added at a mass ratio of Fe3O4 to dopamine hydrochloride of 1:0.5~1 and stirred at room temperature for 12~24 h. After the reaction, the product was collected by magnetic separation, washed with deionized water three times, and vacuum dried to obtain dopamine-coated Fe3O4. (2) The dopamine-coated Fe3O4 was dispersed in anhydrous ethanol, and 3-aminopropyltriethoxysilane was added in a volume ratio of anhydrous ethanol: 3-aminopropyltriethoxysilane = 10:1. Under nitrogen protection, the reaction was stirred at 60°C for 6 hours. The product was collected by magnetic separation, washed with ethanol three times to remove unreacted silane, and vacuum dried to obtain aminosilane-modified Fe3O4.

[0008] As a further improvement of the present invention, in step 2, the temperature of the deionized water is 60° C., the mass ratio of gelatin, deionized water and chitosan-Fe 3 O 4 dispersion is 3-5:100:100, and the relative molecular weight of gelatin is ≥10,000.

[0009] As a further improvement of the present invention, in step three, the liquid layer thickness of the substrate pre-frozen liquid in the polytetrafluoroethylene mold is 2 mm, the bottom of the polytetrafluoroethylene mold is embedded with a copper microneedle array, the intensity of the rotating magnetic field is 50 mT, the rotation speed is 300 rpm, and the gradient freezing parameters are a gradient temperature change from -70°C to -20°C, a cooling rate of 5°C / min, and a gradient insulation of 30 minutes per 5°C.

[0010] As a further improvement of the present invention, in step 4, the concentration of the PLGA solution is 10%, and the PLGA solution contains LL-37 antimicrobial peptide at a concentration of 1 mg / mL; The immersion time is 1 to 2 hours. After immersion, the substrate is hung vertically to drain for 30 to 60 seconds. The PLGA solution after immersion is filtered through a 0.22 μm filter membrane and magnetically stirred for 10 to 20 minutes. The electrospinning method was three-layer gradient uniaxial electrospinning, and the inner layer parameters were set as follows: fiber diameter 100 nm, electrospinning solution flow rate 0.3 ml / h, voltage 16 kV, collection distance 15 cm, needle inner diameter 23 G, and rotation speed 3600 rpm; The parameters of the middle layer were set as follows: fiber diameter 300 nm, electrospinning solution flow rate 0.1 ml / h, voltage 17 kV, collection distance 15 cm, needle inner diameter 21 G, and rotation speed 3300 rpm; The outer layer parameters were set as follows: fiber diameter 500 nm, electrospinning solution flow rate 0.1 ml / h, voltage 17 kV, collection distance 15 cm, needle inner diameter 19 G, and rotation speed 3000 rpm.

[0011] As a further improvement of the present invention, in step five, the first vacuum drying time is 72 hours, the concentration of the genipin solution is 1%, the cross-linking time is 2 days, and the specific washing steps are washing with 80% ethanol solution for 30 minutes and then rinsing the residual ethanol solution with 0.9% NaCl solution. The drying method is vacuum drying for 24 hours, and the sterilization method is ultraviolet radiation sterilization.

[0012] As a further improvement of the present invention, the present invention specifically comprises the following steps: Step 1, preparing a chitosan-Fe3O4 dispersion: dissolving 3-5 parts of chitosan with a relative molecular weight of ≥100,000 and a degree of deacetylation of ≥90% in 100 parts of a 2% acetic acid solution to prepare a chitosan solution, adding 1-5 parts of aminosilane-modified Fe3O4, and ultrasonically dispersing at a power of 300-450 W and a frequency of 20-40 kHz for 20-30 minutes to obtain a chitosan-Fe3O4 dispersion; Step 2: Preparation of substrate pre-frozen solution: Dissolve 3-5 parts of gelatin with a relative molecular weight of ≥10,000 in 100 parts of deionized water at 60°C to prepare a gelatin solution, and mix 100 parts of chitosan-Fe3O4 dispersion with the gelatin solution to obtain a substrate pre-frozen solution; Step 3: Preparation of frozen substrate: The substrate pre-frozen solution was injected into a polytetrafluoroethylene mold with a copper microneedle array embedded in the bottom, with a liquid layer thickness of 2 mm. Gradual freezing was performed from -70°C to -20°C, with a cooling rate of 5°C / min, and the temperature was kept at 5°C for 30 minutes each. During the gradient freezing, a rotating magnetic field with an intensity of 50 mT and a rotation speed of 300 rpm was applied to obtain a freeze-molded substrate; Step 4: Electrospinning: Immerse the freeze-formed substrate in a PLGA solution for 1 to 2 hours. After immersion, hang the substrate vertically to drain for 30 to 60 seconds. Filter the immersed PLGA solution through a 0.22 μm filter membrane and magnetically stir for 10 to 20 minutes to obtain an electrospinning solution. Use the drained substrate as a receiving plate for three-layer gradient uniaxial electrospinning to obtain an electrospun membrane. Among them, the inner layer parameters of the three-layer gradient uniaxial electrospinning were set as follows: fiber diameter 100nm, electrospinning solution flow rate 0.3ml / h, voltage 16kv, collection distance 15cm, needle inner diameter 23G, and rotation speed 3600rpm; the middle layer parameters were set as follows: fiber diameter 300nm, electrospinning solution flow rate 0.1ml / h, voltage 17kv, collection distance 15cm, needle inner diameter 21G, and rotation speed 3300rpm; the outer layer parameters were set as follows: fiber diameter 500nm, electrospinning solution flow rate 0.1ml / h, voltage 17kv, collection distance 15cm, needle inner diameter 19G, and rotation speed 3000rpm; Step 5. Preparation of degradable electrospun dressing: After vacuum drying the electrospun membrane for 72 hours, place it in a 1% genipin solution for cross-linking for 2 days. After cross-linking is completed, wash it with 80% ethanol solution for 30 minutes and then rinse the residual ethanol solution with 0.9% NaCl solution. Vacuum dry it for 24 hours and sterilize it with ultraviolet radiation to obtain a degradable electrospun dressing.

[0013] The present invention further protects a degradable electrospun dressing prepared by the above preparation method.

[0014] The present invention further protects a use of the above-mentioned degradable electrospun dressing in skin repair.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The structurally integrated but functionally gradient skin graft provided by the present invention has good biocompatibility and mechanical properties, and to a certain extent promotes collagen deposition and hair follicle regeneration at the wound site, thereby improving fibrosis at the wound site; the structurally integrated but functionally gradient skin graft provided by the present invention has a three-dimensional structure similar to the extracellular matrix, and its oriented nanostructure has a highly consistent fiber arrangement direction, which can promote cell adhesion and migration through contact guidance, and can further promote cell proliferation and differentiation by combining with cells or growth factors, thereby accelerating skin wound healing; the structurally integrated but functionally gradient skin graft provided by the present invention has a gradient wetting property that enables the dressing to have good management capabilities for exudates at the wound site, avoid excessive drying and hydration, maintain the humidity required for wound healing, and promote wound healing; the structurally integrated but functionally gradient skin graft provided by the present invention has a low cost and can be mass-produced, providing a new idea for the field of skin tissue engineering.

[0016] (2) In the process of preparing aminosilane-modified Fe3O4, dopamine is first oxidized and self-polymerized to form polydopamine in an alkaline environment of pH 8.5. The hydroxyl groups in its catechol structure form coordination bonds with Fe³⁺ / Fe²⁺ on the surface of Fe3O4, thereby firmly coating the surface of Fe3O4 and providing abundant amino and hydroxyl groups, providing active sites for the subsequent cross-linking of aminosilane. In the subsequent reaction, the ethoxy group of 3-aminopropyltriethoxysilane is hydrolyzed into silanol (-Si-OH) in anhydrous ethanol. The silanol undergoes condensation and dehydration reaction with the hydroxyl or amino group of polydopamine coated on the surface of Fe3O4 to form Si-OC or Si-N covalent bonds, thereby fixing the aminosilane on the surface of Fe3O4, so that the surface of Fe3O4 has amino groups, which enhances the surface activity, biocompatibility and dispersion stability of Fe3O4.

[0017] (3) The present invention forms an amino functional layer on the surface of Fe3O4 through dopamine coating and silanization treatment, which significantly improves its dispersibility in the chitosan / gelatin matrix and enhances its chemical bonding ability with PLGA fibers. The aminosilane-modified Fe3O4 forms a directional magnetic domain structure under the induction of a rotating magnetic field, which can produce a local magnetic field effect and inhibit the formation of Staphylococcus aureus biofilm. At the same time, it accelerates local blood circulation in the wound through the magnetothermal effect. The slow-release iron ions of Fe3O4 activate the HIF-1α pathway, increase the expression of VEGF, promote the increase of blood vessel density, and thus accelerate wound healing.

[0018] (4) The present invention uses -70℃ to -20℃ gradient freezing combined with a copper microneedle array in the preparation process of the frozen substrate to form a multi-level pore structure, simulating the fibrous topology of natural skin ECM. The rotating magnetic field synchronously regulates the growth direction of ice crystals, so that the chitosan / gelatin substrate dynamically forms radially arranged nanofiber bundles according to different pore structures, and the tensile strength is greatly improved.

[0019] (5) The present invention adopts a three-layer gradient uniaxial electrospinning structure in the electrospinning process. The inner layer has a diameter of 100nm fiber and uses a high-density nano-network to provide a sustained-release carrier of the antimicrobial peptide LL-37, which effectively inhibits the proliferation of Pseudomonas aeruginosa; the middle layer has a diameter of 300nm fiber, and uses mesoscale fibers to form a mechanical buffer layer, and the elastic modulus matches the dermis layer, reducing the shear damage to the wound caused by movement; the outer layer has a diameter of 500nm fiber, and uses a large pore structure to achieve directional absorption of exudate, and forms a pH-responsive surface through cross-linking with genipin, realizing dynamic response to wound exudate; the gradient cross-linking density design realizes a gradient transition of mechanical properties, and the compression modulus gradually decreases from the surface layer to the bottom layer, adapting to the stress distribution during wound contraction.

[0020] (6) When the substrate is used as a receiving plate, the copper microneedle array induces the PLGA fibers to form a vertically oriented structure, promotes the migration of fibroblasts along the fiber direction, and accelerates wound healing.

[0021] (7) This technology achieves a synergistic improvement in the mechanical properties, antibacterial activity, and tissue regeneration ability of the dressing through the directional assembly of magnetic nanoparticles and biomimetic gradient electrospinning structure, providing a new clinically applicable solution for chronic wound management. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the SEM image of the inner layer of the electrospun membrane in Example 1; Figure 2 This is the SEM image of the middle layer of the electrospun membrane in Example 1; Figure 3 This is the SEM image of the outer layer of the electrospun membrane in Example 1; Figure 4 This is an SEM image of the inner layer of the degradable electrospun dressing in Example 1; Figure 5 This is an SEM image of the middle layer of the degradable electrospun dressing in Example 1; Figure 6 This is an SEM image of the outer layer of the degradable electrospun dressing in Example 1; Figure 7 is a SEM image of a cross section of the degradable electrospun dressing in Example 1; Figure 8This is a graph showing the degradation performance of the degradable electrospun dressing in Example 1, wherein A100 is the inner layer fiber, A300 is the middle layer fiber, and A500 is the outer layer fiber; Figure 9 The digitally recorded photos of the skin wounds on the mice in the Example 1 group in Test Example 3; Figure 10 This is a digitally recorded photo of the skin wound of mice in the control group 1 of Test Example 3; Figure 11 This is a digitally recorded photo of the skin wound of mice in the control group 2 of Test Example 3; Figure 12 This is a digitally recorded photo of the skin wound of mice in the control group 3 of Test Example 3; Figure 13 In Test Example 3, digitally recorded photos of the skin wounds of mice in the blank control group were taken. DETAILED DESCRIPTION

[0023] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] The chitosan used in the examples of this application was from Zhejiang Golden Shell Pharmaceutical Co., Ltd. Example 1: In this example, a degradable electrospun dressing was prepared.

[0025] Step 1: Prepare a chitosan-Fe3O4 dispersion: dissolve 3 parts of chitosan with a relative molecular weight of ≥100,000 and a degree of deacetylation of ≥90% in 100 parts of a 2% acetic acid solution to prepare a chitosan solution, add 5 parts of aminosilane-modified Fe3O4, and ultrasonically disperse at a power of 300 W and a frequency of 20 kHz for 30 minutes to obtain a chitosan-Fe3O4 dispersion; Step 2: Preparation of substrate pre-frozen solution: Dissolve 5 parts of gelatin with a relative molecular weight of ≥10,000 in 100 parts of deionized water at 60°C to prepare a gelatin solution, and mix 100 parts of chitosan-Fe3O4 dispersion with the gelatin solution to obtain a substrate pre-frozen solution; Step 3: Preparation of frozen substrate: The substrate pre-frozen solution was injected into a polytetrafluoroethylene mold with a copper microneedle array embedded in the bottom, with a liquid layer thickness of 2 mm. Gradual freezing was performed from -70°C to -20°C, with a cooling rate of 5°C / min, and the temperature was kept at 5°C for 30 minutes each. During the gradient freezing, a rotating magnetic field with an intensity of 50 mT and a rotation speed of 300 rpm was applied to obtain a freeze-molded substrate; Step 4: Electrospinning: The freeze-formed substrate was immersed in a PLGA solution for 2 hours. After immersion, the substrate was hung vertically to drain for 60 seconds. The immersed PLGA solution was filtered through a 0.22 μm filter membrane and magnetically stirred for 20 minutes to obtain an electrospinning solution. The drained substrate was used as a receiving plate for three-layer gradient uniaxial electrospinning to obtain an electrospun membrane. Among them, the inner layer parameters of the three-layer gradient uniaxial electrospinning were set as follows: fiber diameter 100nm, electrospinning solution flow rate 0.3ml / h, voltage 16kv, collection distance 15cm, needle inner diameter 23G, and rotation speed 3600rpm; the middle layer parameters were set as follows: fiber diameter 300nm, electrospinning solution flow rate 0.1ml / h, voltage 17kv, collection distance 15cm, needle inner diameter 21G, and rotation speed 3300rpm; the outer layer parameters were set as follows: fiber diameter 500nm, electrospinning solution flow rate 0.1ml / h, voltage 17kv, collection distance 15cm, needle inner diameter 19G, and rotation speed 3000rpm; Step 5. Preparation of degradable electrospun dressing: After vacuum drying the electrospun membrane for 72 hours, place it in a 1% genipin solution for cross-linking for 2 days. After cross-linking is completed, wash it with 80% ethanol solution for 30 minutes and then rinse the residual ethanol solution with 0.9% NaCl solution. Vacuum dry it for 24 hours and sterilize it with ultraviolet radiation to obtain a degradable electrospun dressing.

[0026] The preparation method of aminosilane-modified Fe3O4 comprises the following steps: (1) Fe3O4 nanoparticles were dispersed in Tris buffer at pH 8.5 and sonicated for 10 min. Dopamine hydrochloride was added at a mass ratio of 1:1 between Fe3O4 and dopamine hydrochloride, and stirred at room temperature for 24 h. After the reaction, the product was collected by magnetic separation, washed three times with deionized water, and vacuum dried to obtain dopamine-coated Fe3O4. (2) The dopamine-coated Fe3O4 was dispersed in anhydrous ethanol, and 3-aminopropyltriethoxysilane was added in a volume ratio of anhydrous ethanol: 3-aminopropyltriethoxysilane = 10:1. Under nitrogen protection, the reaction was stirred at 60°C for 6 hours. The product was collected by magnetic separation, washed with ethanol three times to remove unreacted silane, and vacuum dried to obtain aminosilane-modified Fe3O4.

[0027] The electrospun dressing produced in this example achieved a tensile strength of 4.52 N, exhibited good biocompatibility, and exhibited a moderate degradation rate. When used as a skin graft, significant collagen deposition and hair follicle regeneration were observed 14 days after wound healing, with some improvement in fibrosis at the wound site.

[0028] Example 2: In this example, a degradable electrospun dressing was prepared.

[0029] Step 1: Prepare a chitosan-Fe3O4 dispersion: dissolve 3 parts of chitosan with a relative molecular weight of ≥100,000 and a degree of deacetylation of ≥90% in 100 parts of a 2% acetic acid solution to prepare a chitosan solution, add 1 part of aminosilane-modified Fe3O4, and ultrasonically disperse at a power of 450 W and a frequency of 40 kHz for 20 minutes to obtain a chitosan-Fe3O4 dispersion; Step 2: Preparation of substrate pre-frozen solution: Dissolve 3 parts of gelatin with a relative molecular weight of ≥10,000 in 100 parts of deionized water at 60°C to prepare a gelatin solution, and mix 100 parts of chitosan-Fe3O4 dispersion with the gelatin solution to obtain a substrate pre-frozen solution; Step 3: Preparation of frozen substrate: The substrate pre-frozen solution was injected into a polytetrafluoroethylene mold with a copper microneedle array embedded in the bottom, with a liquid layer thickness of 2 mm. Gradual freezing was performed from -70°C to -20°C, with a cooling rate of 5°C / min, and the temperature was kept at 5°C for 30 minutes each. During the gradient freezing, a rotating magnetic field with an intensity of 50 mT and a rotation speed of 300 rpm was applied to obtain a freeze-molded substrate; Step 4: Electrospinning: The freeze-formed substrate was immersed in a PLGA solution for 1 hour. After immersion, the substrate was hung vertically to drain for 30 seconds. The immersed PLGA solution was filtered through a 0.22 μm filter membrane and magnetically stirred for 10 minutes to obtain an electrospinning solution. The drained substrate was used as a receiving plate for three-layer gradient uniaxial electrospinning to obtain an electrospun membrane. Among them, the inner layer parameters of the three-layer gradient uniaxial electrospinning were set as follows: fiber diameter 100nm, electrospinning solution flow rate 0.3ml / h, voltage 16kv, collection distance 15cm, needle inner diameter 23G, and rotation speed 3600rpm; the middle layer parameters were set as follows: fiber diameter 300nm, electrospinning solution flow rate 0.1ml / h, voltage 17kv, collection distance 15cm, needle inner diameter 21G, and rotation speed 3300rpm; the outer layer parameters were set as follows: fiber diameter 500nm, electrospinning solution flow rate 0.1ml / h, voltage 17kv, collection distance 15cm, needle inner diameter 19G, and rotation speed 3000rpm; Step 5. Preparation of degradable electrospun dressing: After vacuum drying the electrospun membrane for 72 hours, place it in a 1% genipin solution for cross-linking for 2 days. After cross-linking is completed, wash it with 80% ethanol solution for 30 minutes and then rinse the residual ethanol solution with 0.9% NaCl solution. Vacuum dry it for 24 hours and sterilize it with ultraviolet radiation to obtain a degradable electrospun dressing.

[0030] The preparation method of aminosilane-modified Fe3O4 comprises the following steps: (1) Fe3O4 nanoparticles were dispersed in Tris buffer at pH 8.5 and sonicated for 10 min. Dopamine hydrochloride was added at a mass ratio of 1:1 between Fe3O4 and dopamine hydrochloride, and stirred at room temperature for 24 h. After the reaction, the product was collected by magnetic separation, washed three times with deionized water, and vacuum dried to obtain dopamine-coated Fe3O4. (2) The dopamine-coated Fe3O4 was dispersed in anhydrous ethanol, and 3-aminopropyltriethoxysilane was added in a volume ratio of anhydrous ethanol: 3-aminopropyltriethoxysilane = 10:1. Under nitrogen protection, the reaction was stirred at 60°C for 6 hours. The product was collected by magnetic separation, washed with ethanol three times to remove unreacted silane, and vacuum dried to obtain aminosilane-modified Fe3O4.

[0031] Comparative Example 1: In this comparative example, an electrospun dressing was prepared.

[0032] In this embodiment, three-layer gradient uniaxial electrospinning is not performed, but single-layer uniaxial electrospinning is performed directly. The parameters are set according to the middle layer parameters of Example 1: fiber diameter is 300nm, electrospinning liquid flow rate is 0.1ml / h, voltage is 17kv, collection distance is 15cm, needle inner diameter is 21G, and rotation speed is 3300rpm; other steps are the same as Example 1.

[0033] Comparative Example 2 In this comparative example, an electrospun dressing was prepared.

[0034] In this embodiment, Fe3O4, nanoparticles of dopamine hydrochloride and 3-aminopropyltriethoxysilane are not reacted to generate aminosilane-modified Fe3O4. Instead, Fe3O4, nanoparticles of dopamine hydrochloride and 3-aminopropyltriethoxysilane are directly added, wherein the mass ratio of Fe3O4, nanoparticles of dopamine hydrochloride and 3-aminopropyltriethoxysilane is the same as that in Example 1; other steps are the same as in Example 1.

[0035] Comparative Example 3: In this comparative example, an electrospun dressing was prepared.

[0036] In this embodiment, a rotating magnetic field is not applied simultaneously with gradient freezing. Instead, a rotating magnetic field is applied for 5 hours before gradient freezing. Other steps are the same as those in Example 1.

[0037] Measurement example 1 The cross-sectional structures of the electrospun membrane prepared in step 4 of Example 1 and the degradable electrospun dressing prepared in step 5 were observed using a field emission scanning electron microscope, and the fiber diameter distribution statistics of the inner layer, middle layer and outer layer fibers were analyzed.

[0038] Analysis: If Figures 1 to 3As shown, the nanofibers in the inner, middle and outer layers of the electrospun membrane prepared in step 4 of Example 1 all exhibited smooth, continuous and bead-free uniform morphology and an ordered morphological structure. All groups showed an approximately normal uniform distribution and had a certain orientation. Figures 4 to 6 It can be seen that after the cross-linking operation in step five, the diameter of the nanofibers of the degradable electrospun dressing prepared in step five increased to a certain extent from 79.06±20.12nm, 260.3±89.21nm, 412.63±73.08nm to 140.36±31.1nm, 333.92±68.98nm, 514.29±131.4nm; Figure 7 As shown in the figure, the three layers of fibers of the degradable electrospun dressing of Example 1 are tightly adhered to each other, and there are no pores or uneven connections; SEM results show that compared Figures 1 to 3 It can be seen that the electrospinning parameters have a great influence on the diameter of the electrospun membrane fibers produced, and there is no specific rule. After conducting a large number of experiments, the applicant obtained the electrospinning-related parameters of this application, which can accurately control the diameter of electrospun nanofibers and adjust the porosity of the electrospun support, thereby achieving the technical effect of the technical solution of this application.

[0039] Test Example 2 The degradable electrospun dressing in Example 1 was subjected to a degradation test. The dressing was incubated in a simulated body fluid at 37° C. in an incubator for 14 days, and the weight loss was measured every 7 days.

[0040] Analysis: From Figure 8 The weight loss percentage of each layer in the degradable electrospun dressing was observed: on the 7th day (inner layer: 79.06±4.15%, middle layer: 80.6±7.33%, outer layer: 80.93±3.68%, base material: 85.16±4.48%) and on the 14th day (inner layer: 65.51±2.04%, middle layer: 68.88±1.35%, outer layer: 72.4±2.50%, base material: 74.56±3.37%). The degradation rate of the degradable electrospun dressing of the present invention in the first 7 days was higher than that in the last 7 days. This is because the gelatin content was higher in the initial stage and the degradation rate of gelatin was higher than that of chitosan. The relatively slowly degrading chitosan helped to maintain the integrity of the dressing throughout the regeneration process. The release of gelatin promoted cell adhesion and tissue regeneration, thereby facilitating the wound healing process.

[0041] Test Example 3 Eight-week-old C57BL / 6J mice weighing approximately 20 g were housed in ventilated cages with corn bedding and free access to food and water. The mice were anesthetized intraperitoneally, and their backs were hair removed. A circular, full-thickness skin defect with a diameter of 6 mm was created and secured with a silicone ring with an inner diameter of 6 mm and an outer diameter of 8 mm to prevent back muscle contraction from closing the wound. This allowed for objective evaluation of the skin wound's ability to heal by creeping. The irradiated, sterilized, biodegradable electrospun dressing from Example 1 (with the outer layer facing upward and the inner layer facing the wound tissue, uniformly sized as a 7 mm diameter circular disc) was neatly sewn into the skin defect. Undressed wounds served as a blank control group, while the electrospun dressings prepared in Comparative Examples 1-3 served as Control 1, Control 2, and Control 3, respectively. The skin wounds were covered with 3M Tegaderm transparent wound dressings secured with self-adhesive elastic bandages. Images of the wound area were recorded with a digital camera at predetermined postoperative times (5, 7, and 14 days).

[0042] Analysis: From Figures 9 to 13 It can be seen that the skin wounds of the mice in Example 1 and all the control groups were basically healed, but in comparison, the healing effect of Example 1 was better, the scar area formed was the smallest, and the hair follicle regeneration effect was more obvious than that of other groups. It can be seen that the technical solution 1 of Example 1 of the present application has good biological safety. Among the control groups, the effect of control group 1 was the worst, because no three-layer gradient uniaxial electrospinning was performed in comparative example 1, so no gradient structure was formed, and thus the three-layer gradient effect in the technical solution of the present application was not achieved, resulting in the worst wound repair effect; compared with comparative example 1, control group 2 did not generate aminosilane-modified Fe3O4, so no amino functional layer was generated on the Fe3O4 surface, and its dispersibility in the chitosan / gelatin matrix was poor, and its chemical bonding ability with the PLGA fiber was relatively weak, so the local magnetic field effect generated was weak, the antibacterial effect was reduced, and the effect of accelerating local blood circulation in the wound was poor, and the wound healing speed was slowed down; compared with Example 1, control group 3 did not perform gradient freezing and application of a rotating magnetic field simultaneously, but performed them in steps, and did not synchronously regulate the growth direction of ice crystals, so that the radially arranged nanofiber bundles formed by the chitosan / gelatin substrate could not be adjusted according to the pores of the dressing, and the tensile strength was limited, and thus it could not effectively fit the skin surface, the effect was weakened, and the effect of promoting wound healing was poor.

[0043] In summary, the technical solution of the present application has a significant effect of promoting the healing of biological skin wounds and has a good degradation effect; in the process of preparing the degradable electrospun dressing of the present application, the applicant found that the electrospinning parameters have a great influence on the diameter of the electrospun membrane fiber produced, and there is no specific rule. After conducting a large number of experiments, the applicant obtained the electrospinning related parameters of the present application, which can accurately control the diameter of the electrospun nanofibers and adjust the porosity of the electrospun scaffold, thereby achieving the technical effect of the technical solution of the present application; by modifying the Fe3O4 surface with an amino functional layer, the porosity of the electrospun membrane in the chitosan / gelatin matrix is ​​greatly improved. The dispersion and chemical bonding ability with PLGA fibers are enhanced. The local magnetic field generated by the evenly dispersed Fe3O4 in the dressing interacts with each other, and the antibacterial effect and the effect of accelerating local blood circulation in the wound are significantly improved, which prevents the invasion of foreign bacteria while accelerating the replenishment of internal nutrients, thereby accelerating the healing of the wound; the gradient freezing and the application of the rotating magnetic field are applied simultaneously, and the growth direction of the ice crystals is synchronously regulated during freezing. The radially arranged nanofiber bundles formed by the chitosan / gelatin matrix are adjusted according to the pores of the dressing, and the tensile strength is significantly improved, thereby fully fitting to the skin surface and greatly promoting the healing effect of the wound.

[0044] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for preparing a degradable electrospun dressing, characterized in that: The biodegradable electrospun dressing was obtained by dispersing aminosilane-modified Fe3O4 in a chitosan solution, adding a gelatin solution, stirring and mixing, performing gradient freezing under a rotating magnetic field, infiltrating with a PLGA solution, and then electrospinning. The dressing was cross-linked with a genipin solution, washed, dried, and sterilized.

2. The method for preparing a degradable electrospun dressing according to claim 1, wherein: The following steps are involved: Step 1, preparing a chitosan-Fe3O4 dispersion: dissolving chitosan in an acetic acid solution to prepare a chitosan solution, and adding aminosilane-modified Fe3O4 to obtain a chitosan-Fe3O4 dispersion; Step 2: Preparation of substrate pre-frozen solution: dissolving gelatin in deionized water to prepare a gelatin solution, and mixing the chitosan-Fe3O4 dispersion with the gelatin solution to obtain a substrate pre-frozen solution; Step 3: Preparation of frozen substrate: injecting the substrate pre-frozen solution into a polytetrafluoroethylene mold, performing gradient freezing, and applying a rotating magnetic field during the gradient freezing to obtain a frozen-molded substrate; Step 4: Electrospinning: Immerse the freeze-formed substrate in a PLGA solution, drain the immersed substrate, filter and stir the immersed PLGA solution to obtain an electrospinning solution, and use the drained substrate as a receiving plate for electrospinning to obtain an electrospun membrane. Step 5. Preparation of degradable electrospun dressing: After vacuum drying the electrospun membrane, place it in a genipin solution for cross-linking. After the cross-linking is completed, wash, dry, and sterilize to obtain a degradable electrospun dressing.

3. The method for preparing a degradable electrospun dressing according to claim 1, characterized in that: In the step 1, the concentration of the acetic acid solution is 2%, the mass ratio of chitosan, acetic acid solution and aminosilane-modified Fe3O4 is 3-5:100:1-5, the ultrasonic dispersion power is 300-450W, the time is 20-30min, and the frequency is 20-40kHz. The relative molecular weight of chitosan is ≥100,000 and the degree of deacetylation is ≥90%. The preparation method of aminosilane-modified Fe3O4 comprises the following steps: (1) Fe3O4 nanoparticles were dispersed in Tris buffer at pH 8.5 and ultrasonicated for 10 min. Dopamine hydrochloride was added at a mass ratio of Fe3O4 to dopamine hydrochloride of 1:0.5~1 and stirred at room temperature for 12~24 h. After the reaction, the product was collected by magnetic separation, washed with deionized water three times, and vacuum dried to obtain dopamine-coated Fe3O4. (2) The dopamine-coated Fe3O4 was dispersed in anhydrous ethanol, and 3-aminopropyltriethoxysilane was added in a volume ratio of anhydrous ethanol: 3-aminopropyltriethoxysilane = 10:

1. Under nitrogen protection, the reaction was stirred at 60°C for 6 hours. The product was collected by magnetic separation, washed with ethanol three times to remove unreacted silane, and vacuum dried to obtain aminosilane-modified Fe3O4.

4. The method for preparing a degradable electrospun dressing according to claim 1, wherein: In the step 2, the temperature of the deionized water is 60° C., the mass ratio of gelatin, deionized water, and chitosan-Fe 3 O 4 dispersion is 3-5:100:100, and the relative molecular weight of the gelatin is ≥10,000.

5. The method for preparing a degradable electrospun dressing according to claim 1, characterized in that: In step 3, the thickness of the liquid layer of the substrate pre-frozen liquid in the polytetrafluoroethylene mold is 2 mm, a copper microneedle array is embedded in the bottom of the polytetrafluoroethylene mold, the intensity of the rotating magnetic field is 50 mT, the rotation speed is 300 rpm, and the gradient freezing parameters are a gradient temperature change from -70°C to -20°C, a cooling rate of 5°C / min, and a thermal insulation of 30 minutes per 5°C gradient.

6. The method for preparing a degradable electrospun dressing according to claim 1, characterized in that: In the step 4, the concentration of the PLGA solution is 10%, and the PLGA solution contains LL-37 antimicrobial peptide at a concentration of 1 mg / mL; The immersion time is 1 to 2 hours. After immersion, the substrate is hung vertically to drain for 30 to 60 seconds. The PLGA solution after immersion is filtered through a 0.22 μm filter membrane and magnetically stirred for 10 to 20 minutes. The electrospinning method was three-layer gradient uniaxial electrospinning, and the inner layer parameters were set as follows: fiber diameter 100 nm, electrospinning solution flow rate 0.3 ml / h, voltage 16 kV, collection distance 15 cm, needle inner diameter 23 G, and rotation speed 3600 rpm; The parameters of the middle layer were set as follows: fiber diameter 300 nm, electrospinning solution flow rate 0.1 ml / h, voltage 17 kV, collection distance 15 cm, needle inner diameter 21 G, and rotation speed 3300 rpm; The outer layer parameters were set as follows: fiber diameter 500 nm, electrospinning solution flow rate 0.1 ml / h, voltage 17 kV, collection distance 15 cm, needle inner diameter 19 G, and rotation speed 3000 rpm.

7. The method for preparing a degradable electrospun dressing according to claim 1, characterized in that: In step five, the first vacuum drying time is 72 hours, the concentration of the genipin solution is 1%, the cross-linking time is 2 days, the specific washing steps are to use 80% ethanol solution for washing for 30 minutes and then rinse the residual ethanol solution with 0.9% NaCl solution, the drying method is vacuum drying for 24 hours, and the sterilization method is ultraviolet radiation sterilization.

8. The method for preparing a degradable electrospun dressing according to claim 1, characterized in that: The specific steps include: Step 1, preparing a chitosan-Fe3O4 dispersion: dissolving 3-5 parts of chitosan with a relative molecular weight of ≥100,000 and a degree of deacetylation of ≥90% in 100 parts of a 2% acetic acid solution to prepare a chitosan solution, adding 1-5 parts of aminosilane-modified Fe3O4, and ultrasonically dispersing at a power of 300-450 W and a frequency of 20-40 kHz for 20-30 minutes to obtain a chitosan-Fe3O4 dispersion; Step 2: Preparation of substrate pre-frozen solution: Dissolve 3-5 parts of gelatin with a relative molecular weight of ≥10,000 in 100 parts of deionized water at 60°C to prepare a gelatin solution, and mix 100 parts of chitosan-Fe3O4 dispersion with the gelatin solution to obtain a substrate pre-frozen solution; Step 3: Preparation of frozen substrate: The substrate pre-frozen solution was injected into a polytetrafluoroethylene mold with a copper microneedle array embedded in the bottom, with a liquid layer thickness of 2 mm. Gradual freezing was performed from -70°C to -20°C, with a cooling rate of 5°C / min, and the temperature was kept at 5°C for 30 minutes each. During the gradient freezing, a rotating magnetic field with an intensity of 50 mT and a rotation speed of 300 rpm was applied to obtain a freeze-molded substrate; Step 4: Electrospinning: Immerse the freeze-formed substrate in a PLGA solution for 1 to 2 hours. After immersion, hang the substrate vertically to drain for 30 to 60 seconds. Filter the immersed PLGA solution through a 0.22 μm filter membrane and magnetically stir for 10 to 20 minutes to obtain an electrospinning solution. Use the drained substrate as a receiving plate for three-layer gradient uniaxial electrospinning to obtain an electrospun membrane. Among them, the inner layer parameters of the three-layer gradient uniaxial electrospinning were set as follows: fiber diameter 100nm, electrospinning solution flow rate 0.3ml / h, voltage 16kv, collection distance 15cm, needle inner diameter 23G, and rotation speed 3600rpm; the middle layer parameters were set as follows: fiber diameter 300nm, electrospinning solution flow rate 0.1ml / h, voltage 17kv, collection distance 15cm, needle inner diameter 21G, and rotation speed 3300rpm; the outer layer parameters were set as follows: fiber diameter 500nm, electrospinning solution flow rate 0.1ml / h, voltage 17kv, collection distance 15cm, needle inner diameter 19G, and rotation speed 3000rpm; Step 5. Preparation of degradable electrospun dressing: After vacuum drying the electrospun membrane for 72 hours, place it in a 1% genipin solution for cross-linking for 2 days. After cross-linking is completed, wash it with 80% ethanol solution for 30 minutes and then rinse the residual ethanol solution with 0.9% NaCl solution. Vacuum dry it for 24 hours and sterilize it with ultraviolet radiation to obtain a degradable electrospun dressing.

9. A degradable electrospun dressing prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the degradable electrospun dressing according to claim 9 in skin repair.

Images