A biodegradable electrospun dressing

Abstract

This invention discloses a biodegradable electrospun dressing, belonging to the field of biomaterials technology. The invention involves dispersing aminosilane-modified Fe₃O₄ in a chitosan solution, adding a gelatin solution, stirring and mixing thoroughly, subjecting the mixture to gradient freezing under a rotating magnetic field, permeating with PLGA solution, electrospinning, crosslinking with genipin solution, washing, drying, and sterilization to obtain the biodegradable electrospun dressing. The biodegradable electrospun dressing prepared by this invention uses aminosilane-modified Fe₃O₄ to reinforce the chitosan-gelatin composite substrate, combined with magnetic field-assisted and gradient freezing molding, and undergoes three-layer gradient electrospinning to obtain the electrospun auxiliary material, constructing a biomimetic fiber structure. This achieves dynamic balance of wound exudation and long-lasting antibacterial effects, maintains optimal moisture balance at the wound site, and effectively promotes wound healing.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a biodegradable electrospun dressing. Background Technology

[0002] Modern wound dressings have evolved from traditional gauze into two main categories: interactive and bioactive. Interactive dressings, such as nanofiber membranes and hydrogels, accelerate healing by dynamically regulating wound moisture, antibacterial properties, and breathability. However, they have functional limitations—foam dressings are opaque and unsuitable for dry wounds, while hydrogels, with their high water content, struggle to cope with highly exudative wounds. Bioactive dressings, while capable of real-time monitoring and active intervention in the healing process, are too expensive due to integrated electronic components, limiting their clinical adoption. The current technological challenge lies in balancing exudate management: maintaining a moist environment containing growth factors while preventing excessive exudate from causing infection. This places higher demands on the design of the dressing's wetting gradient and its synergistic antibacterial / healing-promoting mechanisms.

[0003] Electrospinning technology offers a new approach to overcoming the aforementioned bottlenecks. The nanofiber membranes prepared using this technology can precisely mimic the fibrous structure of the natural extracellular matrix. By controlling the composite ratio of gelatin and chitosan, biomimetic scaffolds with both bioactivity and mechanical strength can be constructed. However, existing electrospun membranes still face problems such as limited wettability and insufficient interface control: in deep wounds, due to increased capillary permeability, nutrients and pathogens coexist in the exudate, making it difficult for traditional homogenized dressings to achieve targeted absorption of exudate and targeted release of antibacterial factors. Summary of the Invention

[0004] This invention discloses a biodegradable electrospun dressing. By modifying Fe3O4 with aminosilane to reinforce the chitosan-gelatin composite substrate, and combining magnetic field-assisted and gradient freeze-forming, the electrospun dressing is obtained through three-layer gradient electrospun, constructing a biomimetic fiber structure, achieving dynamic balance of wound exudation and long-lasting antibacterial effect, maintaining the optimal moisture balance at the wound site, and effectively promoting wound healing.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a method for preparing a biodegradable electrospun dressing, which involves dispersing aminosilane-modified Fe3O4 in a chitosan solution, adding a gelatin solution, stirring and mixing evenly, subjecting the mixture to gradient freezing under a rotating magnetic field, permeating it with a PLGA solution, electrospinning it, crosslinking it with a genipin solution, washing, drying, and sterilizing to obtain the biodegradable electrospun dressing.

[0007] As a further improvement to the present invention, the following steps are included:

[0008] Step 1: Preparation of chitosan-Fe3O4 dispersion: Chitosan is dissolved in acetic acid solution to prepare chitosan solution, and aminosilane-modified Fe3O4 is added to obtain chitosan-Fe3O4 dispersion;

[0009] Step 2, Preparation of substrate pre-freezing solution: Dissolve gelatin in deionized water to prepare gelatin solution, mix chitosan-Fe3O4 dispersion with gelatin solution to obtain substrate pre-freezing solution;

[0010] Step 3: Preparation of frozen substrate: The substrate pre-freezing liquid is injected into a polytetrafluoroethylene mold, and gradient freezing is performed. A rotating magnetic field is applied during gradient freezing to obtain a frozen substrate.

[0011] Step 4, Electrospinning process: The frozen-formed substrate is immersed in PLGA solution, the immersed substrate is drained, the immersed PLGA solution is filtered and stirred to obtain electrospinning solution, and the drained substrate is used as a receiving plate for electrospinning to obtain electrospinning film.

[0012] Step 5: Preparation of biodegradable electrospun dressing: After vacuum drying, the electrospun film is placed in a genipin solution for cross-linking. After cross-linking is completed, it is washed, dried, and sterilized to obtain biodegradable electrospun dressing.

[0013] As a further improvement of the present invention, in step one, 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, 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 the aminosilane-modified Fe3O4 includes the following steps:

[0014] (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 Fe3O4 to dopamine hydrochloride of 1:0.5~1. The mixture was stirred at room temperature for 12~24 h. After the reaction was completed, the product was collected by magnetic separation, washed three times with deionized water, and dried under vacuum to obtain dopamine-coated Fe3O4.

[0015] (2) Dopamine-coated Fe3O4 was dispersed in anhydrous ethanol. 3-Aminopropyltriethoxysilane was added at a volume ratio of anhydrous ethanol: 3-aminopropyltriethoxysilane = 10:1. The mixture was stirred at 60°C for 6 hours under nitrogen protection. The product was collected by magnetic separation. Unreacted silane was removed by washing with ethanol three times. The product was then dried under vacuum to obtain aminosilane-modified Fe3O4.

[0016] As a further improvement of the present invention, in step two, the temperature of the deionized water is 60°C, the mass ratio of gelatin, deionized water and chitosan-Fe3O4 dispersion is 3~5:100:100, and the relative molecular weight of gelatin is ≥10,000.

[0017] As a further improvement of the present invention, in step three, the thickness of the pre-freezing liquid layer in the polytetrafluoroethylene mold is 2 mm, the bottom of the polytetrafluoroethylene mold is embedded with a copper microneedle array, the strength of the rotating magnetic field is 50 mT and the rotation speed is 300 rpm, and the gradient freezing parameters are a gradient temperature change from -70℃ to -20℃, a cooling rate of 5℃ / min, and a temperature holding time of 30 min for every 5℃ gradient.

[0018] As a further improvement of the present invention, in step four, the concentration of the PLGA solution is 10%, and the PLGA solution contains LL-37 antimicrobial peptide at a concentration of 1 mg / mL.

[0019] Impregnation time is 1-2 hours. After impregnation, the substrate is hung vertically to drain for 30-60 seconds. The impregnated PLGA solution is filtered through a 0.22μm filter membrane and magnetically stirred for 10-20 minutes.

[0020] The electrospinning method is three-layer gradient uniaxial electrospinning. The inner layer parameters are set as follows: fiber diameter is 100nm, electrospinning solution flow rate is 0.3ml / h, voltage is 16kv, collection distance is 15cm, needle inner diameter is 23G, and rotation speed is 3600rpm.

[0021] The parameters for the middle layer 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.

[0022] 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.

[0023] As a further improvement of the present invention, in step five, the first vacuum drying time is 72 hours, the concentration of genipin solution is 1%, the cross-linking time is 2 days, the washing steps are as follows: wash with 80% ethanol solution for 30 minutes, then rinse with 0.9% NaCl solution to remove residual ethanol solution, the drying method is vacuum drying for 24 hours, and the sterilization method is ultraviolet radiation sterilization.

[0024] As a further improvement to the present invention, the specific steps include:

[0025] Step 1: Preparation of chitosan-Fe3O4 dispersion: Dissolve 3-5 parts of chitosan with a relative molecular weight ≥100,000 and a degree of deacetylation ≥90% in 100 parts of 2% acetic acid solution to prepare chitosan solution. Add 1-5 parts of aminosilane-modified Fe3O4 and ultrasonically disperse at a power of 300-450W and a frequency of 20-40kHz for 20-30 minutes to obtain chitosan-Fe3O4 dispersion.

[0026] Step 2: Preparation of substrate pre-freezing solution: Dissolve 3-5 parts of gelatin with a relative molecular weight ≥10,000 in 100 parts of deionized water at 60℃ to prepare a gelatin solution. Mix 100 parts of chitosan-Fe3O4 dispersion with the gelatin solution to obtain the substrate pre-freezing solution.

[0027] Step 3: Preparation of frozen substrate: The pre-freezing liquid of the substrate is injected into a polytetrafluoroethylene mold with a copper microneedle array embedded at the bottom. The liquid layer thickness is 2 mm. Gradual freezing from -70℃ to -20℃ is carried out, with a cooling rate of 5℃ / min and a temperature holding time of 30 min at every 5℃ gradient. At the same time as the gradient freezing, a rotating magnetic field with an intensity of 50 mT and a rotation speed of 300 rpm is applied to obtain the frozen substrate.

[0028] Step 4, Electrospinning process: The frozen-formed substrate is immersed in PLGA solution for 1-2 hours. After immersion, the substrate is hung vertically to drain for 30-60 seconds. The PLGA solution after immersion is filtered through a 0.22μm filter membrane and magnetically stirred for 10-20 minutes to obtain electrospinning solution. The drained substrate is used as a receiving plate for three-layer gradient uniaxial electrospinning to obtain an electrospinned membrane.

[0029] The parameters for the three-layer gradient uniaxial electrospinning were set as follows: inner layer: 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; middle layer: 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; outer layer: 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.

[0030] Step 5: Preparation of biodegradable electrospun dressing: After vacuum drying the electrospun film for 72 hours, it was placed in a 1% genipin solution for cross-linking for 2 days. After cross-linking, it was washed with 80% ethanol solution for 30 minutes, rinsed with 0.9% NaCl solution to remove residual ethanol solution, vacuum dried for 24 hours, and sterilized by ultraviolet irradiation to obtain biodegradable electrospun dressing.

[0031] The present invention further protects a biodegradable electrospun dressing prepared by the above-described preparation method.

[0032] This invention further protects the application of the above-mentioned biodegradable electrospun dressing in skin repair.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The structure-integrated but functionally graded 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, and improves fibrosis at the wound site; The structure-integrated but functionally graded 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 binding with cells or growth factors, thereby accelerating skin wound healing; The structure-integrated but functionally graded skin graft provided by the present invention has gradient wetting properties, which makes the dressing have good management ability for exudate at the wound site, avoids excessive drying and hydration, maintains the humidity required for wound healing, and promotes wound healing; The structure-integrated but functionally graded skin graft provided by the present invention has low cost and can be mass-produced, providing a new idea for the field of skin tissue engineering.

[0035] (2) In the preparation of aminosilane-modified Fe3O4, dopamine is first oxidized and polymerized to generate polydopamine in an alkaline environment of pH 8.5. The hydroxyl groups in the catechol structure form coordination bonds with Fe³⁺ / Fe²⁺ on the Fe3O4 surface, thus firmly coating the Fe3O4 surface and providing abundant amino and hydroxyl groups, providing active sites for the subsequent crosslinking of aminosilane. In the subsequent reaction, the ethoxy group of 3-aminopropyltriethoxysilane is hydrolyzed in anhydrous ethanol to silanol (-Si-OH). The silanol undergoes condensation and dehydration reaction with the hydroxyl or amino groups of polydopamine coated on the Fe3O4 surface to form Si-OC or Si-N covalent bonds, thereby fixing the aminosilane on the Fe3O4 surface, making the Fe3O4 surface have amino groups, and enhancing the surface activity, biocompatibility and dispersion stability of Fe3O4.

[0036] (3) In this invention, an amino-functionalized layer is formed on the surface of Fe3O4 by dopamine coating and silanization treatment, which significantly improves its dispersibility in chitosan / gelatin matrix and enhances its chemical bonding ability with PLGA fibers. The amino-silane modified Fe3O4 forms a directional magnetic domain structure under the induction of a rotating magnetic field, which can generate a local magnetic field effect, inhibit the formation of Staphylococcus aureus biofilm, and accelerate local blood circulation in the wound through 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.

[0037] (4) In the preparation process of the frozen substrate, the present invention uses a gradient freezing of -70℃ to -20℃ combined with a copper microneedle array to form a multi-level porous structure, which simulates the fibrous topology of natural skin ECM. The rotating magnetic field synchronously controls 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.

[0038] (5) This invention employs a three-layer gradient uniaxial electrospinning structure during the electrospinning process. The inner layer consists of 100 nm diameter fibers, which use a high-density nano-network to provide a sustained-release carrier for the antimicrobial peptide LL-37, effectively inhibiting the proliferation of Pseudomonas aeruginosa. The middle layer consists of 300 nm diameter fibers, which use mesoscale fiber interlacing to form a mechanical buffer layer with an elastic modulus that matches the dermis, reducing shear damage to the wound surface caused by movement. The outer layer consists of 500 nm diameter fibers, which use a large-pore structure to achieve directional absorption of exudate and form a pH-responsive surface through genipin crosslinking, achieving dynamic response to wound exudate. The gradient crosslinking density design achieves a gradient transition in mechanical properties, with the compressive modulus gradually decreasing from the surface to the bottom layer, adapting to the stress distribution during wound contraction.

[0039] (6) In this invention, when a substrate is used as a receiving plate, the copper microneedle array induces PLGA fibers to form a vertical orientation structure, promotes the migration of fibroblasts along the fiber direction, and accelerates wound healing.

[0040] (7) This technology achieves a synergistic improvement in the mechanical properties, antibacterial activity and tissue regeneration capacity of dressings through the directional assembly of magnetic nanoparticles and the biomimetic gradient electrospinning structure, providing a new clinically applicable solution for chronic wound management. Attached Figure Description

[0041] Figure 1 This is a SEM image of the inner layer of the electrospun membrane in Example 1;

[0042] Figure 2 This is a SEM image of the middle layer of the electrospun film in Example 1;

[0043] Figure 3This is a SEM image of the outer layer of the electrospun membrane in Example 1;

[0044] Figure 4 This is a SEM image of the inner layer of the biodegradable electrospun dressing in Example 1;

[0045] Figure 5 This is a SEM image of the middle layer of the biodegradable electrospun dressing in Example 1;

[0046] Figure 6 This is a SEM image of the outer layer of the biodegradable electrospun dressing in Example 1;

[0047] Figure 7 This is a SEM image of the cross-section of the biodegradable electrospun dressing in Example 1;

[0048] Figure 8 The graph shows the degradation performance test results of the biodegradable electrospun dressing in Example 1, where A100 is the inner layer fiber, A300 is the middle layer fiber, and A500 is the outer layer fiber.

[0049] Figure 9 Digital photographs of the skin wounds of mice in the group of Example 1 in Test Example 3;

[0050] Figure 10 These are digital photographs of the skin wounds of control group 1 mice in test case 3;

[0051] Figure 11 These are digital photographs of the skin wounds of control group 2 mice in test case 3;

[0052] Figure 12 These are digital photographs of the skin wounds of control group 3 mice in test case 3;

[0053] Figure 13 Digital photographs of skin wounds in mice of the blank control group were taken in Test Example 3. Detailed Implementation

[0054] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0055] The chitosan used in the embodiments of this application is from Zhejiang Jinke Pharmaceutical Co., Ltd.

[0056] Example 1: This example describes the preparation of a biodegradable electrospun dressing.

[0057] Step 1: Preparation of chitosan-Fe3O4 dispersion: Dissolve 3 parts of chitosan with a relative molecular weight ≥100,000 and a degree of deacetylation ≥90% in 100 parts of 2% acetic acid solution to prepare chitosan solution, add 5 parts of aminosilane modified Fe3O4, and ultrasonically disperse at a power of 300W and a frequency of 20kHz for 30min to obtain chitosan-Fe3O4 dispersion;

[0058] Step 2, Preparation of substrate pre-freezing solution: Dissolve 5 parts of gelatin with a relative molecular weight ≥10,000 in 100 parts of deionized water at 60℃ to prepare a gelatin solution. Mix 100 parts of chitosan-Fe3O4 dispersion with the gelatin solution to obtain the substrate pre-freezing solution.

[0059] Step 3: Preparation of frozen substrate: The pre-freezing liquid of the substrate is injected into a polytetrafluoroethylene mold with a copper microneedle array embedded at the bottom. The liquid layer thickness is 2 mm. Gradual freezing from -70℃ to -20℃ is carried out, with a cooling rate of 5℃ / min and a temperature holding time of 30 min at every 5℃ gradient. At the same time as the gradient freezing, a rotating magnetic field with an intensity of 50 mT and a rotation speed of 300 rpm is applied to obtain the frozen substrate.

[0060] Step 4, Electrospinning process: The frozen-formed substrate is immersed in PLGA solution for 2 hours. After immersion, the substrate is hung vertically to drain for 60 seconds. The PLGA solution after immersion is filtered through a 0.22μm filter membrane and magnetically stirred for 20 minutes to obtain electrospinning solution. The drained substrate is used as a receiving plate for three-layer gradient uniaxial electrospinning to obtain an electrospinned membrane.

[0061] The parameters for the three-layer gradient uniaxial electrospinning were set as follows: inner layer: 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; middle layer: 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; outer layer: 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.

[0062] Step 5: Preparation of biodegradable electrospun dressing: After vacuum drying the electrospun film for 72 hours, it was placed in a 1% genipin solution for cross-linking for 2 days. After cross-linking, it was washed with 80% ethanol solution for 30 minutes, rinsed with 0.9% NaCl solution to remove residual ethanol solution, vacuum dried for 24 hours, and sterilized by ultraviolet irradiation to obtain biodegradable electrospun dressing.

[0063] The preparation method of the aminosilane-modified Fe3O4 includes the following steps:

[0064] (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 Fe3O4 to dopamine hydrochloride of 1:1. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the product was collected by magnetic separation, washed three times with deionized water, and dried under vacuum to obtain dopamine-coated Fe3O4.

[0065] (2) Dopamine-coated Fe3O4 was dispersed in anhydrous ethanol. 3-Aminopropyltriethoxysilane was added at a volume ratio of anhydrous ethanol: 3-aminopropyltriethoxysilane = 10:1. The mixture was stirred at 60°C for 6 hours under nitrogen protection. The product was collected by magnetic separation. Unreacted silane was removed by washing with ethanol three times. The product was then dried under vacuum to obtain aminosilane-modified Fe3O4.

[0066] The electrospun dressing prepared in this embodiment has a tensile strength of 4.52 N, good biocompatibility, and a suitable degradation rate. When used as a skin graft, significant collagen deposition and hair follicle regeneration are observed on day 14 of skin wound healing, and fibrosis at the wound site is improved to some extent.

[0067] Example 2: This example describes the preparation of a biodegradable electrospun dressing.

[0068] Step 1: Preparation of chitosan-Fe3O4 dispersion: Dissolve 3 parts of chitosan with a relative molecular weight ≥100,000 and a degree of deacetylation ≥90% in 100 parts of 2% acetic acid solution to prepare chitosan solution. Add 1 part of aminosilane-modified Fe3O4 and ultrasonically disperse at a power of 450W and a frequency of 40kHz for 20min to obtain chitosan-Fe3O4 dispersion.

[0069] Step 2, Preparation of substrate pre-freezing solution: Dissolve 3 parts of gelatin with a relative molecular weight ≥10,000 in 100 parts of deionized water at 60℃ to prepare a gelatin solution. Mix 100 parts of chitosan-Fe3O4 dispersion with the gelatin solution to obtain the substrate pre-freezing solution.

[0070] Step 3: Preparation of frozen substrate: The pre-freezing liquid of the substrate is injected into a polytetrafluoroethylene mold with a copper microneedle array embedded at the bottom. The liquid layer thickness is 2 mm. Gradual freezing from -70℃ to -20℃ is carried out, with a cooling rate of 5℃ / min and a temperature holding time of 30 min at every 5℃ gradient. At the same time as the gradient freezing, a rotating magnetic field with an intensity of 50 mT and a rotation speed of 300 rpm is applied to obtain the frozen substrate.

[0071] Step 4, Electrospinning process: The frozen-formed substrate is immersed in PLGA solution for 1 hour. After immersion, the substrate is hung vertically to drain for 30 seconds. The PLGA solution after immersion is filtered through a 0.22μm filter membrane and magnetically stirred for 10 minutes to obtain electrospinning solution. The drained substrate is used as a receiving plate for three-layer gradient uniaxial electrospinning to obtain an electrospinned membrane.

[0072] The parameters for the three-layer gradient uniaxial electrospinning were set as follows: inner layer: 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; middle layer: 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; outer layer: 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.

[0073] Step 5: Preparation of biodegradable electrospun dressing: After vacuum drying the electrospun film for 72 hours, it was placed in a 1% genipin solution for cross-linking for 2 days. After cross-linking, it was washed with 80% ethanol solution for 30 minutes, rinsed with 0.9% NaCl solution to remove residual ethanol solution, vacuum dried for 24 hours, and sterilized by ultraviolet irradiation to obtain biodegradable electrospun dressing.

[0074] The preparation method of the aminosilane-modified Fe3O4 includes the following steps:

[0075] (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 Fe3O4 to dopamine hydrochloride of 1:1. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the product was collected by magnetic separation, washed three times with deionized water, and dried under vacuum to obtain dopamine-coated Fe3O4.

[0076] (2) Dopamine-coated Fe3O4 was dispersed in anhydrous ethanol. 3-Aminopropyltriethoxysilane was added at a volume ratio of anhydrous ethanol: 3-aminopropyltriethoxysilane = 10:1. The mixture was stirred at 60°C for 6 hours under nitrogen protection. The product was collected by magnetic separation. Unreacted silane was removed by washing with ethanol three times. The product was then dried under vacuum to obtain aminosilane-modified Fe3O4.

[0077] Comparative Example 1: An electrospun dressing was prepared in this comparative example.

[0078] This embodiment does not perform three-layer gradient uniaxial electrospinning, but directly performs single-layer uniaxial electrospinning. The parameters are set using the middle layer parameters of Example 1: fiber diameter of 300nm, electrospinning solution flow rate of 0.1ml / h, voltage of 17kV, collection distance of 15cm, needle inner diameter of 21G, and rotation speed of 3300rpm; other steps are the same as in Example 1.

[0079] Comparative Example 2: An electrospun dressing was prepared in this comparative example.

[0080] In this embodiment, instead of reacting Fe3O4, nanoparticle dopamine hydrochloride, and 3-aminopropyltriethoxysilane to generate aminosilane-modified Fe3O4, Fe3O4, nanoparticle dopamine hydrochloride, and 3-aminopropyltriethoxysilane are directly added. The mass ratio of Fe3O4, nanoparticle dopamine hydrochloride, and 3-aminopropyltriethoxysilane is the same as in Example 1; other steps are the same as in Example 1.

[0081] Comparative Example 3: An electrospun dressing was prepared in this comparative example.

[0082] In this embodiment, instead of applying a rotating magnetic field during gradient freezing, a rotating magnetic field is applied for 5 hours first, followed by gradient freezing; the other steps are the same as in Example 1.

[0083] Measurement example 1

[0084] The cross-sectional structure of the electrospun membrane prepared in step four of Example 1 and the biodegradable electrospun dressing prepared in step five were observed using field emission scanning electron microscopy, and the fiber diameter distribution of the inner, middle and outer layers was statistically analyzed.

[0085] Analysis: For example Figures 1-3 As shown, the nanofibers in the inner, middle, and outer layers of the electrospun membrane prepared in step four of Example 1 all exhibit a smooth, continuous, and bead-free uniform morphology, and display an ordered structure. All groups show an approximately normal uniform distribution with a certain degree of orientation. Figures 4-6 As can be seen, after the crosslinking operation in step five, the diameter of the nanofibers in the biodegradable electrospun dressing prepared in step five increases to some 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; For example... Figure 7 As shown, the three layers of fibers in the biodegradable electrospun dressing of Example 1 are tightly bonded together, without any porosity or uneven bonding; SEM results show that, compared to... Figures 1-3It is known that electrospinning parameters have a significant impact on the diameter of the electrospinned membrane fibers produced, and there is no specific pattern. After conducting numerous experiments, the applicant has derived the relevant electrospinning parameters for this application, which can precisely control the diameter of the electrospinned nanofibers and adjust the porosity of the electrospinning scaffold, thereby achieving the technical effect of the technical solution of this application.

[0086] Test Example 2

[0087] The biodegradability of the electrospun dressing in Example 1 was tested by incubating it in simulated body fluid at 37°C in an incubator for 14 days, and the weight loss was measured every 7 days.

[0088] Analysis: From Figure 8 The percentage of weight loss of each layer in the biodegradable electrospun dressing was observed on day 7 (inner layer: 79.06±4.15%, middle layer: 80.6±7.33%, outer layer: 80.93±3.68%, substrate: 85.16±4.48%) and day 14 (inner layer: 65.51±2.04%, middle layer: 68.88±1.35%, outer layer: 72.4±2.50%, substrate: 74.56±3.37%). The biodegradable electrospun dressing of the present invention degrades faster in the first 7 days than in the last 7 days because the gelatin content is higher in the initial stage. Gelatin degrades faster than chitosan, while the relatively slow-degrading chitosan helps maintain the integrity of the dressing throughout the regeneration process. The release of gelatin can promote cell adhesion and tissue regeneration, which helps the wound healing process.

[0089] Test Example 3

[0090] Eight-week-old C57BL / 6J mice, weighing approximately 20g, were housed in ventilated cages lined with corn bedding. The animals had free access to food and water. The C57BL / 6J mice were intraperitoneally anesthetized, and their dorsal hair was removed. A circular, full-thickness skin defect with a diameter of 6mm was created and fixed with a silicone ring (6mm inner diameter, 8mm outer diameter) to prevent back muscle contraction and wound closure, thus objectively evaluating the crawling healing ability of the skin wound. The biodegradable electrospun dressing from Example 1 (outer layer facing upwards, inner layer facing the wound tissue, uniformly sized as small round pieces with a diameter of 7mm) sterilized by irradiation was smoothly sutured into the skin defect. A wound without dressing served as a blank control group, and the electrospun dressings prepared in Comparative Examples 1-3 served as control groups 1, 2, and 3, respectively. The skin wound was covered with 3M Tegaderm transparent wound dressing and fixed with self-adhesive elastic bandages. Images of the wound area were recorded using a digital camera at predetermined postoperative times (5 days, 7 days, and 14 days).

[0091] Analysis: From Figures 9-13As can be seen, the skin wounds of mice in Example 1 and all control groups were basically healed. However, compared with the control groups, Example 1 had a better healing effect, the smallest scar area, and a more obvious hair follicle regeneration effect than other groups. It can be seen that the technical solution 1 of Example 1 of this application has good biosafety. In the control groups, control group 1 showed the worst effect because it did not undergo three-layer gradient uniaxial electrospinning, thus failing to form a gradient structure and therefore lacking the three-layer gradient effect of the present application, resulting in the worst wound repair effect. Compared to control group 1, control group 2 did not generate aminosilane-modified Fe3O4, resulting in no amino-functionalized layer on the Fe3O4 surface. This led to poor dispersion in the chitosan / gelatin matrix and relatively weak chemical bonding with PLGA fibers, resulting in a weaker local magnetic field effect, reduced antibacterial effect, and poor acceleration of local blood circulation, thus slowing down wound healing. Compared to example 1, control group 3 did not simultaneously apply gradient freezing and rotating magnetic field, but rather performed them in steps without synchronously controlling the ice crystal growth direction. This prevented the radially arranged nanofiber bundles formed by the chitosan / gelatin matrix from being adjusted according to the dressing pores, limiting the increase in tensile strength and thus failing to effectively adhere to the skin surface, weakening the effect and resulting in poor wound healing.

[0092] In summary, the technical solution of this application has a significant effect on promoting the healing of biological skin wounds and exhibits good degradation performance. During the preparation of the biodegradable electrospun dressing of this application, the applicant discovered that the electrospun parameters have a significant impact on the diameter of the electrospun membrane fibers, and there is no specific pattern. After conducting numerous experiments, the applicant derived the relevant electrospun parameters for this application, which can precisely control the diameter of the electrospun nanofibers and adjust the porosity of the electrospun scaffold, thereby achieving the technical effect of the solution of this application. By modifying Fe3O4 with an amino-functionalized layer on its surface, the biodegradability of the chitosan / gelatin matrix is ​​significantly improved. The dispersibility and enhanced chemical bonding with PLGA fibers, along with the local magnetic field generated by the uniformly dispersed Fe3O4 in the dressing, significantly improve the antibacterial effect and accelerate local blood circulation in the wound. This prevents the invasion of external bacteria while accelerating the replenishment of internal nutrients, thereby speeding up wound healing. The simultaneous application of gradient freezing and a rotating magnetic field simultaneously regulates the direction of ice crystal growth during freezing. The radially arranged nanofiber bundles formed by the chitosan / gelatin substrate are adjusted according to the porosity of the dressing, significantly improving tensile strength and allowing for full adhesion to the skin surface, thus greatly promoting wound healing.

[0093] Although 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 can 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 patent coverage of the present invention.

Claims

1. A method for preparing a biodegradable electrospun dressing, characterized in that, Includes the following steps: Step 1: Preparation of chitosan-Fe3O4 dispersion: Chitosan is dissolved in acetic acid solution to prepare chitosan solution, and aminosilane-modified Fe3O4 is added to obtain chitosan-Fe3O4 dispersion; Step 2, Preparation of substrate pre-freezing solution: Dissolve gelatin in deionized water to prepare gelatin solution, mix chitosan-Fe3O4 dispersion with gelatin solution to obtain substrate pre-freezing solution; Step 3: Preparation of frozen substrate: The substrate pre-freezing liquid is injected into a polytetrafluoroethylene mold, and gradient freezing is performed. A rotating magnetic field is applied during gradient freezing to obtain a frozen substrate. Step 4, Electrospinning process: The frozen-formed substrate is immersed in PLGA solution, the immersed substrate is drained, the immersed PLGA solution is filtered and stirred to obtain electrospinning solution, and the drained substrate is used as a receiving plate for electrospinning to obtain electrospinning film. Step 5: Preparation of biodegradable electrospun dressing: After vacuum drying, the electrospun film is placed in a genipin solution for cross-linking. After cross-linking is completed, it is washed, dried, and sterilized to obtain biodegradable electrospun dressing. In step one, 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 the aminosilane-modified Fe3O4 includes 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 Fe3O4 to dopamine hydrochloride of 1:0.5~1. The mixture was stirred at room temperature for 12~24 h. After the reaction was completed, the product was collected by magnetic separation, washed three times with deionized water, and dried under vacuum to obtain dopamine-coated Fe3O4. (2) Dopamine-coated Fe3O4 was dispersed in anhydrous ethanol. 3-aminopropyltriethoxysilane was added at a volume ratio of anhydrous ethanol: 3-aminopropyltriethoxysilane = 10:

1. The mixture was stirred at 60°C for 6 hours under nitrogen protection. The product was collected by magnetic separation. Unreacted silane was removed by washing with ethanol three times. The product was dried under vacuum to obtain aminosilane-modified Fe3O4. In step two, the temperature of the deionized water is 60°C, the mass ratio of gelatin, deionized water and chitosan-Fe3O4 dispersion is 3~5:100:100, and the relative molecular weight of gelatin is ≥10,000. In step three, the thickness of the pre-freezing liquid layer in the polytetrafluoroethylene mold is 2 mm. The bottom of the polytetrafluoroethylene mold is embedded with a copper microneedle array. The strength of the rotating magnetic field is 50 mT and the rotation speed is 300 rpm. The gradient freezing parameters are a temperature gradient from -70℃ to -20℃, a cooling rate of 5℃ / min, and a holding time of 30 min for every 5℃ gradient. In step four, 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 impregnation time is 1–2 hours; after impregnation, the substrate is vertically suspended to drain for 30–60 seconds; the impregnated PLGA solution is filtered through a 0.22 μm filter membrane and magnetically stirred for 10–20 minutes; the electrospinning method is three-layer gradient uniaxial electrospinning, with the inner layer parameters set as follows: fiber diameter 100 nm, electrospinning solution flow rate 0.3 ml / h. The voltage was 16 kV, the collection distance was 15 cm, the needle inner diameter was 23 G, and the rotation speed was 3600 rpm. The middle layer parameters were set as follows: fiber diameter was 300 nm, electrospinning solution flow rate was 0.1 ml / h, voltage was 17 kV, the collection distance was 15 cm, the needle inner diameter was 21 G, and the rotation speed was 3300 rpm. The outer layer parameters were set as follows: fiber diameter was 500 nm, electrospinning solution flow rate was 0.1 ml / h, voltage was 17 kV, the collection distance was 15 cm, the needle inner diameter was 19 G, and the rotation speed was 3000 rpm.

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

3. The method for preparing a biodegradable electrospun dressing according to claim 1, characterized in that, Specifically, the following steps are included: Step 1: Preparation of chitosan-Fe3O4 dispersion: Dissolve 3-5 parts of chitosan with a relative molecular weight ≥100,000 and a degree of deacetylation ≥90% in 100 parts of 2% acetic acid solution to prepare chitosan solution. Add 1-5 parts of aminosilane-modified Fe3O4 and ultrasonically disperse at a power of 300-450W and a frequency of 20-40kHz for 20-30 minutes to obtain chitosan-Fe3O4 dispersion. Step 2: Preparation of substrate pre-freezing solution: Dissolve 3-5 parts of gelatin with a relative molecular weight ≥10,000 in 100 parts of deionized water at 60℃ to prepare a gelatin solution. Mix 100 parts of chitosan-Fe3O4 dispersion with the gelatin solution to obtain the substrate pre-freezing solution. Step 3: Preparation of frozen substrate: The pre-freezing liquid of the substrate is injected into a polytetrafluoroethylene mold with a copper microneedle array embedded at the bottom. The liquid layer thickness is 2 mm. Gradual freezing from -70℃ to -20℃ is carried out, with a cooling rate of 5℃ / min and a temperature holding time of 30 min at every 5℃ gradient. At the same time as the gradient freezing, a rotating magnetic field with an intensity of 50 mT and a rotation speed of 300 rpm is applied to obtain the frozen substrate. Step 4, Electrospinning process: The frozen-formed substrate is immersed in PLGA solution for 1-2 hours. After immersion, the substrate is hung vertically to drain for 30-60 seconds. The PLGA solution after immersion is filtered through a 0.22μm filter membrane and magnetically stirred for 10-20 minutes to obtain electrospinning solution. The drained substrate is used as a receiving plate for three-layer gradient uniaxial electrospinning to obtain an electrospinned membrane. The parameters for the three-layer gradient uniaxial electrospinning were set as follows: inner layer: 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; middle layer: 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; outer layer: 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. Step 5: Preparation of biodegradable electrospun dressing: After vacuum drying the electrospun film for 72 hours, it was placed in a 1% genipin solution for crosslinking for 2 days. After crosslinking, it was washed with 80% ethanol solution for 30 minutes, then rinsed with 0.9% NaCl solution to remove residual ethanol solution. After vacuum drying for 24 hours, it was sterilized by ultraviolet irradiation to obtain biodegradable electrospun dressing.

4. A biodegradable electrospun dressing prepared by the method described in any one of claims 1 to 3.

5. The application of the biodegradable electrospun dressing as described in claim 4 in the preparation of skin repair materials.

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