Preparation method of biofilm with bionic cuticle'brick-slurry 'structure and application of biofilm in wound dressing

Abstract

The invention relates to a preparation method of a biological membrane with a bionic cuticle'brick-slurry 'structure and a wound dressing application of the biological membrane, and belongs to the technical field of biological auxiliary materials. The preparation method comprises the following steps: dispersing prepared wool keratin microspheres in a fibroin solution, realizing a stable and firm brick-slurry structure by combining solvent volatilization with a photo-crosslinking technology, further crosslinking by utilizing metal ion coordination, and simultaneously endowing the dressing with the capabilities of effectively blocking bacterial permeation and sterilizing. The biological membrane exceeds the design of traditional layered wound dressings, and accurate reproduction of a natural skin structure is preferentially considered. The microspheres are assembled into a densely-arranged masonry structure material in a planned manner, so that the dressing has high moisture permeability, oxygen permeability and strong antibacterial barrier capacity, can promote repair of infectious wounds and burn and scald wounds, and has remarkable clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing keratin artificial skin with a biomimetic stratum corneum microstructure. This material is suitable for infected wounds and burn wounds. Background Technology

[0002] As a vital physical barrier against the external environment, the skin is characterized by a complex three-dimensional structure composed of interconnected cells. At the heart of this protective function is the stratum corneum, the outermost layer of the epidermis, which possesses a unique "brick and mortar" structure. Corneocytes (the "bricks") are embedded within a layered lipid matrix (the "mortar"), forming a dense and semi-permeable barrier that effectively restricts pathogen invasion and prevents excessive moisture loss. Reproducing this structure has become a key challenge in biomimetic material design, especially in advanced dressings designed to promote skin function recovery during wound healing.

[0003] An ideal wound dressing should effectively meet conflicting requirements: it must prevent pathogen invasion, facilitate gas exchange and moisture retention, and prevent fluid buildup. Existing dressings are based on different pore sizes and porosities, with small, dense pores mimicking the epidermis and large, loose pores mimicking the dermis. However, these porous designs, intended to control pore size and density to regulate permeability, inherently reduce barrier effectiveness. Therefore, this invention focuses on overcoming the limitations of traditional dressings with merely layered porous structures, fundamentally resolving the core contradiction between barrier function and water / oxygen permeability.

[0004] To address the aforementioned problems, this invention designs a biomembrane with a biomimetic stratum corneum "brick-mortar" structure, which is applied to wound dressings. This membrane material not only replicates the barrier function of the skin's stratum corneum against environmental pollutants but also regulates the transport of water and oxygen. Through the complexation of keratin with metal ions, the dressing is further functionalized to possess strong antibacterial properties, effectively preventing bacteria from invading the wound site and inhibiting bacterial growth there. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing membrane wound dressings and, by combining the material and structural advantages of natural skin, provide a method for preparing a membrane wound dressing based on wool keratin with a biomimetic "brick-mortar" structure. The resulting membrane wound dressing utilizes keratin microspheres ("bricks") and lipids and silk fibroin ("mortar") to effectively prevent pathogen infiltration while simultaneously achieving optimal water and oxygen exchange.

[0006] To achieve the above objectives, the technical solution used in this invention is as follows: A method for preparing a biofilm with a biomimetic cuticle "brick-mortar" structure, the method comprising the following steps: 1) Keratin microspheres KM were obtained by covalent cross-linking wool keratin in a water-in-oil emulsion using the emulsion method. Then, the thiol groups of KM were activated by a reducing agent and modified by methacrylamide to obtain keratin microspheres MKM with photocrosslinking activity. 2) Silk fibroin was modified by methacrylation to obtain silk fibroin MSF with photocrosslinking activity; 3) MKM is fully dispersed in a solution containing silk fibroin MSF with photocrosslinking activity, transferred to a mold, and evaporated to constant weight. Then, ultraviolet light is used to initiate film crosslinking to obtain a KSF biofilm with a "brick-mortar" structure.

[0007] Furthermore, the preparation method also includes fully immersing the KSF biofilm in a solution containing metal ions to form a metal ion-keratin complex, thereby obtaining an antibacterial KSF biofilm.

[0008] Further, in step 1), the emulsion method specifically involves: preparing an aqueous solution of wool keratin as the aqueous phase, adding an oil phase containing a surfactant, emulsifying for at least 10 minutes, and then adding a crosslinking agent to react and obtain KM; The wool keratin aqueous solution contains wool keratin with a molecular weight of 10-55 kDa and a concentration of 100-500 mg / mL; the oil phase is any one of vegetable oil, dichloromethane, chloroform, and liquid paraffin; the surfactant is any one of Span 20, Span 60, Span 80, lecithin, and ceramide, and the surfactant content is 0.02-0.5 wt% of the oil phase mass; the crosslinking agent is any one of glutaraldehyde, paraformaldehyde, genipin, and tyrosinase.

[0009] Further, in step 1), the specific process of activating KM with the reducing agent is as follows: KM and the reducing agent solution are co-incubated at a ratio of 1g:40mL; The reducing agent is any one of tris(2-carboxyethyl)phosphonic acid hydrochloride, mercaptoethanol, sodium sulfide, and sodium thiosulfate, and the concentration of the reducing agent solution is 20-70 mmol / L; The specific process of methacrylylation modification of activated KM is as follows: the activated KM is placed in a reaction solution containing a methacrylylating agent and reacted fully to obtain MKM. The methacrylylating agent of KM is methacrylic anhydride, glycidyl methacrylate, 3-methacrylamide dopamine or trimethylsilane methacrylate, and the concentration of the reaction solution containing the methacrylylating agent is 0.1-1 mol / L.

[0010] Further, in step 2), the silk fibroin is modified by methacrylation, specifically by mixing the silk fibroin with a reaction solution containing methacrylation reagent at a ratio of 1g:10mL and reacting fully to obtain MSF; The methacrylating agent for silk fibroin is any one of methacrylic anhydride, glycidyl methacrylate, 3-methacrylamide dopamine, and trimethylsilane methacrylate, and the concentration of the reaction solution of the methacrylating agent for silk fibroin is 0.5-2 mol / L.

[0011] Further, in step 3), the solution to be volatilized contains silk fibroin, a humectant, and a photoinitiator; the humectant is any one of glycerin, propylene glycol, urea, and sodium L-pyrrolidone-5-carboxylate; the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone or lithium phenyl(2,4,6-trimethylbenzoyl)phosphate; in the solution to be volatilized, the concentration of silk fibroin is 1-10 mg / mL, and the concentration of the humectant is 2-15 wt%.

[0012] Furthermore, to impart antibacterial properties to KSF, KSF is fully immersed in a solution containing metal ions, causing keratin to complex with the metal ions. The metal ions are Ag. + Cu 2+ Zn 2+ Fe 3+ Zr 4+ The concentration of metal ions in the solution is 0.01-0.2 mol / L, the complexation time is 1-6 hours, and the complexation temperature is 10-40℃.

[0013] Further, in step 1), the reaction temperature of the emulsion method is 10-60℃, the emulsification speed is 400-2000 r / min, and the crosslinking time of the crosslinking agent is 1-12 hours; The conditions for activating KM with a reducing agent are: pH 7-8, reaction time 2-20 minutes, and reaction temperature 20-40℃; the conditions for methacrylylation modification of activated KM are: reaction time 4-12 hours, reaction temperature 10-60℃, and stirring speed 100-300 r / min. In step 2), the reaction time for the methacrylation modification of silk fibroin is 3-12 hours, the reaction temperature is 50-70℃, and the stirring speed is 200-500 r / min. In step 3), the evaporation temperature is 25-60℃ and the UV treatment time is 1-30 min.

[0014] A biomembrane with a biomimetic cuticle "brick-mortar" structure was prepared by the above-described method.

[0015] The aforementioned biofilm is used in wound dressings and is suitable for infected wounds and burn wounds.

[0016] The biofilm prepared by the system, with a biomimetic keratin "brick-mortar" structure, can be used to promote wound healing.

[0017] Compared with the prior art, the present invention has the following advantages: 1) This invention uses wool keratin as the main raw material to prepare wound dressings. As a protein homologous to the main components of the outer layer of skin, it has extremely low immunogenicity, excellent biocompatibility, and is biodegradable. Wool keratin contains abundant cell-binding motifs, such as leucine-aspartic-valine (LDV) and arginine-glycine-aspartic (RGD). These motifs can promote cell adhesion and proliferation, while upregulating the expression of type IV and VII collagen, thus positively impacting wound repair. Furthermore, reusing waste hair not only reduces material costs but also aligns with green and sustainable principles. However, the high cysteine ​​content in WK promotes extensive intra- and inter-chain disulfide bond formation, leading to poor water solubility. In addition, its predominant α-helical conformation often results in poor material processing performance, mechanical fragility, and brittleness. This invention improves the processing properties of wool keratin by controlling the molecular weight of keratin to regulate its water solubility, breaking the disulfide bonds of keratin with a reducing agent, and introducing a thiol group as a nucleophilic reaction site to a methacryloyl functional group. This is the material innovation of this invention.

[0018] 2) This invention cleverly mimics the "brick-and-mortar" structure of the natural stratum corneum from bottom to top in the biofilm wound dressing, where wool keratin microspheres are the "bricks" and silk fibroin is the "mortar." Traditional biomimetic skin dressings partially mimic skin structure; these materials typically have different pore sizes and porosities, with one layer of small, densely packed pores mimicking the epidermis, and another layer of large, loosely packed pores replicating the dermis. However, these porous designs, intended to regulate permeability by controlling pore size and density, inherently weaken barrier function. Interconnected pores facilitate bacterial penetration and fail to replicate the impermeable but dynamic exchange capacity of the skin barrier layer. Although porous dressings can absorb exudate, they often lead to dehydration or erosion due to imbalanced moisture regulation. Conversely, completely closed dressings may cause exudate buildup, exacerbating inflammation and delaying healing. The advantage of the metal-coordinated wool keratin composite membrane wound dressing with a biomimetic "brick-mortar" structure prepared in this invention is that it has similar gas and liquid exchange capabilities to the epidermis, with an oxygen permeability of 361.0 mg / m³. -2 Day 1, water vapor permeability 1879.9 gm -2 day -1 It is 2.8 times that of the commercial membrane dressing Tegaderm™, which is the structural innovation of this invention.

[0019] In summary, the biomembrane prepared by this invention, with a biomimetic stratum corneum "brick-and-mortar" structure, replicates the skin's stratum corneum barrier function against bacteria while regulating vapor and oxygen transport. Furthermore, this composite membrane exhibits good cell compatibility, effective antibacterial activity and anti-inflammatory effects, and can promote the healing of infected and scalded wounds. Beyond its efficacy, the fully biodegradable components align with eco-conscious medical practices, while the simple manufacturing process ensures the feasibility of commercialization. This invention innovatively combines the biomimetic structure of natural skin with multifunctional therapeutic capabilities, harmonizing biofidelity with engineering precision, representing a significant paradigm shift in wound management strategies. This design transcends traditional methods, not only resolving the core contradiction between barrier and permeability but also demonstrating significantly superior overall performance compared to existing commercial products. It lays the foundation for advanced wound management and novel biomimetic structural materials, possessing broad application prospects. Attached Figure Description

[0020] The accompanying drawings for this invention are as follows: Figure 1 This is an image of a biofilm with a biomimetic cuticle "brick-mortar" structure.

[0021] Figure 2 Micrograph of a biofilm with a biomimetic cuticle "brick-and-mortar" structure loaded with copper ions.

[0022] Figure 3 Cu in biofilms with a biomimetic cuticle "brick-mortar" structure loaded with copper ions 2+ Cumulative release rate curve.

[0023] Figure 4 The barrier function of a biofilm with a biomimetic cuticle "brick-mortar" structure loaded with copper ions against Staphylococcus aureus.

[0024] Figure 5 The image shows the therapeutic effects of a biofilm with a biomimetic keratin "brick-mortar" structure loaded with copper ions on bacterial wounds, compared to commercially available wound dressings and gauze.

[0025] Figure 6 The image shows the therapeutic effects of a biomimetic keratin "brick-mortar" structure loaded with copper ions on burn wounds, compared to commercially available wound dressings and gauze. Detailed Implementation

[0026] The following specific embodiments are provided to further illustrate the content of the present invention. It should be noted that the description of these embodiments is for understanding the present invention and should not be construed as limiting the present invention in any way.

[0027] Example 1: Preparation of a biomembrane with a biomimetic cuticle "brick-mortar" structure 1.5 mL of a 400 mg / mL wool keratin solution with a molecular weight of 17 kDa was emulsified in 40 mL of liquid paraffin containing 150 mg lecithin at 500 rpm / min for 1 hour with mechanical stirring. Under continuous stirring, 56 μL of a 50% (v / v) glutaraldehyde aqueous solution was rapidly injected to initiate the crosslinking reaction. After crosslinking at room temperature for 2 hours, KM was collected by centrifugation, washed with an ethanol-water solution to remove organic residues, and stored at 4°C until use. Activated KM was obtained by co-incubating 1 g of KM with 40 mL of a solution containing 50 mmol / L tris(2-carboxyethyl)phosphonic acid hydrochloride. 0.5 g of activated KM was transferred to 5 mL of an aqueous solution containing 0.4 mL of glycidyl methacrylate, and methacrylated at 20°C for 12 hours. MKM was collected by centrifugation, washed 5 times with water, and stored at 4°C until use.

[0028] 50 mg of MKM was dispersed in 5 mL of a solution to be evaporated, which contained 0.25 mg of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, 5 mg of glycerol, and 10 mg of MSF in water, and swelled completely. MSF is a silk fibroin with photocrosslinking activity. 1 g of silk fibroin was mixed with 10 mL of glyceryl methacrylate containing 0.5 mol / L and reacted thoroughly at 60 °C and 300 r / min for 3 hours to obtain MSF.

[0029] After thorough mixing, the mixture was transferred to a 4 cm diameter polytetrafluoroethylene circular mold and dried at 40°C for 48 hours, followed by UV treatment for 5 minutes. The resulting KSF biofilm with a biomimetic cuticle "brick-mortar" structure is shown below. Figure 1 As shown.

[0030] Comparative Example 1: Membranes prepared using unmethacrylated silk fibroin 50 mg of the MKM obtained in Example 1 was dispersed in 5 mL of an aqueous solution containing 0.25 mg of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, 5 mg of glycerol, and 10 mg of unmethacrylated silk fibroin and allowed to swell completely. After thorough mixing, the mixture was transferred to a 4 cm diameter polytetrafluoroethylene circular mold and dried at 40 °C for 48 hours, followed by UV treatment for 5 minutes.

[0031] The membrane material obtained in the comparative example was immersed in PBS buffer. It began to swell violently after 10 minutes and completely disintegrated within 1 hour. The hydrogen bonding induction of the β-sheet secondary structure of silk fibroin on the α-helix of wool keratin was insufficient to support the stable existence of the "brick-and-mortar" structure. The introduction of the methacrylamide crosslinking network stabilized the "brick-and-mortar" structure under the dual effects of physical and chemical processes, and it did not disintegrate in the buffer solution.

[0032] Example 2: Biomembrane with a copper-ion-loaded biomimetic keratinocyte "brick-mortar" structure The film obtained in Example 1 was completely immersed in 5 mL of a solution containing 390 mg of CuSO4·5H2O for 4 h. The microstructure of the resulting Cu@KSF biofilm with a biomimetic cuticle "brick-mortar" structure and loaded with copper ions is shown below. Figure 2 As shown, it exhibits a texture similar to skin.

[0033] Example 3: Test of the bacterial barrier performance of a biofilm with a biomimetic cuticle "brick-mortar" structure loaded with copper ions. (1) Cu 2+ Release test Cu was determined by disulfonate base (BCO) spectrophotometry. 2+ Release. 5 mg of dried copper-loaded biomembrane with a biomimetic cuticle "brick-and-mortar" structure was incubated in 1 mL of PBS at 32°C with shaking. At preset time points, 100 μL of the incubation solution was mixed with 100 μL of BCO solution (2.5 g / L). -1 Mix thoroughly with 100 μL of NH3-NH4Cl buffer (10 mM, pH 9.0) and let stand at room temperature for 10 minutes. Determine Cu from OD600 measurements using a pre-established standard curve. 2+ concentration.

[0034] In moist, warm, and nutrient-rich environments, open wounds provide optimal conditions for bacterial colonization and infection. Such infection can significantly prolong the healing process. Therefore, effective wound dressings must possess antibacterial properties to prevent microbial infection. While keratin exhibits good biocompatibility and promotes cell adhesion, it lacks antibacterial properties. To address this issue, this invention introduces a method in which keratin chelates with metal cations to form a complex using a simple soaking technique, thereby incorporating Cu with strong antibacterial activity. 2+ Effectively incorporated into keratin-based dressings. Releases Cu from the dressing. 2+ The antibacterial effect on wounds is crucial. Under physiological conditions, the keratin-metal coordination complex in Cu@KSF undergoes rearrangement, releasing excess Cu. 2+ .like Figure 3 As shown, Cu within 2 hours at pH 7.4 2+ The cumulative release was 16.16 ± 0.42 μg / mg, and equilibrium was reached within 2 hours, indicating that Cu@KSF can rapidly release Cu. 2+ It inhibits bacterial growth while minimizing prolonged exposure, thus improving the biosafety of the dressing.

[0035] (2) Barrier ability test against Staphylococcus aureus Permeation resistance to Staphylococcus aureus was evaluated under open-environment exposure conditions. The permeation test employed a vertical Franz diffusion cell system, placing the membrane between a pre-sterilized Franz donor and recipient compartment, with the recipient compartment containing 10 ml of nutrient broth. Gauze served as a positive control. All samples were incubated at 32°C for 7 days, followed by turbidity assessment via absorbance measurement at 600 nm. Three measurements were performed for each test condition.

[0036] The results of the permeation experiment are as follows Figure 4 As shown, in an open environment without bacterial inoculation, the optical density of all samples was comparable to that of the sealed negative control, while bacterial growth was significant in the open positive control.

[0037] In summary, the biofilm prepared by this invention has a distinct biomimetic "brick-mortar" structure and, after being chelated with copper ions, possesses a barrier ability to prevent bacterial penetration.

[0038] Example 4: Test of air permeability and oxygen permeability of a biomembrane with a biomimetic cuticle "brick-mortar" structure loaded with copper ions. (1) Water vapor transmission test Water vapor transmission rate (WVTR) was measured according to ASTM standard E96-00, using medical gauze (Qingdao Hainuo Biotechnology Co., Ltd., China) and commercial film (Tegaderm™ film, 3M Corporation, Minnesota, USA) as control materials. The KSF biofilm samples prepared in Example 1 were cut into 30 mm diameter discs and mounted in cylindrical beakers containing 10 mL of distilled water. All edges were sealed with PTFE tape before initial weighing. After incubation for 24 hours in a constant temperature and humidity chamber at 32°C and 30% relative humidity, the samples were weighed again to determine the transmission rate.

[0039] (2) Oxygen permeability test Oxygen permeability was measured similarly to water vapor permeability. Medical gauze, Tegaderm™ membrane, and KSF biofilm samples prepared in Example 1 were cut into 30 mm diameter discs and fixed in cylindrical cups containing 10 mL of freshly prepared deoxygenated water. All edges were immediately sealed with PTFE tape; unsealed cups served as positive controls, and fully sealed cups as negative controls. After exposure to ambient air at 32°C for 24 hours, 1 mL of hexane and 1 mL of water sample were sequentially added using a 2 mL pipette. This two-phase system was carefully transferred to a 20 mL glass bottle pre-filled with 0.5 mL of hexane to minimize air ingress during the bottom deposition process. Following ISO 5813-1983 guidelines, 5 μL of manganese sulfate (380 g / L) and 10 μL of basic iodine reagent were sequentially added to initiate manganese hydroxide precipitation. After a 3-minute reaction, 10 μL of 50% sulfuric acid solution dissolved the flocculent material, releasing iodine in proportion to the dissolved oxygen (DO) content. One mL of the acidified solution was collected below the hexane barrier and titrated with a 0.00625 mol / L sodium thiosulfate solution. The titrant consumption was converted to DO concentration using a stoichiometric equation: 1 mL titrant = 55.5 mg / L DO, thus ensuring trace-level quantitative accuracy.

[0040] Table 1 Membrane material <![CDATA[Water vapor transmission rate (g m -2 day -1 )]]> <![CDATA[Oxygen permeability (mg m -2 day -1 )]]> Cu@KSF 1879.9 ± 26.2 361.0 ± 10.7 Medical gauze 4543.7 ± 121.2 603.3 ± 12.2 Tegaderm 669.0 ± 56.8 407.8 ± 9.6 Table 1 compares the water vapor permeability and oxygen permeability of the copper-loaded biofilm with a biomimetic keratinocyte "brick-mortar" structure with commercially available wound dressings (n = 3). The measurement results are shown in Table 1; the oxygen permeability is 361.0 mg / m³. -2 day -1 The water vapor permeability was 1879.9 gm. -2 day -1 It is 2.8 times more effective than the commercial membrane dressing Tegaderm™. In contrast, the control group gauze was almost completely permeable and lacked barrier properties.

[0041] Example 5: Wound healing effect of copper-loaded biofilm with a biomimetic keratinocyte "brick-mortar" structure. (1) Staphylococcus aureus infection wound treatment model: Male Wistar rats (weighing 190-210 g, 6 weeks old) underwent a 1-week acclimatization period before the experiment. All rats were anesthetized, and their back hair was trimmed with an electric razor and depilatory cream was applied to fully expose the area on both sides of the midline of the back. Subsequently, a full-thickness skin wound (12 mm in diameter) was created, extending to the fascia layer. A silicone ring with bio-adhesive (Golden Ivory, China) prevented early wound contraction. Before the start of treatment, 100 μL of Staphylococcus aureus suspension (1×10⁻⁶) was added to the wound. 8 CFU mL -1 The dressing was applied to the wound and left on for 24 hours. Patients received Tegaderm™, Cu@KSF (Example 2), and a gauze control group, respectively. The dressing was changed three times daily throughout the 14-day study period. Wound progress was photographed on days 0, 3, 5, 7, 10, and 14 post-treatment. Wound closure is shown in the image. Figure 5 As shown.

[0042] (2) Burn wound treatment model: Male Wistar rats (weighing 190-210 g, 6 weeks old) underwent hair removal treatment as described above. A 12 mm diameter burn wound was created on the rats by contacting the skin with a copper rod heated in boiling water (100°C) for 20 seconds. After debridement of necrotic tissue, the rats received treatments including Tegaderm™, Cu@KSF, and a gauze control group. Dressings were changed every three days, and wound progression was monitored by photography on days 1, 4, 7, 14, and 21.

[0043] Wound closure status as follows Figure 6 As shown, the wound healing results indicate that the biofilm with a biomimetic keratin "brick-and-mortar" structure loaded with copper ions can promote wound healing more quickly and leave the least amount of scar tissue and the mildest scarring.

[0044] For anyone skilled in the art, many possible variations and modifications can be made to the technical solutions of this invention, or equivalent embodiments can be modified based on the disclosed technical content, without departing from the scope of the technical solutions of this invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this invention without departing from the content of the technical solutions of this invention should still fall within the protection scope of the technical solutions of this invention.

Claims

1. A method for preparing a biofilm with a biomimetic cuticle "brick-mortar" structure, characterized in that, The method includes the following steps: 1) Keratin microspheres KM were obtained by covalent cross-linking wool keratin in a water-in-oil emulsion using the emulsion method. Then, the thiol groups of KM were activated by a reducing agent and modified by methacrylamide to obtain keratin microspheres MKM with photocrosslinking activity. 2) Silk fibroin was modified by methacrylation to obtain silk fibroin MSF with photocrosslinking activity; 3) MKM is fully dispersed in a solution containing silk fibroin MSF with photocrosslinking activity, transferred to a mold, and evaporated to constant weight. Then, ultraviolet light is used to initiate film crosslinking to obtain a KSF biofilm with a "brick-mortar" structure.

2. The preparation method according to claim 1, characterized in that: The preparation method also includes fully immersing the KSF biofilm in a solution containing metal ions to form a metal ion-keratin complex, thereby obtaining an antibacterial KSF biofilm.

3. The preparation method according to claim 1, characterized in that, In step 1), the emulsion method specifically involves: preparing an aqueous solution of wool keratin as the aqueous phase, adding an oil phase containing a surfactant, emulsifying for at least 10 minutes, and then adding a crosslinking agent to react and obtain KM; The wool keratin aqueous solution contains wool keratin with a molecular weight of 10-55 kDa and a concentration of 100-500 mg / mL; the oil phase is any one of vegetable oil, dichloromethane, chloroform, and liquid paraffin; the surfactant is any one of Span 20, Span 60, Span 80, lecithin, and ceramide, and the surfactant content is 0.02-0.5 wt% of the oil phase mass; the crosslinking agent is any one of glutaraldehyde, paraformaldehyde, genipin, and tyrosinase.

4. The preparation method according to claim 1, characterized in that, In step 1), the specific process of activating KM with the reducing agent is as follows: KM and the reducing agent solution are co-incubated at a ratio of 1g:40mL; The reducing agent is any one of tris(2-carboxyethyl)phosphonic acid hydrochloride, mercaptoethanol, sodium sulfide, and sodium thiosulfate, and the concentration of the reducing agent solution is 20-70 mmol / L; The specific process of methacrylation modification of activated KM is as follows: Activated KM is placed in a reaction solution containing a methacrylation reagent and reacted fully to obtain MKM. The methacrylation reagent of activated KM is methacrylic anhydride, glycidyl methacrylate, 3-methacrylamide dopamine or trimethylsilane methacrylate, and the concentration of the reaction solution containing the methacrylation reagent is 0.1-1 mol / L.

5. The preparation method according to claim 1, characterized in that, In step 2), the silk fibroin is modified by methacrylation, specifically by mixing the silk fibroin with a reaction solution containing methacrylation reagent at a ratio of 1g:10mL and reacting fully to obtain MSF; The methacrylating agent for silk fibroin is any one of methacrylic anhydride, glycidyl methacrylate, 3-methacrylamide dopamine, and trimethylsilane methacrylate, and the concentration of the reaction solution of the methacrylating agent for silk fibroin is 0.5-2 mol / L.

6. The preparation method according to claim 1, characterized in that, In step 3), the solution to be volatilized contains silk fibroin, a humectant, and a photoinitiator; the humectant is any one of glycerin, propylene glycol, urea, and sodium L-pyrrolidone-5-carboxylate; the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone or lithium phenyl(2,4,6-trimethylbenzoyl)phosphate; in the solution to be volatilized, the concentration of silk fibroin is 1-10 mg / mL, and the concentration of the humectant is 2-15 wt%.

7. The preparation method according to claim 2, characterized in that, The metal ion mentioned is Ag. + Cu 2+ Zn 2+ Fe 3+ Zr 4+ The concentration of metal ions in the solution is 0.01-0.2 mol / L, the complexation time is 1-6 hours, and the complexation temperature is 10-40℃.

8. The preparation method according to claim 1, characterized in that, In step 1), the reaction temperature of the emulsion method is 10-60℃, the emulsification speed is 400-2000 r / min, and the crosslinking time of the crosslinking agent is 1-12 hours; The conditions for activating KM with a reducing agent are: pH 7-8, reaction time 2-20 minutes, and reaction temperature 20-40℃; the conditions for methacrylylation modification of activated KM are: reaction time 4-12 hours, reaction temperature 10-60℃, and stirring speed 100-300 r / min. In step 2), the reaction time for the methacrylation modification of silk fibroin is 3-12 hours, the reaction temperature is 50-70℃, and the stirring speed is 200-500 r / min. In step 3), the evaporation temperature is 25-60℃ and the UV treatment time is 1-30 min.

9. A biofilm with a biomimetic cuticle "brick-mortar" structure, characterized in that, Prepared according to the preparation method according to any one of claims 1-8.

10. The application of the biofilm according to claim 9, characterized in that: The biofilm is used in wound dressings.

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