A gelatin / gallic acid antibacterial wound composite dressing based on silk fibroin and a preparation method thereof

Abstract

The application discloses a gelatin / gallic acid antibacterial wound composite dressing based on silk fibroin and a preparation method thereof, and adopts a coaxial electrospinning process to prepare the dressing as follows: 1) preparing an outer layer solution: taking silk fibroin, using formic acid as a solvent, and adding gelatin to obtain electrospinning solution A as the outer layer solution, wherein the concentrations of the silk fibroin and the gelatin are 18-22% g / ml and 0.1-0.5% g / ml respectively; 2) preparing an inner layer solution: taking silk fibroin, using formic acid as a solvent, and adding gallic acid to obtain electrospinning solution B as the inner layer solution, wherein the concentrations of the silk fibroin and the gallic acid are 18-22% g / ml and 0.1-0.5% g / ml respectively; and 3) coaxial electrospinning to prepare the composite dressing. The composite dressing prepared from the silk fibroin, the gelatin and the gallic acid as raw materials has strong mechanical properties for the human body, is non-toxic and harmless, and has anti-inflammatory and antibacterial properties, and can promote wound healing.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a gelatin / gallic acid antibacterial wound composite dressing based on silk fibroin and its preparation method. Background Technology

[0002] Skin is one of the largest and most important organs in the human body, accounting for 4% to 6% of body weight, and together with subcutaneous tissue, it accounts for 15% to 17%. Covering the human body surface, the epidermis, dermis, and subcutaneous tissue together constitute the skin structure. Skin is a crucial barrier against external damage and an important immune organ. It performs vital functions, such as resisting the invasion of pathogens, excreting waste products, preventing water loss, regulating body temperature, and sensing pressure, cold, and heat. Skin defects caused by skin diseases, large-area burns, chronic ulcers, or trauma have long been a challenge for clinicians. Fortunately, antibacterial wound dressings will overcome this difficulty, achieving targeted, long-lasting, and highly efficient sterilization. Furthermore, their significant practical application potential and ease of industrialization give them broad application prospects. Driven by advancements in medical technology, increasingly abundant domestic medical resources, strengthened medical security, accelerated basic medical infrastructure development, rising living standards, increasing surgical volume, and an aging population, the Chinese domestic market for wound care medical dressings is rapidly developing. Dressings, as temporary skin substitutes, play an important role in protecting wounds, stopping bleeding, preventing infection, and promoting wound healing.

[0003] An ideal dressing should maintain a moist environment, release medication, and possess anti-inflammatory, antibacterial, cell-promoting, and skin-rebuilding properties. Based on the properties and characteristics of the materials, dressings are categorized into traditional dressings, interactive dressings, and bioactive dressings. Traditional dressings, such as gauze and bandages, are simple to use and inexpensive, but their function is limited, and they may damage the wound during dressing changes.

[0004] Globally, bacterial infections pose an increasing threat to human health. The emergence of multidrug resistance due to antibiotic overuse has made bacterial infections a major challenge in the biomedical field. To reduce the side effects of antibiotics and combat bacterial attacks, many bactericidal strategies have been proposed, such as photothermal sterilization, photocatalysis, nano-drug delivery systems, and metal or metal oxide-based antibacterial nanomaterials. However, it is difficult to fix these antibacterial nanomaterials onto the wound surface to achieve long-term effective sterilization, which greatly limits their practical application. Topical application of antibiotics to wounds for infection prevention and treatment may lead to bacterial resistance and carries risks of allergies or toxicity. Prevention and treatment of wound infections are particularly important for wound repair. Traditional Chinese medicine (TCM) external application has unique advantages in treating skin wounds. Based on a holistic analysis of the patient and the wound, different treatment methods have been proposed, such as clearing heat and detoxifying, promoting tissue regeneration, and promoting blood circulation and removing blood stasis, reducing swelling and promoting tissue regeneration. These methods can accelerate wound healing, reduce scarring, and improve the quality of wound healing. However, current research on the mechanisms of action of TCM external application drugs that promote wound healing is still insufficient. Therefore, combining traditional Chinese medicine with high-quality polymer carrier antibacterial dressings can enhance the therapeutic effect. Nowadays, traditional medical dressing products can hardly meet the needs of wound treatment. New dressings will become the main driving force for the development of the dressing market. It is foreseeable that with the application of antibacterial and biomaterials, traditional dressing products on the market will gradually decrease, while functional medical dressings, such as hydrogels, hydrocolloids, foam dressings, and transparent dressings, will experience strong growth.

[0005] The increasing market demand for novel wound dressings is inextricably linked to the current medical and social structure. Therefore, there is a need to research flexible, on-demand wound dressings that are harmless to the human body, possess antibacterial, anti-inflammatory, hemostatic, and slow-release drug properties to promote wound healing. However, the addition of antibiotics to wound dressings or the local application of antibiotics to prevent and treat wound infections can lead to bacterial resistance, and some anti-inflammatory agents are even toxic to organisms, potentially causing allergic reactions and nephrotoxicity. Clinically, the addition of functional components such as nanomaterials and antibiotics to enhance the anti-inflammatory properties of dressings can also induce cytotoxicity and drug resistance, threatening the safety of organisms. Therefore, reducing the biotoxicity of anti-inflammatory agents and precisely controlling their release based on the wound's inflammation status are problems that need to be addressed in skin wound dressings. Thus, developing a wound dressing with high biocompatibility, significant anti-inflammatory properties, and the ability to monitor wound healing in real time is an urgent and clinically valuable problem. As an ideal wound dressing, this type of antibacterial wound dressing can maintain a local moist environment, can directly contact human skin to ensure accurate drug delivery to the working area, and can also achieve long-term, continuous biological function. It also has anti-inflammatory, antibacterial, cell proliferation-promoting, and skin-reconstruction-aiding functions, which are of great significance for wound healing.

[0006] Currently, silk fibroin dressings are generally prepared using electrospinning technology, with coaxial electrospinning technology being rarely seen. The principle of coaxial electrospinning is as follows: during electrospinning, the two solutions converge at the capillary opening for a short time, and their diffusion coefficients are low, so the two spinning solutions do not mix before solidification. A high-voltage electric field is applied to the inner and outer liquids, causing the charge in the inner solution to gradually migrate to the surface of the outer solution. As the voltage increases, the electric field force strengthens, and the charge on the surface of the outer solution gradually increases. When the charge accumulates to a certain level, the repulsive force increases, and the outer solution is dragged and stretched, forming a composite Taylor cone at the spinning nozzle. Then, a coaxial composite structure consisting of a shell layer encapsulating a core polymer layer is drawn out from the Taylor cone. The composite Taylor cone is further stretched into a core-shell structure jet, undergoing intense whipping and bending deformation during the stretching process. As the solvent rapidly evaporates during the fine stream stretching process and the jet stream gradually becomes finer, the composite-structured ultrafine fiber membrane is finally collected on the receiving device. Summary of the Invention

[0007] This invention utilizes silk fibroin, gelatin, and gallic acid as raw materials, and through various processes and methods, prepares a skin dressing that possesses strong mechanical properties, is non-toxic and harmless, and has anti-inflammatory and antibacterial properties, promoting wound healing. Therefore, this application protects the following technical solution:

[0008] A method for preparing a gelatin / gallic acid antibacterial wound composite dressing based on silk fibroin, which is prepared by coaxial electrospinning, includes the following steps:

[0009] 1) Preparation of outer layer solution: Take silk fibroin, use formic acid as solvent, add gelatin to obtain electrospinning solution A as outer layer solution, wherein the concentrations of silk fibroin and gelatin in electrospinning solution A are 18-22% g / ml and 0.1-0.5% g / ml, respectively;

[0010] 2) Preparation of inner layer solution: Take silk fibroin, use formic acid as solvent, add gallic acid to obtain electrospinning solution B as inner layer solution, wherein the concentrations of silk fibroin and gallic acid in electrospinning solution B are 18-22% g / ml and 0.1-0.5% g / ml, respectively.

[0011] 3) Coaxial electrospinning: The prepared outer layer solution and inner layer solution are injected into the outer and inner needles of the coaxial electrospinning needle, respectively. Under the conditions of voltage of 15-20KV, receiving distance of 10-20cm, and flow rates of electrospinning solution A, electrospinning solution B and feed inlet of 0.004-0.006ml / min, the electrostatic generator is connected to the coaxial electrospinning needle to perform electrospinning to prepare composite dressing.

[0012] Preferably, the process parameters for coaxial electrospinning in step 3) are as follows: the voltage applied between the spinneret and the receiving plate is 18KV, the distance between the injection needle and the collecting plate is 15cm, and the flow rates of electrospinning solution A, electrospinning solution B and the feed inlet are all 0.005ml / min.

[0013] Preferably, the concentrations of silk fibroin and gelatin in the electrospinning solution A are 18-22% g / ml and 0.3-0.5% g / ml, respectively.

[0014] Preferably, the concentrations of silk fibroin and gallic acid in the electrospinning solution B are 18-22% g / ml and 0.3-0.5% g / ml, respectively.

[0015] Preferably, the concentrations of silk fibroin and gelatin in the electrospinning solution A are 19-21% g / ml and 0.3-0.5% g / ml, respectively;

[0016] The concentrations of silk fibroin and gallic acid in the electrospinning solution B are 19-21% g / ml and 0.3-0.5% g / ml, respectively.

[0017] More preferably, the concentrations of silk fibroin and gelatin in the electrospinning solution A are 20% g / ml and 0.5% g / ml, respectively;

[0018] The concentrations of silk fibroin and gallic acid in the electrospinning solution B are 20% g / ml and 0.5% g / ml, respectively.

[0019] Preferably, the silk fibroin is prepared by degumming silkworm cocoons.

[0020] In the above technical solution, the preparation method of the silk fibroin includes the following steps: degumming silkworm cocoons to obtain degummed silk, washing and drying it, mixing it with anhydrous calcium chloride, adding formic acid to completely dissolve it, centrifuging, casting, air drying, soaking it in ultrapure water to remove impurity ions, and drying it to obtain silk fibroin.

[0021] Preferably, the method for preparing the silk fibroin includes the following steps:

[0022] After the silkworm cocoons are cut into pieces, they are ultrasonically cleaned to remove impurities. Degummed silk is obtained by degumming with sodium carbonate solution. The degummed silk is washed and dried for later use. The dried degummed silk is mixed with anhydrous calcium chloride and formic acid in a ratio of 2.5:1:10 (mass of degummed silk:mass of anhydrous calcium chloride:volume of formic acid). After dissolution, the mixture is centrifuged twice for 20 minutes each time in centrifuge tubes. After centrifugation, the mixture is poured into petri dishes and air-dried for 1-2 days until the mixture is dry. It is then placed in ultrapure water and soaked for at least 12 hours until the color turns pure milk color. After drying, silk fibroin membrane is obtained.

[0023] The present invention also protects the composite dressing prepared by any of the above-described preparation methods.

[0024] The beneficial effects of this invention are:

[0025] 1) The preparation steps for antibacterial dressings are simple, easy to operate, and the reaction conditions are mild, resulting in low costs, making them suitable for large-scale mass production. Silk fibroin is a natural high-molecular-weight fibrous protein with non-toxic and harmless properties. The gallic acid used in this invention is a polyphenolic organic compound combined with silk fibroin. Through coaxial electrospinning technology, the anti-inflammatory, antibacterial, and bioactive properties of gallic acid can be combined, resulting in a dressing with good antibacterial effects, shortening wound healing time and promoting wound healing.

[0026] 2) This invention uses silk fibroin, a natural high-molecular-weight fibrous protein, as a base, and simultaneously incorporates hydrophilic colloids of gelatin macromolecules. The dressing prepared by combining silk fibroin and gelatin has the advantages of good biocompatibility and biodegradability of silk fibroin, which can be well-compatible with wound tissue and will not cause rejection reaction. It also has the hydrophilicity and mechanical strength of gelatin, which improves the mechanical properties of the dressing. At the same time, it can absorb excess exudate at the wound in time, reducing the risk of bacterial infection. In addition, the addition of gelatin can slow down the degradation rate of the dressing and increase the usable time of the dressing.

[0027] 3) The composite dressing of this invention exhibits relatively optimal performance in the combination of silk fibroin and gallic acid / gelatin, with the combination of the two substances maximizing their synergistic effect, compensating for their respective shortcomings while retaining their advantages. Mechanical properties increase with increasing gelatin content, and degradation rate and water absorption have been experimentally demonstrated. Further experiments evaluated the in vitro and in vivo bioactivity of the composite dressing: biocompatibility tests showed that the prepared dressing was non-toxic to NIH-3T3 fibroblasts and had a moderate promoting effect; in a mouse full-thickness skin lesion model, the application of the antibacterial dressing resulted in faster wound healing and a shorter disease course compared to the blank control group. Attached Figure Description

[0028] Figure 1 This is a flowchart of the preparation process of silk fibroin membrane.

[0029] Figure 2 This is a SEM image of the composite dressing obtained by ordinary electrospinning in Example 1.

[0030] Figure 3 A schematic diagram illustrating the principle of preparing the composite dressing of the present invention by coaxial electrospinning.

[0031] Figure 4 The stress-strain tensile curves of composite dressings prepared according to different ratios are shown in the test diagram.

[0032] Figure 5 SEM images of composite dressings with and without gelatin-loaded drugs were captured under different magnifications.

[0033] Figure 6 Degradation curves of different composite dressings in PBS solution containing XIV trypsin.

[0034] Figure 7 This is a graph showing the water absorption rate of different composite dressings in PBS.

[0035] Figure 8 A bar graph showing the cell viability of mouse fibroblasts in a cell biocompatibility test of different ratios of gallic acid in the composite dressing prepared in this invention.

[0036] Figure 9 Fluorescence images of live / dead mouse fibroblasts stained with different ratios of gallic acid in the composite dressing prepared in this invention, used in a cell biocompatibility test.

[0037] Figure 10 This is a comparison of the antibacterial effects of different proportions of gallic acid in the composite dressing prepared in this invention on Staphylococcus aureus.

[0038] Figure 11 Immunohistochemical comparison of the optimal ratio of the composite dressing prepared in this invention applied to wound healing of full-thickness skin lesions in mice. Detailed Implementation

[0039] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0040] Unless otherwise specified, the experimental methods in the following examples are conventional methods; the reagents and materials used are conventional reagents and materials in the art and are commercially available.

[0041] Gelatin (GT): CAS Registry No. 9000-70-8, purity 99%.

[0042] Gallic acid (GA): CAS Registry No. 149-91-7, purity 99%.

[0043] Formic acid: 98% purity.

[0044] Example 1: Preparation of composite dressings by conventional electrospinning

[0045] I. Preparation of Silk Fibroin (SF) Membranes

[0046] Preparation flow chart as follows Figure 1 As shown.

[0047] Select high-quality silkworm cocoons, cut them into small pieces, and ultrasonically clean them for at least 15 minutes to remove impurities. Measure 2.00L of deionized water, add 4.24g of Na2CO3, and heat to boiling (100.00℃). After the Na2CO3 is completely dissolved, put the silkworm cocoons into the solution and stir for 60 minutes. Remove the degummed silk, wash and rub it thoroughly with deionized water, repeating the washing process until the degummed silk is clean. Place it in a drying oven and dry for 24 hours to obtain degummed silk.

[0048] Using anhydrous calcium chloride as the solute and formic acid as the solvent, a certain amount of degummed silk was weighed out and dissolved according to the ratio of degummed silk mass: anhydrous calcium chloride mass: formic acid volume = 2.5:1:10. Specifically, 5g of degummed silk was mixed with 2g of anhydrous calcium chloride, and 20ml of formic acid was added. The mixture was stirred in a magnetic stirrer until the degummed silk was completely dissolved. The solution was then centrifuged twice, 20 minutes each time. After that, it was poured into a 10cm petri dish and placed in a ventilated place to air dry for 1-2 days to allow the formic acid to evaporate. After air drying, it was soaked in ultrapure water until it turned pure milk color to remove the remaining formic acid. After drying, the silk fibroin was obtained.

[0049] II. Preparation of Composite Dressings

[0050] Weigh 2g of silk fibroin, 50mg of gelatin, and 50mg of gallic acid and add them to 10ml of formic acid solution. Stir and dissolve the solution on a magnetic stirrer for 12 hours to obtain a solution for electrospinning. Perform electrospinning with the following process parameters: the voltage applied between the spinneret and the receiving plate is 18KV, the receiving distance (i.e., the distance between the injection needle and the collecting plate) is 15cm, and the flow rate of the spinning solution at the feed inlet is 0.005ml / min.

[0051] The scanning electron microscope (SEM) image of the prepared composite dressing is shown below. Figure 2 As shown: Due to the physicochemical properties and viscosity of gelatin, it is easy to form liquid droplets, resulting in uneven distribution of filament diameter. When mixed with drugs, this leads to uneven distribution of drugs on the composite dressing.

[0052] Example 2: Preparation of composite dressings by coaxial electrospinning

[0053] I. Preparation of Silk Fibroin (SF) Membranes

[0054] The preparation method is the same as in Example 1.

[0055] II. Preparation of Electrospinning Solution A of Silk Fiber Membrane and Gelatin

[0056] Preparation of mixed electrospinning solutions A with different gelatin concentrations:

[0057] 1) Weigh 2g of silk fibroin, add 10mg of gelatin and mix, add 10ml of formic acid solution, stir and dissolve on a magnetic stirrer for 12 hours to obtain a mixed electrospinning solution A1 (20%SF+0.1%GT) with a silk fibroin concentration of 20%g / ml and a gelatin concentration of 0.1%g / ml.

[0058] 2) Weigh 2g of silk fibroin, add 30mg of gelatin and mix, add 10ml of formic acid solution, stir and dissolve on a magnetic stirrer for 12 hours to obtain a mixed electrospinning solution A2 (20%SF+0.3%GT) with a silk fibroin concentration of 20%g / ml and a gelatin concentration of 0.3%g / ml.

[0059] 3) Weigh 2g of silk fibroin, add 50mg of gelatin and mix, add 10ml of formic acid solution, stir and dissolve on a magnetic stirrer for 12 hours to obtain a mixed electrospinning solution A3 (20%SF+0.5%GT) with a silk fibroin concentration of 20%g / ml and a gelatin concentration of 0.5%g / ml.

[0060] III. Preparation of a mixed electrospinning solution B of silk fibroin membrane and gallic acid

[0061] Preparation of mixed electrospinning solutions B with different gallic acid concentrations:

[0062] 1) Weigh 2g of silk fibroin, add 10mg of gallic acid and mix, add 10ml of formic acid solution, stir and dissolve on a magnetic stirrer for 12 hours to obtain a mixed electrospinning solution B1 (20%SF+0.1%GA) with a silk fibroin concentration of 20%g / ml and a gallic acid concentration of 0.1%g / ml.

[0063] 2) Weigh 2g of silk fibroin, add 30mg of gallic acid and mix, add 10ml of formic acid solution, stir and dissolve on a magnetic stirrer for 12 hours to obtain a mixed electrospinning solution B2 (20%SF+0.3%GA) with a silk fibroin concentration of 20%g / ml and a gallic acid concentration of 0.3%g / ml.

[0064] 3) Weigh 2g of silk fibroin, add 50mg of gallic acid and mix, add 10ml of formic acid solution, stir and dissolve on a magnetic stirrer for 12 hours to obtain a mixed electrospinning solution B3 (20%SF+0.5%GA) with a silk fibroin concentration of 20%g / ml and a gallic acid concentration of 0.5%g / ml.

[0065] IV. Preparation of Composite Dressings

[0066] 1) Using coaxial electrospinning technology, composite dressings were prepared by combining mixed electrospinning solution B3 with mixed electrospinning solutions A1, A2 and A3 respectively, resulting in composite dressings loaded with 0.1% gelatin (A1B3), composite dressings loaded with 0.3% gelatin (A2B3) and composite dressings loaded with 0.5% gelatin (A3B3).

[0067] 2) Using coaxial electrospinning technology, composite dressings were prepared by combining mixed electrospinning solution A3 with mixed electrospinning solutions B1, B2 and B3 respectively, resulting in composite dressings loaded with 0.1% gallic acid (A3B1), 0.3% gallic acid (A3B2) and 0.5% gallic acid (A3B3).

[0068] The coaxial electrospinning process parameters are set as follows: the voltage applied between the spinneret and the receiving plate is 18KV; the receiving distance (i.e., the distance between the injection needle and the collecting plate) is 15cm; the flow rates of electrospinning solution A (outer layer solution), electrospinning solution B (inner layer solution), and the feed inlet are all 0.005ml / min; the humidity in the spinning room is maintained at 20%; the spinning time is 26 hours; after spinning, the composite dressing is placed in a dry environment and sealed for later use. The flowchart of coaxial electrospinning is as follows. Figure 3 As shown.

[0069] Example 3, Performance Characterization Test:

[0070] The performance of the composite dressing prepared in Example 2 was tested.

[0071] I. Stress-strain tensile curve testing of composite dressings with different ratios

[0072] The mechanical properties of the composite gelatin / gallic acid-silk fibroin fiber dressing were tested using a universal mechanical testing instrument.

[0073] Composite dressings prepared according to different ratios were made into rectangular samples of 2 cm × 1 cm and fixed on the fixture of a universal testing machine for tensile testing. The sample thickness was measured by a thickness gauge. The tensile speed of the universal testing machine was 100 mm / min during the process. -1 The stress-strain curve is obtained directly from the test using a universal testing machine. For example... Figure 4 As shown, stress and strain gradually increase with increasing gelatin content, indicating that the mechanical properties of the composite dressing increase accordingly.

[0074] II. Scanning electron microscopy observation of composite dressings containing different materials

[0075] The microstructure of the composite gelatin / gallic acid-silk fibroin fiber dressing was examined using scanning electron microscopy (SEM). Prior to examination, the composite gelatin / gallic acid-silk fibroin fiber dressing was vacuum-dried at room temperature, cut into 1cm × 2cm rectangular samples, sputter-coated with gold, and fixed face-up on the scanning mount. The micromorphology of the surface was observed under SEM. The micromorphology of silk fibroin loaded with gelatin (0.5%) and gallic acid (0.5%) was compared at different magnifications to observe the uniformity and diameter of the silk fibroin loaded with gallic acid (0.5%). Figure 5 As shown, compared to dressings that only contain gallic acid without gelatin, the composite dressings containing gallic acid and gelatin have a larger and more uniform diameter, which well explains part of the reason for the increased strain stress.

[0076] III. Testing the antibacterial properties of composite dressings using Staphylococcus aureus.

[0077] The composite dressings A3B1, A3B2, and A3B3 prepared in Example 2 were selected, cut into circles of similar diameter, and irradiated with ultraviolet light for 3 hours for later use. MH culture medium A (solid medium) and culture medium B (liquid medium) powder were placed in bacterial culture flasks, and ultrapure water was added according to the operating instructions to prepare the culture medium. The medium, along with the culture dishes, spreaders, inoculation loops, and inoculation needles, was sterilized for 3 hours. The solid medium was removed and poured into culture dishes, allowed to cool and solidify, and a small amount of bacterial inoculum was spread onto the surface of the medium using a bacterial sampling stick. The flasks were then placed in a 37°C incubator for 24 hours to revive. The liquid medium was stored at 4°C for later use. After 24 hours of bacterial growth, single colonies of suitable size and good growth were selected and placed in the liquid medium. The culture was then placed in a shaker with the following basic parameters: 37°C, 200 rpm, 8-10 hours. After good bacterial growth, 200 μL of the bacterial solution was inoculated onto agar plates, and the experimental samples were gently placed on the surface of the bacterial solution. The culture medium was incubated at 37℃ for 24 hours. Bacterial growth and the formation of inhibition zones around the experimental samples were observed and recorded by photograph. All operations were performed in a laminar flow hood to ensure a sterile environment. Figure 10 As shown, the inhibition zone increases with the increase of gallic acid content, indicating that the antibacterial properties of the composite dressing are positively correlated with the gallic acid content.

[0078] IV. Biocompatibility Testing of Composite Dressings

[0079] The biocompatibility of the composite dressings A3B1, A3B2, and A3B3 prepared in Example 2 was tested (I). The prepared composite skin dressings were soaked in complete cell culture medium, and NIH3T3 fibroblasts were cultured in normal complete culture medium and composite dressing extract (each group was repeated three times). After a period of time, the cell proliferation of cells cultured in the composite dressing extract culture medium was observed by counting and measuring the cells using CCK-8. Figure 8 This is a bar chart showing the cell viability of mouse fibroblasts at different drug concentrations.

[0080] (ii) Place the sterilized material into a 12-well plate, and space the cells at a density of 5 × 10⁶ cells per well. 5 The cells were seeded at a specific density and incubated at 37°C in a 5% CO2 incubator for 36 hours. After 36 hours of incubation, the cells were removed and washed three times with PBS solution. Then, Calcein AM / PI detection working solution from the Living & Dead Cell Kit was added to the cells, and the cells were incubated in the dark for 40 minutes before being photographed under a fluorescence microscope. Figure 9 This is a fluorescence image of live and dead cells. Figure 8 and Figure 9 It can be seen that the cell activity of the composite dressing extract group was higher than that of the complete culture medium group, indicating that the composite dressing prepared in this invention not only does not have cytotoxicity, but can also effectively promote the proliferation of mouse fibroblasts.

[0081] V. Degradation Test of Composite Dressings

[0082] A silk fibroin dressing loaded with gallic acid (0.5%) and gelatin (0.5%) (A3B3) was used as a comparison with a pure silk fibroin dressing without gelatin and gallic acid. ProteaseXIV powder was dissolved in 1×PBS to prepare a 3.5 U mL⁻¹ solution. Samples of similar mass (30 ± 5 mg, recorded as W0) were weighed and tested. Each sample was placed individually in a petri dish, with three samples per group. 500 μL of ProteaseXIV solution was added to each sample, and they were incubated at 37°C. Samples were removed at specific time points, rinsed with ultrapure water, dried at 60°C for 2 h, weighed, and recorded as W1. The ProteaseXIV solution was then replaced, and incubation continued under the same conditions. This process was repeated until the samples were completely degraded. Results are as follows: Figure 6 As shown, dressings loaded with gelatin take longer to degrade than pure silk fibroin dressings without gelatin, around 12 days, which is more in line with the wound healing cycle.

[0083] VI. Water Absorption Rate Test of Composite Dressings

[0084] A dressing containing 0.5% gallic acid and 0.5% gelatin (A3B3) was compared with a pure fibroin dressing without gelatin and gallic acid. Samples of 2cm x 2cm were cut and weighed using an electronic balance, recorded as M1. Each sample was placed in PBS and soaked for 2, 4, 6, 8, 10, and 12 hours. The surface moisture was then absorbed with filter paper, and the mass was recorded as M2. The water absorption rate was calculated as: W = (M2 - M1) / M1 × 100%. Figure 7 As shown, the water absorption rate of the gelatin-loaded dressing is greater than that of the pure silk fibroin dressing without gelatin, which also confirms the beneficial effects of the present invention. It can absorb excess exudate and reduce the risk of wound infection.

[0085] VII. Application of composite dressings in wound healing of full-thickness skin lesions in mice

[0086] Mice were first anesthetized by intraperitoneal injection of 250 μl of 4% (w / v) chloral hydrate solution. Then, a wound approximately 10 mm in diameter was created on the back of the mouse using a biopsy punch. The wound was then treated with either no treatment or a prepared composite dressing. Figure 11 HE-stained images of mouse wound tissue sections at three time points (days 3, 7, and 12) after wound formation show that, compared with the blank control group, the application of the composite dressing (A3B3) selected in this invention improved the wound healing ability and shortened the healing time of the mice.

[0087] In Example 1 of this invention, a composite dressing was prepared using ordinary electrospinning. It was found that due to the viscosity of gelatin's physicochemical properties, it is easy to form liquid droplets, resulting in uneven distribution of the spinning diameter. When mixed with drugs, this leads to uneven distribution of drugs in the dressing. Through the inventors' research, it was found that using coaxial electrospinning to composite silk fibroin / gelatin / gallic acid can turn the disadvantages of gelatin's physicochemical properties into advantages, making the dressing structure more stable: (1) The outer liquid (which is air in ordinary electrospinning) is replaced by a viscoelastic liquid medium. For example, the viscosity of gelatin's physicochemical properties, when the outer liquid is further stretched as a shell, the stretching of the shell liquid gives the interface greater elasticity, thereby achieving the effect of further stabilizing the inner liquid. (2) The relatively high surface tension of liquid-gas in ordinary electrospinning is changed to a lower liquid-gas surface tension, reducing the surface force at the core-liquid boundary and avoiding the "electroporation phenomenon" caused by breaking into droplets. (3) During the electrospinning process, the two solutions merge at the capillary opening in a short time and the diffusion coefficients of the two solutions are low. Therefore, the two spinning solutions will not mix before solidification, and it is not easy to cause changes in chemical properties.

Claims

1. A method for the preparation of a gelatin / gallic acid based silk fibroin antimicrobial wound composite dressing, characterized by, It is prepared by coaxial electrospinning process, including the following steps: 1) Preparation of outer layer solution: Take silk fibroin, use formic acid as solvent, add gelatin to obtain electrospinning solution A as outer layer solution, wherein the concentrations of silk fibroin and gelatin in electrospinning solution A are 18-22% g / ml and 0.5% g / ml, respectively; 2) Preparation of inner layer solution: Take silk fibroin, use formic acid as solvent, add gallic acid to obtain electrospinning solution B as inner layer solution, wherein the concentrations of silk fibroin and gallic acid in electrospinning solution B are 18-22% g / ml and 0.5% g / ml, respectively. 3) Coaxial electrospinning: The prepared outer layer solution and inner layer solution are injected into the outer and inner needles of the coaxial electrospinning needle, respectively. Under the conditions of voltage of 15-20KV, receiving distance of 10-20cm, and flow rate of electrospinning solution A and electrospinning solution B of 0.004-0.006ml / min, the electrostatic generator is connected to the coaxial electrospinning needle to perform electrospinning to prepare composite dressing.

2. The method of claim 1, wherein: The process parameters for coaxial electrospinning in step 3) are as follows: the voltage applied between the spinneret and the receiving plate is 18KV, the distance between the injection needle and the collecting plate is 15cm, and the flow rates of electrospinning solution A and electrospinning solution B are both 0.005ml / min.

3. The preparation method according to claim 1, characterized in that: The concentrations of silk fibroin and gelatin in the electrospinning solution A are 20% g / ml and 0.5% g / ml, respectively. The concentrations of silk fibroin and gallic acid in the electrospinning solution B are 20% g / ml and 0.5% g / ml, respectively.

4. The method of claim 1, wherein: The preparation method of the silk fibroin includes the following steps: degumming silkworm cocoons to obtain degummed silk, washing and drying it, mixing it with anhydrous calcium chloride, adding formic acid to completely dissolve it, centrifuging, casting, air drying, soaking it in ultrapure water to remove impurity ions, and drying it to obtain silk fibroin.

5. The method of claim 4, wherein: The preparation method of the silk fibroin includes the following steps: After the silkworm cocoons are cut into pieces, they are ultrasonically cleaned to remove impurities. Degummed silk is obtained by degumming with sodium carbonate solution. The degummed silk is washed and dried for later use. The dried degummed silk is mixed with anhydrous calcium chloride and formic acid in a ratio of 2.5:1:10 (mass of degummed silk:mass of anhydrous calcium chloride:volume of formic acid). After dissolution, the mixture is centrifuged twice for 20 minutes each time. After centrifugation, it is poured into a petri dish and air-dried for 1-2 days until the mixture is dry. It is then placed in ultrapure water and soaked for at least 12 hours until the color turns pure milk color. After drying, the silk fibroin membrane is obtained.

6. The composite dressing prepared by the preparation method according to any one of claims 1 to 5.

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