Long-lasting antibacterial wound repair micro / nanofiber membrane and its preparation method and application

Abstract

The present invention discloses a long-lasting antibacterial wound repair micro / nanofiber membrane and its preparation method and application. The long-lasting antibacterial wound repair micro / nanofiber membrane comprises coaxial micro / nanofiber and a growth-promoting layer and an antibacterial layer that are self-assembled on the surface of the coaxial micro / nanofiber layer by layer; the coaxial micro / nanofiber comprises a shell layer and a core layer, the core layer comprises an antibacterial component, and the shell layer, the growth-promoting layer and the antibacterial layer all comprise at least a degradable biocompatible polymer material. The growth-promoting layer of the long-lasting antibacterial wound repair micro / nanofiber membrane of the present invention can promote cell proliferation and increase the speed of wound repair; the antibacterial layer on the surface of the coaxial micro / nanofiber can be rapidly released and participate in early antibacterial. After the shell layer, the growth-promoting layer and the antibacterial layer are degraded, the antibacterial components in the core layer are released and participate in late antibacterial. The antibacterial effect lasts for more than 14 days.

Description

Technical Field

[0001] The present invention relates to the field of wound dressings, and in particular to a long-lasting antibacterial wound repair micro / nano fiber membrane and a preparation method and application thereof. Background Art

[0002] As the natural barrier of the human body, the skin has the important function of protecting the human body from microbial invasion. However, skin trauma and skin defects caused by various accidents or diseases will cause a series of problems. For example, chronic wounds caused by skin ulcers in diabetic patients will cause inflammation, leading to long-term bacterial infection and even tissue necrosis. In the face of chronic or long-term bacterial infection problems, it is urgent to repair the skin wound to prevent long-term bacterial infection. Fiber materials with excellent performance can protect the wound and even release antibacterial molecules to achieve long-term antibacterial purposes. They also have certain biological functionality and create an environment conducive to skin cell healing. The existing wound antibacterial materials generally have an antibacterial time of about 72 hours and an antibacterial concentration of 10 4 -10 6 CFU / mL, and the long-term antibacterial ability is not ideal. Summary of the Invention

[0003] In order to solve the problem that the existing wound antibacterial materials have unsatisfactory long-term antibacterial ability, the present invention provides a long-term antibacterial wound repair micro / nano fiber membrane and a preparation method and application thereof.

[0004] The technical solutions provided by the present invention are as follows:

[0005] In the first aspect, the present invention provides a long-lasting antibacterial wound repair micro / nanofiber membrane, which is composed of composite micro / nanofibers, wherein the composite micro / nanofibers include coaxial micro / nanofibers and growth-promoting layers and antibacterial layers self-assembled on the surface of the coaxial micro / nanofibers; the coaxial micro / nanofibers include a shell layer and a core layer, the core layer includes an antibacterial component, and the shell layer, the growth-promoting layer and the antibacterial layer all include at least a degradable biocompatible polymer material.

[0006] In some specific embodiments provided by the present invention, the degradable biocompatible polymer material is at least one of polydopamine, tannic acid, chitosan and its derivatives, chitin, cellulose acetate, polyamide 6, collagen, polyethylene glycol, silk fibroin, silk fibroin, polylactic acid, and polyvinyl alcohol.

[0007] In some specific embodiments provided by the present invention, the degradable biocompatible polymer material used in the growth-promoting layer is one of collagen, tannic acid, and polydopamine; the degradable biocompatible polymer material used in the antibacterial layer is at least one of chitosan derivatives, chitin, cellulose acetate, and polyamide 6; the degradable biocompatible polymer material used in the shell layer is at least one of collagen, polyethylene glycol, silk fibroin, silk fibroin, polylactic acid, and polyvinyl alcohol.

[0008] In some specific embodiments provided by the present invention, the growth-promoting layer further comprises a growth-promoting component, with a degradable biocompatible polymer material serving as a carrier of the growth-promoting component; the antibacterial layer further comprises an antibacterial component, with a degradable biocompatible polymer material serving as a carrier of the antibacterial component; the growth-promoting component comprises one or more of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF).

[0009] In some specific embodiments provided by the present invention, the diameter of the composite micro / nano fiber is 50-500 nm, and the thickness of the long-lasting antibacterial wound repair composite micro / nano fiber membrane is 0.1-0.2 mm.

[0010] In some specific embodiments provided by the present invention, the material used for the shell layer further includes a toughening component, which includes at least one of polyurethane, polystyrene, polyvinyl pyrrolidone, polymethyl methacrylate, polymethyl terephthalate, polyvinyl alcohol, polyvinyl chloride, and polyvinylidene fluoride.

[0011] In some specific embodiments provided herein, the core layer further comprises an antibacterial component carrier, wherein the antibacterial component carrier is one or more of chitosan, collagen, polyethylene glycol, silk fibroin, silk fibroin, polylactic acid, polyvinyl alcohol, and alginic acid. The antibacterial component is a broad-spectrum antibacterial substance, wherein the broad-spectrum antibacterial substance is one or more of lysozyme, plasmin, pancreatic DNase, collagenase, and aprotinin.

[0012] In some specific embodiments provided by the present invention, the number of double layers of the growth-promoting layer and the antibacterial layer self-assembled on the surface of the coaxial micro / nano fiber is 1 to 15, preferably 5 to 11.

[0013] In a second aspect, the present invention provides a method for preparing the above-mentioned long-lasting antibacterial wound repair micro / nanofiber membrane, comprising:

[0014] Coaxial micro / nanofibers are prepared by coaxial spinning technology to form coaxial micro / nanofiber membranes;

[0015] The growth-promoting layer and the antibacterial layer are assembled onto the surface of the coaxial micro / nanofiber using a layer-by-layer self-assembly method.

[0016] In a third aspect, the present invention provides use of the above-mentioned long-acting antibacterial wound repair micro / nanofiber membrane in the preparation of wound dressings.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] The long-acting antibacterial wound repair micro / nanofiber membrane provided by the present invention encapsulates antibacterial components in coaxial micro / nanofibers, and assembles a growth-promoting layer and an antibacterial layer outside the coaxial micro / nanofibers. The growth-promoting layer can enhance cell proliferation and improve the wound repair speed; in the early stage, the antibacterial layer assembled outside the coaxial micro / nanofiber participates in antibacterial treatment; in the middle and late stages, the shell layer, the growth-promoting layer and the antibacterial layer are degraded, and the antibacterial components encapsulated in the core layer are gradually released to participate in antibacterial treatment. During the entire wound repair process, the antibacterial effect lasts for more than 14 days; in addition, the long-acting antibacterial wound repair micro / nanofiber membrane can release antibacterial components at a specific point, avoiding systemic medication. After adding toughening components to the shell layer of the long-lasting antibacterial wound-repairing micro / nanofiber membrane, it also has high dynamic adaptability, and can easily reach a strong tensile stress of 6.7 MPa, with a maximum elongation of up to 103%. Even after 50 cycles, the stress is still maintained well, and the maximum elongation is only lost by 12%. The long-lasting antibacterial wound-repairing micro / nanofiber membrane is used to cover the skin without any deformation or wrinkles. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown (CS / PAD) n Preparation and characterization of typical micro / nanofiber structures; Figure 1 a shows the layer-by-layer self-assembly process of CS(+) and PDA(-) on the surface of core-shell micro / nanofiber; Figure 1 b: TEM image of coaxial micro / nanofiber of SF / PCL. Figure 1 c is the FE-SEM image of SF / PCL, Figure 1 d is (CS / PDA) 10 FE-SEM images of Figure 1 e is (CS / PDA) 10 and (CS / PDA) 10.5 FT-IR spectrum of Figure 1 f is the electrical properties of the raw materials and micro / nanofilms. Error bars represent standard deviations, and the test was repeated three times. Figure 1 g is SF / PCL and (CS / PDA) n Water contact angle curve.

[0020] Figure 2 The results of long-term antibacterial ability studies were presented; Figure 2 a shows (CS / PDA) 5, (CS / PAD) 10and (CS / PDA) 15 Comparison of the cumulative release of LY within 14 days; Figure 2 b shows the growth curve of Staphylococcus aureus. Figure 2 c shows the growth curve of E. coli; Figure 2 d shows SF / PCL and (CS / PDA) n antibacterial activity; Figure 2 e shows a Staphylococcus aureus solution (10 6 / CFU, 4mL), Figure 2 f shows the E. coli solution (10 6 / CFU, 4mL) and different (CS / PDA) n Photos taken after 14 days of incubation; Figure 2 g~ Figure 2 i shows PBS, (CS / PDA)5, (CS / PDA) in turn 10 FE-SEM image of Staphylococcus aureus treated for 14 days; Figure 2 j~ Figure 2 k shows PBS, (CS / PDA)5, (CS / PDA) in turn 10 FE-SEM images of E. coli treated for 14 days, circles indicate morphological changes of cells, insets show agar plates with Staphylococcus aureus and E. coli colonies 24 hours later. Error bars indicate standard deviation (n=3). Figure 2 l is the electron microscopy (FE-SEM) image of Escherichia coli taken after treatment with (CS / PDA)10 for 24 hours.

[0021] Figure 3 Mechanical and adhesive test results are presented. Figure 3 a shows SF / PCL and (CS / PDA) n Stress-strain curves measured in the dry state, Figure 3 b shows SF / PCL and (CS / PDA) n Stress-strain curves measured in the wet state, Figure 3 c Display (CS / PDA) 10 Cyclic stress-strain testing in wet conditions. Figure 3 d~ Figure 3 f showed (CS / PDA) in turn 10 photos glued to wrists, hands, elbows; Figure 3 g showed (CS / PDA) n Adhesion strength; Figure 3 h shows the catechol group enhancement of the PDA layer (CS / PDA) n Principle of adhesion; Note: Error bars represent standard deviation (n=3).

[0022] Figure 4 The results of cell proliferation and migration studies were presented, among which, Figure 4 a shows the L929 cells in SF / PCL and (CS / PDA) n Cell viability, error bars show standard deviation (n=3); significant difference: *p<0.05, **p<0.01; Figure 4 b~ Figure 4 e shows (CS / PDA)5, (CS-PDA) 5.5 、(CS / PDA) 10 、(CS / PDA) 10.5 Fluorescent images of live / dead cell staining after treatment; Figure 4 f~ Figure 4 i shows the (CS / PDA)5, (CS-PDA) 5.5 、(CS / PDA) 10 、(CS / PDA) 10.5 FE-SEM images of L929 cells cultured on . DETAILED DESCRIPTION

[0023] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present invention and the features therein can be combined with each other without conflict.

[0024] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the specific embodiments disclosed below.

[0025] The long-lasting antibacterial wound-repairing micro- / nanofiber membrane provided by the present invention is composed of composite micro- / nanofiber components, including coaxial micro- / nanofiber components and growth-promoting layers and antibacterial layers self-assembled on the coaxial micro- / nanofiber components. The coaxial micro- / nanofiber components include a shell layer and a core layer, the core layer including an antibacterial component, and the shell layer, the growth-promoting layer, and the antibacterial layer all comprising at least a degradable biocompatible polymer material. The shell layer degrades more slowly than the growth-promoting layer and the antibacterial layer.

[0026] In some specific embodiments, the degradable biocompatible polymer material is at least one of polydopamine, tannic acid, chitosan and its derivatives, chitin, cellulose acetate, polyamide 6, collagen, polyethylene glycol, silk fibroin, silk fibroin protein, polylactic acid, and polyvinyl alcohol. Preferably, the degradable biocompatible polymer material used in the growth-promoting layer is one of collagen, tannic acid, and polydopamine; the degradable biocompatible polymer material used in the antibacterial layer is at least one of chitosan derivatives, chitin, cellulose acetate, and polyamide 6; and the degradable biocompatible polymer material used in the shell layer is at least one of collagen, polyethylene glycol, silk fibroin, silk fibroin protein, polylactic acid, and polyvinyl alcohol. The degradability of the degradable biocompatible polymer material ensures that the antibacterial components in the core layer can be released at a later stage, and its biocompatibility can promote wound healing.

[0027] In some specific embodiments provided by the present invention, the growth-promoting layer further comprises a growth-promoting component, with a degradable biocompatible polymer material serving as a carrier of the growth-promoting component, and the growth-promoting component is released during the decomposition of the growth-promoting layer; the antibacterial layer further comprises an antibacterial component, with a degradable biocompatible polymer material serving as a carrier of the antibacterial component, and the antibacterial and antibacterial components are released during the decomposition of the antibacterial layer; the growth-promoting component comprises one or more of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF).

[0028] Further preferably, the growth-promoting layer is a pure polydopamine layer, and the antibacterial layer is a pure chitosan layer. The long-lasting antibacterial wound repair micro / nanofiber membrane can be used as a cell crawling scaffold. The tea phenol group on polydopamine has a specific affinity for amines and thiols on cells and tissues, which can increase the adhesion required for cell proliferation, enhance cell proliferation, and increase the speed of wound repair. The antibacterial components in the chitosan layer and the core layer all have antibacterial effects. The chitosan layer can be quickly released and adsorb negatively charged bacteria (due to the interaction between the positive charge in the chitosan molecules and the negative charge in the microbial cell membrane, the bacterial proteins and other cell components leak out and produce an antibacterial effect), participating in early antibacterial. After the chitosan layer and shell layer are degraded, the antibacterial components in the core layer are released and participate in late antibacterial, ensuring that bacteria are inhibited at a low concentration level. When there are no self-assembled growth-promoting layers and antibacterial layers on the surface of the coaxial micro / nanofiber, the antibacterial components can only be released after the shell layer is degraded. It is impossible to form sufficient antibacterial ability in the early stage and achieve a long-term antibacterial effect. If there are no antibacterial components in the core layer, the antibacterial effect can only be formed in the early stage, and the antibacterial efficiency drops to 50% after three days. The raw materials of the above-mentioned long-term antibacterial wound repair micro / nanofiber membrane are biomass molecules and degradable molecules, which have good biosafety; the long-term antibacterial wound repair micro / nanofiber membrane has good flexibility, can mimic the extracellular matrix, and can be applied to the wound surface to promote cell regeneration; the local synergistic release of antibacterial components avoids systemic medication.

[0029] In some specific embodiments provided by the present invention, the diameter of the composite micro / nano fiber is 50-500 nm, and further preferably 100-400 nm; the thickness of the long-lasting antibacterial wound repair micro / nano fiber membrane is 0.1-0.2 mm.

[0030] In some embodiments provided herein, the shell layer material further comprises a toughening component, comprising at least one of polyurethane, polycaprolactone, polystyrene, polyvinyl pyrrolidone, polymethyl methacrylate, polydimethyl terephthalate, polyvinyl alcohol, polyvinyl chloride, and polyvinylidene fluoride. The toughening component may also be a layered silicate, such as rectorite, capable of reinforcing biodegradable, biocompatible polymers.

[0031] In some embodiments provided herein, the shell layer utilizes a silk fibroin-polycaprolactone mixture with a weight ratio of 1:1 to 5. Preferably, the weight ratio of silk fibroin to polycaprolactone is 1:3. The long-lasting antibacterial wound repair micro / nanofiber membrane prepared under these conditions exhibits a strong tensile stress of 6.7 MPa, with an increase in tensile stress of up to 103%. When the weight ratio is lower than 1:5, the antibacterial components in the core layer are released too quickly, failing to achieve a long-lasting antibacterial effect. When the weight ratio is higher than 1:1, the degradation rate is slow, preventing the release of lysozyme and thus failing to achieve an antibacterial effect. Silk alone degrades too quickly and fails to provide sufficient material toughness. Polycaprolactone alone has poor biocompatibility and is unable to release the antibacterial components in the core layer. In some embodiments provided herein, the core layer includes an antibacterial component and may also include a carrier for the antibacterial component. The antibacterial component is preferably a biomacromolecule to prevent drug resistance. The biomacromolecule is preferably lysozyme, which has a high degree of safety and broad-spectrum antibacterial properties, killing bacteria by hydrolyzing the bacterial membrane. In the present invention, lysozyme is gradually released as the shell and chitosan layers degrade. The antibacterial component carrier can be one or more of chitosan, collagen, polyethylene glycol, silk fibroin, silk fibroin, polylactic acid, polyvinyl alcohol, and alginic acid.

[0032] In some embodiments provided herein, the number of bilayers of the growth-promoting layer and the antibacterial layer self-assembled on the coaxial micro / nanofiber surface is 5 to 15. When the number of bilayers is less than 5, the antibacterial duration of the long-lasting antibacterial wound-healing micro / nanofiber membrane is less than 14 days. When the number of bilayers is greater than 15, the antibacterial duration of the long-lasting antibacterial wound-healing micro / nanofiber membrane is very long, but the economic efficiency is poor. Preferably, the outermost layer of the composite micro / nanofiber is a polydopamine layer.

[0033] In the second aspect, the present invention provides a method for preparing the above-mentioned long-lasting antibacterial wound repair micro / nanofiber membrane, comprising: preparing coaxial micro / nanofiber by coaxial spinning technology to form a coaxial micro / nanofiber membrane; and assembling the growth-promoting layer and the antibacterial layer onto the surface of the coaxial micro / nanofiber by layer-by-layer self-assembly method.

[0034] In some specific embodiments provided by the present invention, coaxial micro / nano fibers are prepared by coaxial spinning technology, and the formation of a coaxial micro / nano fiber membrane includes: providing a shell solution and a core solution; injecting the shell solution into the outer tube of the coaxial needle, and injecting the core solution into the inner tube of the coaxial needle, and advancing the inner tube and the outer tube at a flow rate ratio of 1:3 to perform electrostatic spinning to obtain a coaxial micro / nano fiber membrane.

[0035] Specifically, the concentration of the biodegradable biocompatible polymer in the shell solution is 3 wt.% to 10 wt.%, the concentration of lysozyme in the core solution is 100 mg / mL, and the flow rates of the inner and outer tubes are 0.4 mL / h and 1.2 mL / h, respectively.

[0036] In some specific embodiments provided by the present invention, the use of the layer-by-layer self-assembly method to assemble the growth-promoting layer and the antibacterial layer onto the surface of the coaxial micro / nanofiber includes: alternately immersing the coaxial micro / nanofiber membrane in an acidic chitosan aqueous solution and an alkaline polydopamine solution several times, and washing away the unassembled chitosan or polydopamine with a NaCl solution after each immersion. Specifically, the concentration of the chitosan aqueous solution is 5-20 mg / mL, pH=4-6, preferably, the concentration is 10 mg / mL, pH=5; the concentration of the polydopamine solution is 5-20 mg / mL, pH=8-9, preferably, the concentration is 10 mg / mL, pH=8.5. The immersion time for each time is 10-20 minutes, preferably 15 minutes; the concentration of the NaCl solution is 0.05-0.2M, preferably 0.1M.

[0037] In some embodiments provided herein, the electrospinning voltage is 16 kV, the distance between the coaxial needle tip and the receiver is set at 16 cm, the spinning temperature is 25°C, and the relative humidity is 45%. The coaxial micro / nanofiber membranes prepared under these conditions exhibit uniform core-shell structures, consistent pore sizes, and well-controlled porosity.

[0038] The preparation method of the above-mentioned long-lasting antibacterial wound repair micro / nanofiber membrane is simple and time-saving; the layer-by-layer self-assembly method avoids problems such as structural weakening that may be caused by chemical cross-linking, and can enable the fiber membrane to maintain good mechanical properties and flexibility. The raw materials are economical and easy to obtain, avoiding excessive costs.

[0039] The use of the above-mentioned long-lasting antibacterial wound repair micro / nanofiber membrane in the preparation of wound dressings.

[0040] Unless otherwise specified, dopamine hydrochloride (DA-HCl, Mw = 189.64, purity 98%) used in the following examples was provided by Shanghai Aladdin Biochemical Technology Co., Ltd.; silk fibroin (SF, 99% purity) was purchased from CellMatrix; polycaprolactone (PCL, Mn = 80,000 kDa) was provided by Sigma Chemical Reagent Co., Ltd.; lysozyme (LY, 25,000 U / mg) was provided by Beijing Solebold Technology Co., Ltd.; chitosan (CS, Mw = 2.1 × 10 5 , deacetylation degree ≥ 90%) was provided by Shanghai Ruji Biotechnology Development Co., Ltd. All solvents used in the following examples were analytical grade and ultrapure water (18.2 MΩcm resistivity) without further purification.

[0041] Example 1: Preparation of long-lasting antibacterial wound repair micro / nanofiber membrane

[0042] S1: Preparation of coaxial micro / nanofibers by coaxial spinning technology to form coaxial micro / nanofiber membranes:

[0043] 0.5 g of each silk fibroin / polycaprolactone mixture with weight ratios of 1:1, 1:2, 1:3, 1:4, and 1:5 was dissolved in 9.5 g of hexafluoroisopropanol to prepare a 5% shell solution. Lysozyme was dissolved in 10 g of deionized water to prepare a core solution with a lysozyme concentration of 100 mg / mL. The shell solution was injected into the outer tube of a coaxial needle using a syringe pump, while the core solution was transferred to the inner tube of the coaxial needle using another syringe pump (Longge Precision Pump Co., Ltd., China). The flow rates of the inner and outer tubes were controlled at 0.4 mL / h and 1.2 mL / h, respectively. During coaxial electrospinning, a 16 kV voltage was applied between the needle and collector, and the distance between the needle and collector was 16 cm. The coaxial electrospinning operation was carried out at 25°C and 45% humidity. After spinning, a coaxial micro / nanofiber membrane was obtained. To remove the residual solvent, the prepared coaxial micro / nanofibrous membrane (abbreviated as SF / PCL) was freeze-dried in a freeze drying oven for 24 h.

[0044] S2: Assemble the growth-promoting layer and the antibacterial layer onto the surface of the coaxial micro / nanofiber using a layer-by-layer self-assembly method:

[0045] Chitosan was dissolved in water (10 mg / mL) and adjusted to pH 5 to produce positively charged chitosan molecules, resulting in an acidic chitosan solution. Dopamine hydrochloride was added to Tris buffer (pH 8.5) and stirred at room temperature for 2 hours to form an alkaline polydopamine solution with a concentration of 10 mg / mL. The dried SF / PCL was soaked in the chitosan solution for 15 minutes to form a chitosan layer on the surface of the coaxial micro / nanofiber. The unassembled chitosan was then washed away with 0.1M NaCl solution. The fiber membrane was then immersed in the polydopamine solution for 15 minutes to form a polydopamine layer on the chitosan layer, completing a cycle. The above operation was repeated several times to obtain a long-lasting antibacterial wound repair micro / nanofiber membrane, referred to as (CS / PDA). n , n represents the number of bilayers, and a bilayer consists of an adjacent chitosan layer and a polydopamine layer; for example, (CS / PDA)5 represents a long-lasting antibacterial wound repair micro / nanofiber membrane with 5 bilayers and the outermost layer being a polydopamine layer; (CS / PDA) 5.5 It represents a long-lasting antibacterial wound repair micro / nanofiber membrane with 5.5 double layers and a chitosan layer as the outermost layer.

[0046] Example 2: Verification Experiment

[0047] 1. Morphological Characterization

[0048] After the fiber membrane was gold-plated, field emission scanning electron microscopy (FE-SEM) (Zeiss, Germany) was used to characterize and analyze its surface morphology. Figure 1 As shown, the coaxial micro / nanofibers of SF / PCL exhibited a smooth and upright morphology ( Figure 1 c) and the (CS / PDA) formed after layer-by-layer self-assembly n The composite micro / nanofiber surface shows a network structure ( Figure 1 d), the diameter of the composite micro / nanofibers is 105.3 to 345.9 nm.

[0049] 2. Verify the assembly effect

[0050] The chemical composition of (CS / PDA)n was characterized by Fourier transform infrared spectroscopy (FT-IR) (ThermoNicolet 170-SX) after grinding with KBr and pressing into thin sheets. Figure 1 As shown in Figure e, the chitosan layer and polydopamine layer were successfully assembled on the surface of the coaxial micro / nanofiber.

[0051] 3.Evaluate electrical properties

[0052] (CS / PDA) n After water absorption, zeta potential analysis was performed using a Nano-25 zetasizer (Malvern, UK). When the outermost layer is a chitosan layer, the zeta potential is positive, while when the outermost layer is a polydopamine layer, the zeta potential is negative.

[0053] 4. Hydrophilicity evaluation

[0054] Hydrophilicity is one of the key indicators of cell viability. In this field, the hydrophilicity of fibers is measured by the change of water contact angle (WCA) over time. The present invention uses an optical contact angle goniometer (CAST3.0, KINO Industry Co., Ltd., USA) to record the water contact angle of the fiber to study the change in hydrophilicity of the assembled different numbers of bilayers. After testing, the WCA of SF / PCL was 88.5±4.3°, showing weak hydrophilicity, and became slightly hydrophilic (79.4±3.2°) within 5 seconds ( Figure 1 g). After the chitosan layer and the polydopamine layer are self-assembled layer by layer, the hydrophilic groups of the chitosan layer and the polydopamine layer are (CS / PDA) n The surface effect is internalized and the penetration of water droplets is stimulated, and an increase in hydrophilicity can be observed. (CS / PDA)5 and (CS / PDA) 10 The WCA of the CS / PDA were 58.8±3.8° and 44.9±2.6° at 0s, and (CS / PDA) 10 The water droplets disappeared within 5 seconds. This indicates that the multilayer structure obtained by layer-by-layer self-assembly can increase the accommodation space of the outer layer of the membrane and greatly improve the hydrophilicity.

[0055] 5. Tensile properties and adhesion test

[0056] The present invention uses a physical tester (n=3; ETM502A, China Co., Ltd.) to test (CS / PDA) n Tensile tests were conducted, and adhesion tests were performed on pigskin. Each test sample was 10 cm × 2 cm in size, and the thickness was measured using a thickness gauge. The tensile speed was controlled at 5 mm / min.

[0057] Due to (CS / PDA) n With high affinity structured surface ( Figure 3 h), (CS / PDA) n Shows good adhesion to human skin. After testing, in the dry state, (CS / PDA)5 and (CS / PDA) 10 The tensile strengths were 4.5±0.3MPa and 4.9±0.1MPa ( Figure 3 a); In the wet state, the tensile strength of (CS / PDA)5 is 5.1±0.9MPa, (CS / PDA) 10 The tensile strength is 6.7±0.3MPa, that is, with the increase of the number of double layers, (CS / PDA) n The tensile strength is enhanced ( Figure 3 b). This improvement in tensile strength may be attributed to the promotion of the chitosan layer and the polydopamine layer (CS / PDA) n At the same time, (CS / PDA) was detected in the 10th cycle test. n There is almost no loss in tensile strength. Even after 50 cycles, the tensile strength is still well maintained with only a loss of 12% ( Figure 3 c) indicates (CS / PDA) n Has good toughness. In addition, (CS / PDA) n For covering wrists, hands and elbows without any tightness or wrinkles ( Figure 3 df). Similarly, the adhesion performance test was conducted using pig skin, and it was found that (CS / PDA) n The strongest bonding strength exceeds 15kPa ( Figure 3 g).

[0058] 6. Antibacterial properties

[0059] The present invention uses Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus test (CS / PDA) n The antibacterial activity of the product was confirmed by the shake flask method and bacterial growth curve.

[0060] Shake flask method: First, prepare 4 mL of bacterial suspension (Gram-positive Staphylococcus aureus or Gram-negative Escherichia coli, 10 6 cfu·mL-1 ) solution, irradiate 2×2cm (CS / PDA) with ultraviolet light n After the two sides are stained, they are placed in a bacterial suspension and cultured in a shaking incubator at 37°C for 12 hours. The diluted bacterial suspension is then inoculated on LB (Luria-Bertani) solid medium and cultured at 37°C for 24 hours. Finally, the colonies are counted to evaluate (CS / PDA). n Antibacterial activity. Figure 2 d, CS / PDA) n The antibacterial performance was more than 90% within 24 hours. On the contrary, SF / PCL even promoted the proliferation of bacteria. Figure 2 As shown in g, two spherical Staphylococcus aureus and rod-shaped Escherichia coli cells have smooth and intact surfaces. After treatment with (CS / PDA)5, the cell surfaces showed obvious shrinkage (indicated by circles) ( Figure 2 h and k), in (CS / PDA) 10 After treatment, the cell membranes were severely damaged, and some cell membranes were even completely ruptured (indicated by yellow circles) ( Figure 2 i and l), that is, the increase in the number of double layers can improve (CS / PDA) n antibacterial activity.

[0061] Prepare bacterial growth curve: prepare 4 mL bacterial suspension (Gram-positive Staphylococcus aureus or Gram-negative Escherichia coli, 10 6 cfu·mL -1 ) solution, irradiated both sides of a 2×2 cm fiber membrane with ultraviolet light, placed it in a bacterial suspension, and stirred at 150 rpm at 37°C for 14 days as the experimental group. The control group only stirred the bacterial suspension without fiber membrane at 150 rpm at 37°C for 14 days. All samples were repeated three times. During the culture period, the turbid liquid of the bacterial suspension was collected, and the optical density (OD600) was measured at different time points, and these values ​​were calculated and converted into bacterial growth curves. The bacterial cells of different groups were centrifuged at 6000 rpm for 10 minutes, then washed and fixed with 2.5 wt.% glutaraldehyde solution at 4°C for 2 hours. The fixed samples were dehydrated with a gradient of ethanol solution with a concentration of 20%-100% every 10 minutes, and finally freeze-dried and observed by scanning electron microscopy.

[0062] The results showed that after the bacterial solution was treated with SF / PCL, the bacterial cells were healthy and almost unaffected, indicating that SF / PCL had no cytotoxicity to bacteria. 10 and (CS / PDA) 10.5The treated cell solution showed the smallest OD600 value, which was still clearly visible within 14 days. 5.5 Lower OD600 values ​​were observed in cultured samples after 14 days of signal processing because the positively charged chitosan molecules were rapidly released into the solution to attract negatively charged bacteria, thus controlling bacterial growth at an early stage. As the shell layer continued to degrade, LY was slowly released, ensuring bacterial killing.

[0063] 7. Evaluation of drug release capacity

[0064] In the present invention, 2×2 cm square fiber membranes were incubated with 4 mL PBS in a shaking incubator at 37°C for 14 days, and bacterial solutions without fiber membranes and bacterial solutions with SF / PCL were set as control groups ( Figure 2 b and c). The supernatants were collected and measured three times at each time point using an ELISA kit (ECH0086, China), and the long-term release efficiency of LY in five different fiber membranes was evaluated.

[0065] This example evaluated the effect of LY on SF / PCL and a series of (CS / PDA) n (the number of double layers is 5, 10 and 15 respectively) Figure 2 a) The results showed a good linear correlation between LY release and incubation time over a 14-day period, suggesting that LY release occurs continuously across different bilayers, resulting in long-term inhibition against Gram-positive Staphylococcus aureus and Gram-negative bacteria.

[0066] 8. Cytotoxicity assay

[0067] The fiber membrane was irradiated with UV light for 1 hour and cut into 6 mm diameter round pieces. After washing, they were placed at the bottom of a 96-well microtiter plate. L929 fibroblast suspension was dripped into each well and diluted to 3 × 10 cells per well. 3 cells. The cells were then cultured in a stable incubator at 37°C and 5% CO2 for 1 day, 3 days and 5 days, and cell viability was detected using CCK-8 (Solarbio, China). The absorbance of the samples at 450nm was measured by a microplate absorbance reader (iMark, BioRad, USA). All experiments were performed in triplicate. The p value of the statistical test was calculated by one-way analysis of variance (ANOVA), and any result was less than 0.05, indicating that there was a statistically significant difference between the two groups. The activity of L929 cells after treatment with different fiber membranes was tested using a CCK-8 kit.

[0068] Live / dead assay was performed using Calcein AM / PI double staining kit (Solarbio, China) to investigate the activity of L929 cells in (CS / PDA)n Viability on sterile (CS / PDA) n L929 cells were placed in a 48-well culture plate at a density of 1×10 4 The cells were seeded at a density of 100 cells / mL and incubated for 48 hours (37°C, 5% CO2). After removing the residual culture medium, 150 μL of buffer containing calcein-AM and PI was added and incubated with L929 cells in the dark according to the manufacturer's instructions. The fluorescence of the cells was observed by fluorescence microscopy (Olympus BX53). n After 48 hours of inoculation with cells, the cells were extracted and the cell morphology and attachment status were observed. n The samples were fixed with 4% glutaraldehyde at 4°C for 2 h, washed three times with PBS, and finally dehydrated with ethanol solution with a gradient concentration of 20%-100% for 10 min. FE-SEM observation (CS / PDA) n Cellular changes on.

[0069] The results showed that after 1 day and 5 days of treatment with SF / PCL, the survival rates of L929 cells were 56±5% and 74±4%, respectively ( Figure 4 a), while after 1 day and 5 days of treatment with (CS / PDA)5, the activity was only 61±3% and 82±4%. (CS / PDA) 10 After 1 day and 5 days of treatment, the activity was only 66±3% and 94±4%. n When treated with SF / PCL, the observed cell viability was stronger than that of cells treated with SF / PCL due to the increase of (CS / PDA) 10 The number of layers on the surface can improve its activity, presumably because the self-assembled mesh surface can coat cells to enhance cell proliferation.

[0070] Live cell and dead cell test results Figure 4 As shown in be, compared with SF / PCL, (CS / PDA) 10 and (CS / PAD) 10.5 The density of incubated L929 cells increased, indicating that (CS / PPDA)5 and (CS-PDA)5.5 had good biocompatibility and further verified the detection results of CCK-8 kit. n Due to the presence of multiple double-layered reticular structures, L929 cells can grow on SF / PCL and (CS / PDA) n ( Figure 4 fi) extensively migrates on the surface. PDA on (CS / PDA) nThe catechol groups provided on the surface enhance the active binding ability of cells and promote cell affinity. Combined with the ECM structure and the positively charged CS, (CS / PDA) n It is very suitable for promoting cell proliferation and laying the foundation for cell development.

[0071] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A long-lasting antibacterial wound repair micro / nanofiber membrane, characterized by: The long-lasting antibacterial wound repair micro / nanofiber membrane is composed of composite micro / nanofibers, which include coaxial micro / nanofibers and growth-promoting layers and antibacterial layers that are self-assembled layer by layer on the coaxial micro / nanofibers; the coaxial micro / nanofibers include a shell layer and a core layer, the shell layer uses a silk-polycaprolactone mixture with a weight ratio of silk to polycaprolactone of 1:1 to 5 as the shell material, and the core layer includes an antibacterial component; the growth-promoting layer is a pure polydopamine layer, the antibacterial layer is a pure chitosan layer, and the number of double layers of the growth-promoting layer and the antibacterial layer self-assembled layer by layer on the surface of the coaxial micro / nanofibers is 5 to 15; the outermost layer of the composite micro / nanofibers is a polydopamine layer.

2. The method for preparing the long-lasting antibacterial wound repair micro / nanofiber membrane according to claim 1, characterized in that: include: Coaxial micro / nanofibers are prepared by coaxial spinning technology to form coaxial micro / nanofiber membranes; The growth-promoting layer and the antibacterial layer are assembled onto the surface of the coaxial micro / nanofiber using a layer-by-layer self-assembly method.

3. Use of the long-acting antibacterial wound repair micro / nanofiber membrane according to claim 1 in the preparation of wound dressings.

Images