Coaxial electrostatic spinning nanofiber membrane containing tannic acid coating as well as preparation method and application of coaxial electrostatic spinning nanofiber membrane
By using automated coaxial electrospinning process and impregnation method to prepare nanofiber membranes with tannin coatings, the problem of insufficient antibacterial properties and biocompatibility of existing dressings is solved, and strong mechanical properties are achieved and promote wound healing effects.
Patent Information
- Application Number
- CN202411139804.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-20
- Publication Date
- 2024-12-06
AI Technical Summary
Existing wound dressings have multiple functional problems in promoting wound healing, such as poor antibacterial performance, insufficient biocompatibility, poor mechanical performance and inability to effectively participate in wound healing, and traditional antibacterial materials have risks of drug resistance and cytotoxicity.
Using automated coaxial electrospinning process and simple impregnation method, a fiber membrane with PLGA core and PCL shell was prepared, and tannin (TA) polymerization and cross-linking were induced on the surface of the fiber membrane to form a TA coating to form an antioxidant, Coaxial electrospinned nanofiber membrane with antibacterial effect.
It achieves high biocompatibility, strong mechanical properties, excellent antibacterial and antioxidant abilities, promotes wound healing, reduces the mass loss of fiber membranes during the degradation process, and significantly improves the wound healing speed.
Smart Images
Figure CN119074985A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of medical dressings, and in particular to a coaxial electrostatic spinning nanofiber membrane containing a tannic acid coating, and a preparation method and application thereof. Background Art
[0002] The skin provides a protective function as a barrier, resisting various challenges and responding appropriately to dangers. Disruption or dysfunction of skin integrity can expose sensitive tissues to physical, chemical and pathogenic microbial invasions, which can seriously affect individual health. As a global public health issue, skin trauma has always been the focus of clinical treatment. Wound healing is a complex process consisting of four consecutive physiological stages involving wound bleeding, inflammatory response, cell proliferation and tissue remodeling. During this period, bacteria can easily infect the wound, leading to increased inflammation and hindering wound healing.
[0003] At present, gauze is the main dressing commonly used in clinical practice. During the healing process, due to the large pores of synthetic cellulose, it is unable to prevent external bacteria from entering. More importantly, gauze cannot inhibit bacteria already present in the wound. Traditional wound dressings do not have antibacterial properties and require oral or injected antibiotics to achieve antibacterial effects. However, with the increasing frequency of antibiotic use, antibiotic resistance is becoming more and more serious. In addition, existing antibacterial materials usually have the disadvantages of poor biocompatibility and high cytotoxicity, which is also not conducive to wound healing.
[0004] Electrospinning technology is considered to be the most suitable method for producing nonwoven fiber porous structures. The nanofiber membranes produced by it are characterized by ultrafine continuous fibers, high surface-to-volume ratio, high porosity and variable pore size distribution. Electrospun nanofiber membranes have excellent performance as wound dressings. First, the ductility and tensile strength of electrospun nanofiber membranes are similar to those of human skin, so they can fit well with the wound site. Electrospun nanofiber membranes can simulate the biological functions of the extracellular matrix (ECM), which provides an ideal microenvironment for cell adhesion, proliferation and further differentiation. Due to its high porosity, electrospun wound dressings help tissue cells to exchange gas and liquid, avoiding excessive dehydration and drying of wounds. However, the electrospun fiber membranes currently prepared have a relatively simple structure and a relatively single function. They cannot participate in all stages of wound healing, which limits their ability to heal wounds. An ideal wound dressing should have multiple functions of anti-inflammatory, antibacterial, antioxidant and tissue regeneration to accelerate wound healing.
[0005] The Chinese invention patent with publication number CN106075539A discloses a method for preparing a nanofiber membrane with a core / shell structure loaded with the Chinese medicine Centella asiatica glycoside and its application in wound dressing. Poly(lactic-co-glycolic acid) (PLGA) is used as the shell, and polycaprolactone (PCL) loaded with Centella asiatica glycoside is used as the inner core to prepare a core / shell structure drug-loaded nanofiber membrane by coaxial electrospinning. The inventive method has the characteristics of easy access to raw materials, simple operation, strong controllability, and easy large-scale industrial production. However, for the components of its structure, when PCL is used as the core layer, the good drug loading potential of PLGA is not fully utilized, and when used as the shell layer, the mechanical properties of PLGA are not as good as PCL. The thin and light wound dressing leads to poor mechanical properties, and it is not adapted to the stretching of the skin during use and causes damage. The structure and function of the invention still have room for further optimization.
[0006] The Chinese invention patent with publication number CN107881650A provides a method and application of preparing a nanofiber membrane with a core / shell embedding structure by coaxial double-layer electrospinning. The method adopts coaxial double-layer electrospinning technology to prepare a nanofiber membrane with a core / shell structure, and selects the main components in the skin extracellular matrix as raw materials, wherein the core is a degradable synthetic polymer material carrying type I collagen, and the shell is a degradable synthetic polymer material carrying fibrinogen. The preparation method of the invention is simple and easy, and the prepared nanofiber membrane has good biocompatibility and biodegradability. With the degradation of the shell material in the nanofiber and the exposure of the core material, the dynamic biomimetic of the component changes in the skin wound repair process can be achieved, and the generation of normal skin tissue can be promoted. However, the effect of degradation is also bidirectional. When the porosity of the wound dressing is not suitable (including improper processing method or self-degradation), it is easy to cause the wound to dry. And when the hydrophilicity of the dressing is insufficient, its poor water absorption performance is also easy to cause the wound to be too wet and affect the wound healing.
[0007] In summary, providing a wound dressing with good antibacterial properties, high biocompatibility, low cytotoxicity, the ability to promote angiogenesis and good mechanical properties has positive significance for promoting wound healing. Summary of the invention
[0008] In view of the above-mentioned defects of the prior art, in a first aspect of the present invention, a method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating having a simple process and suitable for large-scale production is provided, comprising the following steps: (1) dissolving poly(lactic acid-co-glycolic acid) copolymer in hexafluoroisopropanol (HFIP) to prepare a core layer spinning solution; dissolving poly(caprolactone) in hexafluoroisopropanol to prepare a shell layer spinning solution; (2) Spinning the core layer spinning solution and the shell layer spinning solution into a fiber membrane formed by fibers having a core layer of polylactic acid-glycolic acid copolymer and a shell layer of polycaprolactone through a coaxial electrospinning device; drying the fiber membrane to obtain a coaxial electrospun fiber membrane; (3) The pH of a tannic acid (TA) aqueous solution is adjusted to alkaline to obtain a coating treatment solution; the coaxial electrospun fiber membrane is immersed in the coating treatment solution for treatment, and then washed with water to remove unpolymerized tannic acid and induce its molecules to form cross-links; and then dried to obtain a coaxial electrospun nanofiber membrane containing a tannic acid coating.
[0009] Based on the above technical scheme, the concept and principle of the present invention is to first prepare a fiber membrane with a PLGA core and a PCL shell by using an automated coaxial electrospinning process and a simple impregnation method, and then induce TA polymerization and cross-linking on the surface of the fiber membrane to form a TA coating to obtain a coaxial electrospun nanofiber membrane containing a tannic acid coating. PCL exhibits excellent mechanical properties and is suitable for use as a shell material in the present invention to improve mechanical strength. TA molecules have hydrophilic groups, and the abundant ortho-hydroxyl groups in the molecules can release hydrogen to scavenge free radicals, block chain reactions and inhibit lipid peroxidation. Its natural polyphenol structure can kill bacteria by destroying bacterial cell membranes and inhibiting bacterial enzyme activity, thereby achieving antioxidant and antibacterial effects.
[0010] It should be noted that in a similar technical field, the prior art has a technical solution (CN118240245A) for preparing chitosan-polyvinyl alcohol-tannic acid linked network hydrogel by impregnation using TA solution. TA partially plays a role in promoting cross-linking and enhancing antioxidant and antibacterial functions in such hydrogels. However, in the present invention, the inventors use TA to prepare the coating, and the design also uses the polyphenol groups of TA to interact with the polyphenols of the cell's mucin to cause molecular interactions, which is manifested as cell adhesion in the macroscopic sense. The TA coating can be used to increase cell adhesion and provide more contact sites for cells, thereby enhancing the migration ability of cells. As a result, the electrospun fiber membrane with TA coating better simulates the structure of ECM, is suitable for cell adhesion and growth, and enables the wound to heal faster. Not only that, the inventors use the TA coating to squeeze the coaxial spinning fiber during the formation process, making the spinning structure tighter, which is manifested as convergence. This phenomenon means that the diameter of the spinning fiber does not increase significantly when a layer of coating is added, which reduces the contact with the solution and reduces the adverse effects caused by degradation. When damage or degradation occurs, the TA coating exhibits self-repairing behavior due to the adsorption between molecules and can also reduce the mass loss of the fiber membrane.
[0011] In the coaxial electrospinning process, the concentration of the spinning solution can affect the morphology, structure and properties of the final fiber. The concentration of the spinning solution directly affects the viscosity and surface tension of the spinning solution, which determine the stability of the spinning solution under the action of the electric field and the fiber formation process. If the concentration is too low, the viscosity of the spinning solution will decrease, which may cause the spinning solution to be unstable at the nozzle, forming irregular fibers or droplets, affecting the continuity and uniformity of the fiber. On the contrary, if the concentration is too high, the viscosity of the spinning solution increases and the surface tension increases, which may hinder the injection of the spinning solution, resulting in difficulties in spinning, or even if the spinning is successful, fibers with larger diameters and uneven structures may be obtained. In addition, the concentration of the spinning solution also affects the mechanical properties and functionality of the fiber. For example, when preparing fibers for drug carriers or bioactive substance delivery systems, a suitable concentration can ensure the effective loading and controlled release of drugs. Therefore, in order to obtain ideal coaxial electrospun fibers, it is appropriate to control the concentrations of the core layer and shell layer spinning solutions within a suitable range to optimize the spinning process and ensure the quality and functionality of the final product.
[0012] Preferably, in step (1), in the core layer spinning solution, the concentration of polylactic acid-glycolic acid copolymer is 15 wt.%-25 wt.%; and in the shell layer spinning solution, the concentration of polycaprolactone is 10 wt.%-12 wt.%.
[0013] During the coaxial electrospinning process, removing bubbles from the spinning solution helps to further optimize the quality of the final fiber. Bubbles may cause discontinuity in the spinning solution, thereby affecting the uniformity and continuity of the fiber. When bubbles pass through the nozzle, they may destroy the uniform electric field of the spinning solution, resulting in interruptions in the spinning process or spinning defects. In addition, the rupture of bubbles in the spinning solution may produce splashes, which not only pollutes the preparation environment but also may damage the electrospinning equipment. Bubbles may also be trapped inside the fiber during the coagulation of the spinning solution, forming voids or weak points, which will weaken the mechanical properties and stability of the fiber. Therefore, before coaxial electrospinning, the spinning solution needs to be degassed to ensure the smooth spinning process and obtain high-quality fiber products.
[0014] Preferably, in the step (1), during the preparation of the core layer spinning solution and the shell layer spinning solution, stirring is performed for 15-45 minutes, and then the solution is allowed to stand to remove bubbles.
[0015] Preferably, the specific operation of step (2) is as follows: the core layer spinning solution and the shell layer spinning solution are respectively sucked into the syringe of the coaxial electrospinning device, and then the syringe is placed in the corresponding micro pump; the feeding speed of the core layer spinning solution and the shell layer spinning solution is controlled by the micro pump, so that the two solutions are transported from the syringe to the coaxial spinning needles of the core layer and the shell layer, and the distance between the coaxial spinning needle and the substrate is adjusted to a set value; then the power supply is turned on, the voltage and spinning time are adjusted, and the temperature and relative humidity of the coaxial electrospinning are controlled; after the coaxial electrospinning is completed, the fiber membrane formed by the fiber whose core layer is polylactic acid-hydroxyacetic acid copolymer and whose shell layer is polycaprolactone is removed from the surface of the substrate, and the coaxial electrospun fiber membrane is obtained after drying.
[0016] In the coaxial electrospinning process, the injection feed rate determines the rate at which the solution flows out, which in turn affects the stability of the spinning jet and the final fiber morphology. If the feed rate is too fast, the solvent may not evaporate in time, causing the fibers to stick together or form irregular structures during the landing process. On the contrary, if the feed rate is too slow, the spinning jet may be unstable, resulting in uneven fiber diameter or spinning interruption. The appropriate feed rate can ensure that the solution has a suitable viscosity and surface tension when it leaves the nozzle, so that a stable Taylor cone can be formed under the action of the electric field, thereby producing continuous and uniform fibers.
[0017] Further preferably, the feeding rate of the core layer spinning solution is 5-15 μL / min; and the feeding rate of the shell layer spinning solution is 15-25 μL / min.
[0018] The distance between the spinneret of the coaxial spinning needle and the base receiver determines the degree of stretching of the fiber during flight, which in turn affects the microstructure and mechanical properties of the fiber. If the distance is too close, the fiber may not be fully stretched, resulting in thicker fibers; if the distance is too far, the fiber may break or sag due to gravity, affecting the continuity of spinning and the orientation of the fiber. Based on the application field of the present invention, the distance is preferably controlled at 14-18 cm.
[0019] Further preferably, the distance between the coaxial spinning needle and the substrate is 14-18 cm.
[0020] Higher equipment voltage can generate stronger electric field forces, which help to stretch and dissociate the spinning solution, thereby forming finer fibers. However, too high a voltage may cause corona discharge, which will not only disrupt the spinning process but also damage the spinning equipment. In addition, too high a voltage may also cause the fibers to have a beaded morphology and inhomogeneity, affecting the quality of the fibers. The spinning time, that is, the duration of the solution or molten polymer forming fibers under the action of the electric field, also needs to be precisely controlled. Spinning time that is too short may result in the fiber not being fully stretched and solidified, thereby affecting the mechanical properties and structural uniformity of the fiber. On the contrary, spinning time that is too long may cause the fiber to be overstretched, increasing the risk of breakage, and may also affect the quality of the fiber due to the accumulation of solvents or heat.
[0021] Further preferably, the voltage is 17-19 kV; and the spinning time is 5-7 h.
[0022] Changes in temperature and relative humidity affect the stability of the Taylor cone during spinning, and thus affect the quality of spinning. Temperature has a significant effect on the solvent evaporation rate and the viscosity of the polymer solution during electrospinning. Higher ambient temperatures accelerate the volatilization of the solvent and reduce the viscosity and surface tension of the polymer solution, which may lead to enhanced stability of the electrospinning jet and facilitate the formation of fibers with smaller diameters. However, if the temperature is too high, the solution may dry out or the viscosity may increase sharply at the spinneret, which may easily clog the spinneret and prevent spinning from proceeding smoothly. Relative humidity affects the solvent evaporation rate during electrospinning and the charge and dielectric constant at the needle tip during electrospinning. Lower ambient humidity increases the solvent volatilization rate, which may lead to faster fiber curing rate, while higher humidity reduces the solvent volatilization rate and affects the collection and quality of the fiber. Changes in humidity also affect charge dissipation and solvent evaporation during electrospinning, which in turn affects the morphology and mechanical properties of the fiber.
[0023] Further preferably, the temperature of the coaxial electrospinning is 22-27° C., and the relative humidity is 45%-55%.
[0024] Preferably, in step (2), the drying temperature is 36-38°C and the drying time is 20-28 h.
[0025] Too low a concentration of TA may result in an incomplete or too thin coating, which cannot provide the expected coverage effect. On the contrary, too high a concentration may result in a coating that is too thick, which increases the burden on the substrate, reduces the adhesion of the coating, and may cause the coating to crack or peel off. An appropriate TA concentration helps ensure that the coating is evenly distributed on the substrate surface. Under the design concept of the present invention, a coating that is too thin will not have an adequate convergence effect on the fiber; while a coating that is too thick will result in a larger overall fiber diameter.
[0026] Preferably, in step (3), the concentration of the tannic acid aqueous solution is 0.8-1.2 mg / mL.
[0027] The cross-linking of TA mainly relies on the self-polymerization phenomenon of forming covalent bonds between TA molecules, which can be carried out at room temperature without external induction. In addition, this covalent bond connection is most efficient at a pH of 8-9, while it is difficult to form a coating structure when the pH is less than 7.5.
[0028] Preferably, in step (3), the pH of the tannic acid aqueous solution is adjusted to 8-9.
[0029] The self-polymerization of TA can be carried out at room temperature. If the immersion treatment time is too short, the coating may not be fully formed or the formed coating may not be uniform enough. If the treatment time is too long, the coating may be too thick or cracks may appear, reducing the adhesion and protective performance of the coating.
[0030] Preferably, in step (3), the coaxial electrospun fiber membrane is immersed in the coating treatment solution for treatment at room temperature, and the treatment time is 20-28 h.
[0031] Preferably, in step (3), the drying temperature is 36-38°C and the drying time is 20-28 h.
[0032] The drying of the present invention is carried out under the normal body temperature range of the human body. The fiber membrane dried at this temperature is closer to the actual biocompatible environment, which helps to reduce the stress caused by temperature differences, thereby improving the stability of the material and the safety of use.
[0033] In the second aspect of the present invention, there is provided a coaxial electrospun nanofiber membrane containing a tannic acid coating which has excellent mechanical properties, hydrophilicity, good antibacterial and antioxidant abilities and can promote angiogenesis. The membrane is prepared by the method of the first aspect of the present invention.
[0034] In the third aspect of the present invention, there is provided an application of the coaxial electrospun nanofiber membrane containing a tannic acid coating according to the second aspect of the present invention, specifically, an application as a dressing in promoting wound healing.
[0035] Compared with the prior art, the present invention has the following advantages and beneficial effects: The invention provides a method for preparing a coaxial electrospinning nanofiber membrane containing a tannic acid coating, which adopts an automated coaxial electrospinning process and a simple impregnation method, has the advantages of simple preparation and a wide source of raw materials, and is suitable for large-scale production.
[0036] The present invention provides a coaxial electrospun nanofiber membrane containing a tannic acid coating, which utilizes the tannic acid coating to converge the fiber membrane structure and has the advantages of good biocompatibility, strong antibacterial and antioxidant capabilities and mechanical properties, high hydrophilicity, and can significantly promote wound healing.
[0037] The present invention provides an application of a coaxial electrospun nanofiber membrane containing a tannic acid coating, which has broad application prospects as a dressing in promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 Fourier transform infrared spectoscopy (FT-IR) of PLGA, PCL, TA, PLGA-PCL, and PLGA-PCL-TA; Figure 2 The images of PLGA-PCL and PLGA-PCL-TA observed under scanning electron microscope (SEM) at different magnifications; Figure 3 This is a transmission electron microscope (TEM) observation image of PLGA-PCL-TA; Figure 4 Degradation curves of PLGA-PCL and PLGA-PCL-TA; Figure 5 are stress-strain curves of PLGA-PCL and PLGA-PCL-TA; Figure 6 The water vapor transmission rate test results of gauze, PLGA-PCL, and PLGA-PCL-TA; Figure 7 The water contact angle test results of PLGA-PCL and PLGA-PCL-TA; Figure 8It is the absorbance at 450 nm of the control group and the intervention group at the set culture days in the cell proliferation experiment; Fig. 9 These are the test results of the control group and the intervention group in the cell migration experiment; Fig.10 These are the test results of the control group and the intervention group in the experiment of active oxygen scavenging ability of 2',7'-dichlorofluoresceindiacetate (DCFH-DA); Fig.11 The control group and the intervention group were tested for their antibacterial properties against Escherichia coli ( E. coli , EC), Staphylococcus aureus ( S. aureus , SA) antimicrobial test results; Fig.12 It is the healing result of the control group and the intervention group in the wound healing experiment after the set recovery days; Fig.13 These are the test results of the control group and the intervention group in the cell adhesion experiment. DETAILED DESCRIPTION
[0039] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0040] In the following embodiments: PLGA (75:25) was purchased from Carbon Water Technology (Guangzhou, China); PCL (80000 MW), TA (1701.2 MW), and HFIP were purchased from McLean Company (Shanghai, China).
[0041] Example 1 The method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating comprises the following steps: (1) PLGA was added to HFIP to prepare a core spinning solution with a concentration of 20 wt.%, and after adding a magnet, the solution was stirred on a magnetic stirrer for 30 min and allowed to stand to remove bubbles. PCL was added to HFIP to prepare a shell spinning solution with a concentration of 12 wt.%, and after adding a magnet, the solution was stirred on a magnetic stirrer for 30 min and allowed to stand to remove bubbles. (2) The core layer spinning solution and the shell layer spinning solution were respectively sucked into syringes with a volume of 5 mL, and the syringes were placed on two different micropumps of the coaxial electrospinning device; the micropump of the core layer spinning solution was set to a feeding rate of 10 μL / min, and the micropump of the shell layer spinning solution was set to a feeding rate of 20 μL / min; the syringes were connected to the coaxial spinning needles of the core layer and the shell layer through catheters, and the distance between the needles and the aluminum foil was set to 16 cm; the power of the equipment was turned on, the voltage was set to 18 kV, the spinning time was 6 h, and the temperature and relative humidity during the electrospinning process were set to 25 ℃ and 50%, respectively; after the spinning was completed, the fiber membrane was peeled off the aluminum foil and dried in a drying oven at 37 ℃ for 24 h to obtain a coaxial electrospun fiber membrane, which was recorded as PLGA-PCL (PP); (3) TA was dissolved in deionized water to prepare a TA aqueous solution with a concentration of 1 mg / mL, and the pH of the solution was adjusted to 8.5 using NaOH to obtain a coating treatment solution; the coaxial electrospun fiber membrane was then placed in the coating treatment solution and stirred and immersed for 24 h at room temperature; after the treatment, it was rinsed with deionized water at pH = 7 to remove the unpolymerized tannic acid and induce its molecules to form cross-links; it was then placed in a drying oven at 37 °C for 24 h to obtain a coaxial electrospun nanofiber membrane containing a tannic acid coating, which was recorded as PLGA-PCL-TA (PPT).
[0042] FT-IR was used to characterize the raw materials PLGA, PCL, TA, and the prepared PLGA-PCL and PLGA-PCL-TA. Figure 1 As shown. Figure 1 It can be seen that PLGA-PCL has the characteristic groups of PLGA and PCL, specifically the PLGA polymer at 1756 cm -1 and 1455 cm -1 The C=O stretching and asymmetric subunit characteristic peaks at 2942 cm -1 and 1723 cm -1 On this basis, PLGA-PCL-TA also has the characteristic peaks of TA, specifically 3290 cm -1 and 1532 cm -1 The characteristic peaks of phenolic hydroxyl group and C=C stretching at , indicating that the coaxial electrospun nanofiber membrane containing tannic acid coating was successfully prepared.
[0043] The microstructure of the prepared PLGA-PCL and PLGA-PCL-TA was observed by SEM. Figure 2 As shown; TEM was used to further observe the microstructure of PLGA-PCL-TA, and the results were as follows Figure 3As shown. Figure 2 It can be seen that the prepared PLGA-PCL and PLGA-PCL-TA have uniform diameters, good orientation, no residual droplets, and good porosity. Figure 3 It can be seen that the PLGA-PCL-TA nanofibers have an obvious three-layer structure, which are the innermost coaxially spun core fiber PLGA, the coaxially spun shell fiber PCL and the coating TA.
[0044] Example 2 The method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating comprises the following steps: (1) PLGA was added to HFIP to prepare a core spinning solution with a concentration of 15 wt.%, and after adding a magnet, the solution was stirred on a magnetic stirrer for 15 min and allowed to stand to remove bubbles; PCL was added to HFIP to prepare a shell spinning solution with a concentration of 10 wt.%, and after adding a magnet, the solution was stirred on a magnetic stirrer for 15 min and allowed to stand to remove bubbles; (2) The core layer spinning solution and the shell layer spinning solution were respectively sucked into syringes with a volume of 5 mL, and the syringes were placed on two different micropumps of the coaxial electrospinning device; the micropump of the core layer spinning solution was set to a feeding rate of 15 μL / min, and the micropump of the shell layer spinning solution was set to a feeding rate of 25 μL / min; the syringes were connected to the coaxial spinning needles of the core layer and the shell layer through catheters, and the distance between the needles and the aluminum foil was set to 16 cm; the power of the equipment was turned on, the voltage was set to 17 kV, the spinning time was 5 h, and the temperature and relative humidity during the electrospinning process were set to 27 ℃ and 55%, respectively; after the spinning was completed, the fiber membrane was peeled off the aluminum foil and dried in a drying oven at 36 ℃ for 28 h to obtain a coaxial electrospinning fiber membrane; (3) TA was dissolved in deionized water to prepare a TA aqueous solution with a concentration of 0.8 mg / mL, and NaOH was used to adjust the pH of the solution to 8 to obtain a coating treatment solution; then the coaxial electrospun fiber membrane was placed in the coating treatment solution at room temperature, stirred and immersed for 28 h; after the treatment, it was rinsed with deionized water with a pH of 7 to remove the unpolymerized tannic acid and induce its molecules to form cross-links; then it was placed in a drying oven at 36 °C for 28 h to obtain a coaxial electrospun nanofiber membrane containing tannic acid coating.
[0045] Example 3 The method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating comprises the following steps: (1) PLGA was added to HFIP to prepare a core spinning solution with a concentration of 25 wt.%, and after adding a magnet, the solution was stirred on a magnetic stirrer for 45 min and allowed to stand to remove bubbles; PCL was added to HFIP to prepare a shell spinning solution with a concentration of 15 wt.%, and after adding a magnet, the solution was stirred on a magnetic stirrer for 45 min and allowed to stand to remove bubbles; (2) The core layer spinning solution and the shell layer spinning solution were respectively sucked into syringes with a volume of 5 mL, and the syringes were placed on two different micro pumps of the coaxial electrospinning device; the micro pump of the core layer spinning solution was set to feed at a rate of 5 μL / min, and the micro pump of the shell layer spinning solution was set to feed at a rate of 15 μL / min; the syringes were connected to the coaxial spinning needles of the core layer and the shell layer through catheters, and the distance between the needles and the aluminum foil was set to 18 cm; the power of the device was turned on, the voltage was set to 19 kV, the spinning time was 7 h, and the temperature and relative humidity during the electrospinning process were set to 27 ℃ and 45%, respectively; after the spinning was completed, the fiber membrane was peeled off the aluminum foil and dried in a drying oven at 38 ℃ for 20 h to obtain a coaxial electrospinning fiber membrane; (3) TA was dissolved in deionized water to prepare a TA aqueous solution with a concentration of 1.2 mg / mL, and NaOH was used to adjust the pH of the solution to 9 to obtain a coating treatment solution; then the coaxial electrospun fiber membrane was placed in the coating treatment solution and stirred and immersed for 20 h at room temperature; after the treatment, it was rinsed with deionized water with a pH of 7 to remove the unpolymerized tannic acid and induce its molecules to form cross-links; then it was placed in a drying oven at 38 °C for 20 h to obtain a coaxial electrospun nanofiber membrane containing tannic acid coating.
[0046] Example 4 This example studies the degradation performance of the coaxial electrospun nanofiber membrane containing tannic acid coating in application. The PLGA-PCL and PLGA-PCL-TA prepared in Example 1 were cut into 5 cm × 5 cm × 0.2 cm cubes, and each was placed in a 50 mL centrifuge tube filled with phosphate buffered saline (PBS). The electrospun nanofiber blocks were taken out at predetermined intervals, and the surface moisture was removed and the mass was weighed. The degradation curves of PLGA-PCL and PLGA-PCL-TA are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that compared with the simple coaxial electrospun fiber PLGA-PCL, the prepared PLGA-PCL-TA nanofibers with TA coating are slower throughout the degradation process. This is because the presence of TA coating converges the structure of electrospinning, and the self-healing properties of TA also slow down the mass loss of degradation.
[0047] Example 5 This example studies the tensile properties of the coaxial electrospun nanofiber membrane containing tannic acid coating in application. The PLGA-PCL and PLGA-PCL-TA prepared in Example 1 were cut into strips of 8 cm × 2 cm × 0.2 cm, respectively, and the stress-strain curves of the two samples were measured using a universal electronic testing machine. The results are shown in Figure 2. Figure 5 As shown. Figure 5 It can be seen that PLGA-PCL-TA exhibits stronger tensile resistance and can resist greater stress under the same strain. The introduction of TA coating helps to enhance the mechanical properties of the nanofiber membrane.
[0048] Example 6 This example studies the water vapor permeability of the coaxial electrospun nanofiber membrane containing tannic acid coating in application. Taking water vapor permeability as the test index, PLGA-PCL and PLGA-PCL-TA prepared in Example 1 were used as test objects, and medical gauze was used as a control; PLGA-PCL, PLGA-PCL-TA, and gauze were cut into 1 cm × 1 cm × 0.2 cm cubes. 5 mL of normal saline was added to a 10 mL open glass bottle, and the cubes made of the sample were covered on the mouth of the glass bottle. The glass bottle was weighed and placed in a 37 ° C oven, and the glass bottle was weighed after 24 h.
[0049] Water vapor transmission rate is measured by the following formula: Water vapor transmission rate (gm -2 day -1 ) = (W1-W0) / S; Where W0 represents the initial mass; W1 represents the mass after 24 h, and S represents the opening area of the glass bottle.
[0050] The test results of water vapor transmission rate are as follows: Figure 6 As shown. The high water vapor permeability of wound dressings makes wound exudate evaporate easily, which cannot maintain a moist environment on the wound surface and hinders wound healing. On the contrary, low water vapor permeability is not conducive to the gas-liquid exchange of tissue cells, slowing down cell proliferation and the growth of granulation tissue. Figure 6 It can be seen that the presence of the TA coating increases the diameter of the nanofibers, slightly weakens the porosity, reduces the evaporated water vapor, and is closer to the ideal water vapor permeability.
[0051] Example 7 This example studies the hydrophilicity of the coaxial electrospun nanofiber membrane containing tannic acid coating in application. The PLGA-PCL and PLGA-PCL-TA prepared in Example 1 were cut into 1 cm × 1 cm × 0.2 cm cubes, and the water contact angles of the two samples were measured using a water contact angle meter. The results are shown in Figure 2. Figure 7 As shown. Figure 7 It can be seen that the water contact angle of PLGA-PCL-TA nanofibers containing TA coating is smaller, indicating that the presence of TA coating increases the hydrophilicity of coaxial electrospun nanofibers.
[0052] Example 8 This example studies the effect of coaxial electrospun nanofiber membranes containing tannic acid coatings on cell proliferation in applications. The design and method of the experiment are as follows: Control group: L929 cells (mouse fibroblasts) were cultured in DMEM (dulbecco's modified eagle medium) high glucose medium with a serum mass fraction of 10%. The medium was changed every 2 days and the cells were passaged every 4 days.
[0053] Intervention group: The PLGA-PCL-TA prepared in Example 1 was cut into small cubes of 2 cm×2 cm×2 cm, immersed in DMEM high-glucose medium for 72 h, and used to culture L929 cells, with the medium changed every 2 days and passaged every 4 days.
[0054] CCK-8 reagent was used to measure the absorbance at 450 nm of cells in the control group and the intervention group (PLGA-PCL-TA) cultured in DMEM high-glucose medium on days 1, 3, and 5. The test results are as follows: Figure 8 As shown. Figure 8 It can be seen that the absorbance of the intervention group on the 3rd and 5th days was significantly greater than that of the control group, reflecting that the cell proliferation rate was significantly improved.
[0055] Example 9 This example studies the effect of coaxial electrospun nanofiber membranes containing tannic acid coatings on cell migration in applications. The design and method of the experiment are as follows: Control group: L929 cells were cultured in DMEM high-glucose medium containing 10% serum mass fraction, the medium was changed every 2 days, and the cells were passaged every 4 days.
[0056] Intervention group: The PLGA-PCL-TA prepared in Example 1 was cut into small cubes of 2 cm×2 cm×2 cm, immersed in DMEM high-glucose medium for 72 h, and used to culture L929 cells, with the medium changed every 2 days and passaged every 4 days.
[0057] The control group cells and the intervention group cells were planted in the upper layer of the Transwell chamber, and 10 hours later, the cells were fixed with paraformaldehyde for 15 hours. Crystal violet stain was used for 30 minutes, and then washed with phosphate buffer. The cells that passed through the Transwell chamber were observed under a microscope. The results are as follows: Fig. 9As shown. Fig. 9 It can be seen that the number of cells passing through the lower chamber in the intervention group was significantly more than that in the control group, indicating that the coaxial electrospun nanofiber membrane containing tannic acid coating has the ability to promote cell migration.
[0058] Example 10 This example uses the DCFH-DA active oxygen scavenging ability experiment to study the antioxidant properties of the coaxial electrospun nanofiber membrane containing tannic acid coating. The design and method of the experiment are as follows: Control group: L929 cells were cultured in DMEM high-glucose medium containing 10% serum mass fraction, the medium was changed every 2 days, and the cells were passaged every 4 days.
[0059] Intervention group: The PLGA-PCL-TA prepared in Example 1 was cut into small cubes of 2 cm×2 cm×2 cm, immersed in DMEM high-glucose medium for 72 h, and used to culture L929 cells, with the medium changed every 2 days and passaged every 4 days.
[0060] The control group and the cells after intervention were incubated with reactive oxygen species staining solution for 6 h, respectively, and then washed with phosphate buffer and observed under an inverted fluorescence microscope. Fig.10 As shown. Fig.10 It can be seen that the amount of green fluorescence in the cells after intervention is significantly less than that in the control group, indicating that the number of reactive oxygen species retained in the cells after intervention is less and the cells after intervention have a stronger ability to clear reactive oxygen.
[0061] Embodiment 11 This example studies the antibacterial properties of the coaxial electrospun nanofiber membrane containing tannic acid coating in application. The design and method of the experiment are as follows: Control group: In a conical flask, Escherichia coli was cultured in LB broth medium, and Staphylococcus aureus was cultured in LB medium. The bacteria were taken out at 72 hours, spread on solid culture medium and cultured for 48 hours, and the growth of bacteria was observed.
[0062] Intervention group: The PLGA-PCL-TA prepared in Example 1 was cut into small cubes of 3 cm × 3 cm × 0.2 cm. One piece was immersed in a conical flask containing bacteria and co-cultured with Staphylococcus aureus and Escherichia coli, respectively. The bacteria were taken out at 72 hours, spread on solid culture medium and cultured for 48 hours, and the growth of bacteria was observed.
[0063] Comparison of antibacterial test results between the control group and the intervention group Fig.11 As shown. Fig.11It can be seen that the growth numbers of the two bacteria in the intervention group were significantly lower than those in the control group, indicating that the prepared coaxial electrospun nanofiber membrane containing tannic acid coating had the ability to kill Staphylococcus aureus and Escherichia coli.
[0064] Example 12 This example studies the application effect of the coaxial electrospun nanofiber membrane containing tannic acid coating based on the wound healing experiment. The design and method of the experiment are as follows: 1) Construction of diabetic rat model: Ten-week-old male SD rats were intraperitoneally injected with 65 mg / kg streptozocin (STZ). One week after the injection, blood glucose was measured for three consecutive days. The blood glucose level was >16.67 mmol / L, indicating that the diabetic rat model was successfully established. 2) Surgery to create skin defects: The diabetic rats were fasted the night before the operation. After being anesthetized, they were fixed on the operating table. The skin was prepared by using surgical scissors to cut the back skin into a circle with a diameter of 25 mm, with a depth of the full layer of skin. 3) Treatment: Intervention group: Use forceps to grasp the PLGA-PCL-TA prepared in Example 1 and place it into the created skin defect; Control group: a 3M Tegaderm hydrophilic dressing was applied on the skin defect; The wound growth was observed on the 7th and 14th days after treatment. The wound healing photos are shown in Fig.12 As shown. Fig.12 By comparison, the wound healing speed in the intervention group was significantly faster than that in the control group, the scab on the wound surface was thicker, there was less bleeding, and the degree of tissue healing was higher.
[0065] Embodiment 13 This example evaluates the adhesion effect of cells on the coaxial electrospun nanofiber membrane containing tannic acid coating by cell adhesion experiment. The design and method of the experiment are as follows: First, the PLGA-PCL and PLGA-PCL-TA prepared in Example 1 were placed in a 24-well plate, and 10,000 L929 cells were seeded on the fibers and incubated for 24 h. The samples were taken out and washed with PBS solution, fixed with 4 wt.% paraformaldehyde and washed with PBS. Then, they were dehydrated with increasing concentrations of ethanol (20 wt.%, 50 wt.%, 70 wt.%, 100 wt.%) and freeze-dried in a freeze dryer. Gold was sprayed on the surface of the sample, and SEM images were taken. The results are shown in Figure 2. Fig.13 shown.
[0066] from Fig.13It can be observed that the number of cells adhering to PLGA-PCL-TA is higher, which is due to the adhesiveness of TA coating, which provides more contact sites for cells, and the molecular interaction between the polyphenol groups of TA and the mucin of cells increases the adhesion of cells.
[0067] In summary, the present invention utilizes an automated coaxial electrospinning process and a simple impregnation method to prepare a fiber membrane having a PLGA core and a PCL shell, and then induces TA polymerization and cross-linking on the surface of the fiber membrane to form a TA coating, thereby obtaining a coaxial electrospun nanofiber membrane containing a tannic acid coating. PCL exhibits excellent mechanical properties and is suitable for being used as a shell material in the present invention to improve mechanical strength. TA molecules have hydrophilic groups, and the abundant ortho-hydroxyl groups in the molecules can release hydrogen to scavenge free radicals, block chain reactions, and inhibit lipid peroxidation. Its natural polyphenol structure can kill bacteria by destroying bacterial cell membranes and inhibiting bacterial enzyme activity, thereby achieving antioxidant and antibacterial effects. The present invention uses the polyphenol groups of TA to interact with the polyphenols of the mucin of the cell, and this effect is manifested as cell adhesion in a macroscopic manner. TA coatings can be used to increase cell adhesion, provide more contact sites for cells, and thus enhance the migration ability of cells. As a result, the electrospun fiber membrane with a TA coating better simulates the structure of ECM, is suitable for cell adhesion and ingrowth, and enables wounds to heal faster. Furthermore, the present invention utilizes the TA coating to extrude the coaxial spun fibers during the formation process, making the spinning structure tighter and showing convergence. This phenomenon means that the diameter of the spun fibers does not increase significantly when a layer of coating is added, which reduces contact with the solution and reduces the adverse effects caused by degradation. When damage or degradation occurs, the TA coating exhibits self-repairing behavior due to adsorption between molecules, and can also reduce the mass loss of the fiber membrane. The coaxial electrospun nanofiber membrane containing a tannic acid coating of the present invention has the advantages of good biocompatibility, strong antibacterial and antioxidant abilities and mechanical properties, and high hydrophilicity. It can significantly promote wound healing and has broad prospects for use as a dressing in promoting wound healing.
[0068] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.
Claims
1. A method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating, characterized in that: The steps include: (1) dissolving polylactic acid-co-glycolic acid in hexafluoroisopropanol to prepare a core layer spinning solution; dissolving polycaprolactone in hexafluoroisopropanol to prepare a shell layer spinning solution; (2) Spinning the core layer spinning solution and the shell layer spinning solution into a fiber membrane formed by fibers having a core layer of polylactic acid-glycolic acid copolymer and a shell layer of polycaprolactone through a coaxial electrospinning device; drying the fiber membrane to obtain a coaxial electrospun fiber membrane; (3) adjusting the pH of the tannic acid aqueous solution to alkaline to obtain a coating treatment solution; The coaxial electrospun fiber membrane is immersed in a coating treatment solution for treatment, and then washed with water to remove unpolymerized tannic acid and induce its molecules to form cross-links; After subsequent drying, a coaxial electrospun nanofiber membrane containing a tannic acid coating was obtained.
2. The method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating according to claim 1, characterized in that: In the step (1), in the core layer spinning solution, the concentration of polylactic acid-glycolic acid copolymer is 15 wt.%-25 wt.%; and in the shell layer spinning solution, the concentration of polycaprolactone is 10 wt.%-12 wt.%.
3. The method for preparing the coaxial electrospun nanofiber membrane containing tannic acid coating according to claim 1, characterized in that: In the step (1), during the preparation of the core layer spinning solution and the shell layer spinning solution, stirring is performed for 15-45 minutes, and then the solution is allowed to stand to remove bubbles.
4. The method for preparing the coaxial electrospun nanofiber membrane containing tannic acid coating according to claim 1, characterized in that: The specific operation of step (2) is as follows: the core layer spinning solution and the shell layer spinning solution are respectively sucked into the syringe of the coaxial electrospinning device, and then the syringe is placed in the corresponding micro pump; the feeding speed of the core layer spinning solution and the shell layer spinning solution is controlled by the micro pump, so that the two solutions are transported from the syringe to the coaxial spinning needles of the core layer and the shell layer, and the distance between the coaxial spinning needle and the substrate is adjusted to a set value; then the power is turned on, the voltage and spinning time are adjusted, and the temperature and relative humidity of the coaxial electrospinning are controlled; after the coaxial electrospinning is completed, the fiber membrane formed by the fiber whose core layer is polylactic acid-hydroxyacetic acid copolymer and whose shell layer is polycaprolactone is removed from the surface of the substrate, and the coaxial electrospun fiber membrane is obtained after drying.
5. The method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating according to claim 4, characterized in that: The feeding rate of the core layer spinning solution is 5-15 μL / min; the feeding rate of the shell layer spinning solution is 15-25 μL / min; the distance between the coaxial spinning needle and the substrate is 14-18 cm; the voltage is 17-19 kV; the spinning time is 5-7 h; the temperature of the coaxial electrospinning is 22-27 ° C, and the relative humidity is 45%-55%.
6. The method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating according to claim 1, characterized in that: In the steps (2) and (3), the drying temperature is 36-38°C and the drying time is 20-28 hours.
7. The method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating according to claim 1, characterized in that: In the step (3), the concentration of the tannic acid aqueous solution is 0.8-1.2 mg / mL; and the pH of the tannic acid aqueous solution is adjusted to 8-9.
8. The method for preparing a coaxial electrospun nanofiber membrane containing a tannic acid coating according to claim 1, characterized in that: In the step (3), the coaxial electrospun fiber membrane is immersed in the coating treatment solution for treatment at room temperature for 20-28 hours.
9. A coaxial electrospun nanofiber membrane containing a tannic acid coating, characterized in that: The method is prepared according to any one of claims 1 to 8.
10. An application of the coaxial electrospun nanofiber membrane containing tannic acid coating as claimed in claim 9, characterized in that: Application as a dressing in promoting wound healing.