Composite nanofiber capable of stopping bleeding, resisting bacteria and promoting vascular wound repair as well as preparation method and application of composite nanofiber

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a composite nanofiber capable of stopping bleeding, resisting bacteria and promoting vascular wound repair as well as a preparation method and application of the composite nanofiber. The composite nanofiber comprises a fiber matrix and copper tannic acid nanoparticles, wherein the fiber matrix is composed of a hydrophilic polymer and zein, and the copper tannic acid nanoparticles are loaded on the fiber matrix. The composite nanofiber can generate active oxygen and release copper ions for synergistic sterilization, angiogenesis is facilitated, wound healing is promoted, excellent biocompatibility is shown, the composite nanofiber is suitable for wounds of different sizes and depths, safe, hemostatic, antibacterial and wound repair customized management promotion can be achieved, and the application prospect is wide. A solution is provided for rapid hemostasis, antibiosis and wound healing of acute wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to a composite nanofiber that has hemostatic, antibacterial and vascular wound repair-promoting properties, as well as its preparation method and application. Background Technology

[0002] Trauma, burns, surgical wounds, traffic accidents, and battlefield injuries pose a significant threat to people's lives and place a huge economic burden on social medical systems. In these open wounds, some severe injuries exceed the skin's regenerative capacity, making the wounds susceptible to bacterial infection and hindering healing.

[0003] Traditional wound dressings such as gauze only serve to cover and physically shield wounds during the healing process, and their standardized packaging makes it difficult to achieve full coverage of wounds in emergency situations. Furthermore, traditional wound dressings have poor antibacterial properties, failing to meet the needs for rapid hemostasis, antibacterial action, and wound healing promotion. Therefore, researchers are developing a wide range of wound dressings for emergency situations to accelerate wound healing.

[0004] Electrospun nanofibers possess high surface area and high porosity, enhancing contact with damaged tissue and exhibiting excellent interpore connectivity, allowing for air permeability. Furthermore, compared to traditional wound dressings, nanofiber dressings can load bioactive molecules or drugs into the nanofibers, facilitating in-situ drug loading.

[0005] Furthermore, traditional antibiotics face the problem of drug-resistant bacteria, a situation that has become increasingly severe in recent years. Widely used silver nanoparticles also face further biosafety concerns. Given the complexity of the wound's physiochemical environment, the urgent need for hemostasis, antibacterial properties, and repair of large, irregular wounds, and the limitations of existing technologies in terms of biosafety and functional singularity, it is particularly necessary to conduct in-depth research on a wound dressing that integrates hemostasis, antibacterial properties, and repair functions for large, irregular wounds. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a composite nanofiber for hemostasis, antibacterial activity, and promoting vascular wound repair, along with its preparation method and applications. The composite nanofiber of this invention can generate reactive oxygen species and release copper ions for synergistic bactericidal action, promote angiogenesis, and thus accelerate wound healing. It exhibits excellent biocompatibility and is suitable for wounds of varying sizes and depths. It enables safe, hemostatic, antibacterial, and wound-healing-promoting customized management, providing a solution for rapid hemostasis, antibacterial action, and wound healing in acute wounds.

[0007] In a first aspect, the present invention provides a composite nanofiber that has hemostatic, antibacterial, and vascular wound repair-promoting properties. The composite nanofiber comprises a fiber matrix composed of a hydrophilic polymer and zein, and copper tannic acid nanoparticles loaded on the fiber matrix; wherein the mass ratio of the hydrophilic polymer to zein is 0.1:1 to 3:1, and the copper tannic acid nanoparticles account for 0.1% to 5% of the mass percentage of the fiber matrix.

[0008] Preferably, the molar ratio of tannic acid to copper in the copper tannic acid nanoparticles is 0.1:1 to 0.4:1.

[0009] Preferably, the hydrophilic polymer is one or more of polyvinylpyrrolidone, sodium alginate, polyvinyl alcohol, gelatin, carboxymethyl chitosan, and polyethylene glycol.

[0010] Secondly, the present invention provides a method for preparing the aforementioned composite nanofibers that have hemostatic, antibacterial, and vascular wound repair-promoting effects. The preparation method includes the following steps: After dissolving tannic acid and copper source in water, a hydrothermal reaction was carried out to obtain copper tannic acid nanoparticles. Hydrophilic polymers, zein, and copper tannic acid nanoparticles were dissolved in a solvent to obtain a spinning precursor solution. Electrospinning of the spinning precursor solution yields composite nanofibers that have hemostatic, antibacterial, and vascular wound repair-promoting properties.

[0011] Preferably, the copper source is one or a mixture of several of copper chloride, copper nitrate, copper acetate, copper carbonate, and copper sulfate.

[0012] Preferably, the hydrothermal reaction is carried out at a temperature of 40–70°C for 3–6 hours; more preferably, the pH of the solution is adjusted to 7.2–8 before the hydrothermal reaction.

[0013] Preferably, the solvent is one or a mixture of water, ethanol, acetic acid, and acetone.

[0014] Preferably, the parameters of the electrospinning are: a high voltage electrostatic field of 10-20 kV, a flow rate of 0.5-10 mL / h, a distance of 8-15 cm between the spinneret and the receiving device, and a needle type of 20-30 G.

[0015] Thirdly, the present invention provides the application of the aforementioned hemostatic, antibacterial and vascular wound-healing composite nanofibers in the preparation of fibrous dressings for wound repair.

[0016] Preferably, the wound is an open wound that is prone to infection.

[0017] Preferably, the fiber dressing is a fiber dressing deposited at the wound site by in-situ electrospinning.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) The composite nanofibers described in this invention utilize zein and polyvinylpyrrolidone to promote blood clotting, and are suitable for multiple electrospinning technologies such as in-situ electrospinning, which can achieve rapid hemostasis of large-area irregular wounds.

[0020] (2) The composite nanofibers described in this invention achieve wound antibacterial effect by loading copper tannic acid nanoparticles and generating ROS in an acidic environment created by bacterial metabolic acid products, and releasing copper ions in synergy with the release of copper ions. At the same time, the release of copper ions promotes wound repair.

[0021] (3) The composite nanofibers described in this invention have good biocompatibility, which ensures the possibility of using them as a safe and long-lasting treatment option for wound management as a fiber dressing. Attached Figure Description

[0022] Figure 1 Transmission electron microscopy (TEM) image of Cu@TA nanoparticles prepared in Example 1; Figure 2 Photograph of ZP fiber tissue adhesion prepared in Example 1; Figure 3 Scanning electron microscope images and elemental distribution diagrams of CTZP fibers prepared in Example 1; Figure 4 In vitro coagulation results of ZP fibers and CTZP fibers prepared in Example 1; Figure 5 The results of tail-cutting experiment on SD rats with CTZP fibers prepared in Example 1 are shown; (A) is a rapid hemostasis characteristic image of rat tail-cutting experiment, and (B) is the hemostasis rate obtained by weighing the change in paper mass. Figure 6 TMB colorimetric experiment of Cu@TA nanoparticles prepared in Example 1; Figure 7 The reactive oxygen species detection experiment of Cu@TA nanoparticles prepared in Example 1 under EPR test; Figure 8 The results of ICP ion release from the CTZP fibers prepared in Example 1; Figure 9 The results of culturing bacteria on CTZP fibers prepared in Example 1; Figure 10 The results of in vitro endothelial cell angiogenesis of ZP fibers and CTZP fibers prepared in Example 1; Figure 11The healing results of infected wounds in SD rats using ZP fibers and CTZP fibers prepared in Example 1; Figure 12 Results of in vitro coagulation experiments on fibrinogen with different ratios of zein and polyvinylpyrrolidone; Figure 13 The results of in vitro endothelial cell proliferation of ZP fibers and fibers with different concentrations of CTZP fibers; Figure 14 The results of in vitro antibacterial experiments on ZP fibers and CTZP fibers of different concentrations are shown; (A) is an optical image of colonies on agar plates, and (B) is the antibacterial rate of colonies on agar plates. Detailed Implementation

[0023] The present invention is further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention. The following exemplary description illustrates the hemostatic, antibacterial, and vascular wound repair-promoting composite nanofibers of the present invention, their preparation methods, and applications.

[0024] The composite nanofibers of this invention comprise a fiber matrix composed of a hydrophilic polymer and zein, and copper tannin nanoparticles loaded onto the fiber matrix. This invention introduces the amphiphilic fiber component zein, which has hemostatic activity, and also utilizes the tissue adhesion properties of the hydrophilic polymer (e.g., polyvinylpyrrolidone). Through the synergistic effect of these two components, cell adhesion and blood coagulation are promoted, achieving rapid hemostasis. Furthermore, the amphiphilic nature of zein is more conducive to drug loading. In addition, unlike the antibacterial effect of copper nanoparticles alone, this invention utilizes the acidic catalysis of copper tannin nanoparticles to generate hydroxyl radicals. The release of reactive oxygen species in conjunction with copper ion release achieves antibacterial effects, rather than the release of copper ions alone, significantly improving antibacterial efficiency. Moreover, unlike immune regulation that promotes wound healing, this invention promotes angiogenesis through copper ion release, and the fiber matrix promotes cell migration, synergistically promoting wound healing.

[0025] Unlike standalone drug-loaded applications, this invention uses hydrophilic polymers and zein as the matrix for electrospun fibers. Functional applications in wound repair include, but are not limited to, adhesion, hemostatic properties, retention of drug activity, antibacterial properties, and promotion of wound healing. This discovery significantly expands the functionality of hydrophilic polymers and zein as fiber spinning matrices. Furthermore, unlike traditional drug loading methods, this invention loads the drug as novel copper tannic acid nanoparticles, which possess catalytic and active ion release functions. Their activity retention within the fiber matrix achieves excellent in vivo wound healing capabilities.

[0026] Zeatin, as a self-assembling superhydrophobic molecule, can promote blood coagulation and thus achieve wound hemostasis. The amphiphilic nature of the hydrophilic polymer (e.g., polyvinylpyrrolidone) lays the foundation for the dressing to achieve good wound adhesion and drug sustained release functions. The mass ratio of the hydrophilic polymer to zeatin is 0.1:1 to 3:1. Controlling the ratio of hydrophilic polymer to zeatin within this range ensures the spinnability of the composite nanofibers and their effective adhesion to the wound surface, while also guaranteeing excellent hemostatic properties. A low ratio of hydrophilic polymer to zeatin weakens wound adhesion and reduces the blood coagulation-promoting effect. A high ratio weakens the blood coagulation-promoting hemostatic effect. In some embodiments, the mass ratio of the hydrophilic polymer to zeatin is 0.1:1 to 2:1.

[0027] The hydrophilic polymers include, but are not limited to, one or more of polyvinylpyrrolidone, sodium alginate, polyvinyl alcohol, gelatin, carboxymethyl chitosan, and polyethylene glycol.

[0028] Copper tannic acid nanoparticles can generate reactive oxygen species and release copper ions under acidic conditions, achieving potent antibacterial effects. Simultaneously, the introduction of copper tannic acid nanoparticles also has the potential to promote angiogenesis and thus accelerate wound healing. The copper tannic acid nanoparticles constitute 0.1% to 5% of the fiber matrix by mass. By limiting the content of copper tannic acid nanoparticles within this range, not only is the vascular wound repair function of the composite nanofibers guaranteed, but it also possesses good antibacterial properties and is non-toxic. If the mass percentage of copper tannic acid nanoparticles in the fiber matrix is ​​low, it cannot exert sufficient antibacterial effects and its cell proliferation-promoting ability is insufficient. If the mass percentage of copper tannic acid nanoparticles in the fiber matrix is ​​high, it will be toxic to cells.

[0029] Tannic acid molecules contain multiple ortho- and posterior phenolic hydroxyl groups, which can act as ligands to complex with a central copper ion, self-assembling to form a cyclic chelate coordination network, namely a copper ion-polyphenol network. Therefore, copper tannic acid nanoparticles are complexes formed by the coordination bonding of copper ions and tannic acid. Specifically, the tannic acid ligands complex with copper ions to form a cyclic chelate coordination network. The molar ratio of tannic acid to copper in the copper tannic acid nanoparticles is 0.1:1 to 0.4:1. By controlling the molar ratio of copper to tannic acid within this range, the anti-inflammatory properties of tannic acid and the acidic release capacity of copper ions can be balanced: on the one hand, if the molar ratio of copper ions is too low, insufficient copper ions cannot be provided to crosslink tannic acid, resulting in low yield; on the other hand, if the proportion of copper ions is too high, excessive crosslinking of tannic acid by copper ions leads to larger copper tannic acid nanoparticle sizes, affecting the dispersion effect of the nanoparticles.

[0030] The composite nanofibers described in this invention exhibit excellent biocompatibility, while the copper tannic acid nanoparticles loaded with them can generate reactive oxygen species and release copper ions for synergistic bactericidal action. In addition, the release of a certain amount of copper ions is also beneficial to angiogenesis and thus promotes wound healing. This invention can be used to prepare multifunctional in-situ wound dressings that integrate hemostasis, antibacterial and wound healing functions for large-area irregular wounds, providing a solution for rapid hemostasis, antibacterial and wound healing of acute wounds.

[0031] The following exemplarily illustrates the preparation method of the composite nanofibers of the present invention.

[0032] Copper tannic acid nanoparticles were prepared using a one-step method. In some embodiments, tannic acid and a copper source were dissolved in water and then subjected to a hydrothermal reaction to obtain copper tannic acid nanoparticles. The copper source includes, but is not limited to, one or a mixture of copper chloride, copper nitrate, copper acetate, copper carbonate, and copper sulfate. For example, tannic acid and copper chloride were dissolved in ultrapure water to obtain a solution. The pH of the solution was adjusted to 7.2–8 before the hydrothermal reaction was carried out. The temperature of the hydrothermal reaction could be 40–70°C, and the time could be 3–6 hours. The resulting suspension was collected, centrifuged, washed, and dried to obtain copper tannic acid nanoparticles, named Cu@TA.

[0033] A spinning precursor solution is obtained by dissolving a hydrophilic polymer, zein, and copper tannic acid nanoparticles in a solvent. For example, a fiber matrix solution is obtained by dissolving the hydrophilic polymer and zein in a solvent, and then the copper tannic acid nanoparticles and the fiber matrix solution are mixed to obtain the spinning precursor solution. Alternatively, the copper tannic acid nanoparticles can be incorporated into the fiber matrix solution to obtain the spinning precursor solution.

[0034] The solvent includes, but is not limited to, one or more of pure water, ethanol, acetic acid, and acetone. Preferably, the solvent is pure water, ethanol (medical alcohol), or a mixture of both. By using medical alcohol as a solvent, the volatile alcohol is used to inhibit bacteria during the spinning process, quickly creating a sterile environment, avoiding the biosafety hazards caused by the volatilization of organic solvents, and achieving sterile treatment of the wound environment.

[0035] As an example, in the spinning precursor solution, the mass-to-volume ratio of hydrophilic polymer to solvent can be 5–50 g / mL; the mass-to-volume ratio of zein to solvent can be 5–50 g / mL. The concentrations of hydrophilic polymer and zein affect the electrospinning process: (1) They affect the viscosity and surface tension of the spinning precursor solution, thus affecting the spinnability of the solution. If the concentration is too low, electrospinning is impossible, and only electrospinning can be formed; if the concentration is too high, the needle will be easily blocked. (2) The fiber morphology is controlled by the concentration. If the concentration is too low, bead formation will occur; if the concentration is too high, the fiber will become coarser and the fiber thickness distribution will be uneven, affecting the specific surface area of ​​the fiber, drug release capacity, etc. Preferably, the mass-to-volume ratio of hydrophilic polymer to solvent can be 7.5 g / mL, and the mass-to-volume ratio of zein to solvent can be 22.5 g / mL.

[0036] Electrospinning of the spinning precursor solution yields composite nanofibers (labeled CTZP) with hemostatic, antibacterial, and vascular wound repair-promoting properties. The spinning can be in-situ electrospinning. For example, the spinning precursor solution can be electrospinned in situ to collect the composite nanofibers (film). The electrospinning parameters can be conventionally selected according to the needs of those skilled in the art. As an example, the electrospinning parameters are: a high-voltage electrostatic field of 10–20 kV, a flow rate of 0.5–10 mL / h, a distance of 8–15 cm between the spinneret and the receiving device, and a needle type of 20–30 G.

[0037] This invention also relates to the application of composite nanofibers in the preparation of fibrous dressings for wound repair. Specifically, this invention provides the application of composite nanofiber dressings in wound repair.

[0038] Preferably, the wound is an open wound that is prone to infection.

[0039] The composite nanofiber product of this invention can be directly applied to the wound site. Alternatively, the spinning precursor solution can be directly deposited at the wound site to form a fiber membrane via in-situ electrospinning. In other words, this invention allows for in-situ electrospinning to the wound surface, enabling the composite nanofiber dressing to be directly electrospun and deposited at the wound site. This method, utilizing in-situ electrospinning technology, can be used for wounds of varying sizes and depths, achieving customized management that provides safety, hemostasis, antibacterial properties, and promotes wound healing.

[0040] Preferably, a fibrous dressing film is formed at the wound site using handheld in-situ electrospinning. This can be achieved using a handheld electrospinning machine. Handheld in-situ electrospinning technology not only offers the advantages of portability and ease of operation, but also boasts good drug compatibility, preserving the efficacy of drugs (such as copper tannin nanoparticles). Furthermore, the high porosity and high specific surface area of ​​the electrospun fibers promote good wound contact, providing a feasible method for wound management.

[0041] In summary, the composite nanofibers of this invention achieve rapid hemostasis through the physical blockage of spun fibers and the formation of blood cell clots. In the acidic environment created by bacterial metabolic acidic products, the acidic response generates ROS (reactive oxygen species) and releases copper ions to achieve wound antibacterial effects. It also promotes angiogenesis to accelerate wound healing, promotes blood cell adhesion, cell migration and hematopoiesis, thereby safely achieving hemostasis, antibacterial and antimicrobial effects, and promoting wound healing.

[0042] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0043] Unless otherwise defined or stated, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the methods of this invention.

[0044] Example 1

[0045] CTZP fiber membranes loaded with copper tannic acid nanoparticles were prepared by electrospinning: 0.341 g of copper chloride dihydrate and 0.425 g of tannic acid were dissolved in 100 mL of ultrapure water, the pH was adjusted to 7.2, and the mixture was subjected to a hydrothermal reaction in a 50 °C water bath for 210 min. After centrifugation and washing, copper tannic acid nanoparticles (labeled Cu@TA) were obtained. 2.25 g of zein, 0.75 g of polyvinylpyrrolidone, and 0.116 g of Cu@TA were dissolved in 10 mL of solvent (a mixture of alcohol and water in a 3:1 volume ratio) to form a homogenized spinning precursor solution. The homogenized spinning precursor solution was electrospun to obtain the CTZP membrane loaded with copper tannic acid nanoparticles. The electrospinning parameters were: a high-voltage electrostatic field of 15 kV, a flow rate of 1.5 mL / h, a distance of 12 cm between the spinneret and the receiving device, and a 25G needle type.

[0046] ZP fiber membranes were prepared by in-situ electrospinning: 2.25 g of zein and 0.75 g of polyvinylpyrrolidone were dissolved in 10 mL of solvent (a mixture of alcohol and water in a 3:1 volume ratio) to form a homogenized spinning precursor solution. The homogenized spinning precursor solution was then electrospun to obtain the ZP membrane. The electrospinning parameters were: a high-voltage electrostatic field of 15 kV, a flow rate of 1.5 mL / h, a distance of 12 cm between the spinneret and the receiving device, and a 25G needle type.

[0047] Figure 1 The image shows the transmission electron microscope (TEM) morphology of the Cu@TA nanoparticles prepared in Example 1. As shown in the figure, the Cu@TA nanoparticles are uniform in size, approximately in the nanometer range, with an average particle size of about 3–60 nm.

[0048] The spinning precursor solution used to prepare ZP fibers was spun onto the subcutaneous tissue surface of SD rats using a handheld electrospinner via a one-step in-situ method. Then, one end of the tissue was lifted with tweezers and photographed to obtain a tissue adhesion image. Figure 2 The image shows a tissue adhesion photograph of the ZP fibers prepared in Example 1. As shown, the ZP fibers bind well to the tissue and exhibit good wettability and affinity. This demonstrates that the ZP fibers possess excellent tissue adhesion properties.

[0049] Figure 3 Scanning electron microscope (SEM) images and elemental distribution maps of the CTZP fibers prepared in Example 1 are shown. As shown, the Cu@TA-loaded CTZP fibers exhibit a uniform diameter distribution and contain particles enriched with large atomic numbers. Elemental analysis results indicate that the main components of the fibers are C and O, with copper being the enriched region of large atomic number particles. This demonstrates the successful preparation of Cu@TA-loaded CTZP fibers, with uniform distribution of C and O elements and a small amount of Cu-enriched regions within the composite nanofibers.

[0050] 200 μL of sodium citrate-modified whole blood was added to a blank centrifuge tube (control), a centrifuge tube loaded with 30 mg ZP fiber membrane, and a centrifuge tube loaded with 30 mg CTZP fiber membrane. After storage at 37°C for 1 minute, the tubes were inverted and images were taken. Figure 4 The in vitro coagulation results of the ZP and CTZP fibers prepared in Example 1 are shown in the figure. As shown, the ZP and CTZP fiber membranes can promote blood coagulation and resist gravity, demonstrating that the ZP and CTZP fiber membranes have good in vitro coagulation properties.

[0051] Prepare pre-weighed filter paper. Cut off the tail of the SD rat 4 cm from the tail end and cover it with a CTZP fiber membrane. Take a picture every minute until 5 minutes later, then weigh the filter paper. The difference in filter paper weight before and after the experiment is taken as the amount of bleeding. The control group is not covered with a CTZP fiber membrane. Figure 5 The results of tail-cutting of SD rats with CTZP fibers prepared in Example 1 are shown in the figure. As shown, the prepared CTZP fiber membrane can promote rapid coagulation of blood from severed tails in mice in vivo and reduce bleeding, demonstrating that the CTZP fiber membrane has good in vivo hemostatic properties.

[0052] Example 2

[0053] Experimental group: TMB was dissolved in acetate buffer to obtain a 20 mM TMB solution, 1 mg / mL Cu@TA was added, and then the pH was adjusted to 7.4.

[0054] Experimental group: TMB was dissolved in acetate buffer to obtain a 20 mM TMB solution, 1 mg / mL Cu@TA was added, and then the pH was adjusted to 4.5.

[0055] Control group: TMB was dissolved in acetate buffer to obtain a 20 mM TMB solution without adding Cu@TA.

[0056] After the solutions of the experimental group and the control group were allowed to stand at 37℃ for 30 min, 100 μL of the solution was collected, and the absorbance change curve of the solution in the range of 550–750 nm was detected.

[0057] Figure 6 The results of the colorimetric experiment on Cu@TA nanoparticles prepared in Example 1 for TMB are shown in the figure. As shown, the TMB solution in the pH=4.5 experimental group gradually turned blue, and a significant peak in absorbance intensity was observed at 600 nm. The absorbance curves of the solutions in the pH=7.4 experimental group and the control group did not show significant changes. This indicates that Cu@TA nanoparticles can effectively catalyze the generation of reactive oxygen species from hydrogen peroxide under acidic conditions.

[0058] Example 3

[0059] 1 mg Cu@TA was mixed with 1 mL of acetate buffer solution at pH 7.4 and 1 mL of acetate buffer solution at pH 4.5, respectively. Then, 200 μL of hydrogen peroxide was added to the reaction mixture. 50 μL of the resulting mixture was taken, 5 μL of DMPO was added, and the mixture was placed in an inductively coupled plasma atomic emission spectrometer (ICP-AES) to detect the characteristic peak of the hydroxyl radical (·OH) in the reaction system.

[0060] Figure 7 The figure shows the results of reactive oxygen species (ROS) detection under EPR on the Cu@TA nanoparticles prepared in Example 1. As shown in the figure, the Cu@TA nanoparticles can capture obvious hydroxyl radical peaks, indicating that the Cu@TA nanoparticles can catalyze the generation of ·OH from hydrogen peroxide under acidic conditions. The results indicate that the Cu@TA nanoparticles can catalyze the generation of hydroxyl radicals from hydrogen peroxide under acidic conditions.

[0061] Example 4

[0062] The release of copper ions from CTZP fibers was detected by inductively coupled plasma atomic emission spectrometry. 100 mg of CTZP fiber was dissolved in acetate buffer solutions at pH 4.5 and pH 7.4 at a concentration of 10 mg / mL. Solutions were collected at specific time points, and the amount of copper ions released was measured and accumulated.

[0063] Figure 8The figure shows the ICP ion release results of the CTZP fibers prepared in Example 1. As shown in the figure, the copper ion release concentration of the CTZP fibers under acidic conditions (pH=4.5) is significantly higher than that under neutral conditions (pH=7.4), which proves that the CTZP composite nanofiber membrane has the ability to release copper ions in response to acidity. This indicates that the CTZP fibers can effectively release copper ions in response to acidity.

[0064] Example 5

[0065] Add 2 mg of CTZP fiber membrane to a concentration of 10 in 2 mL. 6 Staphylococcus aureus was cultured in a CFU / mL saline solution in 24-well plates with titanium sheets placed at the bottom, and incubated at 37°C for 6 hours. After the experiment, the titanium sheets were collected, and the bacteria on the surface of the titanium sheets were fixed with a mixture of alcohol and hexamethyldisilazane at varying concentrations. After gold sputtering, the bacterial morphology was observed using scanning electron microscopy (SEM). The control group did not add CTZP fiber membranes, but only 2 mL of a 10 CFU / mL solution. 6 Staphylococcus aureus physiological saline solution at cfu / mL.

[0066] Figure 9 The image shows the results of culturing bacteria with the CTZP fiber prepared in Example 1. As shown in the figure, the SEM results indicate that the Staphylococcus aureus treated with CTZP no longer retains its regular spherical bacterial morphology; its bacterial membrane is damaged, and intracellular substances leak out, making it unable to maintain normal life activities. This demonstrates that the CTZP fiber can effectively disrupt the bacterial membrane structure, causing cytoplasmic leakage, proving that CTZP has good antibacterial properties through the release of reactive oxygen species and copper ions.

[0067] Example 6

[0068] In a pre-cooled 24-well plate, 200 μL of Matrigel (base adhesive) was added to each well and cured at 37°C for 30 min. Subsequently, 2 × 10⁻⁶ ppm was applied to each well. 4 HUVECs were seeded with the total number of cells. 1 mg / mL ZP fiber and 1 mg / mL CTZP fiber were added to each well of the plate, and the cells were co-cultured for 6 hours. The control group received no fiber, only the necessary culture medium for cell growth.

[0069] Figure 10 The results of in vitro endothelial cell angiogenesis using ZP fibers and CTZP fibers prepared in Example 1 are shown in the figure. As shown, the number of blood vessels formed by both ZP fibers and CTZP fibers was significantly increased compared to the control group, demonstrating that both ZP and CTZP fiber membranes have good angiogenesis-promoting capabilities. This indicates that ZP fibers and CTZP fibers can promote endothelial cell angiogenesis. Among them, CTZP fibers have a better angiogenesis-promoting ability.

[0070] Example 7

[0071] A 1 cm diameter wound was created on the back of anesthetized SD rats, and 20 μL of a 10% concentration was dripped into each wound. 6 A Staphylococcus aureus culture at CFU / mL was cultured for one day to obtain an infected wound model. The following day, 200 μL of the spinning precursor solution from Example 1, used to prepare ZP or CTZP fibers, was taken and applied to the wound surface via in-situ electrospinning to cover the wound with ZP or CTZP fiber membranes. Wound healing was subsequently observed at given time points. The control group received no fiber addition and was only covered with a commercially available sterile dressing.

[0072] Figure 11 The results of ZP and CTZP fibers in the healing of infected wounds in SD rats are shown in the figure. As indicated, both ZP and CTZP fibers demonstrated a strong ability to promote the healing of infected wounds. Based on previous results, we can conclude that ZP fibers promote wound healing in vivo through good tissue adhesion, hemostasis, and vascular healing; CTZP further promotes wound healing by catalyzing the metabolism of acidic bacterial products and releasing copper ions to kill bacteria, achieving excellent wound management and repair effects in vivo. This indicates that CTZP fibers can promote the healing of infected wounds in vivo.

[0073] Example 8

[0074] 4g, 3g, 2g, 1g, and 0g of polyvinylpyrrolidone and 0g, 1g, 2g, 3g, and 4g of zein were mixed separately. The total amount of polyvinylpyrrolidone and zein was 4g. Then, 10mL of 75% medical ethanol was added, and PVP, P3Z3, P2Z2, P1Z3, and Zein fiber membranes were obtained by electrospinning.

[0075] Figure 12 The results of in vitro coagulation experiments on fibrin with different ratios of zein and polyvinylpyrrolidone were presented. Fibrin was placed in 6-well plates. 100 μL of 0.1M calcium chloride and 900 μL of rat whole blood were mixed, and 50 μL of the mixture was added to the surface of each fiber. The control group served as a blank control, with no additional fiber added. At given time points, 5 mL of ultrapure water was added, and the absorbance at 540 nm was measured to obtain the coagulation factor (BCI). A lower BCI indicates better procoagulant and hemostatic function of the material. As shown in the figure, the fiber membrane exhibited the best procoagulant and hemostatic effect when the ratio of polyvinylpyrrolidone to zein was 1:3.

[0076] Example 9

[0077] According to 2*10 per hole 5Endothelial cells were seeded into 24-well plates and cultured at 37°C for 24 hours. After cell attachment, 1 mg / mL ZP fiber membrane and 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL CTZP fiber membranes were added, respectively. Fluorescence intensity was measured using Alamar Blue reagent after 1, 3, and 5 days of co-culturing. The control group received no fiber, only endothelial cell culture medium. Figure 13 The results show the in vitro endothelial cell proliferation of ZP fibers and different concentrations of CTZP fibers. Higher fluorescence intensity indicates stronger cell viability. As shown in the figure, when the cells reached day 5 of culture, the cell viability of both the ZP fiber membrane group and the CTZP fiber membrane group was higher than that of the control group; however, the promoting effect of the CTZP fiber membrane group was better than that of the ZP fiber membrane group, with the 0.5 mg / mL CTZP group showing the best effect in promoting endothelial cell proliferation.

[0078] Take 2 mL of a concentration of 1*10 5 CFU / mL Staphylococcus aureus culture was added to 5 mL centrifuge tubes, followed by the addition of 2 mg ZP fiber membrane, and then 1 mg, 2 mg, and 3 mg CTZP fiber membranes, respectively. After co-culturing for 6 hours, 100 μL of the culture was evenly spread onto an agar plate and incubated for 24 hours before observing the colony count. The control group did not add fiber, but only 2 mL of a 10% concentration. 6 Staphylococcus aureus physiological saline solution at cfu / mL. Figure 14 The results of in vitro antibacterial experiments on ZP fibers and CTZP fibers at different concentrations are shown. A is an optical photograph of bacterial colonies on an agar plate, and B is the antibacterial rate calculated relative to the control group after counting the number of bacterial colonies on the agar plate. As shown in the figure, the introduction of ZP fibers promoted bacterial proliferation, but the presence of Cu@TA immediately inhibited this promoting effect and even showed a significant ability to kill bacteria. Among them, 0.5 mg / mL CTZP achieved an antibacterial effect of 59%, while 1.0 mg / mL CTZP achieved an antibacterial effect of 90.9%, and 1.5 mg / mL CTZP almost completely killed bacteria, with an antibacterial rate of 98.4%.

[0079] In summary, this invention provides a composite nanofiber with hemostatic, antibacterial, and vascular wound repair-promoting functions, along with its preparation method and applications. It is suitable for preparing multifunctional in-situ wound dressings that integrate hemostasis, antibacterial, and vascular repair-promoting functions for large-area irregular wounds. It is portable and easy to operate, which not only broadens the application field of in-situ electrospinning but also provides a new solution for wound management.

Claims

1. A composite nanofiber that has hemostatic, antibacterial, and vascular wound repair-promoting effects, characterized in that, The composite nanofiber comprises a fiber matrix composed of a hydrophilic polymer and zein and copper tannic acid nanoparticles loaded on the fiber matrix; wherein the mass ratio of the hydrophilic polymer and zein is 0.1:1 to 3:1, and the copper tannic acid nanoparticles account for 0.1% to 5% of the mass of the fiber matrix.

2. The composite nanofiber according to claim 1, characterized in that, The molar ratio of tannic acid to copper in the copper tannic acid nanoparticles is 0.1:1 to 0.4:

1.

3. The composite nanofiber according to claim 1 or 2, characterized in that, The hydrophilic polymer is one or more of the following: polyvinylpyrrolidone, sodium alginate, polyvinyl alcohol, gelatin, carboxymethyl chitosan, and polyethylene glycol.

4. The method for preparing composite nanofibers with hemostatic, antibacterial, and vascular wound repair-promoting effects according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: After dissolving tannic acid and copper source in water, a hydrothermal reaction was carried out to obtain copper tannic acid nanoparticles. Hydrophilic polymers, zein, and copper tannic acid nanoparticles were dissolved in a solvent to obtain a spinning precursor solution. Electrospinning of the spinning precursor solution yields composite nanofibers with hemostatic, antibacterial, and vascular wound repair properties.

5. The preparation method according to claim 4, characterized in that, The copper source is one or a mixture of several of copper chloride, copper nitrate, copper acetate, copper carbonate, and copper sulfate.

6. The preparation method according to claim 4 or 5, characterized in that, The hydrothermal reaction is carried out at a temperature of 40–70°C for 3–6 hours; preferably, the pH of the solution is adjusted to 7.2–8 before the hydrothermal reaction.

7. The preparation method according to any one of claims 4 to 6, characterized in that, The solvent is one or a mixture of water, ethanol, acetic acid, and acetone.

8. The preparation method according to any one of claims 4 to 7, characterized in that, The parameters for electrospinning are as follows: high voltage electrostatic field of 10-20kV, flow rate of 0.5-10mL / h, distance between spinneret and receiving device of 8-15cm, and needle type of 20-30G.

9. The use of the composite nanofibers with hemostatic, antibacterial and vascular wound repair properties according to any one of claims 1 to 3 in the preparation of fibrous dressings for wound repair.

10. The application according to claim 9, characterized in that, The wound in question is an open wound that is prone to infection.

11. The application according to claim 9 or 10, characterized in that, The fiber dressing is a fiber dressing deposited at the wound site through in-situ electrospinning.

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