A nanofiber membrane with a wound healing-promoting effect and a preparation method thereof

Abstract

The application belongs to the technical field of biological medicine, and provides a nano-fiber membrane with a wound healing promotion effect and a preparation method thereof. An acidic intermediate is obtained through condensation reaction of baicalein and glutaric anhydride, the acidic intermediate and chitosan are crosslinked through a carbodiimide chemical crosslinking reaction to prepare a baicalein grafted chitosan polymer, the drug and the electrospinning material are organically combined, and a baicalein-chitosan nano-fiber membrane rich in drugs is obtained through electrospinning. The preparation method is controllable, simple and effective, the nano-fiber membrane prepared by the method can reduce or prevent bacterial and fungal infection of skin, mucosa and other tissues, reduce inflammation and oxidation reaction, reduce wound infection, promote wound tissue growth, improve wound healing efficiency, and has a good protective effect on the wound. Therefore, the application has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a nanofiber membrane with wound-healing properties and its preparation method. Background Technology

[0002] Human skin is damaged by external factors, resulting in the loss of skin tissue; this wound is called a lesion, and it is part of trauma. Surgical treatment, acute trauma, chronic diseases, or familial genetic diseases are all major causes of wounds. Wounds can be classified as acute or chronic wounds based on their healing time. Chronic wounds caused by conditions such as diabetes or tumors often take a long time to heal and may even lead to amputation, endangering the patient's life and health. Reactive oxygen species (ROS) are important participants in wound healing; high ROS levels cause oxidative damage, such as extracellular matrix (ECM) destruction and cell damage. These abnormal cells prevent the formation of granulation tissue and ECM deposition, leading to non-healing wounds. Simultaneously, during the natural wound healing process, contaminating pathogens can colonize the site of skin injury. The bacteria that make up the skin microbiota at the wound site can prevent pathogen colonization. However, when pathogens exceed critical levels and produce a significant amount of biofilm, the healing process is delayed. Staphylococcus aureus is the most common colonizing pathogen, affecting the initial stages of wound healing; Escherichia coli is commonly found in chronic wounds, affecting deeper layers of the skin. Infections of exposed skin associated with these pathogens can lead to severe inflammatory responses and incomplete wound healing. Therefore, it is essential to use appropriate antimicrobial wound dressings to prevent bacterial infection and to aid in the natural wound healing process.

[0003] Electrospinning is a technique that uses electrostatic force to generate polymer-based protofibrils with fiber diameters ranging from nanometers to micrometers. The resulting fiber membranes possess a highly interconnected porous structure and partial extracellular matrix structure, meeting various needs for skin wound repair. Chitosan is a commonly used material in electrospinning matrices. The amino and hydroxyl groups on chitosan side chains can serve as active sites for modification reactions. The active groups of chitosan can be grafted onto monomers to impart their functional properties. Baicalein (BAI) possesses anti-inflammatory, antiviral, antitumor, antioxidant, and antibacterial effects. Baicalein exhibits good inhibitory effects on various bacteria and fungi, including Staphylococcus aureus. Studies have found that baicalein has strong scavenging activity against DPPH free radicals and superoxide anions. However, baicalein has poor stability. Against this backdrop, it is imperative to develop a wound dressing material that offers superior resistance to oxidative damage and antibacterial effects, is biocompatible, semi-permeable to water and oxygen, has low allergenicity, and is also green, environmentally friendly, biodegradable, non-toxic, has no side effects, and is non-irritating. Summary of the Invention

[0004] The purpose of this invention is to provide a nanofiber membrane with wound-healing properties and its preparation method, thereby addressing the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing a nanofiber membrane with wound-healing properties includes the following steps:

[0007] Step 1: Weigh out baicalein, 4-dimethylaminopyridine and glutaric anhydride in a certain proportion, add 1-10 mL of triethylamine and dry tetrahydrofuran to the reaction flask, and stir magnetically under 254 nm ultraviolet light for 14-24 h.

[0008] Step 2: After rotary evaporating the reaction solution from Step 1, add ethyl acetate to the concentrate, add 5 mL of 1M HCl, extract with water, and extract with ethyl acetate 1-5 times. Combine the ethyl acetate layers, extract with saturated NaCl 1-5 times to obtain the ethyl acetate layer, and dry with anhydrous sodium sulfate. Remove the solvent using a rotary evaporator, mix the product with silica gel, load it onto a silica gel column, and elute with petroleum ether, ethyl acetate, and formic acid to separate the acidic intermediate.

[0009] Step 3: Dissolve 1-50g of the acidic intermediate in methanol, add 1-10g of N-hydroxysuccinimide to the solution, stir at room temperature for 10-50min, dissolve chitosan in 1% acetic acid aqueous solution, add 1-10g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir in the dark for 14-30h, dialyze the reaction solution using a dialysis bag for 10-80h, freeze-dry for 10-60h to obtain BAC-g-CS;

[0010] Step 4: Weigh out BAC-g-CS and PVA powder in a certain proportion, dissolve them in 90% acetic acid aqueous solution, stir the mixture with a magnetic stirrer at room temperature for 12-24 hours, let the prepared solution stand for 1-8 hours, and store it in a sealed container at room temperature.

[0011] Step 5: Pour the prepared spinning solution into the syringe, connect the positive terminal of the high-voltage power supply to the needle, and connect the negative terminal of the high-voltage power supply to the collection plate with the equipotential paper attached. After the nanofiber membrane is spun, remove it and place it in an oven to dry at 80℃ for 12-24 hours.

[0012] Furthermore, in step 1, the ratio of baicalein, 4-dimethylaminopyridine, and glutaric anhydride is 1-10: 1-10: 1-10.

[0013] Furthermore, in step 2, the ratio of petroleum ether, ethyl acetate, and formic acid is 1-50:1-50:1-50.

[0014] Furthermore, in step 3, the molecular weight cutoff of the dialysis bag is 3000-10000 Da.

[0015] Furthermore, in step 4, the ratio of BAC-g-CS to PVA powder is 1-10:1-10.

[0016] Furthermore, in step 5, the distance between the needle and the collecting plate is 15-18cm, the spinning voltage is 10-30kV, the spinning time for each nanofiber membrane is 0.1-24h, the positive voltage is 5-20kV, the negative voltage is 1-10kV, the propulsion speed is 0.1-1mL / h, and the spinning temperature is 10-29℃.

[0017] A nanofiber membrane prepared by the above method.

[0018] Application of the above-mentioned nanofiber membrane in the preparation of wound dressings.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. This invention utilizes chitosan, a natural polymer material, as a drug carrier to load baicalein, an effective drug component obtained from traditional Chinese medicine, which effectively improves wound healing efficiency and regeneration effect.

[0021] 2. This invention obtains an acidic intermediate through the condensation reaction of baicalein and glutaric anhydride. This acidic intermediate is then cross-linked with chitosan via a carbodiimide chemical cross-linking reaction to prepare a baicalein-grafted chitosan polymer. This achieves the goal of linking baicalein and chitosan molecules, ensuring the stability of baicalein. The drug is organically combined with electrospun materials to obtain a drug-rich baicalein-chitosan nanofiber membrane. The drug components are released through the degradation ability of the material itself, preventing rapid drug loss from the wound. It also has antioxidant and antibacterial effects, and plays a good role in promoting wound healing.

[0022] 3. This invention possesses numerous advantages, including good biocompatibility, high safety, low toxicity, and antioxidant and antibacterial effects. Furthermore, the preparation method is controllable, relatively simple, and highly effective. The nanofiber membrane prepared by the method provided by this invention can reduce or prevent bacterial and fungal infections of skin, mucous membranes, and other tissues, reduce inflammation and oxidative reactions, decrease wound infection rates, promote wound tissue growth, and improve wound healing efficiency while also providing excellent protection. Therefore, this invention has broad application prospects. Attached Figure Description

[0023] Figure 1 This is the Fourier transform infrared spectrum of Embodiment 1 of the present invention.

[0024] Figure 2 This is a first-order mass spectrum of the acidic intermediate in positive ion mode in Example 1 of the present invention.

[0025] Figure 3 This is the 1H NMR spectrum of the acidic intermediate in Example 1 of the present invention.

[0026] Figure 4 This is the 1H NMR spectrum of the baicalin-chitosan graft copolymer in Example 1 of the present invention.

[0027] Figure 5 This is the BAC content standard curve of Example 1 of the present invention.

[0028] Figure 6 In the image, (a) is a scanning electron microscope image of Bac-CS in Example 2; (b) is a fiber diameter distribution diagram of Bac-CS in Example 2.

[0029] Figure 7 This is the stretching curve of the fiber in Example 2 of the present invention.

[0030] Figure 8 This is the Fourier transform infrared spectrum of Embodiment 2 of the present invention.

[0031] Figure 9 This is a graph showing the DPPH scavenging activity results of baicalin, vitamin C, graft copolymer and chitosan in Example 3 of the present invention.

[0032] Figure 10 The diagram shows the reducing power results of chitosan, baicalin, graft copolymer and Vc in Example 3 of the present invention.

[0033] Figure 11 This is a graph showing the toxicity test results of different concentrations of baicalin-chitosan graft copolymer on cells in Example 3 of the present invention.

[0034] Figure 12 This is a graph showing the toxicity test results of different concentrations of H2O2 on cells in Example 3 of the present invention.

[0035] Figure 13 These are laser confocal images of ROS in macrophages treated with different concentrations of Bac-CS in Example 3 of this invention.

[0036] Figure 14 The data are flow cytometry data of ROS in macrophages treated with different concentrations of Bac-CS in Example 3 of this invention.

[0037] Figure 15This is a plate colony diagram of different concentrations of polymerized Staphylococcus aureus and Escherichia coli in Example 4 of the present invention. From left to right, S. aureus concentrations are 18.75 μg / mL, 37.5 μg / mL, and 75 μg / mL; E. coli concentrations are 18.75 μg / mL, 37.5 μg / mL, and 75 μg / mL.

[0038] Figure 16 This is a colony diagram of Staphylococcus aureus in different concentrations of polymer and chitosan solutions in Example 4 of the present invention. (a) is a 37.5 μg / mL chitosan solution; (b) is a 50 μg / mL chitosan solution; (c) is a 75 μg / mL chitosan solution; (d) is a 37.5 μg / mL polymer solution; (e) is a 50 μg / mL polymer solution; (f) is a 75 μg / mL polymer solution; and (g) is a negative control.

[0039] Figure 17 The images show colony diagrams of *E. coli* obtained from different concentrations of polymer and chitosan solutions in Example 4 of this invention. (a) is a 18 μg / mL chitosan solution, (b) is a 37 μg / mL chitosan solution, (c) is a 50 μg / mL chitosan solution, (d) is an 18 μg / mL polymer solution, (e) is a 37 μg / mL polymer solution, (f) is a 50 μg / mL polymer solution, and (g) is a negative control. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0042] An embodiment of the present invention provides a method for preparing a nanofiber membrane with wound-healing properties, comprising the following steps:

[0043] Step 1: Weigh baicalein, 4-dimethylaminopyridine and glutaric anhydride in a ratio of 1-10:1-10:1-10 (g:g:g). Add 1-10 mL of triethylamine and dry tetrahydrofuran (THF) to the reaction flask and stir magnetically under 254 nm ultraviolet light for 14-24 h.

[0044] Step 2: After rotary evaporating the reaction solution from Step 1, add ethyl acetate to the concentrate, add 5 mL of 1M HCl, extract with water, and extract with ethyl acetate 1-5 times. Combine the ethyl acetate layers, extract with saturated NaCl 1-5 times to obtain the ethyl acetate layer, and dry with anhydrous sodium sulfate. Remove the solvent using a rotary evaporator, mix the product with silica gel, load it onto a silica gel column, and elute with petroleum ether, ethyl acetate, and formic acid (1-50:1-50:1-50) to separate the acidic intermediate.

[0045] Step 3: Dissolve 1-50g of the acidic intermediate in methanol, add 1-10g of N-hydroxysuccinimide (NHS) to the solution, stir at room temperature for 10-50min, dissolve chitosan (90% deacetylated, Mw 200kDa) in 1% (v / v) aqueous acetic acid solution, add 1-10g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), stir in the dark for 14-30h, dialyze the reaction solution using a dialysis bag (Mw 3000-10000) for 10-80h, freeze-dry for 10-60h to obtain the polymer BAC-g-CS;

[0046] Step 4: Weigh BAC-g-CS and PVA powder in a ratio of 1-10:1-10, dissolve them in a 90% acetic acid aqueous solution, and stir the mixture with a magnetic stirrer at room temperature for 12-24 hours to ensure that the polymer is completely dissolved and a homogeneous solution is obtained; let the prepared solution stand for 1-8 hours to degas, and store it in a sealed container at room temperature.

[0047] Step 5: Pour the prepared spinning solution into the syringe. Connect the positive terminal of the high-voltage power supply to the needle and the negative terminal of the high-voltage power supply to the collecting plate with equipotential paper attached. The distance between the needle and the collecting plate should be 15-18 cm. The spinning voltage should be 10-30 kV. The spinning time for each nanofiber membrane should be 0.1-24 h. The positive voltage should be 5-20 kV and the negative voltage should be 1-10 kV. The feed rate should be 0.1-1 mL / h and the spinning temperature should be 10-29℃. After the nanofiber membrane is spun, remove it and place it in an oven to dry at 80℃ for 12-24 h.

[0048] An embodiment of the present invention provides a nanofiber membrane prepared by the above preparation method.

[0049] One embodiment of the present invention provides the application of the above-mentioned nanofiber membrane in the preparation of a wound dressing.

[0050] Example 1: Characterization of baicalin-chitosan graft copolymer

[0051] (1) Fourier transform infrared spectroscopy analysis: Weigh 5 mg each of acidic intermediate, chitosan, baicalin, and graft copolymer, mix with KBr, compress into tablets, and spectroscopy at 400–4000 cm⁻¹. -1 The infrared absorption spectrum was obtained by scanning the area using an infrared spectrometer.

[0052] The results are as follows: See Figure 1 3414cm -1 and 3102cm -1 The peak represents the stretching vibration of the hydroxyl groups at positions 5 and 6 of the baicalin molecule, at 1657 cm⁻¹. -1 This is the stretching vibration peak at the 4-position carbonyl group of the baicalin molecule, at 1619 cm⁻¹. -1 1586cm -1 and 1492cm -1 This is the stretching vibration peak of the carbon-carbon double bond in the baicalin skeleton. Compared to baicalin, the peak at 1707 cm⁻¹ in the figure is [missing value]. -1 1754cm -1 The stretching vibration peak of the carbonyl group on the carboxyl group of the intermediate can be used to determine the structure of the acidic intermediate. (At 3446 cm⁻¹) -1 The broad peaks nearby are the stretching vibration peaks of OH and NH in the CS molecule, at 2919 cm⁻¹. -1 The nearby peak represents the methyl CH peak in the CS molecular unit, at 1636 cm⁻¹. -1 and 1558cm -1 The peaks at 1658 cm⁻¹ represent the absorption peaks of amide I (C=O) and amide II (NH) bands in the CS molecule, respectively. Compared to chitosan, the peak at 1658 cm⁻¹ is significantly higher. -1 The peaks are stretching vibrations of amide bonds, which can be used to determine the structure of the graft copolymer.

[0053] (2) Mass spectrometry analysis:

[0054] A. Chromatographic conditions: A Waters ACQUITY UPLC HSS T3 (100 mm x 2.1 mm, 1.8 μm) column was used. The mobile phase was acetonitrile (A) and 0.1% formic acid in water (B), with gradient elution: 0–5 min, 5%–20% A; 5–30 min, 20%–30% A; 30–40 min, 30%–95% A. The flow rate was 0.30 mL / min; the column temperature was 35 °C; and the injection volume was 2 μL.

[0055] B. Mass spectrometry conditions: Electrospray ionization (ESI) source, scanning in positive and negative ion modes respectively. Ultra-high purity gas (He) and high-purity nitrogen (N) were used as the collision gas and nebulizing gas, respectively. The parameters for positive ion mode were set as follows: capillary temperature 350°C; nitrogen gas, flow rate 413.7 kPa; auxiliary gas, flow rate 68.95 kPa. The parameters for negative ion mode were set as follows: capillary temperature 350°C; nitrogen gas, flow rate 275.8 kPa; auxiliary gas, flow rate 68.95 kPa. Mass axis calibration was performed using the external standard method (mass error less than 1 × 10⁻⁶). -5 The first-order mass spectrometer was fully scanned in FT mode (resolution R = 30000, scan range m / z 100–1200).

[0056] The results are as follows: See Figure 2 In positive ion mode, the peak at 271.0620 [MH]+ in the mass spectrum is the peak of baicalin. The expected peak at 385.0925 [MH]+ in the mass spectrum confirms the presence of the acidic intermediate.

[0057] (3) Nuclear magnetic resonance hydrogen spectrum analysis: The structure of the acidic intermediate was identified using a 400MHz nuclear magnetic resonance spectrometer. An appropriate amount of the intermediate was dissolved in deuterated DMSO at a concentration of 5mg / mL, loaded into a nuclear magnetic resonance tube, and then detected by the spectrometer.

[0058] The results are as follows: See Figure 3 13.01 ppm corresponds to the chemical shift of the hydroxyl group on the carboxyl group, 8.10 ppm corresponds to the chemical shifts of the hydrogens at the 2' and 6' positions, 7.60 ppm corresponds to the chemical shifts of the hydrogens at the 3', 4', and 5' positions, 7.04 ppm corresponds to the chemical shift of the hydrogen at the 3' position, 6.70 ppm corresponds to the chemical shift of the hydrogen at the 8' position, and 2.66 ppm, 2.39 ppm, and 1.91-1.82 ppm correspond to the chemical shifts of the hydrogens on the aliphatic chain. The structure of the acidic intermediate can be deduced from this. See also... Figure 4 The chemical shifts of hydrogen in the hydroxyl groups on the acidic intermediate were 7.93, 7.71, 7.63-7.59, 7.51 and 6.72 ppm, respectively; and the chemical shifts of hydrogen in the chitosan were 4.27, 2.36, 1.81, 1.67-1.60, 1.33 and 0.84 ppm, respectively, which proved that the polymer synthesis was successful.

[0059] (4) Determination of Bac-CS grafting degree:

[0060] A. Solution preparation: Accurately weigh 10 mg each of BAC, the Bac-CS sample to be tested, and CS, and dilute to 10 mL with 1% (v / v) acetic acid aqueous solution to obtain a 1 mg / mL reference solution, test solution, and negative sample solution.

[0061] B. Content determination method: Accurately take 5.00 mg of baicalin into a 50 ml volumetric flask, add methanol to dilute to volume, and obtain a 0.1 mg / mL reference solution. Prepare three solutions for each concentration, and measure the absorbance at a wavelength of 275 nm.

[0062] C. Linearity test: Prepare a series of reference solutions with concentrations of 0.1–0.9 mg / mL and measure the absorbance.

[0063] D. Determination of Bac-CS grafting degree: Accurately weigh the Bac-CS sample and determine the Bac content (mg / mL) according to the method in (4)D.

[0064] Linearity assessment: A linear regression equation was plotted with absorbance as the Y-axis and concentration as the X-axis. The regression equation for BAC in the concentration range of 0.1–0.9 mg / mL was y = 0.087x - 0.0101, with R² = 0.9996, indicating a good linear relationship. (See results below.) Figure 5 .

[0065] The grafting degree of Bac in the product was calculated (mg / g). The grafting degree of baicalin in the product was calculated to be 114.5 mg / g.

[0066] Example 2: Characterization of baicalin-chitosan nanofiber membrane

[0067] (1) Scanning electron microscopy observation: Nanofiber membranes of different compositions were cut into 1×1cm pieces. 2 The nanofiber membrane was adhered to the sample stage using carbon conductive adhesive and dried in an infrared lamp chamber. An Au film was then deposited on the sample surface using a vacuum coating instrument. The membrane morphology and surface topography were analyzed using a scanning electron microscope with an accelerating voltage of 20 kV. The diameter of the fiber membrane was observed through electron microscopy images, and the diameter distribution of the nanofibers in the membrane was measured and calculated.

[0068] Microscopic morphological characterization: See Figure 6 As shown in (a) and (b), the fiber membrane has uniform size, smooth surface, and good morphology. When the CS:PVA ratio is 1:4, the fiber diameter is mainly distributed between 88±19 nm, as detailed in Table 1.

[0069] Table 1. Fiber diameter of Bac-CS

[0070]

[0071] (2) Mechanical property testing: The mechanical properties of the nanofiber membrane were studied using a universal testing machine. The samples were cut into 10×100mm pieces. 2 A rectangular spline of a certain size is used, with a gauge length of 25 mm and a test speed of 5 mm / min.

[0072] The tensile test data are shown in Table 2. The experimental results show that when the CS:PVA ratio is 1:4, the average tensile strength of the nanofiber membrane is 5.00±0MPa and the average elongation at break is 8.00±4%, which meets the mechanical strength requirements for its use as a wound dressing.

[0073] Table 2 Tensile test data

[0074]

[0075] from Figure 7 As can be seen, in the initial stage of tensile deformation, the stress increases sharply with the increase of strain. When the stress reaches a certain value, the stress increases only slightly with the increase of strain. Then the stress increases slowly with the increase of strain until the fiber breaks.

[0076] (3) Fourier transform infrared spectroscopy: 5 mg of nanofiber membrane was weighed, mixed with KBr, and compressed into a tablet. The light was then spectroscopy was performed at 400–4000 cm⁻¹. -1 The infrared absorption spectrum was obtained by scanning the area using an infrared spectrometer.

[0077] like Figure 8 As shown, at 3446cm -1 The broad peaks nearby are the stretching vibration peaks of OH and NH in the CS molecule, at 2920 cm⁻¹. -1 The nearby peak represents the methyl CH peak in the CS molecular unit, at 1638 cm⁻¹. -1 and 1558cm -1 The peaks at 1658 cm⁻¹ represent the absorption peaks of amide I (C=O) and amide II (NH) bands in the CS molecule, respectively. -1 The peaks are stretching vibrations of amide bonds, which helps determine the structure of the nanofiber membrane.

[0078] Example 3: Evaluation of the in vitro antioxidant activity of Bac-CS

[0079] (1) Determination of DPPH free radical scavenging rate: Mix DPPH solution with 200 μl of sample solutions of different concentrations (0.2-1 mg / ml), follow the kit instructions, vortex to mix, and let stand at room temperature in the dark for 30 min. Measure the absorbance of the reaction solution at 515 nm. Use Vc as a positive control, and label them as A blank, A assay, A control, and A positive control, respectively. Set up one control tube for each assay tube. Calculate the DPPH free radical scavenging ability of the sample solution according to the following formula.

[0080] Formula for calculating the free radical scavenging rate of the positive control:

[0081] DPPH free radical scavenging rate % = [(A blank - A positive control) ÷ A blank] × 100%.

[0082] Formula for calculating the free radical scavenging rate of a sample:

[0083] DPPH free radical scavenging rate % = {[A blank - (A assay - A control)] ÷ A blank} × 100%.

[0084] DPPH radicals are stable nitrogen-centered free radicals and are an important indicator of a sample's antioxidant capacity. DPPH radicals have unpaired electrons, and their alcoholic solution is purple with strong absorption at 515 nm. In the presence of antioxidants, DPPH radicals are scavenged, the solution color lightens, and the absorbance decreases. Within a certain range, the change in absorbance is directly proportional to the degree of free radical scavenging. Figure 9 As shown, the scavenging activity against DPPH free radicals gradually increased with increasing sample concentration. At the same concentration, the scavenging activity of the samples from largest to smallest was: Vc > baicalin > graft copolymer > chitosan, indicating that grafting baicalin onto chitosan can improve the antioxidant activity of chitosan.

[0085] (2) Determination of reducing power: Take 0.15 ml of sample solutions of different concentrations, 0.2 ml of PB buffer (0.05 M, pH = 6.6) and 0.2 ml of potassium ferricyanide solution (1%, w / v), mix well, and incubate in a 50℃ water bath in the dark for 20 min. Add 0.2 ml of trichloroacetic acid aqueous solution (10%, w / v), and centrifuge the mixture at 5000 rpm for 10 min. Take 0.5 ml of supernatant, 0.5 ml of deionized water and 0.1 ml of ferric chloride (0.1%, w / v) aqueous solution, mix well. Add 200 μL / well / ×3 to a 96-well plate, and use a microplate reader to measure the absorbance at 700 nm. The higher the absorbance, the stronger the reducing power of the surface sample. Calculate the reducing power of the sample solution using the following formula:

[0086] Reducing force = A S -A0

[0087] The absorbance of the sample group is denoted as A. S Use deionized water instead of the sample, denoted as A0.

[0088] Antioxidants can reduce Fe 3+ / Reduction of ferricyanide complex to Fe 2+ The strength of an antioxidant's reducing power is related to its hydrogen and electron-donating abilities. For example... Figure 10 As shown, the reducing power of chitosan, baicalin, graft copolymer, and vitamin C is positively correlated with the sample concentration. When the concentration is the same, the reducing power of the samples from highest to lowest is: vitamin C > baicalin > graft copolymer > chitosan. Compared with chitosan, the reducing power of the graft copolymer is significantly increased.

[0089] (3) Cell culture: RAW264.7 macrophages were revived and seeded in 25 cm⁻¹ cells. 2 Add 5 mL of complete culture medium (90% DMEM: high glucose + 10% fetal bovine serum + 1% antibiotics) to the cell culture flask and place it in a constant temperature incubator at 37°C and saturated humidity with 5% CO2. Change the culture medium every other day. When the cell density reaches about 70%–80%, digest and passage the cells (digestion solution: 1 mL of 0.125% Trypsin-EDTA).

[0090] (4) In vitro cell toxicity test: The growth activity of cells in solution was detected using a cell proliferation and toxicity assay kit. Cell viability was calculated using the following formula:

[0091] Cell viability (%) = (As-Ab) / (Ac-Ab) × 100%

[0092] In the formula, As is the absorbance of the experimental well (the culture medium containing cells, CCK-8 and the sample); Ac is the absorbance of the control well (the culture medium containing cells and CCK-8); and Ab is the absorbance of the blank well (the culture medium containing CCK-8 but without cells and the sample).

[0093] In this experiment, cells were cultured with sample solutions of different concentrations (25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml), with a negative control group (i.e., complete culture medium). Each group was replicated with 10 wells. Cells in the logarithmic growth phase were collected, digested with 0.125% Trypsin-EDTA for 3 min, and then digestion was terminated with complete culture medium containing serum. The cells were resuspended and transferred to centrifuge tubes for centrifugation (800 rpm, 3 min). After centrifugation, the supernatant was removed, and fresh complete culture medium was added. The cells were resuspended and stained with 60 μL of 0.4% trypan blue solution. Cells were counted using a hemocytometer. Complete culture medium was added to adjust the cell density to approximately 3 × 10⁴ cells / mL. The cells were seeded into 96-well plates at 100 μL / well (approximately 3000 cells per well). No cells were seeded for four weeks. An equal volume of PBS solution was added. After inoculation, the culture plates were placed in a constant temperature incubator and cultured at 37°C and saturated humidity (5% CO2) for 24 hours. The old culture medium was gently aspirated, and 100 μL of blank medium containing 10% CCK-8 was added. The plates were then incubated for another 1 hour in a cell culture incubator. The absorbance (A value) at 450 nm was measured using a microplate reader to determine the number of surviving cells. During the microplate reader measurement, it was ensured that there were no air bubbles in the wells. Ten replicates were performed for each sample group. The cell viability of each experimental group was calculated, and the degree of toxicity was assessed.

[0094] like Figure 11As shown, the in vitro cytotoxicity test results indicated that none of the samples at any concentration exhibited significant cytotoxicity against RAW 264.7 macrophages.

[0095] (5) Establishment of the H2O2 oxidative damage model of cells: First, RAW 264.7 macrophages were seeded on the surface of 96-well cell plates at a cell concentration of 3×10⁻⁶ cells / well. 4 Cells / wells were placed in a cell culture incubator and cultured for 24 hours. Afterward, the culture medium was removed, and different concentrations of H2O2 (12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) were added for stimulation. After 2 hours, 10 μL of LCK-8 assay reagent was added to each well, and the cells were incubated for another 1 hour. The absorbance (A value) at 450 nm was measured using a microplate reader to determine the number of surviving cells. During the microplate reader measurement, ensure there are no air bubbles in the wells. Ten replicates were performed for each sample group. The cell viability of each experimental group was calculated.

[0096] To determine the optimal concentration of H2O2 in the oxidative stress model, cells were treated with H2O2 at concentrations of 12.5, 25, 50, 100, and 200 μM for 2 h, and the degree of cell damage was then assessed. Cell viability was measured using the CCK-8 assay as follows: Figure 12 As shown in the figure, the damaging effect of H2O2 on cells is dose- and time-dependent. Compared with the control group, stimulation with 200 μM for 2 hours resulted in a loss of more than 20% of cell viability.

[0097] (6) Laser confocal microscopy study of reactive oxygen species in RAW 264.7 macrophages: The cell grafting process is as follows: First, RAW 264.7 macrophages were seeded on the surface of a 24-well cell plate at a cell concentration of 3 × 10⁻⁶ cells / well. 5 Cells / wells were cultured in a cell culture incubator for 12 hours. The culture medium was removed, and samples of different concentrations were added to the cells. H2O2 (50 μM) was added for stimulation. After 2 hours, the samples and H2O2 were removed, followed by washing three times with sterile PBS solution. Then, 500 μL of 2',7'-dichlorofluorescein diacetate (DCFH-DA) was added, and the cells were incubated in the dark for 20 minutes. After washing three times with serum-free culture medium, the cells were finally photographed using a laser confocal scanning microscope. The control group included a blank and a blank-H2O2 sample, with four replicates per group.

[0098] Analysis of RAW 264.7 macrophages using 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining showed that the polymer possessed good antioxidant activity. Figure 13As shown, almost no fluorescent staining was observed in the Control group without H2O2 stimulation, indicating a low ROS content under oxidative stimulation. Upon addition of H2O2 stimulation, a strong fluorescent signal was observed intracellularly, as H2O2 can cross the cell membrane and activate macrophages to produce ROS. After Bac-CS treatment, the DCFH fluorescence intensity of cells in the H2O2-stimulated group began to decrease, and the intracellular fluorescence intensity continued to decrease with increasing Bac-CS concentration, reaching a minimum at a Bac-CS concentration of 400 μg / ml.

[0099] (7) Flow cytometry study of reactive oxygen species (ROS) in RAW 264.7 macrophages: Flow cytometry was used to study the effect of Bac-CS on ROS production in RAW 264.7 macrophages damaged by H2O2. RAW 264.7 macrophages were first seeded on the surface of 6-well cell plates at a cell concentration of 8 × 10⁻⁶ cells / well. 5 Cells / wells were placed in a cell culture incubator and cultured for 12 hours. After that, the culture medium was removed, and samples of different concentrations were added to the cells. H2O2 (200 μM) was added for stimulation. After 2 hours, the samples and H2O2 were removed, and the cells were washed three times with sterile PBS solution. Then, 500 μL of 2',7'-dichlorofluorescein diacetate (DCFH-DA) (S0033, Beyotime) was added. The cells were incubated in the cell culture incubator for 20 minutes in the dark. After washing three times with serum-free culture medium, the cells were centrifuged and resuspended in 300 μL of PBS (pH 7.4). The cells were then analyzed using flow cytometry. 10,000 cells were recorded for each sample, with three replicates per group.

[0100] Flow cytometry data of ROS in macrophages treated with different concentrations of Bac-CS are as follows: Figure 14 As shown in Table 3, the proportions of ROS-producing macrophages treated with different concentrations of Bac-CS are as follows:

[0101] Table 3. Proportion of ROS-producing macrophages treated with different concentrations of Bac-CS

[0102]

[0103] Example 4: Evaluation of the antibacterial activity of Bac-CS

[0104] (1) Preparation of bacterial culture medium: Add the broth culture medium powder to a 500mL Erlenmeyer flask, mix it with deionized water in a fixed ratio to form a solution, stir and dissolve it in a water bath at 50-80℃, seal it with a sterile sealing film, sterilize it in a high-temperature sterilizer at 121℃ for 15-20min, cool it to room temperature, and place it on a sterile operating table sterilized by ultraviolet light. Prepare and use it as soon as possible.

[0105] Add agar medium powder to a 500mL Erlenmeyer flask, mix with deionized water at a fixed ratio to prepare a solution, and stir to dissolve in a 50-80℃ water bath. Seal with sterile sealing film and sterilize in a 121℃ autoclave for 15-20 minutes. In a sterile operating table, pour the agar medium cooled to 40℃ into 90mm diameter sterile Petri dishes, about 15mL of medium per dish, and immediately rotate to ensure thorough and even dispersion. Place in a laminar flow hood, cool to room temperature, and sterilize with UV light for 20 minutes. Store the resulting agar plates at 4℃ and use as soon as possible.

[0106] (2) Bacterial Culture: Antibacterial activity was assessed using *Escherichia coli* and *Staphylococcus aureus*. The specific method is described below using *E. coli* as an example. First, a nutrient solution culture medium was prepared using 4.0 g peptone, 2.0 g beef dip powder, 2.0 g sodium chloride, and 400.0 g ultrapure water as raw materials. Following the above steps, 8.0 g agar was added to prepare a solid culture medium. Then, bacterial colonies were inoculated into the liquid culture medium and incubated in an air bath shaker (37℃, 130 rpm) for 24 h to obtain a fresh bacterial suspension with good activity. Before the experiment, the bacterial suspension concentration was diluted to 10. 5 CFU / mL.

[0107] (3) Determination of MIC and MBC: The MIC and MBC were determined using the double dilution method. The polymer concentration was diluted to 600 μg / mL as the starting concentration for the antibacterial experiment. The polymer concentration in the broth medium was serially diluted from well 1 to well 9 of a 96-well plate, resulting in concentrations of 300, 150, 75, 37.5, 18.75, 9.38, 4.68, 2.34, and 1.17 μg / mL, respectively. 10 μL of bacterial culture was added to each of the broth medium containing 600 μg / mL polymer. The 10th tube without polymer served as a blank control. The volume of broth medium and drug solution in each tube was 100 μL. After incubation at 37℃ with shaking for 12 h, the tubes were removed and bacterial growth was observed. Compared with the blank control, the lowest concentration at which the medium was observed to be clear and inhibited bacterial growth was determined as the minimum inhibitory concentration of the traditional Chinese medicine. Take 10 μL of bacterial suspensions at concentrations of 300, 150, 75, 37.5, 18.75, and 9.38 μg / mL and add them to agar medium. After culturing for 24 hours, take them out and observe the bacterial growth. The lowest concentration at which no bacterial growth is observed is determined as the minimum bactericidal concentration of the traditional Chinese medicine.

[0108] The volume of broth and herbal solution in each test tube was 100 μL. After incubation at 37℃ with shaking for 12 h, the tubes were removed to observe bacterial growth. Compared with the blank control, the lowest concentration at which the culture medium became clear and bacterial growth was inhibited was determined as the minimum inhibitory concentration (MIC) of the herbal medicine. The MIC of Bac-CS against *S. aureus* was 37.5 μg / mL, and the MIC against *E. coli* was also 37.5 μg / mL. 10 μL of bacterial solutions at concentrations of 300, 150, 75, 37.5, 18.75, and 9.38 μg / mL were added to agar medium and incubated for 24 h. Bacterial growth was then observed. Figure 15 As shown, the lowest concentration at which no bacterial growth was observed was determined as the minimum bactericidal concentration of traditional Chinese medicine. The minimum bactericidal concentration of Bac-CS against S. aureus was 75 μg / mL, and the minimum bactericidal concentration against E. coli was 75 μg / mL.

[0109] (4) Study on the antibacterial effect of plate colony method on Bac-CS: Under aseptic conditions, 100 μL each of polymer and chitosan solutions of different concentrations were added to the wells of a 96-well plate, followed by 10 μL of Staphylococcus aureus suspension (1×10⁻⁶). 5 The negative control was 100 μL of 1% acetic acid-water solution. Plates were incubated at 37℃ for 12 h. 100 μL of Staphylococcus aureus bacterial suspension (1×10⁻⁶ CFU / mL) was then used. 5 The CFU / mL solution was evenly spread on nutrient agar medium, and the medium was incubated in a 37℃ biochemical incubator for 24 hours.

[0110] observe Figure 16 It was found that polymer solutions of 37.5, 50, and 75 μg / mL all exhibited inhibitory effects on Staphylococcus aureus, and the effects were superior to those of chitosan solutions of the same concentration. (Observation) Figure 17 It can be seen that polymers of 18, 37, and 50 μg / mL have an inhibitory effect on Escherichia coli, and the effect is better than that of chitosan solution of the same concentration.

[0111] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing a nanofiber membrane with wound-healing properties, characterized in that, Includes the following steps: Step 1: Weigh out baicalein, 4-dimethylaminopyridine and glutaric anhydride in a certain proportion, add 1-10 mL of triethylamine and dry tetrahydrofuran to the reaction flask, and stir magnetically under 254 nm ultraviolet light for 14-24 h. Step 2: After rotary evaporating the reaction solution from Step 1, add ethyl acetate to the concentrate, add 5 mL of 1M HCl, extract with water, and extract with ethyl acetate 1-5 times. Combine the ethyl acetate layers, extract with saturated NaCl 1-5 times to obtain the ethyl acetate layer, and dry with anhydrous sodium sulfate. Remove the solvent using a rotary evaporator, mix the product with silica gel, load it onto a silica gel column, and elute with petroleum ether, ethyl acetate, and formic acid to separate the acidic intermediate. Step 3: Dissolve 1-50g of the acidic intermediate in methanol, add 1-10g of N-hydroxysuccinimide to the solution, stir at room temperature for 10-50min, dissolve chitosan in 1% acetic acid aqueous solution, add 1-10g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stir in the dark for 14-30h, dialyze the reaction solution using a dialysis bag for 10-80h, the molecular weight cutoff of the dialysis bag is 3000-10000Da, freeze dry for 10-60h to obtain BAC-g-CS; Step 4: Weigh out BAC-g-CS and PVA powder in a certain proportion, dissolve them in 90% acetic acid aqueous solution, stir the mixture with a magnetic stirrer at room temperature for 12-24 hours, let the prepared solution stand for 1-8 hours, and store it in a sealed container at room temperature. Step 5: Pour the prepared spinning solution into the syringe, connect the positive terminal of the high-voltage power supply to the needle, and connect the negative terminal of the high-voltage power supply to the collection plate with the equipotential paper attached. After the nanofiber membrane is spun, remove it and place it in an oven to dry at 80℃ for 12-24 hours.

2. The method for preparing the nanofiber membrane with wound-healing effect according to claim 1, characterized in that, In step 1, the ratio of baicalin, 4-dimethylaminopyridine, and glutaric anhydride is 1-10:1-10:1-10.

3. The method for preparing the nanofiber membrane with wound-healing effect according to claim 1, characterized in that, In step 2, the ratio of petroleum ether, ethyl acetate and formic acid used for elution is 1-50:1-50:1-50.

4. The method for preparing the nanofiber membrane with wound-healing effect according to claim 1, characterized in that, In step 4, the ratio of BAC-g-CS to PVA powder is 1-10:1-10.

5. The method for preparing the nanofiber membrane with wound-healing effect according to claim 1, characterized in that, In step 5, the distance between the needle and the collecting plate is 15-18cm, the spinning voltage is 10-30kV, the spinning time for each nanofiber membrane is 0.1-24h, the positive voltage is 5-20kV, the negative voltage is 1-10kV, the propulsion speed is 0.1-1mL / h, and the spinning temperature is 10-29℃.

6. A nanofiber membrane prepared by the preparation method according to claim 1.

7. The use of the nanofiber membrane according to claim 6 in the preparation of a wound dressing.

Images