An antibacterial and wound healing-promoting nanocomposite film and a preparation method and application thereof

Abstract

The application discloses an antibacterial and wound healing promoting nanocomposite film as well as a preparation method and application thereof. The nanocomposite film is obtained by repeatedly freezing and thawing a mixed solution of polyvinyl alcohol, lignin and MXene to form a crosslinked network. The mass ratio of the polyvinyl alcohol, the lignin and the MXene is 1:4:(0.0025-0.04). The application introduces MXene and LS into a PVA hydrogel system, endows the composite film with good mechanical properties, biocompatibility and antibacterial activity, and the composite film has moderate swelling performance and conductivity. The nanocomposite film of the application is non-toxic and has good chemical stability. The nanocomposite film can promote wound healing, significantly reduce inflammatory response, promote the formation of new blood vessels, strengthen nutrient transport, and can be combined with electric stimulation to further promote wound healing, and has a broad application prospect in the field of wound dressings.

Description

Technical Field

[0001] This invention relates to a nanocomposite membrane, specifically to an antibacterial and wound-healing nanocomposite membrane, its preparation method, and its application. Background Technology

[0002] The treatment of skin wounds is crucial to human health, making the development of novel, high-performance wound dressings a hot topic in the medical field in recent years. Traditional wound dressings, such as bandages and gauze, are passive dressings made of natural fibers. Their fiber structure can absorb exudate to keep the wound dry, but they are prone to adhesion, lack antibacterial properties, cannot provide sufficient drainage, and do not significantly promote wound healing. An ideal wound dressing should possess characteristics such as the ability to remove excess exudate, protect the wound from microbial infection, have good mechanical properties, be biocompatible, and be inexpensive. Polyvinyl alcohol (PVA) meets most of the characteristics of an ideal wound dressing and is currently being used in the development of novel wound dressings. However, the low mechanical strength and poor antibacterial properties of PVA limit its widespread use in wound dressings.

[0003] Recently, researchers have attempted to improve the mechanical properties of PVA hydrogel systems by adding different nanofillers. MXene, a novel two-dimensional nanomaterial, possesses numerous excellent properties, such as high hydrophilicity, high specific surface area, and superior mechanical properties. Furthermore, MXene exhibits antibacterial properties; its sharp edges can disrupt bacterial cell membranes to achieve an antibacterial effect. In addition, MXene can promote cell adhesion and proliferation. Therefore, MXene shows broad application potential in the biomedical field. Moreover, currently, antibiotics are often added to wound dressings to improve their antibacterial properties; however, antibiotic resistance is becoming an increasingly serious problem.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide an antibacterial and wound-healing nanocomposite membrane, its preparation method, and its application. This invention solves the problems of low mechanical strength and poor antibacterial properties of existing PVA. The nanocomposite membrane of this invention has good mechanical properties, biocompatibility, conductivity, and antibacterial activity.

[0006] To achieve the above objectives, the present invention provides an antibacterial and wound-healing nanocomposite membrane, which is obtained by repeatedly freezing and thawing a mixed solution of polyvinyl alcohol, lignin and MXene to form a cross-linked network; wherein the mass ratio of polyvinyl alcohol, lignin and MXene is 1:4:(0.0025~0.04).

[0007] Preferably, the repeated freeze-thaw conditions involve freezing at -20°C for 22 hours, followed by thawing at room temperature for 2 hours.

[0008] A second objective of this invention is to provide a method for preparing the aforementioned antibacterial and wound-healing nanocomposite membrane. This method comprises: adding polyvinyl alcohol to water, heating and stirring until the PVA is completely dissolved to obtain a PVA aqueous solution; then, adding lignin to the PVA solution and stirring to ensure that the lignin is uniformly dissolved in the PVA aqueous solution to obtain a PVA / LS mixed solution; adding an MXene dispersion aqueous solution to the PVA / LS mixed solution, stirring, and allowing it to stand to obtain a PVA / LS / MXene mixed solution; and subjecting the PVA / LS / MXene mixed solution to repeated freeze-thaw cycles to form a cross-linked network.

[0009] Preferably, the concentration of the PVA aqueous solution is 10 wt%.

[0010] Preferably, the concentration of the MXene dispersion aqueous solution is 1–16 mg / mL.

[0011] Preferably, polyvinyl alcohol is added to water, heated to 95°C and stirred until the PVA is completely dissolved to obtain a PVA aqueous solution.

[0012] Preferably, an aqueous dispersion of MXene is added to the PVA / LS mixed solution, stirred for 2 hours, and allowed to stand for 6 hours to obtain the PVA / LS / MXene mixed solution.

[0013] Preferably, the PVA / LS / MXene mixed solution is poured into a mold and frozen at -20°C for 22 hours, then thawed at room temperature for 2 hours, and the freeze-thaw cycle is repeated 2 to 6 times to form a stable cross-linked network.

[0014] A third objective of the present invention is to provide the application of the aforementioned antibacterial and wound-healing-promoting nanocomposite membrane in antibacterial and wound-healing-promoting aspects.

[0015] The antibacterial and wound-healing-promoting nanocomposite membrane of the present invention, its preparation method, and its application solve the problems of low mechanical strength and poor antibacterial properties of existing PVA, and have the following advantages:

[0016] (1) The nanocomposite membrane of the present invention introduces MXene and LS into the PVA hydrogel system, which endows the composite membrane with good mechanical properties, biocompatibility and antibacterial activity, and it also has moderate swelling properties and conductivity. Moreover, the nanocomposite membrane of the present invention is non-toxic and has good chemical stability, and it will not be oxidized after being placed at room temperature for 28 days.

[0017] (2) The nanocomposite membrane of the present invention has a loose and porous structure on its surface and cross-section, which can provide abundant cell adhesion sites and nutrient delivery channels, thereby achieving the effect of promoting wound healing. Moreover, the PLM composite membrane has excellent mechanical properties, good flexibility and stretchability.

[0018] (3) The nanocomposite membrane of the present invention, the PLM composite membrane can promote wound healing. Animal experiments show that the PLM composite membrane can heal wounds in a short time. The PLM composite membrane can downregulate the expression of pro-inflammatory factors IL-6 and TNF-α genes, significantly reduce inflammatory response, reduce the distribution of inflammatory cells and promote epithelial tissue formation. The PLM composite membrane can also promote the formation of new blood vessels and enhance the delivery of nutrients. Moreover, the PLM composite membrane has no obvious biotoxicity in vivo and has no significant effect on the vital organs of mice.

[0019] (4) The nanocomposite membrane of the present invention can be combined with electrical stimulation to further promote wound healing, showing broad application prospects in the field of wound dressings.

[0020] (5) The preparation method of the present invention is a strategy for green synthesis of hydrogel wound dressing, that is, using the sol-gel method to prepare a composite membrane (PLM) of PVA, LS and MXene with excellent antibacterial activity and mechanical properties. Through repeated freeze-thaw cycles, the mixed solution forms a stable and uniform cross-linked network. At the same time, the -OH on the PVA chain and LS forms hydrogen bonds with functional groups such as -OH, -F, and =O on the surface of MXene. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of the PLM composite membrane of the present invention.

[0022] Figure 2 Characterization results of MXene nanosheets prepared in Example 1 of this invention; (a) transmission electron microscopy; (b) Tyndall effect.

[0023] Figure 3 Characterization results of the PLM composite film prepared in Example 1 of the present invention: (a) XRD spectrum; (b) FTIR spectrum.

[0024] Figure 4SEM images of the PLM composite film of the present invention with PVA and PL; (a) surface; (b) cross-section.

[0025] Figure 5 The flexibility (a) and stretchability (b-d) of the PLM8 composite membrane of the present invention under wet conditions are shown.

[0026] Figure 6 The images show the following: (a) Staphylococcus aureus and Escherichia coli plate coating of the PLM composite membrane with PVA and PL in bacterial experiments of this invention; (b) Staphylococcus aureus colony count; (c) Escherichia coli colony count; and (d) inhibition zones of Staphylococcus aureus and Escherichia coli.

[0027] Figure 7 The conductivity of the PLM composite film of this invention is shown.

[0028] Figure 8 The swelling curves of the PLM composite membrane of the present invention with PVA and PL are shown in (a); the swelling rate of the composite membrane after 24 hours of immersion is shown in (b); and optical photographs of the four mixed solutions before and after standing at room temperature for 28 days are shown in (c).

[0029] Figure 9 The results of CCK-8 assays on days 1, 3, and 5 show the PLM composite membrane of this invention with PVA and PL.

[0030] Figure 10 Images of L929 cells cultured on the PLM composite membrane of this invention with PVA and PL for 1, 3, and 5 days (scale bar: 300 μm).

[0031] Figure 11 The images show optical images (a) of the wound after electrical stimulation using the PLM composite membrane of this invention, and H&E stained images (b) collected on postoperative days 3, 9, and 15.

[0032] Figure 12 The images show the expression of TNF-α (a), IL-6 (b), and representative images of TNF-α immunofluorescence staining on day 3 and day 9, and representative images of CD31 immunofluorescence staining on day 9 and day 15, when the PLM composite membrane of the present invention was applied to mouse wound tissue.

[0033] Figure 13 The images show H&E staining of vital organs (heart, liver, spleen, lung, and kidney) on day 15 after the PLM composite membrane of this invention was applied to the wound tissue of mice. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] It should be noted that: for conditions not specifically specified in the examples, standard conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0036] In this invention, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used for simplicity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible secondary ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0037] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0038] This invention provides an antibacterial and wound-healing-promoting nanocomposite membrane, its preparation method, and its application. The preparation method of the antibacterial and wound-healing-promoting nanocomposite membrane includes:

[0039] (1) Add PVA to deionized water and stir magnetically at 95°C until PVA is completely dissolved to obtain a 10wt% PVA aqueous solution; then, add an appropriate amount of LS (lignin) powder to the PVA solution and stir magnetically for 0.5h to ensure that the LS powder is uniformly dissolved in the PVA solution.

[0040] (2) Prepare MXene dispersions of different concentrations (1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, 12 mg / mL and 16 mg / mL), then add them to a PVA / LS mixed solution and stir magnetically for 2 h and let stand for 6 h;

[0041] (3) Pour the mixed solution into a mold and freeze it in a -20℃ freezer for 22 hours. Then thaw it at room temperature for 2 hours. Repeat the freeze-thaw cycle 2 to 6 times to form a stable cross-linked network (non-covalent cross-linking) to obtain a PLM composite membrane.

[0042] Because the final product is used as a wound dressing to promote wound healing, it must be in the form of a film. The mass ratio of PVA, LS, and MXene in the final composite film is 1:4:0.005-0.04.

[0043] The following examples and comparative examples provide a detailed description of the antibacterial and wound-healing nanocomposite membrane, its preparation method, and its application.

[0044] The raw materials and reagents used in the following examples and comparative examples are as follows:

[0045] 1) MXene solution: prepared by our laboratory using 400-mesh MAX phase as raw material, according to the fluorine etching method (HCl+LiF), or commercially available MXene solution can be used directly;

[0046] 2) PVA: PVA powder from Thermo Scientific was used;

[0047] 3)LS: LS powder from BOSF is used.

[0048] Example 1

[0049] A nanocomposite membrane with antibacterial and wound-healing properties is prepared by the following method:

[0050] Add 2.0 g PVA to 20 mL of deionized water and stir at 95 °C until the PVA is completely dissolved to obtain a 10 wt% PVA aqueous solution. Then add 8.0 g LS powder to the prepared PVA solution and stir magnetically for 0.5 h.

[0051] Prepare 5 mL of 1 mg / mL MXene dispersion aqueous solution, add it to the above mixed solution and stir magnetically for 2 h. After stirring, let stand for 6 h, pour the mixed solution into a mold and freeze in a -20℃ freezer for 22 h, thaw for 2 h, and repeat the freeze-thaw cycle 4 times to form a cross-linked network to obtain the PLM-1 composite membrane.

[0052] Example 2

[0053] An antibacterial and wound-healing-promoting nanocomposite membrane is prepared using a method essentially the same as in Example 1, with the difference being:

[0054] The concentration of the prepared MXene dispersion aqueous solution was 2 mg / mL.

[0055] The resulting composite membrane is designated as PLM-2 composite membrane.

[0056] Example 3

[0057] An antibacterial and wound-healing-promoting nanocomposite membrane is prepared using a method essentially the same as in Example 1, with the difference being:

[0058] The concentration of the prepared MXene aqueous solution was 4 mg / mL.

[0059] The resulting composite membrane is designated as PLM-4 composite membrane.

[0060] Example 4

[0061] An antibacterial and wound-healing-promoting nanocomposite membrane is prepared using a method essentially the same as in Example 1, with the difference being:

[0062] The concentration of the prepared MXene dispersion aqueous solution was 8 mg / mL.

[0063] The resulting composite membrane is designated as PLM-8 composite membrane.

[0064] Example 5

[0065] An antibacterial and wound-healing-promoting nanocomposite membrane is prepared using a method essentially the same as in Example 1, with the difference being:

[0066] The concentration of the prepared MXene dispersion aqueous solution was 12 mg / mL.

[0067] The resulting composite membrane is designated as PLM-12 composite membrane.

[0068] Example 6

[0069] An antibacterial and wound-healing-promoting nanocomposite membrane is prepared using a method essentially the same as in Example 1, with the difference being:

[0070] The concentration of the prepared MXene dispersion aqueous solution was 16 mg / mL.

[0071] The resulting composite membrane is designated as PLM-16 composite membrane.

[0072] Comparative Example 1

[0073] A PVA composite film is prepared by the following method:

[0074] Add 1.0 g PVA to 10 mL of deionized water and stir at 90 °C until the PVA is completely dissolved to obtain a 10 wt% PVA aqueous solution.

[0075] Subsequently, 2.5 mL of deionized water was added to the above mixed solution and magnetically stirred for 6 hours. After stirring, the mixed solution was poured into a mold and placed in a -20°C freezer for 22 hours. After thawing for 2 hours, the freeze-thaw cycle was repeated 4 times to form a cross-linked network. Then, the PVA composite film was obtained by freeze drying.

[0076] Comparative Example 2

[0077] A PL composite membrane is prepared by the following method:

[0078] Add 1.0 g PVA to 10 mL of deionized water and stir at 90 °C until the PVA is completely dissolved to obtain a 10 wt% PVA aqueous solution. Then add 4.0 g LS powder to the PVA solution and sonicate for 0.5 h.

[0079] Subsequently, 2.5 mL of deionized water was added to the above mixed solution and magnetically stirred for 6 hours. After stirring, the mixed solution was poured into a mold and placed in a -20°C freezer for 22 hours. After thawing for 2 hours, the freeze-thaw cycle was repeated 4 times to form a cross-linked network. Then, the PL composite membrane was obtained by freeze drying.

[0080] Experimental Example 1: Material Structure Characterization

[0081] 1. Characterization of the prepared MXene nanosheets

[0082] like Figure 2 As shown in (a), MXene exhibits a clean and smooth two-dimensional layered structure under a transmission electron microscope. Figure 2 As shown in (b), the Tyndall effect of the MXene solution demonstrates that the MXene flakes have good dispersibility, uniformity and stability in water.

[0083] 2. Characterization of PLM composite membrane

[0084] like Figure 3 As shown, (a) XRD patterns and (b) FTIR patterns of the raw materials and composite membranes are presented, where MAX represents the MXene precursor material. From... Figure 3 This indicates that hydrogen bonds have formed between PVA, LS, and MXene.

[0085] 3. Morphology and structure of PLM composite membrane

[0086] like Figure 4 As shown, SEM images of the surface (a) and cross-section (b) of the composite membrane are presented, illustrating that the surface of the composite membrane becomes rough with the addition of LS and MXene, and the cross-section exhibits a loose and porous morphology.

[0087] Experiment Example 2 Performance Test

[0088] 1. Mechanical property testing

[0089] (1) Qualitative test of tensile properties of PLM composite film

[0090] like Figure 5As shown, the PLM8 composite membrane exhibits good (a) flexibility and (b-d) stretchability in a wet state (hydrogel obtained through freeze-thaw technology without freeze-drying). (b) and (d) are images before and after stretching, and (c) is an image during stretching.

[0091] (2) Quantitative testing of the mechanical properties of PLM composite membrane

[0092] All mechanical properties of the PLM composite hydrogel were tested at room temperature using an electronic universal testing machine (AGS-X, Shimadzu, Japan). Each test was repeated three times for each sample, and the average value was calculated.

[0093] Evaluation of tensile properties: The PLM composite membrane samples prepared in each embodiment were cut into standard rectangular shapes (size: 30mm × 20mm × 2mm). Tensile tests were conducted at a constant rate of 5mm / min. The tensile strength was calculated based on the maximum applied load / cross-sectional area of ​​the sample, and the elongation at break was calculated using the following formula: (length of tensile fracture / initial gauge length) × 100%. The elastic modulus (i.e., Young's modulus) was calculated based on the slope of the initial linear portion of the stress-strain curve.

[0094] Compression strength test: The PLM composite membrane samples prepared in each embodiment were made into cylinders with a diameter of 15.5 mm and a height of 5 mm. During freeze-thaw cycles, the uniformly mixed solution was poured into the cylindrical mold, and after freeze-thaw gelation, cylindrical samples were obtained. Under room temperature conditions, each sample was subjected to three repeated compression tests, and the compressive strength was calculated based on the load / cross-sectional area.

[0095] The results are shown in Table 1. The experimental results show that MXene concentration affects both the tensile strength and elongation at break of the composite film. With increasing MXene concentration, both elongation at break and tensile strength first increase and then decrease, while the elastic modulus shows an increasing trend.

[0096] Table 1 shows a performance comparison of the composite membranes prepared in the various embodiments and comparative examples of the present invention.

[0097]

[0098]

[0099] 2. Antibacterial activity test

[0100] The antibacterial properties of the composite membrane were systematically evaluated using the inhibition zone test and colony counting method. The experimental procedure is as follows:

[0101] Sterilized composite membranes (PLM-4, PLM-8, PL, or PVA) were cut into circles (1 cm in diameter). Staphylococcus aureus and Escherichia coli were then diluted to appropriate concentrations in sterilized Luria-Bertani (LB) medium, and the diluted bacterial solutions were evenly spread onto agar medium using a sterile spreader. Next, different sterilized composite membrane samples (a control group was set up in the experiment, which did not contain any materials) were placed on the medium and incubated at 37°C for 18–22 hours. The formation and size of inhibition zones were then observed.

[0102] In the colony counting experiment, diluted bacterial solutions and different sterilized composite membranes (a Control group was set up in the experiment, which did not contain any materials) were co-cultured on a constant temperature shaker at 37°C for 18–22 hours. Finally, the obtained bacterial solution was further diluted to an appropriate concentration and evenly spread on agar medium, and then incubated at 37°C for another 18–22 hours. The bacterial colonies were then counted to evaluate the degree of inhibition of bacterial growth by the composite membrane.

[0103] See results Figure 6 The images show the following bacterial experiments using the PLM composite membrane of this invention: (a) Staphylococcus aureus and Escherichia coli plate coating; (b) Staphylococcus aureus colony count; (c) Escherichia coli colony count; and (d) inhibition zones for Staphylococcus aureus and Escherichia coli. It can be seen that the PLM composite membrane of this invention has good antibacterial properties. Both the colony counting method and the inhibition zone experiment results show that the composite membrane has good antibacterial ability and exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria.

[0104] Table 1 shows the colony counts of Staphylococcus aureus and Escherichia coli on plate coatings. It can be seen that MXene concentration also affects antibacterial activity. With increasing MXene concentration, the antibacterial performance of the PLM composite membrane against Staphylococcus aureus and Escherichia coli is enhanced.

[0105] 3. Conductivity test

[0106] The PLM composite film prepared in Example 1 was connected to the positive electrode of the battery on one side and to one end of the lamp strip on the other side. The other end of the lamp strip was then connected to the negative electrode of the battery, and the light emission of the lamp strip was observed.

[0107] See results Figure 7 The PLM composite film of the present invention has good electrical conductivity.

[0108] 4. Chemical stability test

[0109] 1) Swelling experiment

[0110] Accurately weigh and record the weight (W0) of the freeze-dried PVA, PL, PLM-4, and PLM-8 composite membranes. Then, immerse each group of samples in a predetermined volume of PBS solution at room temperature. Remove the composite membranes at preset time points (0.5h, 1h, 3h, 6h, 12h, and 24h), gently wipe away any residual moisture with filter paper, and immediately weigh the composite membrane (Ws). Each experiment is repeated three times. The swelling rate is calculated based on the following formula:

[0111] Swelling rate (%) = (Ws - W0) / W0 × 100

[0112] 2) Oxidative stability test

[0113] To evaluate the effects of PVA and LS on the oxidative stability of the freshly prepared MXene solution, an oxidation inhibition experiment was conducted, as detailed below:

[0114] First, solutions containing different raw materials were prepared: (a) MXene dispersion; (b) MXene-PVA mixed solution; (c) MXene-LS mixed solution; (d) MXene-PVA-LS mixed solution. Each solution was left to stand at room temperature for 28 days, and then the oxidation of the solution was observed.

[0115] See results Figure 8 (a) shows the swelling curve of the PLM composite membrane of the present invention; (b) shows the swelling rate of the PLM composite membrane of the present invention after immersion for 24 hours; and (c) shows photographs of the four mixed solutions before and after standing at room temperature for 28 days. It can be seen that the PLM composite membrane of the present invention has a low swelling rate and good stability when used in atmospheric environments. Furthermore, the MXene-PVA-LS mixed solution exhibits good chemical stability at room temperature and does not oxidize after being placed at room temperature for 28 days.

[0116] Experiment Example 3: Biocompatibility Experiment

[0117] The biocompatibility of the composite membrane was evaluated using the CCK-8 assay, as detailed below:

[0118] Mouse fibroblasts (L-929) were selected as the model cells. L929 cells were cultured in MEM (modified Eagle medium) containing 10% horse serum at 37°C, 95% humidity, and 5% CO2. 1.2 × 10⁶ cells were seeded onto different sterilized composite membranes. 4L929 cells were cultured for 1, 3, and 5 days, and cell proliferation was assessed. At each time point, all the original culture medium was gently aspirated from the wells, and PBS solution was added. The cells were washed three times, and the PBS solution was then aspirated. Subsequently, CCK-8 reagent was added to the wells under dark conditions, and the cells were incubated at 37°C in a cell culture incubator for 4 hours. After incubation, the liquid in the wells was transferred to 96 cell culture plates, and the absorbance was measured at 450 nm using a multi-mode microplate reader to evaluate cell number and proliferation status.

[0119] See results Figure 9 The figures show the CCK-8 assay results of the PLM composite membrane of this invention on days 1, 3, and 5. The CCK-8 results show a higher cell count on the composite membrane, indicating that the PLM composite membrane of this invention can promote cell proliferation compared with the control group (PVA, PL), and that the PLM composite membrane of this invention has good biocompatibility. Moreover, with the increase of MXene concentration, cell proliferation showed a trend of first increasing and then decreasing.

[0120] Experiment Example 4: Cytotoxicity Test

[0121] Based on the analysis of the above mechanical properties and CCK-8 experimental results, PLM-4 and PLM-8 composite membranes, which possess both excellent mechanical properties and biocompatibility, were selected as the experimental group for Calcein-AM / PI double staining experiments to evaluate their cytotoxicity, as detailed below:

[0122] With 1.5×10 per hole 4 L929 cells were seeded at different densities on sterilized composite membranes (NC group consisted of cells only without the composite membrane). A control group was established where L929 cells were directly seeded into wells. Cell viability was observed after 1, 3, and 5 days of culture. On each fixed day, all original culture medium was aspirated from the wells, and PBS solution was added. The cells were washed three times, and the PBS solution was then removed. Next, Calcein-AM and PI staining solutions were added to each well to stain the cells. The cells were then incubated at 37°C in the dark for 30 minutes, and the number of viable cells was observed and photographed under a fluorescence microscope.

[0123] See results Figure 10 Images of L929 cells cultured on the PLM composite membrane of this invention for 1, 3, and 5 days, showing double staining of live / dead cells. Calcein-AM / PI double staining results show that most cells on the composite membrane are green, indicating that these cells are alive, demonstrating that the PLM composite membrane of this invention is non-toxic to cells.

[0124] Experiment Example 5: Animal Experiment

[0125] To verify the wound-healing promoting effect of PLM composite membrane, the following animal experiments were conducted:

[0126] Thirty-six female Kunming mice weighing between 27 and 30 g were selected as subjects for the experiment. After acclimatization in the laboratory environment for two weeks, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg). Subsequently, the fur on the backs of the mice was shaved, the exposed skin was cleaned, and a full-thickness skin wound with a diameter of 7 mm was created on their backs.

[0127] Wound models were randomly divided into four groups: control group (only wound model treatment, without composite membrane as wound dressing or electrical stimulation), ES (electrical stimulation) group, PLM group (using PLM-8), and PLM+ES group (using PLM-8+ES). The ES group and PLM+ES group received 3.6V DC stimulation for 1 hour daily, with the negative electrode placed on the wound site and the positive electrode attached to the normal skin area. During the experiment, wound size was observed and recorded at regular intervals on days 0, 3, 6, 9, 12, and 15 to compare wound healing under different conditions. In addition, mouse wound tissue was collected on days 3, 9, and 15 for H&E staining to observe histological changes in the skin, and the expression of common inflammatory factor TNF-α and vascular endothelial cell marker CD31 was detected by immunofluorescence staining. Simultaneously, the mRNA levels of inflammatory factors IL-6 and TNF-α were measured using RT-PCR to further analyze cytokine changes in mouse wound tissue. Finally, on day 15 of the experiment, vital organs (including heart, liver, spleen, lungs, and kidneys) of mice were collected and H&E staining was performed to assess the biocompatibility of the PLM composite membrane in vivo.

[0128] See results Figures 11-13 , Figure 11 The images show (a) of the wound after the PLM composite membrane combined with electrical stimulation (ES) of the present invention, and (b) H&E stained images acquired on postoperative days 3, 9, and 15. Figure 12 The images show the analysis of TNF-α expression (a) and IL-6 expression (b) in mouse wound tissue when the PLM composite membrane of the present invention was applied, as well as representative images of TNF-α immunofluorescence staining on days 3 and 9 (c) and representative images of CD31 immunofluorescence staining on days 9 and 15 (d). Figure 13 The images show H&E staining of vital organs (heart, liver, spleen, lung, and kidney) on day 15 after the PLM composite membrane of this invention was applied to the wound tissue of mice.

[0129] from Figure 11 It can be seen that the PLM composite membrane of the present invention can promote wound healing, and animal experiments show that the PLM composite membrane can heal wounds in a shorter time.

[0130] from Figure 12 It can be seen that the PLM composite membrane can downregulate the expression of pro-inflammatory factors IL-6 and TNF-α genes. RT-PCR experiments showed that the PLM composite membrane can reduce the expression of pro-inflammatory factors and significantly reduce the inflammatory response. Immunofluorescence results of CD31, a marker of vascular endothelial cells, showed that the PLM composite membrane can promote angiogenesis at the wound site, indicating that the PLM composite membrane of the present invention can promote angiogenesis and enhance nutrient delivery. The PLM composite membrane can reduce the distribution of inflammatory cells. HE staining experiments showed that the PLM composite membrane can reduce inflammatory cells in connective tissue and promote epithelial tissue formation.

[0131] from Figure 13 It can be seen that the PLM composite membrane has no obvious biotoxicity in vivo, and the PLM composite membrane has no significant effect on the important organs (heart, liver, spleen, lungs, and kidneys) of mice.

[0132] In summary, the PLM composite membrane of the present invention not only has good mechanical properties, chemical stability and conductivity, but also has antibacterial effects, good biocompatibility, and is non-toxic. It can promote wound healing and angiogenesis, and has no effect on vital organs. Furthermore, it can be combined with electrical stimulation to further promote wound healing, showing broad application prospects in the field of wound dressings.

[0133] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A nanocomposite membrane for antibacterial and wound-healing purposes, characterized in that, This nanocomposite membrane was obtained by repeatedly freezing and thawing a mixed solution of polyvinyl alcohol, lignin, and MXene to form a cross-linked network. The mass ratio of polyvinyl alcohol, lignin and MXene is 1:4:(0.0025~0.04).

2. The antibacterial and wound-healing-promoting nanocomposite membrane according to claim 1, characterized in that, The repeated freeze-thaw conditions involved freezing at -20°C for 22 hours, followed by thawing at room temperature for 2 hours.

3. The method for preparing the antibacterial and wound-healing-promoting nanocomposite membrane as described in claim 1 or 2, characterized in that, The method includes: Polyvinyl alcohol is added to water and heated and stirred until PVA is completely dissolved to obtain a PVA aqueous solution; then, lignin is added to the PVA solution and stirred to ensure that the lignin is uniformly dissolved in the PVA aqueous solution to obtain a PVA / LS mixed solution. An aqueous dispersion of MXene was added to the PVA / LS mixed solution, stirred, and allowed to stand to obtain a PVA / LS / MXene mixed solution. The PVA / LS / MXene mixed solution was subjected to repeated freeze-thaw cycles to form a cross-linked network.

4. The preparation method according to claim 3, characterized in that, The concentration of the PVA aqueous solution is 10 wt%.

5. The preparation method according to claim 3, characterized in that, The concentration of the MXene dispersion aqueous solution is 1~16 mg / mL.

6. The preparation method according to claim 3, characterized in that, Polyvinyl alcohol is added to water, heated to 95°C and stirred until PVA is completely dissolved to obtain a PVA aqueous solution.

7. The preparation method according to claim 3, characterized in that, An aqueous dispersion of MXene was added to the PVA / LS mixed solution, stirred for 2 hours, and allowed to stand for 6 hours to obtain the PVA / LS / MXene mixed solution.

8. The preparation method according to claim 3, characterized in that, The PVA / LS / MXene mixed solution was poured into a mold and frozen at -20°C for 22 h, then thawed at room temperature for 2 h. This process was repeated 2 to 6 times to form a stable cross-linked network.

9. The use of the antibacterial and wound-healing-promoting nanocomposite membrane as described in claim 1 or 2 in the preparation of antibacterial and wound-healing-promoting drugs.

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