Active biomedical fabric based on plant filamentous fungi as well as preparation method and application of active biomedical fabric

Abstract

The invention discloses an active biomedical fabric based on plant filamentous fungi and a preparation method and application thereof, and belongs to the technical field of biological materials. The plant fungus-based active biomedical fabric (FgMLM) is successfully prepared by using a transboundary plant filamentous fungus fusarium graminearum (Fg) capable of specifically infecting plant cells but not infecting animal cells through a filtering process; the fabric has no infectivity and toxicity to human and animals, and has the characteristics of natural antibacterial property, self-growth, self-repair, biological safety and the like; according to the invention, exogenous genes are introduced through gene editing, various active eukaryotic protein drugs can be expressed by using a powerful eukaryotic expression system of Fg, and more biomedical functions are introduced for FgMLM, so that the FgMLM has remarkable characteristics of infection resistance, inflammation resistance, wound healing acceleration and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly to an active biomedical fabric based on plant filamentous fungi, a preparation method thereof, and an application thereof. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art known to those of ordinary skill in the art.

[0003] As a vibrant field, active materials make full use of the unique capabilities of organisms and have inherent properties such as sensing, self - growth, self - adaptation, and the introduction of exogenous multifunctions. Active materials have developed rapidly in the cross - field of synthetic biology and materials science and have attracted much attention. In recent years, by using the unique capabilities of complex bioactive substances such as synthesis, environmental sensing, signal transduction, and genetic and surface modifiability, organisms have been widely used to construct active materials through synthetic biology, chemical, and physical methods. Active materials have inherent properties such as sensing, self - growth, self - adaptation, and regeneration, and can also be engineered to introduce exogenous multifunctions, such as responses to various environmental stimuli (such as molecular effectors, light, temperature, pH, mechanical stress). Active materials with both life and intelligent attributes have great potential in applications as active biomedical materials.

[0004] Compared with single living cells such as bacteria, yeast, and mammalian cells, filamentous fungi are composed of multiple elongated cells, forming an adaptable colonial living system. This special composition and morphology endow them with unique properties, such as inherent mechanical strength, high adjustability, and the ability to form porous three - dimensional network structures. Recently, the mycelia of filamentous fungi have been widely used to construct macroscopic active materials, such as hydrogels, scaffolds, and biosensors. For example, mycelia are processed into mycelial inks for 3D printing active mycelial structures and complex objects, and are also designed as environmental monitors. However, due to safety concerns about infection and toxicity, the application of these active mycelial materials in the biomedical field is less.

[0005] Plant filamentous fungi are specifically parasitic on plants and cannot recognize and infect animals and humans, which indicates their good biosafety, which is a key attribute of active materials in biomedical applications. However, so far, the application of active materials based on plant filamentous fungi in the biomedical field has not been explored. In addition, due to the shortage of editing tools, most plant filamentous fungi cannot be modified by genetic engineering, so new exogenous functions cannot be introduced as needed.

[0006] Therefore, it is necessary to develop a new active biomedical fabric based on plant filamentous fungi to solve the above problems. Summary of the Invention

[0007] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide an active biomedical fabric based on plant filamentous fungi. This fabric is non-infectious and non-toxic to humans and animals, and has characteristics such as self-growth, self-repair, anti-infection, anti-inflammation, and accelerating wound healing. It can express various active eukaryotic protein drugs.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] An active biomedical fabric based on plant filamentous fungi, characterized in that the active biomedical fabric is composed of the mycelium of Fusarium graminearum after gene editing. The gene editing includes the following steps:

[0010] Step 1: Construct a plasmid containing an exogenous gene and linearize the plasmid;

[0011] Step 2: Prepare protoplasts of Fusarium graminearum and transfer the linearized plasmid into the protoplasts.

[0012] The present invention also provides a preparation method of the above-mentioned active biomedical fabric based on plant filamentous fungi, including the following steps:

[0013] S1: Cultivate Fusarium graminearum to obtain a bacterial suspension containing Fusarium graminearum;

[0014] S2: Vacuum filter and dry the bacterial suspension to obtain the active biomedical fabric FgMLM. The pressure during the filtration process is 0.1 MPa, and the pore size of the filter membrane used is 5 μm and the diameter is 50 mm. FgMLM has a porous structure, good toughness and flexibility, and significant hydrophilicity.

[0015] Through gene editing, the active biomedical fabric can be used to express fluorescent proteins and eukaryotic protein drugs, such as interleukin-22 (IL-22) and chemokine (C-X-C motif) ligand 12 (CXCL12), and has the ability for bioimaging and treatment.

[0016] The active biomedical fabric can also secrete a variety of effective antibacterial substances (such as antibacterial peptides and metabolic compounds), has good antibacterial ability and anti-inflammatory characteristics, and can be applied to the preparation of anti-infection drugs or anti-inflammatory drugs. The infections include pathogenic bacterial infections and pathogenic fungal infections. The pathogenic bacteria include Pseudomonas aeruginosa, Propionibacterium acnes, Staphylococcus aureus, Salmonella typhimurium. The pathogenic fungal infection includes Candida albicans.

[0017] The active biomedical fabric can also be used to prepare drugs for treating wounds, which can prevent wound infections, inhibit wound inflammation, and accelerate wound healing.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention uses the cross-kingdom plant filamentous fungus Fusarium graminearum (Fg), which can specifically infect plant cells but not animal cells, and successfully prepares an active biomedical fabric based on plant fungi (FgMLM) through a filtration process; this fabric is non-infectious and non-toxic to humans and animals, and has characteristics such as natural antibacterial properties, self-growth, self-repair, and biosafety; the present invention introduces exogenous genes through gene editing, and can use the powerful eukaryotic expression system of Fg to express various active eukaryotic protein drugs, introducing more biomedical functions into FgMLM, making it have significant anti-infection, anti-inflammatory, and wound healing acceleration characteristics.

[0020] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following takes the preferred embodiments of the present invention and describes them in detail in conjunction with the accompanying drawings. Description of the Drawings

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, some of the following drawings are embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 Preparation and characterization of living materials based on plant filamentous fungi (FgMLM): a, Schematic diagram of the preparation process of FgMLM. b, Appearance inspection of FgMLM. c, Evaluation of the compressibility and bendability of FgMLM. For detailed mechanical properties, see d, Tensile stress-strain curve; e, Compression stress-strain curve; f, Rheological stress-strain curve of FgMLM. g, Hydrophobicity evaluation of FgMLM. A: Aerial hyphae of Fusarium graminearum, B: FgMLM. Data are presented as mean ± standard deviation (n = 3). h, Scanning electron microscope image of FgMLM. Scale bar: 50 μm, 10 μm. i, Cumulative pore size distribution and differential pore size distribution of FgMLM.

[0023] Figure 2Demonstration of the biosafety of FgMLM: a, Schematic diagram of the specific infection of Fusarium graminearum mycelium, indicating its preferential infection of plant cells rather than animal cells. b, Representative laser scanning confocal microscopy (CLSM) images of wheat coleoptiles and mouse skin co-incubated with Fusarium graminearum. Scale bars: 20 μm, 50 μm. c, CCK-8 assays of human umbilical vein endothelial cells (HUVECs) and d, 293T cells after co-incubation with Fusarium graminearum. HUVECs and 293T cells not treated with Fusarium graminearum were used as controls (n = 3). e, Calcein / Propidium Iodide (PI) staining of HUVECs and 293T cells cultured with Fusarium graminearum. Untreated HUVECs and 293T cells were used as controls. Scale bar: 50 μm. e, Growth of Fusarium graminearum at 25 °C and 37 °C (n = 3). f, Growth trend of Fusarium graminearum under aerobic and anaerobic conditions (n = 3). g, In vivo biosafety assessment of FgMLM in a mouse wound model. h, Quantification of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in mouse serum on day 7 (n = 5). i, Body weights of mice treated with FgMLM and Candida albicans (n = 5). Mice treated with PBS were used as controls. Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test, and p-values were given, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns indicates no significant difference.

[0024] Figure 3 Hematoxylin and eosin (H&E) staining results of normal organs of mice treated with FgMLM, Candida albicans (C. albicans), and phosphate buffered saline (PBS), scale bar: 2.5 mm.

[0025] Figure 4Showing the active characteristics of FgMLM: a, Self-growth of FgMLM on agar. b, Self-repair of FgMLM on agar. Circular samples of FgMLM with sizes from 1 to 4 were made in the center and placed on agar for 3 days (n = 3). c, Self-repair of FgMLM on pig skin. Circular FgMLM was made in the center and stored on agar for 3 days. d, Gene editability of FgMLM. Construction and observation of green fluorescent protein (GFP)-Fusarium graminearum, red fluorescent protein (mCherry)-Fusarium graminearum, and GFP-mCherry-Fusarium graminearum fluorescent hyphae. Scale bar: 50 μm. e, FgMLM expressing GFP in different shapes. f, FgMLM expressing mCherry in different shapes. g, Schematic diagram showing the construction and PCR verification of IL-22-FgMLM. h, Quantification of IL-22 in the supernatant of Fusarium graminearum on day 2 (n = 4). i, Schematic diagram showing the construction and PCR verification of CXCL12-FgMLM. j, Quantification of CXCL12 in the supernatant of Fusarium graminearum on day 2 (n = 4).

[0026] Figure 5 : a, CLSM images of spores and hyphae of engineered Fg. Scale bar: 20 μm, 50 μm. b, Growth of engineered Fg (n = 3). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test, and p values were given. ns indicates no significant difference.

[0027] Figure 6 Showing the antibacterial property of FgMLM: a-b, Observation of liquid cultures co-cultured with bacteria after removing Fusarium graminearum. Pathogenic bacteria Staphylococcus aureus, PA14, VNP, and pathogenic fungus Candida albicans were co-cultured with Fusarium graminearum at 37 °C for 6 hours, and then Fusarium graminearum was removed. Cultures not treated with Fusarium graminearum were used as controls. c-d, Confirmation of bacterial attachment on the mycelium of Fusarium graminearum. Pathogens were co-cultured with Fusarium graminearum at 37 °C for 6 hours. The mycelium was taken out, resuspended in PBS, homogenized, and the homogenate was diluted and spread on agar plates. e-f, Antibacterial activity of the secreted substances of Fusarium graminearum. Pathogens were treated with the supernatant containing the secreted substances of Fusarium graminearum at 37 °C for 6 hours (n = 3). Statistical analysis was performed using a two-tailed unpaired t-test between the two groups, and p values were given, *p < 0.05, **p < 0.01, ***p < 0.001. g-h, Typical transmission electron microscopy (TEM) images of the morphology of pathogens after co-culture with Fusarium graminearum. Scale bar: 1 μm.

[0028] Figure 7Demonstration of the antibacterial property of FgMLM: a-b, Observation of liquid cultures co-cultured with bacteria after removal of Fusarium graminearum. The pathogenic bacteria Staphylococcus aureus, PA14, VNP, and the pathogenic fungus Candida albicans were co-cultured with Fusarium graminearum at 37 °C for 6 h, and then Fusarium graminearum was removed. Cultures without treatment with Fusarium graminearum were used as controls. c-d, Confirmation of bacterial attachment on the mycelia of Fusarium graminearum. The pathogens were co-cultured with Fusarium graminearum at 37 °C for 6 h. The mycelia were taken out and resuspended in PBS for homogenization. The homogenate was diluted and spread on agar plates. e-f, Antibacterial activity of the secreted substances of Fusarium graminearum. The pathogens were treated with the supernatant containing the secreted substances of Fusarium graminearum at 37 °C for 6 h (n = 3). Statistical analysis was performed using two-tailed unpaired t-tests between the two groups, and p-values were given, *p < 0.05, **p < 0.01, ***p < 0.001. g-h, Representative transmission electron microscopy (TEM) images of the pathogen morphology after co-culture with Fusarium graminearum. Scale bar: 1 μm.

[0029] Figure 8 Demonstration of the efficacy of IL-22-FgMLM in acne treatment: a, Release of IL-22 by IL-22-FgMLM in simulated artificial body fluid (n = 4). b, In vitro anti-infective effect of IL-22-FgMLM against Propionibacterium acnes. c, In vitro anti-infective effect of IL-22-FgMLM against Staphylococcus aureus. The survival of the pathogens treated with IL-22-FgMLM was measured (n = 3). d, In vitro anti-inflammatory effect of IL-22-FgMLM. Macrophage polarization was analyzed by flow cytometry (n = 3). e, Schematic diagram of the mouse experimental design for evaluating the efficacy of IL-22-FgMLM in acne treatment. f, Photographs of acne lesions in different treatment groups on day 2, day 4, and day 7 (n = 6). g, Detection and analysis of neutrophils in sectioned skin tissues using anti-Ly-6G antibody (red) and 4',6-diamidino-2-phenylindole (DAPI, blue) (n = 5). h, Hematoxylin and eosin (H&E) staining of wound tissues on day 2. Scale bar: 200 μm. i, Thickness of abnormal epithelium and size of sebaceous glands (n = 5). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test, and p-values were given, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0030] Figure 9Identification results of IL-22 and CXCL12 secreted in the supernatant of IL-22-CXCL12 Fg on days 0.5, 1, 2, 3, 4, and 10 (n = 4). Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test to obtain p-values, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

[0031] Figure 10 Acne area scores of mice treated with IL-22-FgMLM. Untreated mice and mice treated with erythromycin and FgMLM were used as controls (n = 6). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test to give p-values, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0032] Figure 11 : a, Body weights of acne mice in different treatment groups (n = 6). b, Survival of Staphylococcus aureus and Propionibacterium acnes after treatment with IL-22-FgMLM (n = 6). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test to give p-values, *p < 0.05, **p < 0.01, ***p < 0.001.

[0033] Figure 12Demonstrating the efficacy of IL-22-CXCL12-FgMLM in the treatment of wound infection: a, construction of IL-22-CXCL12-Fg. b, quantification of IL-22 and CXCL12 secreted by IL-22-CXCL12-Fg after 2 days of culture (n = 4). c, curves of IL-22 and CXCL12 released by IL-22-CXCL12-FgMLM in simulated artificial body fluid (n = 4). d, in vitro anti-infective effect of IL-22-CXCL12-FgMLM against PA14. The survival of PA14 was measured after co-incubating the cultured IL-22-CXCL12-Fg supernatant with PA14 at 37 °C for 6 hours (n = 3). e, in vitro anti-inflammatory effect of IL-22-CXCL-12-FgMLM. Macrophage polarization was analyzed by flow cytometry (n = 3). f, wound healing scratch assay was performed on human umbilical vein endothelial cells (HUVECs) treated with IL-22-CXCL12-FgMLM. Scale bar: 100 μm. g, hematoxylin-eosin (H&E) staining of wound tissues on day 2. h, Masson staining of wound tissues on day 2. Scale bar: 200 μm. i, photographs of wounds in different treatment groups on day 0 and day 14. j, quantification of wound healing rates in different treatments during the treatment period (n = 5). Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test, and p values were given, *p < 0.05, **p < 0.01, ***p < 0.001.

[0034] Figure 13 : a, body weights of acne mice in different treatment groups. Data are presented as mean ± standard deviation (n = 6). b, survival rates of Staphylococcus aureus and Propionibacterium acnes after IL-22-XXX treatment. Data are presented as mean ± standard deviation (n = 6). Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test, and p values were obtained, *p < 0.05, **p < 0.01, ***p < 0.001. Detailed implementation manners

[0035] To better describe the present invention, the following is further illustrated by specific examples. The methods in the following examples are conventional methods unless otherwise specified.

[0036] The technical solutions described in the present invention are conventional solutions in the art unless otherwise specified; the reagents or materials are commercially available unless otherwise specified.

[0037] The following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0038] Example 1 Preparation of Fusarium graminearum mycelial live material (FgMLM)

[0039] 1.1 Materials and strains

[0040] Strains: The plant filamentous fungus Fusarium graminearum (Fg, NRRL.31084) was kindly provided by Professor Tang Weihua of the University of Chinese Academy of Sciences. Pseudomonas aeruginosa PA14 was provided by Professor Wang Jianxin of Fudan University in Shanghai and cultured in LB medium at 37 °C. VNP20009 was purchased from the China General Microbiological Culture Collection Center and cultured in LB medium at 37 °C. Staphylococcus aureus was provided by Professor Li Min of Shanghai Jiao Tong University and cultured in LB medium at 37 °C. Candida albicans was purchased from the Shanghai Biological Resource Collection Center (SHBCC, Shanghai) and cultured in YPD medium at 37 °C. Propionibacterium acnes was purchased from SHBCC and cultured under anaerobic conditions (10% H 2 、10% CO 2 and 80% N 2 ) at 37 °C. Human umbilical vein endothelial cells (HUVEC) were provided by Dr. He Caiping of Southern University of Science and Technology and cultured in Roswell Park Memorial Institute 1640 (RPMI1640) medium at 37 °C. 293T cells were purchased from the American Type Culture Collection (ATCCCRL-3216) and cultured in Dulbecco's Modified Eagle Medium (DMEM) at 37 °C.

[0041] Reagents: PCR Master Mix, HotStart PCR Genotyping MasterMix (with dye), Gentamicin Sulfate (G418), and Hygromycin B were purchased from Yeasen Biotech Co., Ltd. (Shanghai). The One Step Cloning Kit (ClonExpress Ultra One Step Cloning Kit) was provided by Novoprotein Scientific Inc. (Nanjing). LB broth, LB agar, YPD broth, YPD agar, and PBS buffer were purchased from Sangon Biotech Co., Ltd. (Shanghai). Other reagents and solvents were used as received.

[0042] 1.2 Preparation of Growth Medium and Fungal Cultivation

[0043] The preparation method of V8 juice agar is as follows: Add 100 mL of V8 juice made from 8 vegetables, 1 g of calcium carbonate, and 2% agar to 1 L of deionized water. The preparation method of mung bean liquid medium is as follows: Add 40 g of mung beans to 1 L of deionized water, and remove the mung bean residue after boiling. The above media were autoclaved at 121 °C for 20 minutes.

[0044] The conidia of Fusarium graminearum were stored in a glycerol stock solution at -80 °C. The conidia were inoculated on V8 juice agar plates and cultured at 25 °C for 3 to 5 days, and then subsequent inoculation was carried out.

[0045] 1.3 Preparation of Fusarium graminearum Mycelium Live Material (FgMLM)

[0046] The mycelium grown on V8 juice solid medium was transformed into spores using mung bean liquid medium, and the spore concentration was adjusted to 2×106 / mL. Add 1 mL of spore suspension to 200 mL of yeast extract peptone dextrose (YEPD) liquid medium. After culturing in YEPD medium for 2 days, take 30 mL of the culture for vacuum filtration to prepare the fungus-based live material. The filtration conditions were: passing through a filter membrane with a pore size of 5 μm and a diameter of 50 mm for 30 seconds under a pressure of 0.1 MPa. After filtration, it was dried at room temperature for 1 hour, and the Fusarium graminearum mycelium live material (FgMLM) was successfully prepared.

[0047] Example 2 Characterization and Property Analysis of Fusarium graminearum Mycelium Live Material (FgMLM)

[0048] 2.1 Macroscopic Property Characterization:

[0049] Through the preparation process described in Example 1, the FgMLM active fabric with a thickness of 0.76 - 0.86 mm and a weight of 0.46 - 0.53 g was successfully prepared ( Figure 1 b). The macroscopic properties of FgMLM, including mechanical properties and hydrophilicity / hydrophobicity, were characterized:

[0050] All mechanical tests of FgMLM were carried out on a universal material testing machine (Instron 3342, USA). For the compression test, a strain rate of 5 mm / min was used, and cylindrical samples were used with the compression strain set at 90% or 95%. For the tensile test, rectangular samples were used at a strain rate of 100 mm / min. Each test was repeated three times. As Figure 1 shown in c, FgMLM has good toughness and bendability. Mechanical tests showed that the tensile stress of this active fabric material was 256.48 kPa at 10.19% strain and the compression stress was 1273.12 kPa at 40% strain ( Figure 1 d, e).

[0051] The rheological properties of FgMLM were measured by frequency sweep tests. According to Figure 1 f, the spectra of the elastic modulus (G') and viscous modulus (G") were relatively stable, showing a typical hydrogel spectrum, and G' was greater than G".

[0052] The hydrophilicity and hydrophobicity of FgMLM were evaluated by contact angle experiments. To evaluate the hydrophobic properties of F. graminearum aerial hyphae and FgMLM, the samples were adhered to glass slides. 5 μL of water droplets were carefully dropped onto random areas of the sample surface using a micropipette and kept stationary. Subsequently, the contact angle was measured using a contact angle measuring instrument (KRÜSS DSA100, Germany). The water absorption rate at different time intervals (5, 10, and 30 s) was evaluated by calculating the percentage increase in weight. Before weighing, the excess water on the sample was gently blotted with filter paper to ensure accurate measurement. As Figure 1 shown in g, the contact angle of Fg hyphae growing on solid medium was greater than 90°, indicating strong hydrophobicity, while when the filamentous hyphae were processed into FgMLM, the contact angle decreased to 0°, showing significant hydrophilicity.

[0053] 2.2 Microscopic property characterization:

[0054] To further evaluate the microscopic characteristics of this mycelial active material, its fiber network was observed by imaging with a scanning electron microscope (SEM, Zeiss 1550VP field emission scanning electron microscope) ( Figure 1 h). Accordingly, the cumulative pore size distribution and differential pore size distribution in the range of 0.5 - 5 μm were analyzed by mercury intrusion porosimetry ( Figure 1 i). The porosity of FgMLM was determined to be 66.13%, which means it has a porous structure and good water absorption potential.

[0055] Example 3 Biosafety identification of FgMLM

[0056] Biosafety is a key criterion for evaluating therapeutic materials. Therefore, the biosafety of FgMLM was tested in vitro and in vivo. Fg is a plant-derived fungus that can specifically infect crops such as wheat and corn, and it shows strong invasiveness to wheat coleoptiles ( Figure 2 a).

[0057] 3.1 In vitro biosafety assessment

[0058] The cytotoxicity of the substances secreted by Fg to animal cells (human embryonic kidney 293T cells (293T) and human umbilical vein endothelial cells (HUVEC)) was evaluated using the Cell Counting Kit-8: HUVEC and 293T cells (5×105 cells / mL) were seeded in 12-well plates for 12 hours until the cells adhered. Subsequently, the medium was replaced with fresh medium. HUVEC cells were cultured in RPMI1640 medium, and 293T cells were cultured in DMEM medium, and both media contained 10% Fusarium graminearum culture solution. After culturing for 24 and 48 hours, CCK-8 reagent (Beyotime, China) was added to detect cell viability. The specific steps were as follows: First, the original medium was replaced with 300 μL of fresh medium containing 10% CCK-8 solution, and then incubated in the dark for 2 hours. Finally, the OD value at 450 nm was recorded ( Figure 2 ).

[0059] The biocompatibility of Fusarium graminearum was evaluated using the calcein / PI cytotoxicity assay. The cells were cultured in a medium containing 10% Fusarium graminearum culture solution at 37 °C for 48 hours. The cell suspension was collected and gently mixed with calcein / PI (Beyotime, China) staining solution in the dark. Then, 1 mL of the stained cell suspension was added to a confocal microscopy culture dish for 30 minutes. Fluorescence was observed under a confocal laser scanning microscope (CLSM) to evaluate cell viability and cytotoxicity.

[0060] As Figure 2 shown by the cell viability data in c, Fg had no effect on the cells within 24 or 48 hours. The live / dead cell staining results showed strong green fluorescence and no obvious red fluorescence, which also indicated that Fg had good cell biocompatibility and biosafety for animal cells ( Figure 2 d).

[0061] In addition, the in vitro growth curves and colony sizes under different conditions showed that the fungus could not grow at 37 °C or in an anaerobic environment ( Figure 2 e, 2f), which proved that FgMLM is a safe biomaterial with the potential to be designed into biomedical active materials and applied to animals and even humans.

[0062] 3.2 In vivo biosafety assessment

[0063] Female Balb / c and C57 mice (6 - 8 weeks old) specific pathogen free were purchased from Beijing SPF Biotechnology Co., Ltd. All animal experiments were conducted according to institutional guidelines and approved by the Animal Care and Use Committee of Shanghai Yishang Biotechnology Co., Ltd. (IACUC - 2023 - Mi - 218).

[0064] A wound with a diameter of 1 cm was made on the back of female Balb / c mice (6 - 8 weeks old), and then FgMLM was applied to the wound site. Two days later, FgMLM was removed. After 15 days, the skin tissues in contact with the fungus, as well as the heart, liver, spleen, lungs, and kidneys, were removed from the mice. To evaluate the biosafety of the fungus, the samples were stained with calcofluor white (18909, Sigma) and hematoxylin - eosin (H&E) staining (C0105M, Beyotime Biotechnology) according to the manufacturer's instructions. Mouse tissues treated with PBS and Candida albicans were used as controls.

[0065] Based on the results of in vitro biosafety tests, its performance was further evaluated in a mouse wound model( Figure 2 g). A skin wound with a diameter of 1.5 cm was made on the back of the mice, and then the wound was treated with FgMLM with medical glue for 3 days, with phosphate - buffered saline (PBS) and the human pathogenic fungus Candida albicans (C. albicans) as controls. Seven days after treatment, the levels of the representative inflammatory factors interleukin - 6 (IL - 6) and tumor necrosis factor - α (TNF - α) in the blood were measured by enzyme - linked immunosorbent assay (ELISA).

[0066] The evaluation results showed that no infection was observed in animal cells when the mouse skin was treated with Fg( Figure 2 b). As Figure 2 shown in h, the levels of these inflammatory factors in mice treated with PBS and FgMLM were not affected. On the contrary, compared with the PBS and FgMLM groups, the levels of IL - 6 and TNF - α in the group treated with C. albicans were significantly increased, indicating that the plant - fungal - based active fabric material does not trigger any inflammatory response. In addition, hematoxylin - eosin (H&E) staining showed that no obvious pathological changes were observed in the major organs of mice treated with FgMLM( Figure 3 ). The body weights of all mice were also recorded in the in - vivo biosafety assessment, Figure 2 as shown in i, the body weights of mice in the FgMLM group were significantly higher than those in the PBS group, while the body weights of mice in the C. albicans group were significantly lower. Overall, in vitro and in - vivo evaluations showed that, different from human pathogenic fungi, Fg does not infect animal cells and does not produce toxic side effects on mice, and has good application potential as a safe biomedical active fabric.

[0067] Example 4 Self - growth and self - repair activity characteristics of FgMLM

[0068] Mycelium - based active materials usually possess self - repair and self - adaptation capabilities. The vigorous metabolic activity and physiological functions of mycelial cells endow them with remarkable active characteristics, such as self - growth, self - repair, and gene - editing abilities. Therefore, it is necessary to identify whether this active fabric constructed from pure filamentous mycelium and containing no nutritional components has these active characteristics.

[0069] First, the active fabric was placed on a solid agar plate to observe its morphological changes. As Figure 4 shown in a, from day 0 to day 3, the active fabric underwent regrowth and the colony area expanded. Its self - regeneration ability was demonstrated by culturing active fabrics with circular incisions of different sizes (diameter 0.1 - 1 cm) at the center. It was noted that regardless of the incision size, they gradually self - repaired ( Figure 4 b). In addition, during the self - regeneration process of FgMLM, the newly grown mycelia gradually connected with each other to form a mycelial network, which then developed into a tight and dense network.

[0070] The self - growth and self - regeneration abilities of FgMLM were further explored through an ex - vivo animal model. The active fabric with a central circular hole was placed on the skin of pigs and rats, and it was found that the missing circular hole was filled and the colony area expanded ( Figure 4 c). All these data reflect the strong guerrilla - growth characteristics and curiosity of the mycelial network, as well as good potential for future in - vivo applications.

[0071] Example 5 Gene - editing activity characteristics of FgMLM

[0072] Compared with synthetic materials, filamentous fungi can be designed as intelligent active materials because they have a powerful eukaryotic expression system and show the ability to serve as an effective chassis for producing various proteins. To introduce the desired foreign functions into FgMLM, first, a plasmid driving the expression of relevant genes by the EF1 - α promoter was constructed, and then the plasmid was linearized. At the same time, protoplasts of Fusarium graminearum were prepared, and the linearized plasmid was transferred into the protoplasts for the expression of relevant genes. FgMLM can continuously express different proteins. Compared with other physical and chemical materials, Fusarium graminearum can be designed as a more intelligent bioactive material.

[0073] To verify the gene editing ability of Fg, plasmids with the EF1-α promoter were constructed to drive gene expression and achieve single and dual expression of fluorescent proteins. For the construction of fluorescent strains, the green fluorescent protein (gfp) gene was cloned into ZN1 (a vector driven by the EF1-α promoter with a nourseothricin resistance cassette), and the red fluorescent protein (mcherry) gene was inserted into ZN2 (a vector driven by the EF1-α promoter with a neomycin resistance cassette). The resulting constructs (ZN1-GFP, ZN2-mcherry) were transformed into the protoplasts of Fusarium graminearum. To construct dual-fluorescent strains, ZN2-mcherry was transformed into the protoplasts of Fg-GFP.

[0074] As Figure 4 shown in Figure 4 d, the typical confocal laser scanning microscopy (CLSM) images showed that engineered Fg successfully produced green fluorescent protein (GFP, green) and mCherry (red) either alone or simultaneously, and the hyphae and spores showed green, red, and yellow fluorescence. FgMLM made from engineered filamentous hyphae was also equipped with fluorescent proteins regardless of its shape (

[0075] e, f). Figure 4 The versatility of gene editing of Fg was tested by producing eukaryotic protein drugs. Constructs for expressing the well-studied cytokine IL-22 and chemokine CXCL12 were assembled in Fg (

[0076] g, i). The cDNA of interleukin-22 (IL-22) was amplified and inserted into the ZN2 vector. The cDNA of CXC chemokine ligand 12 (CXCL12) was amplified and inserted into ZN3 (a vector driven by the EF1-α promoter with a hygromycin resistance cassette). The resulting constructs (ZN2-IL-22, ZN3-CXCL12) were transformed into the protoplasts of Fg-GFP. For the construction of bifunctional strains, ZN3-CXCL12 was transformed into the protoplasts of Fg-IL-22. The plasmids and primers used in this example are listed in Tables 1 and 2, respectively.

[0076] Table 1 Plasmids used in the Fg gene editing process

[0077]

[0078]

[0079] Table 2 Primers used in the Fg gene editing process

[0080]

[0081] The expression levels of these two proteins were measured by ELISA, as Figure 4 h andFigure 4 As shown in Figure j, IL-22 and CXCL12 were successfully produced and secreted into the supernatant as designed, with concentrations of approximately 20,000 pg / mL and 300 pg / mL, respectively. In addition, the expression of these proteins had no significant effect on the morphological characteristics and germination process of their spores and the growth of hyphae ( Figure 5 a, b). Furthermore, through further experiments, it was found that when fluorescent proteins (GFP or mcherry) and eukaryotic protein drugs (IL-22 and CXCL12) were co-expressed, the expression level of eukaryotic protein drugs by Fg was significantly increased (see Table 3), and the introduction of fluorescent protein genes had a synergistic effect on the expression of eukaryotic protein drugs.

[0082] Table 3 Expression levels of eukaryotic protein drugs

[0083]

[0084] Through gene editing, FgMLM was able to successfully express fluorescent proteins and eukaryotic protein drugs, showing potential for bioimaging and therapy.

[0085] Example 6 Antibacterial properties of FgMLM

[0086] The mycelia of Fg were co-cultured with various pathogenic bacteria, such as Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa PA14 (PA14), and Salmonella typhimurium VNP20009 (VNP), as well as the pathogenic fungus C. albicans, to examine whether filamentous Fg had natural antibacterial activity.

[0087] Specifically, the pathogenic bacteria S. aureus, PA14, and VNP were cultured overnight in LB liquid medium, while the pathogenic fungus C. albicans was cultured with shaking at 37 °C in YPD liquid medium. After collecting these four microorganisms, they were washed with PBS and diluted to an optical density (OD) value of 1 at a wavelength of 600 nm. Subsequently, 50 μL of the microbial suspension and 0.1 g of mycelia were added to 5 mL of the corresponding liquid medium. After co-culturing the mycelia with these pathogenic microorganisms at 25 °C (the optimal growth temperature of Fg) for 24 hours, turbidity comparisons were made, using pathogenic bacteria without Fusarium graminearum as a control. After removing the mycelia, it was found that almost no growth of all these pathogens occurred in the presence of the mycelia, and the culture medium remained relatively clear. After co-culturing the mycelia with these pathogenic microorganisms at 37 °C (the body temperature in humans) for 6 hours, turbidity comparisons (also removing the mycelia) were made, using pathogenic bacteria without Fusarium graminearum as a control. AsFigure 6 As shown in Figs. 7a and 7b, after co-culturing with Fg for 6 hours, the growth of the pathogen was inhibited. Therefore, Fg exhibits natural antibacterial properties against pathogens within a wide temperature range.

[0088] The isolated mycelia were transferred to a tissue grinder containing two steel beads with a diameter of 3 mm, 5 mL of PBS buffer was added, and the mixture was ground at 70 Hz for 2 minutes. 50 μL of the solution was spread on LB and YPD agar plates and cultured overnight at 37 °C for colony counting. As Figure 6 shown in Figs. 7c and 7d, the bacterial count indicated that the Fg mycelia could not adsorb the pathogen, which means that its filamentous morphology is not the reason for inhibiting the growth of pathogenic microorganisms by adsorbing them.

[0089] The pathogen was cultured in a medium containing Fg secretions at 37 °C for 6 hours, and then diluted and plated. The results of bacterial counting showed that the secretions of Fg had an inhibitory effect on these pathogens ( Figure 6 Figs. 7e and 7f). Transmission electron microscopy (TEM) was used to observe the resulting microscopic morphological changes to explore how these secretions exert antibacterial effects. Typical TEM images showed that the phenotypic changes of these pathogens were different, which means that Fg secretes different substances to combat different pathogens, and the corresponding destruction mechanisms are also different ( Figure 6 Fig. 7g). For example, in the case of Staphylococcus aureus, certain secreted substances can cause a significant overall increase in cell size, accompanied by cell rupture and leakage of intracellular contents. The results of particle size and zeta potential measurements further confirmed the microscopic changes shown in the TEM images ( Figure 7 ). Leakage and vacuole formation were observed inside the treated PA14 cells by TEM, although there was no significant difference in the overall size. For the treated VNP, cytoplasmic shrinkage and a gap between the cell membrane and cell wall were noted. The results of zeta potential tests further confirmed the severe alteration of the VNP cell membrane. In the case of the treated Candida albicans, the cell surface presented an uneven and rough appearance with a large number of surface damages.

[0090] It can be seen from this that Fg has good antibacterial ability, and the antibacterial mechanism is that Fg can secrete a variety of effective antibacterial substances, such as antibacterial peptides and metabolic compounds.

[0091] Example 7 Anti-infective and anti-inflammatory effects of interleukin-22 modified Fusarium graminearum mycelial live material (IL-22-FgMLM)

[0092] After studying various characteristics of FgMLM, including biosafety, self-repair, gene editability, and natural antibacterial properties, its potential in in vivo biomedical applications was further explored. Specifically, FgMLM that produces interleukin-22 (IL-22) was selected to evaluate its therapeutic value in treating acne, which is caused by co-infection of Propionibacterium acnes and Staphylococcus aureus; interleukin-22 (IL-22) is a member of the interleukin-10 family and plays an important role in physiological processes such as anti-infection and anti-inflammation.

[0093] 7.1 Release ability of IL-22 from IL-22-FgMLM

[0094] Before evaluating the therapeutic effect, the release of IL-22 from IL-22-FgMLM was tested in vitro. A piece of FgMLM was placed in simulated artificial body fluid, and the level of IL-22 in the fluid was measured by enzyme-linked immunosorbent assay (ELISA) at specified time points (0.5, 1, 2, 3, 4, and 10 days). As Figure 8 a and Figure 9 shown, IL-22 began to be rapidly released from IL-22-FgMLM after being placed in simulated artificial body fluid; thereafter, the level of IL-22 in the artificial body fluid showed a sharp upward trend, reaching 4000 picograms per milliliter on the 1st day, and then gradually reaching a peak of approximately 6000 picograms per milliliter; on the 4th day, the secretion of IL-22 began to decrease, and new secretion could still be detected on the 10th day. The above results indicate that IL-22-FgMLM can release protein drugs for a long time in a simulated environment, showing the potential for sustainable development and providing a solid foundation for subsequent in vivo applications.

[0095] 7.2 Anti-infection ability of IL-22-FgMLM

[0096] The anti-infection ability of IL-22-FgMLM against acne-related pathogens Propionibacterium acnes and Staphylococcus aureus was verified in vitro: The supernatant containing IL-22 secretion was collected and used to culture Propionibacterium acnes or Staphylococcus aureus at 37°C for 6 hours, and the supernatant of unengineered Fg and fresh LB were used as controls. Due to the natural antibacterial properties of Fg, its supernatant significantly inhibited the growth of Propionibacterium acnes and Staphylococcus aureus. However, the supernatant containing IL-22 was able to further enhance the inhibitory effect on these two bacteria, reducing the survival rates of Propionibacterium acnes and Staphylococcus aureus by 3.4-fold and 6.6-fold, respectively ( Figure 8 b, c).

[0097] 7.3 Anti-inflammatory ability of IL-22-FgMLM

[0098] The M2 / M1 macrophage polarization ratio is a key indicator for evaluating the inflammatory condition, and the in vitro anti-inflammatory effect of IL-22-FgMLM was explored through macrophage polarization. First, bone marrow-derived macrophages (BMDMs) were isolated from the bone marrow of C57 mice and then cultured in DMEM medium supplemented with macrophage colony-stimulating factor (concentration 50 ng / mL, Sino Biological, China), 10% fetal bovine serum (FBS), streptomycin (100 mg / mL), and penicillin (100 U / mL) in vitro for 7 days. 500 μL of the supernatant of the fungal culture solution was added to BMDMs together with 2 mg / mL lipopolysaccharide (Sigma) and cultured at 37 °C for 12 hours. After the cells were washed three times, they were stained with anti-CD11b (M1 / 70), anti-F4 / 80 (BM8), and anti-CD86 (24F, M1 macrophage marker) antibodies. After the cells were washed again, they were fixed and permeabilized, and then intracellular staining was performed with anti-CD206 antibody (M2 macrophage marker) to evaluate the activation of BMDMs. The Foxp3 / Transcription Factor Fixation / Permeabilization Kit (eBioscience) was used to fix and permeabilize the cells. All antibodies were purchased from BioLegend and eBioscience. Data were collected on a FACSVerse flow cytometer (BD Biosciences, USA) and analyzed using FlowJo software (TreeStar Inc., USA). The polarization of M1 and M2 macrophages was detected by flow cytometry using anti-CD11b (M1 / 70), anti-F4 / 80 (BM8), and anti-CD86 antibodies and anti-CD206 antibody. As Figure 8 shown in d, the M2 / M1 ratio in the IL-22-FgMLM group was approximately 1.5 times higher than that in the FgMLM group, indicating that IL-22-FgMLM has anti-inflammatory properties.

[0099] Example 8 Therapeutic effect of IL-22-FgMLM in the treatment of acne

[0100] Based on the detection of the performance of IL-22-FgMLM in vitro (including the long-acting release of protein drugs and enhanced anti-infection and anti-inflammatory capabilities), its therapeutic effect in an acne model was further studied:

[0101] 8.1 Test animals

[0102] Specific pathogen-free female Balb / c and C57 mice (6 - 8 weeks old) were purchased from Beijing SPF Biotechnology Co., Ltd. All animal experiments were conducted according to institutional guidelines and approved by the Animal Care and Use Committee of Shanghai Yishang Biotechnology Co., Ltd. (IACUC-2023-Mi-218).

[0103] 8.2 In vivo biosafety assessment

[0104] A wound with a diameter of 1 cm was made on the back of female Balb / c mice (6 - 8 weeks old), and then FgMLM was applied to the wound site. Two days later, FgMLM was removed. After 15 days, the skin tissues in contact with the fungus, as well as the heart, liver, spleen, lungs, and kidneys, were removed from the mice. To evaluate the biosafety of the fungus, the samples were stained with Calcofluor White (18909, Sigma) and hematoxylin - eosin (H&E) staining (C0105M, Beyotime Biotechnology) according to the manufacturer's instructions. Mouse tissues treated with PBS and Candida albicans were used as controls.

[0105] 8.3 Establishment of a mouse acne model

[0106] Propionibacterium acnes and Staphylococcus aureus were cultured and resuspended in PBS to prepare a bacterial suspension. These bacteria were mixed in a ratio of 7:3, and 50 μL of this mixture was intradermally injected into the left and right sides of the back of Balb / c mice. After injecting the bacteria, a mouse acne model was successfully established 2 days later.

[0107] 8.4 Treatment of acne with IL - 22 - FgMLM

[0108] The mice with induced acne were randomly divided into four groups. The first group served as a negative control, with only gauze applied and no other treatment. The second group served as a positive control, wiped with erythromycin ointment and then gauze was applied. The third group was treated with FgMLM, and the fourth group was treated with IL - 22 - FgMLM. Two days after treatment, the materials of all groups were removed before subsequent applications. Three days later, the mice were euthanized for further analysis.

[0109] 8.5 Evaluation of treatment effects

[0110] The lesions were observed and recorded daily. Photos of mouse acne were taken, and its development was analyzed by evaluating the severity of the induced lesions (using the clinical and histological scores described in the Leeds acne scoring system) Figure 8 f). This improvement was reflected in a decrease in the lesion area and a reduction in severity Figure 8 f).

[0111] According to the scoring results, compared with the mice treated with erythromycin and non - engineered FgMLM, the mice treated with IL - 22 - FgMLM obtained the lowest score two days after treatment Figure 10 ). The body weights of the mice were recorded daily, and no effects were detected Figure 11a). To deeply analyze the treatment effect, the mice were euthanized for tissue biopsy and histopathological evaluation. Local inflammation, as well as abnormal epithelium and sebaceous glands, are the main features of skin changes caused by acne. Lesion tissues were extracted and stained with anti-Ly-6G antibody to detect neutrophils, which are an important indicator of the inflammatory response. The results showed that IL-22-FgMLM effectively inhibited the inflammation in the lesion area ( Figure 8 g).

[0112] The H&E staining results further confirmed this finding, which showed a significant reduction in the infiltration of inflammatory cells in the skin tissue ( Figure 8 h). At the same time, it should be noted that in the mice treated with IL-22-FgMLM, both the thickness of the abnormal epithelium and the size of the sebaceous glands were significantly reduced ( Figure 8 i). In addition, the tissue biopsy samples were homogenized to quantify the bacteria Propionibacterium acnes and Staphylococcus aureus in the lesion site, indicating that IL-22-FgMLM has effective antibacterial activity in the treatment of acne ( Figure 11 b). In summary, these findings suggest that IL-22-FgMLM can effectively reduce the inflammatory response and eliminate acne, and its performance is superior to the antibiotic treatment in current clinical practice.

[0113] Example 9 Therapeutic effect of IL-22-CXCL12-FgMLM in wound treatment

[0114] The living fabric materials were further engineered, and their therapeutic effects were evaluated using a mouse wound model.

[0115] 9.1 Preliminary preparation

[0116] Clinically, wound treatment mainly focuses on preventing bacterial infection and promoting healing. CXCL12 belongs to the chemokine family and has been shown to accelerate wound healing in mice, pigs, and even humans. First, an engineered Fg capable of dual production of IL-22 and CXCL12 was constructed for the preparation of IL-22-CXCL12-FgMLM ( Figure 12 a). Then, the expression of IL-22 and CXCL12 was confirmed by ELISA. As Figure 12 shown in b, IL-22 and CXCL12 were detectable in the dual-engineered Fg, with concentrations of approximately 20,000 picograms per milliliter and 300 picograms per milliliter, respectively. The co-expression of IL-22 and CXCL12 was also verified to have a negligible effect on the physiological activity of Fg ( Figure 5 ).

[0117] In addition, the release curves of IL-22 and CXCL12 in IL-22-CXCL12-FgMLM were studied in vitro. Specifically, the material was placed in simulated artificial body fluid at time points of 0.5, 1, 2, 3, 4, and 10 days, and ELISA( Figure 12 c and Figure 9 ) was used. Two separately engineered Fgs (IL-22-Fg and CXCL12-Fg) were used as controls. The release curve of IL-22 showed that the secretion of IL-22 gradually increased, reaching a peak of approximately 8000 picograms per milliliter. Thereafter, its concentration began to decline and reached the lowest level on the tenth day. The concentration of CXCL12 peaked at approximately 200 picograms per milliliter and remained detectable within 10 days( Figure 12 c). In summary, this bifunctional living fabric material can continuously produce and release protein drugs under in vitro simulated conditions, indicating good potential for further in vivo applications.

[0118] After confirming the secretion of IL-22 and CXCL12 in the dual-engineered Fg, their functions in antibacterial, anti-inflammatory, and healing aspects were studied in vitro. As expected, the presence of IL-22 enhanced its antibacterial ability against the skin infection-related bacterium PA14 and its anti-inflammatory ability by regulating macrophage polarization( Figure 12 d, e).

[0119] 9.2 Cell Scratch Assay

[0120] HUVEC cells were cultured in RPMI1640 medium in a 12-well cell culture plate to achieve confluent growth. A bidirectional scratch wound was created on the bottom surface using a 200 μL micropipette tip, and then gently washed twice with 1 mL PBS to remove unattached cell debris. Subsequently, 1 mL of RPMI1640 medium containing 10% IL-22-CXCL12-Fg culture solution was added to each well. After wound treatment, the healing progress in each well was recorded using an inverted microscope at 0, 12, and 48 hours.

[0121] Through the above experiments, it was found that both IL-22 and CXCL12 accelerated the migration of human umbilical vein endothelial cells (HUVECs). More importantly, when both were present, the healing effect was more significant, indicating that after treatment with IL-22-CXCL12-FgMLM for 48 hours, the cell scratch completely disappeared, indicating a significant enhancement in wound healing ability( Figure 12 f).

[0122] 9.3 Establishment of Wound Infection Model

[0123] To construct a wound infection model, a wound with a diameter of 1.5 cm was made on the back of Balb / c mice, and then infected with 107 CFU of PA14.

[0124] 9.4 Treatment of wound infection with IL-22-CXCL12-FgMLM

[0125] Short-term wound infection treatment: Mice with PA14-infected wounds were treated with IL-22-CXCL12-FgMLM for 2 days, and mice treated with IL-22-FgMLM, CXCL12-FgMLM, FgMLM, and the clinically commonly used iodine treatment group were used as controls. The mice were sacrificed for bacterial counting and histopathological evaluation. The diluted homogenate was spread on plates, and the remaining bacteria in the wounds were counted. Wound tissues were also collected and immersed in 4% paraformaldehyde fixative, and then embedded in paraffin for H&E and Masson staining. Subcutaneous lymph nodes were removed from the mice. Subsequently, the tissues were homogenized through a 70-μm cell strainer to generate a single-cell suspension in PBS buffer. After incubation with purified anti-CD16 / 32 (93) at 4°C for 15 minutes, the obtained cells were stained with antibodies. To evaluate macrophage activation, the cells were stained with anti-CD11b (M1 / 70), anti-F4 / 80 (BM8), and anti-CD86 (24F) antibodies, and then intracellular staining was performed with anti-CD206 antibody. To analyze neutrophil infiltration, the cells were stained with anti-CD11b (M1 / 70) and anti-Gr1 antibody (RB6-8C5). Data were collected on a FACSVerse flow cytometer and analyzed using FlowJo software.

[0126] Long-term wound treatment: Mice with PA14-infected wounds were treated with IL-22-CXCL12-FgMLM, and mice treated with iodine, IL-22-FgMLM, CXCL12-FgMLM, FgMLM, and the clinically commonly used iodine treatment group were used as controls. The infected wounds were observed and recorded at different time intervals (days 4, 8, and 14). The wound area was analyzed using ImageJ software. The body weight of the mice was monitored daily during the in vivo experiment.

[0127] H&E staining of skin tissues showed that the number of inflammatory cells such as neutrophils and lymphocytes in mice treated with IL-22-CXCL12-FgMLM was significantly lower than that in the control group ( Figure 12 g). In addition, Masson's trichrome staining showed that the treatment with IL-22-CXCL12-FgMLM promoted the deposition level of new collagen and the formation of an intertwined network structure ( Figure 12 h). Wound images were taken to analyze the size changes at different time points ( Figure 12i). In particular, the results showed that on the fourth day of treatment, the wound healing effect of the IL-22-CXCL12-FgMLM group was significantly better than that of all control groups ( Figure 12 j). The bacterial quantification of PA14 in the wounds was also analyzed, and the results showed that the number of PA14 in the wounds of the IL-22-CXCL12-FgMLM group remained the lowest compared with the control group ( Figure 13 a). Meanwhile, macrophage polarization and neutrophil granulocytes were detected by flow cytometry, indicating that IL-22-CXCL12-FgMLM has anti-inflammatory ability ( Figure 13 b, c).

[0128] In summary, the present invention explores the potential of developing a new type of fungal-based living biomedical nonwoven fabric using the cross-kingdom filamentous fungus Fg. Through the vacuum filtration process, the filamentous mycelium was successfully made into a living fabric material, and its mechanical properties and strength were verified. Taking advantage of its unique characteristics that it cannot infect animals and humans, this living fabric exhibits excellent biosafety and has the characteristics of self-growth and self-repair. In addition, it also has the life characteristics of producing a variety of eukaryotic proteins through genetic engineering to achieve exogenous multifunctionality. The present invention found that due to the bioactive substances secreted by Fg, this living fabric material has natural antibacterial properties against common clinical pathogens. In order to explore the versatility and potential biomedical applications of this fungal-based living fabric, the present invention further created different engineered living fabric materials and evaluated their therapeutic value in two mouse models. Single engineered IL-22-FgMLM showed satisfactory acne cleaning ability and safety in the treatment of acne. At the same time, dual engineered IL-22-CXCL12 showed enhanced ability to promote wound healing and fight bacterial infection in the treatment of wound infection. Given the flexibility of genetic engineering to produce different bioactive proteins and the simplicity of the manufacturing process, the present invention anticipates that this plant-fungus-based living textile material will pave the way for new living biomedical biomaterials.

[0129] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0130] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An active biomedical fabric based on plant filamentous fungi, characterized in that: The active biomedical fabric is composed of mycelium of Fusarium graminearum that has been gene-edited, and the gene editing comprises the following steps: Step 1, constructing a plasmid containing an exogenous gene and linearizing the plasmid; Step 2: prepare Fusarium graminearum protoplasts, and transfer the linearized plasmid into the protoplasts.

2. A method for preparing an active biomedical fabric based on plant filamentous fungi as claimed in claim 1, characterized in that: The steps include: S1. culturing Fusarium graminearum to obtain a bacterial suspension containing Fusarium graminearum; S2. The bacterial suspension is vacuum filtered and dried to obtain an active biomedical fabric. The pressure during the filtration process is 0.1 MPa, and the pore size of the filter membrane used is 5 μm and the diameter is 50 mm.

3. An application of the active biomedical fabric based on plant filamentous fungi as claimed in claim 1, characterized in that: The active biomedical fabric is used for expressing fluorescent protein and eukaryotic protein drugs.

4. The use according to claim 3, characterized in that The eukaryotic protein drug is interleukin-22 (IL-22).

5. The use according to claim 3, characterized in that The eukaryotic protein drug is chemokine (CXC motif) ligand 12 (CXCL12).

6. A use of the active biomedical fabric based on plant filamentous fungi as claimed in claim 1 in the preparation of anti-infective drugs, characterized in that: The infections include pathogenic bacterial infections and pathogenic fungal infections.

7. A use according to claim 6, characterized in that The pathogenic bacteria include Pseudomonas aeruginosa, Propionibacterium acnes, Staphylococcus aureus, and Salmonella typhimurium.

8. A use according to claim 6, characterized in that: The pathogenic fungal infections include Candida albicans.

9. Use of the active biomedical fabric based on plant filamentous fungi as claimed in claim 1 in the preparation of anti-inflammatory drugs.

10. A use of the active biomedical fabric based on plant filamentous fungi as claimed in claim 1 in the preparation of a medicine for treating wounds, characterized in that: The drug treats wounds by preventing wound infection, inhibiting wound inflammation and accelerating wound healing.

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