Lentinan hydrogel film as well as preparation method and application thereof

Abstract

The invention discloses a lentinan hydrogel film as well as a preparation method and application thereof, the lentinan hydrogel film comprises the following raw materials: lentinan, polyvinyl alcohol and tannic acid, and the lentinan, the polyvinyl alcohol and the tannic acid are mutually crosslinked. The lentinan hydrogel film is applied to preparation of an antibacterial material, an antioxidant material or a skin defect repair material. The lentinan hydrogel film disclosed by the invention is relatively good in viscosity and mechanical property, has very good biological activity and biocompatibility, and has application values in the aspects of antibiosis, antioxidation and skin defect repair.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and particularly relates to a lentinan hydrogel film, a preparation method thereof, and an application thereof. Background Art

[0002] As the largest organ of the human body, the skin is one of the most important organs of the human body. It is in direct contact with the external environment and has functions such as protection, excretion, body temperature regulation, and perception of external stimuli. However, this exposure makes the integrity of the skin extremely vulnerable to damage. The destruction of skin tissue integrity or dysfunction is usually referred to as a wound.

[0003] Traditional wound dressings, such as bandages, have the advantages of protecting the wound surface, absorbing exudate, wide raw material sources, simple operation, and low price. However, their disadvantages are also obvious, such as unsatisfactory hemostatic effect, easy adhesion to tissues, and easy to cause exogenous infections when soaked, which makes the research on new dressings more urgent. Summary of the Invention

[0004] The main object of the present invention is to provide a lentinan hydrogel film with good biological activity and biocompatibility, better viscosity and mechanical properties, and having application value in antibacterial, antioxidant, and skin defect repair, as well as a preparation method and an application thereof.

[0005] To achieve the above object, the present invention provides a lentinan hydrogel film, the raw materials of which include lentinan, polyvinyl alcohol, and tannic acid, and the lentinan, the polyvinyl alcohol, and the tannic acid are cross-linked with each other.

[0006] Further, the mass ratio of the lentinan to the polyvinyl alcohol is 1-3:1-3. More preferably, the mass ratio is 3:1.

[0007] Further, the molecular weight of the polyvinyl alcohol is 89000-98000.

[0008] The present invention also provides a preparation method of the above lentinan hydrogel film, which includes the following steps: mixing a lentinan solution and a polyvinyl alcohol solution evenly, then pouring them into a container, drying to form a film, then taking it out for freeze-thaw cycling treatment, and finally soaking it in a tannic acid solution, taking it out and drying to obtain the lentinan hydrogel film.

[0009] Further, the lentinan solution, the polyvinyl alcohol solution, and the tannic acid solution are all aqueous solutions.

[0010] Further, the concentration of the lentinan polysaccharide solution is 2.0 - 5.0 wt.%, more preferably 3.5 - 4 wt.%; the concentration of the polyvinyl alcohol solution is 5.0 - 15.0 wt.%, more preferably 10 - 15 wt.%; the concentration of the tannic acid solution is 5.0 - 15.0 wt.%, more preferably 10 - 15 wt.%.

[0011] Further, the volume ratio of the lentinan polysaccharide solution to the polyvinyl alcohol solution is 1 - 9:1, more preferably 9:1.

[0012] Further, the temperature for air-drying into a film is 20 - 35 °C, more preferably 25 °C, and the time is 2 - 3 d.

[0013] Further, for the freeze-thaw cycle treatment, the freezing temperature is -20 °C, the freezing time is 24 h, the thawing temperature is room temperature, and the thawing time is 4 h.

[0014] Further, the temperature for the soaking treatment is room temperature and the time is 48 h.

[0015] The present invention also provides the application of the above-mentioned lentinan polysaccharide hydrogel film in the preparation of antibacterial materials, antioxidant materials or skin defect repair materials.

[0016] Polyvinyl alcohol (PVA) is an environmentally friendly hydrophilic vinyl polymer, generally prepared on a large scale by the hydrolysis of polyvinyl acetate. It is inexpensive and has good solvent resistance, film-forming property, low protein adsorption property, compatibility and degradability, etc., and it is non-toxic, transparent and has good mechanical properties.

[0017] Tannic acid (TA) is a polyphenolic compound widely present in plants. The polyphenolic hydroxyl group structure it contains has a strong hydrogen-donating ability, can scavenge oxygen free radicals, has strong antioxidant effects, has good antibacterial, antiviral, antioxidant and hemostatic properties, and at the same time shows excellent adhesion. In addition, due to the presence of polyphenol groups, compounds can crosslink with each other through covalent and non-covalent interactions. By incorporating tannic acid into polymer materials, it has better biocompatibility and biological activity.

[0018] Lentinan polysaccharide (LNT) is an effective active ingredient extracted from the fruiting bodies of Lentinula edodes, which has functions such as antiviral, anti-tumor, regulating immune function and stimulating interferon formation. The lentinan glucan therein is mainly composed of β-1,3-glucose and β-1,6-glucose chains. This unique structure makes lentinan glucan have good biocompatibility and biological activity. Research shows that β-glucan can be absorbed by intestinal cells in vivo, thereby affecting the function of the immune system.

[0019] The design principle of the present invention is:

[0020] Since PVA has excellent film-forming properties, PVA films can be made using this characteristic. However, PVA films have a high degree of crystallinity, which gives them a relatively high tensile strength but a very small elongation at break and poor toughness, limiting the application of the films in the flexible field. Therefore, PVA needs to be modified. Among them, chemical cross-linking or physical cross-linking is the most commonly used method for modifying PVA (because the hydroxyl groups in the PVA chain are relatively active and very easy to react with compounds containing functional groups such as carboxyl, aldehyde, and hydroxyl groups to form a cross-linked network structure, thereby enhancing its toughness and reducing the swelling degree of the film in water). Therefore, the present invention selects to use lentinan and tannic acid for modification, with the expectation of having better biocompatibility and bioactivity on the basis of improving the structural properties of the PVA film.

[0021] However, the inventors found during the research process that due to the low chemical reactivity of LNT, the cross-linking process is difficult. At the same time, the chemical cross-linking agent has certain biological toxicity and will affect the biological activity of LNT. Therefore, the use of chemical cross-linking was rejected; when using physical cross-linking, since LNT is mainly composed of a β-1,3-D-glucan backbone, which is characterized by two β-1,6-linked glucose side branches for every 5 glucose residues on the main chain, this structural feature significantly enhances its water solubility. In view of the fact that both PVA and LNT are strongly hydrophilic substances, simply mixing LNT and PVA by mechanical force is difficult to change the hydrophilicity of the film or improve its stability, which will limit its application in the field of wound repair.

[0022] To solve the above problems, the present invention adopts double physical cross-linking. First, using the film-forming property of PVA, PVA and LNT are co-blended into a film to compress the intermolecular gaps to make their physical interactions more compact, and then a certain amount of water molecules are introduced using their hydrophilicity. When LNT interacts with water molecules, it will undergo a gelation process. At this time, by the method of freeze-thaw cycling, the hydrogen bond interactions within and between PVA and LNT molecules can be enhanced to form a hydrogel structure with a three-dimensional network inside, which solves the problem of its instability in aqueous solution to a certain extent. At this time, the film formed still has a certain room for improvement in terms of viscosity and mechanical strength; then, TA is indirectly introduced in an immersion manner to simulate the polyhydroxy structure of mussel foot silk (the inventors found during the research that if direct mixing is selected, the acidity of the rich TA solution may damage the gel structure, and at the same time, TA is easily oxidized by air during mixing, which may cause it to lose its antioxidant ability), which can endow the material with excellent adhesion performance. TA can have certain interactions with both PVA and LNT, mainly in the form of hydrogen bond interactions and adsorption electrostatic interactions. As a secondary cross-linking agent, TA can endow the film with antibacterial and antioxidant abilities while also enhancing the mechanical strength and tissue adhesion of the film.

[0023] Furthermore, by ingeniously designing the ratio of LNT to PVA, the present invention constructs a system in which PVA serves as the gel skeleton and polysaccharide molecules are embedded therein, enabling LNT to be more uniformly distributed in the system, thereby better playing the main role of LNT in wound repair.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention first constructs a lentinan hydrogel film with a three-dimensional network structure by using polyvinyl alcohol and tannic acid through a freeze-thaw cycle technique, and then immerses the film in a tannic acid solution. The obtained lentinan hydrogel film has adjustable physicochemical properties (the relevant properties are adjusted by changing the ratio of polyvinyl alcohol to lentinan). Moreover, the lentinan hydrogel film of the present invention has good viscosity and mechanical properties, has excellent biological activity and biocompatibility, has application value in antibacterial, antioxidant, and skin defect repair, and the preparation method of the present invention is simple, the raw materials are easy to obtain, which is conducive to large-scale popularization and application. Description of the Drawings

[0026] Figure 1 It is a physical diagram of the films of Example 1, 2, 3, 4, 5 and Comparative Example 1;

[0027] Figure 2 It is an infrared spectrum diagram of the films of Example 1-5 and Comparative Example 1 and tannic acid (TA);

[0028] Figure 3 It is the degradation experiment result of the films of Example 1-5 and Comparative Example 1;

[0029] Figure 4 It is the scavenging rate of DPPH by vitamin C and the film of Example 5 at different times;

[0030] Figure 5 It is the tensile test (A) and Young's elastic modulus (B) test results of the films of Example 1-5 and Comparative Example 1 in the wet state;

[0031] Figure 6 It is the test result of the adhesion strength of the films of Example 1-5 and Comparative Example 1;

[0032] Figure 7 It is the physical diagram (A) and experimental result (B) of the inhibition zone method experiment of the films of Example 5 and Comparative Example 1 against Escherichia coli;

[0033] Figure 8 It is the physical diagram (A) and experimental result (B) of the inhibition zone method experiment of the films of Example 5 and Comparative Example 1 against Staphylococcus aureus;

[0034] Figure 9 It is the physical diagram of the bacterial plate counting method experiment of the film of Example 5 against Escherichia coli and Staphylococcus aureus;

[0035] Figure 10 Photographs of adding water, PBS and the membrane of Example 5 to whole blood and incubating for different times, and diagrams of the proportion of non-lysed red blood cells in whole blood with different concentrations of the membrane of Example 5 added;

[0036] Figure 11 Photographs of the repair effect of defective skin after different times for the membranes of Example 5 and Comparative Example 1 (A), wound closure rate (B), and HE and Masson staining results 14 days after defective skin repair;

[0037] Figure 12 Swelling rates of the membranes of Examples 1-5 and Comparative Example 1 at different times;

[0038] Figure 13 Water retention rates of the membranes of Examples 1-5 and Comparative Example 1 at different times;

[0039] Figure 14 TA release rates of the membranes of Examples 1-5 and Comparative Example 1 at different times;

[0040] Figure 15 Tensile test diagram of Example 5 (A) and adhesion strength test diagram of Example 5 (B);

[0041] Figure 16 Adhesion test diagrams of Example 5 to different materials after wetting (A) and adhesion force test diagrams of Example 5 to centrifuge tubes of different weights (B). Detailed implementation manners

[0042] To make the technical solutions of the present invention clearer and more understandable to those skilled in the art, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0043] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods; unless otherwise specified, the methods used in the examples of the present invention are all methods mastered by those skilled in the art. Among them:

[0044] Polyvinyl alcohol: Purchased from Shanghai Macklin Biochemical Co., Ltd., with a molecular weight of 89,000-98,000 and a purity of 95%.

[0045] Tannic acid: Purchased from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.5%.

[0046] The lentinan used was extracted by the alcohol precipitation and alkali extraction method, and the steps are as follows:

[0047] Take 500 g of dried Lentinus edodes fruiting bodies and process them into 60-mesh powder by a pulverizer. Then dissolve the powder in 3 L of 0.9 wt% NaCl solution (normal saline) for three times of extraction and purification. Specific operation: Mix the powder and the salt solution at a ratio of 1:6 (w / v), stir at a constant speed with a magnetic stirrer for 24 hours, and then centrifuge at 4500 rpm for 20 minutes at 4 °C. Discard the supernatant containing small molecule impurities and water-soluble proteins, and repeat the salting-out process twice to fully remove the surface adsorbed substances.

[0048] Resuspend the precipitate after salting-out in 3 L of deionized water and place it in an autoclave for gradient extraction. Set the parameters: High-temperature treatment at 121 °C for 30 minutes (steam pressure 0.1 MPa), and destroy the hyphal cell wall structure through thermally induced swelling. After centrifuging to collect the supernatant, repeat the hot extraction process three times to fully release the bound polysaccharides.

[0049] Alkali treatment: 0.4% NaOH solution: Solid-liquid ratio 1:6, stir at room temperature for 24 h, centrifuge at 4500 rpm and discard the supernatant to remove low-alkalinity soluble impurities. 0.8% NaOH solution: Treat under the same conditions to further dissolve hemicellulose-like substances. 5% NaOH solution: Core extraction stage, repeat the extraction twice, and combine the supernatant rich in Lentinus edodes polysaccharides.

[0050] Neutralization and precipitation: Gradually adjust the pH to 7.0 by dropping 36% glacial acetic acid to dissociate the polysaccharide-protein complex.

[0051] Centrifugal purification: Centrifuge at 4500 rpm for 20 min to remove the precipitated denatured proteins and undissolved impurities.

[0052] Rotary evaporation treatment: Rotary evaporate the supernatant at 60 °C to half of its original volume.

[0053] Dialysis treatment: Inject the liquid into a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyze with flowing deionized water for 48 h, changing the dialysis solution every 24 hours to completely remove the inorganic salts.

[0054] Freeze-drying and forming: Pre-cool to -80 °C and then transfer to a freeze dryer, and dry under a vacuum of <10 Pa for 24 h to obtain yellow sponge-like crude polysaccharides.

[0055] Purification: Dissolve 10 g of dry crude polysaccharide in 800 mL of deionized water, add 200 mL of Sevage reagent (chloroform: n-butanol = 4:1), stir at 300 r / min for 30 min, and then centrifuge the mixed solution at 8000 rpm for 5 min. Then, add 50 mL of 30% hydrogen peroxide and decolorize at 50 °C for 2 h. Then, collect the aqueous phase by centrifugation and dialyze it (cut-off Mw 8-10 kDa) in deionized water at 4 °C for 48 h, changing the water every 8 h. Then, add 5 volumes of 95% ethanol to precipitate for 24 h. After filtration and vacuum drying, refined lentinan is obtained, and the composition of lentinan is shown in Table 1 below:

[0056] Table 1

[0057]

[0058] Example 1

[0059] Preparation of lentinan hydrogel film

[0060] The preparation method is as follows:

[0061] (1) Disperse 5 g of polyvinyl alcohol in 50 g of pure water and stir at 70 °C for 2 h to completely dissolve the polyvinyl alcohol, obtaining a 10 wt.% polyvinyl alcohol aqueous solution; disperse 3.5 g of lentinan in 100 g of pure water and stir at 90 °C until it is completely dissolved, obtaining a 3.5 wt.% lentinan aqueous solution; disperse 2 g of tannic acid in 20 ml of pure water and stir until it is completely dissolved, obtaining a 10 wt.% tannic acid aqueous solution.

[0062] (2) Add 10 ml of the 10 wt.% polyvinyl alcohol aqueous solution prepared in step (1) to 10 ml of the 3.5 wt.% lentinan aqueous solution prepared in step (1), stir (500 revolutions per second) for 15 min, add 1 drop of glycerol, and then stir for 5 min to completely mix the two. Then, use a homogenizer to shear the mixed solution, specifically shear at 10000 rpm for 1 min, shear 3 times in total to make the mixed solution homogeneous, then centrifuge at 6000 rpm for 5 min to defoam, and finally pour it into a petri dish (diameter 60 mm), dry it at 25 °C for 2 d to form a film in a dry and soft state, and then take it out for freeze-thaw cycle treatment. The freezing temperature of the freeze-thaw cycle treatment is -20 °C, the freezing time is 24 h, the thawing temperature is room temperature, the thawing time is 4 h, and the number of cycles is five.

[0063] (3) Immerse the film prepared in step (2) in the 10 wt.% tannic acid aqueous solution prepared in step (1) for 48 h, take it out and dry it to obtain the lentinan hydrogel film, denoted as LPTM-1.

[0064] Example 2

[0065] Preparation of Lentinan Hydrogel Film

[0066] The preparation method is as follows:

[0067] (1) Disperse 5 g of polyvinyl alcohol in 50 g of pure water, stir at 70 °C for 2 h to completely dissolve the polyvinyl alcohol, and obtain a 10 wt.% polyvinyl alcohol aqueous solution; disperse 3.5 g of lentinan in 100 g of pure water, stir at 90 °C until it is completely dissolved, and obtain a 3.5 wt.% lentinan aqueous solution; disperse 2 g of tannic acid in 20 ml of pure water, stir until it is completely dissolved, and obtain a 10 wt.% tannic acid aqueous solution.

[0068] (2) Add 8 ml of the 10 wt.% polyvinyl alcohol aqueous solution prepared in step (1) to 12 ml of the 3.5 wt.% lentinan aqueous solution prepared in step (1), stir (500 revolutions per second) for 15 min, add 1 drop of glycerol, and then stir for 5 min to completely mix the two. Subsequently, use a homogenizer to shear the mixture, specifically shear at 10000 rpm for 1 min, and shear a total of 3 times to make the mixture texture uniform. Then centrifuge at 6000 rpm for 5 min to defoam, and finally pour it into a petri dish (diameter 60 mm), dry it at 25 °C for 2 d to form a film in a dry and soft state, and then take it out for freeze-thaw cycling treatment. The freezing temperature of the freeze-thaw cycling treatment is -20 °C, the freezing time is 24 h, the thawing temperature is room temperature, the thawing time is 4 h, and the number of cycles is five.

[0069] (3) Immerse the film prepared in step (2) in the 10 wt.% tannic acid aqueous solution prepared in step (1) for 48 h, take it out and dry it to obtain the lentinan hydrogel film, denoted as LPTM-2.

[0070] Example 3

[0071] Preparation of Lentinan Hydrogel Film

[0072] The preparation method is as follows:

[0073] (1) Disperse 5 g of polyvinyl alcohol in 50 g of pure water, stir at 70 °C for 2 h to completely dissolve the polyvinyl alcohol, and obtain a 10 wt.% polyvinyl alcohol aqueous solution; disperse 3.5 g of lentinan in 100 g of pure water, stir at 90 °C until it is completely dissolved, and obtain a 3.5 wt.% lentinan aqueous solution; disperse 2 g of tannic acid in 20 ml of pure water, stir until it is completely dissolved, and obtain a 10 wt.% tannic acid aqueous solution.

[0074] (2) Add 6 ml of the 10 wt.% polyvinyl alcohol aqueous solution prepared in step (1) to 14 ml of the 3.5 wt.% lentinan aqueous solution prepared in step (1). After stirring (500 revolutions per second) for 15 min, add 1 drop of glycerol, and then stir for another 5 min to completely mix the two. Subsequently, use a homogenizer to shear the mixture. Specifically, shear at 10,000 rpm for 1 min, and shear a total of 3 times to make the mixture texture uniform. Then centrifuge at 6,000 rpm for 5 min to defoam. Finally, pour it into a petri dish (diameter 60 mm) and air-dry at 25 °C for 2 d to form a film in a dry and soft state. Then take it out for freeze-thaw cycle treatment. The freezing temperature of the freeze-thaw cycle treatment is -20 °C, the freezing time is 24 h, the thawing temperature is room temperature, the thawing time is 4 h, and the number of cycles is five times.

[0075] (3) Immerse the film prepared in step (2) in the 10 wt.% tannic acid aqueous solution prepared in step (1) for 48 h, take it out and air-dry to obtain the lentinan hydrogel film, denoted as LPTM-3.

[0076] Example 4

[0077] Preparation of Lentinan Hydrogel Film

[0078] The preparation method is as follows:

[0079] (1) Disperse 5 g of polyvinyl alcohol in 50 g of pure water and stir at 70 °C for 2 h until the polyvinyl alcohol is completely dissolved to obtain a 10 wt.% polyvinyl alcohol aqueous solution; disperse 3.5 g of lentinan in 100 g of pure water and stir at 90 °C until it is completely dissolved to obtain a 3.5 wt.% lentinan aqueous solution; disperse 2 g of tannic acid in 20 ml of pure water and stir until it is completely dissolved to obtain a 10 wt.% tannic acid aqueous solution.

[0080] (2) Add 3 ml of the 10 wt.% polyvinyl alcohol aqueous solution prepared in step (1) to 17 ml of the 3.5 wt.% lentinan aqueous solution prepared in step (1). After stirring (500 revolutions per second) for 15 min, add 1 drop of glycerol, and then stir for another 5 min to completely mix the two. Subsequently, use a homogenizer to shear the mixture. Specifically, shear at 10,000 rpm for 1 min, and shear a total of 3 times to make the mixture texture uniform. Then centrifuge at 6,000 rpm for 5 min to defoam. Finally, pour it into a petri dish (diameter 60 mm) and air-dry at 25 °C for 2 d to form a film in a dry and soft state. Then take it out for freeze-thaw cycle treatment. The freezing temperature of the freeze-thaw cycle treatment is -20 °C, the freezing time is 24 h, the thawing temperature is room temperature, the thawing time is 4 h, and the number of cycles is five times.

[0081] (3) Immerse the membrane prepared in step (2) in the 10 wt.% tannic acid aqueous solution prepared in step (1) for 48 h, take it out and air-dry to obtain the lentinan hydrogel membrane, denoted as LPTM-4.

[0082] Example 5

[0083] Preparation of Lentinan Hydrogel Membrane

[0084] The preparation method is as follows:

[0085] (1) Disperse 5 g of polyvinyl alcohol in 50 g of pure water, stir at 70 °C for 2 h to completely dissolve the polyvinyl alcohol, and obtain a 10 wt.% polyvinyl alcohol aqueous solution; disperse 3.5 g of lentinan in 100 g of pure water, stir at 90 °C until it is completely dissolved to obtain a 3.5 wt.% lentinan aqueous solution; disperse 2 g of tannic acid in 20 ml of pure water, stir until it is completely dissolved to obtain a 10 wt.% tannic acid aqueous solution.

[0086] (2) Add 2 ml of the 10 wt.% polyvinyl alcohol aqueous solution prepared in step (1) to 18 ml of the 3.5 wt.% lentinan aqueous solution prepared in step (1), stir (500 revolutions per second) for 15 min, then add 1 drop of glycerol, and stir for another 5 min to completely mix the two. Subsequently, use a homogenizer to shear the mixture, specifically shear at 10000 rpm for 1 min, shear a total of 3 times to make the mixture texture uniform, then centrifuge at 6000 rpm for 5 min to defoam, and finally pour it into a petri dish (diameter 60 mm), air-dry at 25 °C for 2 d to form the mixture into a dry and soft state membrane, then take it out for freeze-thaw cycle treatment. The freezing temperature of the freeze-thaw cycle treatment is -20 °C, the freezing time is 24 h, the thawing temperature is room temperature, the thawing time is 4 h, and the number of cycles is five.

[0087] (3) Immerse the membrane prepared in step (2) in the 10 wt.% tannic acid aqueous solution prepared in step (1) for 48 h, take it out and air-dry to obtain the lentinan hydrogel membrane, denoted as LPTM-5.

[0088] Comparative Example 1

[0089] Preparation of Polyvinyl Alcohol - Tannic Acid Crosslinked Membrane

[0090] The preparation method is as follows:

[0091] (1) Disperse 5 g of polyvinyl alcohol in 50 g of pure water, stir at 70 °C for 2 h to completely dissolve the polyvinyl alcohol, and obtain a 10 wt.% polyvinyl alcohol aqueous solution; disperse 2 g of tannic acid in 20 ml of pure water, stir until it is completely dissolved to obtain a 10 wt.% tannic acid aqueous solution.

[0092] (2) Pour the 10 wt.% polyvinyl alcohol aqueous solution prepared in step (1) into a petri dish (60 mm in diameter), and dry it at 25 °C for 2 days to form a PVA film in a dry and soft state. Then take it out and perform freeze-thaw cycle treatment. The freezing temperature of the freeze-thaw cycle treatment is -20 °C, the freezing time is 24 h, the thawing temperature is room temperature, the thawing time is 4 h, and the number of cycles is five times.

[0093] (3) Immerse the PVA film prepared in step (2) in the 10 wt.% tannic acid aqueous solution prepared in step (1) for 48 h, take it out and dry it to obtain a polyvinyl alcohol tannic acid cross-linked film, denoted as PTM.

[0094] Structural Characterization and Performance Testing of the Membrane:

[0095] (1) Appearance

[0096] The actual pictures of the films prepared in Examples 1-5 and Comparative Example 1 are as Figure 1 shown. It can be seen that as the proportion of lentinan gradually increases, the yellow component of the sample is more, and the light transmittance is also smaller.

[0097] (2) Microstructure

[0098] The infrared spectra of LPTM-5, LNT, TA, and PVA are as Figure 2 shown. In the pure TA spectrum, a broad band of stretching vibration of O-H was observed at about 3370 cm -1 . The peaks at 1710 cm -1 and 1610 cm -1 correspond to the C=O stretching and C=C symmetric stretching vibration peaks in the TA molecule, respectively. At 1540 cm -1 , the corresponding peak is the stretching vibration peak of C=C in the aromatic ring of the TA molecule; in the pure LNT spectrum, a peak due to the stretching vibration of O-H was found at 3310 cm -1 . The peak at about 2910 cm -1 is the characteristic absorption peak of C-H stretching vibration of carbohydrates. The peak at 1650 cm -1 is the water absorption peak of sugar. The peak at about 1460 cm -1 is attributed to the bending vibration of C-O-H. The peaks at about 891 cm -1 and 1156 cm -1 are the characteristic peaks of β-D glucan, and the peak at about 920 cm -1 is the characteristic peak of α-D glucan; in the pure PVA spectrum, it was observed that PVA has peaks at 3300 cm -1 and 2930 cm -1There are characteristic bands on both sides here, which are caused by the stretching vibrations of O-H and aliphatic C-H. In addition, PVA exhibits a typical secondary alcohol group at 1087 cm -1 and shows a typical secondary alcohol group at 1730 cm -1 and the absorption bands at 1579 cm -1 are the C-O stretching vibrations of carboxylic acid and acetate groups respectively. In the spectrum of Example 5, characteristic absorption peaks of TA were observed at around 1710 cm -1 and 1610 cm -1 ; a characteristic absorption peak of PVA appeared at 1087 cm -1 ; while the absorption peak at 891 cm -1 in the original corresponding LNT shifted to 871 cm -1 here, the peak at 1156 cm -1 shifted to 1180 cm -1 , and the peak at 1400 cm -1 shifted to 1440 cm -1 . These shifts indicate that the addition of TA and PVA has changed the vibration mode of the C-O bond in LNT, which may be due to the formation of an interconnected network structure between them and the interaction between the three molecules. This cross-linked structure not only enhances the stability of the membrane but may also have a positive impact on the mechanical and electrochemical properties of the membrane. These results indicate the successful synthesis of the lentinan hydrogel membrane.

[0099] (3) Degradation performance

[0100] The membrane was placed in a phosphate buffer solution at 37 °C, and its degradation rate was detected at different time points. The results are shown in Figure 3 . The results show that LPTM-5, LPTM-4, and PTM have relatively fast degradation rates, while other samples maintain similar degradation speeds. This phenomenon may be related to the ratio of LNT and PVA. The alcohol hydroxyl group in PVA reacts with water to form alcohol and acid, and the acid is further hydrolyzed to alcohol and CO2; or its alcohol hydroxyl group forms hydrogen bonds in water, dissolves in water, and further reacts in water. Therefore, the degradation rate of PTM is very fast. While LNT is very likely to be released into the external environment in molecular form. The more LNT components there are, the more they are distributed on the surface of the hydrogel membrane, and the easier it is to diffuse into the liquid environment in molecular form. At the same time, the hydroxyl group in the polysaccharide is oxidized to form an aldehyde group and then further forms a carboxyl group, causing the degradation of the polysaccharide. This is also one of the reasons for the relatively fast degradation rates of LPTM-4 and 5. The dressing scaffold should degrade at an appropriate rate that matches the growth rate of the new tissue. A suitable degradation speed is beneficial to its application in the biomedical field.

[0101] (4) Water retention and swelling properties

[0102] The membrane was immersed in water to obtain its swelling ratio with respect to water at different times. After reaching stable swelling, it was taken out of the water and its water retention rate after being placed in air for different times was measured. The results are as Figure 12 and 13 shown. The results indicate that PTM, LPTM-4, and LPTM-3 swell rapidly within 90 min, followed by LPTM-5, LPTM-2, and finally LPTM-1. After 90 min, it was observed that PTM continued to lose weight, indicating its poor swelling property. After 120 min, the swelling ratios of LPTM-4 and LPTM-1 started to show a downward trend, but the amplitude was not large. After 150 min, the swelling ratios of LPTM-3, LPTM-5, and LPTM-2 showed a slight decrease. Generally speaking, LPTM-3 has the strongest swelling ability, reaching 289.12 ± 24.805%; while LPTM-1 has the weakest swelling ability, with a maximum of only 158.95 ± 34.195%. It should be noted that although the maximum swelling ability of LPTM-5 is 232.26 ± 22.2%, it has a relatively long swelling retention ability and is more stable; as time goes by, the water content of the hydrogel membrane continuously decreases. Among them, LPTM-5 has the strongest water retention ability, with the water content only decreasing by 126.49% ± (9.105 - 34.995) within 3 h, which is the slowest decrease rate among all samples; in contrast, the water content of LPTM-3 decreases the fastest, decreasing by 193.48% ± (17.705 - 34.255) within 3 h. Corresponding to the swelling ratio, due to the uniform ratio between molecules in LPTM-3, the pore density generated by hydrogen bond cross-linking is small, and the pore size is large, resulting in more water loss. In addition to the strong cross-linking strength and denser pores in LPTM-5, LNT, which accounts for 80% in the system, also has a certain water retention property, reducing water loss. Furthermore, the three-dimensional network structure of the polysaccharide molecules further increases the hindrance to water molecules, making the combination of polysaccharide and water very firm. Appropriate water absorption ability helps to accelerate wound healing by absorbing wound exudate and reduce the risk of infection; while good water retention ability can keep the wound environment moist, accelerate wound healing, and reduce scar formation.

[0103] (5) Tensile mechanical characteristics

[0104] The membrane was subjected to a tensile test in a wet state (i.e., without drying, directly taken out and tested after being immersed in tannic acid aqueous solution). The results are as Figure 5As shown, it can be seen that compared with Comparative Example 1, the Young's modulus of elasticity of the examples has been significantly reduced. Among them, the Young's modulus of elasticity of Examples 3 and 4 is relatively large. Generally speaking, the larger the Young's modulus of elasticity of the hydrogel, the smaller its elongation at break. The Young's modulus of elasticity presented by the sample in the wet state corresponds exactly to its elongation at break. It is worth noting that the elongation at break of LPTM-1 reaches nearly 500%, approaching that of PTM, showing a significant difference from other samples. Under wet conditions, the sample has a certain tensile resistance, which can prevent the wound from deforming due to external forces.

[0105] (6) Adhesion performance

[0106] For the adhesion strength test of the film, the specific method is to attach a wet film (1x2 cm, that is, without drying, directly taken out for testing after soaking in tannic acid aqueous solution) with two long strip filter papers (4×2 cm), and then test it with a tensile machine. The test results are as Figure 6 shown, and the test process is as Figure 15 (B) shown. As shown in the figure, the adhesion strength of LPTM-5 is the largest, reaching 250.56±15.69 Kpa, and the adhesion strength of LPTM-3 is the smallest, only 92.721±5.698 Kpa. It can be seen that when the PVA fraction in the system is greater than LNT, the adhesion strength of the hydrogel film decreases continuously with the introduction of LNT; once the PVA fraction in the system is less than LNT, the adhesion strength of the hydrogel film begins to increase again. Generally speaking, the adhesion strength of the hydrogel is usually related to the matrix of the synthesized hydrogel. PVA, LNT, and TA are all biological macromolecules. The larger the molecular weight, the stronger the intermolecular force, resulting in an increase in viscosity, which is the basis for the viscosity of the hydrogel film. At the same time, the viscosity of the hydrogel is also related to the concentration of the substance. The higher the concentration, the smaller the entropy of the particles in the hydrogel, and the more obvious the intermolecular force, resulting in an increase in viscosity. In addition, TA is a common catechol derivative that can be used to enhance tissue adhesion and is considered to provide strong adhesion to the hydrogel through hydrogen bonds and π-π interactions. Therefore, the ability of the hydrogel film to bind TA also has a certain impact on its viscosity. The phenolic hydroxyl group in TA can form hydrogen bonds with the hydroxyl groups in PVA and LNT and thus bind. At the same concentration, LNT contains more hydroxyl groups, so its ability to bind TA is relatively stronger. The above can reasonably explain the trend of the adhesion strength of the hydrogel film.

[0107] The adhesion performance test of LPTM-5 with different materials was carried out, as Figure 16 (A) shown, and the results show that LPTM-5 shows adhesion performance to a variety of materials; the load-bearing test of LPTM-5 was carried out, as Figure 16 (B) shown, and the results show that LPTM-5 has good adhesion load-bearing capacity.

[0108] (7) Release performance of TA

[0109] Take 200 mg of the film and soak it in 50 ml of PBS. Oscillate it in an incubator at 37 °C. At 30 min, 60 min, 90 min, 120 min, and 150 min, take 1 ml of the medium solution, centrifuge it at 5000 rpm for 5 min, extract the supernatant, and quantitatively measure the absorbance of the supernatant using a UV spectrophotometer. Use the absorbance to characterize the release amount of TA. The results are as Figure 14 shown. The results show that within 0 - 60 min, the release rate of TA from the hydrogel film is relatively slow; within 60 - 90 min, the release rate rises rapidly, with the fastest increase of about 50.47%; after 90 min, the release rate gradually slows down and tends to be stable. It should be noted that LPTM-5 still has an upward trend, and its TA release rate at 150 min is the highest, reaching 85.12 ± 0.35%. The release rate of TA is related to the pores of the hydrogel and the ability of the matrix to bind TA. When first contacting the aqueous environment, the hydrogel film has not fully swollen. At this time, the TA free on the surface first interacts with water to form hydrogen bonds and is released into the environment. When the hydrogel swells and the pores open, sufficient water enters the interior of the gel, thus rapidly releasing the internal TA. As the concentration of TA in the system gradually decreases, the release rate of TA also gradually slows down. Due to the polyhydroxy structure of LNT, its ability to capture TA is stronger, so the TA release rate of the gel film with a large proportion of LNT is also relatively high.

[0110] (8) Antioxidant performance

[0111] The DPPH (1,1-diphenyl-2-picrylhydrazyl) method was used to detect the free radical scavenging rate of the film at different time points to illustrate the antioxidant effect of the film. Specifically, at 37 °C, the film was co-cultured with DPPH in PBS. At different time points, the liquid was aspirated, and the absorbance value was measured at 517 nm, and then compared and calculated. The scavenging rates of vitamin C and LPTM-5 of Example 5 for DPPH (500 μg / mL) at different times are as Figure 4 shown. The results show that at 30 min, the DPPH free radical scavenging rate of LPTM-5 reached 41.26 ± 0.5291%. Although the free radical scavenging rate of LPTM-5 is lower than that of vitamin C throughout the test process, its overall scavenging level is close to that of vitamin C, indicating that LPTM-5 has good antioxidant effects. It should be noted that in the time period of 60 - 90 min, the free radical scavenging rate of LPTM-5 increased significantly by 6.94 ± (0.495 - 2.475)%, which is consistent with the release law of TA. This may be because the high antioxidant ability of the hydrogel film comes from TA with high reducing ability. And at the initial stage of the test, the release rate of TA is very low, and the antioxidant ability at this time may come from the combined action of LNT and TA.

[0112] (9) Antibacterial performance

[0113] Taking LPTM-5 of Example 5 as an example, the inhibition effects of the membrane on Escherichia coli and Staphylococcus aureus were tested by the inhibition zone method, and the results are as Figure 7 、 8 shown; at the same time, the antibacterial performance was also tested by the bacterial plate counting method, as Figure 9 shown. The results showed that obvious antibacterial effects of LPTM-5 could be observed on the petri dishes paved with Escherichia coli and Staphylococcus aureus. Measured with a ruler, the diameter of the inhibition zone of LPTM-5 against Escherichia coli was 9.667 ± 1.133 mm, and the diameter of the inhibition zone against Staphylococcus aureus was 10.5 ± 0.667 mm. It should be noted that these results were all smaller than the diameter of the inhibition zone of the positive control group TA. The diameter of the inhibition zone of TA against Escherichia coli was 11.833 ± 0.567 mm, and the diameter of the inhibition zone against Staphylococcus aureus was 12.67 ± 0.567 mm. The reason for this difference may be due to the slow-release effect of the hydrogel. However, the phenomenon that there were differences in the antibacterial effects of TA against Escherichia coli and Staphylococcus aureus was consistent with that of LPTM-5. According to the research, the antibacterial effect of TA against Gram-positive bacteria was significantly better than that against Gram-negative bacteria. This may be because Gram-negative bacteria have an outer membrane that can limit the diffusion of hydrophobic compounds, while Gram-positive bacteria do not. Therefore, TA is more easily penetrated to interfere with the normal function of the cytoplasmic membrane and disrupt the normal transport of substances inside and outside the cell. At the same time, Escherichia coli can modify TA and play a degradation role. In addition, it should be noted that PTM showed a certain degree of disintegration on the petri dishes for culturing the two different bacteria, affecting the evaluation of the antibacterial effect. This may be related to the combined action of water vapor and temperature for more than a dozen hours, further proving that the introduction of polysaccharides enhanced the stability of the hydrogel membrane. To further verify the effect of LPTM-5 on bacterial viability, by observing the final number of CFUs on the co-culture plate, we surprisingly found that the antibacterial effect of LPTM-5 was significantly higher than that of PTM, which highly likely indicated that LNT had a certain antibacterial effect.

[0114] (10) Hemolytic performance

[0115] Different concentrations of LPTM-5 of Example 5 were added to whole blood, and the hemolysis of red blood cells at different time points was detected. Water and PBS were set as comparisons, and the results are as Figure 10 shown, Figure 10 (A) are the physical pictures of adding water, PBS, and LPTM-5 to whole blood at each time period, Figure 10(B) is the proportion of undissolved red blood cells after adding different concentrations of LPTM-5 to whole blood. The results show that the hemolysis rate of LPTM-5 at 720 min is only 1.275 ± 0.565%, which is lower than 5%, meeting the international threshold of 5% for the blood compatibility of biomaterials.

[0116] (11) Performance in skin defect repair

[0117] The LPTM-5 of Example 5 and the PTM of Comparative Example 1 were used for repairing the defective skin of mice. The wound recovery conditions at different times are as Figure 11 shown. The results show that all wounds significantly shrank within 14 days. After 14 days, the wounds in the drug group and the LPTM-5 group were almost healed, leaving a little pigmentation and scar; the scab in the blank control group had not completely fallen off; the wound in the PTM group was nearly healed but still showed slight redness and swelling. Further staining analysis found that a large number of inflammatory cells appeared in the blank control group, and at the same time, the upper epidermis was not fully covered, the granulation tissue was loose, and the collagen sequence was disordered, indicating that its wound healing effect was poor; fewer or no inflammatory cell infiltrations were shown in the other three groups. It can be observed that the drug group showed a thicker new epithelium and denser granulation tissue, but its new epithelium was discontinuous and the epidermal layer was broken; the same situation occurred in the PTM group and showed multiple breaks; through Masson staining, it was observed that its collagen fibers were disorderly and overly dense, which might lead to scar hyperplasia. However, the thickness of the new epithelium in the LPTM-5 group was second only to that in the drug group, its epithelialization showed a certain continuity, and at the same time, new capillaries and denser granulation tissue could be observed; Masson staining analysis found that the arrangement of its collagen fibers was also relatively orderly. These results indicate that LPTM-5 can accelerate wound healing by inhibiting excessive inflammation, promoting epithelialization and collagen remodeling.

[0118] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An Lentinan hydrogel film, characterized in that, The raw materials include lentinan, polyvinyl alcohol and tannic acid, and the lentinan, the polyvinyl alcohol and the tannic acid are crosslinked with each other.

2. The lentinan hydrogel film according to claim 1, wherein The mass ratio of the lentinan to the polyvinyl alcohol is 1-3:1-3.

3. The lentinan hydrogel film according to claim 1 or 2, wherein The molecular weight of the polyvinyl alcohol is 89000-98000.

4. The preparation method of the lentinan hydrogel film according to claim 1 or 2 or 3, characterized in that, It includes the following steps: Mix the lentinan solution and the polyvinyl alcohol solution evenly, then pour them into a container, dry to form a film, then take it out for freeze-thaw cycle treatment, and finally soak it in the tannic acid solution, take it out and dry it to obtain the lentinan hydrogel film.

5. The preparation method of the lentinan hydrogel film according to claim 4, wherein, The lentinan solution, the polyvinyl alcohol solution and the tannic acid solution are all aqueous solutions.

6. The preparation method of the lentinan hydrogel film according to claim 4 or 5, characterized in that, The concentration of the lentinan solution is 2.0-5.0 wt.%, the concentration of the polyvinyl alcohol solution is 5.0-15.0 wt.%, and the concentration of the tannic acid solution is 5.0-15.0 wt.%.

7. The preparation method of the lentinan hydrogel film according to claim 4 or 5, characterized in that, The temperature for drying to form a film is 20-35 °C, and the time is 2-3 d.

8. The preparation method of the lentinan hydrogel film according to claim 4 or 5, characterized in that, For the freeze-thaw cycle treatment, the freezing temperature is -20 °C, the freezing time is 24 h, the thawing temperature is room temperature, and the thawing time is 4 h.

9. The preparation method of the lentinan hydrogel film according to claim 4 or 5, characterized in that, The temperature for the soaking treatment is room temperature, and the time is 48 h.

10. Use of the lentinan hydrogel film according to any one of claims 1 to 3 in the preparation of an antibacterial material, an antioxidant material or a skin defect repair material.

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