A protein-based antibacterial film, its preparation method and application

Abstract

The application belongs to the field of biomedicine, and particularly relates to a protein-based bacteriostatic film as well as a preparation method and application thereof. The SF / PVA / CTS / PcoC@CuNPs blending film is a blending film taking silk fibroin as a base material, cross-linking polyvinyl alcohol, chitosan and copper chaperone, and loading nano-copper. The preparation method of the blending film is mixing a silk fibroin / polyvinyl alcohol aqueous solution and a chitosan solution, adding a nano-copper solution and copper chaperone, magnetically stirring and defoaming, uniformly coating the mixed solution, and finally air-drying to obtain a film. The SF / PVA / CTS / PcoC@CuNPs blending film has good biocompatibility, can inhibit bacteria, keep moist, ventilate and stop bleeding, and has the potential as a wound dressing.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a protein-based antibacterial film, its preparation method, and its application. Background Technology

[0002] In recent years, protein materials have developed rapidly in the biomedical field. Among them, silk fibroin (SF) is a protein material with good biocompatibility and certain mechanical properties. SF membranes processed from SF have good biocompatibility and excellent light transmittance and insulation properties. However, the molecular conformation of single-component SF membranes is mainly an unstable, randomly coiled structure, which leads to problems such as poor flexibility, brittleness, and low strength, which seriously limits the application of SF membranes.

[0003] Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer with good density and high crystallinity. Films made from PVA are flexible, smooth, oil-resistant, and solvent-resistant. PVA is also easy to form films, possesses good tensile strength and flexibility, and is widely used due to its non-toxic, harmless, chemically stable, biocompatible, and low-cost characteristics.

[0004] Chitosan (CTS) is a natural polysaccharide with good biocompatibility, biodegradability, and antibacterial properties. It is a positively charged alkaline polysaccharide found in nature. CTS is a biomaterial with excellent hemostatic effects, antibacterial and adsorption capabilities, which can promote wound healing. It is also inexpensive to manufacture, biodegradable, and breathable.

[0005] Copper is one of the essential trace elements for the human body and has certain bactericidal properties. Multiple studies have shown that nano-copper is chemically more reactive than ordinary copper and possesses broad-spectrum microbial activity, inhibiting the growth of bacteria, fungi, viruses, and algae. Therefore, it can be used as an antibacterial agent.

[0006] Studies have shown that blending SF with other polymers (such as PVA, water-soluble polyurethane, sodium alginate, etc.) can improve the flexibility of SF membranes, thus blending modification has been widely applied in the field of polymer modification. However, using PVA and SF alone to form membranes results in poor hemostatic and antibacterial effects; using CTS and SF alone results in membranes with unsatisfactory flexibility and stability. Currently, there are no reports on blended SF membranes using PVA and CTS, crosslinked with copper chaperone proteins, and loaded with copper nanoparticles. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention modifies SF by blending it with PVA and CTS, improving the membrane's flexibility and elongation at break. Using SF as the base material, CTS and PVA are added to improve the properties of a single SF membrane. Copper chaperone protein is crosslinked, and copper nanoparticles are loaded, thus preparing an SF / PVA / CTS / PcoC@CuNPs blend film with good biocompatibility, capable of antibacterial, moisturizing, breathable, and hemostatic properties.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0009] A protein-based antibacterial film, wherein the antibacterial film is a blend film with silk fibroin as the base material, crosslinked with polyvinyl alcohol, chitosan and copper chaperone protein, and loaded with copper nanoparticles.

[0010] A method for preparing a protein-based antibacterial film as described above includes the following steps:

[0011] Mix silk fibroin / polyvinyl alcohol aqueous solution with chitosan solution, add nano copper solution; take the above mixed solution, add copper chaperone protein, stir magnetically to defoam, coat the mixed solution evenly, and air dry to form a film.

[0012] Furthermore, the molar ratio of silk fibroin to polyvinyl alcohol in the silk fibroin / polyvinyl alcohol aqueous solution is 0.12:1.

[0013] Furthermore, the mixing conditions for the silk fibroin / polyvinyl alcohol aqueous solution and the chitosan solution are: maintaining a temperature of 90°C and stirring at 350 rpm / min.

[0014] Furthermore, the final concentration of the chitosan is 7 g / L.

[0015] Furthermore, the final concentration of the nano-copper is 6.2 mg / L.

[0016] Furthermore, the final concentration of the copper chaperone protein is 2.5 × 10⁻⁶. -7 mol / L.

[0017] Furthermore, the air-drying film formation refers to natural air drying at room temperature for two days, followed by the addition of an appropriate amount of deionized water to help remove the film, and storage at 4°C.

[0018] Application of a protein-based antibacterial film as described above as a wound dressing.

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

[0020] 1. The protein-based antibacterial film of the present invention has good biocompatibility and the advantages of moisturizing, breathability and hemostasis.

[0021] 2. The protein-based antibacterial film of the present invention has good tensile and flexibility properties and is not easily broken.

[0022] 3. The protein-based antibacterial film of the present invention has good antibacterial properties and its antibacterial properties are relatively long-lasting. Attached Figure Description

[0023] Figure 1 This is a SEM image of the blend membrane of the present invention.

[0024] Figure 2 This is a tensile stress-strain diagram of the blend film of the present invention.

[0025] Figure 3 This is a comparison diagram of the antibacterial effect of the blended membrane in test tube culture according to the present invention.

[0026] Figure 4 This is a comparison chart of the antibacterial effects of the blended membrane plate culture of the present invention. Detailed Implementation

[0027] To facilitate understanding of the present invention, a more comprehensive description will be provided below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0028] Example 1 Solution Preparation

[0029] (1) Preparation of SF / PVA aqueous solution

[0030] At room temperature, 0.5g of SF powder was weighed and slowly poured into 30mL of deionized water, while stirring with a magnetic stirrer at 400rpm / min for 5min to obtain an SF solution. Then, 1.5g of PVA particles were weighed and added to the SF solution in small amounts several times to prevent the PVA particles from agglomerating. The mixture was stirred with a magnetic stirrer at 350rpm / min for 30min to ensure that the PVA particles were fully dispersed and swollen, allowing the SF aqueous solution to fully penetrate into the center of the PVA particles. The solution was then heated in a water bath at 90℃ for 30min and maintained at that temperature for 15min. During this process, the stirring speed was 350rpm / min, finally yielding an SF / PVA aqueous solution.

[0031] (2) Preparation of CTS solution

[0032] Prepare a 5 mL acetic acid solution with a concentration of 50 g / L. Weigh 0.25 g of CTS into the acetic acid solution and stir slowly until all CTS is dissolved. Then place the solution in a 4°C refrigerator to defoam and use it for later use.

[0033] (3) Preparation of nano-copper solution

[0034] Prepare 25 mL of a 0.8 g / L nano-copper solution. Weigh 0.2061 g of nano-copper and place it in a 250 mL volumetric flask. Add an appropriate amount of deionized water, shake well, and then dilute to volume.

[0035] Example 2

[0036] Preparation of SF / PVA / CTS / PcoC@CuNPs blend membrane

[0037] (1) Mix the above SF / PVA aqueous solution with the CTS solution while it is still hot;

[0038] (2) Take 10 mL of the solution obtained in step (1), cool it slightly, and add 77.5 μL of nano copper solution to make the final concentration of nano copper solution reach 6.2 mg / L. Stir carefully until evenly mixed.

[0039] (3) Take 1 mL of the blended solution containing nano-copper into a container, and add 250 μL of a 10% concentration solution. -6 Add mol / L PcoC, stir carefully to reduce bubble formation, and air dry at room temperature for two days. After film formation, add an appropriate amount of deionized water, remove the film, and store it in a 4℃ refrigerator to obtain the SF / PVA / CTS / PcoC@CuNPs blend membrane.

[0040] Comparative Example 1

[0041] Preparation of SF / PVA / CTS blend membrane

[0042] Mix the above SF / PVA aqueous solution with the CTS solution while it is still hot, maintain the temperature, stir with a magnetic stirrer at 350 rpm / min for 5 minutes to remove bubbles, and while it is still hot, evenly spread a portion of the solution into a container, taking care to reduce air bubbles. Let it air dry naturally at room temperature for one day to form a film. After adding an appropriate amount of deionized water, remove the film and store it in a refrigerator at 4℃ to obtain the SF / PVA / CTS blend membrane.

[0043] Preparation of SF / PVA / CTS@CuNPs blend membrane

[0044] (1) Mix the above SF / PVA aqueous solution with the CTS solution while it is still hot;

[0045] (2) Take 10 mL of the blend solution, cool it slightly and add 77.5 μL of nano copper solution to make the final concentration of nano copper solution reach 0.62 g / L. After stirring carefully and evenly, spread the solution into a container and air dry it at room temperature for two days. After the film is formed, add an appropriate amount of deionized water, take out the film and store it in a refrigerator at 4℃ to obtain the SF / PVA / CTS@CuNPs blend film.

[0046] SF / PVA / CTS@Cu 2+ Preparation of blended membranes

[0047] (1) Mix the above SF / PVA aqueous solution with the CTS solution while it is still hot;

[0048] (2) Take 10 mL of the mixed solution, let it cool slightly, then add 0.013 g of copper chloride to make Cu 2+ The final concentration reached 1×10 -4 After carefully stirring the solution to a concentration of mol / L, spread it into a container and allow it to air dry at room temperature for two days. Once the film has formed, add an appropriate amount of deionized water, remove the film, and store it in a 4°C refrigerator to obtain SF / PVA / CTS@Cu. 2+ Blend membrane.

[0049] SF / PVA / CTS / PcoC@Cu 2+ Preparation of blended membranes

[0050] (1) Mix the above SF / PVA aqueous solution with the CTS solution while it is still hot;

[0051] (2) Take 10 mL of the mixed solution, let it cool slightly, then add 0.013 g of copper chloride to make Cu 2+ The final concentration reached 1×10 -4 mol / L, stir carefully until homogeneous;

[0052] (3) Take 1 mL containing Cu 2+ The blended solution was placed in a container, and 250 μL of a 10% concentration was added to each. -6 Add mol / L PcoC, stir carefully to reduce bubble formation, and air dry at room temperature for two days. After film formation, add an appropriate amount of deionized water, remove the film, and store it in a 4°C refrigerator to obtain SF / PVA / CTS / PcoC@Cu. 2+ Blend membrane.

[0053] Example 3: Basic Property Test

[0054] (1) Scanning electron microscopy

[0055] A small amount of SF / PVA / CTS / PcoC@CuNPs blend film sample was attached to conductive adhesive and sputtered with gold at 10mA for 45s using a Quorum-SC7620 sputtering coating instrument. Subsequently, the morphology of the sample was photographed using a TESCAN-MIRA-LMS scanning electron microscope with an accelerating voltage of 3kV and an SE secondary electron detector.

[0056] (2) Mechanical testing

[0057] The tensile strength, breaking strength and elastic modulus of the SF / PVA / CTS / PcoC@CuNPs blend film were obtained by using an INSTRON-3343 high-performance mechanical testing machine.

[0058] Example 4

[0059] (1) Scanning electron microscopy

[0060] Small amounts of the four blend film samples obtained in Comparative Example 1 were attached to conductive adhesive and sputtered with gold at 10mA for 45s using a Quorum-SC7620 sputtering coating instrument. Subsequently, the morphology of the samples was photographed using a TESCAN-MIRA-LMS scanning electron microscope with an accelerating voltage of 3kV and an SE secondary electron detector.

[0061] (2) Mechanical testing

[0062] The tensile strength, breaking strength and elastic modulus of the SF / PVA / CTS blend film obtained in Comparative Example 1 were obtained by using an INSTRON-3343 10,000 kJ / m² mechanical testing machine.

[0063] Electron microscopy results as follows Figure 1 As shown in the figure, Figure A represents the SF / PVA / CTS blend film, Figure B represents the SF / PVA / CTS@CuNPs blend film, Figure C represents the SF / PVA / CTS / PcoC@CuNPs blend film, and Figure D represents the SF / PVA / CTS@CuNPs blend film. 2+ The blend membrane, E diagram is SF / PVA / CTS / PcoC@Cu 2+ Blend membrane;

[0064] Cu was added. 2+ The film after the addition of nano-copper showed uneven pores, while the film after the addition of nano-copper did not show significant changes. The blend film after the addition of PcoC contained a large number of microporous structures, which enhanced the air permeability of the film. This indicates that the micropores can be distributed relatively evenly on the surface of the SF / PVA / CTS / PcoC@CuNPs blend film. When the film is used as a wound dressing, the microporous structure can absorb a large amount of wound exudate, which provides a certain guarantee for wound healing.

[0065] Mechanical test results as follows Figure 2 As shown in Table 1, the tensile stress of the SF / PVA / CTS blend film is 11.23 MPa, and the tensile stress of the SF / PVA / CTS / PcoC@CuNPs blend film is 7.44 MPa. In comparison, the tensile stress of the SF / PVA / CTS / PcoC@CuNPs blend film is lower.

[0066] The tensile strain (displacement) of the SF / PVA / CTS blend film was 127.3%, while the tensile stress of the SF / PVA / CTS / PcoC@CuNPs blend film was 533.0%. In comparison, the tensile strain (displacement) of the SF / PVA / CTS / PcoC@CuNPs blend film increased.

[0067] The data above indicate that the SF / PVA / CTS / PcoC@CuNPs blend film has reduced breaking strength but good tensile and flexibility properties, making it more suitable as a wound dressing.

[0068] Table 1 Changes in mechanical properties

[0069]

[0070] Example 5

[0071] Antibacterial function test of SF / PVA / CTS / PcoC@CuNPs blend membrane

[0072] (1) Antibacterial test in test tube culture

[0073] Take a test tube containing 5 mL of liquid culture medium that has been sterilized at high temperature, add 200 μL of E. coli bacterial suspension and 5 μL of Ampicillin, add SF / PVA / CTS / PcoC@CuNPs blend membrane (labeled No. 5), and then place it in a constant temperature shaker at 37℃ and a shaking frequency of 150 r / min for 2 h, 4 h, 6 h, and 8 h, and take photos and record them in time.

[0074] (2) Plate culture antibacterial test

[0075] After the test tube described in step (1) is shaken and cultured for 2 hours, take out 100 μL of bacterial solution under sterile conditions, spread it on a culture dish marked No. 5, and then place it in an electric thermostatic incubator at 37°C for 12 hours. Take a picture and record the results.

[0076] Example 6

[0077] (1) Antibacterial test in test tube culture

[0078] Take a test tube containing 5 mL of liquid culture medium that has been sterilized by high temperature, add 200 μL of E. coli bacterial suspension and 5 μL of Ampicillin, and label them 1, 2, 3, and 4 respectively. Add an SF / PVA / CTS blend membrane to test tube 1; add an SF / PVA / CTS@Cu membrane to test tube 2. 2+ Blended membrane; SF / PVA / CTS@CuNPs blended membrane added to test tube 3; SF / PVA / CTS / PcoC@CuNPs blended membrane added to test tube 4. 2+The blended membranes were then placed in a constant temperature shaker and cultured at 37°C and a shaking frequency of 150 r / min for 2 h, 4 h, 6 h, and 8 h, with photos taken and recorded in a timely manner.

[0079] (2) Plate culture antibacterial test

[0080] After the test tubes described in step (1) are shaken and cultured for 2 hours, take out 100 μL of bacterial solution from test tubes 1, 2, 3 and 4 respectively under sterile conditions, spread them onto culture dishes labeled 1, 2, 3 and 4, and then place them in an electric thermostatic incubator at 37℃ for 12 hours and take pictures to record.

[0081] Results of test tube culture antibacterial test as follows Figure 3 As shown in the figure, A, B, C, and D represent the conditions after 2h, 4h, 6h, and 8h of cultivation, respectively. The good bacterial growth in tube 1 indicates that the SF / PVA / CTS blend membrane itself has a weak antibacterial effect. The good bacterial growth in tubes 2 and 3 indicates that the blend membrane has a weak loading capacity for nano-copper, and the nano-copper and Cu... 2+ The particle loading on the blend membrane was low; the solutions in test tubes 4 and 5 were clear, indicating that the addition of PcoC improved the loading of nano-copper and Cu on the blend membrane. 2+ Its load-bearing capacity is enhanced, and its antibacterial effect is improved.

[0082] After 8 hours, the solution in test tube 4 became cloudy, while test tube 5 remained clear. This indicates that over time, the Cu bound to PcoC... 2+ The antibacterial effect gradually weakens, but the antibacterial effect of copper nanoparticles in the SF / PVA / CTS / PcoC@CuNPs blend film is highly sustainable. In other words, the antibacterial effect of SF / PVA / CTS / PcoC@CuNPs blend film is relatively sustainable. 2+ The blended membranes of the former have a weak effect on the sustained release of copper ions and poor antibacterial durability; while the blended membranes of SF / PVA / CTS / PcoC@CuNPs have a strong effect on the sustained release of copper nanoparticles and good antibacterial durability.

[0083] The results of the plate culture antibacterial test are as follows: Figure 4 As shown, compared to culture dishes 1, 2, and 3, culture dishes 4 and 5 are more transparent, meaning that almost no colonies have grown in them. This indicates that the blended membrane after adding PcoC is more effective than the nano-copper and Cu. 2+ It has a stronger load-bearing capacity and a better antibacterial effect.

[0084] The embodiments described above are merely specific examples of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A protein-based antibacterial film, characterized in that, The antibacterial film is a blended film made of silk fibroin as the base material, cross-linked with polyvinyl alcohol, chitosan and copper chaperone protein, and loaded with copper nanoparticles. The copper chaperone protein is PcoC.

2. A method for preparing a protein-based antibacterial film as described in claim 1, characterized in that, Includes the following steps: Mix silk fibroin / polyvinyl alcohol aqueous solution with chitosan solution, then add nano copper solution; take the above mixed solution, add copper chaperone protein, stir magnetically to defoam, coat the mixed solution evenly, and air dry to form a film.

3. The method for preparing the protein-based antibacterial film according to claim 2, characterized in that, The molar ratio of silk fibroin to polyvinyl alcohol in the silk fibroin / polyvinyl alcohol aqueous solution is 0.12:

1.

4. The method for preparing the protein-based antibacterial film according to claim 2, characterized in that, The mixing conditions for the silk fibroin / polyvinyl alcohol aqueous solution and the chitosan solution are: maintaining a temperature of 90°C and stirring at 350 rpm.

5. The method for preparing the protein-based antibacterial film according to claim 2, characterized in that, The final concentration of the chitosan was 7 g / L.

6. The method for preparing the protein-based antibacterial film according to claim 2, characterized in that, The final concentration of the nano-copper was 6.2 mg / L.

7. The method for preparing the protein-based antibacterial film according to claim 2, characterized in that, The final concentration of the copper chaperone protein was 2.5 × 10⁻⁶. -7 mol / L.

8. The method for preparing the protein-based antibacterial film according to claim 2, characterized in that, The air-drying film formation refers to air-drying naturally at room temperature for two days, adding an appropriate amount of deionized water after film formation to help remove the film, and storing it at 4°C.

9. The application of the protein-based antibacterial film as described in claim 1 in the preparation of wound dressings.

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