Fibroin nanofiber-bismuth nanoparticle composite membrane and preparation method thereof

Abstract

The invention discloses a fibroin nanofiber-bismuth nanoparticle composite membrane and a preparation method thereof. The composite membrane is formed by compounding bismuth nanoparticles and fibroin nanofibers; the preparation method comprises the following steps: dispersing fibroin nanofibers in deionized water, heating and stirring until the fibroin nanofibers are dissolved, then adding bismuth nitrate powder, continuously stirring uniformly, and then adding a reducing agent for treatment until no bubble overflows, so that bismuth elementary substances grow on the fibroin nanofibers to obtain a bismuth-based fibroin nanofiber solution; and drying the bismuth-based fibroin nanofiber solution to obtain the bismuth-based fibroin nanofiber composite membrane. The obtained membrane product has excellent hydrophilicity, biodegradability, anti-inflammatory performance and antibacterial performance, is wide in application, can simulate an extracellular matrix structure, is used as an infectious skin wound dressing, can be combined with ion slow release / photo-thermal / photodynamic therapy to promote wound repair, and is safe and effective.

Description

Technical Field

[0001] The invention relates to the technical field of medical clinical treatment, in particular to a method for preparing a silk fibroin nanofiber-bismuth nanoparticle composite film used as an infectious wound dressing. Background Art

[0002] Injury to the skin, oral cavity, bone tissue and other parts or organs caused by acute trauma or chronic diseases, as well as wound infection, are core issues in clinical medicine, bringing economic burden and mental stress to countless patients around the world. Currently, clinical treatment mainly uses dressings such as gauze and cotton wool, whose main function is to maintain moisture in the wound and protect it from infection by pathogenic microorganisms. However, wound healing under these treatments is still a slow and passive process, which is ineffective in severe trauma that has already caused infection. Therefore, in order to further promote wound healing, it is urgent to develop a class of multifunctional dressings and scaffold materials that are bioactive and can simulate the composition and structure of the extracellular matrix, and introduce antibacterial components that can kill pathogens to achieve the effect of coordinated promotion of wound healing.

[0003] Biomaterials for wound healing applications should have biocompatibility, low cytotoxicity, excellent biodegradability and antibacterial properties. Silk protein has many ideal physicochemical properties, such as good biocompatibility, biodegradability, bioabsorbability, low immunogenicity and adjustable mechanical properties, and has been widely used as a raw material for the preparation of various types of biomaterials. Based on the current understanding of silk structure, nanofibers extracted from silk fibers can retain the original complex hierarchical structure of silk to maintain the stability of silk, and can also retain the excellent mechanical properties of silk. Therefore, it has the potential to replace electrospun nanofibers and become a new preparation method for nanofiber antibacterial dressings. Although nanofiber dressings are a kind of biomaterial with excellent performance, they lack antibacterial properties themselves, which limits their clinical use. With the rise of research in the field of metal-based nanomedicine, certain categories of metal nanoparticles are good antibacterial agents because they have high activity and diverse bactericidal effects at low doses, providing an ideal possibility for reducing or eliminating the evolution of drug-resistant strains. Among these antibacterial metal materials, bismuth and its derivatives stand out for their low cost, wide availability, clinical application precedents, unique antibacterial properties and excellent biocompatibility. Very importantly, bismuth-based materials have excellent photothermal / photodynamic properties, which mainly play a bactericidal effect through the active oxygen generated under high temperature or light irradiation.

[0004] Therefore, combining silk nanofibers with bismuth would be a very promising way to prepare a new type of skin dressing. Summary of the invention

[0005] In view of the shortcomings of traditional wound dressing materials, the present invention has improved a bismuth elemental / silk fibroin nanofiber antibacterial composite film, which is a new material that is simple to prepare, has powerful functions, and can synergistically repair infectious wounds.

[0006] The present invention utilizes the unique physical and chemical properties of silk fibroin and combines it with bismuth with antibacterial function to prepare a composite membrane wound dressing that simulates the structure of the extracellular matrix. After the composite membrane is applied to mice with skin infection and then irradiated with visible light or near-infrared laser, the infected bacteria are basically eliminated. In the later repair, the mice show a rapid healing effect. Therefore, the material can be used in the field of infectious skin wound repair and can be used as an ideal wound dressing.

[0007] The specific technical solutions of the present invention are as follows:

[0008] 1. A silk nanofiber-bismuth nanoparticle composite film:

[0009] The composite film is mainly composed of bismuth nanoparticles and silk nanofibers.

[0010] The mass ratio between the bismuth nanoparticles and the silk nanofibers is 0.1:1-2000:1.

[0011] 2. A method for preparing a silk nanofiber-bismuth nanoparticle composite film, the specific preparation steps adopted are as follows:

[0012] (1) The silk nanofibers were dispersed in deionized water, and the morphology regulator and Bi(NO) were added successively. 3 ) 3 ·5H 2 O;

[0013] (2) Add NaBH to the solution obtained in step (1) 4 reaction;

[0014] (3) transferring the solution obtained in step (2) to an ultrasonic crusher for ultrasonic treatment, followed by centrifugation to obtain monodisperse bismuth-based silk composite nanofibers;

[0015] (4) dispersing the bismuth-based silk fibroin composite nanofibers obtained in step (3) into deionized water, and after being evenly dispersed, using a vacuum solution filter to filter and form a membrane, and drying to obtain a bismuth-based silk fibroin nanofiber composite membrane.

[0016] The bismuth-based silk nanofiber composite membrane prepared by the method can not only slowly release bismuth for wound antibacterial treatment, but also combine photothermal / photodynamic antibacterial treatment and wound treatment.

[0017] In the step (1), the collected silk nanofibers have a length of 20 nm-50 μm and a diameter of 5 nm-2 μm.

[0018] The silk fibroin nanofibers are derived from degummed silk fibers and are obtained by centrifugation after ultrasonic crushing and deionized water dialysis.

[0019] In the step (1), the added mass of the silk nanofibers relative to the deionized water is 0.01 wt %.

[0020] In the step (1), the morphology control agent is one of carbonates, EDTA, disorbitol, phosphates, citric acid, tartaric acid, and silicates, and the added mass of the morphology control agent relative to the silk nanofibers is 0.01wt%-4wt%.

[0021] In the step (1), the morphology control agent is first added, and Bi(NO 3 ) 3 ·5H 2 O, making Bi(NO 3 ) 3 ·5H 2 The final O concentration is 0.01wt%-20wt%.

[0022] In step (2), NaBH is added after continued stirring. 4 , making NaBH 4 The final concentration is 0.01wt%-4wt%, and the reaction is carried out until no bubbles overflow so that the bismuth element grows on the silk nanofibers to obtain a bismuth-based silk nanofiber solution.

[0023] In the step (3), ultrasound is performed at a power of 100-1000 W for 1-30 min, followed by centrifugation at a speed of 8000 rpm for 10 min.

[0024] The innovation of the present invention lies in the use of ultrasonic treatment-in-situ reduction-filtration drying-near-infrared irradiation process steps, which brings about precise regulation and multimodal functional integration of the bismuth-based silk nanofiber composite membrane structure, and realizes the effect / advantage of bismuth sustained-release-photothermal-photodynamic synergistic therapy to quickly promote wound healing.

[0025] Compared with the prior art, the present invention has the following outstanding features:

[0026] (1) Excellent biocompatibility. The silk fibroin used in the raw material is a natural active material that has been approved for clinical use by the Food and Drug Administration of many countries. It has passed biocompatibility and safety tests and meets medical standards. The bismuth metal used in the raw material has low toxicity and antibacterial properties. Related bismuth-containing agents have been approved by many countries for clinical cancer treatment and in vivo imaging, or have been developed into drugs for the treatment of gastrointestinal diseases, and will not cause harm to the human body.

[0027] (2) The preparation and storage are simple. By collecting silk nanofibers and generating bismuth nanoparticles in situ, bismuth-based silk nanofibers that are firmly bound and evenly dispersed can be obtained. The composite membrane can then be prepared in large quantities by filtering and forming a membrane. The composite membrane can be stored in a low-temperature, dry state and can be used as needed.

[0028] (3) Multimodal antibacterial effect. Under the synergistic effect of bismuth ion sustained release / photothermal / photodynamic, it can achieve efficient sterilization, thereby promoting the healing of infected wounds. It can be used for antibacterial treatment of superficial surface sites, such as the mouth, skin, and deep tissues, such as bone tissue and internal organs. It also has a good inhibitory effect on the growth of both Gram-positive and Gram-negative bacteria.

[0029] (4) It is easy to use and does not require complicated bandaging procedures. It can be applied to the skin. There are a variety of light sources to choose from during photothermal treatment. In response to the "passive and slow" problem of existing wound dressings, it provides an "active and fast" method for sterilization and promotion of wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a transmission electron microscope image of the bismuth / silk nanofibers prepared in Example 1;

[0031] Figure 2 The actual pictures are the bismuth / silk nanofiber antibacterial composite membrane prepared in Example 1 and the single silk nanofiber membrane without adding bismuth;

[0032] Figure 3 This is a cross-sectional electron micrograph of the bismuth / silk nanofiber antibacterial composite membrane prepared in Example 1;

[0033] Figure 4 This is a diagram showing the wound healing condition after 11 days of near-infrared light treatment using the bismuth / silk nanofiber antibacterial composite membrane prepared in Example 4;

[0034] Figure 5 This is a diagram showing the wound healing condition of the bismuth / silk nanofiber antibacterial composite film prepared in Example 4 after 11 days without near-infrared light treatment.

[0035] Figure 6 This is a diagram showing the wound healing of the single silk nanofiber membrane group prepared for comparison in Example 4 after being treated with near-infrared light for 11 days. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described in detail below through specific embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments.

[0037] Embodiments of the present invention are as follows:

[0038] Example 1

[0039] (1) 0.5 g of degummed silk fibers were immersed in 50 mL of 0.5 mol / L NaOH solution at 37 °C for 6 h, and ultrasonically disrupted in a cell disruptor with a power of 600 W for 10 min. The resulting silk nanofiber solution was transferred to a dialysis bag, dialyzed in deionized water for two days, and centrifuged at 8000 rpm for 10 min to collect the silk nanofibers.

[0040] (2) Disperse 1 mg of the silk nanofibers prepared in step (1) in 10 mL of deionized water, add 0.09 g of EDTA-2Na, and stir at 37° C. until dissolved;

[0041] (3) The solution obtained in step (2) was stirred for 30 min, and then 0.09 g of Bi(NO 3 ) 3 5H2O;

[0042] (4) The solution in step (3) was stirred for 30 min, and then 0.01 g of NaBH was added. 4 , react until no bubbles overflow;

[0043] (5) transferring the solution obtained in step (4) to an ultrasonic crusher, ultrasonicating at a power of 300 W for 10 min, and then centrifuging at a rate of 8000 rpm for 10 min to obtain bismuth-based silk composite nanofibers;

[0044] (6) Take 2 mg of the bismuth-based silk composite nanofibers obtained in step (5), disperse them in 10 mL of deionized water, and after uniform dispersion, use a vacuum solution filter to filter and form a membrane, and naturally dry to obtain a bismuth-based silk nanofiber composite membrane.

[0045] The bismuth element / silk fibroin nanofiber monodisperse particles prepared in this example are as follows: Figure 1 As shown above Figure 1 It can be seen that dense two-dimensional bismuth nanosheets are loaded on the silk nanofibers.

[0046] Bismuth / silk nanofiber composite membrane and single silk nanofiber membrane Figure 2 As shown above Figure 2 It can be seen that the composite film is black with a smooth surface, and the bismuth is evenly and densely distributed, while the single film is white with a smooth surface.

[0047] The cross-sectional electron microscopy image of the bismuth / silk nanofiber composite membrane is shown in Figure 3 As shown above Figure 3 It can be seen that the composite nanofibers are stacked into a uniform film after filtration and drying.

[0048] Example 2

[0049] (1) 0.5 g of degummed silk fibers were taken and immersed in 50 mL of 3 mol / L NaOH solution at 37°C for 0.2 h, and ultrasonically disrupted in a cell disruptor with a power of 100 W for 30 min. The resulting silk nanofiber solution was transferred to a dialysis bag, dialyzed in deionized water for two days, and centrifuged at 8000 rpm for 10 min to collect the silk nanofibers.

[0050] (2) Disperse 1 mg of the silk nanofibers prepared in step (1) in 10 mL of deionized water and add 0.09 g of Na 2 CO 3 , stir at 37°C until dissolved;

[0051] (3) The solution obtained in step (2) was stirred for 30 min, and then 2 g of Bi(NO 3 ) 3 5H2O;

[0052] (4) The solution in step (3) was stirred for 30 min, and then 0.4 g of NaBH was added. 4 , react until no bubbles overflow;

[0053] (5) transferring the solution obtained in step (4) to an ultrasonic crusher, ultrasonicating at a power of 100 W for 30 min, and then centrifuging at a rate of 8000 rpm for 10 min to obtain bismuth-based silk composite nanofibers;

[0054] (6) Take 2 mg of the bismuth-based silk composite nanofibers obtained in step (5), disperse them in 10 mL of deionized water, and after uniform dispersion, use a vacuum solution filter to filter and form a membrane, and naturally dry to obtain a bismuth-based silk nanofiber composite membrane.

[0055] Example 3

[0056] (1) 0.5 g of degummed silk fibers were taken and immersed in 50 mL of 0.01 mol / L NaOH solution at 37°C for 12 h, and ultrasonically disrupted in a cell disruptor with a power of 1000 W for 1 min. The resulting silk nanofiber solution was transferred to a dialysis bag, dialyzed in deionized water for two days, and centrifuged at 8000 rpm for 10 min to collect the silk nanofibers;

[0057] (2) Disperse 1 mg of the silk nanofibers prepared in step (1) in 10 mL of deionized water, add 0.09 g of disorbitol, and stir at 37° C. until dissolved;

[0058] (3) The solution obtained in step (2) was stirred for 30 min, and then 0.001 g of Bi(NO3 ) 3 5H2O;

[0059] (4) The solution in step (3) was stirred for 30 min, and then 0.001 g of NaBH was added. 4 , react until no bubbles overflow;

[0060] (5) transferring the solution obtained in step (4) to an ultrasonic crusher, ultrasonicating at a power of 1000 W for 1 min, and then centrifuging at a speed of 8000 rpm for 10 min to obtain bismuth-based silk composite nanofibers;

[0061] (6) Take 2 mg of the bismuth-based silk composite nanofibers obtained in step (5), disperse them in 10 mL of deionized water, and after uniform dispersion, use a vacuum solution filter to filter and form a membrane, and naturally dry to obtain a bismuth-based silk nanofiber composite membrane.

[0062] Example 4

[0063] (1) 0.5 g of degummed silk fibers were immersed in 50 mL of 0.5 mol / L NaOH solution at 37 °C for 6 h, and ultrasonically disrupted in a cell disruptor with a power of 600 W for 10 min. The resulting silk nanofiber solution was transferred to a dialysis bag, dialyzed in deionized water for two days, and centrifuged at 8000 rpm for 10 min to collect the silk nanofibers.

[0064] (2) Disperse 1 mg of the silk nanofibers prepared in step (1) in 10 mL of deionized water, add 0.09 g of EDTA-2Na, and stir at 37° C. until dissolved;

[0065] (3) The solution obtained in step (2) was stirred for 30 min, and then 0.03 g of Bi(NO 3 ) 3 5H2O;

[0066] (4) The solution in step (3) was stirred for 30 min, and then 0.01 g of NaBH was added. 4 , react until no bubbles overflow;

[0067] (5) transferring the solution obtained in step (4) to an ultrasonic crusher, ultrasonicating at a power of 300 W for 10 min, and then centrifuging at a rate of 8000 rpm for 10 min to obtain bismuth-based silk composite nanofibers;

[0068] (6) Take 2 mg of the bismuth-based silk composite nanofibers obtained in step (5), disperse them in 10 mL of deionized water, and after uniform dispersion, use a vacuum solution filter to filter and form a membrane, and naturally dry to obtain a bismuth-based silk nanofiber composite membrane.

[0069] (7) Infectious skin wounds were established in experimental mice, and the obtained composite film was then applied to the back wounds. Eleven days after treatment, the wounds of the bismuth / silk nanofiber antibacterial composite film group combined with photothermal / photodynamic therapy were almost completely covered by newly formed skin ( Figure 4 ), the wound size of the bismuth / silk nanofiber antibacterial composite membrane group without photothermal / photodynamic therapy became smaller ( Figure 5 ), while the wound size of the single silk nanofiber membrane group remained almost unchanged ( Figure 6 ). It can be seen that the wound dressing of the present invention can quickly promote wound healing, and the effect of combining bismuth ion sustained release / photothermal / photodynamic therapy is better.

[0070] It can be seen from this implementation that the product obtained by the present invention has excellent hydrophilicity, biodegradability, anti-inflammatory and antibacterial properties, is widely used, can also simulate the extracellular matrix structure, and be used as a dressing for infected skin wounds. At the same time, it can also combine ion sustained release / photothermal / photodynamic therapy to promote wound repair, which is safe and effective.

[0071] Finally, it should be noted that the above examples are only specific implementation examples of the present invention. Obviously, the present invention is not limited to the above examples, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A silk nanofiber-bismuth nanoparticle composite film, characterized in that: The composite film is mainly composed of bismuth nanoparticles and silk nanofibers.

2. The silk nanofiber-bismuth nanoparticle composite film according to claim 1, characterized in that: The mass ratio between the bismuth nanoparticles and the silk nanofibers is 0.1:1-2000:

1.

3. A method for preparing the silk nanofiber-bismuth nanoparticle composite film according to any one of claims 1 to 2, characterized in that: The specific preparation steps adopted are as follows: (1) Dispersing silk nanofibers into deionized water, and then adding morphology control agent and Bi(NO3)3·5H2O; (2) adding NaBH4 to the solution obtained in step (1) for reaction; (3) transferring the solution obtained in step (2) to an ultrasonic crusher for ultrasonic treatment, followed by centrifugation to obtain monodisperse bismuth-based silk composite nanofibers; (4) dispersing the bismuth-based silk fibroin composite nanofibers obtained in step (3) into deionized water, and after being evenly dispersed, using a vacuum solution filter to filter and form a membrane, and drying to obtain a bismuth-based silk fibroin nanofiber composite membrane.

4. A preparation method according to claim 3, characterized in that: In the step (1), the collected silk nanofibers have a length of 20 nm-50 μm and a diameter of 5 nm-2 μm.

5. A preparation method according to claim 3, characterized in that: In the step (1), the added mass of the silk nanofibers relative to the deionized water is 0.01 wt %.

6. A preparation method according to claim 3, characterized in that: In the step (1), the morphology control agent is one of carbonates, EDTA, disorbitol, phosphates, citric acid, tartaric acid, and silicates, and the added mass of the morphology control agent relative to the silk nanofibers is 0.01wt%-4wt%.

7. A preparation method according to claim 3, characterized in that: In the step (1), the morphology control agent is first added, and Bi(NO3)3·5H2O is added after continued stirring, so that the final concentration of Bi(NO3)3·5H2O is 0.01wt%-20wt%.

8. A preparation method according to claim 3, characterized in that: In the step (2), NaBH4 is added after continued stirring to make the final concentration of NaBH4 be 0.01wt%-4wt%, and the reaction is continued until no bubbles overflow.

9. A preparation method according to claim 3, characterized in that: In the step (3), ultrasound is performed at a power of 100-1000 W for 1-30 min, followed by centrifugation at a speed of 8000 rpm for 10 min.

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