阅读说明：本技术 一种单侧负载银的丝素蛋白抗菌敷料及制备方法和应用 (Silk fibroin antibacterial dressing with silver loaded on one side, and preparation method and application thereof ) 是由 葛少华 邵金龙 马保金 崔雅婷 商玲玲 于 2021-09-17 设计创作，主要内容包括：本发明具体涉及一种单侧负载银的丝素蛋白抗菌敷料及制备方法和应用。银具有良好的抗菌性能可应用于创面的抗菌剂进行添加,但有研究表明银可能同时具有细胞毒性作用从而降低创面细胞活力。针对上述技术问题,本发明提供了一种单侧负载原位生成AgCl颗粒的丝素蛋白抗菌敷料,该抗菌敷料包括银负载层及丝素蛋白层,丝素蛋白层直接接触创面,为创面细胞提供良好的生长环境,与此同时,银负载层通过丝素蛋白层的缓冲作用持续释放Ag～(+),降低对创面细胞毒性的同时还发挥抗菌剂的作用。所述抗菌敷料具有制备工艺简单、促进伤口愈合的优点。(The invention particularly relates to a silk fibroin antibacterial dressing with silver loaded on one side, and a preparation method and application thereof. Silver has good antibacterial performance and can be applied to the addition of an antibacterial agent of a wound surface, but researches show that the silver possibly has a cytotoxic effect at the same time so as to reduce the activity of cells of the wound surface. Aiming at the technical problem, the invention provides a silk fibroin antibacterial dressing with single-side loading and in-situ generation of AgCl particles, which comprises a silver loading layer and a silk fibroin layer, wherein the silk fibroin layer is directly contacted with a wound surface to provide a good growth environment for cells of the wound surface, and meanwhile, the silver loading layer continuously releases Ag through the buffering effect of the silk fibroin layer + Reducing cytotoxicity to wound surfaceAnd also acts as an antimicrobial. The antibacterial dressing has the advantages of simple preparation process and promotion of wound healing.)

1. The silk fibroin antibacterial dressing with silver loaded on one side is characterized by comprising a silk fibroin layer and a silver-silk fibroin layer which are attached to each other.

2. The silver-loaded silk fibroin antibacterial dressing of claim 1, wherein the layer thickness ratio of the silk fibroin layer to the silver-silk fibroin layer is 1: 1-100: 1; preferably, the layer ratios are 1:1, 3:1, 5:1, 7:1, 10:1, 20:1, 40:1, 80:1 or 100: 1.

3. The preparation method of the silk fibroin antibacterial dressing with silver loaded on one side, which is characterized by comprising the following steps of:

adding sodium carbonate into boiling distilled water for dissolving, adding cut silkworm cocoon shell, continuously stirring until degumming, cleaning and soaking the degummed silk fibroin with distilled water to remove residual sodium carbonate on the surface, and completely drying the cleaned silk fibroin at room temperature; dissolving calcium chloride in formic acid to form formic acid-calcium chloride mixed solution, then adding silk fibroin into the mixed solution, and stirring until the silk fibroin is completely dissolved to form silk fibroin solution; adding a silver nitrate aqueous solution into a silk fibroin solution to form a silver-silk fibroin solution, uniformly dispersing the silver-silk fibroin solution on a mold, volatilizing, adding the silk fibroin solution on a dispersion interface after volatilizing for a certain time, and continuously volatilizing to obtain the silver-silk fibroin/silk fibroin antibacterial dressing.

4. The method for preparing the silver-loaded silk fibroin antibacterial dressing with one side as claimed in claim 3, wherein the silk fibroin solution is prepared in the following manner: adding silk fibroin into a mixed solution of formic acid and calcium chloride for dissolving;

preferably, the formic acid is 85-90% of formic acid solution in mass fraction; or the concentration of the calcium chloride is 0.2-1 g/10-15 mL; or the concentration of the silk fibroin solution is 1-2 g/10-15 mL.

5. The method for preparing the silk fibroin antimicrobial dressing with silver loaded on one side as claimed in claim 3, wherein the silk fibroin is degummed by an alkaline method.

6. The method for preparing the silk fibroin antibacterial dressing with silver loaded on one side as claimed in claim 5, wherein the degumming mode of the silk fibroin is as follows: heating water to boil, adding sodium carbonate to dissolve, and adding silkworm cocoon shell to boil until degumming; after washing, the degummed silk fibroin was soaked for a period of time and dried.

7. The method for preparing the silver-loaded silk fibroin antibacterial dressing with one side loaded as claimed in claim 1, wherein the preparation method of the silver-silk fibroin solution is as follows: adding the degummed silk fibroin into a formic acid-calcium chloride solution, stirring and dissolving to prepare a silk fibroin solution, and then adding silver nitrate into the silk fibroin solution and uniformly mixing.

8. The preparation method of the silk fibroin antibacterial dressing with silver loaded on one side as claimed in claim 7, wherein the silver-silk fibroin solution is uniformly dispersed in a mold, the silk fibroin solution is added into the mold after the silver-silk fibroin solution is volatilized for 0.4-0.6 h under ventilation condition and continuously volatilized for 2-3h, and the mold is sequentially immersed into water and ethanol solution to release the mold, so that the silver-silk fibroin/silk fibroin antibacterial dressing is obtained.

9. A wound dressing comprising the silver-loaded silk fibroin antimicrobial dressing of claim 1 or 2 on one side.

10. The wound dressing of claim 9, further comprising a backing layer, wherein the backing layer is one of release paper, non-woven fabric, gauze, synthetic fiber, or polymeric film material; or the wound dressing also comprises other active ingredients, wherein the other active ingredients are water-absorbing ingredients, anti-inflammatory ingredients or antimicrobial ingredients.

Technical Field

The invention belongs to the technical field of antibacterial wound dressing, and particularly relates to a silk fibroin antibacterial dressing with silver loaded on one side, a preparation method of the antibacterial dressing and application of the antibacterial dressing as a wound dressing.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Silk Fibroin (SF) is a natural protein extracted from silk, which has the ability to promote cell migration, proliferation, angiogenesis and re-epithelialization and a structure similar to that of extracellular matrix and is widely used in wound dressings. Dressings with silk fibroin as a scaffold material generally have excellent biocompatibility and degradability, low immunogenicity, and weak inflammatory response, and further, it has been reported that silk fibroin dressings can promote wound healing. Since most wounds are exposed directly to the environment and are highly susceptible to bacterial attack, prevention and reduction of bacterial infection is an important factor in promoting healing. However, silk fibroin itself has no antibacterial effect.

Silver (Ag) has a broad spectrum of antimicrobial properties and does not exhibit as broad a resistance as antibiotics and is therefore often used as an antimicrobial agent added to wound dressings. Research shows that eukaryotic cells have higher tolerance to silver than prokaryotic cells, and in prokaryotic cells, important biochemical pathways such as respiratory chain and DNA replication are located on a cytoplasmic membrane, while the biochemical pathways of eukaryotic cells are located in mitochondria and cell nucleus protected by organelles. Thus, the difference between the cytotoxic and antimicrobial concentrations of silver can serve as a therapeutic window for its antimicrobial application.

Nevertheless, the use of silver is controversial. Some studies found that silver-containing dressings delayed wound healing, while others demonstrated that silver-containing dressings promoted wound healing. The current silver-containing dressing has no consistent conclusion on the effect of wound healing, and the possible reason is that silver is a double-edged sword, has antibacterial property and is toxic to cells. Therefore, how to reduce the side effect of silver on cell viability while maintaining high antibacterial ability is a problem to be solved urgently.

At present, methods for reducing the cytotoxicity of silver are mostly methods such as a green biosynthesis method, a method for reducing active oxygen in cells, a method for coating a collagen film on the surface of a silver-containing titanium plate and the like. Layer-by-layer assembly techniques are a method for depositing thin films to produce functional materials that have controllable structure, properties, and functionality for a variety of applications.

Disclosure of Invention

Aiming at the research background, the invention provides a simplified layer-by-layer assembly technology, a silk fibroin layer is compounded on a silver-loaded silk fibroin film, the cytotoxicity of silver is reduced by utilizing a two-layer structure, and meanwhile, the antibacterial effect of the silver is kept. Therefore, the invention designs and prepares a composite membrane material (Ag-SF/SF) which simultaneously has a silver-loaded silk fibroin (Ag-SF) layer and a pure Silk Fibroin (SF) layer, and the shape, the physicochemical property and the Ag of the composite membrane⁺The release profile was characterized. The antibacterial activity of the Ag-SF/SF composite membrane loaded with different silver contents is evaluated through an antibacterial experiment. Human Foreskin Fibroblasts (HFFs) were seeded onto Ag-SF/SF composite membranes to explore the cellular compatibility of the Ag-SF side and SF side. The effect of different thickness ratios of the Ag-SF layer to the SF layer on cell activity was further studied to elucidate the effect of the bilayer structure on reducing silver cytotoxicity. The influence of the SF side of the Ag-SF/SF composite membrane on wound healing in vitro is detected through a scratch experiment. Furthermore, by determining in real timeQuantitative polymerase chain reaction (qRT-PCR) was used to measure the expression levels of collagen type I (Collagen I, Col I) and transforming growth factor-beta (TGF-beta) mRNA in cells on the SF side of composite membranes. Finally, the Ag-SF/SF composite membrane was applied to a dorsal skin excision wound splint model in staphylococcus aureus infected rats to evaluate its effect on infected wound healing.

Based on the above research, the present invention provides the following technical solutions:

the invention firstly provides a silk fibroin antibacterial dressing with silver loaded on one side, and the antibacterial dressing is provided with a silk fibroin layer and a silver-silk fibroin layer which are attached.

Silver has antimicrobial properties that make it widely used in wound dressings, but its resulting cytotoxicity has also attracted considerable attention. The present invention proposes a novel design of an antimicrobial dressing with a single-sided silver distribution to reduce cytotoxicity while maintaining antimicrobial properties, thereby extending its therapeutic range. The Ag-SF/SF composite membrane is prepared by adopting a simplified layer-by-layer assembly technology, the SF side of the composite membrane shows lower cytotoxicity in vitro than the Ag-SF side, and meanwhile, the equivalent antibacterial effect is kept on both sides. Further in vivo results indicate that Ag-SF/SF antimicrobial dressings can accelerate healing of infected wounds.

The skin is elastic and has a range of motion, and therefore the wound dressing should have a certain tensile strength. In the research of the invention, the prepared SF film (7.0 MPa) and Ag-SF/SF composite film (7.6 MPa) both have higher Young modulus, and the beta folding structure of the silk fibroin in the composite film enhances the mechanical property through the shape determination.

In addition, the addition of silver imparts antimicrobial activity to the composite film in the form of Ag⁺The form exists. The nano silver has high preparation cost and insufficient stability, and the nano silver essentially serves as a silver simple substance and has insufficient release effect. In order to overcome the technical defects, the invention adopts the following solving method: provides Ag⁺An antibacterial product in a state. The invention specifically adopts the mode that silver nitrate is used as a raw material and reacts with a reactantThe calcium chloride generates a replacement reaction to produce AgCl, thereby realizing that the silver in the antibacterial dressing can maintain Ag for a long time⁺The form is released. Furthermore, the research result of the invention shows that in the composite membrane, both the silk fibroin side and the silver-silk fibroin side can play an antibacterial role, the antibacterial effect is equivalent, and the silk fibroin side has a good healing promotion effect when being contacted with a wound to dye the wound.

Based on the research conclusion, the composite membrane provided by the invention has multiple physiological activities when being applied to wound healing, has important production significance, is simple and convenient in preparation process, and is easy to realize industrial expanded production. Therefore, in a second aspect, the present invention provides a preparation method of the silver-loaded silk fibroin antibacterial dressing with one side, which comprises the following steps:

adding sodium carbonate into boiled distilled water for dissolving, adding cut silkworm cocoon shells, continuously stirring until degumming, cleaning and soaking the degummed silk fibroin by using distilled water to remove residual sodium carbonate on the surface, completely drying the cleaned silk fibroin at room temperature, dissolving calcium chloride into formic acid to form formic acid-calcium chloride mixed solution, then adding the silk fibroin into the mixed solution, and stirring until the silk fibroin is completely dissolved to form silk fibroin solution; adding a silver nitrate aqueous solution into a silk fibroin solution to form a silver-silk fibroin (Ag-SF) solution, uniformly dispersing the silver-silk fibroin solution on a mold, volatilizing for a certain time, adding the silk fibroin solution on a dispersing interface, and continuously volatilizing to obtain the silver-silk fibroin/silk fibroin (Ag-SF/SF) antibacterial dressing.

Finally, the invention provides an application of the silk fibroin antibacterial dressing with silver loaded on one side as a wound dressing, wherein the wound dressing comprises the silk fibroin antibacterial dressing with silver loaded on one side in the first aspect.

The beneficial effects of one or more technical schemes are as follows:

1. the Ag-SF/SF composite membrane is successfully prepared by a simplified layer-by-layer assembly technology; both sides of the Ag-SF/SF composite membrane play an antibacterial role, and the SF side can effectively promote the healing of wounds; when the concentration of the loaded silver exceeds 0.06mg/mL, the Ag-SF/SF composite membrane can play an effective and equivalent antibacterial property on both sides; the result of cell activity detection shows that the Ag0.12-SF/SF1:3 composite membrane has good cell compatibility at the SF side, and simultaneously has strong antibacterial performance at the SF side of the Ag0.12-SF/SF1:3 composite membrane; in addition, the Ag0.12-SF/SF1:3 composite membrane can promote expression of Col I and TGF-beta mRNA in vitro and can remarkably promote healing of infected wounds in vivo.

2. The research of the invention provides a new strategy, namely, the single-side distribution of silver is realized by adopting a simplified layer-by-layer assembly technology, so that the cytotoxicity of the material is reduced, the treatment window of the silver-containing wound dressing is further expanded, and the prepared Ag-SF/SF bifunctional composite membrane as the wound dressing can effectively repair the damaged skin, thereby having great application potential. In addition, the simplified layer-by-layer assembly technique is not limited to the preparation of a two-layer structure composite membrane, but also includes two or more layers.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing morphological characteristics and elemental analysis results of the SF film and Ag-SF/SF composite film described in example 1;

(A) SEM images of the Ag-SF side and the SF side of the SF film and the Ag-SF/SF composite film under different magnification factors;

(B) element distribution;

(C) atomic ratio of particles.

FIG. 2 shows the phases and mechanical properties of the SF film and Ag-SF/SF composite film and Ag of the Ag-SF/SF composite film in example 1⁺Release result graph;

(A) XRD patterns of the SF film and the Ag-SF/SF composite film;

(B) FTIR spectra of SF films and Ag-SF/SF composite films;

(C-E) mechanical property analysis of SF films and Ag-SF/SF composite films;

(F) of Ag-SF/SF composite filmsAg⁺Release profile.

FIG. 3 is a graph showing the results of the measurement of the antibacterial activity against Staphylococcus aureus (A-D) and Escherichia coli (E, F) of the samples described in example 1;

(A) representative pictures of the Ag-SF side and SF side bacteriostatic rings of Ag0.03-SF/SF, Ag0.06-SF/SF, Ag0.12-SF/SF and Ag0.24-SF/SF samples;

(B) quantitative analysis of Ag-SF side and SF side antibacterial rings in the Ag-SF/SF sample;

(C) representative pictures of Ag-SF side, SF side and control group antibacterial rings in the Ag0.12-SF/SF1:3, Ag0.12-SF/SF1:5 and Ag0.12-SF/SF1:7 samples, and a single-layer film with uniformly distributed silver is taken as a control group;

(D) quantitative analysis of Ag-SF side, SF side and control group antibacterial ring in samples with different thickness ratios of Ag0.12-SF/SF;

(E) representative pictures of the Ag-SF side and SF side bacteriostatic rings of Ag0.03-SF/SF, Ag0.06-SF/SF, Ag0.12-SF/SF and Ag0.24-SF/SF samples;

(F) quantitative analysis of Ag-SF-side and SF-side antibacterial rings in Ag-SF/SF samples. The diameter of the sample was 10 mm.^*P<0.05，^**P<0.01，^***P<0.001，^****P<0.0001。

FIG. 4 is a graph showing the results of the cytocompatibility test described in example 1;

(A) staining of live and dead cells after 24h incubation of HFFs on the Ag-SF side of TCP, SF membranes and Ag-SF/SF membranes, yellow arrows indicate dead cells;

(B, C) CCK-8 analysis results of HFFs cultured on TCP, SF membrane and Ag-SF/SF membrane for 24 h;

(B) HFFs are cultured on the Ag-SF side of the Ag-SF/SF membrane;

(C) HFFs were cultured on the SF side of Ag-SF/SF membranes. Compared with the SF, the method has the advantages that,^**P<0.01，^****P<0.0001。

FIG. 5 is a graph of the results of the Ag0.12-SF/SF1:3, Ag0.12-SF/SF1:5, and Ag0.12-SF/SF1:7 membrane cell compatibility assays described in example 1;

(A) staining of live and dead cells after 24h incubation of HFFs on Ag0.12-SF/SF1:3, Ag0.12-SF/SF1:5, and Ag0.12-SF/SF1:7 membranes, the yellow arrows indicate dead cells. A single-layer film with uniformly distributed silver is used as a control group;

(B) and (3) analyzing the CCK-8 analysis result of the HFFs after being cultured for 24 hours on different surfaces of the SF film and the Ag-SF/SF film with different thickness ratios. Compared with the SF, the method has the advantages that,^**P<0.01，^***P<0.001，^****P<0.0001。

FIG. 6 is a graph showing the results of the scratch test and the gene expression results of example 1 for evaluating the healing of wounds in vitro;

(A, B) are microscopic images of the TCP group under different magnifications after scratching for 0h (A) and 24h (B);

(C, D) are microscopic images of the SF film group under different magnifications after scratching for 0h (C) and 24h (D);

(E, F) micrographs of the SF-side group of Ag0.12-SF/SF1:3 films at different magnifications after scoring 0h (E) and 24h (F);

(G) expression levels of Col I after 7 days of HFFs culture;

(H) expression levels of TGF-. beta.after 7 days of culture of HFFs. In contrast to TCP,^****P<0.0001。

FIG. 7 is a graph showing the results of the macroscopic image and Masson staining image analysis of the wound surface after operation described in example 1;

(A) macroscopic images of wounds in sham, SF and Ag0.12-SF/SF1 groups at 0, 4, 7, 11 and 14 days post-surgery;

(B) quantitatively analyzing the percentage of the remaining wound area in the wound macroscopic image;

(C) masson staining images of the sham surgery group, SF group and Ag0.12-SF/SF1:3 groups of wound surface tissues 4 and 14 days after surgery;

(D) collagen formation in Masson images was quantitatively analyzed.^*P<0.05，^**P<0.01，^****P<0.0001。

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As introduced in the background technology, silver has good broad-spectrum antibacterial effect when being used as an antibacterial agent, is not easy to generate drug resistance, and can effectively inhibit the breeding of bacteria on a wound surface and promote the healing of the wound. At the same time, silver also has cytotoxic effects, and studies have shown that the addition of silver to dressings can interfere with wound healing. In order to solve the technical problems, the invention provides the silk fibroin antibacterial dressing with silver loaded on one side, the silver is isolated by the silk fibroin layer and directly acts on the wound surface, and the cytotoxicity effect of the silk fibroin antibacterial dressing is reduced while the antibacterial effect of the silk fibroin antibacterial dressing is kept.

In a first aspect of the invention, a silver-loaded silk fibroin antimicrobial dressing with a single side is provided, wherein the antimicrobial dressing comprises a silk fibroin layer and a silver-silk fibroin layer which are attached to each other.

Further, the layer thickness ratio of the silk fibroin layer to the silver-silk fibroin layer is 1:1 to 100:1, for example, 1:1, 3:1, 5:1, 7:1, 10:1, 20:1, 40:1, 80:1, and 100:1, but not limited to the exemplified ratios.

Research shows that the optimal layer thickness ratio of Ag-SF to SF is 1:3, and the SF layer can balance cytotoxicity and antibacterial effect of the composite membrane.

In the Ag-SF/SF antibacterial dressing, a plurality of particles are formed only on the side surface of Ag-SF, and element analysis is carried out on the particles to prove that the particles are AgCl particles; the observation of SEM shows that the Ag-SF/SF composite membrane has different performances on different sides, which proves that the composite membrane with a two-layer structure is successfully prepared by a simplified layer-by-layer assembly technology and provides a foundation for subsequent research. The XRD pattern showed that the Ag-SF side of the composite film showed typical AgCl diffraction peaks at 2 theta of 27.8 °, 32.2 ° and 46.2 °,which point to the (111), (200) and (220) planes, respectively (JCPDS No. 85-1355); meanwhile, similar to the SF film, the XRD pattern of the Ag-SF/SF composite film also shows a broad peak at 2 θ ═ 21 °, which is a crystal diffraction peak of silk II having a β sheet structure. Consistent with XRD pattern, the SF film and Ag-SF/SF composite film in FTIR spectrum are 1517cm^-1And 1623cm^-1The characteristic peaks at (a) belong to the oscillation of amide II and amide I, respectively, which also confirms that the beta sheet structure exists in the Ag-SF/SF composite membrane. Previous studies have shown that the beta sheet structure can serve as a physical cross-link connecting different protein chains to form a continuous network, which gives SF scaffolds higher mechanical strength. The research of the invention proves that the silk fibroin in the antibacterial dressing can also form a beta-folded structure, thereby providing good mechanical properties. XRD and FTIR results show that the Ag-SF/SF composite membrane retains the characteristic peak of silk fibroin, and the structural property of the silk fibroin is not changed by adding silver.

In a second aspect of the present invention, a preparation method of the silver-loaded silk fibroin antibacterial dressing with one side of the dressing is provided, wherein the preparation method comprises the following steps:

adding sodium carbonate into boiling distilled water for dissolving, adding cut silkworm cocoon shell, continuously stirring until degumming, cleaning and soaking the degummed silk fibroin with distilled water to remove residual sodium carbonate on the surface, and completely drying the cleaned silk fibroin at room temperature; dissolving calcium chloride in formic acid to form formic acid-calcium chloride mixed solution, then adding silk fibroin into the mixed solution, and stirring until the silk fibroin is completely dissolved to form silk fibroin solution; adding a silver nitrate aqueous solution into a silk fibroin solution to form a silver-silk fibroin (Ag-SF) solution, uniformly dispersing the silver-silk fibroin solution on a mold, volatilizing for a certain time, adding the silk fibroin solution on a dispersing interface, and continuously volatilizing to obtain the silver-silk fibroin/silk fibroin (Ag-SF/SF) antibacterial dressing.

Preferably, the silk fibroin solution is prepared as follows: adding silk fibroin into mixed solution of formic acid-calcium chloride for dissolving.

In an embodiment of the above preferred technical solution, the formic acid is 85 to 90% by mass of a formic acid solution.

Furthermore, the concentration of the calcium chloride is 0.2-1 g/10-15 mL.

Further, the concentration of the silk fibroin solution is 1-2 g/10-15 mL.

In this series of embodiments, the silk fibroin is degummed as follows: heating water to boil, adding sodium carbonate to dissolve, and adding silkworm cocoon shell to boil until degumming; after washing, the degummed silk fibroin was soaked for a period of time and dried.

Preferably, the preparation method of the silver-silk fibroin (Ag-SF) solution is as follows: adding the degummed silk fibroin into a formic acid-calcium chloride solution, stirring and dissolving to prepare a silk fibroin solution, and then adding silver nitrate into the silk fibroin solution and uniformly mixing.

In a specific embodiment of the preparation method, the silver-silk fibroin (Ag-SF) solution is placed in a mold to be uniformly dispersed, the solution is volatilized for 0.4-0.6 h under ventilation conditions, the silk fibroin solution is added into the mold to be continuously volatilized for 2-3h, and the mold is sequentially immersed in water and ethanol solution to release the mold, so that the silver-silk fibroin/silk fibroin (Ag-SF/SF) antibacterial dressing is obtained.

In a third aspect of the invention, a wound dressing is provided, and the wound dressing comprises the silver-loaded silk fibroin antibacterial dressing with one side of the first aspect.

Preferably, the wound dressing further comprises a backing layer, wherein the backing layer includes, but is not limited to, one of release paper, non-woven fabric, gauze, synthetic fiber or a polymeric membrane material.

Preferably, the wound dressing further comprises other active ingredients, such as a water-absorbing ingredient, an anti-inflammatory ingredient or an antimicrobial ingredient.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

Experimental materials and methods:

preparation and characterization of SF film and Ag-SF/SF antibacterial dressing

1. Preparation of fibroin solution

Heating 1.2L distilled water to boil, adding 2.55g sodium carbonate to dissolve, adding 3g cut silkworm cocoon shell, and boiling for 40min to remove pectin. The degummed silk fibroin was rinsed three times with distilled water, soaked overnight in 1L of distilled water to remove residual sodium carbonate on the surface, taken out and completely dried at room temperature. Dissolving and weighing 0.2-1 g of calcium chloride, dissolving the calcium chloride in 10-15 mL of formic acid (mass fraction is 85-90%), adding 1-2 g of degummed silk fibroin into the solution, and stirring until the calcium chloride is completely dissolved for further use.

Preparation of SF films and Ag-SF/SF antimicrobial dressings

The petri dish served as a mold for preparing the membrane. Firstly, respectively preparing 0.03-0.24mg/mL Ag-SF solution, uniformly dispersing 200 mu L of 0.03-0.24mg/mL Ag-SF solution in the whole mould, placing the mould in a fume hood to volatilize for 0.5h until no liquid fluidity exists on the surface, and then adding 600 mu L SF solution to continuously volatilize for 2.5h to form Ag0.03-SF/SF, Ag0.06-SF/SF, Ag0.12-SF/SF and Ag0.24-SF/SF composite membranes.

Preparing Ag0.12-SF/SF1:3, Ag0.12-SF/SF1:5 and Ag0.12-SF/SF1:7 composite films according to the steps, adding 200 mu L of 0.12mg/mL Ag-SF solution into a mold until the surface has no liquid fluidity, and then respectively adding 600 mu L, 1000 mu L or 1400 mu L SF solution; the SF film is prepared by directly adding 800 μ L SF solution into a mold and volatilizing for 3 h. The mould with the membrane was immersed in distilled water for 0.5h and then in absolute ethanol for 1-2h to release the mould. Finally, a portion of the prepared material was cut into 10mm diameter samples and stored in absolute ethanol at 4 ℃ for further use.

Characterization of SF films and Ag-SF/SF antimicrobial dressings

The material characterization is carried out in the section by using an Ag0.12-SF/SF1:3 film. The morphology of the SF film and Ag-SF/SF antimicrobial dressing was observed under a Scanning Electron Microscope (SEM). The elemental distribution of the Ag-SF/SF antimicrobial dressing was analyzed by energy dispersive X-ray (EDX). The physicochemical properties of the Ag-SF/SF antimicrobial dressing were analyzed by X-ray diffraction (XRD) spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. The tensile properties of the SF film and the Ag-SF/SF composite film (n ═ 3) were measured by a universal tester, and the strain at break, young's modulus and tensile strength were counted and analyzed.

Ag-SF/SF antibiotic dressing Ag⁺Release detection

Ag-SF/SF (ag0.12-SF/SF1:3) antimicrobial dressing (n ═ 3) was soaked in 2mL phosphate-buffered solution (PBS) with constant stirring at 37 ℃. At 1, 6, 24, 72, 168, 240 and 336h, 2mL of supernatant was collected and replaced with fresh PBS solution, respectively. Estimation of Ag by Atomic Absorption Spectroscopy (AAS) analysis⁺To evaluate the release profile.

(II) in vitro experiment detection of antibacterial performance of Ag-SF/SF antibacterial dressing and influence thereof on cell biological activity

1. In vitro antibacterial testing

The antibacterial effect of the antibacterial dressing on staphylococcus aureus and escherichia coli was evaluated by a bacteriostatic ring test. Bacterial suspension was prepared by mixing the strain with sterile saline (0.85% NaCl) and the OD was measured using UV spectrophotometer_600nmThe value was 0.123 and then diluted 10-fold. Subsequently, the bacterial suspension was spread evenly on nutrient agar plates, and a sample (n-3) having a diameter of 10mm was placed on the bacteria-coated agar plates. After incubation at 36 ℃ for 20h, images were taken with a ruler as a calibrator, and the diameter of the transparent zone was measured and analyzed.

Cell compatibility testing on SF films and Ag-SF/SF antimicrobial dressings

Samples of each group were soaked in 75% ethanol overnight, then sterilized by ultraviolet irradiation in a 48-well plate for 1h, rinsed three times with PBS, added with PBS for soaking and continued irradiation under an ultraviolet lamp for 1h, and after removal of PBS, added with complete medium for soaking overnight. HFFs were scaled up to 3X 10⁴The density of individual cells/well was seeded on SF membranes or Ag-SF/SF antimicrobial dressings, with the TCP group as a control. Staining live and dead cells: after 24h of cell culture, the cell status was observed using a live-dead cell staining kit according to the manufacturer's instructions. CCK-8 experiment: after the cells are cultured for 24 hours, the cells on the surface of the material are digested by using trypsin-EDTA digestive juice, cell suspension is collected and is transferred to a new 96-well plate again, after incubation for 12 hours, the original culture solution is discarded, 10 mu L of CCK-8 solution and 100 mu L of DMEM are added into each well (n is 3), and after the cells are cultured for 2-3 hours in the dark at 37 ℃, an enzyme linked immunosorbent spectrophotometer is used for detecting the absorbance value at the wavelength of 450 nm.

3. Scratch experiment for detecting influence of SF film and Ag-SF/SF antibacterial dressing on HFFs in-vitro wound healing

HFFs were scaled up to 3X 10⁵Inoculating the density of each cell/hole on SF membrane or Ag-SF/SF antibacterial dressing, taking TCP group as a control, culturing the cells the next day, drawing a vertical line perpendicular to the plate bottom by using a 10 mu L sterile gun head after the cells are paved on the plate bottom, and continuously culturing for 24h in an incubator at 37 ℃. After scratching for 0 and 24h, the wound was observed under a microscope and a wound healing photograph was taken.

qRT-PCR analysis

HFFs were scaled up to 1 × 10⁵The density of each cell/well was inoculated on SF membrane or Ag-SF/SF antimicrobial dressing (n ═ 3) in 6-well plates and cultured for 7 days to examine Col I and TGF- β gene expression. Total RNA was extracted using TRIzol reagent, and mRNA concentration of each sample was measured using a Nanodrop 2000 microspectrophotometer, and then mRNA was reverse transcribed into cDNA for real-time quantitative PCR analysis. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the mRNA levels of Col I and TGF- β.

(III) in vivo experiment detection of influence of Ag-SF/SF antibacterial dressing on infected wound healing

1. Establishment of skin wound excision splint fixing model

12 healthy male Wistar rats aged 8 weeks were used for the experiment and were fasted for 12h before surgery. Blinding is performed by independent researchers retaining information on all sample sets, and grouping information is not disclosed until all data is collected. All wounds were randomly divided into 3 groups, i.e. sham control, SF and ag0.12-SF/SF1:3 (n ═ 8), where the SF side of the ag0.12-SF/SF1:3 composite membrane touched the skin infected wound defect area and no material was placed in the defect area of the sham control. After isoflurane inhalation anesthesia, large part of the body is shaved offThe skin of rat back was disinfected by iodophor and 75% ethanol, four circular wounds 8mm in diameter were punched on both sides of rat back skin with a disposable skin punch, a red circular silicone ring (inner diameter 12mm, outer diameter 22mm, thickness 1mm) was sutured over the defect to prevent wound closure due to skin contraction, and then 10. mu.L of Staphylococcus aureus (1X 10) dissolved in 0.85% sterile saline was applied (1X 10)⁸CFU) bacterial suspension was injected onto the wound surface and the wound was covered with a different film and further fixed using 3M transparent dressing and bandage. Wound healing photographs were taken at 0, 4, 7, 11 and 14 days post-surgery, respectively. On days 4 and 14, rats were euthanized with an excess of anesthetic and wounds and surrounding normal skin tissue were collected and fixed in 4% paraformaldehyde. Wound reduction was quantified according to previous studies by the inventors. Wound area (%) was expressed as wound reduction and calculated using the following formula:

2. histological analysis

After 4% paraformaldehyde fixation, the samples were gradient dehydrated and embedded in paraffin, and 5 μm thick serial sections were prepared from the top to the deep layer of the skin, and Masson staining was performed every nineteenth section. All samples were observed under an olympus microscope and measured using ImageJ software. Collagen formation was quantified using the Collagen Volume Fraction (CVF), the blue stained tissue area was considered as the collagen area and the CVF was expressed as collagen area/total area (%), calculated specifically as the area occupied by blue stained tissue divided by the total area of tissue under direct vision.

The experimental results are as follows:

morphological characterization and elemental analysis of SF films and Ag-SF/SF antimicrobial dressings

And analyzing the morphology and the element composition of the SF film and the Ag-SF/SF composite film through SEM-EDX. As shown in fig. 1A, both the SF film and the Ag-SF/SF antimicrobial dressing show a smooth surface. In Ag-SF-Many particles were visible on the Ag-SF side of the SF antimicrobial dressing, EDX results confirmed that the particles contained C, N, O, Cl and Ag (fig. 1B), and the atomic ratio of Ag to Cl element was close to 1:1 (FIG. 1C), indicating that AgNO is responsible for₃And CaCl₂The reaction between the two forms AgCl.

Phases, mechanical Properties and Ag of SF films and Ag-SF/SF antibacterial dressings⁺Release Profile

The XRD patterns were used to study the crystalline phases of SF and AgCl (fig. 2A). The XRD pattern of the SF film has a typical diffraction peak at 2 θ ═ 21 °, and this characteristic diffraction peak is also observed on the Ag-SF side of the Ag-SF/SF antibacterial dressing, while the Ag-SF/SF antibacterial dressing shows characteristic peaks of AgCl at 2 θ ═ 27.8 °, 32.2 ° and 46.2 °. FTIR analysis shows that the SF film and the Ag-SF/SF antibacterial dressing are 1517cm^-1And 1623cm^-1The peaks characteristic of amide II and amide I oscillations are shown (fig. 2B). The mechanical properties results show that young's modulus and tensile strength are similar for SF films and Ag-SF/SF antimicrobial dressings (fig. 2C-E). Detection of Ag Using AAS⁺Release concentration of Ag⁺The release profile shows that Ag is in the early stage⁺The release rate is relatively fast, and internal Ag is in later stage⁺The release rate was relatively slow (fig. 2F).

Detection of antibacterial Properties of Ag-SF/SF antibacterial dressings

The antibacterial performance of the Ag-SF/SF antibacterial dressing is quantitatively evaluated by a bacteriostatic ring test. As shown in FIG. 3A, in the bacteriostatic ring test for Staphylococcus aureus, the Ag0.03-SF/SF composite membrane showed no bacteriostatic ring, but a significant bacteriostatic ring was observed from the Ag0.06-SF/SF composite membrane. In the Ag-SF/SF composite film of the same silver content, the Ag-SF side and the SF side showed similar antibacterial effects, and further, the antibacterial effect of the Ag-SF/SF composite film was gradually enhanced as the silver content was increased, but the antibacterial effect of the Ag0.12-SF/SF composite film was equivalent to that of the Ag0.24-SF/SF composite film (FIG. 3B). To further clarify the effect of different thickness ratios between the two layers on the antimicrobial efficacy, for different Ag-SF layers: the test was carried out on the Ag-SF/SF film having the SF layer thickness ratio, and the film in which the single layer of silver was uniformly distributed was used as a control, and the results showed that both the film having the double-layered structure and the film in which the single layer of silver was uniformly distributed exhibited significant antibacterial rings (FIG. 3C), and the antibacterial performance of the material was gradually decreased as the thickness of the SF layer was increased, but the antibacterial performance was not significantly different between the single-layer film control group, the Ag-SF side of the double-layer film, and the SF-side group at the same layer thickness ratio (FIG. 3D). To demonstrate the broad-spectrum antibacterial activity of the Ag-SF/SF composite membrane, the antibacterial effect of Ag-SF/SF1:3 membranes with different silver contents on escherichia coli was tested. Similar to the results of inhibiting Staphylococcus aureus, both the Ag-SF side and the SF side of the Ag0.12-SF/SF1:3 membranes had better inhibiting effects on Escherichia coli (FIG. 3E, F).

Detection of cellular Activity of SF Membrane and Ag-SF/SF antimicrobial dressing

To study the cellular compatibility of the SF membrane and Ag-SF/SF antimicrobial dressing, the state of the cells seeded on the different membranes was observed using live and dead cell staining, as shown in FIG. 4A, live cells were stained green, while dead cells were stained red, and the number of live cells was significantly reduced and the number of dead cells appeared higher in the Ag0.24-SF/SF group compared to the other groups. In addition, the CCK-8 experiment further quantitatively evaluated the cell activity of different sides of the Ag-SF/SF antibacterial dressing, as shown in FIGS. 4B and C, the cytotoxicity of both sides of the Ag-SF/SF antibacterial dressing increased with the increase of the silver content, however, the SF side of the Ag0.12-SF/SF group was observed to have almost no cytotoxicity, while the Ag-SF side showed relatively significant cytotoxicity, thus demonstrating that the cell activity of HFFs is higher on the SF side than on the Ag-SF side. The SF side based on Ag-SF/SF antimicrobial dressings showed better cellular compatibility than the Ag-SF side, further discussing ag0.12-SF: effect of SF different layer thickness ratio material on cell viability, a single layer membrane with uniform distribution of silver was used as a control. As shown in FIGS. 5A and B, the SF side of the Ag0.12-SF/SF film showed better cell compatibility and certain cytotoxicity at the Ag-SF side compared to the monolayer film having the same thickness in which silver was uniformly distributed, regardless of the layer thickness ratio of the Ag0.12-SF/SF film.

5. Scratch experiment for detecting influence of SF film and Ag-SF/SF antibacterial dressing on HFFs in-vitro wound healing

As shown in fig. 6A-F, after creating the scratch, the SF-side groups of TCP, SF film and ag0.12-SF/SF1:3 film all exhibited a scratch line without cells, but due to the flexibility of the SF film material, the scratch lines created on the SF-side of the SF film and ag0.12-SF/SF1:3 film were wider than those of the TCP group. Despite these differences, wound closure in vitro was similar for the SF-side group of SF films and Ag0.12-SF/SF1:3 films to that of the TCP group.

Effect of SF membranes and Ag-SF/SF antimicrobial dressings on the expression of Col I and TGF-beta genes from HFFs

Col I is the main component of extracellular matrix, and is the natural matrix for cell attachment, growth and differentiation; TGF- β can regulate cell proliferation, migration, differentiation and extracellular matrix production, and play a diverse role in wound healing; thus, the expression levels of Col I and TGF- β were further evaluated to explore the potential of Ag-SF/SF antimicrobial dressings for skin regeneration. As shown in FIGS. 6G and H, the expression levels of Col I and TGF-. beta.were higher in the SF-side group of SF membrane and Ag0.12-SF/SF1:3 composite membrane than in the TCP group after 7 days of cell culture. Thus, increased expression levels of Col I and TGF- β may indicate that the Ag-SF/SF antimicrobial dressing may promote wound healing.

Effect of Ag-SF/SF antimicrobial dressings on wound healing in vivo

In order to explore the influence of the Ag-SF/SF composite membrane on the healing of skin infection wound, an in vivo experiment is carried out by adopting a rat wound excision splint fixed model infected by staphylococcus aureus. All splints were normalized to the wound area within 14 days of the experiment (fig. 7A), although the rate of healing was over 90% for all wounds at 14 days, the rate of healing was significantly faster for the ag0.12-SF/SF1:3 group than for the sham and SF groups (fig. 7B). Masson staining showed that the collagen formation in the Ag0.12-SF/SF1:3 group was higher than that in the sham and SF groups (FIG. 7C, D), and that the amount of nascent collagen in the Ag0.12-SF/SF1:3 group reached 50% on day 4 and 80% on day 14.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.