阅读说明：本技术 丝素纳米纤维和基于丝素纳米纤维的载银抗菌敷料的制备方法 (Silk fibroin nanofiber and preparation method of silver-loaded antibacterial dressing based on silk fibroin nanofiber ) 是由 江奇锋 李杨 熊兴良 查小英 章艳 于 2019-10-30 设计创作，主要内容包括：丝素纳米纤维和基于丝素纳米纤维的载银抗菌敷料的制备方法。本发明公开了一种丝素纳米纤维的制备方法：1)将蚕茧放入碳酸钠溶液中脱胶得到丝素蛋白；2)将丝素蛋白置于氢氧化钠/尿素水溶液中溶解得到溶解液；3)将透析袋置于碳酸氢钠/EDTA溶液中沸腾处理,然后取出透析袋放入EDTA溶液中沸腾处理；4)将步骤2)得到的溶解液倒入处理好的透析袋中透析,超声处理,冷冻离心,得到丝素纳米纤维水溶液。还公开了一种载银抗菌敷料的制备方法：1)采前述方法制备丝素纳米纤维水溶液；2)取硝酸银溶解于得到的丝素纳米纤维水溶液中,硝酸银与丝素纳米纤维水溶液的质量体积比为0.02％～0.08％,在紫外条件下从杯底照射；3)紫外照射完毕后先预冷,再冷冻,最后将其真空冷冻干燥,即得。(A preparation method of fibroin nano-fiber and a silver-loaded antibacterial dressing based on the fibroin nano-fiber. The invention discloses a preparation method of fibroin nano-fiber, which comprises the following steps: 1) degumming silkworm cocoons in a sodium carbonate solution to obtain silk fibroin; 2) dissolving silk fibroin in a sodium hydroxide/urea aqueous solution to obtain a dissolved solution; 3) placing the dialysis bag in a sodium bicarbonate/EDTA solution for boiling treatment, and then taking out the dialysis bag and placing the dialysis bag in the EDTA solution for boiling treatment; 4) pouring the solution obtained in the step 2) into a treated dialysis bag for dialysis, carrying out ultrasonic treatment, and carrying out refrigerated centrifugation to obtain the fibroin nanofiber aqueous solution. Also discloses a preparation method of the silver-loaded antibacterial dressing, which comprises the following steps: 1) preparing a fibroin nanofiber aqueous solution by adopting the method; 2) dissolving silver nitrate into the obtained fibroin nanofiber aqueous solution, wherein the mass volume ratio of the silver nitrate to the fibroin nanofiber aqueous solution is 0.02-0.08%, and irradiating from the bottom of a cup under an ultraviolet condition; 3) precooling and then freezing after the ultraviolet irradiation is finished, and finally, freezing and drying the mixture in vacuum to obtain the ultraviolet-curing.)

1. A preparation method of fibroin nanofiber is characterized by comprising the following steps:

1) cutting silkworm cocoon, cleaning, degumming in sodium carbonate solution, cleaning, and oven drying to obtain silk fibroin;

2) preparing a sodium hydroxide/urea aqueous solution from sodium hydroxide and urea, and dissolving the silk fibroin obtained in the step 1) in the sodium hydroxide/urea aqueous solution to obtain a dissolved solution;

3) preparing an EDTA solution and a sodium bicarbonate solution, mixing the sodium bicarbonate solution with part of the EDTA solution to obtain a sodium bicarbonate/EDTA solution, placing a dialysis bag in the sodium bicarbonate/EDTA solution for boiling treatment for 8-12 min, then taking out the dialysis bag, placing the dialysis bag in the EDTA solution for boiling treatment for 8-12 min, and sealing the solution for later use;

4) pouring the solution obtained in the step 2) into the dialysis bag treated in the step 3) for dialysis, taking out the dialysate after the dialysis is finished, performing ultrasonic treatment to remove the silk fibroin nanofiber ribbons only partially dissolved in the completely dissolved silk fibroin, and performing freezing centrifugation to obtain the silk fibroin nanofiber aqueous solution.

2. The preparation method of the silk fibroin nanofiber as claimed in claim 1, wherein in the step 2), the total mass of sodium hydroxide and urea is 12 g-36 g per 100g of sodium hydroxide/urea aqueous solution, the mass ratio of sodium hydroxide to urea is 0.19-1.38: 1, the silk fibroin obtained in the step 1) is placed in the sodium hydroxide/urea aqueous solution to be dissolved at-24 ℃ to-8 ℃ for 72-120 h to obtain a dissolved solution, and the mass ratio of the silk fibroin to the sodium hydroxide/urea aqueous solution is 2-6: 100.

3. The method for preparing the silk fibroin nanofibers according to claim 2, wherein in the step 2), the silk fibroin obtained in the step 1) is placed in a sodium hydroxide/urea aqueous solution for dissolution, and multiple times of stirring are performed to promote dissolution.

4. The method for preparing the silk fibroin nanofiber as claimed in claim 1, wherein the concentration of the EDTA solution prepared in the step 3) is 1mM, the concentration of sodium bicarbonate in the sodium bicarbonate solution is 0.02g/ml, and the EDTA solution and the sodium bicarbonate solution are mixed according to the volume ratio of 1: 0.8-1.2.

5. The method for preparing the silk fibroin nanofiber of claim 4, wherein the pH of the EDTA solution is 7.5-8.5.

6. The preparation method of the silk fibroin nanofiber as claimed in claim 1, wherein in the step 4), the dissolved solution obtained in the step 2) is poured into the dialysis bag treated in the step 3) for dialysis for 60-90 h, the power of ultrasonic treatment is 300W, the ultrasonic treatment is performed for 20-30 min, and the freezing centrifugation is performed for 20-30 min.

7. A preparation method of a silver-loaded antibacterial dressing based on fibroin nanofibers is characterized by comprising the following steps: 1) preparing an aqueous solution of silk fibroin nanofibers by the method of claim 1; 2) dissolving silver nitrate into the obtained fibroin nanofiber aqueous solution, wherein the mass volume ratio of the silver nitrate to the fibroin nanofiber aqueous solution is 0.02-0.08%, sealing a beaker, and irradiating from the bottom of the beaker under an ultraviolet condition to reduce silver ions in the solution into nano-silver particles; 3) precooling and then freezing after the ultraviolet irradiation is finished, and finally, freezing and drying the mixture in vacuum to obtain the ultraviolet-curing.

8. The preparation method of the silver-loaded antibacterial dressing based on the fibroin nanofibers according to claim 7, wherein the specific treatment method after the completion of ultraviolet irradiation in the step 3) is as follows: precooling at-24 to-18 ℃ for 2-4 h, freezing at-80 to-70 ℃ for 12-24 h, and finally freeze-drying in vacuum at the temperature of-5 to 0 ℃ under the pressure of 20 to 50pa for 30-40 h.

9. The preparation method of the silver-loaded antibacterial dressing based on the fibroin nanofibers according to claim 7, wherein the wavelength of ultraviolet irradiation in the step 2) is 254-365 nm, and the irradiation time is 4-6 h.

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a fibroin nanofiber and a preparation method of a silver-loaded antibacterial dressing based on the fibroin nanofiber.

Background

Silk fibroin (silk fibroin, abbreviated as SF) is a natural, biocompatible, biodegradable, low-cost polymer fibrin extracted from silk. SF in silkworm cocoon is 5263 amino acid residues consisting of glycine, alanine, serine, tyrosine and valine, and the other 15 amino acids account for only 4.7%. Due to its tunable mechanical properties and good physical structure, silk proteins are very attractive for many biomedical applications, including drug delivery, tissue engineering, implantable devices, biosensors and wound dressings.

The skin, the largest organ in the human body, not only protects our normal metabolism, but also plays an important role in protecting against ultraviolet radiation, chemicals, foreign organisms and other harmful environmental factors. However, wounds and burns often cause tissue necrosis and destroy the skin's defense function. Infection is one of the most common complications in burn wound care, and can lead to microbial invasion, impede regeneration and repair of epidermal tissue, delay wound healing, promote scarring, and even threaten the life safety of the patient. The traditional dressing has poor air permeability and biocompatibility and slow wound healing, so that a novel material is urgently needed to replace the traditional dressing. The silk fibroin is used as a natural high molecular polymer, has wide source, good biocompatibility, biodegradability and controllable degradation time, can promote wound healing, and is a good substitute.

Because the biocompatibility of the silk fibroin is good, the silk fibroin can be used for the fixed-point delivery of the drugs in vivo; the silk fibroin is made into gel, and the aim of controlling the drug release is achieved by controlling the strain capacity of the silk fibroin to the environment; such as pH, osmotic pressure, temperature, etc.; has great significance for clinical treatment. The silk fibroin is used as a natural high polymer with good biocompatibility and can be made into a medical bracket; biological molecules such as growth factors and the like can be added into the implant to promote the growth and healing of the cartilage; it can also be used as the substrate for cell growth, and corresponding cell growth factor is added for cell directed differentiation culture, such as vascular endothelial cells.

The two main proteins contained in silkworm cocoon are silk fibroin and sericin, which exist in a complex form and are wrapped by the sericin. It has been shown that the protein in this complex form produces an adaptive immune response in vivo, and that the remaining silk fibroin does not produce this immunogenicity when the sericin is removed. Therefore, the research is based on the technology of extracting and separating silk fibroin, and has practical significance for the application of the silk fibroin.

The traditional extraction method of silk fibroin comprises dissolving, dialyzing and centrifuging a solution of inorganic salt (lithium bromide, calcium chloride/ethanol/water), high-concentration acid and high-ion concentration to obtain a silk fibroin aqueous solution. However, this approach has the following drawbacks: the silk fibroin is excessively sheared by dissolution, most of original hydrogen bonds are cut off, and the performance of the regenerated silk fibroin is weaker than that of the original silk fibroin. Another common extraction method involves using hydrochloric acid/formic acid, formic acid/calcium chloride, formic acid/lithium bromide and HFIP (hexafluoroisopropanol) as solvents to obtain a silk fibroin solution. In the method, only a small amount of hydrogen bonds are destroyed, so that the fiber structure and the performance of the natural silk fibroin can be greatly protected, but the defects that the aqueous solution state cannot be kept in a formic acid/calcium chloride system and protein is separated out by adding water are overcome; in a formic acid/lithium bromide system, the obtained aqueous solution must be stored in an environment at 4 ℃ to avoid premature gelling; while HFIP is a toxic agent. In summary, the conventional extraction method has different limitations in different aspects, and therefore a new extraction method is needed.

Disclosure of Invention

Aiming at the problems, the invention provides a preparation method of fibroin nano-fiber, which comprises the following steps:

1) cutting silkworm cocoon, cleaning, degumming in sodium carbonate solution, cleaning, and oven drying to obtain silk fibroin;

2) preparing a sodium hydroxide/urea aqueous solution from sodium hydroxide and urea, and dissolving the silk fibroin obtained in the step 1) in the sodium hydroxide/urea aqueous solution to obtain a dissolved solution;

3) preparing an EDTA solution and a sodium bicarbonate solution, mixing the sodium bicarbonate solution with part of the EDTA solution to obtain a sodium bicarbonate/EDTA solution, placing a dialysis bag in the sodium bicarbonate/EDTA solution for boiling treatment for 8-12 min, then taking out the dialysis bag, placing the dialysis bag in the EDTA solution for boiling treatment for 8-12 min, and sealing the solution for later use;

4) pouring the solution obtained in the step 2) into the dialysis bag treated in the step 3) for dialysis, taking out the dialysate after the dialysis is finished, performing ultrasonic treatment to remove the silk fibroin nanofiber ribbons only partially dissolved in the completely dissolved silk fibroin, and performing freezing centrifugation to obtain the silk fibroin nanofiber aqueous solution.

In the step 2), the total mass of the sodium hydroxide and the urea is 12 g-36 g per 100g of the sodium hydroxide/urea aqueous solution, the mass ratio of the sodium hydroxide to the urea is 0.19-1.38: 1, the silk fibroin obtained in the step 1) is placed in the sodium hydroxide/urea aqueous solution to be dissolved for 72-120 h at-24 ℃ to-8 ℃ to obtain a dissolved solution, and the mass ratio of the silk fibroin to the sodium hydroxide/urea aqueous solution is 2-6: 100.

In the step 2), the silk fibroin obtained in the step 1) is placed in a sodium hydroxide/urea aqueous solution for dissolving, and is stirred for many times to promote dissolution.

The concentration of the EDTA solution prepared in the step 3) is 1mM, the mass fraction of sodium bicarbonate in the sodium bicarbonate solution is 2%, and the EDTA solution and the sodium bicarbonate solution are mixed according to the volume ratio of 1: 0.8-1.2.

The pH value of the EDTA solution is 7.5-8.5.

And in the step 4), the solution obtained in the step 2) is poured into the dialysis bag treated in the step 3) for dialysis for 60-90 hours, the power of ultrasonic treatment is 300W, the ultrasonic treatment lasts for 20-30 min, and the freezing and centrifuging time lasts for 20-30 min.

The invention also provides a preparation method of the silver-loaded antibacterial dressing based on the fibroin nanofiber, which comprises the following steps: 1) preparing a fibroin nanofiber aqueous solution by adopting the method; 2) dissolving silver nitrate into the obtained fibroin nanofiber aqueous solution, wherein the mass volume ratio of the silver nitrate to the fibroin nanofiber aqueous solution is 0.02-0.08%, sealing a beaker, and irradiating from the bottom of the beaker under an ultraviolet condition to reduce silver ions in the solution into nano-silver particles; 3) precooling and then freezing after the ultraviolet irradiation is finished, and finally, freezing and drying the mixture in vacuum to obtain the ultraviolet-curing.

In the above technical solution, the specific treatment method after the completion of the ultraviolet irradiation in step 3) is as follows: precooling at-24 to-18 ℃ for 2-4 h, freezing at-80 to-70 ℃ for 12-24 h, and finally freeze-drying in vacuum at the temperature of-5 to 0 ℃ under the pressure of 20 to 50pa for 30-40 h.

In the technical scheme, the wavelength of the ultraviolet radiation in the step 2) is 254-365 nm, and the irradiation time is 4-6 h.

The invention has the beneficial effects that:

the fibroin nano-fiber preparation method of the invention can not completely dissolve the fibroin, only destroys a small amount of original hydrogen bonds and Van der Waals force compared with the traditional extraction method, and can more reserve the initial structure and performance of the fibroin; so that the fibers in the solution are peeled into smaller fibroin nanofiber bands which are uniformly dispersed in the solution; and the fibroin nano-fiber solution can be kept for at least 15 days without gelling or coagulation after being placed at the normal temperature (25 ℃), and a milky white uniformly dispersed solution state is always kept. The method utilizes a sodium hydroxide/urea mixed solvent system to dissolve the silk fibroin, so that the structure and the characteristics of natural silk fibroin are greatly preserved on the premise that the silk fibroin nano-fiber can be modified, for example, the original beta-sheet content is greatly preserved by reducing the damage of hydrogen bonds, and the content of the beta-sheet is closely related to the mechanical property of the material.

The preparation method of the silver-loaded antibacterial dressing based on the fibroin nanofibers is simple in steps, mild in reaction conditions, simple and easy to operate, and suitable for large-scale batch production, and the prepared dressing not only has good biocompatibility, but also has good biodegradability, mechanical properties and antibacterial properties, and overcomes a series of rejection reactions such as inflammatory reaction, immune reaction and the like of the existing commercial dressing to wounds. The antibacterial dressing has excellent biodegradable performance of silk fibroin, and a series of problems of secondary damage and the like to patients when the dressing such as the existing commercial gauze is removed are solved. The nano silver particles loaded in the dressing have excellent antibacterial performance, the release is slow and lasting, the explosive release of the medicines in the common dressing is overcome, the dressing has a lasting antibacterial effect, and the dressing is more beneficial to wound healing. The silver-loaded antibacterial dressing prepared by the invention has obvious inhibition effect on the growth of gram-positive bacteria and gram-negative bacteria, and overcomes the problem of single antibacterial.

Drawings

Fig. 1 is a schematic diagram of a preparation method of the silk fibroin nanofiber of the present invention.

Fig. 2 is a comparison graph of the beta-sheet content of the fibroin nanofibers prepared by the method of the present invention and the conventional extraction method, wherein a is the method of the present invention, and b is the conventional extraction method.

Fig. 3 is a flow chart of the preparation method of the silver-loaded antibacterial dressing based on the fibroin nanofibers.

FIG. 4 is an electron microscope scanning image of the silver-loaded antimicrobial dressing based on the fibroin nanofiber prepared by the method of the present invention, wherein a is the concentration of the fibroin nanofiber as the raw material for preparation of 0.03g/mL, and b is the concentration of the fibroin nanofiber as the raw material for preparation of 0.06 g/mL.

Fig. 5 is a mechanical performance test chart of the silver-loaded antibacterial dressing based on silk fibroin nano-fibers prepared by the method of the invention compared with the dressing prepared by silk fibroin of the traditional extraction method.

Fig. 6 is a comparative degradation performance test diagram of the silver-loaded antimicrobial dressing prepared by the method of the present invention based on the fibroin nanofibers and the dressing prepared by the traditional extraction method based on the fibroin protein, wherein a is the concentration of the fibroin nanofiber as the raw material for preparation of 0.03g/mL, and b is the concentration of the fibroin nanofiber as the raw material for preparation of 0.06 g/mL.

Fig. 7 is the result of an antibacterial test using the silver-loaded antibacterial dressing of the present invention, in which a, b, and c are the inhibitory effects of the silver-loaded antibacterial dressing on the growth of escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus, respectively.

Fig. 8 is the result of a cell compatibility test using the silver-loaded antimicrobial dressing of the present invention.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

The experimental procedures in the following examples are conventional unless otherwise specified.