CN105862144A - Efficient preparing method for fibroin nano-fiber and application of fibroin nano-fiber - Google Patents

Abstract

The present invention aims at the shortcomings of ultrasonic preparation of silk fibroin nanofibers, and provides an improved high-efficiency preparation method of silk fibroin nanofibers. All silk fibers are immersed in the solution, the power is about 200W~1750W, the frequency is 5kHz~5MHz, the duration of ultrasound is 1~24 hours, and the interval of ultrasound is 3~60s and stop 1~10s. The temperature of the ultrasonic solution is controlled between 0°C and 90°C; the ultrasonic silk fiber is fixed on a square hollow or circular hollow plane fixture, and the vertical distance from the ultrasonic probe to the plane of the silk fiber is controlled to be 0.1 to 5cm , move horizontally the clamp where the ultrasonic silk fibroin fiber is located at a speed of 0-5 cm/Min, thereby preparing silk nanofiber material. The silk fibroin nanofiber prepared by the invention has good hydrophilicity, water vapor permeability and drug slow-release performance, and the invention protects its application in medical dressings.

Description

A kind of silk fibroin nanofiber efficient preparation method and its application

technical field

The invention relates to an improved high-efficiency preparation method and application of silk fibroin nanofibers, belonging to the technical field of biomedical materials.

Background technique

With the in-depth research on the structure and properties of silk and the development of new silk materials, the application of silk at home and abroad is expanding from the traditional textile field to tissue engineering, drug release, biomedicine, cosmetics, food and other fields. Silk fibroin nanofiber is a typical nano silk material. In addition to the excellent properties of natural silk fibre, it also has a larger specific surface area, high surface energy, small size effect, and surface effect.

At present, silk fibroin nanofibers are mainly prepared by electrospinning. This method requires a regenerated silk fibroin solution as a raw material. The preparation of the regenerated silk fibroin solution requires dissolving and dialysis processes. The process is complicated and cannot be mass-produced.

Ultrasound is a simple and efficient method for directly preparing silk nanofibers from silk fibers, which has the advantages of simple process flow and suitable for large-scale preparation (patent 200510086251.7). Since silk fiber, as a long fiber material, cannot do random Brownian motion in the solution, it is affected by the physical characteristics of ultrasonic waves, such as the directionality of sound wave transmission, the attenuation of sound waves with distance caused by reflection, refraction, scattering and absorption, etc. Ultrasonic treatment of silk fibroin fibers has a problem of inhomogeneity in which a part is split, while the other part is hardly affected, which results in uneven fiber splitting and extremely low yield of silk fibroin nanofibers prepared by ultrasonic method. In response to this problem, this patent optimizes the method of ultrasonically preparing silk fibroin nanofibers, and characterizes the properties of silk fibroin nanofibers prepared by the optimized process when applied to medical dressings.

Contents of the invention

The purpose of the present invention is to provide a method for efficiently preparing silk fibroin nanofibers to overcome the shortcomings and deficiencies of the prior art in order to solve the problems in the ultrasonic preparation of silk fibroin nanofibers.

The technical problem to be solved in the present invention, a method for efficiently preparing silk fibroin nanofibers, can be realized through the following technical solutions:

It is characterized in that a convex array or linear array probe is used, the ultrasonic probe and the silk fiber to be ultrasonicated are immersed in the solution, the power is about 200W~1750W, the frequency is 5kHz~5MHz, and the duration of ultrasound is 1~24 Hours, the ultrasonic interval time is 3-60s and stop for 1-10s. During the process, the temperature of the ultrasonic solution is controlled between 0°C and 90°C; the ultrasonic silk fiber is fixed on a square hollow or circular hollow plane fixture, Controlling the vertical distance from the ultrasonic probe to the plane where the silk fibers are located is 0.1-5 cm, moving the plane where the ultrasonic silk fibers are located horizontally at a speed of 0-5 cm/Min, thereby preparing silk nanofiber materials.

The silk fibroin fibers include silk fibroin as the main body to form fibers and silk fabric materials produced by spinning silkworms, tussah silkworms, celestial silkworms, wild silkworms and transgenic silkworms, and degumming cocoon shells.

The solution refers to any one or more of weak acid, weak base, pure water and ultrapure water, or any 2 to 3 of sodium carbonate, trisodium phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate mixture.

The ultrasonic power and frequency are to ensure that the prepared silk fibroin nanofibers have relatively uniform size and 90% diameter distribution within 300nm.

The purpose of fixing the silk fiber is to facilitate the control of the direction and distance of the ultrasonic material relative to the ultrasonic probe, so that the ultrasonic wave will continue to act on the silk fiber from a specific direction, so that the energy generated by the ultrasonic wave will be on the surface of the silk fiber in time and space. A superposition effect is formed to avoid the phenomenon of uneven fiber distribution existing on the surface of the ultrasonically treated silk fibroin fiber.

Said moving the silk fibroin fiber horizontally at a certain speed is to realize the large-scale preparation of the silk fibroin nanofiber technologically.

The present invention can realize the direct splitting of silk fibroin fibers with a diameter of micron scale from a single root into hundreds of silk fibroin nanofibers under the action of ultrasonic through fine control of ultrasonic process conditions.

The silk fibroin nanofiber prepared by the invention has good hydrophilicity, water vapor permeability and drug slow-release performance, and this patent protects its application in medical dressings.

Description of drawings

Fig. 1. Scanning electron micrograph of a single fiber after ultrasonic splitting under the condition that the ultrasonic conditions are not optimized;

Figure 2. The scanning electron microscope micrograph of the sample treated with the optimized ultrasonic method in this patent, a single silk fibroin fiber can be directly split into hundreds of nanofibers;

Fig. 3. Using the optimized ultrasonic treatment method in this patent, the statistical diagram of the diameter distribution interval of the prepared silk fibroin nanofibers;

Figure 4. In the case of unoptimized ultrasonic conditions, after ultrasonic, the surface fiber distribution of silk fibroin is not uniform;

Figure 5. The scanning electron microscope micrograph of the sample treated by the optimized ultrasonic method in this patent shows that the surface of the silk fiber fiber is uniform;

Figure 6. The scanning electron microscope micrograph of the sample treated by the optimized ultrasonic method in this patent shows that the surface of the silk fiber fiber is uniform;

Figure 7. Using the optimized ultrasonic treatment method in this patent, the statistical diagram of the diameter distribution interval of the prepared silk fibroin nanofibers;

Figure 8. The scanning electron microscope micrograph of the sample treated by the optimized ultrasonic method in this patent can realize the large-scale preparation of silk fibroin nanofibers;

Figure 9. Comparison of static contact angles between silk fibroin fibers and silk fibroin nanofibers measured by a droplet with a diameter of 2 μL;

Figure 10. The static contact angle comparison between silk fibroin fibers and silk fibroin nanofibers under the drop of 8 μL diameter;

Figure 11. Comparison of water vapor transmission properties of silk fibers, silk nanofibers and bacterial cellulose (BC-Foam);

Figure 12. Comparison of cumulative drug release (CRP) percentages between silk fibers and silk nanofibers.

detailed description

Example 1 Comparison of fiber splitting of a single fiber before and after optimization of ultrasonic conditions

(1) When the ultrasonic conditions are not optimized, the fiber splitting of a single silk fibroin fiber after ultrasonic treatment

Using the method of patent 200510086251.7, put the silk fiber into the ultrasonic solution, use 1250 W power, 20 KHz frequency, stop for 1 second after super 3 seconds, and the time is 15 minutes, ultrasonically treat the silk fiber, and ultrasonically treat the surface of a single silk fiber The result of fiber splitting is shown in the scanning electron microscope picture shown in Figure 1. The silk fibroin nanofibers fall off from the fiber surface layer by layer in the form of ribbons, which will easily cause the silk fibroin nanofibers that first entered the solution to dissolve into the solution under the action of ultrasonic waves. , with the extension of ultrasonic time, the yield of silk fibroin nanofibers prepared by ultrasonic is less than 1% and has no obvious increasing trend. It can be seen from Figure 1 that when the ultrasonic conditions are not optimized, the silk nanofibers are separated layer by layer from the surface of silk fibers, and the influence of ultrasonic waves on the separation of silk fibers (diameters between 10 and 30 microns) stays at within 1 micron of the surface.

(2) Using this patent to optimize the ultrasonic conditions, the fiber separation of a single silk fiber after ultrasonic treatment

Using the method of this patent, put the silk fiber into the ultrasonic solution, use a convex array probe, use 1250 W power, 20 KHz frequency, the ultrasonic duration is 15 minutes, and stop for 1 second after exceeding 3 seconds. During the process, the ultrasonic solution is controlled The temperature is 50°C, the ultrasonic silk fiber will be fixed on the circular hollow plane fixture, the vertical distance from the ultrasonic probe to the plane where the silk fiber is located is controlled to be 1cm, the silk fiber is ultrasonically treated, and the result of fiber splitting on the surface of a single silk fiber is scanned Electron microscope picture is shown in accompanying drawing 2. It can be seen from the figure that since the ultrasonic waves continue to act on the silk fibers from a specific direction, a single silk fiber can be directly split into hundreds of nanofibers, avoiding the uneven fiber distribution on the surface of the ultrasonically treated silk fiber phenomenon, making the yield of silk fibroin nanofibers higher than 50%. It can be seen from accompanying drawing 3 that about 99% of the prepared silk fibroin nanofibers have a diameter below 300 nm.

Example 2 Comparison of surface fiber distribution inhomogeneity of silk fibroin fiber before and after ultrasonic condition optimization

(1) Using the method of patent 200510086251.7, put the silk fiber into the ultrasonic solution, use 1750 W power, 20KHz frequency, stop for 1 second after super 3 seconds, and the time is 20 minutes, ultrasonically treat the silk fiber, and ultrasonically treat the silk fiber surface The result of fiber splitting is shown in the figure, and the surface of the ultrasonic silk fibroin fiber is shown in Figure 4. Due to the instantaneous burst of ultrasonic action and the different positions of silk fibroin fibers relative to the ultrasonic probe, the fibers affected by ultrasonic are prone to fiber splitting, while the remaining fibers seem to be unaffected by ultrasonic action. This kind of fiber splitting effect occurs in a part of the fibers, while the non-uniform fiber splitting phenomenon of another part of unaffected silk fibroin fibers seriously affects the yield of ultrasonically generated silk fibroin nanofibers. Within 20 minutes, the yield of silk fibroin nanofibers was only 1.2%.

(2) Using the method of this patent, put the silk fibroin fiber into the ultrasonic solution, use a convex array probe, use 1750 W power, 20 KHz frequency, the ultrasonic duration is 15 minutes, stop for 1 second after exceeding 3 seconds, and control during the process The temperature of the ultrasonic solution is 50°C, the ultrasonic silk fiber is fixed on a circular hollow plane fixture, the vertical distance from the ultrasonic probe to the plane where the silk fiber is located is controlled to be 0.5 cm, and the silk fiber is ultrasonically treated. Because the silk fiber is fixed, the ultrasonic wave continues to act on the silk fiber from a specific direction, and the surface of the ultrasonic fiber can realize the uniform distribution of nanofibers, avoiding the uneven fiber distribution existing on the surface of the ultrasonically treated silk fiber, making the silk The productive rate of silk nanofibers is higher than 50%. As shown in accompanying drawing 5, the scanning electron microscope images of silk nanofibers generated at magnifications of 100 times, 1000 times and 10000 times, 90% of the diameters of the obtained silk nanofibers are distributed below 300nm .

Example 3 Using the method of this patent, put the silk fiber into the mixed ultrasonic solution of sodium carbonate and trisodium phosphate for pretreatment for 5 hours, fix the ultrasonic silk fiber on the square hollow plane fixture, and control the ultrasonic probe to the silk fibroin The vertical distance of the plane where the fibers are located is 0.5cm. Ultrasonic treatment of silk fibroin fibers, using a convex array probe, with 500 W power and 20 KHz frequency, the duration of ultrasound is 15 minutes, and the ultrasonic solution temperature is controlled during the process. is 60°C. The yield of the obtained silk fibroin nanofibers is higher than 50%. As shown in accompanying drawings 6 and 7, the scanning electron micrographs of the generated silk fibroin nanofibers at magnifications of 100 times and 10000 times, the diameter distribution of the obtained silk fibroin nanofibers is about 99% below 300nm.

Example 4 Using the method of this patent, put the silk fiber into the mixed ultrasonic solution of sodium carbonate and trisodium phosphate, fix the ultrasonic silk fiber on a circular hollow plane fixture, and control the ultrasonic probe to where the silk fiber is located. The vertical distance of the plane is 0.5cm, and the plane where the ultrasonic silk fiber is located is moved horizontally at a speed of 0.2cm/Min. Using a convex array probe, with 500W power and 20KHz frequency, stop for 1 second after 4 seconds, and control the ultrasonic solution during the process. The temperature is 60° C., and the ultrasonic duration is 1 h, and a silk nanofiber cloth with a diameter of about 8 cm can be obtained (the area inside the black circle in FIG. 8B ).

Example 5 Comparison of hydrophilic and hydrophobic properties between silk nanofibers prepared by this method and silk fibers

Randomly cut out 5 pieces of samples, drop 5 drops of water with a diameter of 2 μl and 8 μl on different places on the surface of the medium, and after 5 seconds, the instrument automatically measures the angle formed by the contour of the water droplet in contact with the surface of the medium to determine the hydrophilic and hydrophobic properties of the surface of the medium. Left and Right represent the left and right contact angles θ1 and θ2 respectively. Generally, the angle greater than 150 degrees indicates that the surface of the medium is superhydrophobic, and the contact angle comparison is as follows. The contact angle is an important parameter to characterize the hydrophilicity of the surface of solid planar materials. The smaller the contact angle of the material surface, the better its hydrophilicity, and vice versa, the worse its hydrophilicity. Figure 9 and Figure 10 are the contact angle data of the droplets automatically taken by the system after the water droplets with a diameter of 2 μl and 8 μl touch the surface of the fiber for 1.0 s, respectively. It can be seen from the table that the water droplets with diameters of 2 μl and 8 μl are nearly circular on the surface of silk fibroblast after degumming, and the static contact angles are 140.7° and 135.8° respectively, showing hydrophobicity and belonging to hydrophobic materials. Water droplets with a diameter of 2 μl and 8 μl became flattened on the surface of silk fibroin nanofibers after ultrasonic treatment, and the static contact angles were 9.7° and 4.0° respectively, showing hydrophilicity and belonging to hydrophilic materials. Ultrasonic treatment makes the silk fiber split and become finer, and at the same time introduces a large number of hydroxyl groups on the surface of the fiber, which promotes the silk fiber from hydrophobic fiber to silk nanofiber with good hydrophilicity, effectively improving the silk fiber. biocompatibility.

Example 6 Comparison of water vapor permeability between silk nanofibers prepared by this method and silk fibers

Medical dressings need to have an appropriate water vapor transmission rate, that is, have a certain moisturizing performance in order to maintain the "moist environment" of the wound. If the water vapor transmission rate is too high, it will easily lead to the loss of wound exudate and excessive dehydration. If the water vapor transmission rate is too low, it will affect the normal metabolism of the body and lead to wound deterioration. At 35°C, the moisture volatilization of normal human skin is 240±12 g·m ^-2 ·d ^-1 , while the moisture volatilization of damaged skin is relatively large, depending on the degree of damage, from 279±26 g·m ^-2 ·d ^-1 The ideal water vapor transmission rate of recommended medical dressings ^{is between 2000 and 2500 g·m -2 ·} d ^-1 . Attached Figure 10 shows the water vapor transmission rate values of silk fibers and silk nanofibers. The results show that the average water vapor transmission rate of silk fibers is 1912.41 g·m ^-2 ·d ^-1 , and the water vapor transmission rate of silk nanofibers The average vapor transmission rate is 2133.74 g·m ^-2 ·d ^-1 . The water vapor transmission rate of silk fibroin nanofibers is within the range of ideal dressings, and has better moisture permeability than bacterial cellulose (BC-Foam in Figure 11).

Example 7 Comparison of drug sustained-release properties between silk nanofibers prepared by this method and silk fibers

The representative antibiotic amoxicillin was selected as a model drug, and the adsorption and sustained-release performance of the prepared nanofibers on amoxicillin was determined. The drug release curve is shown in the figure below. Fiber has better drug sustained release performance, and its 72-hour cumulative drug release percentage is between 8% and 16%.

Claims (4)

1. the method efficiently preparing fibroin nanofiber, it is characterised in that use convex battle array or linear array probe, ultrasonic probe and All being immersed in solution by ultrasonic fibroin fiber, power is about between 200W ~ 1750W, and frequency is 5 KHz～5 megahertzs, super The sound persistent period is 1～24 hour, and ultrasonic interval time stops 0～10s for surpassing 3～60s, during control by ultrasonic solution temperature Degree is between 0 DEG C～90 DEG C；To be fixed on by ultrasonic fibroin fiber on the level clamp of square hollow or circular hollow, control Ultrasonic probe is 0.1～5cm to the vertical dimension of fibroin fiber place plane, moves horizontally with the speed of 0～5cm/Min and is surpassed The fixture at sound fibroin fiber place, thus prepare fibroin nano-fiber material.

2. the fibroin fiber described in claim 1 includes that silkworm, Antherea pernyi Guerin-Meneville, giant silkworm, Bombyx mandarina Moore and transgenic silkworm are weaved silk, cocoond What case degumming post-treatment was made forms fiber-like material with fibroin albumen for main body.

3. the solution described in claim 1 refers to any one of weak acid, weak base, pure water and ultra-pure water or multiple, or Person is any 2 in sodium carbonate, tertiary sodium phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate～3 kind of mixed solution.

4. fibroin nanofiber prepared by the present invention has good hydrophilic, water vapo(u)r transmission energy and medicament slow release performance, This patent protects its application in terms of medical dressing.