Silk fibroin / chitosan-micron silver core-shell functional particle as well as preparation method and application thereof

Abstract

The invention discloses a silk fibroin / chitosan-micron silver core-shell functional particle as well as a preparation method and application thereof, and belongs to the technical field of biological composite materials. The preparation method comprises the following steps: putting silkworm cocoon silk into a sodium carbonate solution to prepare degummed fibroin; degummed fibroin is dissolved in lithium bromide, and silk fibroin is obtained through ultrasonic treatment, dilution, washing, centrifugation and freeze drying; the preparation method comprises the following steps: dissolving a mixture of silk fibroin and chitosan in an alkali solution, adding the mixture into a mixed solution in which silver nitrate and polyvinylpyrrolidone are dissolved, and fully reacting to obtain the functional composite particle with silk fibroin / chitosan shell micron silver. According to the prepared silk fibroin / chitosan shell micron silver composite particle, the diameter of the micron silver particle is 3.569 microns, after the micron silver particle is placed for three months, the size, the morphology and the core-shell structure of the outermost layer of the micron silver particle are almost unchanged, and high stability is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological composite materials, and particularly relates to a silk fibroin / chitosan-microsilver core-shell functional particle, a preparation method thereof and an application thereof. Background Art

[0002] Silk, as a natural multi-level structured biological material, has a precise spatial distribution of its chemical components: the outer layer is composed of 20%-30% sericin to form a protective matrix, the inner core is 70%-80% fibroin, and is accompanied by about 1%-3% pigment and carbohydrate impurities. The purified silk fibroin obtained by degumming treatment with sodium carbonate solution is a water-insoluble polymer formed by self-assembly of 18 amino acids through a β-sheet layered structure.

[0003] Silk fibroin has good biocompatibility, degradability, mechanical stability and optical properties, and also has good processability. It can be processed into various forms such as films, scaffolds, gels, etc., and has been widely used in the field of tissue repair. Silk fibroin exhibits multiple advantages: firstly, it has biodegradability and low immune rejection with human tissues; secondly, excellent mechanical stability and processability support its transformation into various forms such as films, three-dimensional scaffolds, hydrogels, etc.; thirdly, its optical properties and structural tunability enable its in-depth application in the fields of tissue repair (such as wound suture, bone regeneration engineering) and artificial tendon construction. In biomedicine, although silk fibroin has advantages such as good biocompatibility, it is relatively lacking in antibacterial properties.

[0004] Chitosan is a natural polymer compound with various excellent properties. In terms of stability, the functional groups such as amino and hydroxyl groups in its molecular structure interact with each other to form a relatively stable structure, enabling it to maintain its own properties under certain conditions, which is beneficial to maintaining the properties of materials based on chitosan and ensuring the product quality and application effect. In terms of biocompatibility, the chemical structure of chitosan is similar to some polysaccharide components in human tissues, and it can interact with various cells in the organism without causing obvious immune rejection reactions, which lays a foundation for its applications in the field of biomedicine such as drug carriers and tissue engineering scaffolds, and can better integrate into the organism to play a role.

[0005] Due to their special physical and chemical properties, micron silver particles have broad application prospects in the fields of optoelectronics, biosensors, drug delivery, information storage, industrial catalysis, biological labeling, molecular recognition, and medicine. However, the application of micron silver still faces certain limitations. The preparation process is complex and costly. Micron silver prepared by traditional physical gas-phase methods has problems such as irregular sizes and expensive equipment. Under the same mass, compared with nano silver, micron silver has a smaller specific surface area and a slower silver ion release rate, and may require a higher concentration to achieve the same antibacterial effect, increasing the material usage and potential cost. Summary of the Invention

[0006] To solve the problems of existing materials, the purpose of the present invention is to provide a silk fibroin / chitosan-micron silver core-shell functional particle, a preparation method, and an application thereof; in order to obtain micron silver particles with excellent performance, after compounding micron silver with a composite material of silk fibroin / chitosan, the biocompatibility of the protein matrix, the stability and antibacterial properties of chitosan, and the antibacterial properties of silver itself can be combined to develop an intelligent dressing with the function of promoting tissue repair.

[0007] To achieve the purpose of the present invention, the specific solutions of the present invention are as follows:

[0008] (1) Remove the pupae from the silkworm cocoons, wash them, and peel them into sheet-like shapes. Place them in a sodium carbonate (Na2CO3) solution and stir. After taking out the silk that has become filamentous and centrifuging it to dry, continue to place it in the sodium carbonate solution, and repeat the above operation until there is no obvious sheet-like silk. Thoroughly wash the filamentous silk with deionized water, and place it in an oven to dry to obtain degummed silk fibroin.

[0009] (2) Place the degummed silk fibroin obtained in step (1) in a lithium bromide (LiBr) solution and perform ultrasonic treatment. After the degummed silk fibroin is fully dissolved and dispersed in the solution, dilute the solution with deionized water and then centrifuge and wash it. Finally, freeze-dry it to obtain silk fibroin.

[0010] (3) Grind and compound the silk fibroin obtained in step (2) with chitosan, then dissolve it with deionized water, adjust the pH value of the solution with a sodium hydroxide (NaOH) solution, and stir to obtain an aqueous solution of silk fibroin / chitosan.

[0011] (4) Dissolve silver nitrate (AgNO3) in deionized water to obtain an aqueous solution of silver nitrate.

[0012] (5) Dissolve polyvinylpyrrolidone (PVP) in the aqueous solution of silver nitrate obtained in step (3) to obtain an aqueous solution of polyvinylpyrrolidone-silver nitrate.

[0013] (6) Drop the aqueous solution of silk fibroin / chitosan obtained in step (5) into the aqueous solution of PVP-AgNO3 obtained in step (4). After fully reacting at room temperature, composite particles with a silk fibroin / chitosan outer shell and micron silver are prepared.

[0014] Preferably, the addition amount of silkworm cocoons in the sodium carbonate solution in step (1) is 0.2 g / mL, and the concentration of sodium carbonate is 0.5-0.8%; the stirring temperature is 50-100 °C, and the stirring time is 20-40 min; the oven temperature is 60-80, and the drying time is 2-4 h.

[0015] Preferably, in step (2), the concentration of lithium bromide is 8-10 mol / L, and the addition amount of degummed silk fibroin in lithium bromide is 0.12 g / mL; the ultrasonic temperature is 30-50 °C, and the ultrasonic time is 1-3 h; the centrifugation conditions are 6000-8000 rpm, and the centrifugation time is 30-50 min, and the freeze-drying temperature is -60 to -40 °C, and the freeze-drying time is 36-60 h.

[0016] Preferably, in step (3), the dosage of chitosan is 0.004-0.035 g / mL, the dosage of silk fibroin is 0.04-0.045 g / mL, and the grinding time is 1 h-3 h; the dissolution temperature is 30-60 °C, and the dissolution time is 1-3 h; the pH value of the mixed solution is adjusted to 9-11 with sodium hydroxide.

[0017] Preferably, in step (4), the concentration of the silver nitrate solution is 0.02-0.07 g / mL.

[0018] Preferably, in step (5), the dosage of polyvinylpyrrolidone is 0.002-0.007 g / mL.

[0019] Preferably, in step (6), the silk fibroin and the polyvinylpyrrolidone-silver nitrate mixed solution are mixed at a volume ratio of 1:4-1:1; the reaction time is 30-50 min, and the reaction condition is room temperature; centrifugally wash until the pH of the supernatant is 7, and the centrifugation conditions are 6000-8000 rpm, and the centrifugation time is 30-50 min.

[0020] In the present invention, the tyrosine residues in the purified silk fibroin obtained after degumming treatment with sodium carbonate solution contain active phenolic hydroxyl groups, which can reduce Ag+ to micro-nano particles through redox reactions and stably load them on the protein network by means of hydrogen bonds and van der Waals forces. This in-situ synthesis technology avoids the toxic residue of chemical reducing agents.

[0021] The silk fibroin / chitosan-micron silver core-shell functional particles prepared in the present invention have a core-shell structure, and the outermost layer of micron silver is wrapped by silk fibroin and chitosan. This invention can be applied in the field of biomedicine.

[0022] Compared with the prior art, what are the advantages of the present invention:

[0023] (1) By optimizing the degumming and purification processes and abandoning the traditional dialysis process, the present invention realizes the intensification of the operation process, effectively shortens the production cycle and reduces the energy consumption cost. The obtained silk fibroin forms a porous solid material after freeze-drying, and can maintain the structural integrity for about 12 months or more under the conventional room temperature environment, breaking through the technical limitation that traditional silk fibroin needs to be stored at low temperature. More importantly, the surface of this material is rich in phenolic hydroxyl groups of tyrosine residues, which can directly act as a green reducing agent to undergo an oxidation-reduction reaction with silver nitrate in an alkaline environment.

[0024] (2) The present invention prepares micron silver particles with controllable sizes. According to the crystal nucleation and growth kinetics regulation, by controlling the silver nitrate concentration gradient, the dosage of the dispersant and the reaction temperature, the particle size of the micron silver particles can be accurately regulated within the range of 1.5 - 5.8 μm; within a placement time of up to three months, the size, morphology and the outermost core-shell structure of the micron silver particles can be kept almost unchanged, with high stability, which is beneficial for the micron-sized carrier to achieve lesion targeting through surface modification, such as tumor tissues or inflammatory sites, reducing the side effects on healthy tissues, and can realize the gradient release of drugs or the intelligent release in response to the environment (pH, temperature, etc.), prolonging the curative effect. This technical system provides a new strategy for the development of low-cost and high-stability antibacterial biological composite materials.

[0025] (3) The present invention prepares micron silver particles with excellent and stable performance. In the preparation method of the core-shell functional particles, a grinding mixture of silk fibroin and chitosan is added to ensure the stability, antibacterial property and biocompatibility of the micron silver core-shell functional particles; by introducing polyvinylpyrrolidone (PVP), a micron silver system with certain dispersibility and silk fibroin / chitosan attached to the surface is successfully constructed. The amide groups in the PVP molecular chain are selectively adsorbed on the Ag(111) crystal plane, effectively inhibiting the particle agglomeration phenomenon. At the same time, the three-dimensional steric hindrance protection layer formed by PVP can significantly delay the oxidation release rate of silver ions, while maintaining the long-term antibacterial activity, ensuring the interfacial stability of the core-shell structure.

[0026] (4) In the composite system constructed by the present invention, the bioactivity of silk fibroin, the stability and antibacterial property of chitosan are perfectly integrated with the structural function of micro silver, providing a new solution with both controllability and stability for the development of biomedical materials such as wound repair scaffolds and antibacterial bone cements; the antibacterial property of micro silver combined with the cell adhesion-promoting property of silk fibroin can avoid the rejection reaction caused by the combination of metal and the human body, and can simultaneously achieve anti-infection, promote wound healing (such as burn dressings) or functionalize the surface of implants; after introducing chitosan, the negatively charged surface of the bacterial cell membrane and the positive charge of chitosan are strongly combined through electrostatic interaction, prompting chitosan to insert into the cell membrane and destroy the integrity of the membrane structure, resulting in the leakage of intracellular substances and ultimately leading to the death of bacteria, enhancing the antibacterial property of silver particles. Description of the Drawings

[0027] Figure 1 Scanning electron microscope image of the silk fibroin / chitosan-micro silver composite particles prepared in Example 1.

[0028] Figure 2 Scanning electron microscope image of the silk fibroin / chitosan-micro silver composite particles prepared in Example 2.

[0029] Figure 3 Scanning electron microscope image of the silk fibroin / chitosan-micro silver composite particles prepared in Example 3.

[0030] Figure 4 Scanning electron microscope image of the silver particles prepared in Comparative Example 1.

[0031] Figure 5 Scanning electron microscope image of the silver particles prepared in Comparative Example 2.

[0032] Figure 6 Scanning electron microscope image of the silver particles prepared in Comparative Example 3.

[0033] Figure 7 Scanning electron microscope image of the silver particles prepared in Comparative Example 4.

[0034] Figure 8 Scanning electron microscope image of the silver particles prepared in Comparative Example 5.

[0035] Figure 9 Scanning electron microscope image of the silver particles prepared in Comparative Example 6. Detailed Embodiments

[0036] The present invention will be further described in detail below with reference to the drawings and specific embodiments, but the protection scope of the present invention is not limited to the content described.

[0037] Example 1

[0038] A preparation method of silk fibroin / chitosan-micron silver core-shell functional particles specifically includes the following steps:

[0039] (1) Remove the pupae from 16 g of silkworm cocoons and peel them into several layers. Put them into 80 mL of a sodium carbonate solution with a concentration of 0.5%, stir at 50 °C for 20 min. Take out the silk that has become filamentous and spin-dry it, then put it back into the sodium carbonate solution. Repeat the above operation until there is no obvious flaky silk. Wash the filamentous silk thoroughly with deionized water, put it into an oven at 60 °C for 1 h, and obtain degummed silk fibroin after drying.

[0040] (2) Place 5 g of the degummed silk fibroin obtained in step (1) in an 8.0 mol / L lithium bromide solution, and ultrasonicate at 50 °C for 1 h. After the degummed silk fibroin is fully dissolved and dispersed in the solution, dilute the solution with deionized water and centrifuge and wash for 30 min, where the centrifugation speed is 8000 rpm. Finally, obtain silk fibroin through freeze-drying at -60 °C for 36 h.

[0041] (3) Dissolve 0.2 g of AgNO3 in 10 mL of deionized water to obtain an AgNO3 solution.

[0042] (4) Dissolve 0.02 g of PVP in the AgNO3 solution obtained in step (3) to obtain an aqueous PVP-AgNO3 solution.

[0043] (5) Manually grind 0.2 g of the silk fibroin obtained in step (2) and 0.02 g of chitosan for 1 h, then dissolve it with deionized water, stir at 40 °C for 2 h, and adjust the pH value of the solution to 11 with a 1 mol / L sodium hydroxide solution to obtain an aqueous solution of silk fibroin / chitosan.

[0044] (6) Drop the aqueous solution of silk fibroin / chitosan obtained in step (5) into the PVP-AgNO3 solution in step (4). After reacting at room temperature for 30 min, centrifuge and wash the precipitate with deionized water and absolute ethanol for 30 min each until the pH of the upper layer liquid is 7, where the centrifugation speed with deionized water is 8000 rpm and the centrifugation speed with absolute ethanol is 8000 rpm, to obtain micron silver particles coated with silk fibroin / chitosan.

[0045] Dilute and disperse the obtained silk fibroin / chitosan-micron silver core-shell functional particles on a silicon wafer and observe their surface morphology through a scanning electron microscope. As Figure 1 can be seen, it can be observed that the prepared product is irregular micron silver flakes with silk fibroin / chitosan.

[0046] Example 2

[0047] A preparation method of silk fibroin / chitosan-micron silver composite particles with a core-shell structure specifically includes the following steps:

[0048] (1) Remove the pupae from 16 g of silkworm cocoons and peel them into several layers. Place them in 80 mL of a 0.6% sodium carbonate solution, stir at 100 °C for 30 min. After taking out the silk that has become filamentous and spinning it dry, put it back into the sodium carbonate solution and repeat the above operation until there is no obvious flaky silk. Wash the filamentous silk thoroughly with deionized water, place it in an oven at 70 °C for 1 h, and obtain degummed silk fibroin after drying.

[0049] (2) Place 5 g of the degummed silk fibroin obtained in step (1) in a 9.0 mol / L lithium bromide solution, sonicate at 40 °C for 2 h. After the degummed silk fibroin is fully dissolved and dispersed in the solution, dilute the solution with deionized water and centrifuge and wash for 40 min, with a centrifuge speed of 7000 rpm. Finally, obtain silk fibroin protein by freeze-drying at -50 °C for 44 h.

[0050] (3) Dissolve 0.4 g of AgNO3 in 10 mL of deionized water to obtain an AgNO3 solution.

[0051] (4) Dissolve 0.04 g of PVP in the AgNO3 solution obtained in step (3) to obtain an aqueous PVP-AgNO3 solution.

[0052] (5) Manually grind 0.4 g of the silk fibroin protein obtained in step (2) and 0.04 g of chitosan for 2 h, then dissolve it with deionized water, stir at 30 °C for 3 h, and adjust the pH value of the solution to 9 with 1 mol / L sodium hydroxide solution to obtain an aqueous solution of silk fibroin protein / chitosan.

[0053] (6) Drop the aqueous solution of silk fibroin protein / chitosan obtained in step (5) into the PVP-AgNO3 solution in step (4). After reacting at room temperature for 30 min, centrifuge and wash the precipitate with deionized water and absolute ethanol for 40 min each until the pH of the upper layer liquid is 7, with a centrifuge speed of 7000 rpm for deionized water and 7000 rpm for absolute ethanol, to obtain micron silver particles coated with silk fibroin protein / chitosan.

[0054] Dilute and disperse the obtained silk fibroin protein / chitosan - micron silver core-shell functional particles on a silicon wafer and observe their surface morphology through a scanning electron microscope. After testing, it can be seen from Figure 2 that the size of the formed micron silver particles is about 3.569 μm, and the outer layer is evenly coated with silk fibroin protein, becoming monodisperse micron silver core-shell functional particles with a size of about 3.569 μm and having silk fibroin protein / chitosan.

[0055] The prepared micron silver core-shell functional particles of silk fibroin / chitosan in this example use silk fibroin dissolved at 30 °C. If dissolved at a higher temperature, it is extremely easy to cause protein denaturation. If dissolved at room temperature, the silk fibroin is not completely dissolved, which will affect the reduction effect of silk fibroin. At the same time, both a small amount and an excessive amount of PVP will cause aggregation and uneven morphology of micron silver particles. In addition, silk fibroin has a higher solubility in alkaline solutions with a higher pH value, while chitosan has a lower solubility in alkaline solutions with a higher pH value. If in a higher alkaline environment, a certain amount of the protein structure of silk fibroin will be damaged and it is difficult for chitosan to be completely dissolved.

[0056] Example 3

[0057] A preparation method of silk fibroin / chitosan-micron silver composite particles with a core-shell structure specifically includes the following steps:

[0058] (1) Remove the pupae from 16 g of silkworm cocoons and peel them into several layers. Put them into 80 mL of a 0.8% sodium carbonate solution and stir at 75 °C for 40 min. Take out the formed silk and spin it dry, then put it back into the sodium carbonate solution and repeat the above operation until there is no obvious flaky silk. Wash the silk filaments thoroughly with deionized water and put them into an oven at 80 °C for 2 h. After drying, degummed silk fibroin is obtained.

[0059] (2) Place 5 g of the degummed silk fibroin obtained in step (1) into a 10.0 mol / L lithium bromide solution and ultrasonicate at 30 °C for 3 h. After the degummed silk fibroin is fully dissolved and dispersed in the solution, dilute the solution with deionized water and centrifuge and wash for 50 min, where the centrifugation speed is 6000 rpm. Finally, freeze-dry at -40 °C for 60 h to obtain silk fibroin.

[0060] (3) Dissolve 0.7 g of AgNO3 in 10 mL of deionized water to obtain an AgNO3 solution.

[0061] (4) Dissolve 0.07 g of PVP in the AgNO3 solution obtained in step (3) to obtain an aqueous PVP-AgNO3 solution.

[0062] (5) Manually grind 0.7 g of the silk fibroin obtained in step (2) and 0.07 g of chitosan for 3 h, then dissolve them with deionized water, stir at 50 °C for 1 h, and adjust the pH value of the solution to 10 with a 1 mol / L sodium hydroxide solution to obtain an aqueous solution of silk fibroin / chitosan.

[0063] (6) The aqueous solution of silk fibroin / chitosan obtained in step (5) was dropped into the PVP-AgNO3 solution in step (4). After reacting at room temperature for 30 min, the precipitate was centrifugally washed with deionized water and absolute ethanol for 50 min in sequence until the pH of the upper layer solution was 7. The centrifugation speed for deionized water was 6000 rpm, and the centrifugation speed for absolute ethanol was 6000 rpm, obtaining micron silver particles coated with silk fibroin / chitosan.

[0064] The obtained silk fibroin / chitosan-micron silver core-shell functional particles were diluted and dispersed on a silicon wafer, and their surface morphology was observed by scanning electron microscopy. After detection, it was Figure 3 found that micron silver core-shell functional particles with silk fibroin / chitosan were prepared.

[0065] Comparative Example 1

[0066] The difference between this example and Example 2 is that PVP was not added during the particle preparation reaction. The specific steps are as follows:

[0067] (1) 16 g of silkworm cocoons were depupated and peeled into several layers, and placed in 80 mL of a 0.6% sodium carbonate solution. Stirred at 100 °C for 30 min. After the silk that had become filamentous was taken out and spun dry, it was continued to be placed in the sodium carbonate solution, and the above operation was repeated until there was no obvious flaky silk. The filamentous silk was thoroughly washed with deionized water and placed in an oven at 70 °C for 1 h. After drying, degummed silk fibroin was obtained.

[0068] (2) 5 g of the degummed silk fibroin obtained in step (1) was placed in a 9.0 mol / L lithium bromide solution and ultrasonicated at 40 °C for 2 h. After the degummed silk fibroin was fully dissolved and dispersed in the solution, the solution was diluted with deionized water and centrifugally washed for 40 min, with a centrifugation speed of 7000 rpm. Finally, freeze-dried at -50 °C for 44 h to obtain silk fibroin.

[0069] (3) 0.4 g of AgNO3 was dissolved in 10 mL of deionized water to obtain an AgNO3 solution.

[0070] (4) 0.4 g of the silk fibroin obtained in step (2) and 0.04 g of chitosan were manually ground for 2 h, then dissolved with deionized water, stirred at 30 °C for 3 h, and the pH value of the solution was adjusted to 9 with 1 mol / L sodium hydroxide solution to obtain an aqueous solution of silk fibroin / chitosan.

[0071] (5) The aqueous solution of silk fibroin / chitosan obtained in step (4) was dropped into the AgNO₃ solution obtained in step (3). After reacting at room temperature for 30 min, the precipitate was centrifugally washed successively with deionized water and absolute ethanol for 40 min until the pH of the upper layer solution was 7. The centrifugation speed with deionized water was 7000 rpm, and the centrifugation speed with absolute ethanol was 7000 rpm, obtaining micron silver particles coated with silk fibroin / chitosan.

[0072] The obtained silk fibroin / chitosan - micron silver core - shell functional particles were diluted and dispersed on a silicon wafer, and their surface morphology was observed by scanning electron microscopy. After detection, Figure 4 it can be seen that the prepared product is micron silver core - shell functional particles with severe agglomeration of silk fibroin / chitosan, and the dispersion performance is not as good as that of Example 2. The reason is that PVP was not added during the reaction process. The amide groups in the PVP molecular chain cannot selectively adsorb on the Ag(111) crystal plane, and thus cannot effectively inhibit the particle agglomeration phenomenon and play a dispersing role, making the prepared particles have monodispersity. The micron particles prepared without a dispersant will agglomerate.

[0073] Comparative Example 2

[0074] The difference between this example and Example 2 is that silk fibroin was not added, but ascorbic acid was used as a reducing agent. The specific steps are as follows:

[0075] (1) 0.4 g of AgNO₃ was dissolved in 10 mL of deionized water to obtain an AgNO₃ solution.

[0076] (2) 0.04 g of PVP was dissolved in the AgNO₃ solution obtained in step (1) to obtain a PVP - AgNO₃ aqueous solution.

[0077] (3) 0.4 g of ascorbic acid obtained in step (2) and 0.04 g of chitosan were manually ground for 2 h, and then dissolved with deionized water. After stirring at 30 °C for 3 h, the pH value of the solution was adjusted to 9 with 1 mol / L sodium hydroxide solution to obtain an ascorbic acid / chitosan aqueous solution.

[0078] (4) The ascorbic acid / chitosan aqueous solution obtained in step (3) was dropped into the PVP - AgNO₃ solution in step (2). After reacting at room temperature for 30 min, the precipitate was centrifugally washed successively with deionized water and absolute ethanol for 40 min until the pH of the upper layer solution was 7. The centrifugation speed with deionized water was 7000 rpm, and the centrifugation speed with absolute ethanol was 7000 rpm.

[0079] The obtained chitosan - micron silver core - shell functional particles were diluted and dispersed on a silicon wafer, and their surface morphology was observed by scanning electron microscopy. After detection,Figure 5 It can be seen that the prepared product has a smaller particle size than that in Example 2. The reason is that the ascorbic acid added during the reaction has a stronger reducibility than silk fibroin, and its reduction rate is relatively fast. The particles do not have enough time to grow, so the reduced particles have a smaller particle size. At the same time, compared with the micron silver particles prepared by silk fibroin, the micron silver particles reduced by ascorbic acid are only coated with chitosan and do not have the coating of silk fibroin on the outer layer. Therefore, they are not as widely used in the medical and biological fields as the particles prepared with the addition of silk fibroin.

[0080] Comparative Example 3

[0081] The difference between this example and Example 2 is that in step (5), the silk fibroin / chitosan was not dissolved with an alkali solution. The specific steps are as follows:

[0082] (1) Remove the pupae from 16 g of silkworm cocoons and peel them into several layers. Put them into 80 mL of a sodium carbonate solution with a concentration of 0.6%, stir at 100 °C for 30 min. Take out the formed silk threads and spin-dry them, then put them back into the sodium carbonate solution. Repeat the above operation until there is no obvious flaky silk. Wash the silk threads thoroughly with deionized water, put them into an oven at 70 °C for 1 h, and obtain degummed silk fibroin after drying.

[0083] (2) Place 5 g of the degummed silk fibroin obtained in step (1) into a 9.0 mol / L lithium bromide solution, and ultrasonicate it at 40 °C for 2 h. After the degummed silk fibroin is fully dissolved and dispersed in the solution, dilute the solution with deionized water and centrifuge and wash it for 40 min, with a centrifugation speed of 7000 rpm. Finally, obtain silk fibroin by freeze-drying at -50 °C for 44 h.

[0084] (3) Dissolve 0.4 g of AgNO3 in 10 mL of deionized water to obtain an AgNO3 solution.

[0085] (4) Dissolve 0.04 g of PVP in the AgNO3 solution obtained in step (3) to obtain a PVP-AgNO3 aqueous solution.

[0086] (5) Manually grind 0.4 g of the silk fibroin obtained in step (2) and 0.04 g of chitosan for 2 h, then dissolve them with deionized water and stir at 30 °C for 3 h to obtain an aqueous solution of silk fibroin / chitosan.

[0087] (6) Drop the aqueous solution of silk fibroin / chitosan obtained in step (5) into the PVP-AgNO3 solution in step (4). After reacting at room temperature for 30 min, centrifuge and wash the precipitate with deionized water and absolute ethanol for 40 min in sequence until the pH of the upper layer liquid is 7, with a centrifugation speed of 7000 rpm for deionized water and 7000 rpm for absolute ethanol.

[0088] The centrifuged solution was diluted and dispersed on a silicon wafer, and its surface morphology was observed by scanning electron microscopy. After detection, it was known from Figure 6 that a mixture of AgNO3 particles and trace amounts of unstable micro-silver core-shell functional particles with silk fibroin / chitosan was obtained. The reason is that the PVP-AgNO3 solution is an acidic solution. Silk fibroin is difficult to dissolve in an acidic environment, while chitosan is easily soluble in an acidic solution. And in an acidic environment, silk fibroin will undergo ion exchange, that is, the negative charges on the surface of silk fibroin molecules attract the positive charges of ions to form a colloidal structure. PVP has a certain reducing property, but due to the short reaction time, its reducing property cannot be fully exerted. Therefore, a mixture of unreacted AgNO3 particles and trace amounts of unstable micro-silver core-shell functional particles with silk fibroin / chitosan was obtained.

[0089] Comparative Example 4

[0090] The difference between this example and Example 2 is that the solutions in steps (3) and (4) were not combined. The specific steps are as follows:

[0091] A preparation method of silk fibroin / chitosan-micro silver composite particles with a core-shell structure specifically includes the following steps:

[0092] (1) Remove the pupae from 16 g of silkworm cocoons and peel them into several layers. Put them into 80 mL of a 0.6% sodium carbonate solution and stir at 100 °C for 30 min. Take out the formed silk threads and spin them dry, then put them back into the sodium carbonate solution. Repeat the above operation until there is no obvious flaky silk. Wash the silk threads thoroughly with deionized water and put them into an oven at 70 °C for 1 h. After drying, degummed silk fibroin is obtained.

[0093] (2) Place 5 g of the degummed silk fibroin obtained in step (1) in a 9.0 mol / L lithium bromide solution and sonicate at 40 °C for 2 h. After the degummed silk fibroin is fully dissolved and dispersed in the solution, dilute the solution with deionized water and centrifuge and wash for 40 min, where the centrifugation speed is 7000 rpm. Finally, freeze-dry at -50 °C for 44 h to obtain silk fibroin.

[0094] (3) Dissolve 0.4 g of AgNO3 in 10 mL of deionized water to obtain an AgNO3 solution.

[0095] (4) Dissolve 0.04 g of PVP in 2.5 mL of deionized water to obtain a PVP aqueous solution.

[0096] (5) Manually grind 0.4 g of the silk fibroin obtained in step (2) and 0.04 g of chitosan for 2 h, then dissolve them with deionized water, stir at 30 °C for 3 h, and adjust the pH value of the solution to 9 with 1 mol / L sodium hydroxide solution to obtain an aqueous solution of silk fibroin / chitosan.

[0097] (6) The PVP aqueous solution obtained in step (4) was dropped into the AgNO3 solution obtained in step (3), and then immediately the aqueous solution of silk fibroin / chitosan obtained in step (5) was dropped into the PVP-AgNO3 solution in step (4). After reacting at room temperature for 30 min, the precipitate was centrifugally washed with deionized water and absolute ethanol for 40 min in sequence until the pH of the supernatant was 7. The centrifugal speed for deionized water was 7000 rpm, and the centrifugal speed for absolute ethanol was 7000 rpm, obtaining micron silver particles coated with silk fibroin / chitosan.

[0098] The obtained silk fibroin / chitosan-micron silver core-shell functional particles were diluted and dispersed on a silicon wafer, and their surface morphology was observed by scanning electron microscopy. After detection, Figure 7 it can be seen that the prepared product is micron silver core-shell functional particles with serious aggregation of silk fibroin / chitosan, and the dispersion performance is not as good as that of Example 2. Although PVP was added during the reaction process, the addition time of PVP was too late, and the PVP molecules had no time to disperse, so the aggregation of particles could not be effectively inhibited. Therefore, the prepared micron particles would aggregate.

[0099] Comparative Example 5

[0100] The difference between this example and Example 2 is that the reaction temperature in step (6) is 50 °C, and the specific steps are as follows:

[0101] A preparation method of silk fibroin / chitosan-micron silver composite particles with a core-shell structure specifically includes the following steps:

[0102] (1) 16 g of silkworm cocoons were depupated and peeled into several layers, and put into 80 mL of a sodium carbonate solution with a concentration of 0.6%. Stir at 100 °C for 30 min. After taking out the formed silk filaments and spinning them dry, put them back into the sodium carbonate solution, and repeat the above operation until there is no obvious flaky silk. The silk filaments were thoroughly washed with deionized water and put into an oven at 70 °C for 1 h. After drying, degummed silk fibroin was obtained.

[0103] (2) 5 g of the degummed silk fibroin obtained in step (1) was placed in a 9.0 mol / L lithium bromide solution and ultrasonicated at 40 °C for 2 h. After the degummed silk fibroin was fully dissolved and dispersed in the solution, the solution was diluted with deionized water and centrifugally washed for 40 min, with a centrifugal speed of 7000 rpm. Finally, freeze-drying was carried out at -50 °C for 44 h to obtain silk fibroin.

[0104] (3) 0.4 g of AgNO3 was dissolved in 10 mL of deionized water to obtain an AgNO3 solution.

[0105] (4) Dissolve 0.04 g of PVP into the AgNO3 solution obtained in step (3) to obtain an aqueous PVP-AgNO3 solution.

[0106] (5) Manually grind 0.4 g of the silk fibroin obtained in step (2) and 0.04 g of chitosan for 2 h, then dissolve them in deionized water, stir at 30 °C for 3 h, and adjust the pH value of the solution to 9 with 1 mol / L sodium hydroxide solution to obtain an aqueous solution of silk fibroin / chitosan.

[0107] (6) Drop the aqueous solution of silk fibroin / chitosan obtained in step (5) into the PVP-AgNO3 solution in step (4). After reacting at 50 °C for 30 min, centrifuge and wash the precipitate with deionized water and absolute ethanol for 40 min until the pH of the upper layer solution is 7. The centrifugation speed for deionized water is 7000 rpm, and the centrifugation speed for absolute ethanol is 7000 rpm to obtain micron silver particles coated with silk fibroin / chitosan.

[0108] Dilute and disperse the obtained silk fibroin / chitosan-micron silver core-shell functional particles on a silicon wafer and observe their surface morphology through a scanning electron microscope. After detection, Figure 8 it can be seen that the prepared product is micron silver core-shell functional particles with severe agglomeration and uneven shapes of silk fibroin / chitosan, and the dispersibility and shape uniformity are not as good as those in Example 2. The reason is that at a higher reaction temperature, the particle movement is accelerated, and silver ions are extremely likely to collide and then agglomerate to form aggregates when growing into micron silver particles, and the formed shapes and sizes are different.

[0109] Comparative Example 6

[0110] The difference between this example and Example 2 is that: chitosan is not added in step (5), and the specific steps are as follows:

[0111] A preparation method of silk fibroin / chitosan-micron silver composite particles with a core-shell structure specifically includes the following steps:

[0112] (1) Remove the pupae from 16 g of silkworm cocoons and peel them into several layers, put them into 80 mL of a 0.6% sodium carbonate solution, stir at 100 °C for 30 min, take out the formed silk filaments and spin-dry them, then continue to put them into the sodium carbonate solution, repeat the above operation until there is no obvious flaky silk, wash the silk filaments thoroughly with deionized water, and put them into an oven at 70 °C for 1 h. After drying, degummed silk fibroin is obtained.

[0113] (2) The degummed silk fibroin obtained in step (1) (5 g) was placed in a 9.0 mol / L lithium bromide solution and sonicated at 40 °C for 2 h. After the degummed silk fibroin was fully dissolved and dispersed in the solution, the solution was diluted with deionized water and centrifugally washed for 40 min at a centrifugal speed of 7000 rpm. Finally, silk fibroin protein was obtained by freeze-drying at -50 °C for 44 h.

[0114] (3) 0.4 g of AgNO3 was dissolved in 10 mL of deionized water to obtain an AgNO3 solution.

[0115] (4) 0.04 g of PVP was dissolved in the AgNO3 solution obtained in step (3) to obtain an aqueous PVP-AgNO3 solution.

[0116] (5) 0.4 g of the silk fibroin protein obtained in step (2) was dissolved in deionized water and stirred at 30 °C for 3 h. The pH value of the solution was adjusted to 9 with 1 mol / L sodium hydroxide solution to obtain an aqueous solution of silk fibroin / chitosan.

[0117] (6) The aqueous silk fibroin protein solution obtained in step (5) was dropped into the PVP-AgNO3 solution in step (4). After reacting at 50 °C for 30 min, the precipitate was centrifugally washed with deionized water and absolute ethanol for 40 min in sequence until the pH of the upper layer solution was 7. The centrifugal speed for using deionized water was 7000 rpm, and the centrifugal speed for using absolute ethanol was 7000 rpm, obtaining micron silver particles coated with silk fibroin protein.

[0118] The obtained silk fibroin-micron silver core-shell functional particles were diluted and dispersed on a silicon wafer, and their surface morphology was observed through a scanning electron microscope. After detection, it was known that the prepared product was micron silver core-shell functional particles coated with silk fibroin protein. Its stability, antibacterial property, and dispersibility were not as good as those in Example 2. The reason was that chitosan was not added during the process. During the reaction, only silk fibroin protein coated the silver particles, and there were only obvious biocompatibility and the antibacterial property of silver itself, so the applicable scenarios were limited. And after observing its morphology, size, and surface core-shell structure after three months of placement, it was found that there were certain changes and the core-shell structure disappeared. Figure 9

[0119] The present invention is further described in detail; it should be understood that the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other; based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.​

Claims

1. A preparation method of silk fibroin / chitosan-micron silver core-shell functional particles, characterized in that: It includes the following steps: (1) Remove the pupae from the silkworm cocoons, wash them, peel them into laminar flakes, put them into a sodium carbonate solution and stir to prepare degummed silk fibroin; (2) Place the degummed silk fibroin obtained in step (1) in a lithium bromide solution for ultrasonic treatment. After fully dissolving, perform centrifugal washing and freeze-drying to obtain silk fibroin protein; (3) Grind and compound the silk fibroin protein obtained in step (2) with chitosan and dissolve it in water to obtain a mixed solution. Adjust the pH value of the mixed solution and stir to obtain an aqueous solution of silk fibroin protein / chitosan; (4) Dissolve silver nitrate in water to obtain an aqueous silver nitrate solution; (5) Dissolve polyvinylpyrrolidone in the aqueous silver nitrate solution obtained in step (4) to obtain an aqueous solution of polyvinylpyrrolidone-silver nitrate; (6) Add the aqueous solution of silk fibroin protein / chitosan obtained in step (3) to the aqueous solution of polyvinylpyrrolidone-silver nitrate obtained in step (5). After fully reacting at room temperature, prepare composite particles with a silk fibroin protein / chitosan outer shell of micron silver.

2. The preparation method of the silk fibroin / chitosan-micron silver core-shell functional particles according to claim 1, wherein: In step (1), the addition amount of silkworm cocoons in the sodium carbonate solution is 0.2 g / mL, and the mass percentage concentration of sodium carbonate is 0.5-0.8%; the specific preparation method is as follows: after adding sodium carbonate and stirring, the stirring temperature is 50-100 °C, the stirring time is 20-40 min. Take out the formed silk-like silk and spin it dry, then continue to put it into the sodium carbonate solution, repeat the above operation until there is no obvious flaky silk. Wash the silk-like silk thoroughly with deionized water, put it into an oven for drying, the oven temperature is 60-80 °C, and the drying time is 1-4 h to obtain degummed silk fibroin.

3. The preparation method of the silk fibroin / chitosan-micron silver core-shell functional particles according to claim 1, wherein: In step (2), the lithium bromide concentration is 8-10 mol / L, and the addition amount of degummed silk fibroin in lithium bromide is 0.12 g / mL; the ultrasonic temperature is 30-50 °C, and the ultrasonic time is 1-3 h; the centrifugation conditions are 6000-8000 rpm, the centrifugation time is 30-50 min, the freeze-drying temperature is -60 to -40 °C, and the freeze-drying time is 36-60 h.

4. The preparation method of the silk fibroin / chitosan-micron silver core-shell functional particles according to claim 1, characterized in that: In step (3), the dosage of chitosan is 0.004-0.035 g / mL, and the dosage of silk fibroin protein is 0.04-0.045 g / mL; the pH value of the mixed solution is adjusted to 9-11 with sodium hydroxide.

5. The preparation method of the silk fibroin / chitosan-micron silver core-shell functional particles according to claim 1, characterized in that: In step (4), the concentration of the silver nitrate solution is 0.02-0.07 g / mL.

6. The preparation method of the silk fibroin / chitosan-micron silver core-shell functional particles according to claim 1, characterized in that: In step (5), the dosage of polyvinylpyrrolidone is 0.002-0.007 g / mL.

7. The preparation method of the silk fibroin / chitosan-micron silver core-shell functional particles according to claim 1, wherein: In step (6), the silk fibroin protein / chitosan and the polyvinylpyrrolidone-silver nitrate mixed solution are mixed at a volume ratio of 1:4-1:1; the reaction time at room temperature is 30-50 min. After the reaction, centrifuge and wash until the pH of the supernatant solution = 7, the centrifugation conditions are 6000-8000 rpm, and the centrifugation time is 30-50 min.

8. Silk fibroin protein / chitosan-micron silver core-shell functional particles prepared by the method according to any one of claims 1 to 7.

9. Use of the silk fibroin protein / chitosan-micron silver core-shell functional particles according to claim 8 in the field of biocomposite materials technology.

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