Nanosilica reinforced silk fibroin composite material and preparation method thereof

Abstract

The application discloses a preparation method of a nanometer silicon dioxide reinforced silk fibroin composite material, and comprises the following steps: nanometer silicon dioxide sol preparation: after deionized water and tetraethyl orthosilicate are uniformly mixed, lysine is added, the mass ratio of the tetraethyl orthosilicate and the lysine is 10-20:0.3-0.6, stirring and dissolving are conducted to obtain a mixed solution, and the pH value of the mixed solution is adjusted to 8.5-9.5; the mixed solution is heated to 50-70 DEG C, continues to be stirred for 2-5 hours, the pH value of the system is adjusted to 6.5-7.5, and ultrasonic dispersion is adopted; aging is conducted at room temperature to obtain nanometer silicon dioxide sol; nanometer reinforced material preparation: silk fibroin solution and the nanometer silicon dioxide sol are uniformly mixed, drying is conducted after gelation is formed, and the nanometer silicon dioxide reinforced silk fibroin composite material is obtained. The nanometer silicon dioxide reinforced silk fibroin composite material prepared by the preparation method has a great improvement in mechanical properties compared with pure silk fibroin material.

Description

Technical Field

[0001] This invention relates to the fields of bone tissue repair and organic / inorganic material composite technology, specifically to a method for preparing a nano-silica-reinforced silk fibroin composite material and the nano-silica-reinforced silk fibroin composite material prepared by the method. Background Technology

[0002] Human bone tissue serves multiple functions, including support, protection, movement, and metabolism. However, millions of patients suffer bone damage each year due to various accidents. While some bone injuries can be repaired by the body itself, more severe bone damage requires the implantation of bone repair materials for treatment. Existing bone repair materials often suffer from insufficient biodegradability, high processing costs, or difficulty in obtaining them.

[0003] Silk fibroin materials have wide applications in bone tissue engineering and bone repair materials. For example, Chinese invention patent CN201010288285.5 discloses a silk fibroin / calcium phosphate bone cement composite material and its preparation method. The prepared composite material has good mechanical properties and biocompatibility and can be used as a filler material for bone repair in the medical field. Chinese invention patent CN201910277658.X discloses a silk fibroin / cellulose derivative composite material and its preparation method. The composite silk fibroin matrix material prepared by mixing regenerated silk fibroin and cellulose into a gel has good biocompatibility and improves the mechanical properties of the regenerated silk fibroin material, which can be applied in the biomedical field.

[0004] However, limitations remain in the direct processing of silk fibroin fibers, and the processing conditions for composite materials are either too demanding or too expensive. The application of silk fibroin-based bone repair materials requires further in-depth and extensive research, and using nanoparticles to reinforce silk fibroin materials is a potentially feasible direction.

[0005] Silica is a substance that can improve the mechanical properties of materials and has good biocompatibility. The functional groups of silica inorganic sol can combine with organic macromolecules with specific functions through hydrogen bonds or electrostatic interactions, thereby enabling silk fibroin to bind and be uniformly distributed with silica molecules, thus improving the mechanical properties of silk fibroin materials. For example, Chinese invention patent CN201910269969.1 discloses a method for preparing a composite porous membrane for bone regeneration guided by nano-silica / polymer materials. The prepared composite material has the characteristics of high mechanical strength, good biocompatibility, and moderate degradation rate, and can be applied to tissue repair. Chinese invention patent CN202110359379.5 discloses a silk fibroin / silica composite material and its preparation method, using tetraethyl orthosilicate and ethanol to prepare the silk fibroin / silica composite material. The results show that the composite material has excellent mechanical properties and good biocompatibility. However, this patent uses a large amount of silica, accounting for 30% to 40% of the composite material, when combining silica with silk fibroin through blending. This is because the agglomeration of nano-silica leads to a decrease in composite performance. Another example is Chinese invention patent CN202111008612.1, which discloses a worm-shaped silica abrasive particle, its preparation method, and its application. It uses tetraethyl orthosilicate and L-lysine to prepare worm-shaped silica abrasive particles, and adds calcium chloride to control agglomeration, resulting in abrasives with better stability. However, the addition of calcium chloride limits its application in biomaterials; and the worm-shaped silica abrasive particles lack active groups, making them difficult to use for material reinforcement, and the prepared worm-shaped silica abrasive particles have a large particle size. Therefore, it is necessary to seek a better method for reinforcing silk fibroin materials. Summary of the Invention

[0006] In view of this, in order to overcome the defects of the prior art and achieve the above objectives, the purpose of this invention is to provide a method for preparing a nano-silica reinforced silk fibroin composite material.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A nano-silica reinforced silk fibroin composite material includes the following steps: preparing nano-silica sol and silk fibroin solution respectively; mixing the silk fibroin solution and nano-silica sol evenly to form a gel; and drying to obtain the nano-silica reinforced silk fibroin composite material.

[0009] According to some preferred embodiments of the present invention, the nano-silica sol is prepared by the following method: deionized water and tetraethyl orthosilicate are mixed evenly, lysine is added, and the mixture is stirred to dissolve, thereby obtaining a mixed solution. The pH of the mixed solution is adjusted to 8.5-9.5. The mixed solution is heated to 50-70°C and stirred for 2-5 hours until the mixed solution no longer separates into layers. The pH of the system is adjusted to 6.5-7.5, and the mixture is dispersed by ultrasonication. The mixture is aged at room temperature for 2-3 days to obtain the nano-silica sol.

[0010] According to some preferred embodiments of the present invention, the particle size of the nano-silica sol is 10-15 nm, which is small and has a narrow particle size distribution.

[0011] According to some preferred embodiments of the present invention, the mass ratio of tetraethyl orthosilicate to lysine is 10-20:0.3-0.6, and the amount of lysine used is relatively high, which can increase the content of lysine on the surface of nano-silica, so as to improve the binding force between silica and silk fibroin when preparing composite materials.

[0012] According to some preferred embodiments of the present invention, the mass ratio of deionized water to tetraethyl orthosilicate is 1-2:0.1-0.2.

[0013] According to some preferred embodiments of the present invention, the lysine is selected from one or more of L-lysine, D-lysine, DL-lysine, and hydroxylysine.

[0014] According to some preferred embodiments of the present invention, after adding lysine and stirring to dissolve it, the pH of the system is adjusted using a sodium hydroxide solution of 0.1 to 1 mol / L.

[0015] According to some preferred embodiments of the invention, the pH of the system is adjusted with 0.1 to 1 mol / L hydrochloric acid before ultrasonic dispersion.

[0016] According to some preferred embodiments of the present invention, the ultrasonic dispersion uses an ultrasonic power of 100-300W and a processing time of 5-30 minutes.

[0017] According to some preferred embodiments of the present invention, the silk fibroin solution is prepared by the following method: sodium carbonate and / or sodium bicarbonate are added to deionized water to adjust the pH of the solution to 9.0-9.5; silkworm cocoon shells are added and the solution is kept at a gentle boil for 30-35 minutes while stirring to maintain the uniform distribution of the cocoon shells; after removing the cocoon shells, residual sericin on the surface is washed away with deionized water. The degummed silk is dried, cut into pieces, and dissolved in a saturated lithium bromide solution, followed by dialysis to obtain the silk fibroin solution.

[0018] According to some preferred embodiments of the present invention, after repeating the degumming operation 2 to 3 times, the degummed cocoon shells are placed in an oven at 55 to 60°C and dried for 10 to 24 hours to obtain degummed silk.

[0019] According to some preferred embodiments of the present invention, the mass concentration of the silk fibroin solution is 3-7%.

[0020] According to some preferred embodiments of the present invention, the specific steps for preparing the nano-reinforced material are as follows: nano-silica sol is added dropwise to a silk fibroin solution, ultrasonically oscillated, poured into a mold, and placed at 35-45°C for 1-5 hours until a gel is formed; after removing the gel, it is washed with deionized water, aged, and dried to obtain a nano-silica-reinforced silk fibroin composite material.

[0021] According to some preferred embodiments of the present invention, the mass ratio between silica in the nano silica sol and silk fibroin in the silk fibroin solution is 1 to 10:100.

[0022] According to some preferred embodiments of the invention, the gel is aged for 3 to 7 days under constant temperature and humidity conditions of 30 to 50°C and 75 to 90% relative humidity.

[0023] According to some preferred embodiments of the invention, the drying temperature is 60–80°C and the time is 2–5 days.

[0024] In some specific embodiments of this application, the preparation method of the nano-silica reinforced silk fibroin composite material, using silkworm cocoon shells as raw materials, specifically includes the following steps:

[0025] (1) Preparation of nano-silica

[0026] Deionized water and tetraethyl orthosilicate were mixed and stirred until homogeneous. Lysine was added and stirred until dissolved. The pH of the system was adjusted to 8.5–9.5 using sodium hydroxide solution. The mixture was heated to 50–70°C and stirred for 2–5 hours until no further stratification occurred. The pH of the system was then adjusted to 6.5–7.5 using hydrochloric acid, followed by ultrasonic dispersion. The mixture was aged at room temperature for 2–3 days to obtain nano-silica sol. This aging process is to obtain more uniformly distributed nanoparticles.

[0027] (2) Preparation of silk fibroin solution

[0028] Add 0.01 mol / L sodium carbonate and sodium bicarbonate to deionized water to adjust the pH to 9.0–9.5. Add silkworm cocoon shells and maintain a gentle boil for 30–35 minutes, stirring to ensure uniform distribution of the cocoon shells. Remove the cocoon shells and wash away any residual sericin with deionized water. Repeat the degumming process three times. Dry the degummed silk, cut it into pieces, and dissolve it in a saturated lithium bromide solution. Dialyze the solution to obtain a silk fibroin solution.

[0029] (3) Preparation of nano-reinforced materials

[0030] Following a mass ratio of 1–10:100 between silica in the nano-silica sol and silk fibroin in the silk fibroin solution, the nano-silica sol was dropwise added to the silk fibroin aqueous solution, and the liquid was mixed thoroughly using a vortex mixer. The mixture was then placed in an ultrasonic cleaner and ultrasonically vibrated at 100–300W for 10–30 minutes to ensure uniform mixing of the nano-silica particles and the silk fibroin solution. The mixture was then poured into a mold and placed in an oven at 35–45℃ for 1–5 hours until gel formation. After removing the gel, it was washed with deionized water and aged in a constant temperature and humidity chamber at 30–50℃ and 75–90% relative humidity for 3–7 days. Finally, it was removed and dried in an oven at 60–80℃ for 2–5 days to obtain the nano-silica-reinforced silk fibroin composite material.

[0031] The present invention also provides a nano-silica reinforced silk fibroin composite material prepared by the preparation method described above, wherein the mass percentage of nano-silica is 1-10% and the mass percentage of silk fibroin is 90-99%.

[0032] The technical principle of this invention is as follows: Nano-silica sol is prepared by catalyzing tetraethyl orthosilicate with lysine, resulting in small-sized nano-silica particles. Lysine contains a large number of amino groups and has a certain degree of alkalinity, which can catalyze the hydrolysis of tetraethyl orthosilicate. Lysine contains both acidic carboxyl groups and basic amino groups, forming a buffer system to stabilize the pH of the reaction system and ensure stable reaction. Furthermore, lysine carries a positive charge, while the silica sol formed after the hydrolysis of tetraethyl orthosilicate carries a partial negative charge, allowing lysine to serve as a nucleation core for nanoparticle formation. Due to the high concentration of lysine, the number of nuclei is large, significantly increasing the number of nanoparticles formed, thus reducing the space for subsequent nanoparticle growth and resulting in nano-silica with a smaller particle size (below 15 nm). This nano-silica with partial lysine content, formed using lysine as a template, exhibits strong binding forces when combined with silk fibroin. These forces include the electrostatic interaction between the positive charge of lysine and silk fibroin, as well as hydrogen bonds between the nano-silica and silk fibroin. These forces effectively promote the formation of strong cross-linked structures in silk fibroin, thereby achieving an enhancing effect.

[0033] Due to the adoption of the above technical solutions, this invention has the following advantages compared with the prior art: The nano-silica reinforced silk fibroin composite material of this invention generates very small nano-silica particles through a lysine-catalyzed reaction, which penetrate into the weak points of the silk fibroin material, thereby enhancing the mechanical properties of the composite material. During deformation under external force, the nano-silica filler can absorb some energy. Furthermore, the hydrogen bonding between the numerous hydroxyl groups on the surface of the nano-silica particles and the silk fibroin also enhances the interaction between macromolecular chains, resulting in a significant improvement in the mechanical properties of the composite material. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 These are morphological photographs of the nano-silica-reinforced silk fibroin composite materials prepared in Examples 2-6;

[0036] Figure 2 The sol particle size diagrams are shown for the nano-silica materials prepared in Example 2 and Comparative Examples 1 and 2.

[0037] Figure 3The image shows a comparison of the compressive strength of the nano-silica reinforced silk fibroin composite materials prepared in Examples 2-6 and Comparative Example 3. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0039] Addressing the issue of insufficient mechanical properties in existing silk fibroin materials, this invention utilizes lysine to catalyze a mixture of tetraethyl orthosilicate to prepare nano-silica sol with a particle size less than 15 nm. Using β-sheet silk fibroin as a substrate, the nano-silica sol is dropwise added to an aqueous silk fibroin solution at a solid-to-silica mass ratio of 1–10 / 100. After uniform mixing and drying, a nano-silica-reinforced silk fibroin composite material is obtained.

[0040] The preparation method of the nano-silica reinforced silk fibroin composite material in this embodiment uses silkworm cocoon shells as raw materials and specifically includes the following steps:

[0041] Step (1) Preparation of nano-silica

[0042] Deionized water and tetraethyl orthosilicate were mixed and stirred until homogeneous. Lysine powder was added and stirred until dissolved. The pH of the system was adjusted to 8.5–9.5 using a 0.1–1 mol / L sodium hydroxide solution. The mixture was heated to 50–70°C and stirred for 2–5 hours until the mixture no longer separated into layers. The pH of the system was then adjusted to 6.5–7.5 using a 0.1–1 mol / L hydrochloric acid solution, followed by ultrasonic dispersion. The mixture was aged at room temperature for 2–3 days to obtain nano-silica sol.

[0043] The mass ratio of deionized water to tetraethyl orthosilicate is 1–2:0.1–0.2; the mass ratio of tetraethyl orthosilicate to lysine is 10–20:0.3–0.6. Lysine is selected from one or more of L-lysine, D-lysine, DL-lysine, and hydroxylysine. The ultrasonic dispersion uses an ultrasonic power of 100–300 W for 5–30 minutes.

[0044] Step (2): Preparation of silk fibroin solution

[0045] Add 0.01 mol / L sodium carbonate and sodium bicarbonate to deionized water to adjust the pH to 9.0–9.5. Add cocoon shells and maintain a gentle boil for 30–35 minutes, stirring to ensure uniform distribution of the cocoon shells. Remove the cocoon shells and wash away any residual sericin with deionized water. Repeat the degumming process 2–3 times. Then, dry the degummed cocoon shells in an oven at 55–60°C for 10–24 hours to obtain degummed silk. Dry the degummed silk, cut it into pieces, and dissolve it in a saturated lithium bromide solution. After dialysis, obtain a silk fibroin solution.

[0046] Step (3): Preparation of nano-reinforced materials

[0047] The mass ratio of silica in the nano-silica sol to silk fibroin in the silk fibroin solution is 1–10:100. The nano-silica sol is dropwise added to the silk fibroin aqueous solution, and the liquid is mixed using a vortex mixer. The mixture is then placed in an ultrasonic cleaner and ultrasonically vibrated at 100–300W for 10–30 minutes to ensure uniform mixing of the nanofiller and the silk fibroin solution. The mixture is then poured into a mold and placed in an oven at 35–45℃ for 1–5 hours until a gel forms. The gel is removed, washed with deionized water, and aged in a constant temperature and humidity chamber at 30–50℃ and 75–90% relative humidity for 3–7 days. It is then removed and dried in an oven at 60–80℃ for 2–5 days to obtain a nano-silica-reinforced silk fibroin composite material. The mass percentage of nano-silica is 1–10%, and the mass percentage of silk fibroin is 90–99%.

[0048] Example 1

[0049] The preparation method of the silk fibroin solution in this embodiment specifically includes the following steps:

[0050] Step (1): Select silkworm cocoon shells and remove silkworm pupae and other impurities. Weigh 80 grams of silkworm cocoon shells using a balance. Measure 4 liters of deionized water and boil it using an induction cooker. Add 1 gram of sodium carbonate and 3 grams of sodium bicarbonate to adjust the pH of the solution to 9.5. Add the cocoon shells and keep the solution simmering for 30 minutes, stirring to ensure even distribution of the cocoon shells. Remove the cocoon shells and wash them with deionized water to remove any residual sericin on the surface. Repeat the above operation three times, then place the degummed cocoon shells in a 60℃ oven to dry for 24 hours to obtain degummed silk.

[0051] Step (2): Take the dried, degummed silk and cut it into small pieces. Dissolve it in a 9.3 mol / L lithium bromide aqueous solution at a mass ratio of 15 / 100. The dissolution conditions are a water bath at 65°C with gentle stirring using a magnetic stirrer for about 1 hour. After dissolution, place it at room temperature to allow the silk fibroin / lithium bromide solution to cool. Fill the solution into dialysis bags with a molecular weight cutoff of 8–14 kDa and dialyze using deionized water for 3 days, changing the dialysis water every 2 hours. After filtration, centrifuge to remove impurities, and finally obtain a silk fibroin aqueous solution.

[0052] Example 2

[0053] The preparation method of the nano-silica reinforced silk fibroin composite material in this embodiment specifically includes the following steps:

[0054] Step (1) Preparation of nano-silica

[0055] Weigh 100g of deionized water and 10g of tetraethyl orthosilicate, and mix them using a vortex mixer to obtain a mixed solution; add 300mg of L-lysine powder, dissolve it, and then adjust the pH of the system to 8.5 using 0.4mol / L sodium hydroxide solution.

[0056] The mixture was reacted for 5 hours under magnetic stirrer speed of 600 r / min and water bath heating at 70℃ until the mixture no longer separated into layers. The pH of the system was adjusted to 6.5 with 0.5 mol / L hydrochloric acid. The mixture was then dispersed using an ultrasonic cell disruptor at 100W and shaken for 30 minutes. It was then aged at room temperature for 2 days to obtain L-lysine-catalyzed nano-silica sol.

[0057] Step (2): Prepare silk fibroin solution using the method in Example 1.

[0058] Step (3), Preparation of nano-reinforced materials:

[0059] The mass ratio of silica in the nano silica sol to silk fibroin in the silk fibroin solution is 1:100. The nano silica sol is added dropwise to the silk fibroin aqueous solution, and the liquid is mixed evenly using a vortex mixer.

[0060] The mixture was placed in an ultrasonic cleaner and ultrasonically oscillated at 100W for 30 minutes to ensure uniform mixing of the nanoparticles and silk fibroin solution. The mixture was then poured into a mold and placed in a 45°C oven for 1 hour until a gel was formed.

[0061] After removing the gel and washing it with deionized water, it was placed in a constant temperature and humidity chamber at 30℃ and 90% relative humidity for 7 days to slowly dry and obtain the composite material. The composite material was then placed in a 60℃ oven for 5 days to obtain the nano-silica reinforced silk fibroin composite material.

[0062] Example 3

[0063] The preparation method of the nano-silica reinforced silk fibroin composite material in this embodiment specifically includes the following steps:

[0064] Step (1) Preparation of nano-silica sol

[0065] Weigh 120g of deionized water and 12g of tetraethyl orthosilicate, and mix them using a vortex mixer to obtain a mixed solution; add 350mg of D-lysine powder, dissolve it, and then adjust the pH of the system to 8.8 using 0.5mol / L sodium hydroxide solution.

[0066] The mixture was reacted for 4 hours under magnetic stirrer speed of 650 r / min and water bath heating at 65℃ until the mixture no longer separated into layers. 0.6 mol / L hydrochloric acid was added to adjust the pH of the system to 6.7. The mixture was then dispersed using an ultrasonic cell disruptor at 150W for 25 minutes. Finally, it was aged at room temperature for 2 days to obtain D-lysine-catalyzed nano-silica sol.

[0067] Step (2): Prepare silk fibroin solution using the method in Example 1.

[0068] Step (3) Preparation of nano-reinforced materials

[0069] The mass ratio of silica in the nano silica sol to silk fibroin in the silk fibroin solution is 2.5:100. The nano silica sol is added dropwise to the silk fibroin aqueous solution, and the liquid is mixed evenly using a vortex mixer.

[0070] The mixture was placed in an ultrasonic cleaner and ultrasonically oscillated at 150W for 25 minutes to ensure uniform mixing of the nanoparticles and silk fibroin solution. The mixture was then poured into a mold and placed in a 42℃ oven for 2 hours until a gel was formed.

[0071] After removing the gel and washing it with deionized water, it was placed in a constant temperature and humidity chamber at 35℃ and 85% relative humidity for 6 days to slowly dry and obtain the composite material. The composite material was then placed in a 65℃ oven for 4 days to obtain the nano-silica reinforced silk fibroin composite material.

[0072] Example 4

[0073] The preparation method of the nano-silica reinforced silk fibroin composite material in this embodiment specifically includes the following steps:

[0074] Step (1) Preparation of nano-silica sol

[0075] Weigh 150g of deionized water and 15g of tetraethyl orthosilicate, and mix them using a vortex mixer to prepare a mixed solution; add 400mg of hydroxylysine powder, dissolve it, and then adjust the pH to 9.0 using 0.6mol / L sodium hydroxide solution.

[0076] The mixture was reacted for 3 hours under magnetic stirrer speed of 700 r / min and water bath heating at 60℃ until the mixture no longer separated into layers. 0.7 mol / L hydrochloric acid was added to adjust the pH of the system to 6.9. The mixture was then dispersed using an ultrasonic cell disruptor at 200W and shaken for 20 minutes. Finally, it was aged at room temperature for 2 days to obtain hydroxylysine-catalyzed nano-silica sol.

[0077] Step (2): Prepare silk fibroin solution using the method in Example 1.

[0078] Step (3) Preparation of nano-reinforced materials

[0079] The mass ratio of silica in the nano silica sol to silk fibroin in the silk fibroin solution is 5:100. The nano silica sol is added dropwise to the silk fibroin aqueous solution, and the liquid is mixed evenly using a vortex mixer.

[0080] The mixture was placed in an ultrasonic cleaner and ultrasonically oscillated at 200W for 20 minutes to ensure uniform mixing of the nanoparticles and silk fibroin solution. The mixture was then poured into a mold and placed in a 40℃ oven for 3 hours until a gel was formed.

[0081] After removing the gel and washing it with deionized water, it was placed in a constant temperature and humidity chamber at 40℃ and 80% relative humidity for 5 days to slowly dry and obtain the composite material. The composite material was then placed in a 70℃ oven for 3 days to obtain the nano-silica reinforced silk fibroin composite material.

[0082] Example 5

[0083] The preparation method of the nano-silica reinforced silk fibroin composite material in this embodiment specifically includes the following steps:

[0084] Step (1) Preparation of nano-silica sol

[0085] Weigh 180 g of deionized water and 18 g of tetraethyl orthosilicate, and mix them using a vortex mixer to obtain a mixed solution; add 500 mg of L-lysine powder, dissolve it, and adjust the pH to 9.2 using 0.7 mol / L sodium hydroxide solution.

[0086] The mixture was reacted for 2 hours under magnetic stirrer speed of 750 r / min and water bath heating at 55℃ until the mixture no longer separated into layers. 0.8 mol / L hydrochloric acid was added to adjust the pH of the system to 7.2. The mixture was then dispersed using an ultrasonic cell disruptor at 250W and shaken for 15 minutes. Finally, it was aged at room temperature for 3 days to obtain L-lysine-catalyzed nano-silica sol.

[0087] Step (2): Prepare silk fibroin solution using the method in Example 1.

[0088] Step (3) Preparation of nano-reinforced materials

[0089] The mass ratio of silica in the nano silica sol to silk fibroin in the silk fibroin solution is 7.5:100. The nano silica sol is added dropwise to the silk fibroin aqueous solution, and the liquid is mixed evenly using a vortex mixer.

[0090] The mixture was placed in an ultrasonic cleaner and ultrasonically oscillated at 250W for 15 minutes to ensure uniform mixing of the nanoparticles and silk fibroin solution. The mixture was then poured into a mold and placed in a 38°C oven for 4 hours until a gel was formed.

[0091] After removing the gel and washing it with deionized water, it was placed in a constant temperature and humidity chamber at 45℃ and 78% relative humidity for 4 days to slowly dry and obtain the composite material. The composite material was then placed in a 75℃ oven to dry for 2 days to obtain the nano-silica reinforced silk fibroin composite material.

[0092] Example 6

[0093] The preparation method of the nano-silica reinforced silk fibroin composite material in this embodiment specifically includes the following steps:

[0094] Step (1) Preparation of nano-silica sol

[0095] Weigh 200g of deionized water and 20g of tetraethyl orthosilicate, and mix them using a vortex mixer to obtain a mixed solution; add 600mg of L-lysine powder, dissolve it, and then adjust the pH to 9.5 using 0.8mol / L sodium hydroxide solution.

[0096] The mixture was stirred at 800 rpm in a magnetic stirrer and heated in a 50°C water bath for 2 hours until the mixture no longer separated into layers. 0.9 mol / L hydrochloric acid was added to adjust the pH of the system to 7.5. The mixture was then dispersed using an ultrasonic cell disruptor at 300W for 10 minutes and aged at room temperature for 3 days to obtain L-lysine-catalyzed nano-silica sol.

[0097] Step (2): Prepare silk fibroin solution using the method in Example 1.

[0098] Step (3) Preparation of nano-reinforced materials

[0099] The mass ratio of silica in the nano silica sol to silk fibroin in the silk fibroin solution is 10:100. The nano silica sol is added dropwise to the silk fibroin aqueous solution, and the liquid is mixed evenly using a vortex mixer.

[0100] The mixture was placed in an ultrasonic cleaner and ultrasonically oscillated at 300W for 10 minutes to ensure uniform mixing of the nanoparticles and silk fibroin solution. The mixture was then poured into a mold and placed in a 35°C oven for 5 hours until a gel was formed.

[0101] After removing the gel and washing it with deionized water, it was placed in a constant temperature and humidity chamber at 50℃ and 75% relative humidity for 3 days to slowly dry and obtain the composite material. The composite material was then placed in an oven at 80℃ for 2 days to obtain the nano-silica reinforced silk fibroin composite material.

[0102] Comparative Example 1

[0103] The preparation method of the nano-silica sol in this comparative example includes the following steps:

[0104] Step (1): Take 100g of deionized water and 10g of tetraethyl orthosilicate, and use a vortex mixer to blend them to obtain a mixed solution.

[0105] Step (2): Adjust the pH of the mixed solution to 2.0 using a 0.1 mol / L hydrochloric acid solution.

[0106] Step (3): React under the conditions of magnetic stirrer speed of 600r / min and water bath heating at 60℃ until the mixture no longer separates into layers. After filtration, age at room temperature for 2 days to obtain hydrochloric acid catalyzed nano silica sol.

[0107] Comparative Example 2

[0108] The preparation method of the nano-silica sol in this comparative example includes the following steps:

[0109] Step (1): Take 100g of deionized water and 10g of tetraethyl orthosilicate, and use a vortex mixer to blend them to obtain a mixed solution.

[0110] Step (2): Adjust the pH of the system to 9.0 using 0.1 mol / L ammonia solution;

[0111] Step (3): React under the conditions of magnetic stirrer speed of 600r / min and water bath heating at 60℃ until the mixture no longer separates into layers. After filtration, age at room temperature for 2 days to obtain ammonia-catalyzed nano silica sol.

[0112] Comparative Example 3

[0113] This comparative example demonstrates a method for preparing pure silk fibroin material without the addition of nano-silica, comprising the following steps:

[0114] Step (1), Preparation of silk fibroin solution: The silk fibroin solution was prepared using the method in Example 1.

[0115] Step (2): Add the ethanol solution dropwise to the silk fibroin aqueous solution and mix the liquid thoroughly using a vortex mixer. Pour the mixture into a mold and place it in a 40°C oven for 1 hour until a gel forms. Remove the gel, wash it with deionized water, and then place it in a constant temperature and humidity chamber at 40°C and 75% relative humidity for 5 days to slowly dry and obtain the composite material. Place the composite material in a 60°C oven for 2 days to obtain pure silk fibroin material.

[0116] Performance Comparison and Analysis

[0117] 1. The sol particle size of the nano-silica materials prepared in Example 2 and Comparative Examples 1 and 2 was tested and analyzed, such as... Figure 2 As shown.

[0118] Depend on Figure 2 The particle size distribution shows that the nano-silica particles prepared by the hydrochloric acid-catalyzed hydrolysis of tetraethyl orthosilicate in Comparative Example 1 are mainly distributed in the ranges of 50–100 nm and 500–1000 nm, with a large difference in average particle size distribution and many agglomerates larger than 1000 nm. The overall distribution is uneven and the particle size is large. Therefore, the nano-silica prepared by hydrochloric acid catalysis is prone to agglomeration, providing a limited number of surface hydroxyl groups, thus limiting the application of the nano-size effect and making it unsuitable for subsequent use as a nano-reinforcing filler for silk fibroin.

[0119] In Comparative Example 2, the ammonia-catalyzed sol was mainly distributed around 34 nm with no obvious agglomeration. The nano-silica formed by ammonia catalysis was relatively uniformly distributed with less severe agglomeration. However, its particle size was still relatively large, resulting in insufficient binding force with silk fibroin, and the improvement in the mechanical properties of the composite material formed by adding silk fibroin was not ideal.

[0120] Compared to the hydrochloric acid and ammonia catalytic methods, the nano-silica sol prepared by L-lysine catalysis in Example 2 has a size of only about 12 nm and a very narrow particle size distribution. Considering that lysine can further enhance the binding force between nanoparticles and silk fibroin, the nano-silica formed by lysine catalysis has the potential to serve as a nano-reinforcing filler for silk fibroin.

[0121] 2. Mechanical properties of nano-silica / silk fibroin composite materials

[0122] The compressive strength of the nano-silica reinforced silk fibroin composites prepared in Examples 2-6 and Comparative Example 3 was tested, and the results are as follows: Figure 3 .

[0123] according to Figure 3 In the examples, adding a small amount of silica significantly enhanced the compressive strength of silk fibroin. In Example 4, the composite material with a silica / silk fibroin mass ratio of 5 / 100 increased its compressive strength from approximately 19 MPa to approximately 45 MPa compared to pure silk fibroin, achieving a significant enhancement in compressive strength. This is likely because the nano-silica particles filled the weak gaps in the silk fibroin polymer material, increasing intermolecular forces through hydrogen bonding with the protein molecular chains, thus achieving the reinforcing effect.

[0124] However, further increasing the silica content did not improve the compressive strength of the material (silica / silk fibroin ratio 7.5 / 100) and even decreased the strength (silica / silk fibroin ratio 10 / 100). This may be because excessive nano-silica particles agglomerate, reducing the specific surface area and the number of hydroxyl groups on the nano-silica surface. This weakens the hydrogen bonding between the organic silk fibroin and the inorganic silica, thus reducing the enhancement effect on the mechanical properties of the silk fibroin material.

[0125] The test results above show that the nano-silica reinforced silk fibroin composite material prepared by the method of the present invention can significantly improve the compressive strength of pure silk fibroin material.

[0126] The preparation method of the nano-silica reinforced silk fibroin composite material of the present invention includes the following steps:

[0127] (1) A lysine-catalyzed tetraethyl orthosilicate solution was used to prepare nano-silica sol; (2) Silkworm cocoon shells were immersed in a solution of sodium carbonate and sodium bicarbonate for degumming; (3) The degummed silk was dried, shredded, and dissolved in lithium bromide solution, and then dialyzed to obtain a silk fibroin solution; (4) The silk fibroin solution was mixed with the nano-silica sol to obtain a nano-silica-reinforced silk fibroin composite material. The above preparation method is simple and can be used for batch production. By adding lysine, the diameter of silica particles is reduced, and their composite performance with silk fibroin is improved. The mechanical properties of the nano-silica-reinforced silk fibroin composite material prepared are significantly improved compared with pure silk fibroin material. Compared with the prior art, the present invention has at least the following beneficial effects:

[0128] 1. The nano-silica reinforced silk fibroin composite material of the present invention is obtained by solution blending and drying; the preparation method is simple to operate, the materials are readily available and the cost is low, making it suitable for industrial mass production.

[0129] 2. To address the problem that the extremely high surface energy of nanoparticles easily leads to particle agglomeration, resulting in uneven distribution and reduced specific surface area of ​​the nanoparticle powder material when finally combined with other materials, which is detrimental to the material's reinforcing effect, this invention uses tetraethyl orthosilicate as the silicon source and high-concentration lysine as the catalyst to prepare nano-silica sol. The resulting nano-silica particles have small particle size and narrow distribution. Due to the reduction in particle size and agglomeration, the amount of nano-silica used can be significantly reduced, achieving a significant enhancement of mechanical properties even at extremely low dosages.

[0130] 3. This invention generates very small nano-silica particles through a lysine-catalyzed reaction, which then penetrate the weak points of silk fibroin materials, thereby enhancing the mechanical properties of the silk fibroin composite material. During deformation under external force, the rigid nano-silica filler can absorb some of the energy. Furthermore, the hydrogen bonding between the numerous hydroxyl groups on the surface of the nano-silica particles and the silk fibroin also enhances the interactions between macromolecular chains, resulting in a significant improvement in the mechanical properties of the composite material.

[0131] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a nanosilica-reinforced silk fibroin composite material, characterized by, The preparation method comprises the following steps: Preparation of nano-silica sol: After mixing the deionized water and tetraethyl orthosilicate uniformly, lysine is added, the mass ratio of tetraethyl orthosilicate to lysine is 10-20:0.3-0.6, and the mixture is stirred and dissolved to obtain a mixed solution, and the pH value of the mixed solution is adjusted to 8.5-9.5; the mixed solution is heated to 50-70 DEG C, and stirring is continued for 2-5 hours until the mixed solution is no longer layered, the pH value of the system is adjusted to 6.5-7.5, and ultrasonic dispersion is used; the gel is aged at room temperature for 2-3 days to obtain the nano-silica sol; Preparation of nano-reinforced material: The fibroin solution and the nano-silica sol are mixed uniformly under ultrasonic oscillation, the gel is aged and dried to obtain the nano-silica reinforced fibroin composite material. The mass ratio between the silica in the nano-silica sol and the fibroin in the fibroin solution is 1-10:

100.

2. The production method according to claim 1, characterized by, The particle size in the nano-silica sol is 10-15 nm.

3. The preparation method according to claim 1, characterized in that, The lysine is one or more selected from L-lysine, D-lysine, DL-lysine and hydroxylysine.

4. The method of claim 1, wherein, After stirring and dissolving the lysine, the pH value of the system is adjusted using a 0.1-1 mol / L sodium hydroxide solution; before ultrasonic dispersion, the pH value of the system is adjusted using a 0.1-1 mol / L hydrochloric acid.

5. The preparation method according to claim 1, characterized in that, The specific steps for preparing the nano-reinforced material are as follows: the nano-silica sol is dropped into the fibroin solution, ultrasonic oscillation is performed, then the mixture is poured into a mold, and the mold is placed at 35-45 DEG C for 1-5 hours until a gel is formed; the gel is taken out, washed with deionized water, aged and dried to obtain the nano-silica reinforced fibroin composite material.

6. The production method according to claim 5, wherein The aging of the gel is performed under constant temperature and humidity conditions of 30-50 DEG C and 75-90% relative humidity for 3-7 days.

7. The preparation method according to claim 5, characterized in that, The drying temperature is 60-80 DEG C, and the drying time is 2-5 days.

8. The method of any one of claims 1-7, wherein, The fibroin solution is prepared by the following method: sodium carbonate and / or sodium bicarbonate are added to deionized water, the pH value of the solution is adjusted to 9.0-9.5, cocoon shells are added to keep the solution slightly boiling for 30-35 minutes and stirring is performed, the cocoon shells are taken out and washed with deionized water to remove the surface residual sericin; after repeated multiple times, the degummed silk is dried, cut and dissolved in a saturated lithium bromide solution, and the solution is dialyzed to obtain the fibroin solution.

9. A nano-silica reinforced fibroin composite material prepared by the preparation method according to any one of claims 1-8.

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