Nanosilica / silane coupling agent / fibroin composite material and preparation method thereof

Abstract

The application discloses a preparation method of a nano-silica / silane coupling agent / fibroin composite material, and comprises the following steps: nano-silica sol preparation: uniformly mixing deionized water and tetraethyl orthosilicate, then adding lysine, the mass ratio of the tetraethyl orthosilicate and the lysine being 10-20:0.3-0.6, stirring and dissolving to obtain a mixed solution, and adjusting the pH value of the mixed solution to 8.5-9.5; heating the mixed solution to 50-70 DEG C, continuously stirring for 2-5 hours until the mixed solution does not separate any more, adjusting the pH value of the system to 6.5-7.5, and dispersing by using ultrasonic waves; aging at room temperature to obtain the nano-silica sol; nano-reinforced material preparation: uniformly mixing a fibroin solution, the nano-silica sol and gamma-glycidoxypropyltrimethoxysilane, aging and drying after forming a gel to obtain the nano-silica / silane coupling agent / fibroin composite material.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bone tissue repair and organic / inorganic material compounding, and in particular to a preparation method of a nano-silica / silane coupling agent / silk fibroin composite material and a nano-silica / silane coupling agent / silk fibroin composite material prepared by the preparation method. BACKGROUND

[0002] Silk fibroin is widely used in bone repair materials, but there are still problems such as limited direct processing of silk fibroin fibers, harsh processing conditions of composite materials, or high cost. The application of silk fibroin-based bone repair materials still needs further research, and the use of nanoparticles to enhance silk fibroin materials is a potential and feasible direction. Silica particles can combine with silk fibroin to improve the mechanical properties of silk fibroin materials, and also have certain biocompatibility.

[0003] For example, CN201910269969.1 discloses a preparation method of a nano-silica / high polymer material guided bone regeneration composite porous membrane. The prepared composite material has the characteristics of high mechanical strength, good biocompatibility, and moderate degradation rate, and can be applied to tissue repair. For example, CN202110359379.5 discloses a silk fibroin / silica composite material and a preparation method thereof. Tetraethyl orthosilicate and ethanol are used to prepare a silk fibroin / silica composite material. The results show that the composite material has excellent mechanical properties and good biocompatibility. However, when the silica is compounded with the silk fibroin by the blending method in the patent, the amount of silica is large, accounting for about 30% or even 40% of the composite material. This is because the nano-silica agglomerates, reducing the organic / inorganic interfacial force, resulting in a decrease in the composite performance. For example, CN202111008612.1 discloses a worm-shaped silica abrasive particle and a preparation method and application thereof. Tetraethyl orthosilicate and L-lysine are used to prepare worm-shaped silica abrasive particles, and calcium chloride is added to control agglomeration to obtain abrasive particles with good stability. However, the addition of calcium chloride limits the application of biological materials. The worm-shaped silica abrasive particles also lack active groups, making it difficult to be used 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. Considering the conditions when used, the silk fibroin composite material particularly needs to enhance its strength in the wet state. SUMMARY

[0004] In view of the above, in order to overcome the defects of the prior art and achieve the above-mentioned purposes, the purpose of the present application is to provide a preparation method of nano-silica / silane coupling agent / fibroin composite material, which enhances the combination of nano-silica and fibroin by preparing nano-silica with small particle size and adding silane coupling agent, so as to obtain nano-silica / silane coupling agent / fibroin composite material with excellent mechanical properties in wet state.

[0005] In order to achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0006] A nano-silica / silane coupling agent / fibroin composite material comprises the following steps: preparing nano-silica sol and fibroin solution respectively, mixing the fibroin solution, nano-silica sol and gamma-glycidoxypropyltrimethoxysilane (KH560) uniformly, and forming a gel, and then washing, aging and drying to obtain the nano-silica / silane coupling agent / fibroin composite material.

[0007] According to some preferred embodiments of the present application, the nano-silica sol is prepared by the following method: mixing deionized water and tetraethyl orthosilicate uniformly, then adding lysine, stirring and dissolving to obtain a mixed solution, adjusting the pH value of the mixed solution to 8.5-9.5, heating the mixed solution to 50-70℃, continuing to stir for 2-5h until the mixed solution no longer separates into layers, adjusting the pH value of the system to 6.5-7.5, and dispersing by ultrasonic wave; aging at room temperature to obtain the nano-silica sol.

[0008] According to some preferred embodiments of the present application, the particle size of the nano-silica sol is 10-15nm, which is small and has a narrow particle size distribution.

[0009] According to some preferred embodiments of the present application, the mass ratio of tetraethyl orthosilicate to lysine is 10-20:0.3-0.6, and the amount of lysine 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 fibroin when preparing the composite material.

[0010] According to some preferred embodiments of the present application, the mass ratio of deionized water to tetraethyl orthosilicate is 1-2:0.1-0.2.

[0011] According to some preferred embodiments of the present application, the lysine is one or more selected from L-lysine, D-lysine, DL-lysine and hydroxylysine.

[0012] According to some preferred embodiments of the present application, after adding lysine and stirring and dissolving, a 0.1-1 mol / L sodium hydroxide solution is used to adjust the pH value of the system.

[0013] According to some preferred embodiments of the present application, the pH value of the system is adjusted to 0.1-1 mol / L hydrochloric acid before the ultrasonic dispersion.

[0014] According to some preferred embodiments of the present application, the ultrasonic dispersion is performed at a power of 100-300 W for 5-30 minutes.

[0015] According to some preferred embodiments of the present application, the silk fibroin solution is prepared by adding sodium carbonate and / or sodium bicarbonate into deionized water to adjust the pH value of the solution to 9.0-9.5, adding cocoon shells to keep the solution at a slight boil for 30-35 minutes and keeping the uniform distribution of the cocoon shells by stirring, washing the surface residual silk gum with deionized water after removing the cocoon shells, drying the degummed silk, cutting it, dissolving it in saturated lithium bromide solution, and obtaining the silk fibroin solution after dialysis treatment.

[0016] According to some preferred embodiments of the present application, the degummed cocoon shells are dried in an oven at 55-60°C to obtain the degummed silk after repeating the degumming operation for 2-3 times.

[0017] According to some preferred embodiments of the present application, the mass concentration of the silk fibroin solution is 3-7%.

[0018] According to some preferred embodiments of the present application, the specific steps for preparing the nano-reinforced material are mixing the silk fibroin solution, the nanosilica sol, and γ-glycidoxypropyltrimethoxysilane uniformly, placing the formed gel at 60-80°C for 3-6 hours, removing the unreacted γ-glycidoxypropyltrimethoxysilane after sufficient reaction, aging and drying to obtain the nanosilica / silane coupling agent / silk fibroin composite material.

[0019] According to some preferred embodiments of the present application, the mass ratio between the nanosilica and the silk fibroin in the silk fibroin solution is 1-7:100; and the mass ratio between the γ-glycidoxypropyltrimethoxysilane and the silk fibroin in the silk fibroin solution is 0.5-3:10.

[0020] According to some preferred embodiments of the present application, the specific steps for forming the gel are adding the nanosilica sol dropwise into the silk fibroin solution, mixing uniformly, adding the γ-glycidoxypropyltrimethoxysilane dropwise into the mixed solution, treating by ultrasonic oscillation, pouring into a mold, and placing in a constant-temperature water bath oscillation box at 35-45°C for 2-12 hours until the gel is formed. The γ-glycidoxypropyltrimethoxysilane is added after the nanosilica sol and the silk fibroin solution are mixed, which prevents the local over-crosslinking caused by rapid addition and forms a lump of gel, which is difficult to disperse uniformly.

[0021] According to some preferred embodiments of the present application, the removal of incompletely reacted γ-glycidoxypropyltrimethoxysilane is achieved by immersing the gel in deionized water at a bath ratio of 1-3:100 for 3-4 days, with water changes every 2-3 hours.

[0022] According to some preferred embodiments of the present application, the aging of the gel is performed at a temperature of 40-60°C and a relative humidity of 75-90% for 3-7 days.

[0023] According to some preferred embodiments of the present application, the drying temperature is 60-80°C and the drying time is 1-5 days.

[0024] In some embodiments of the present application, a method for preparing a nanosilica / silane coupling agent / fibroin composite material is provided, which comprises the following steps:

[0025] (1) Preparation of nanosilica

[0026] The deionized water and tetraethyl orthosilicate are mixed and stirred until uniform, then lysine is added and stirred until dissolved. The pH of the system is adjusted to 8.5-9.5 using a sodium hydroxide solution. The mixed solution is heated to 50-70°C and stirred for 2-5 hours until the mixture is no longer layered. The pH of the system is adjusted to 6.5-7.5 using hydrochloric acid, and then the solution is dispersed using ultrasonic waves. The solution is aged at room temperature for 2-3 days to obtain a nanosilica sol.

[0027] (2) Preparation of a fibroin solution

[0028] 0.01 mol / L sodium carbonate and sodium bicarbonate are added to deionized water to adjust the pH of the solution to 9.0-9.5. Bombyx mori silkworm cocoons are added to the solution and kept at a slight boil for 30-35 minutes while stirring to maintain uniform distribution of the cocoons. The cocoons are removed and washed with deionized water to remove residual sericin. The degumming process is repeated 3 times. The degummed silk is dried, cut into pieces, and dissolved in a saturated lithium bromide solution. The solution is dialyzed to obtain a fibroin solution.

[0029] (3) Preparation of a nanoreinforced material

[0030] According to the mass ratio between the silica in the nanometer silica sol and the silk fibroin in the silk fibroin solution being 1-7:100, the nanometer silica sol is added dropwise into the silk fibroin aqueous solution, and the liquid is mixed uniformly by using a vortex mixer. According to the mass ratio between KH560 and the silk fibroin in the silk fibroin solution being 0.5-3:10, KH560 is added dropwise into the mixed solution. Then, ultrasonic oscillation treatment is carried out under the power of 100-300 W for 10-30 minutes, and after mixing uniformly, the mixture is poured into a mold and placed in a 35-45℃ constant-temperature water bath oscillation box for 2-12 hours until gelation, and then placed in a 60-80℃ oven for 3-6 hours to promote the reaction between the epoxy group of KH560 and the active groups of silk fibroin.

[0031] Then, the gel is demolded, and immersed in deionized water with a bath ratio of 1-3:100 to remove the remaining KH560 and hydrolysis product ethanol which are not completely reacted, for 3-4 days and the water is changed every 2-3 hours. Then, it is placed in a constant-temperature and constant-humidity box with a temperature of 40-60℃ and a relative humidity of 75-90% for 3-7 days for slow drying (aging). Then, the temperature is increased to 60-80℃ to continue drying in the oven for 1-5 days, and finally, the nanometer silica / silane coupling agent / silk fibroin composite material is obtained.

[0032] The application also provides a nanometer silica / silane coupling agent / silk fibroin composite material prepared by the preparation method described above, wherein the mass ratio of nanometer silica is 0.5-7%, the mass ratio of KH560 is 5-30%, and the mass ratio of silk fibroin is 70-95%.

[0033] The technical principle of the present application is that: the nano-silica sol is prepared by lysine catalyzing tetraethyl orthosilicate, and the generated nano-silica particles have a small particle size (less than 15 nm). Lysine contains a large number of amino groups and has a certain basicity, which can catalyze the hydrolysis of tetraethyl orthosilicate. In addition, lysine has a positive charge, and the silica sol formed after the hydrolysis of tetraethyl orthosilicate has a partial negative charge, which can form a nucleation core with lysine to form nano-particles. Due to the high concentration of lysine, the number of nucleation is large, and the number of nano-particles formed is greatly improved, so that the space for subsequent growth of nano-particles is reduced, and nano-silica with a small particle size (less than 15 nm) can be obtained. The nano-silica formed by lysine as a template and having a partial lysine can produce a relatively large binding force when combined with silk fibroin. Including the electrostatic force between the positive charge of lysine and the silk fibroin and the hydrogen bond formed between the nano-silica and the silk fibroin. These forces effectively promote the formation of a strong cross-linked structure of the silk fibroin, and thus the dry-state enhancement effect is achieved. The epoxy silane coupling agent KH560 is added as an organic / inorganic interface reinforcing agent to improve the bonding between the organic silk fibroin and the inorganic nano-silica particles in the composite material. The epoxy group therein can react with the active groups of the silk fibroin, such as amino, hydroxyl, phenolic hydroxyl, and carboxyl, and the other end of the siloxane can react with the hydroxyl on the nano-silica, so that more covalent bond cross-linking is formed between the nano-silica and the silk fibroin. The lysine in the nano-silica can catalyze and promote the reaction between the active epoxy group in the silane coupling agent and the active groups of the silk fibroin. In addition, as the content of the active groups on the silk fibroin decreases after the reaction, the water absorption of the composite material is also reduced, and the compression performance of the material in a wet state is enhanced.

[0034] Due to the adoption of the above technical solutions, the present application has the following advantages compared with the prior art: the nano-silica / silane coupling agent / silk fibroin composite material of the present application generates nano-silica particles with a very small particle size through lysine catalytic reaction, enters the weak link of the silk fibroin material, and adds the silane coupling agent KH560 to enhance the connection between the nano-silica particles and the silk fibroin, thereby realizing the enhancement effect of the silk fibroin material. During the deformation process of the composite material under external force, the rigid nano-silica filler can absorb part of the energy. On the other hand, the hydrogen bond interaction between the large number of hydroxyl groups on the surface of the nano-silica particles and the silk fibroin also enhances the interaction between the macromolecular chains, thereby increasing the mechanical strength of the material. In particular, the wet-state enhancement is realized, so that the composite material can be applied to the wet environment of the biological body. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to make the technical solutions in the embodiments of the present application clearer, the accompanying drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort based on these drawings.

[0036] Figure 1 The morphology photos of the nanosilica / silane coupling agent / fibroin composite materials prepared in Examples 2-5 and Comparative Examples 1-3;

[0037] Figure 2 The sol particle size diagram of the nanosilica materials prepared in Example 2 and Comparative Examples 4, 5;

[0038] Figure 3 The dry compression strength comparison diagram of the nanosilica / silane coupling agent / fibroin composite materials prepared in Examples 2-5 and Comparative Examples 1-3;

[0039] Figure 4 The wet compression strength comparison diagram of the nanosilica / silane coupling agent / fibroin composite materials prepared in Examples 2-5 and Comparative Examples 1-3. DETAILED DESCRIPTION

[0040] In order to make the technical solutions in the embodiments of the present application clearer, the accompanying drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort based on these drawings.

[0041] The preparation method of the nanosilica / silane coupling agent / fibroin composite material in the present embodiment can prepare nanosilica with small particle size by a special method, reduce agglomeration and reduce the amount of nanosilica; at the same time, by adding an interface coupling agent, the bonding force between the organic-inorganic interfaces of the composite material is enhanced, the amount of nanosilica is further reduced, and the mechanical properties of the fibroin composite material are significantly improved.

[0042] Specifically, the present embodiment uses silkworm cocoon shells as raw materials, and the preparation method includes the following steps:

[0043] Step (1), preparation of nanosilica

[0044] The deionized water and tetraethyl orthosilicate are blended and stirred until uniform, then the lysine powder is added and stirred until dissolved. The pH of the system is adjusted to 8.5-9.5 using 0.1-1 mol / L sodium hydroxide solution. The mixed solution is heated to 50-70°C and stirred for 2-5 hours until the mixture is no longer layered. The pH of the system is adjusted to 6.5-7.5 using 0.1-1 mol / L hydrochloric acid, and then the solution is dispersed using ultrasonic waves. The solution is aged at room temperature for 2-3 days to obtain a nanometer silica sol.

[0045] wherein the mass ratio of deionized water to tetraethyl orthosilicate is 1-2:0.1-0.2, and the mass ratio of tetraethyl orthosilicate to lysine is 10-20:0.3-0.6. The lysine is one or more selected from L-lysine, D-lysine, DL-lysine, and hydroxylysine. The ultrasonic power for ultrasonic dispersion is 100-300 W, and the time is 5-30 minutes.

[0046] Step (2), preparation of a silk fibroin solution

[0047] 0.01 mol / L sodium carbonate and sodium bicarbonate are added to deionized water to adjust the pH of the solution to 9.0-9.5. Cocoon shells are added and the solution is kept at a boil for 30-35 minutes while stirring to keep the cocoon shells uniformly distributed. The cocoon shells are removed and the surface residual sericin is washed off with deionized water. The degumming operation is repeated 2-3 times. The degummed cocoon shells are dried in an oven at 55-60°C for 10-24 hours to obtain degummed silk. The degummed silk is dried, cut into pieces, and dissolved in a saturated lithium bromide solution. The solution is dialyzed to obtain a silk fibroin solution.

[0048] Step (3), preparation of a nanometer-reinforced material with an epoxy silane coupling agent

[0049] The mass ratio of silica in the nanometer silica sol to silk fibroin in the silk fibroin solution is 1-7:100. The nanometer silica sol is added dropwise to the silk fibroin aqueous solution and mixed using a vortex mixer. KH560 is added dropwise to the mixture in a mass ratio of KH560 to silk fibroin in the silk fibroin solution of 0.5-3:10. The mixture is then treated by ultrasonic oscillation at a power of 100-300 W for 10-30 minutes. The mixture is poured into a mold and placed in a constant-temperature water bath oscillation box at 35-45°C for 2-12 hours until a gel is formed. The gel is then placed in an oven at 60-80°C for 3-6 hours to promote further reaction of the epoxy groups of KH560 with the active groups of the silk fibroin.

[0050] After that, the gel is demolded and soaked in deionized water with a bath ratio of 1-3:100 to remove the remaining KH560 and hydrolysis product ethanol which is not fully reacted, for 3-4 days and the water is changed every 2-3 hours. Then it is placed in a constant temperature and humidity box at a temperature of 40-60℃ and a relative humidity of 75-90% for 3-7 days for slow drying (aging). Then the temperature is increased to 60-80℃ and the drying in the oven is continued for 1-5 days, and finally the nano-silica / silane coupling agent / fibroin composite material is obtained. Among them, the mass ratio of nano-silica is 0.5-7%, the mass ratio of KH560 is 5-30%, and the mass ratio of fibroin is 70-95%.

[0051] Preferably, the mass ratio of nano-silica, silane coupling agent, and fibroin in the nano-silica / silane coupling agent / fibroin composite material is 5:20:100.

[0052] Example 1

[0053] The preparation method of the fibroin solution of the present embodiment specifically includes the following steps:

[0054] Step (1), select the cocoon shell, remove the pupae and other impurities. Use a balance to weigh 80 grams of cocoon shell. Measure 4 liters of deionized water and use an electromagnetic oven to boil, add 1 gram of sodium carbonate and 3 grams of sodium bicarbonate to adjust the solution pH to 9.5. Add the cocoon shell and keep the solution slightly boiling for 30 minutes and keep the uniform distribution of the cocoon shell by stirring. After taking out the cocoon shell, wash it with deionized water to remove the surface residual silk gum. Repeat the above operation three times and place the degummed cocoon shell in a 60℃ oven to dry for 24 hours to obtain degummed silk.

[0055] Step (2), take the above dried and degummed silk and cut it into pieces, dissolve it in a 9.3 mol / L lithium bromide aqueous solution with a mass ratio of 15 / 100, and dissolve it under the condition of water bath heating at a temperature of 65℃ and gentle stirring by a magnetic stirrer for about 1 hour. After dissolution, place it at room temperature to cool the silk / fibroin lithium bromide solution, fill it into a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyze it with deionized water for 3 days, during which the dialysis water is changed every 2 hours. After filtration, centrifuge to remove impurities, and finally obtain a fibroin aqueous solution.

[0056] Example 2

[0057] The preparation method of the nano-silica / silane coupling agent / fibroin composite material of the present embodiment specifically includes the following steps:

[0058] Step (1), preparation of nano-silica

[0059] Take 100 grams of deionized water, 10 grams of tetraethyl orthosilicate, and blend them using a vortex mixer to obtain a mixed solution; add 300 milligrams of L-lysine powder, dissolve it, and then use a 0.4 molar / liter sodium hydroxide solution to adjust the pH value of the system to 8.5.

[0060] Under the condition of a magnetic stirrer rotating at 600 r / min and a 70°C water bath heating for 5 hours until the mixed solution is no longer layered, use a 0.5 molar / liter hydrochloric acid to adjust the pH value of the system to 6.5, and use an ultrasonic cell disruptor to disperse it at a power of 100 W for 30 minutes. Then, under room temperature conditions, age it for 2 days to obtain an L-lysine-catalyzed nanosilica sol.

[0061] Step (2), prepare a silk fibroin solution using the method in Example 1.

[0062] Step (3), preparation of a nanoreinforced material added with an epoxy silane coupling agent

[0063] According to the mass ratio of the nanosilica sol silica to the silk fibroin in the silk fibroin solution being 1:100, add the nanosilica sol dropwise into the silk fibroin aqueous solution, and use a vortex mixer to mix the liquid.

[0064] According to the mass ratio of the γ-glycidoxypropyltrimethoxysilane to the silk fibroin in the silk fibroin solution being 5:100, add KH560 dropwise into the mixed solution, and use an ultrasonic oscillator to treat it uniformly at a power of 100 W for 30 minutes. After mixing uniformly, pour it into a mold, and place it in a 37°C constant-temperature water bath oscillation box for 2 hours until it gels. After the mixed solution gels, take it out and place it in a 60°C oven for 6 hours to promote the reaction of the KH560 epoxy group and the silk fibroin amino group.

[0065] Then, demold the gel, and use deionized water to soak it at a bath ratio of 1 / 100 to remove the remaining KH560 and hydrolysis product ethanol that is not completely reacted, for 3 days and change the water every 2 hours. Then, slowly age and dry it in a constant-temperature and constant-humidity box at 40°C and 90% relative humidity for 7 days, and then place it in a 60°C oven for drying treatment for 5 days to obtain a nanosilica / silane coupling agent / silk fibroin composite material.

[0066] Example 3

[0067] The preparation method of the nanosilica / silane coupling agent / silk fibroin composite material of this example specifically includes the following steps:

[0068] Step (1), preparation of a nanosilica sol

[0069] Take 120 grams of deionized water, 12 grams of tetraethyl orthosilicate, and blend them using a vortex mixer to obtain a mixed solution; add 350 milligrams of D-lysine powder, dissolve it, and then use a 0.5 mol / L sodium hydroxide solution to adjust the pH value of the system to 8.8.

[0070] Under the condition of a magnetic stirrer rotating at 650 r / min and a 65°C water bath heating, react for 4 hours until the mixed solution is no longer layered; add 0.6 mol / L hydrochloric acid to adjust the pH value of the system to 6.7, and disperse it using an ultrasonic cell disruptor at a power of 150 W for 25 minutes. Then, age it at room temperature for 2 days to obtain a D-lysine-catalyzed nanosilica sol.

[0071] Step (2), prepare a silk fibroin solution using the method in Example 1.

[0072] Step (3), preparation of a nanoreinforced material added with an epoxy silane coupling agent

[0073] According to the mass ratio of the nanosilica sol to the silk fibroin in the silk fibroin solution being 5:100, add the nanosilica sol dropwise into the silk fibroin aqueous solution, and mix the liquid using a vortex mixer.

[0074] According to the mass ratio of γ-glycidoxypropyltrimethoxysilane to the silk fibroin in the silk fibroin solution being 10:100, add KH560 dropwise into the mixed solution, and ultrasonically oscillate it at a power of 150 W for 25 minutes to uniformly mix it. After mixing, pour it into a mold, and place it in a 38°C constant-temperature water bath oscillation box for 6 hours to gel. After the mixed solution gels, take it out, and place it in a 70°C oven for 5 hours to promote the reaction of the epoxy group of KH560 with the amino group of the silk fibroin.

[0075] Then, demold the gel, and use deionized water to soak it at a bath ratio of 2 / 100 to remove the remaining KH560 and hydrolysis product ethanol that is not completely reacted, for 3 days and change the water every 2 hours. Then, slowly age and dry it in a constant-temperature and constant-humidity box at 45°C and 85% relative humidity for 6 days, and then place it in a 65°C oven for drying treatment for 4 days, to finally obtain a nanosilica / silane coupling agent / silk fibroin composite material.

[0076] Example 4

[0077] The method for preparing the nanosilica-reinforced silk fibroin composite material of this example specifically includes the following steps:

[0078] Step (1), preparation of a nanosilica sol

[0079] Take 180 grams of deionized water, 18 grams of tetraethyl orthosilicate, and blend them using a vortex mixer to obtain a mixed solution; add 500 milligrams of hydroxylysine powder, dissolve it, and then use a 0.7 molar / liter sodium hydroxide solution to adjust the pH value to 9.2.

[0080] Under the condition of a magnetic stirrer rotating at 750 r / min and a water bath heating at 55°C, react for 2 hours until the mixed solution is no longer layered, add 0.8 molar / liter hydrochloric acid to adjust the pH value of the system to 7.2, disperse using an ultrasonic cell disruptor at a power of 250 W for 15 minutes. Then, age at room temperature for 3 days to obtain a hydroxylysine catalyzed nanosilica sol.

[0081] Step (2), prepare a silk fibroin solution by using the method in Example 1.

[0082] Step (3), preparation of a nanoreinforced material added with an epoxy silane coupling agent

[0083] According to the mass ratio of the nanosilica sol to the silk fibroin in the silk fibroin solution being 5:100, add the nanosilica sol dropwise into the silk fibroin aqueous solution, and mix the liquid using a vortex mixer.

[0084] According to the mass ratio of γ-glycidoxypropyltrimethoxysilane to the silk fibroin in the silk fibroin solution being 20:100, add KH560 dropwise into the mixed solution, and ultrasonically oscillate at a power of 250 W for 15 minutes to uniformly mix them. After uniform mixing, pour the mixture into a mold, and place it in a constant-temperature water bath oscillation box at 39°C for 9 hours to gel. After the mixed solution is gelled, take it out, and place it in an oven at 75°C for 4 hours to promote the reaction of the epoxy group of KH560 with the amino group of the silk fibroin.

[0085] Then, demold the gel, and use deionized water to soak it at a bath ratio of 2.5 / 100 to remove the remaining KH560 and the hydrolysis product ethanol that is not completely reacted, for 4 days and change the water every 3 hours. Then, slowly age and dry it in a constant-temperature and constant-humidity box at 50°C and a relative humidity of 80% for 4 days, and then place it in an oven at 70°C for drying treatment for 3 days, to finally obtain a nanosilica / silane coupling agent / silk fibroin composite material.

[0086] Example 5

[0087] The method for preparing the nanosilica reinforced silk fibroin composite material in this example specifically includes the following steps:

[0088] Step (1), preparation of a nanosilica sol

[0089] Take 200 grams of deionized water, 20 grams of tetraethyl orthosilicate, and use a vortex mixer to blend to obtain a mixed solution; add 600 milligrams of L-lysine powder, dissolve, and then use 0.8 moles / liter of sodium hydroxide solution to adjust the pH value to 9.5.

[0090] Under the condition of 800 r / min of the rotation speed of the magnetic stirrer and 50°C water bath heating, react for 2 hours until the mixed solution is no longer layered, add 0.9 moles / liter of hydrochloric acid to adjust the pH value of the system to 7.5, disperse using an ultrasonic cell disruptor at a power of 300 W for 10 minutes, and then age at room temperature for 3 days to obtain the L-lysine catalyzed nanosilica sol.

[0091] Step (2), prepare the silk fibroin solution by the method in Example 1.

[0092] Step (3), preparation of the nanoreinforced material added with the epoxy silane coupling agent

[0093] According to the mass ratio of the nanosilica sol to the silk fibroin in the silk fibroin solution of 5:100, drop the nanosilica sol into the silk fibroin aqueous solution and mix the liquid using a vortex mixer.

[0094] According to the mass ratio of γ-glycidoxypropyltrimethoxysilane to the silk fibroin in the silk fibroin solution of 30:100, drop KH560 into the mixed solution, and uniformly mix by ultrasonic oscillation at a power of 300 W for 10 minutes. After mixing, pour into a mold and place in a 40°C constant-temperature water bath oscillation box for 12 hours to gel. After the mixed solution gels, take it out and place it in an 80°C oven for 3 hours to promote the reaction of the epoxy group of KH560 with the amino group of the silk fibroin.

[0095] Then demold the gel and use deionized water to soak at a bath ratio of 3 / 100 to remove the remaining KH560 and hydrolysis product ethanol that is not completely reacted, for 4 days and change the water every 3 hours. Then slowly age and dry at 60°C and 75% relative humidity in a constant-temperature and humidity box for 3 days, and then place in an 80°C oven for drying treatment for 2 days to finally obtain the nanosilica / silane coupling agent / silk fibroin composite material.

[0096] Comparative Example 1

[0097] This comparative example is a method for preparing pure silk fibroin material without adding nanosilica, including the following steps:

[0098] Step (1), preparation of a silk fibroin solution: prepare the silk fibroin solution by the method in Example 1.

[0099] Step (2), ethanol solution was added dropwise into the aqueous solution of silk fibroin, and the liquid was mixed uniformly using a vortex mixer. The mixed solution was poured into a mold and placed in a 40°C oven for 1 hour until a gel was formed. The gel was removed and washed with deionized water, and then placed in a constant temperature and humidity chamber at a temperature of 40°C and a relative humidity of 75% for 5 days to slowly dry to obtain a composite material. The composite material was placed in a 60°C oven for 2 days to obtain a pure silk fibroin material.

[0100] Comparative Example 2

[0101] The preparation method of the nano-silica / silk fibroin material without adding KH560 in this comparative example comprises the following steps:

[0102] Step (1), preparation of a silk fibroin solution: the silk fibroin solution was prepared by the method in Example 1.

[0103] Step (2), preparation of a nano-silica sol: the nano-silica sol was prepared by the method in step (1) of Example 2.

[0104] Step (3), according to the mass ratio between the silica in the nano-silica sol and the silk fibroin in the silk fibroin solution being 5:100, the nano-silica sol was added dropwise into the aqueous solution of silk fibroin, and the liquid was mixed uniformly using a vortex mixer. The mixed solution was placed in a 40°C oven until gelation. The gel was removed and washed with deionized water, and then placed in a constant temperature and humidity chamber at a temperature of 40°C and a relative humidity of 75% for 5 days to slowly dry to obtain a composite material. The composite material was placed in a 60°C oven for 2 days to obtain a nano-silica / silk fibroin material.

[0105] Comparative Example 3

[0106] The preparation method of the silane coupling agent / silk fibroin material without adding nano-silica in this comparative example comprises the following steps:

[0107] Step (1), preparation of a silk fibroin solution: the silk fibroin solution was prepared by the method in Example 1.

[0108] Step (2), KH560 was added dropwise into the silk fibroin solution according to a mass ratio of KH560 / silk fibroin of 20 / 100, and then mixed uniformly using ultrasonic oscillation and placed in a 37°C constant temperature water bath oscillation box for 12 hours until gelation. After the mixed solution was gelled, it was removed and placed in a 60°C oven for 3 hours to promote the reaction between the epoxy groups of KH560 and the amino groups of silk fibroin.

[0109] Step (3), after the gel was removed, the remaining incompletely reacted KH560 and hydrolysis product ethanol were removed by soaking with deionized water at a bath ratio of 1 / 100 for 3 days and water was changed every 2 hours. The material was slowly aged and dried at 40°C and 75% relative humidity in a constant temperature and humidity chamber for 5 days, and then dried in an oven at 60°C for 1 day, to obtain a silane coupling agent / silk fibroin material.

[0110] Comparative Example 4

[0111] The preparation method of the nanosilica sol of the present comparative example comprises the following steps:

[0112] Step (1), 100 grams of deionized water and 10 grams of tetraethyl orthosilicate were blended using a vortex mixer to obtain a mixed solution.

[0113] Step (2), the pH value of the mixed solution was adjusted to 2.0 using a 0.1 mol / L hydrochloric acid solution.

[0114] Step (3), the reaction was carried out under the conditions of a magnetic stirrer speed of 600 r / min and a 60°C water bath heating, until the mixed solution no longer separated, and the hydrochloric acid catalyzed nanosilica sol was obtained after filtration and aging at room temperature for 2 days.

[0115] Comparative Example 5

[0116] The preparation method of the nanosilica sol of the present comparative example comprises the following steps:

[0117] Step (1), 100 grams of deionized water and 10 grams of tetraethyl orthosilicate were blended using a vortex mixer to obtain a mixed solution.

[0118] Step (2), the pH value of the system was adjusted to 9.0 using a 0.1 mol / L ammonia solution.

[0119] Step (3), the reaction was carried out under the conditions of a magnetic stirrer speed of 600 r / min and a 60°C water bath heating, until the mixed solution no longer separated, and the ammonia catalyzed nanosilica sol was obtained after filtration and aging at room temperature for 2 days.

[0120] Performance comparison and analysis

[0121] 1. The sol particle size of the nanosilica material prepared in Example 2 and Comparative Examples 4 and 5 was tested and analyzed, as shown in Table 1. Figure 2

[0122] Figure 2 ​​The particle size distribution of the silica nanoparticles prepared in Example 2 is shown in FIG. 2. The particle size distribution of the silica nanoparticles prepared in Comparative Example 4 is shown in FIG. 4. The particle size distribution of the silica nanoparticles prepared in Comparative Example 5 is shown in FIG. 5. The particle size distribution of the silica nanoparticles prepared in Comparative Example 6 is shown in FIG. 6.

[0123] The particle size distribution of the silica nanoparticles prepared in Comparative Example 5 is shown in FIG. 5. The particle size distribution of the silica nanoparticles prepared in Comparative Example 6 is shown in FIG. 6.

[0124] Compared with the hydrochloric acid and ammonia catalysis methods, the silica nanoparticles prepared in Example 2 using L-lysine as a catalyst have a size of only about 12 nm, and the particle size distribution is very narrow. Considering that lysine can further enhance the binding force between the nanoparticles and the silk fibroin, the silica nanoparticles prepared by lysine catalysis have the potential to be used as a nano-enhanced filler for silk fibroin.

[0125] 2. Morphology of the composite material

[0126] Figure 1 The photographs of the prepared pure silk fibroin material and the composite material are shown in FIG. 7. Compared with the pure silk fibroin material, the composite material added with the silane coupling agent has a good forming and a uniform columnar shape.

[0127] 3. Mechanical properties of the silica nanoparticle / silane coupling agent / silk fibroin composite material

[0128] The composite materials prepared in each example and comparative example were taken out after being placed in a 60°C oven for 3h, and were tested using a universal testing machine to obtain the compression strength in a dry state.

[0129] The dry-state compression strength of the composite material prepared in Example 2 is shown in FIG. 8. Figure 3 As can be seen from the dry-state compression strength of the composite material prepared in Example 2, compared with the pure silk fibroin material, the addition of the silica nanoparticles and the silane coupling agent KH560 can significantly improve the compression strength of the silk fibroin material in a dry state. The dry-state compression strength of Comparative Example 2, Comparative Example 3 and each example has been significantly increased. Among them, each example has more increase in the compression strength compared with the comparative examples due to the addition of the silane coupling agent. From the dry-state compression strength of the composite material prepared in Example 2, it can be seen that the addition of the silica nanoparticles and the silane coupling agent KH560 can significantly improve the compression strength of the silk fibroin material in a dry state. Figure 3It can be seen that the dry compressive strength of the nanosilica / silane coupling agent / fibroin composite material increases with the increase of the mass ratio of KH560. When the mass ratio of silane coupling agent / fibroin reaches 20 / 100, the dry compressive strength increases from 44.6 MPa of the silica / fibroin composite material to 72.3 MPa. The reason may be that the epoxy group in KH560 reacts with the amino group or side chain residue on the molecular chain of fibroin to form covalent crosslinking, which enhances the interaction between the fibroin macromolecular chains, increases the crosslinking degree, and enhances the compression performance. However, when the mass ratio is further increased to 30 / 100, the mechanical properties of the material do not change significantly. The reason may be that the reaction crosslinking point provided by the fibroin is close to complete reaction, and the excess KH560 cannot further enhance the crosslinking effect between the macromolecular chains of the material, and cannot further enhance the dry compressive performance of the material.

[0130] The composite materials prepared in each example and comparative example were placed in a PBS buffer with pH = 7.4 at a bath ratio of 1 / 100, and were placed in a constant temperature water bath oscillator at a temperature of 37°C for 48 hours. After taking out, the water on the outer surface of the material was wiped off with a water absorption paper, and a universal testing machine was used for testing to obtain the compressive strength in the wet state.

[0131] From the mechanical property test results of the wet state of Figure 4 It can be seen from the wet state mechanical property test results of the wet state of Figure 4 The wet compressive strength of the silane coupling agent / fibroin composite material with a mass ratio of more than 10 / 100 is significantly improved. The wet compressive strength of the composite material with a mass ratio of 20 / 100-30 / 100 increases from 9.9 MPa of the silica / fibroin material to about 67.1 MPa, which significantly improves the defect that the mechanical properties of the fibroin material in the wet state decrease significantly. The reason may be that the hydrophobicity of KH560 reduces the water absorption rate of the composite material in the wet state, and the process of water molecules entering between the macromolecules is limited, which reduces the slippage of the fibroin macromolecular chains caused by water molecules, and improves the mechanical properties of the material in the wet state. The mechanical properties of the composite material can be significantly enhanced when the mass ratio of KH560 / fibroin reaches 20 / 100, and the enhancement effect is not significant when the mass ratio of KH560 is further increased to 30 / 100.

[0132] The test results show that the nanometer silicon dioxide / silane coupling agent / fibroin composite material prepared by the method can significantly improve the compression strength of pure fibroin material in dry and wet states.

[0133] The preparation method of the nanometer silicon dioxide / silane coupling agent / fibroin composite material comprises the following steps: (1) selecting lysine to catalyze a tetraethyl orthosilicate mixed solution to prepare a nanometer silicon dioxide sol; (2) immersing a domestic silkworm cocoon shell in a solution compounded by sodium carbonate and sodium bicarbonate for degumming treatment; (3) drying, cutting and dissolving the degummed silk in a lithium bromide solution, and then performing dialysis treatment to obtain a fibroin solution; (4) mixing the fibroin solution with the nanometer silicon dioxide sol to prepare a nanometer silicon dioxide / fibroin mixed solution; and (5) adding γ-glycidyl ether propyltrimethoxysilane (KH560) dropwise into the nanometer silicon dioxide / fibroin mixed solution to prepare the nanometer silicon dioxide / silane coupling agent / fibroin composite material. The preparation method is simple and can be batch prepared, and the mechanical properties of the prepared nanometer silicon dioxide / silane coupling agent / fibroin composite material are greatly improved compared with those of pure fibroin material. Compared with the prior art, the nanometer silicon dioxide / silane coupling agent / fibroin composite material has at least the following beneficial effects:

[0134] 1. The nanometer silicon dioxide / silane coupling agent / fibroin composite material provided by the application is obtained by a solution blending and drying method, and has the advantages of simple operation, easy-to-obtain material and low cost, and is suitable for industrialized mass production.

[0135] 2. In order to solve the problem that the extremely high surface energy of nanometer particles is prone to cause particle aggregation, resulting in uneven distribution of nanometer particle powder material and reduction of specific surface area when finally combined with the material, which is not conducive to the enhancement effect of the material, tetraethyl orthosilicate is selected as a silicon source, and high-concentration lysine is selected as a catalyst to catalytically prepare a nanometer silicon dioxide sol, so that the obtained nanometer silicon dioxide particles have small particle size and narrow distribution. Due to the reduction of particle size and the reduction of aggregation, the amount of nanometer silicon dioxide can be greatly reduced, and the mechanical properties can be significantly enhanced at a very low amount. The addition of silane coupling agent KH560 realizes uniform dispersion of the silicon dioxide particles in the composite material and reduces the hydrophilic performance of the composite material, reduces the water absorption of the material, and improves the wet mechanical properties of the material.

[0136] 3. The present application generates nano-silica particles with very small particle size (less than 15 nm) through lysine catalytic reaction, enters the weak link of silk fibroin material, and adds silane coupling agent KH560 to enhance the connection between nano-silica particles and silk fibroin, thereby realizing the reinforcing effect of silk fibroin material. During the deformation process of the composite material under external force, the rigid nano-silica filler can absorb part of the energy. On the other hand, through the hydrogen bond interaction between the large number of hydroxyl groups on the surface of the nano-silica particles and the silk fibroin, the interaction between the macromolecular chains is also enhanced, realizing the increase of the mechanical strength of the material. Especially, the reinforcement in the wet state is realized, which enables the composite material to be applied in the wet environment of the biological body.

[0137] The above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the spirit and essence of the present application shall be covered within the protection scope of the present application.

Claims

1. A method for preparing a nano-silica / silane coupling agent / silk fibroin composite material, characterized in that, Includes the following steps: Preparation of nano-silica sol: After thoroughly mixing deionized water and tetraethyl orthosilicate, lysine is added at a mass ratio of tetraethyl orthosilicate to lysine of 10-20:0.3-0.

6. The mixture is stirred until dissolved to obtain a mixed solution, and the pH of the mixed solution is adjusted to 8.5-9.

5. The mixed solution is then 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 then adjusted to 6.5-7.5, and the mixture is dispersed using ultrasound. The mixture is then aged at room temperature for 2-3 days to obtain the nano-silica sol. Preparation of nano-reinforced materials: The silk fibroin solution, the nano silica sol, and γ-glycidyl etheroxypropyltrimethoxysilane were mixed evenly to form a gel. The mixture was then placed at 60-80 °C for 3-6 hours. After that, the unreacted γ-glycidyl etheroxypropyltrimethoxysilane was removed, and the mixture was aged and dried to obtain the nano silica / silane coupling agent / silk fibroin composite material. The mass ratio of silica in the nano silica sol to silk fibroin in the silk fibroin solution is 1~7:

100.

2. The preparation method according to claim 1, characterized in that, The particle size of the nano-silica sol is 10~15nm.

3. The preparation method according to claim 1, characterized in that, The mass ratio of the γ-glycidoxypropyltrimethoxysilane to the silk fibroin in the silk fibroin solution is 0.5~3:

10.

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

5. The preparation method according to claim 1, characterized in that, After adding lysine and stirring to dissolve, adjust the pH of the system using a 0.1-1 mol / L sodium hydroxide solution; Before ultrasonic dispersion, the pH of the system is adjusted using 0.1~1 mol / L hydrochloric acid.

6. The preparation method according to claim 1, characterized in that, The specific steps for forming the gel are as follows: add nano-silica sol dropwise into the silk fibroin solution, mix evenly, add γ-glycidoxypropyltrimethoxysilane dropwise to the mixture, treat with ultrasonic vibration, pour into a mold, and place at 35-45℃ for 2-12 hours until the gel is formed.

7. The preparation method according to claim 1, characterized in that, To remove unreacted γ-glycidyl etheroxypropyltrimethoxysilane and hydrolysis product ethanol, the following steps are taken: After the gel is demolded, soak it in deionized water at a bath ratio of 1~3:100 for 3~4 days, changing the water every 2~3 hours.

8. The preparation method according to claim 1, characterized in that, The gel is aged for 3 to 7 days under constant temperature and humidity conditions of 40 to 60°C and 75 to 90% relative humidity; the drying temperature is 60 to 80°C and the drying time is 1 to 5 days.

9. The preparation method according to any one of claims 1-8, characterized in that, The silk fibroin solution was prepared by the following method: sodium carbonate and / or sodium bicarbonate were added to deionized water to adjust the pH of the solution to 9.0-9.

5. Silkworm cocoon shells were added and the solution was kept at a gentle boil for 30-35 minutes while stirring. After removing the cocoon shells, the residual sericin on the surface was washed off with deionized water. After repeating this process several times, the degummed silk was dried, cut into pieces, and dissolved in a saturated lithium bromide solution. After dialysis, the silk fibroin solution was obtained.

10. A nano-silica / silane coupling agent / silk fibroin composite material prepared by the preparation method according to any one of claims 1-9.

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