A functional silk fibroin / titanium dioxide nanotube composite membrane and its preparation method

Abstract

This invention discloses a functional silk fibroin / titanium dioxide nanotube composite membrane and its preparation method. The composite membrane is a three-layer structure: a lower layer is a support membrane based on silk fibroin, a middle layer is a titanium dioxide nanotube membrane separated from a metal substrate, and an upper layer is a polymer electrospun fiber layer. The preparation method involves coating a silk fibroin mixture solution onto the surface of a titanium-based metal substrate, drying it, peeling off the membrane, treating it with an etchant, immersing it in a drug solution, drying it, and using it as a receiving electrode. Polymer electrospun fibers are then grown on the membrane using electrospinning technology to seal the titanium dioxide nanotubes, resulting in the composite membrane. The composite membrane of this invention ensures that the titanium dioxide nanotube membrane layer is not prone to cracking or curling, and while achieving the sealing of the drug-loaded titanium dioxide nanotube openings, it does not affect the structure and performance of the support membrane.

Description

Technical Field

[0001] This invention relates to a functional composite membrane and its preparation method, and more particularly to a functional silk fibroin / titanium dioxide nanotube composite membrane and its preparation method. Background Technology

[0002] In the biomedical field, titanium dioxide (TiO2) nanotubes can promote protein adsorption, influence cell signaling pathways, and promote osteoblast differentiation, making them suitable for bone tissue repair. In recent years, anodized TiO2 nanotubes have attracted considerable attention due to their controllable size and high degree of order. Traditional anodizing methods typically produce TiO2 nanotube arrays attached to a metal substrate, but the underlying metal matrix prevents direct application under certain conditions. To address this, researchers have directly peeled individual TiO2 nanotube films from titanium-based metal substrates. However, they have found that in dry environments, TiO2 nanotube films are prone to cracking or curling, limiting their widespread application. A feasible approach is to prepare a polymer self-supporting film and use it as a binder to separate TiO2 nanotubes from the metal substrate, leveraging the toughness of the polymer film to maintain the ordered structure of the TiO2 nanotubes.

[0003] Furthermore, the TiO2 nanotube structure is advantageous for loading drug molecules. Therefore, by loading antibacterial drugs and growth factors, it is possible to achieve multiple effects such as bactericidal function and accelerated tissue repair. However, it should be noted that the openings of the drug-loaded TiO2 nanotubes need to be sealed; otherwise, a good sustained-release effect cannot be achieved. However, for the aforementioned polymer self-supporting membranes with TiO2 nanotube structures, the high temperature or chemical environment during the sealing process will damage the self-supporting membrane structure, leading to a deterioration in mechanical properties. Therefore, how to seal the TiO2 nanotube openings without affecting the structure and performance of the self-supporting membrane is another technical problem that needs to be solved. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a functional silk fibroin / titanium dioxide nanotube composite membrane that can seal TiO2 nanotubes without damaging the polymer support membrane structure.

[0005] The second objective of this invention is to provide a method for preparing the above-mentioned functional silk fibroin / titanium dioxide nanotube composite membrane.

[0006] Technical solution: The functional silk fibroin / titanium dioxide nanotube composite membrane of the present invention is a three-layer membrane, the lower layer is a support membrane with silk fibroin as the matrix, the middle layer is a titanium dioxide nanotube membrane separated from the metal substrate, and the upper layer is a polymer electrospun fiber layer.

[0007] The preparation method of the above-mentioned functional silk fibroin / titanium dioxide nanotube composite film includes the following steps:

[0008] (1) Coat the surface of the titanium-based metal substrate with a silk fibroin mixture solution, and after drying, peel off the film;

[0009] (2) Treat the stripped membrane with a corrosive agent, then immerse the membrane in a drug solution, and then remove and dry it;

[0010] (3) The dried membrane is used as the receiving electrode, and polymer electrospun fibers are grown on the membrane by electrospinning to seal the titanium dioxide nanotubes on the membrane, thus obtaining a composite membrane.

[0011] In step (1), the silk fibroin mixed solution includes a regenerated silk fibroin solution, a crosslinking agent, short fibers, and nanoparticles; the silk fibroin solution contains both short fibers and nanoparticles, which improves the strength and toughness of the support membrane, making the support membrane less prone to rupture.

[0012] The mass ratio of regenerated silk fibroin, cross-linking agent, short fibers, and nanoparticles is 1-0.8:0-0.1:0.01-0.1:0.01-0.1. The preparation process of the silk fibroin mixed solution involves blending the regenerated silk fibroin, cross-linking agent, short fibers, and nanoparticles; after mixing, the solution is concentrated to 3-20%. The concentration of the mixed solution determines its viscosity and flow rate, which in turn affects the integrity, flexibility, and mechanical properties of the self-supporting membrane. Selecting an appropriate concentration is crucial for the preparation of self-supporting membranes.

[0013] The regenerated silk fibroin solution is a silkworm fibroin and / or tussah silkworm fibroin solution; the concentration of the regenerated silk fibroin solution is 1-10%.

[0014] The crosslinking agent is at least one of genipin, a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide, horseradish peroxidase, and glutaraldehyde; the short fiber is degummed silk filament after shearing, with an average fiber length of 0.1-5 mm; the nanoparticles are black scale nanoparticles and / or two-dimensional transition metal carbon nitride compounds, with an average particle size of 40-400 nm.

[0015] In step (1), the titanium-based metal substrate is preferably anodized titanium sheet / foil or titanium alloy; the surface of the anodized titanium sheet / foil or titanium alloy has a uniform titanium dioxide nanotube structure, and the average inner diameter of the titanium dioxide nanotubes is 50-500 nm, and the average tube length is 1-6 μm. During the transfer of titanium dioxide nanotubes from the surface of titanium sheet / foil or titanium alloy to the self-supporting film, the average inner diameter and tube length affect whether the entire titanium dioxide nanotube layer can be completely transferred to the self-supporting film. If the average inner diameter and tube length of the titanium dioxide nanotubes are too small, it is difficult to transfer; if the average inner diameter and tube length are too large, the titanium dioxide nanotubes are easily broken during the transfer process.

[0016] The membrane is peeled off by immersion in an alcohol solution; the alcohol solution is one of methanol, ethanol, methanol-water mixed solvent, or ethanol-water mixed solvent, and the treatment time is 0.5-2 hours.

[0017] The corrosive agent is a mixed solution of hydrofluoric acid and phytic acid, with a volume ratio of 1-0.8:0-0.2, and the treatment time is 5-40 minutes.

[0018] The drug solution includes antimicrobial peptides and bone morphogenetic protein-2, with a mass ratio of 1:0.1 to 0.5, and an immersion time of 0.5 to 8 hours.

[0019] In step (2), the drying environment is room temperature.

[0020] In step (3), during electrospinning, the side with the titanium dioxide nanotube membrane is facing upwards, and polymer fibers are stacked on top of it. The polymer is at least one of silk fibroin, collagen, gelatin, chitin and its derivatives, and alginate. The spinning time is controlled to be 2-8 hours. The spinning time should not be too long or too short. If it is too long, the spun fiber layer will be too thick, and the slow release will be too slow. If it is too short, the sealing effect will not be achieved. The selected polymers are all natural biodegradable polymer materials. On the one hand, the degradation of the polymer itself helps to achieve sustained drug release. On the other hand, these materials have good biocompatibility and hydrophilicity, making it easier for cells to adhere to them during application, thus facilitating drug absorption.

[0021] Beneficial effects: Compared with the prior art, the present invention achieves the following significant effects: (1) The composite membrane of the present invention has good strength and toughness in the bottom layer of the regenerated silk fibroin support membrane, which can effectively prevent the TiO2 nanotube membrane from cracking or curling and breaking in a dry environment; the black scale nanoparticles or MXene nanoparticles in the support membrane have good photothermal effect, which can give the self-supporting membrane a certain antibacterial property and help improve the mechanical properties of the support membrane; the TiO2 nanotubes loaded with antibacterial drugs and growth factors are sealed by polymer electrospun fibers, so as to achieve the purpose of both slow release of drugs and no damage to the support membrane. While giving the composite membrane antibacterial and tissue growth promotion functions, it can also ensure the structural stability and long-lasting function of the composite membrane. (2) This method does not require special equipment, is easy and simple to operate, and has low cost. It can be applied to the field of bone tissue repair. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the silk fibroin / titanium dioxide nanotube composite membrane structure of the present invention;

[0023] Figure 2 The sustained-release curves of the composite membrane samples in Examples 1-6 and Comparative Examples 5-6 are shown.

[0024] Figure 3 CCK-8 images of osteoblasts cultured on composite membrane samples from Examples 1-6 and Comparative Examples 1-4 for 6 days. Detailed Implementation

[0025] The present invention will now be described in further detail.

[0026] Example 1

[0027] A 1% concentration of regenerated silkworm fibroin solution, genipin, degummed short silk fibers with an average length of approximately 5 mm, and black scale nanoparticles with an average particle size of 40 nm were mixed in a ratio of 1:0.05:0.01:0.01. After uniform mixing, the mixture was further concentrated to 3%, and then uniformly coated onto the surface of anodized TA2 type pure titanium sheet, wherein the average inner diameter of TiO2 was 500 nm and the average tube length was 6 μm. After drying, the composite film was peeled off and immersed in methanol for 0.5 h. Then, the composite film was treated with a mixed solution of hydrofluoric acid and phytic acid with a volume ratio of 1:0.05 for 5 min. After cleaning and drying, the composite membrane was immersed in a mixed solution of antimicrobial peptide and bone morphogenetic protein-2 at a mass ratio of 1:0.1 for 0.5 h. After removal and drying at room temperature, the resulting composite membrane was used as the receiving electrode, with the side containing the TiO2 nanotube structure facing upwards. Using a 33% concentration of regenerated silkworm fibroin solution as the spinning solution, electrospinning technology was employed for 2 h to seal the TiO2 nanotubes in the electrospun fibers. Figure 1This is a schematic diagram of the structure of the functional silk fibroin / titanium dioxide nanotube composite membrane of the present invention.

[0028] Example 2

[0029] The basic steps are the same as in Example 1, except that: a 3% concentration of regenerated silkworm fibroin solution, a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide, degummed silk short fibers with an average length of about 4 mm, and black scale nanoparticles with an average particle size of 100 nm are mixed in a ratio of 1:0.08:0.05:0.03, and the mixed solution is further concentrated to 8%; the mixed solution is then uniformly coated onto anodized TA2 type pure titanium sheet. On the surface, the average inner diameter of TiO2 is 400 nm and the average tube length is 5 μm. The composite membrane is immersed in a 90% methanol aqueous solution for 1.0 h. The composite membrane is treated with a mixed solution of hydrofluoric acid and phytic acid with a volume ratio of 1:0.1 for 15 min. The composite membrane is immersed in a mixed solution of antimicrobial peptide and bone morphogenetic protein-2 with a mass ratio of 1:0.2 for 1.0 h. A 30% mixed solution of regenerated silkworm fibroin / collagen is used as the spinning solution, and the spinning time is 3 h.

[0030] Example 3

[0031] The basic steps are the same as in Example 1, except that: a 5% concentration of regenerated tussah silk fibroin solution, horseradish peroxidase, degummed silk short fibers with an average length of about 3 mm, and MXene with an average particle size of 150 nm are mixed in a ratio of 1:0.1:0.06:0.04, and the mixed solution is further concentrated to 12%; the mixed solution is uniformly coated onto the anodized TC4 titanium alloy surface, wherein the average inner diameter of TiO2 is 300 nm and the average tube length is 4.2 μm; the composite membrane is immersed in ethanol for 1.5 h; the composite membrane is treated with a mixed solution of hydrofluoric acid and phytic acid with a volume ratio of 9:1 for 20 min; the composite membrane is immersed in a mixed solution of antimicrobial peptide and bone morphogenetic protein-2 with a mass ratio of 1:0.2 for 2.0 h; a 20% concentration of collagen / sodium alginate solution is used as the spinning solution, and the spinning time is 4 h.

[0032] Example 4

[0033] The basic steps are the same as in Example 1, except that: a 6% concentration of regenerated tussah silk fibroin solution, glutaraldehyde, degummed silk short fibers with an average length of about 2.1 mm, and MXene with an average particle size of 200 nm are mixed in a ratio of 1:0.1:0.07:0.05, and the mixed solution is further concentrated to 15%; the mixed solution is uniformly coated onto the anodized TC4 titanium alloy surface, wherein the average inner diameter of TiO2 is 220 nm and the average tube length is 3.1 μm; the composite membrane is immersed in a 90% ethanol aqueous solution for 80 min; the composite membrane is treated with a mixed solution of hydrofluoric acid and phytic acid with a volume ratio of 8:1 for 25 min; the composite membrane is immersed in a mixed solution of antimicrobial peptide and bone morphogenetic protein-2 with a mass ratio of 1:0.4 for 3.0 h; a 15% concentration of chitosan / silk fibroin / gelatin mixed solution is used as the spinning solution, and the spinning time is 5 h.

[0034] Example 5

[0035] The basic steps are the same as in Example 1, except that: an 8% concentration of a mixed solution of silkworm fibroin / tussah fibroin, genipin / horseradish peroxidase, degummed short silk fibers with an average length of approximately 1.2 mm, and MXene with an average particle size of 300 nm are mixed in a ratio of 0.8:0.1:0.08:0.06, and the mixed solution is further concentrated to 18%; the mixed solution is then uniformly coated onto the anodized TC4 titanium alloy surface, wherein TiO2... The average inner diameter of the composite membrane was 100 nm and the average tube length was 2.1 μm. The composite membrane was immersed in an 80% methanol aqueous solution for 1.5 h. The composite membrane was treated with a mixed solution of hydrofluoric acid and phytic acid with a volume ratio of 9:2 for 30 min. The composite membrane was immersed in a mixed solution of antimicrobial peptide and bone morphogenetic protein-2 with a mass ratio of 1:0.45 for 4.0 h. A 12% collagen / gelatin / silk fibroin mixed solution was used as the spinning solution, and the spinning time was 7 h.

[0036] Example 6

[0037] The basic steps are the same as in Example 1, except that: a 10% mixed solution of silkworm fibroin / tussah fibroin, genipin / horseradish peroxidase / glutaraldehyde, degummed short silk fibers with an average length of approximately 0.1 mm, and black scale nanoparticles with an average particle size of 100 nm / MXene with an average particle size of 400 nm are mixed in a ratio of 0.8:0.1:0.08:0.1, and the mixed solution is further concentrated to 20%; the mixed solution is then uniformly coated onto anodized TC4 titanium alloy. On the gold surface, the average inner diameter of TiO2 is 50 nm and the average tube length is 1 μm. The composite membrane is immersed in an 80% ethanol aqueous solution for 2 h. The composite membrane is treated with a mixed solution of hydrofluoric acid and phytic acid with a volume ratio of 4:1 for 40 min. The composite membrane is immersed in a mixed solution of antimicrobial peptide and bone morphogenetic protein-2 with a mass ratio of 1:0.5 for 6.0 h. A 10% chitosan / collagen / gelatin / silk fibroin mixed solution is used as the spinning solution, and the spinning time is 8 h.

[0038] Comparative Example 1

[0039] The basic steps are the same as in Example 1, except that no degummed silk short fibers and black scale nanoparticles are added.

[0040] Comparative Example 2

[0041] The basic steps are the same as in Example 1, except that no degummed silk short fibers are added.

[0042] Comparative Example 3

[0043] The basic steps are the same as in Example 1, except that no black scale nanoparticles were added.

[0044] Comparative Example 4

[0045] The basic steps are the same as in Example 1, except that the mixed solution is coated onto the surface of a pure titanium sheet without anodizing.

[0046] Comparative Example 5

[0047] The basic steps are the same as in Example 1, except that the TiO2 nanotubes were not sealed by electrospinning.

[0048] Comparative Example 6

[0049] The basic steps are the same as in Example 1, except that the TiO2 nanotubes were not sealed by electrospinning, but were instead sealed in deionized water at 90°C for 40 minutes.

[0050] The mechanical properties of the composite films in Examples 1-6 and Comparative Examples 1-3 and 5-6 were tested, and the results are shown in Tables 1 and 2.

[0051] The sustained-release performance of the composite membrane samples prepared in Examples 1-6 and Comparative Examples 5-6 was tested, and the results are as follows: Figure 2 As shown.

[0052] The antibacterial properties of the composite films prepared in Examples 1-6 and Comparative Examples 1-4 were tested under both light-free and light-illuminated conditions, and the results are shown in Tables 3 and 4.

[0053] The biocompatibility of the composite membranes prepared in Examples 1-6 and Comparative Examples 1-4 was tested. Osteoblasts were seeded onto the composite membranes and cultured for 6 days. The results are as follows: Figure 3 As shown.

[0054] Table 1. Mechanical properties of the composite membranes in Examples 1-6 of the present invention

[0055]

[0056] Table 2. Mechanical properties of the composite membranes in Comparative Examples 1-3 and 5-6 of the present invention

[0057]

[0058] Table 3. Antibacterial rate test results of samples from Examples 1-6 of the present invention.

[0059]

[0060] Table 4. Antibacterial rate test results of the composite films in Comparative Examples 1-4 of the present invention

[0061]

[0062] As shown in Tables 1 and 2, the composite film prepared by the present invention has good strength and toughness, which makes the TiO2 nanotube film layer less prone to cracking or curling.

[0063] Depend on Figure 2 It can be seen that the composite membrane prepared by the present invention has good sustained-release performance and will not exhibit burst release, indicating that the composite membrane can ensure the long-term effectiveness of the drug.

[0064] As shown in Tables 3 and 4, the composite membrane prepared by this invention has certain antibacterial properties, and the antibacterial performance of the composite membrane is further improved under near-infrared laser irradiation. This indicates that the drug sustained-release carried by TiO2 nanotubes can endow the composite membrane with antibacterial properties. Furthermore, the black scale nanoparticles or MXene particles in the bottom support membrane can exert their photothermal effect under laser irradiation, further promoting the antibacterial performance of the composite membrane.

[0065] Depend on Figure 3It is evident that the composite membrane prepared in this invention, by loading and releasing bone morphogenetic protein-2, can better promote osteoblast growth and proliferation, indicating that the prepared composite membrane has good biocompatibility.

Claims

1. A functional silk fibroin / titanium dioxide nanotube composite membrane, characterized in that, The composite membrane is a three-layer structure: the lower layer is a support membrane with silk fibroin as the matrix, the middle layer is a titanium dioxide nanotube membrane separated from the metal substrate, and the upper layer is a polymer electrospun fiber layer. The preparation method of the functional silk fibroin / titanium dioxide nanotube composite film includes the following steps: (1) Coating the silk fibroin mixture solution onto the surface of a titanium-based metal substrate, drying it, and then peeling off the film; the titanium-based metal substrate is anodized titanium sheet / foil or titanium alloy, the surface of the anodized titanium sheet / foil or titanium alloy has a uniform titanium dioxide nanotube structure, and the average inner diameter of the titanium dioxide nanotubes is 50-500 nm and the average tube length is 1-6 μm; the entire titanium dioxide nanotube layer is transferred to a self-supporting film; the silk fibroin mixture solution includes regenerated silk fibroin solution, crosslinking agent, short fibers and nanoparticles; the mass ratio of the regenerated silk fibroin, crosslinking agent, short fibers and nanoparticles is 1-0.8:0-0.1:0.01-0.1:0.01-0.1; the preparation process of the silk fibroin mixture solution is as follows: after mixing the regenerated silk fibroin solution, crosslinking agent, short fibers and nanoparticles, the mixture solution is concentrated to 3-20%; the short fibers are degummed silk fibers after shearing, and the average fiber length is 0.1~5 mm; (2) The membrane after stripping is treated with a corrosive agent, then the membrane is immersed in a drug solution and then dried. (3) The dried membrane is used as the receiving electrode, and polymer electrospun fibers are grown on the membrane by electrospinning to seal the titanium dioxide nanotubes on the membrane to obtain a composite membrane; the spinning time is 2-8h.

2. A method for preparing the functional silk fibroin / titanium dioxide nanotube composite membrane according to claim 1, characterized in that, Includes the following steps: (1) Coating the silk fibroin mixture solution onto the surface of a titanium-based metal substrate, drying it, and then peeling off the film; the titanium-based metal substrate is anodized titanium sheet / foil or titanium alloy, the surface of the anodized titanium sheet / foil or titanium alloy has a uniform titanium dioxide nanotube structure, and the average inner diameter of the titanium dioxide nanotubes is 50-500 nm and the average tube length is 1-6 μm; the entire titanium dioxide nanotube layer is transferred to a self-supporting film; the silk fibroin mixture solution includes regenerated silk fibroin solution, crosslinking agent, short fibers and nanoparticles; the mass ratio of the regenerated silk fibroin, crosslinking agent, short fibers and nanoparticles is 1-0.8:0-0.1:0.01-0.1:0.01-0.1; the preparation process of the silk fibroin mixture solution is as follows: after mixing the regenerated silk fibroin solution, crosslinking agent, short fibers and nanoparticles, the mixture solution is concentrated to 3-20%; the short fibers are degummed silk fibers after shearing, and the average fiber length is 0.1~5 mm; (2) The membrane after stripping is treated with a corrosive agent, then the membrane is immersed in a drug solution and then dried. (3) The dried membrane is used as the receiving electrode, and polymer electrospun fibers are grown on the membrane by electrospinning to seal the titanium dioxide nanotubes on the membrane to obtain a composite membrane; the spinning time is 2-8h.

3. The method for preparing the functional silk fibroin / titanium dioxide nanotube composite membrane according to claim 2, characterized in that, The regenerated silk fibroin solution is a silkworm fibroin and / or tussah silkworm fibroin solution, and the concentration of the regenerated silk fibroin solution is 1-10%.

4. The method for preparing the functional silk fibroin / titanium dioxide nanotube composite membrane according to claim 2, characterized in that, The crosslinking agent is at least one of genipin, a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide, horseradish peroxidase, and glutaraldehyde.

5. The method for preparing the functional silk fibroin / titanium dioxide nanotube composite membrane according to claim 2, characterized in that, The nanoparticles are black scale nanoparticles and / or two-dimensional transition metal carbon-nitrogen compounds, and the average particle size of the nanoparticles is 40-400 nm.

6. The method for preparing the functional silk fibroin / titanium dioxide nanotube composite membrane according to claim 2, characterized in that, In step (3), the polymer is at least one of silk fibroin, collagen, gelatin, chitin and its derivatives, and alginate.

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