Method for producing ultra-high modulus protein fiber

Abstract

To provide a protein fiber having high elasticity usable for producing a decellularized artificial organ.SOLUTION: A method for producing an ultra-high elasticity protein fiber comprises discharging fibrous protein into an aqueous solution of an organic solvent having a polarity of 5 to 6 and drying the obtained fiber. The ultra-high elasticity protein fiber is obtained by the method.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a protein fiber having an extremely high elastic modulus, and to a protein fiber obtained thereby. [Background technology]

[0002] In regenerative medicine, constructing tissues and organs with complex three-dimensional structures and functions similar to those of living organisms outside the body remains a major challenge. Therefore, attention has been focused on decellularized tissues obtained by treating animal tissues and organs with surfactants or ultra-high pressure to remove cellular components. Decellularized tissues are mainly composed of extracellular matrix (ECM) components and retain the complex tissue structure of living tissues, so they are expected to be useful as transplant organs in regenerative medicine by filling them with human cells (Non-Patent Document 1). However, decellularized organs have problems such as immunogenicity and size mismatch with human organs. Therefore, it is expected that these problems can be solved if decellularized organs can be artificially produced. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] K. Uygun et al., Nat. Med. 2008, 16, 814 Summary of the Invention [Problem to be solved by the invention]

[0004] In order to artificially create decellularized organs, it is first necessary to create fibers that have a high elastic modulus similar to that of biological fibers such as biological collagen fibers and are also biocompatible. [Means for solving the problem]

[0005] The present inventors have conducted extensive research to solve the above problems, and have found that protein fibers having an extremely high elastic modulus can be obtained by discharging a fibrous protein solution into an aqueous solution containing an organic solvent having a polarity within a specific range and drying the obtained fibers, thereby completing the present invention.

[0006] (1) A method for producing protein fibers, comprising discharging a fibrous protein solution into an aqueous solution containing an organic solvent and drying the obtained fibers. (2) The method according to (1), wherein the polarity of the organic solvent is 5 to 6. (3) The method according to (1) or (2), wherein the concentration of the organic solvent in the aqueous solution is 50 to 80 vol %. (4) The method according to (1) or (2), wherein the concentration of the organic solvent in the aqueous solution is 60 to 80 vol %. (5) The method according to any one of (1) to (4), wherein the organic solvent is selected from the group consisting of acetonitrile, acetone and methanol. (6) The method according to any one of (1) to (4), wherein the organic solvent is acetonitrile, and the concentration of acetonitrile in the aqueous solution is 60 to 70 vol %. (7) The method according to any one of (1) to (4), wherein the organic solvent is acetone, and the concentration of acetone in the aqueous solution is 60 to 80 vol %. (8) The method according to any one of (1) to (4), wherein the organic solvent is methanol and the concentration of methanol in the aqueous solution is 60 vol %. (9) The method according to any one of (1) to (8), wherein the fibrous protein is selected from the group consisting of type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, keratin, myosin, and elastin. (10) The method described in (9), wherein the fibrous protein is type I collagen. (11) The method according to any one of (1) to (10), wherein the drying is carried out by drying under reduced pressure. (12) The method according to any one of (1) to (11), wherein the obtained protein fiber has an elastic modulus of 3 GPa or more. (13) The method described in (11), wherein the obtained protein fiber has an elastic modulus of 10 GPa or more. Effect of the Invention

[0007] According to the present invention, protein fibers with a very high elastic modulus can be easily obtained. For example, when producing collagen fibers, protein fibers with a high elastic modulus comparable to or exceeding that of spider silk can be obtained. The method of the present invention can also be applied to a submerged printing method to create decellularized organs and blood vessels with high strength. Since many fibrous proteins are derived from living organisms, the protein fibers obtained by the present invention are also excellent in biocompatibility. [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows the effect of the concentration of organic solvents on the elastic modulus of collagen fibers. The numbers in parentheses indicate the polarity of each solvent. [Diagram 2] Figure 2 shows the relationship between the maximum elastic modulus of collagen fibers obtained using an aqueous solution of an organic solvent and the polarity of the organic solvent. The numbers in parentheses indicate the polarity of each solvent. [Diagram 3] The upper panel of Fig. 3 shows the results of X-ray diffraction measurement of collagen fibers obtained using a 60% acetonitrile aqueous solution, and the lower panel of Fig. 3 shows the results of X-ray diffraction measurement of collagen fibers obtained using a 100% acetonitrile aqueous solution. [Figure 4] The upper panel of Fig. 4 shows a photograph of collagen fibers obtained after freeze-drying (left) and collagen fibers swollen in a 60% acetonitrile aqueous solution (right). The lower panel of Fig. 4 shows the elastic modulus of the fibers obtained by discharging a 1 wt% collagen aqueous solution and a 2 wt% collagen aqueous solution into a 60% acetonitrile aqueous solution, drying in air, and then drying under reduced pressure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] In this specification, unless otherwise specified, the concentration of an organic solvent is expressed in volume percent (vol%). Furthermore, in this specification, unless otherwise specified, a numerical value representing a concentration or strength may include a range of that numerical value ±5% or that numerical value ±10%.

[0010] The present invention provides a method for producing protein fibers, comprising discharging a fibrous protein solution into an aqueous solution containing an organic solvent, and drying the obtained fibers.

[0011] The elastic modulus of the protein fiber obtained in the present invention varies depending on the polarity and concentration of the organic solvent, and the type and concentration of the fibrous protein. In order to obtain a protein fiber with a high elastic modulus, the polarity of the organic solvent used is preferably 4.5 to 6.5, and more preferably 5 to 6. The organic solvent used in the present invention is a polar organic solvent. Examples of organic solvents preferably used in the present invention include, but are not limited to, acetonitrile, acetone, and methanol. The polarity of many organic solvents is known in the art. In this specification, the polarity of the organic solvent is Snyder's polarity index (Snyder, LR Classification of the Solvent Properties of Common Liquids. Journal of Chromatographic Science 16, 223-234 (1978)).

[0012] The preferred concentration of the organic solvent for obtaining high modulus protein fibers varies depending on the type of organic solvent and the type and concentration of the fibrous protein. Usually, the preferred concentration of the organic solvent is 50% to 90%, more preferably 50% to 80%, and even more preferably 60 to 80%. For example, in the case of an acetonitrile aqueous solution, the preferred acetonitrile concentration is 50 to 80%, more preferably 60 to 70%. In the case of an acetone aqueous solution, the preferred acetone concentration is 50 to 90%, more preferably 60 to 80%. In the case of a methanol aqueous solution, the preferred methanol concentration is 50 to 70%, more preferably 60%. The above preferred organic solvents and organic solvent concentrations are merely examples and are not limited thereto.

[0013] The organic solvent in the aqueous organic solvent solution may be of one type or of multiple types.

[0014] The relationship between the polarity of the organic solvent and the elastic modulus of the protein fiber is considered as follows. When the organic solvent has a moderate polarity, it penetrates into the fiber and easily interacts with water molecules, and is volatilized together with the water molecules during the drying process. When the organic solvent has a moderate polarity, the removal of water molecules from the fiber is promoted, so the orientation of the protein in the fiber increases, and the elastic modulus of the fiber increases. When the polarity of the organic solvent is too low, the organic solvent does not easily penetrate into the protein fiber, so the water molecules inside the fiber are difficult to remove. When the polarity of the organic solvent is too high, the organic solvent remains in the fiber and swells the fiber together with the water molecules. When the polarity of the organic solvent is too low or too high, the orientation of the protein in the fiber does not increase, and the elastic modulus of the fiber does not increase.

[0015] A person skilled in the art can select the type and concentration of the solvent according to the type of fibrous protein by ejecting a fibrous protein solution from a nozzle or the like into an aqueous organic solvent solution and measuring the strength of the obtained protein fibers using a known tensile test.

[0016] Fibrous proteins are proteins with long fibrous structures, and many fibrous proteins constitute connective tissues, tendons, bones, and skeletal muscles in living organisms. Examples of fibrous proteins include, but are not limited to, collagen, keratin, myosin, elastin, resilin, silk protein, and amyloid. Fibrous protein families are also included. For example, collagen includes types I, II, III, V, and XI collagen, and atelocollagen. Fibrous proteins may be derived from any part of any animal. Methods for obtaining and purifying fibrous proteins from living organisms are known. Fibrous proteins may be wild-type proteins, mutant proteins, or modified proteins. Fibrous proteins may be extracted from living tissues, or may be artificially produced by methods such as chemical synthesis of peptides or genetic recombination. Depending on the physical properties, such as the strength and shape, and the chemical and biological properties of the desired protein fibers, the type of fibrous protein can be selected, or mutations and modifications can be added by known methods.

[0017] The fibrous protein solution can be prepared by dissolving the fibrous protein in a solvent. The type of solvent for dissolving the fibrous protein is not particularly limited, and can be appropriately selected depending on the type and concentration of the fibrous protein, the required fiber strength, and the like. Examples of the solvent include water, ionic liquid, phosphate buffered saline, culture solution, and mixtures thereof. The concentration of the fibrous protein in the fibrous protein solution can be appropriately selected depending on the type of the fibrous protein, the type of solvent, the required fiber strength, and the like. The concentration of the fibrous protein may be, for example, 0.5 to 20% by weight, 1 to 10% by weight, 2 to 7% by weight, and the like. The solvent may contain a buffer, a salt, an ionic liquid electrolyte, and the like.

[0018] The ejection of the fibrous protein solution into the aqueous solution of the organic solvent can be performed by a known method, for example, using a nozzle or a syringe needle. Usually, the fibrous protein is ejected as an aqueous solution. The conditions such as the size and shape of the ejection port and the ejection speed can be changed according to the desired shape and strength of the protein fiber. The ejection of the fibrous protein solution may be performed according to a known wet spinning method. The fibrous protein may be ejected by using a combination of a known liquid printing method and a 3D printer to mold it according to the shape of a blood vessel or an organ.

[0019] The temperature of the aqueous solution of the organic solvent is not particularly limited, but is preferably a temperature that does not reduce the strength of the resulting fiber. The temperature of the aqueous solution of the organic solvent can be appropriately selected depending on the type of fibrous protein, the type and concentration of the organic solvent, etc. Examples of such temperatures include, but are not limited to, 0°C to 10°C, 10°C to 20°C, 20°C to 30°C, 30°C to 40°C, 40°C to 50°C, and room temperature.

[0020] The aqueous solution of the organic solvent may contain, in addition to the organic solvent and water, for example, inorganic salts, organic salts, proteins, growth factors, organic low molecular weight compounds, inorganic substances, etc., provided that these substances are soluble or dispersible in the aqueous solution of the organic solvent used.

[0021] The fibers of the fibrous protein discharged into the aqueous solution of the organic solvent may be stretched before drying. The stretching can be performed by a known method. The stretching is preferably performed during fiber formation in the aqueous solution of the organic solvent or during fiber drying.

[0022] The fibers of fibrous protein may be crosslinked. Crosslinking methods are known, including, but not limited to, crosslinking using a crosslinking agent such as formaldehyde, glutaraldehyde, carbodiimide, etc., and crosslinking using transglutaminase. Crosslinking the fibers can not only increase the strength of the fibers, but also prevent the fibers from redissolving in an aqueous solution.

[0023] In the method of the present invention, the fibers obtained by discharging the fibrous protein solution into an aqueous organic solvent solution are dried. Drying can be performed by a known method. Air drying, ventilation drying, reduced pressure drying, freeze drying, etc. can be used, but the drying method is not limited to these.

[0024] The shape of the protein fibers produced by the method of the present invention can vary depending on the application of the fibers, etc. In addition to threads, the protein fibers may be formed into rods, needles, columns, plates, films, spheres, blocks, etc. The strength of the fibers may be increased by twisting or weaving the fibers.

[0025] The method of the present invention can obtain protein fibers having a high elastic modulus. By using reduced pressure drying in the drying step, protein fibers with an even higher elastic modulus can be obtained. The elastic modulus of the protein fibers obtained by the method of the present invention can be 2 GPa to 3 GPa or more. In the method of the present invention, fibers with a higher elastic modulus can be obtained by thoroughly drying the protein fibers using reduced pressure drying or the like. In the method of the present invention, the elastic modulus of the protein fibers when drying is performed by reduced pressure drying can be 5 GPa to 10 GPa or more. In addition, in the method of the present invention, the tensile strength of the protein fibers when drying is performed by reduced pressure drying can be 1 GPa to 2 GPa or more. The tensile strength and elastic modulus can be measured and calculated using a tensile strength tester. Various tensile strength testers have been devised and are commercially available, and can be used as appropriate. EXAMPLES

[0026] 1. Consideration of the type and concentration of organic solvent A type I atelocollagen derived from porcine skin (provided by Nippon Ham Co., Ltd.) was used as a fibrous protein, and its aqueous solution (1 weight percent (wt%)) was prepared. 0.5 mL of the type I atelocollagen aqueous solution was discharged into 50 mL of each organic solvent aqueous solution using a syringe pump to obtain fibers. The organic solvents tested were 2-propanol, ethanol, methanol, acetone, acetonitrile, N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO). The organic solvent concentrations were 50%, 60%, 70%, 80%, 90%, and 100%. The obtained fibers were air-dried for 5 minutes, and the tensile modulus was measured. The results are shown in Figure 1. The elastic modulus of the fibers was high when acetonitrile, acetone, and methanol were used. In the case of acetonitrile, the elastic modulus of the fibers was relatively high at a concentration of 50 to 80%, and further increased at a concentration of 60 to 70%, and the elastic modulus of the fibers was the highest at a concentration of 60% (about 3 GPa). In the case of acetone, the elastic modulus of the fiber was relatively high at a concentration of 50-90%, and further increased at a concentration of 60-80%, with the elastic modulus of the fiber reaching its maximum at a concentration of 80% (approximately 2.7 GPa). In the case of methanol, the elastic modulus of the fiber was relatively high at a concentration of 50-70%, and reached its maximum at a concentration of 60% (approximately 2.7 GPa). The relationship between the maximum elastic modulus of the collagen fiber obtained using the aqueous solution of each organic solvent and the polarity of the organic solvent is summarized in Figure 2. These results show that the elastic modulus of the collagen fiber tends to be high when the polarity of the organic solvent is 5-6. On the other hand, when the polarity of the organic solvent is lower or higher than 5-6, the elastic modulus of the collagen fiber tends to be low.

[0027] 2. Examination of protein orientation in fibers X-ray diffraction measurements were performed on type I collagen fibers prepared using 60% acetonitrile and type I collagen fibers prepared using 100% acetonitrile. The fibers used were air-dried. The results are shown in Figure 3. In type I collagen fibers prepared using 60% acetonitrile, characteristic peaks were observed in the left-handed helical chains of the three-helix collagen molecules indicated by the arrows, indicating high orientation of the collagen molecules in the fibers. On the other hand, in type I collagen fibers prepared using 100% acetonitrile, these peaks disappeared, indicating denaturation of the fibers. These results indicate that fibers with a high elastic modulus have a high degree of orientation.

[0028] 3. Collagen concentration and drying method An aqueous solution (2 wt%) of type I atelocollagen derived from pig skin was prepared and extruded into a 60% aqueous acetonitrile solution in the same manner as above. The resulting fiber was air-dried for 5 minutes and then dried under reduced pressure overnight, and the tensile modulus was measured. As a result, an ultra-high modulus fiber of approximately 10 GPa was obtained (graph in Figure 4). This modulus was comparable to or exceeded that of spider silk. The photograph in the upper left of Figure 4 shows the fiber after drying under reduced pressure, and the photograph in the upper right shows the fiber swollen in a 60% aqueous acetonitrile solution. The modulus and tensile stress of the protein fiber obtained in this experiment are shown in Table 1, in comparison with existing fibers, bone, and tendon. [Table 1] [Industrial Applicability]

[0029] The protein fibers obtained by the present invention exhibit extremely high elastic modulus and excellent biocompatibility, and therefore can be applied to regenerative medicine and biodegradable high-strength materials.

Claims

1. A method for producing a protein fiber, comprising discharging a fibrous protein solution into an aqueous solution containing an organic solvent, and drying the obtained fiber, (a) the organic solvent is acetonitrile, and the concentration of acetonitrile in the aqueous solution is 50 to 80 vol %, or (b) the organic solvent is acetone, and the concentration of acetone in the aqueous solution is 60 to 80 vol %; The fibrous protein is selected from the group consisting of type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, keratin, and elastin; method.

2. 2. The method of claim 1, wherein the fibrous protein is type I collagen.

3. 3. The method according to claim 1, wherein the drying is carried out by vacuum drying.

Images