JP6717459B2 - Nanofiber manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a nanofiber using a biodegradable material and a method for producing the nanofiber.

In recent years, it has been feared that plastic waste has a great impact on the global environment, such as the impact on the ecosystem, the generation of harmful gases during combustion, and the global warming caused by a large amount of combustion heat. Biodegradable plastics are being actively developed as plastics that can solve the above problems.

Among them, plant-derived biodegradable plastics are said to have carbon dioxide produced when they were burned originally in the air, and carbon dioxide in the atmosphere is said not to increase. This is called carbon neutral, and it is emphasized under the Paris Agreement that imposes a carbon dioxide reduction target value, and active use of plant-derived biodegradable plastics is desired.

Recently, from the viewpoint of biodegradability and carbon neutrality, aliphatic polyester-based resins have attracted attention as plant-derived biodegradable plastics. Among them, polyhydroxyalkanoate (hereinafter sometimes abbreviated as PHA), of which poly(3-hydroxybutyrate) homopolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) are particularly preferable. ) Copolymer, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer (hereinafter sometimes referred to as PHBH), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Rate) copolymers, polylactic acid, and the like are drawing attention.

Of these PHA, studies are underway to make poly-3-hydroxyalkanoic acid and the like into nanofibers, but as such biodegradable nanofibers, the market demand is satisfied compared to general nanofibers. Those having such mechanical properties have not been obtained. Further, among PHA, PHBH is slow to crystallize, and thus it is difficult to form fibers by a usual melt spinning method, and in addition, the produced nanofibers have variations in shape, and uniform nanofibers cannot be obtained. There was a problem.

Therefore, fiberization using an electrospinning method (electrospinning method) instead of the melt spinning method has been studied. This is because the electrospinning method enables spinning even with a material such as PHBH that is slow to crystallize. Non-Patent Documents 1 and 2 disclose a method for producing a fiber mat by an electrospinning method using various types of PHA. Further, it is disclosed that the produced fiber mat can be used as a scaffold having biocompatibility and bioabsorbability in medical material applications.

“Polymer”, 2007, 48, p.1419-1427 “Biomaterials”, 2008, 29, p.1307-1317

Since PHA is a polymer showing hydrophobicity and poor hydrophilicity, how to control cell adhesiveness has been a problem in applying nanofibers using PHA as a material to medical materials.

In view of the above situation, an object of the present invention is to provide a nanofiber having a highly uniform fiber shape in which hydrophilicity and hydrophobicity are controlled while using polyhydroxyalkanoate as a raw material.

The present inventors have conducted extensive studies to solve such problems, and by mixing polyvinyl alcohol as a hydrophilic material in a polyhydroxyalkanoate copolymer to form nanofibers by an electrospinning method, The present inventors have found that nanofibers having a highly uniform fiber shape can be obtained while controlling hydrophilicity and hydrophobicity, and the present invention has been completed. That is, the present invention relates to nanofibers formed from a mixture of a polyhydroxyalkanoate copolymer and polyvinyl alcohol.

Preferably, the polyhydroxyalkanoate copolymer is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).

Preferably, the mass ratio of the polyhydroxyalkanoate copolymer to polyvinyl alcohol is 95:5 to 5:95.

Preferably, the fiber diameter of the nanofibers is 100 nm or more and less than 1,000 nm.

The present invention also relates to a fiber laminated structure formed by laminating a plurality of sheets of nanofibers.

Further, the present invention relates to a method for producing the nanofiber, which comprises a step of electrospinning a solution containing a mixture of a polyhydroxyalkanoate copolymer and polyvinyl alcohol.

Preferably, the solvent constituting the solution is a mixed liquid of fluoroalcohol and water.

Preferably, the content of water with respect to the entire liquid mixture of fluoroalcohol and water is 50% by mass or less.

ADVANTAGE OF THE INVENTION According to this invention, while using polyhydroxyalkanoate as a raw material, the nanofiber which has a highly uniform fiber shape whose hydrophilicity and hydrophobicity are controlled can be provided.

An electron micrograph of the nanofibers produced in Example 1. An electron micrograph of the nanofibers produced in Example 2 An electron micrograph of the nanofibers produced in Example 3 An electron micrograph of the nanofibers produced in Example 4 An electron micrograph of the nanofibers produced in Example 5. An electron micrograph of the nanofibers produced in Example 6 An electron micrograph of the nanofibers produced in Example 7. Electron micrograph of the nanofiber produced in Reference Example 1 An electron micrograph of the nanofiber produced in Reference Example 2

The present invention will be described in detail below.

The present invention relates to nanofibers formed from a mixture of polyhydroxyalkanoate copolymer and polyvinyl alcohol. In the present invention, by using a hydrophobic polyhydroxyalkanoate copolymer and hydrophilic polyvinyl alcohol in combination, it is possible to provide nanofibers whose hydrophilicity and hydrophobicity are controlled.

[Polyhydroxyalkanoate copolymer]
Polyhydroxyalkanoate copolymer employed in the present invention (hereinafter sometimes abbreviated as PHA copolymer) represented by the general formula _{(1): [- CHR-} CH 2 -CO-O -] ( wherein, R Is an alkyl group represented by C _n H _2n+1 , and n is an integer of 1 or more and 15 or less.) is an aliphatic polyester copolymer containing two or more kinds of repeating units represented by The form of copolymerization is not particularly limited, and may be random copolymerization, alternating copolymerization, block copolymerization, graft copolymerization, etc., but random copolymerization is preferred because it is easily available. PHA copolymers have the advantages of lower melting point, higher ductility and higher flexibility than poly(3-hydroxybutyrate) homopolymer.

The PHA copolymer used in the present invention is not particularly limited, but examples thereof include PHBH [poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyric acid-co-3-hydroxy). Hexanoic acid)], PHBV [poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid)], P3HB4HB[poly(3-hydroxybutyrate) -Co-4-hydroxybutyrate), poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid)], poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid-co-3-hydroxyhexanoic acid), Examples thereof include poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) and poly(3-hydroxybutyrate-co-3-hydroxyoctadecanoate). Among these, PHBH, PHBV, and P3HB4HB are mentioned as those that are industrially easily produced. Among these copolymers, PHBH is particularly preferable. PHBH has a wide range of processing temperatures that can be applied during heat processing, and therefore has good processability when secondary processing a sheet made of nanofibers, and has low crystallinity, and therefore has high biodegradability and PVAH. This is because it has the advantage of being easily mixed uniformly.

The average composition ratio of the repeating units in the PHA copolymer, particularly PHBH, is 80 mol% to 99 mol% when the composition ratio of the 3-hydroxybutyrate repeating units is 80 mol% from the viewpoint of the balance between flexibility and strength of the obtained nanofibers. It is preferably present, and more preferably 85 mol% to 97 mol %. If the composition ratio of the 3-hydroxybutyrate repeating unit is less than 80 mol %, the rigidity tends to be insufficient, and if it exceeds 99 mol %, the flexibility tends to be insufficient.

In the present invention, the PHA copolymer may be used alone or in combination of at least two kinds. For example, two or more PHA copolymers having different composition ratios of 3-hydroxybutyrate repeating units can be mixed and used. The molecular weight of the PHA copolymer used in the present invention is not particularly limited as long as it shows substantially sufficient physical properties for the intended use. When the molecular weight is low, the strength of the obtained nanofiber may decrease. On the other hand, if it is too high, it may be difficult to form fibers. Considering these, the range of the weight average molecular weight of the PHA copolymer used in the present invention is preferably 50,000 to 3,000,000, more preferably 100,000 to 1,500,000. In addition, the weight average molecular weight here means what was measured from polystyrene conversion molecular weight distribution using the gel permeation chromatography (GPC) which used a chloroform eluent. As a column in the GPC, a column suitable for measuring the molecular weight may be used.

The PHA copolymer is preferably one produced by a microorganism, and the microorganism producing the PHA copolymer is not particularly limited as long as it is a microorganism capable of producing PHA. For example, as a copolymer-producing bacterium of hydroxybutyrate and other hydroxyalkanoate, PHBV and PHBH-producing bacteria Aeromonas caviae, P3HB4HB-producing bacterium Alcaligenes eutrophus, etc. Are known. In particular, regarding PHBH, in order to improve the productivity of PHBH, an Alcaligenes eutrophus AC32 strain (Alcaligenes eutrophus AC32, FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol) was introduced. , 179, p4821-4830 (1997)) and the like are more preferable. In addition to the above, genetically modified microorganisms into which various PHA synthesis-related genes have been introduced can be used according to the PHA copolymer to be produced.

After culturing these microorganisms under appropriate conditions to accumulate the PHA copolymer in the cells, the cells can be crushed to separate the PHA copolymer.

[Polyvinyl alcohol]
Commercially available PVA can be used as polyvinyl alcohol (hereinafter sometimes abbreviated as PVA) used in the present invention, and is not particularly limited. The degree of saponification of the PVA which can be used may be in the range of, for example, 88 to 100%, preferably 88 to 99.8%. The degree of polymerization may be in the range of 800 to 8000. The stereoregularity of PVA is not particularly limited, and PVA having any structure of atactic, isotactic and syndiotactic can be used. Further, a PVA copolymer such as an ethylene-vinyl alcohol copolymer or a modified PVA in which a substituent is introduced into PVA can also be used as the PVA of the present invention. Since PVA has high hydrophilicity and excellent biocompatibility, nanofibers containing PVA as one of the polymer components are suitable for medical material applications.

In the nanofiber of the present invention, the ratio of PHA copolymer and PVA is not particularly limited, but is preferably 95:5 to 5:95 on a mass basis, and 90:10 to 10:90, for hydrophilicity and hydrophobicity control. Is more preferable. The hydrophilicity and hydrophobicity of the nanofibers can be controlled by adjusting the ratio of both polymers, and the hydrophilicity and hydrophobicity suitable for the application can be imparted to the nanofibers. Further, according to the production method using a specific mixed solvent described below, it is possible to produce nanofibers having a relatively high hydrophilicity, in which the ratio of PVA to the total of PHA copolymer and PVA exceeds 50% by mass. In this case, the ratio of PHA copolymer to PVA is preferably 40:60 to 5:95 on a mass basis, and more preferably 30:70 to 10:90.

The nanofiber of the present invention may contain components other than the PHA copolymer and PVA within the range in which the effects of the present invention are exhibited. Examples of such components include other polymer components; antioxidants, ultraviolet absorbers, colorants such as dyes and pigments, plasticizers, lubricants, inorganic fillers, antistatic agents, and other additive components; crystallization rate. Examples include a nucleating agent for adjusting. The content of these other components is not particularly limited as long as the effects of the present invention are not impaired.

[Nanofiber]
The nanofiber of the present invention preferably means a nanosize fiber having a fiber diameter of less than 1,000 nm. Nano-sized fibers are known to have excellent filtration performance, wiping performance, liquid retention performance, etc. due to their morphology such as low porosity and large specific surface area. The fiber diameter is preferably less than 1000 nm, more preferably less than 800 nm, still more preferably less than 700 nm. Furthermore, according to the present invention, it is possible to produce extremely thin nanofibers having a fiber diameter of 200 nm or less. The lower limit of the fiber diameter is not particularly limited, but is preferably 100 nm or more.

The nanofibers of the present invention can be produced by the electrospinning method described later, and thereby a sheet (nonwoven fabric) made of nanofibers can be formed. Since the nanofibers produced by the electrospinning method are porous, they are suitable as biocompatible and bioabsorbable scaffolds.

Further, by stacking a plurality of such sheets, it is possible to manufacture a thick fiber laminated structure. The thickness of such a fiber laminated structure can be easily adjusted depending on the number of laminated layers, and it can be suitably used as a biodegradable medical material.

[Production method]
The nanofibers of the present invention can be produced by electrospinning a solution containing a mixture of PHA copolymer and PVA. To perform electrospinning, a commercially available electrospinning device can be used. The conditions for electrospinning are not particularly limited and may be appropriately selected so as to obtain a desired nanofiber.

A solvent that can dissolve the PHA copolymer and PVA is selected as a solvent for forming the solution. Examples of such a solvent include HFIP (formal name: 1,1,1,3,3,3-hexafluoro-2-propanol), TFE (CF ₃ CH ₂ OH), TFP (HCF ₂ CF ₂ CH). ₂ OH), C ₆ F ₁₃ C ₂ H ₄ OH and other fluoroalcohols; dioxane; dimethylsulfoxide; water and the like. Of these, fluoroalcohol is preferable, and HFIP is particularly preferable. One or more of these may be used, or two or more of them may be mixed and used in consideration of the solubility of both the PHA copolymer and PVA. Further, when the ratio of PVA to the total amount of PHA copolymer and PVA exceeds 50% by mass and the hydrophilicity of the polymer component becomes high, the solubility in fluoroalcohol decreases, so that the solvent may be a mixture of fluoroalcohol and water. It is preferable to use a liquid, and it is more preferable to use a mixed liquid of HFIP and water. By using such a mixed solution as a solvent during electrospinning, it becomes possible to produce nanofibers having a PVA ratio of more than 50% by mass and high hydrophilicity.

In the mixed liquid of fluoroalcohol and water, the ratio of fluoroalcohol and water is not particularly limited, and may be appropriately determined depending on the degree to which the mixture of PHA copolymer and PVA is dissolved. However, since the production of nanofibers is easy, it is preferable that the content of water is 50% by mass or less based on the entire mixed liquid of fluoroalcohol and water. Furthermore, the ratio by mass of fluoroalcohol to water is more preferably in the range of 95:5 to 50:50, further preferably in the range of 95:5 to 60:40, and 90:10 to 70. It is particularly preferably in the range of :30. Further, by adjusting the ratio of fluoroalcohol and water to make the ratio of water relatively large, it becomes possible to manufacture extremely thin nanofibers having a fiber diameter of 200 nm or less. The mass-based ratio of fluoroalcohol and water in this case depends on the ratio of the PHA copolymer and PVA and therefore cannot be specified unconditionally, but for example, 80:20 to 50:50 is preferable, and 80:20 to 60:40 is more preferable, and 80:20 to 70:30 is further preferable.

The concentration of the solution containing the mixture of the PHA copolymer and PVA may be appropriately selected so that it can be formed into fibers by electrospinning. Generally, when the concentration is high, the fiber diameter of the nanofibers tends to be large, but when the concentration is too high, the fibers do not become fibers and tend to become lumps. On the other hand, if the concentration is too low, it does not become a fiber and tends to be in the form of particles. From these, the concentration of the solution is preferably about 0.5 to 5% by mass, more preferably about 0.5 to 3% by mass.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

<Production Example 1> Production of PHBH KNK-005 strain (see US Pat. No. 7,384,766) was used for culture production.

The composition of Tanehaha medium 1w / v% Meat-extract, 1w / v% Bacto-Tryptone, 0.2w / v% Yeast-extract, 0.9w / v% Na 2 HPO 4 · 12H 2 O, 0.15w / v% KH ₂ PO _4, and the (pH6.8).

The composition of the preculture medium is 1.1 w/v% Na ₂ HPO ₄ ·12H ₂ O, 0.19 w/v%
KH ₂ PO ₄ , 1.29 w/v% (NH ₄ ) ₂ SO ₄ , 0.1 w/v% MgSO ₄ .7H ₂ O, 0.5 v/v% Trace metal salt solution (1. 6 w/v% FeCl ₃ .6H ₂ O, 1 w/v% CaCl ₂ .2H ₂ O, 0.02 w/v% CoCl ₂ .6H ₂ O, 0.016 w/v% CuSO ₄ .5H ₂ O, 0. 012 w/v% NiCl ₂ .6H ₂ O dissolved therein.). As a carbon source, palm oil was added all at once at a concentration of 10 g/L.

The composition of the production medium was 0.385 w/v% Na ₂ HPO ₄ ·12H ₂ O, 0.067 w/v% KH ₂ PO ₄ , 0.291 w/v% (NH ₄ ) ₂ SO ₄ , 0.1 w/v. % MgSO ₄ .7H ₂ O, 0.5 v/v% trace metal salt solution (1.6 w/v% FeCl ₃ .6H ₂ O in 0.1N hydrochloric acid, 1 w/v% CaCl ₂ .2H ₂ O, 0. 02 w/v% CoCl ₂ .6H ₂ O, 0.016 w/v% CuSO ₄ .5H ₂ O, 0.012 w/v% NiCl ₂ .6H ₂ O dissolved therein), 0.05 w/v% BIOSPUREX 200K (Antifoaming agent: manufactured by Cognis Japan).

First, a glycerol stock (50 μl) of the KNK-005 strain was inoculated into a seed culture medium (10 ml) and cultured for 24 hours to perform seed culture. Next, the seed culture medium was inoculated at 1.0 v/v% into a 3 L jar fermenter (MDL-300 type manufactured by Maruhishi Bioengine) containing 1.8 L of the preculture medium. The operating conditions were a culturing temperature of 33° C., a stirring speed of 500 rpm, and an aeration rate of 1.8 L/min. A 14% aqueous ammonium hydroxide solution was used for pH control.

Next, the preculture liquid was inoculated at 1.0 v/v% into a 10 L jar fermenter (MDS-1000 type manufactured by Marubishi Bioengine) containing 6 L of the production medium. The operating conditions were a culture temperature of 28° C., a stirring speed of 400 rpm, and an aeration rate of 6.0 L/min, and the pH was controlled between 6.7 and 6.8. A 14% aqueous ammonium hydroxide solution was used for pH control. Palm oil was used as the carbon source. The culture was carried out for 64 hours, and after the culture was completed, the cells were collected by centrifugation, washed with methanol, freeze-dried, and the weight of the dried cells was measured.

100 ml of chloroform was added to 1 g of the obtained dry cells, and the mixture was stirred at room temperature for 24 hours to extract PHBH in the cells. The bacterial cell residue was filtered off, concentrated with an evaporator until the total volume reached 30 ml, 90 ml of hexane was gradually added, and the mixture was allowed to stand for 1 hour with slow stirring. The precipitated PHBH was filtered off and vacuum dried at 50° C. for 3 hours to obtain PHBH.

The 3HH composition analysis of the obtained PHBH was measured by gas chromatography as follows. To 20 mg of dried PHBH, 2 ml of a sulfuric acid-methanol mixed solution (15:85) and 2 ml of chloroform were added, and the mixture was sealed and heated at 100° C. for 140 minutes to obtain a methyl ester of a PHBH decomposition product. After cooling, 1.5 g of sodium hydrogen carbonate was added little by little to neutralize, and the mixture was allowed to stand until generation of carbon dioxide gas stopped. After adding 4 ml of diisopropyl ether and mixing well, the mixture was centrifuged, and the monomer unit composition of the polyester degradation product in the supernatant was analyzed by capillary gas chromatography. The gas chromatograph used was Shimadzu GC-17A, and the capillary column used was GL Science's NEUTRA BOND-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). He was used as a carrier gas, the column inlet pressure was 100 kPa, and 1 μl of the sample was injected. The temperature conditions were such that the initial temperature was raised from 100 to 200° C. at a rate of 8° C./min, and further from 200 to 290° C. at a rate of 30° C./min. As a result of analysis under the above conditions, PHBH had a monomer ratio of 3-hydroxyhexanoate (3HH) of 5.4 mol %. The weight average molecular weight Mw measured by GPC was 350,000, and the melting point was 141°C.

(Examples 1 to 7 and Reference Examples 1 and 2)
In each of the examples and reference examples, PHBH produced in Production Example 1 was used as PHBH.

The following PVA was used. Atactic polyvinyl alcohol, manufactured by Unitika Ltd., saponification degree 99.5 mol%, polymerization degree DP=1730.

PHBH and/or PVA with a weight ratio shown in Table 1 was dissolved in HFIP (manufactured by Wako Pure Chemical Industries, Ltd.) and/or water with a weight ratio shown in Table 1 as a solvent at a concentration shown in Table 1. Each polymer solution was obtained. In preparing each polymer solution, a homogenous solution was obtained by adding each polymer component to a solvent and then stirring at room temperature for 24 hours.

A glass syringe (capacity 10 cc, As One Co., Ltd.) containing each polymer solution was attached to an electrospinning device (Nanofiber Electrospinning Unit), and electrospinning was performed on a cooking sheet or aluminum foil wound on a target. A sheet composed of nanofibers having different polymer compositions was obtained. The syringe used was one in which the tip of an injection needle (0.6 mmφ×26 mm) (subcutaneous needle 1/2, Narita Injection Needle Mfg. Co., Ltd.) was scraped and flattened with a double-edged file. The conditions for electrospinning were: injection angle 30°, distance to target 15 cm, extrusion rate 0.3-0.4 mm/min, applied voltage 20 kV, ambient temperature 23-25°C, humidity 20-30%.

Each of the obtained nanofibers was photographed with a scanning electron microscope (JSM-6010LA, manufactured by JEOL Ltd.). The photographs taken are shown in FIGS. Obtain the fiber diameter of a plurality of fibers by comparison with the scale bar in each photograph, from the obtained value, determine the minimum fiber diameter in the nanofibers of each Example and Comparative Example, and the maximum fiber diameter, the average fiber diameter , And standard deviation were calculated, and the results are shown in Table 1.

In Examples 1 to 7, nanofibers having a fiber diameter of less than 1000 nm were successfully produced from a mixture of PHBH and PVA by electrospinning. It can be seen from FIGS. 1 to 7 that the nanofibers of any of the examples have uniform fiber shapes and fiber diameters and have high uniformity. In Examples 4 to 7 with high PVA content, nanofibers were obtained by using a mixed solvent of HFIP and water as a solvent. Further, from the comparison between Example 4 and Example 5 and the comparison between Example 6 and Example 7, the fiber diameter of the nanofibers can be reduced by increasing the mixing ratio of water in the mixed solvent of HFIP and water. I understand. As a result, in Examples 5 and 7, it was possible to obtain extremely thin nanofibers having a fiber diameter of 200 nm or less.

The nanofiber of the present invention can be used as a scaffold having biocompatibility and bioabsorbability for medical material applications.

Claims (4)

A method of making nanofibers formed from a mixture of a polyhydroxyalkanoate copolymer and polyvinyl alcohol , comprising:
A step of solution electrospinning comprising a mixture of a polyhydroxyalkanoate copolymer and polyvinyl alcohol seen including,
The solvent constituting the solution is a mixed liquid of fluoroalcohol and water, and the mass ratio of the fluoroalcohol and water is 80:20 to 50:50,
The production method , wherein the average fiber diameter of the nanofibers is 200 nm or less . The production method according to claim 1, wherein the polyhydroxyalkanoate copolymer is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). The production method according to claim 1 or 2, wherein the mass-based ratio of the polyhydroxyalkanoate copolymer to polyvinyl alcohol is 95:5 to 5:95. The manufacturing method according to claim 1, wherein the average fiber diameter of the nanofibers is 100 nm or more and 200 nm or less .