Method for producing hyaluronic acid salt-containing fine fibers

Abstract

To provide a method for producing hyaluronate-containing fine fibers that are excellent in moisture retention and hygroscopicity and contain high-molecular-weight hyaluronate at a high concentration, hyaluronate-containing fine fibers, and a textile product.SOLUTION: A method for producing hyaluronate-containing fine fibers comprises: a step of preparing a mixed aqueous solution containing a hyaluronate having a weight average molecular weight of 500,000 or more, a polymer having a hydrophilic group and water; and an electro-spinning of injecting the mixed aqueous solution charged from a spinning nozzle on which a high voltage is applied to spin hyaluronate-containing fine fibers. The hyaluronate-containing fine fibers contain a hyaluronate having a weight average molecular weight of 500,000 or more and a polymer having a hydrophilic group, and the hyaluronate is contained in an amount of 10 mass% or more based on a total amount of the fibers.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing hyaluronate-containing fine fibers for use in medical materials, cosmetic materials, etc., hyaluronate-containing fine fibers, and textile products. [Background technology]

[0002] Hyaluronic acid is a high molecular weight polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine units. It is one of the major extracellular matrix (ECM) components of various tissues and has attracted attention for its potential medical applications due to its high biocompatibility.

[0003] In regenerative medicine and medical devices, there is a demand for the development of materials that can replace ECM using hyaluronic acid. Microfibers containing hyaluronic acid or its salts, such as sodium hyaluronate, are attracting attention as potential ECM substitutes. Microfibers containing hyaluronic acid have a high affinity with living organisms and may be able to easily mimic the structure of ECM. Furthermore, electrospinning has attracted attention as a means for producing fine fibers containing hyaluronic acid. Here, the electrospinning method is a technique for spinning fine fibers by spraying a polymer solution from a nozzle while applying a high voltage to the polymer solution.

[0004] To spin fine fibers containing hyaluronic acid using the electrospinning method, a solution containing hyaluronic acid is required. However, when hyaluronic acid is dissolved in water, it forms a hydrogel in the aqueous solution due to interactions caused by electric charges and hydrogen bonds based on the carboxyl groups in the side chains, and therefore the hydrogelled hyaluronic acid aqueous solution cannot be spun directly by the electrospinning method. Therefore, it has been proposed to produce fine fibers containing hyaluronic acid by using hyaluronic acid with modified side chains or hyaluronic acid with chemical crosslinking to prepare an aqueous solution of hyaluronic acid that suppresses hydrogelation, and then spinning the aqueous solution by electrospinning (e.g., Non-Patent Document 1). There are also reports that the spinnability of fine fibers containing hyaluronic acid has been improved by spinning them by electrospinning using a solution of unmodified hyaluronic acid dissolved in an aprotic organic solvent such as dimethylformamide or dimethyl sulfoxide (Patent Document 1, Non-Patent Documents 2 and 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 056937 [Non-patent literature]

[0006] [Non-Patent Document 1] Int. J. Pharm., 456, 125-134, 2013 [Non-patent document 2] Macromol. Rapid Commun., 27, 114-120, 2006 [Non-patent document 3] Carbohydr. Polym., 87, 926-929, 2012 Summary of the Invention [Problem to be solved by the invention]

[0007] In the methods described in Patent Document 1 and Non-Patent Documents 1-3, there are limitations on the molecular weight of hyaluronic acid that can be used in electrospinning and the concentration of hyaluronic acid in the solution, making it difficult to spin a solution containing a high concentration of high molecular weight hyaluronic acid using the electrospinning method. Therefore, it has been difficult to produce fine fibers containing high molecular weight hyaluronic acid or nonwoven fabrics using such fine fibers using conventional techniques. Furthermore, hyaluronic acid that has been modified at the side chain or chemically crosslinked may have poorer moisture absorption and moisture retention properties than unmodified hyaluronic acid. Furthermore, when used in medical materials, cosmetic materials, etc., it is desirable to avoid organic solvents as much as possible.

[0008] Therefore, a primary object of the present invention is to provide a method for producing hyaluronate-containing fine fibers that have excellent moisture retention and moisture absorption properties and contain a high concentration of high-molecular-weight hyaluronate. Another object of the present invention is to provide a method for producing hyaluronate-containing fine fibers that can be produced using an aqueous solvent without using substantially any organic solvent. A further object of the present invention is to provide hyaluronate-containing fine fibers which have excellent moisture retention and moisture absorption properties and contain a high concentration of high-molecular-weight hyaluronate, and textile products using the same. [Means for solving the problem]

[0009] The method for producing hyaluronate-containing fine fibers according to the present invention comprises the steps of preparing a mixed aqueous solution containing hyaluronate having a weight-average molecular weight of 500,000 or more, a polymer having a hydrophilic group, and water, and an electrospinning step of spraying the charged mixed aqueous solution from a spinning nozzle to which a high voltage is applied to spin hyaluronate-containing fine fibers.

[0010] The hyaluronate-containing fine fibers according to the present invention contain hyaluronate having a weight-average molecular weight of 500,000 or more and a polymer having a hydrophilic group, and the hyaluronate is contained in an amount of 10% by mass or more relative to the total amount of the fibers. The textile product of the present invention uses hyaluronate-containing fine fibers. [Effects of the Invention]

[0011] According to the method for producing hyaluronate-containing fine fibers of the present invention, there is no need to use organic solvents as the solvent of the solution used in the electrospinning process, and the production process is highly safe.Furthermore, the obtained hyaluronate-containing fine fibers and textile products using the same are also highly safe, and are suitable for use as medical materials and cosmetic materials. The hyaluronate-containing fine fibers and textile products of the present invention contain high concentrations of high molecular weight hyaluronate, making them suitable for use as substitute materials for ECM. The hyaluronate-containing fine fibers and textile products of the present invention do not require the use of modified or crosslinked hyaluronate, and therefore have excellent moisture retention and moisture absorption properties, making them suitable for use as medical materials and cosmetic materials. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is an explanatory diagram showing an electrospinning step in one embodiment of the method for producing hyaluronate-containing fine fibers of the present invention. [Figure 2] 1 is a graph showing the relationship between the pH and complex viscosity (η*) of a mixed aqueous solution. [Figure 3] (a) to (d) are scanning electron microscope (SEM) photographs showing the morphology of hyaluronate-containing fine fibers obtained from mixed aqueous solutions with different pH values. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, hyaluronic acid salt-containing fine fibers (hereinafter sometimes simply referred to as fine fibers) according to one embodiment of the present invention, a method for producing the same, and textile products using the fine fibers of the present invention will be described.

[0014] First, the method for producing the fine fibers of the present invention will be described. The method for producing hyaluronate-containing fine fibers of the present invention includes the steps of preparing a mixed aqueous solution containing hyaluronate having a weight-average molecular weight (Mw) of 500,000 or more, a polymer having a hydrophilic group, and water, and an electrospinning step of spraying the charged mixed aqueous solution from a spinning nozzle to which a high voltage is applied to spin hyaluronate-containing fine fibers. This makes it possible to produce hyaluronate-containing fine fibers that have excellent moisture retention and moisture absorption properties and contain a high concentration of high-molecular-weight hyaluronate. This also allows production using an aqueous solvent, substantially without using an organic solvent.

[0015] <Mixed aqueous solution preparation process> A method for producing fine fibers according to one embodiment of the present invention includes a step of preparing a mixed aqueous solution for use in electrospinning, the mixed aqueous solution containing a hyaluronate, a polymer having a hydrophilic group, and water. The hyaluronic acid salt has a weight-average molecular weight (Mw) of 500,000 or more, preferably 1,000,000 or more. By using such a high-molecular-weight hyaluronic acid salt, the obtained fine fibers can be suitably used as an alternative material to ECM. Examples of the hyaluronic acid salt include sodium salt, potassium salt, calcium salt of hyaluronic acid, etc. The reason for using a salt of hyaluronic acid is that hyaluronic acid, which has a property of being hardly soluble in water, becomes more soluble in water when it is made into a salt of hyaluronic acid.

[0016] In the present invention, the polymer having hydrophilic groups (hereinafter sometimes referred to as hydrophilic polymer) has the effect of improving the spinnability of fine fibers when using an aqueous solution containing a high concentration of high molecular weight hyaluronate. That is, when the hydrophilic polymer and the high molecular weight hyaluronate are present in the mixed aqueous solution, the viscosity of the solution increases, and the spinning property improves. Examples of the hydrophilic group include a hydroxyl group, an amino group, a carboxyl group, and an ether bond. Representative examples of such hydrophilic polymers include polyacrylic acid (PAA), polyethyleneimine (PEI), and mixtures and copolymers thereof. For example, polyethylene oxide preferably has a weight-average molecular weight (Mw) of 100,000 or more. The mixed aqueous solution contains at least water as a solvent. The mixed aqueous solution may contain other water-soluble solvents, such as methanol, ethanol, acetone, formic acid, acetic acid, butyric acid, and hydrochloric acid, as long as the other water-soluble solvents do not adversely affect the resulting fine fibers.

[0017] The content of the hyaluronic acid salt in the mixed aqueous solution is preferably 10% by mass or less, which allows the solution to have a viscosity suitable for spinning. The content of the hydrophilic polymer in the mixed aqueous solution is preferably 0.1% by mass or more and 20% by mass or less, which allows the solution to have a viscosity suitable for spinning. The ratio of hyaluronic acid salt to hydrophilic polymer in the mixed aqueous solution is set so that the ratio of hyaluronic acid salt in the obtained fine fibers is 10% by mass or more.

[0018] The molecular weight of the hyaluronic acid salt used in the method for producing hyaluronic acid salt-containing fine fibers of the present invention has a significant effect on the solution viscosity of the mixed aqueous solution, and the higher the molecular weight of the hyaluronic acid salt, the more likely it is that the viscosity of the mixed aqueous solution will increase even if it is contained in a small amount. In one embodiment of the present invention, the method for producing hyaluronate-containing fine fibers can suppress an increase in viscosity by adjusting the pH of the mixed aqueous solution to a predetermined pH, thereby allowing the mixed aqueous solution to contain a larger amount of high-molecular-weight hyaluronate having a weight-average molecular weight (Mw) of 500,000 or more while maintaining the viscosity of the mixed aqueous solution suitable for electrospinning. Specifically, the pH of the mixed aqueous solution is preferably adjusted to 6 or less, and more preferably 5 or less. The mixed aqueous solution also preferably has a pH of 3 or more. This allows the viscosity of the mixed aqueous solution to be reduced even when it contains a high concentration of high-molecular-weight hyaluronate salts with a weight-average molecular weight (Mw) of 500,000 or more, making it possible to spin fibers using an electrospinning method using an aqueous solution containing a higher concentration of high-molecular-weight hyaluronate salts. Furthermore, reducing the viscosity of the mixed aqueous solution improves the spinnability of hyaluronate-containing fine fibers. Furthermore, reducing the viscosity of the mixed aqueous solution allows the diameter of the resulting fine fibers to be reduced, making it possible to spin finer fibers. The pH can be adjusted by adding an acidic or alkaline substance to the mixed aqueous solution. Examples of the acidic substance include inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as citric acid and oxalic acid.

[0019] The preparation procedure when preparing the above-mentioned mixed solution is not particularly limited, for example, can be prepared by preparing hyaluronate aqueous solution and hydrophilic polymer aqueous solution in advance, and then mixing them.Also, for example, can be prepared by first preparing hyaluronate aqueous solution, and then adding hydrophilic polymer to prepare mixed solution.

[0020] The mixed aqueous solution may also contain various additives such as salts and surfactants, provided that they do not adversely affect the mixed aqueous solution or the resulting fine fibers. The term "salt" as used herein refers to a salt of a metal ion such as sodium, potassium, calcium, or magnesium with an acid such as hydrochloric acid, citric acid, acetic acid, phosphoric acid, carbonic acid, or boric acid. Specific examples of the surfactant include Triton X, SDS, Pluronic (registered trademark), and Tween.

[0021] <Electrospinning process> The method for producing fine fibers of the present invention includes a step of spinning fine fibers by electrospinning using the mixed aqueous solution. In the electrospinning step, as shown in Fig. 1, for example, mixed aqueous solution 1 is contained in a syringe 2, and is extruded from this syringe 2 at a constant speed and sent to a spinning nozzle 3.

[0022] A high voltage is applied to the spinning nozzle 3 from a high-voltage power supply 4, and by spraying the charged mixed aqueous solution 1 from the spinning nozzle 3, the solvent (water) contained in the mixed aqueous solution 1 gradually evaporates and is spun to form fine fibers 6, and the spun fine fibers 6 adhere to the surface of the collector 5.

[0023] The collector 5 is disk-shaped or cylindrical, and rotates around a central axis 7. As a result, the fine fibers 6 sprayed and spun from the spinning nozzle 3 are wound onto the circumferential surface of the rotating collector 5.

[0024] In the process of producing fine fibers by electrospinning the mixed aqueous solution 1, the rotation speed of the collector 5 is preferably 2 m / s or more and 50 m / s or less, and more preferably 20 m / s or less, which results in a strong traction force and allows for the production of thinner fibers. Other conditions for producing fine fibers by electrospinning the mixed aqueous solution 1 include, for example, the flow rate during spraying, the applied voltage, and the distance from the nozzle 3 to the collector. These are not particularly limited, but optimal conditions can be selected from, for example, a flow rate of 0.05 to 1 mL / hour, an applied voltage of 10 to 40 kV, and a distance from the nozzle 3 to the collector 5 of 4 to 20 cm.

[0025] A fine fiber nonwoven fabric can be formed by depositing the fine fibers 6 spun by electrospinning on the surface of the collector 5. That is, the fine fibers 6 are fused or entangled with each other to form a fine fiber nonwoven fabric. Furthermore, a flat collector (not shown) may be used instead of the disk-shaped or cylindrical collector 5, and the fine fibers may be deposited on this collector to form a fine fiber nonwoven fabric. In this way, according to the method for producing fine fibers of the present invention, a textile product such as a nonwoven fabric containing fine fibers can be obtained.

[0026] <Fine fibers containing hyaluronate> The hyaluronate-containing fine fibers of one embodiment of the present invention contain a hyaluronate having a weight-average molecular weight of 500,000 or more and a polymer having a hydrophilic group, and the hyaluronate is contained in an amount of 10% by mass or more relative to the total amount of the fibers. Thus, the fine fibers of the present invention contain a high concentration of high-molecular-weight hyaluronate, making them suitable for use as an alternative material to ECM.

[0027] The hyaluronate contained in the fine fibers of the present invention has a weight-average molecular weight (Mw) of 500,000 or more, preferably 1,800,000 or more and 2,200,000 or less. By containing such a high-molecular-weight hyaluronate, the fine fibers of the present invention can be suitably used as a substitute for ECM.

[0028] The hydrophilic polymer contained in the fine fibers of the present invention has functional groups (hydroxyl groups, carbonyl groups, carboxyl groups, amino groups, amide bonds, ether bonds, etc.) that can interact with hyaluronic acid in the fine fibers.

[0029] Specific examples of the hyaluronate and hydrophilic polymer contained in the fine fibers of the present invention are as described in the production method.

[0030] In this embodiment, if necessary, the above-mentioned hyaluronic acid salt and hydrophilic polymer may contain other components such as salt, surfactant, etc. Here, the salt refers to the salt of metal ions such as sodium, potassium, calcium, magnesium, etc. with acids such as hydrochloric acid, citric acid, acetic acid, phosphoric acid, carbonic acid, boric acid, etc. Specific examples of the surfactant include Triton X, SDS, Pluronic (registered trademark), Tween, etc.

[0031] The fine fibers of this embodiment contain 10% by mass or more, preferably 50% by mass or more, of hyaluronic acid salt relative to the total amount of the fine fibers. This allows the fine fibers to have high moisture retention and moisture absorption. The upper limit of the hyaluronic acid salt content is not particularly limited, but is preferably 98% by mass.

[0032] The average fiber diameter of the fine fibers is not particularly limited, but is preferably 0.1 μm or more and 8.0 μm or less, and more preferably 0.3 μm or less. This increases the specific surface area and improves the moisturizing effect and moisture absorption effect. The average fiber diameter of the fine fibers can be determined, for example, from an SEM photograph.

[0033] Hyaluronic acid is a major ECM component in various biological tissues and has high biocompatibility, so the resulting hyaluronate-containing fine fibers are expected to be applied in the medical field, for example, in regenerative medicine. Furthermore, the fine fibers of the present invention are expected to be applied in the cosmetics field, for example, as materials for cosmetic packs (face packs), taking advantage of their moisturizing properties. In particular, the fine fibers of the present invention are substantially free of organic solvents, making them suitable for use in the medical and cosmetics fields. Furthermore, the fine fibers of the present invention can be used for artificial extracellular matrices, scaffolds for cell culture, and the like. As described above in the manufacturing method, the fine fibers according to the present invention can be formed into a sheet-like nonwoven fabric when spun by electrospinning, but are not limited thereto and may also be formed into three-dimensional structures having various shapes. Such three-dimensional structures can be used, for example, as artificial extracellular matrices or scaffolds for cell culture.

[0034] The fine fiber membrane formed from the fine fibers of the present invention can be given anisotropy in tensile strength at desired locations by controlling the orientation of the fine fibers. In this way, by controlling the geometric structure of the membrane containing fine fibers (e.g., density, fiber orientation strength, degree of fiber fusion, porosity, etc.), the surface roughness of the fine fiber membrane, membrane thickness, tension, fiber diameter, beaded fiber, etc., desired fine fiber membrane properties can be obtained.

[0035] By using the fine fibers of the present invention, the textile product of the present invention can be endowed with high moisture retention, moisture absorption, etc., due to the hyaluronate at desired locations of the textile product. Examples of the form of the textile product according to the present invention include, but are not limited to, nonwoven fabrics and the like, and may be three-dimensional structures having various shapes. Furthermore, the textile product according to the present invention may be any product that contains at least a portion of the fine fibers of the present invention, and may be a laminate or composite with other textile materials or different materials.

[0036] In the textile product according to the present invention, by controlling the orientation of the fine fibers, it is possible to impart anisotropy in tensile strength to desired portions of the textile product, for example. In this way, by controlling the geometric structure of the membrane containing fine fibers (e.g., density, fiber orientation strength, degree of fiber fusion, porosity, etc.), the surface roughness of the fine fiber membrane, membrane thickness, tension, fiber diameter, beaded fiber, etc., desired fine fiber membrane properties can be obtained.

[0037] Specific examples of the textile products according to the present invention include, but are not limited to, face packs in the cosmetics field, wound dressings in the medical field that maintain a moist environment on the wound surface, and cell culture substrates (artificial extracellular matrices, scaffolding materials for cell culture, etc.) that can be used in the bioindustry (e.g., culturing antibody-producing cell lines) and regenerative medicine. The textile product of the present invention is highly safe because it uses hyaluronate-containing fine fibers that are substantially free of organic solvents, and is therefore suitable for use in the medical and cosmetic fields. [Example]

[0038] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0039] <Preparation process of mixed aqueous solution> Sodium hyaluronate with a weight-average molecular weight (Mw) of 2 million was used as the hyaluronate salt. Polyethylene oxide (PEO) with a weight-average molecular weight (Mw) of 2 million was used as the hydrophilic polymer. An aqueous solution of sodium hyaluronate and an aqueous solution of polyethylene oxide were mixed to prepare a mixed aqueous solution containing 1.5% by mass of sodium hyaluronate and 1.0% by mass of PEO. The resulting mixed aqueous solution had a pH of 8.5. It was then adjusted to pH 3, 4, 5, and 6 with HCl. The viscosity at each pH was then measured using a rheometer (a viscoelasticity measuring device). The results are shown in Figure 2. As shown in Figure 2, the viscosity decreases as the pH decreases. This means that at high pH, ​​sodium hyaluronate molecules electrostatically interact with each other, becoming entangled and less fluid, whereas at lower pH, the fluidity of sodium hyaluronate increases and its miscibility with PEO improves.

[0040] <Electrospinning process> Using each of the pH-adjusted mixed aqueous solutions, a fine fiber nonwoven fabric was produced by electrospinning.The resulting fine fibers each contained 60% by mass of sodium hyaluronate. The spinning conditions for the electrospinning method were as follows: flow rate of the mixed aqueous solution 1 shown in Figure 1 to the spinning nozzle 3 was 0.4 to 0.6 mL / h; electric field applied during spinning was 2.0 to 2.5 kV / cm; rotation speed of the collector 5 was 13 m / s; diameter of the spinning nozzle 3 was 23 G; distance from the tip of the spinning nozzle 3 to the collector 5 was 10 cm; and spinning time was 10 minutes. SEM images of the obtained fine fibers are shown in Figures 3(a) to 3(d). The adjusted pH of each mixed aqueous solution, the flow rate to the spinning nozzle 3, and the fiber diameter of each obtained fine fiber are shown in Table 1. The fiber diameter was determined from the SEM images. [Table 1] As shown in Figures 3(a) to (d) and Table 1, fine fibers could be spun from mixed aqueous solutions with a pH of 6.0 or less. In particular, fine fibers with a small diameter could be obtained from mixed aqueous solutions with a pH of 5.0 or less, and spinnability was also excellent. [Explanation of symbols]

[0041] 1 Mixed aqueous solution 2 syringes 3 Spinning nozzle 4 High-voltage power supply 5 Collector 6 Fine Fiber 7 Center axis

Claims

[Claim 1] A step of preparing a mixed aqueous solution containing a hyaluronic acid salt having a weight average molecular weight of 500,000 or more, a polymer having a hydrophilic group, and water; adjusting the pH of the mixed aqueous solution to 4 or more and 6 or less; an electrospinning step in which the charged mixed aqueous solution is sprayed from a spinning nozzle to which a high voltage is applied to spin hyaluronate-containing fine fibers; A method for producing hyaluronic acid salt-containing fine fibers, comprising:

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