JP2005290610A - Nanoscale fiber and formed product of polysaccharides - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nanoscale fiber which is made of polysaccharides as a material and usable as a scaffold or a support of a cell tissue and as a part of a substrate for biological tissue culture and a biological material (an artificial valve, an artificial organ, an artificial blood vessel, a wound covering material, etc.) for the purpose of repairing, regenerating and treating substitute skin or a defective tissue as a medical material in the medical field, especially in regeneration medicine, has excellent supply of oxygen or nutrients and is expectable of efficient proliferation and differentiation of cells and to further provide a nonwoven thin film composed of the fiber and a formed product processed therewith. <P>SOLUTION: The nanoscale fiber which is made of the polysaccharides as a main raw material, obtained by a method for electrostatic spinning and characterized as having ≤500 nm diameter. The nanoscale fiber of the polysaccharides which is the fiber of a composite composition containing an additive other than the polysaccharides or fiber of the polysaccharides alone. The nanoscale fiber of the polysaccharides has 1-100 nm diameter. The nonwoven thin film is composed of the fiber and the nonwoven thin film is composed of an aggregate of the fiber and a microfiber having >500 nm diameter. The medical material comprises the fiber and the medical material comprises the nonwoven thin film. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a molded article such as a nanoscale fiber of a polysaccharide obtained by an electrospinning method and a thin film thereof. More specifically, the present invention can be used as a medical material in the medical field, in particular, as a scaffold for culturing living tissue in the field of regenerative medicine, and a biomaterial (artificial valve, artificial organ, artificial blood vessel, wound dressing, etc.) As a part of this, it can be used for repairing, regenerating and treating skin and organs of living tissues. The present invention also relates to a thin film having a non-woven three-dimensional structure constituted by polysaccharide nanoscale fibers.

Regenerative medicine was established in 1998 in the United States, where human embryonic stem (ES) cells with the potential to regenerate bone, cartilage, skin, nerves, muscles, blood vessels and other organs were established. This is a field that is attracting attention. Regenerative medicine is a new medical technique in the field of biological tissue engineering that cultivates cells and tissues, regenerates biological tissue that has lost its function, or substitutes for organ function. An important approach in tissue engineering for regenerative medicine is the establishment of a regenerative field. Many water-soluble polymer materials and biomaterials are used for regeneration. The development of scaffold materials with excellent functionality is required more than ever.
In general, the requirements for biomaterials as medical materials are biofunctionality (performance and effects according to the application), biosafety (non-toxic), sterilizable (can be sterilized to prevent infection), There is biocompatibility (affinity to the living body). In addition to the above conditions, as a part of biological tissue culture and biomaterials, there are 1) adequate mechanical strength, 2) bioabsorbability, and 3) sufficient cell adhesion. Securing a specific surface area, and 4) a gap structure capable of supplying nutrients and oxygen.

Cellular tissue scaffolds in vivo are extracellular substances (extracellular matrix) typified by collagen and mucopolysaccharides (which bind to proteins and exist as proteoglycans in vivo), and cells proliferate and perform normal functions. It is deeply involved in cell adhesion necessary to do.
As a natural polymer, polysaccharides are attracting attention as raw materials for medical materials because they are not only highly biocompatible and non-cytotoxic, but also have moldability. If this polysaccharide can be used as a raw material, a material having a suitable mechanical strength, a high affinity with cells, a sufficient specific surface area for cell adhesion, and a density capable of supplying nutrients and oxygen can be obtained. Not only can it be used as a substitute for conventional collagen and synthetic polymer materials, but also the possibility of interfacial control with cells is expanded more than ever.

  Nanotechnology is beginning to be used as a means to control the interface with cells. Nanotechnology is attracting attention as an innovative technology that can manipulate and control atoms and molecules on the scale of 1 nm to 100 nm, change the structure and arrangement of materials, and create new functions and superior properties. Its development has been remarkable, and its practical application is steadily being promoted in various fields such as IT, electronics, and biotechnology (Non-patent Documents 1 and 2). In the fiber world, if the conventional practical fiber is 20-50 μm, it is a so-called microfiber with an average diameter of 2-5 μm, which is recently called a synthetic polymer material called ultra-fine fiber or ultra-fine fiber by a composite spinning technique such as sea-island type. Development is underway for commercialization of products and practical application of finer fibers (submicron size or less). In general, when the average diameter is 100 nm or less (less than 1 / 10,000 of 1 mm), so-called nano-size, it is known that the specific surface area increases in each step (Non-patent Document 3). However, with the current technology, in the field of natural materials, a technology capable of spinning nanoscale to submicron scale fibers on an industrial scale has not yet been established.

Recently, a method using an electrospinning technique has attracted attention as a means for obtaining nanoscale fibers. The electrospinning method was devised more than 70 years ago, and was developed as a technique for producing fibers, films, and non-woven fabrics. Currently, it is also applied to mass spectrometry. In particular, a production method for spinning by this method is also called an electrospray spinning (ESP) method, and a method for producing a thin film or nonwoven fabric obtained is also called an electrospray deposition (ESD) method (Non-patent Document 4).
Its characteristic is that, unlike the conventional commercial spinning method, the polymer material is charged under high voltage and spun by surface repulsion. The materials that have been studied for application in the electrospinning method so far include polyvinyl alcohol, polyethylene oxide, polyvinyl pyrrolidone, polyacrylamide, polyurethane, polycarbonate, polytetrafluoroethylene, polyethylene, polypropylene, polyacrylate, polyacrylate for synthetic polymer systems. There are hydroxybutyrate, polyaniline, aramid and the like, and natural polymers include deoxyribonucleic acid, collagen, α-lactalbumin, invertase, silk fibroin and the like. However, many formed fibers have different concentrations, molecular weights, viscosities, surface tensions, electrical conductivities, dielectric constants, applied voltages, humidity, etc. as physical properties parameters of solutions depending on the materials, and predict the conditions of polysaccharides. It was extremely difficult to do.

Special table 2003-521493 gazette Introduction to nanotechnology (Ohm, 2002) Special issue on nanotechnology (Nihon Keizai Shimbun, October 6, 2003) Challenges to general-purpose fiber innovation by ultra-thinning (Chemical Engineering, Vol.68, No.1, p36-37, 2004) Nanofiber Technology Research and Development Cases that Change the Fiber World Production of Polymer Nanofibers by ESD Method (Industrial Materials, vol51, No.9, p29-33, 2003)

  Polysaccharides such as chitin, chitosan, hyaluronic acid, and chondroitin sulfate are important as an extracellular substance as a scaffold for cell growth and differentiation, like collagen, and may be used as medical materials. Although there are some references to the electrospinning of polysaccharides that have excellent functions in natural polymers, they are not supported by the examples (Japanese Patent Publication No. 2003-521493), and natural organic ultrafine fibers In regard to the above, it is possible to obtain nanoscale fibers having a uniform fiber diameter obtained by dispersing fibers in a solvent and shearing force (Japanese Patent Laid-Open No. 2003-155349), and there is a description of nanoscale particle chitosan However, the production method is not electrostatic spinning (Japanese Patent Publication No. 2002-536392). As described above, there are various nanoscale objects having different production methods and properties, but there have been no nanoscale fibers and thin films of polysaccharides produced by the electrospinning method so far. According to the electrospinning method, not only nanoscale particles and fibers can be produced, but these can be integrated to form a porous or non-woven thin film. In addition, various coating processes can be applied to the surface and application development is possible.

  The present invention relates to medical materials in the medical field, in particular, biological materials for biological tissue culture in regenerative medicine and biological materials for the purpose of defect, repair, regeneration, and treatment of biological tissues (artificial valves, artificial organs, artificial blood vessels, wound coverings). As a part of the material, etc., it is a scaffold and support for cellular tissue, excellent in supplying oxygen and nutrients, and can be expected to grow and differentiate efficiently, and the material is polysaccharide and nanoscale fiber Furthermore, it is an object to provide a non-woven thin film that can be formed from the thin film, and a molded body processed with the non-woven thin film.

  As a result of diligent studies to solve the above-mentioned problems, the present inventor uses polysaccharides contained in animals, plants, seaweeds, microorganisms, etc. as materials, and does not use conventional commercial spinning methods, but the present invention. The present inventors have found that nanoscale fibers (nanofibers) made of polysaccharides can be obtained by the electrospinning method using the device (application number; Japanese Patent Application No. 2003-040642) filed earlier. According to this technology, it was confirmed that a thin film composed of nanoscale fibers made of polysaccharides, showing a thin, dense and non-woven property, and having a high specific surface area with a three-dimensional structure was obtained. . That is, the present invention is characterized by polysaccharide nanoscale fibers and molded bodies by an electrospinning method.

The gist of the present invention is the nanoscale fiber of the following polysaccharides (1) to (3).
(1) A nanoscale fiber of a polysaccharide, which is a fiber mainly composed of a polysaccharide obtained by an electrospinning method and having a diameter of 500 nm or less.
(2) A nano-scale fiber of a polysaccharide according to the above (1), which is a composite composition containing additives other than polysaccharides or a single fiber of polysaccharides.
(3) The nanoscale fiber of the polysaccharide according to the above (2) having a diameter of 1 to 100 nm.

The gist of the present invention is the following non-woven thin film (4) to (5).
(4) A non-woven thin film comprising the fiber of (1) above.
(5) A non-woven thin film comprising an assembly of the fibers of (1) above and microfibers having a diameter exceeding 500 nm.

The gist of the present invention is the following medical materials (6) to (7).
(6) A medical material containing the fiber of (1) above.
(7) A medical material comprising the non-woven thin film of (4) or (5) above.

The present invention provides polysaccharide nano-scale fibers obtained by an electrospinning method, which is highly safe, non-cytotoxic, has a large specific surface area, a thin film thereof, and a molded article such as a medical material. be able to.
In particular, it is used as a part of biological tissue culture substrate in regenerative medicine and biological materials (artificial valve, artificial organ, artificial blood vessel, wound dressing, etc.) for the purpose of defect, repair, regeneration, treatment of biological tissue, It is a scaffold and support for cellular tissues, and is excellent in supplying oxygen and nutrients, and can be expected to proliferate and differentiate cells efficiently.

Hereinafter, although this invention is demonstrated in detail, it is not restrained at all regarding this invention.
The present invention is a part of a biological tissue culture base material in regenerative medicine and a biomaterial (an artificial valve, an artificial organ, an artificial blood vessel, a wound dressing material, etc.) for the purpose of repair, regeneration, and treatment of alternative skin and defective tissue. Yes, it is a scaffold or support for cellular tissue, and is excellent in supplying oxygen and nutrients, and can be expected to proliferate and differentiate cells efficiently. The present invention relates to polysaccharides and nanoscale fibers, and further relates to a non-woven thin film, and a molded article such as a medical material processed with them.
That is, the polysaccharide fiber of the present invention is a nanoscale fiber having an average diameter of several nanometers to several hundred nanometers, so-called nanofiber, which can be continuously spun on a large scale by an electrostatic spinning method. This can be achieved by using a device such as The apparatus is equipped with a capillary (such as glass) into which an electrode (platinum wire or the like) is inserted, a conductive substrate (such as aluminum), or the like. The principle is that when a high voltage of 2,000 V to 20,000 V is applied between the nozzle tip of the capillary containing the polymer solution and the substrate under atmospheric pressure, the electric repulsive force of the polymer solution at the nozzle tip causes surface tension. The particles, spindles, and fibrous structures are dried and accumulated on the substrate depending on conditions.

  For fibers formed by electrostatic spinning, the molecular weight of the polymer material, solution concentration, viscosity, temperature, surface tension, electrical conductivity, physical properties such as dielectric constant, applied voltage, capillary tip diameter and other parameter conditions Depending on the polymer material. Also in the present invention, as a result of finding an optimum range in which a fibrous material can be obtained by a combination of these conditions and the solvent and additives used, it is a polysaccharide material, and a nanoscale fiber having a diameter of several nanometers to several hundred nanometers. A non-woven thin film with a two-dimensional and three-dimensional extent composed of them could be obtained stably.

  The polymer material according to the present invention is a polysaccharide. The polysaccharide is present in plants, seaweeds, shells, microorganisms, mushrooms and the like and is not particularly limited. The types of polysaccharides are generally classified into natural products and semi-synthetic products, but are not particularly limited. A polysaccharide is a form in which monosaccharides such as glucose (there are also neutral sugars having hydroxyl groups, amino sugars having functional groups such as amino groups and carboxyl groups, and acidic sugars) linked together by glycosidic bonds. It is non-cytotoxic, has unique properties depending on the constituent sugar, functional group, and molecular weight, and is rich in resources. Polysaccharides are important components that exist in all living organisms, but besides simple cell-to-cell filling and role as tissue supports, intercellular communication, cell recognition (self-nonself, immunity), tissues Deeply involved in construction, development and cell division. In addition, other functions such as anti-cancer, anti-virus, immunostimulation, and anti-inflammation are known. Since these polysaccharides are highly evaluated in terms of safety, they are widely used for food materials, medical materials, pharmaceutical materials, and the like.

  For example, as materials used in the electrospinning method, there are mucopolysaccharides such as chitin, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and keratosulfuric acid. In plant systems, there are cellulose, pectin, xylan, lignin, glucomannan, galacturon, psyllium seed gum, tamarind seed gum, gum arabic, tragacanth gum, water soluble polysaccharides of soybeans, and the like. In the seaweed system, there are alginic acid, carrageenan, laminaran, agar (agarose), fucoidan and the like. Microbial systems including molds and mushrooms include pullulan, dextran, curdlan, lentinan, xanthan gum and the like. In addition, derivatives of these polysaccharides can also be used.

  These polysaccharide materials may be dissolved or suspended as they are, but it is preferable to dissolve them. The reason is that in the case of a solution, the polysaccharide is uniformly dispersed from the center to the surface of the nanofiber, and the function of the fiber can be exhibited to the maximum. On the other hand, when suspending polysaccharides, uniform particles are required that are smaller than nanofibers, and it is very difficult and impractical to produce such particles first. Examples of the polysaccharide solvent include water, acid, alkali, ethanol, methanol, acetone, toluene, and the like. The polysaccharide may be selected in consideration of the solubility of the polysaccharide, and at least one kind or the physical properties of the solution is changed. For this reason, it is not a problem even if it is used multiple times, but it is preferably a weak acid-neutral-weak alkali solvent, purified water, ethanol water, physiological saline, various buffers (phosphate buffer solution, acetate buffer) Liquid etc.) is desirable. Further, the molecular weight of the polysaccharide is not particularly limited depending on the type of polysaccharide and the extraction method, and there are several molecular weights and several tens of thousands to several millions. In general, the higher the molecular weight, the easier it is to form a fiber structure that is easy to form, and the smaller the molecular weight, the easier it is to form the particle structure, preferably the molecular weight is 10,000 to 5,000,000, most preferably 30,000 to 3,000,000. is there. As the viscosity of the solution, the higher the molecular weight, the higher the concentration, the higher the viscosity, and the temperature and pH of the solution also vary. Generally, the higher the viscosity, the easier to obtain thin and long fibers, and the lower the viscosity, the thinner and shorter fibers and particles. It is easy to obtain, preferably 1,000 mPa · s or less, and most preferably 500 mPa · s or less. The concentration of the polysaccharide is not particularly limited as long as the viscosity does not exceed 1,000 mPa · s. In general, the higher the concentration, the easier it is to obtain a fiber structure, and the lower the concentration, the easier to obtain fine and short fibers and particles. Is 0.5% to 50%, most preferably 1% to 30%. In addition, physical properties such as the electrical conductivity and dielectric constant of the solution affect the charged state at the time of spinning, but generally the lower the electrical conductivity, the more suitable for the electrospinning method. It is desirable to use after desalting with electrodialysis, UF membrane, NF membrane, RO membrane or the like, or washing the polysaccharide with ethanol or acetone for precipitation. The electric conductivity of the solution is preferably 600 mS / cm or less, and most preferably 100 mS / cm or less. The temperature of the solution is not particularly limited as long as the function and activity of the polysaccharide and the additive contained therein are not impaired, and may be cooled or heated at room temperature, but preferably 0 to 100. ° C, most preferably 4-80 ° C. These polysaccharide solutions may be filtered through a centrifugal separator or a membrane filter if there are insolubles. For example, it may be processed with a pore size of 3,000 rpm for 10 minutes or more for centrifugation and 0.2 μm to 10 μm for a membrane filter.

  Next, the polysaccharide to be used can be used singly or as a mixture of a plurality of polysaccharides. Of course, the combination of neutral polysaccharides is good during mixing. In other cases, the neutral polysaccharide is preferably a combination of a basic polysaccharide or an acidic polysaccharide with respect to the neutral polysaccharide, and the combination of the basic polysaccharide and the acidic polysaccharide causes aggregation and precipitation and should be avoided. In addition, it is possible to make fibers with polysaccharides alone, but as necessary, synthetic polymers and fiber modifiers, bioactive substances (cell adhesion activity) can be used to maximize performance. Factor, cell growth factor, fibroblast growth factor, immune activity factor, nerve action factor, etc.), serum component, biological tissue component, surfactant and the like are desirable. For example, additives other than polysaccharides include polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polylactic acid, silk fibroin, proteoglycan, fibronectin, vitronectin, enterectin, elastin, laminin, selectin, galectin, lectin (WGA, concanavalin). A), collagen, gelatin, enzyme (collagenase, trypsin, glucosidase, protein kinase, urokinase, SOD, etc.), albumin, fibrin, fibrinogen, poly-L-lysine, poly-L-glutamic acid, extracellular matrix protein, integrin, Amino acids (glycine, proline, hydroxyproline, alanine, serine, asparagine, glutamic acid, threonine, cysteine, leucine, methionine, etc.), dipipe Tide, tripeptide, peptide protein (including tyrosine-isoleucine-glycine-serine-arginine sequence, arginine-glutamic acid-aspartic acid-valine sequence, arginine-glycine-aspartic acid sequence), glycosaminoglycan, mucin-type glycan , Asparagine-type binding sugar chain, oligosaccharide (trehalose, chitin oligosaccharide, chitosan oligosaccharide, galactooligosaccharide, fructooligosaccharide, xylooligosaccharide, cyclodextrin, etc.), monosaccharide (mannose, N-acetyl-D-glucosamine, glucosamine, N -Acetyl-D-galactosamine, sialic acid, muramic acid, glucose, galactose, lactose, fucose, arabinose, etc.), ganglioside, sphingolipid, glycolipid (alkyl glycoside, galactosylceramide, etc.) Fatty acid ester, fatty acid ester (DHA, EPA, etc.), long chain fatty acid (arachidonic acid, linoleic acid, retinoic acid, etc.), glycerol, polyhydric alcohol, cholesterol, squalene, prostaglandin, thromboxane, leukotriene, steroid, insulin, Transferrin, dexamethasone, hydrocortisone, thyroxine, triiodotyrosine, β-mercaptoethanol, acetylcholine, acids (lactic acid, malic acid, ascorbic acid, gluconic acid, glucuronic acid, pantothenic acid, pyruvic acid, etc.), glycyrrhizin, rutin, stevioside, saponin , Arbutin, polyphenol (catechin), SOD-like active substance, interferon, interleukin, cytokine, chemokine, TNF-α, monoclonal antibody, polyclonal antibody, Body factor, gene, DNA, RNA, adenine, bovine calf serum, vitamins, inorganic salts, silicon, ceramics, apatite, polyoxyethylene sorbitan monooleate, is ethylenediaminetetraacetic acid or the like. The additive may be dissolved or used as a powder, but is preferably dissolved. Examples of the solvent for the additive include water, acid, alkali, ethanol, methanol, acetone, toluene, and the like, which may be selected in consideration of the solubility of the additive, and at least one kind or a plurality may be used. Although it does not cause any problem, it is preferably a weak acid to neutral to weak alkali solvent, and purified water, ethanol water, physiological saline, and various buffers (phosphate buffer, acetate buffer, etc.) are desirable.

  The diameter of the capillary may be freely selected according to the purpose, and is not particularly limited. In the embodiment, a diameter of 50 μm is used. The substrate may be any conductive material, and aluminum or the like is often used, but other conductive materials may be used. The distance from the capillary to the substrate may be freely selected and is not particularly limited, but is set to 30 mm to 100 mm in the embodiment. The applied voltage may be changed depending on the properties of the solution. The higher the voltage, the easier to obtain elongated fibers, and when it is low, the fiber structure is less likely to form, making it easier to obtain short fibers and particles, preferably 2,000 v to 50,000 v. Most preferably, it is 4,000v to 20,000v. If the viscosity of the solution is high, the applied voltage may be increased, but it may be supplementarily pressurized from above with a syringe pump, etc., and there is no significant effect on the properties of the resulting fiber or coating, and there is no problem. . Other factors that affect the fiber and thin film include the humidity inside the device, but if the humidity exceeds 70%, it cannot be stably spun, so the application is less than 70%, preferably 10% to 30%. It is necessary to keep it constant.

  The fibers and non-woven thin film integrated on the substrate may be cross-linked to obtain strength. Examples of the crosslinking agent include glutaraldehyde, carbodiimide, carbonyl imidazole, diepoxy compound, dicarboxylic acid anhydride, epichlorohydrin and the like. After these cross-linkings, washing is performed with acetone, ethanol, purified water, physiological saline, various buffer solutions or the like so that the cross-linking agent does not remain.

  By applying the principle of this apparatus, substrates of any shape and size can be jetted and nanoscale fibers can be deposited on a substrate. Further, instead of spraying directly on the substrate, it is possible to directly spray glass, plastic, nonwoven fabric or the like as a base material to coat the surface. For example, it can be directly sprayed on the surface of a cell culture medium material or a biomaterial (artificial valve, artificial organ, artificial blood vessel, artificial bone, artificial tooth, artificial skin, etc.). Also, different solutions can be prepared and sprayed on the same substrate, so that thin films with different properties can be stacked several times. For example, a thin film having a two-layer structure including a synthetic polymer layer (support layer) and a polysaccharide layer is also possible. Further, the thin film can be peeled off from the substrate, and can be affixed to or embedded in a living tissue that is a wounded part as a covering material.

  Polysaccharide nanoscale fibers and non-woven thin films made of the fibers are finer than conventional polysaccharide fibers, and therefore have a large specific surface area and an increased adhesion area to cells. It becomes an optimal scaffold for cell proliferation and differentiation, and can be used as a base material for biological tissue culture, which is a medical material in the field of regenerative medicine, and as a part of biological materials such as artificial organs and wound dressings.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

  The electrospinning apparatus shown in FIG. 1 was used. The sample used was chitosan (Mw 120,000, manufactured by Kyowa Technos Co., Ltd.) separated and purified from crab shells. The sample concentration is 10% (w / v, containing 5% acetic acid as solubilizer). The sample station (viscosity 120 cp / 20 ° C.) was filled into a capillary with a tip diameter of 50 μm, and voltage was applied (at room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 2).

  The electrospinning apparatus shown in FIG. 1 was used. The sample used was chitosan (Mw 70,000, manufactured by Kyowa Technos Co., Ltd.) separated and purified from crab shell. The concentration of the sample solution is 10% (w / v, containing 5% acetic acid as a solubilizer). 5% polyethylene glycol (Mw 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added to this sample solution at a weight ratio of 6: 4 (viscosity 120 cp / 20 ° C), and the capillary was filled with a tip diameter of 50 μm. The applied voltage was 9.13 kV (room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers with a diameter of 98-386 nm was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 3).

  The electrospinning apparatus shown in FIG. 1 was used. The sample used was sodium chondroitin sulfate (Mw 110,000, manufactured by Sakai Shoji Co., Ltd.) separated and purified from shark cartilage. The concentration of the sample solution is 10% (w / v, dissolved in distilled water). To this sample solution, 5% polyethylene glycol (Mw 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added at a weight ratio of 8: 2 (viscosity 78 cp / 70 ° C.) and filled into a capillary with a tip diameter of 50 μm. The applied voltage was 16.53 kV (room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers having a diameter of 115 to 304 nm was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 4).

  The electrospinning apparatus shown in FIG. 1 was used. As a sample, pectin separated from citrus fruits (Mw 510,000, manufactured by Wako Pure Chemical Industries, Ltd.) was used. The concentration of the sample solution is 5% (w / v, dissolved in distilled water). To this sample solution, 5% polyethylene glycol (Mw 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added at a weight ratio of 6: 4 (viscosity 26 cp / 70 ° C.), and filled into a capillary with a tip diameter of 50 μm. The applied voltage was 10.89 kV (at room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers having a diameter of 75 to 267 nm was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 5).

  The electrospinning apparatus shown in FIG. 1 was used. As a sample, chitosan (Mw 30,000, manufactured by Kyowa Technos Co., Ltd.) separated and purified from crab shells was used. The concentration of the sample solution is 10% (w / v, containing 5% acetic acid as a solubilizer). 15% polyvinylpyrrolidone (Mw 360,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added to this sample solution at a weight ratio of 1: 1 (viscosity 120 cp / 20 ° C), and the capillary was filled with a tip diameter of 50 μm. The applied voltage was 14.00 kV (at room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers having a diameter of 70-210 nm was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 6).

(Production of free-standing thin film)
The electrospinning apparatus shown in FIG. 1 was used. As a sample, chitosan (Mw 30,000, manufactured by Kyowa Technos Co., Ltd.) separated and purified from crab shells was used. The concentration of the sample solution is 10% (w / v, containing 5% acetic acid as a solubilizer). To this sample solution, 5% polyethylene glycol (Mw 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added at a weight ratio of 8: 2 (viscosity 115 cp / 22 ° C.), and filled in a capillary with a tip diameter of 50 μm. An aluminum foil was laid on the substrate, and the applied voltage was 16 kV (room temperature, atmospheric pressure). As a result of carrying out under conditions where the humidity in the apparatus was different, two types of thin films with different fiber diameters were obtained. When the fiber diameter was measured for each thin film, it was a thin film with a diameter of 100-300 nm (humidity 20%) and a thin film with a diameter of 500-1000 nm (humidity 35%). Next, the thin film was immersed in a mixed solvent of methanol and 1N caustic soda (weight ratio 99: 1) at room temperature for 2 hours while being attached to the aluminum foil, and then peeled off from the aluminum foil. Further, it was washed with pure water and air-dried at room temperature to obtain an insoluble self-supporting film.

(Protein adsorption test)
In this test, bovine serum albumin (manufactured by Sigma) was used as a protein, and a 1% bovine serum albumin solution was prepared with distilled water. The solution was immersed in a 37-fold amount (weight) of the dried thin film (humidity 20% condition, fiber diameter 100 nm-300 nm). The soaking solution was diluted 10 times with distilled water, free protein was measured by an absorbance method using 280 nm, and the amount of protein adsorbed on the thin film was determined from a calibration curve of bovine serum albumin. Also, add 50% (weight) of 1% bovine serum albumin solution to the dried thin film (humidity 35% condition, fiber diameter 500nm-1000nm), soak it and dilute it 10 times. The amount of protein adsorbed on the thin film was determined from the bovine serum albumin calibration curve by measuring the absorbance at 280 nm.
As a result, a thin film with a fiber diameter of 100 nm to 300 nm adsorbed 452 mg of bovine serum albumin per 1 g of the dry thin film, and a thin film with a fiber diameter of 500 nm to 1000 nm adsorbed 171 mg of bovine serum albumin per 1 g of the dry thin film. The thin film with a small fiber diameter was about 2.6 times more adsorbed than the thin film with a large fiber diameter.

Comparative example

[Comparative example]
As a comparative example, a commercially available chitosan fiber (nonwoven fabric) was obtained, and the fiber diameter was 12-38 μm (FIG. 7).

  The nano-scale fibers of polysaccharides by the electrospinning method of the present invention and the molded body constituted by the fibers are highly safe, non-cytotoxic, and have a large specific surface area. As a medical material in the field, it can be used as a base material for biological tissue culture, a scaffold for biological materials (artificial organs, artificial blood vessels, artificial bones, etc.), a wound dressing, and the like.

Outline of electrospinning equipment Image (magnification: 10,000 times, 100 μm × 100 μm, bar in the figure: 1.0 μm) of the thin film of chitosan obtained in Example 1 by atomic force microscope (nanopics1000, Seiko Instruments Inc.) Image by scanning electron microscope (SM-200, Topcon Co., Ltd.) of the thin film of chitosan / polyethylene glycol obtained in Example 2 (magnification: 10,000 times, 10 μm × 10 μm, bar in the figure: 1.0 μm) Image by scanning electron microscope (SM-200, Topcon Co., Ltd.) of the chondroitin sulfate / polyethylene glycol thin film obtained in Example 3 (magnification: 10,000 times, 10 μm × 10 μm, bar in the figure: 1.0 μm) Image of pectin / polyethylene glycol thin film obtained in Example 4 by scanning electron microscope (SM-200, Topcon Co., Ltd.) (magnification: 10,000 times, 10 μm × 10 μm, bar in the figure: 1.0 μm) Image by scanning electron microscope (SM-200, Topcon Co., Ltd.) of the thin film of chitosan / polyvinylpyrrolidone obtained in Example 5 (magnification: 10,000 times, 10 μm × 10 μm, bar in the figure: 1.0 μm) An image of the polysaccharide fiber (chitosan / wet spinning) shown in the comparative example by scanning electron microscope (SM-200, Topcon Co., Ltd.) (magnification: 100 times, bar in the figure: 100 μm)

Explanation of symbols

1 Voltage (Controlling applied voltage)
2 Electrode 3 Capillary 4 Substrate 5 Ground

Claims (7)

  A polysaccharide nano-scale fiber characterized in that it is a fiber mainly composed of a polysaccharide obtained by an electrospinning method and having a diameter of 500 nm or less.   2. The polysaccharide nanoscale fiber according to claim 1, which is a composite composition containing additives other than polysaccharides, or a single fiber of polysaccharides.   The polysaccharide nanoscale fiber of claim 2 having a diameter of 1 to 100 nm.   A non-woven thin film comprising the fibers of claim 1.   A non-woven thin film comprising an assembly of the fibers of claim 1 and microfibers having a diameter of more than 500 nm.   A medical material comprising the fiber of claim 1. A medical material comprising the nonwoven film of claim 4 or 5.