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WO2011018995A1 - Hydrogel composite organique/inorganique et son procédé de production - Google Patents

Hydrogel composite organique/inorganique et son procédé de production Download PDF

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Publication number
WO2011018995A1
WO2011018995A1 PCT/JP2010/063371 JP2010063371W WO2011018995A1 WO 2011018995 A1 WO2011018995 A1 WO 2011018995A1 JP 2010063371 W JP2010063371 W JP 2010063371W WO 2011018995 A1 WO2011018995 A1 WO 2011018995A1
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organic
compound
inorganic composite
composite hydrogel
clay mineral
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PCT/JP2010/063371
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English (en)
Japanese (ja)
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和敏 原口
三惠子 深澤
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財団法人川村理化学研究所
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Publication of WO2011018995A1 publication Critical patent/WO2011018995A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/02—Applications for biomedical use

Definitions

  • the present invention relates to an organic / inorganic composite hydrogel in which an organic polymer and a clay mineral form a three-dimensional network, and an organic / inorganic composite hydrogel exhibiting degradability in water or in vivo.
  • Polymer hydrogels are organic polymer three-dimensional cross-linked products that contain water and swell, and as soft materials with swelling and rubber-like elasticity, such as medical / medicine, food, civil engineering, bioengineering, sports-related, etc. Widely used in the field.
  • a polymer hydrogel having a three-dimensional network formed by combining a water-soluble organic polymer and a layered clay mineral has characteristics such as excellent water absorption and extremely high extensibility.
  • a polymer of a water-soluble organic monomer having an amide bond is mainly used, and it has been difficult to apply it to polyethylene glycol, which is a polymer having excellent biocompatibility.
  • the synthesis of polyethylene glycol hydrogels has been performed using macromers that are structurally designed so that regular chemical crosslinks are formed.
  • Non-Patent Document 1 It has been reported that a greatly improved polyethylene glycol hydrogel can be obtained (see, for example, Non-Patent Document 1).
  • the resulting polyethylene glycol hydrogel exhibited about 280% stretch and about 50 kPa strength at a polymer concentration of 120 mg / ml (see Comparative Example 1). This is achieved by reacting two kinds of reactive four-chain polyethylene glycols by mixing to obtain a chemically crosslinked polyethylene glycol hydrogel having a uniform molecular weight between crosslinking points.
  • the mechanical properties of the obtained polyethylene glycol hydrogel are not yet sufficiently high for practical use, and it is desired to further improve the draw ratio and tensile strength and to control the mechanical properties over a wide range. It was.
  • a polymer material that gradually decomposes by being kept in a predetermined atmosphere For example, materials that are gradually decomposed and metabolized after being implanted in the living body, materials that decompose after use for a certain period of time in soil, materials that decompose gradually when placed in a high-temperature or high-humidity atmosphere, use for a certain period of time Examples include materials that later lose shape or mechanical strength due to decomposition, and materials that signal the end of use, and materials that can be decomposed correspondingly when the atmosphere changes.
  • the polymer material before decomposing needs to have excellent performance according to the purpose, especially high mechanical properties, flexibility, transparency, safety, processability to various shapes.
  • the decomposition conditions are not severe conditions such as high temperature and high pressure, but can be performed under mild conditions as much as possible.
  • the decomposition conditions are not severe conditions such as high temperature and high pressure, but can be performed under mild conditions as much as possible.
  • biodegradable polymers that are degraded by microorganisms and hydrolyzable polymers that are degraded in water or at high humidity.
  • biodegradable polymers that are degraded by microorganisms
  • hydrolyzable polymers that are degraded in water or at high humidity.
  • polylactic acid, polycaptolactone, polyglycolic acid, polydioxanone, modified polyvinyl alcohol, Polybutylene succinate, glycolic acid / lactic acid copolymer, lactide / caprolactone copolymer, cellulose, hyaluronic acid, cellulose acetate, starch, casein and the like are used.
  • polymer gels those using the above-mentioned polymers and polymer gels made of collagen, chitosan, proteins, etc. that are derived from living bodies and are absorbed by living bodies are known. However, it satisfies the above-mentioned high transparency, mechanical properties controlled in a wide range, flexibility, safety, biocompatibility, processability to various shapes, etc. as well as degradability under mild conditions, In addition, there is no one that satisfies all conditions such as non-toxicity after decomposition, and its development has been strongly demanded.
  • Non-Patent Document 2 An organic-inorganic composite gel composed of a monomer polymer and a layered peelable inorganic clay mineral has been reported (Patent Document 2, Non-Patent Document 2). However, although the obtained gel has excellent properties, it did not have degradability in water or in vivo. In addition, none of the polymer gels and organic / inorganic composite gels having excellent mechanical properties, flexibility, and transparency reported to others have been known (Non-Patent Documents 1, 3 to 5).
  • a protein is a string-like polymer in which amino acids are connected one-dimensionally, and the string is folded to create a specific three-dimensional structure.
  • the protein exhibits various functions. Typical examples are enzymes and antibodies.
  • Specific examples of functional proteins include amylase, protease, lipase, cellulase, oxidase, dehydrogenase, gelatin, collagen, fibronectin, albumin, IgG, macroglobulin, blood coagulation factor, monoclonal antibody, polyclonal antibody and the like.
  • protein functions are expressed on the basis of three-dimensional structures stabilized by non-covalent bonds such as hydrophobic interactions, van der Waals interactions, and hydrogen bonds. Deactivating the function has become a major issue.
  • Patent Document 3 a method for obtaining an improved protein by substituting amino acid residues in a protein
  • Patent Document 4 a method for obtaining a thermostable protein by obtaining a mutant enzyme
  • Patent Document 5 a protein using a cationic surfactant
  • sufficient results are not always obtained for effective heat resistance improvement.
  • Patent Document 2 US6710104 JP 2009-118749 A JP 2001-78786 A Special table 2004-500005
  • the problems to be solved by the present invention include excellent mechanical properties, flexibility, transparency, safety, and in particular, polyethylene glycol whose tensile strength and stretch ratio are greatly improved or controlled over a wide range is an organic component.
  • a polymer hydrogel and a method for producing the same are excellent mechanical properties, flexibility, transparency, safety, and in particular, polyethylene glycol whose tensile strength and stretch ratio are greatly improved or controlled over a wide range is an organic component.
  • Another object of the present invention is to provide a degradable organic material that satisfies the above-mentioned conditions, which solves the above-mentioned problems and has a property that can be decomposed in a mild atmosphere and that the decomposed components are also non-toxic.
  • An object of the present invention is to provide an inorganic composite hydrogel, a method for producing the same, and a biological implant material using the same.
  • Another object of the present invention is to provide an organic-inorganic composite hydrogel containing a protein that can increase the thermal stability of the protein and exhibit its function even at a higher temperature, a method for producing the same, and a method for stabilizing the protein. There is.
  • the present inventors have uniformly dispersed finely dispersed clay minerals in a cross-linked polyethylene glycol so that they form a three-dimensional network.
  • the organic / inorganic composite hydrogel is excellent in mechanical properties such as tensile strength and stretch ratio and controlled in a wide range while maintaining uniformity, and such an organic / inorganic composite. It has been found that the thermal stability of the protein is improved in the hydrogel containing the protein and the degradation product of the gel, and the present invention has been completed.
  • the present invention is characterized in that a polymer compound (A) having a branched structure or network structure in which a plurality of polyethylene glycol chains are chemically crosslinked is combined with a layered exfoliated clay mineral (B).
  • An organic-inorganic composite hydrogel is provided.
  • the present invention also provides an organic-inorganic composite hydrogel containing the protein (C) in the organic-inorganic composite hydrogel or a decomposition product thereof.
  • the present invention provides a living body implanting material using the organic-inorganic composite hydrogel.
  • the present invention also provides an aqueous dispersion of the clay mineral (B) by layering the clay mineral (B) in an aqueous medium.
  • a method for producing the organic-inorganic composite hydrogel described above is provided.
  • the present invention also provides a protein stabilization method characterized by improving the stability of the protein (C) by containing the protein (C) in the organic-inorganic composite hydrogel or the decomposition product thereof. To do.
  • the organic-inorganic composite hydrogel obtained by the present invention provides an organic / inorganic composite hydrogel having excellent uniformity and stretch mechanical properties by forming a three-dimensional network of layered exfoliated clay mineral and polyethylene glycol.
  • an organic / inorganic composite hydrogel comprising polyethylene glycol having excellent transparency, uniformity, stretching strength and elongation as an organic component can be obtained.
  • the organic-inorganic composite hydrogel of the present invention is degradable by using, as the polymer compound (A), a compound having an ester group at least in part and having a polyethylene glycol chain chemically cross-linked by an amide bond. Gel. This gel confirms that the degraded product is safe in biological testing. In this case, a degradable gel suitable for in vivo degradation can be obtained by changing the composition such as the content of clay minerals or ester groups.
  • the thermal stability of the protein is improved, and for example, the enzyme activity of the enzyme is maintained at a higher temperature for a long time.
  • the organic / inorganic composite hydrogel having the above characteristics can be obtained in various shapes including cylindrical, rod-like, film-like and thread-like, and is an artificial valve excellent in biocompatibility and flexibility, especially in the medical and pharmaceutical fields.
  • Artificial organ materials such as artificial blood vessels and artificial cartilage, therapeutic materials such as catheters, and single or other biocompatible materials and biodegradable materials, which can be embedded in a living body with excellent safety after degradation. It is used effectively as a material. In addition, it is also used in fields such as agriculture, industry, electronic materials, civil engineering, and packaging materials as various industrial materials having excellent elasticity.
  • FIG. 3 is a diagram showing stress-strain curves in a tensile test of organic / inorganic composite hydrogels obtained in Examples 1 to 4 and Comparative Example 1. It is a figure which shows the cytotoxicity test result of the decomposition solution of the hydrogel obtained in Example 9 and Comparative Example 4. It is a figure which shows the stress-strain curve by the extending
  • the polymer compound (A) used in the present invention is a polymer compound having a branched structure or network structure in which a plurality of polyethylene glycol chains are chemically crosslinked, and a plurality of linear polyethylene glycols are a plurality of crosslinked molecules. It has a structure connected by points or branch points. Such a structure can be used without particular limitation as long as the effects of the present invention are not impaired.
  • a part of the polymer chain has a functional group that causes an interaction with a clay mineral, such as a hydroxyl group, a carboxyl group, an amino group, a sulfonic acid group, an amide group, an ester group, or a quaternary ammonium ion.
  • a group into which one or a plurality of ionic groups such as a group is introduced is used.
  • the chemical cross-linking of the polymer compound (A) is based on an amide bond, and a compound having a structure having an ester group in a part of the molecular chain is preferable because it is excellent in decomposability. More specifically, it is a polymer compound having a branched structure or network structure in which a polyethylene glycol chain containing an ester group at least partially is chemically cross-linked by an amide bond, and a plurality of linear polyethylene glycols are plural. A compound having a structure connected by a crosslinking point or a branching point.
  • the polymer compound (A) used in the present invention includes a compound (a1) having a polyethylene glycol chain and a plurality of reactive functional groups (Q1) in the same molecule, and the reactive functional group (Q1) It can be produced by reacting a compound (a2) having a plurality of reactive functional groups (Q2) capable of reacting.
  • compound (a1) or compound (a2) for example, a compound represented by the following formula (4) or formula (5) can be used.
  • R is a group represented by the following formula (3), formula (6) to formula (11), and n is an integer of 1 or more. Further, the total of four n in one molecule is preferably 50 to 1000, more preferably 100 to 800, and particularly preferably 150 to 500.
  • the weight average molecular weight of the compounds represented by the above formulas (4) and (5) is preferably from 1,000 to 100,000, more preferably from 5,000 to 50,000, and particularly preferably from 5,000 to 40,000.
  • R is the type SUNBRIGHT PTE-050GS (weight average molecular weight 5000), PTE-100GS (weight average molecular weight 10,000), PTE-150GS (weight average molecular weight 15000), PTE-200GS (weight average molecular weight) of the above formula (3) 20000), PTE-400GS (weight average molecular weight 40000)
  • R is type PTE-100HS (weight average molecular weight 10000), PTE-200HS (weight average molecular weight 20000), PTE-400HS (weight average molecular weight 40000) of the above formula (6)
  • R is type PTE-100MA (weight average molecular weight 10000), PTE-200MA (weight average molecular weight 20000), PTE-400MA (weight average molecular weight 40000) of the above formula (8)
  • R is type PTE-100PA (weight average molecular weight 10,000), PTE-150PA (weight average molecular weight 15000), PTE-200PA (weight average molecular weight
  • any one of the groups represented by the above formulas (3) and (6) to (11) is selected as the reactive functional group (Q1), and can react with the reactive functional group (Q1).
  • Reactive functional group (Q2) is selected. And if the compound which has these reactive functional groups (Q1) and (Q2) is made to react as a compound (a1) and a compound (a2), respectively, the high molecular compound (A) which can be used by this invention will be manufactured. Can do.
  • any compound is selected from the compounds represented by the above formulas (4) and (5), and this is used as the compound (a1) having a reactive functional group (Q1).
  • Compound (a2) is a known compound (C) having a plurality of reactive functional groups (Q2) other than the compounds represented by 4) and formula (5) and having a plurality of reactive functional groups (Q2) capable of reacting with this reactive functional group (Q1).
  • the polymer compound (A) that can be used in the present invention can also be produced.
  • Examples of such a compound (C) include ethylenediamine, hexamethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, phenylenediamine, butanediamine, pentanediamine, and amino acids such as arginine, asparagine, lysine, and the like. Protein etc. are used.
  • polymer compound (A) used in the present invention more preferably, a compound that has been chemically cross-linked so that the molecular weight between cross-linking points is uniform is effectively used.
  • polyethylene glycol chemically cross-linked so that the molecular weight between the cross-linking points is uniform include, for example, two reactive 4-chain polyethylene glycols described in Non-Patent Documents 2 and 7 (amine end having an amine at the end)
  • examples thereof include polyethylene glycol obtained by mixing and reacting 4-chain polyethylene glycol (TAPEG) and polyethylene glycol having an N-hydroxysuccinimide glutarate terminal at the terminal (TNPEG). In this case, an amide group or an ester group exists in the polyethylene glycol chain.
  • the molecular weight between cross-linking points of chemical cross-linking is 1 ⁇ 2 of the molecular weight of the 4-chain polyethylene glycol, but the value is preferably 1000 to 50000, more preferably 2000 to 30000, and particularly preferably 3000 to 20000.
  • the value of the molecular weight between cross-linking points is less than 1000, the flexibility is insufficient or the draw ratio is reduced.
  • it is larger than 50000 the elastic modulus is too low or the handleability is deteriorated.
  • the compound (a1) is a compound represented by the following formula (1)
  • the compound (a2) is represented by the following formula (2). It is particularly preferable to use compounds which are both compounds and have a weight average molecular weight of 1,000 to 100,000.
  • n is an integer.
  • Y is a group represented by —CH 2 CH 2 CH 2 NH 2 , and n is an integer.
  • a swellable clay mineral that can be peeled in layers is used, and particularly preferably dispersed in water in a molecular (single layer) or within 1 to 10 layers for uniform dispersion.
  • Possible clay minerals are used.
  • water-swellable smectite or water-swellable mica is used.
  • water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, water-swellable synthetic mica, etc. Can be mentioned.
  • the layered exfoliated clay mineral (B) and the polymer compound (A) interact to form a three-dimensional network.
  • the interaction between the clay mineral and the polymer compound (A) is one or more of ionic bond, hydrogen bond, hydrophobic bond, coordination bond, and covalent bond.
  • the amide group and / or ester group of the polymer compound (A) and the clay mineral (B) form a three-dimensional network by hydrogen bonding.
  • organic or inorganic functional molecules and particles can be added for the purpose of improving or controlling physical properties, as long as such three-dimensional network formation is not hindered.
  • inorganic particles it is effective to use nanoparticles such as silica, titania, zirconia, palladium, silver, gold, and platinum together.
  • the mass ratio (B / A) of the clay mineral (B) to the polymer compound (A) is preferably 0.03 to 3, more preferably 0.04 to 1. .5, particularly preferably 0.05 to 0.5.
  • the mass ratio is 0.03 or less, improvement of mechanical properties tends to be insufficient, and when it is 3 or more, uniform fine dispersion of the clay mineral is often difficult.
  • any of simple proteins including oligopeptides and polypeptides and complex proteins such as glycoproteins, lipoproteins, and phosphorylated proteins can be used. These may be natural products derived from living organisms, artificial synthetic products, or products produced from bacterial cells or the like by DNA recombination. Furthermore, these are preferably functional proteins having some physiological activity, and examples thereof include serum proteins, enzymes, immunoactive substances, antithrombotic substances, cell adhesion factors, hormones, cytokines, bacteria, viruses and the like. .
  • functional proteins include amylase, protease, lipase, cellulase, peroxidase, oxidase, dehydrogenase, gelatin, collagen, fibronectin, albumin, IgG, macroglobulin, blood coagulation factor, monoclonal antibody, polyclonal antibody, etc. Is done. One or more of these are selected and used.
  • the content of the protein (C) used in the present invention in the organic / inorganic composite hydrogel can be uniformly dispersed but can be set according to the purpose within a concentration range.
  • the solvent used for the formation of the protein-containing organic / inorganic composite hydrogel gel of the present invention is water, but as long as the protein-containing gel is formed, it is miscible with water and mixed with water-soluble monomers, clay minerals and proteins in a uniform solution. It is also possible to include an organic solvent that is miscible with the water that forms. Further, as long as a uniform solution is formed, an aqueous solution containing a salt or the like can also be used.
  • organic solvent miscible with water examples include methanol, ethanol, propanol, acetone, methyl ethyl ketone, methyl isobutyl ketone, tetrahydrofuran, dimethylacetamide, dimethylformamide, dimethyl sulfoxide, and a mixed solvent thereof.
  • the protein-containing organic / inorganic composite gel of the present invention is a gel containing water and protein in a three-dimensional network formed by complexing crosslinked polyethylene glycol chains and clay minerals. That is, proteins interact with each other (ionic interactions, hydrogen bonds, hydrophobic interactions, chelates) in a three-dimensional network formed by complexing crosslinked polyethylene glycol chains and clay minerals in water at the molecular level. It has the characteristics of being dispersed finely by the action of formation, polymer entanglement, etc.
  • protein stability is improved by supporting the protein in the organic / inorganic composite gel.
  • a protein (enzyme) -containing organic / inorganic composite gel is held in 37 ° C. water and the enzyme activity in the surrounding liquid is measured, the enzyme is simply placed in the 37 ° C. water. In comparison, high enzyme activity is observed over a long period of time. This means that the enzyme was stabilized by supporting the enzyme in the organic / inorganic composite gel and by supporting the enzyme in the degradation product. .
  • the organic / inorganic composite hydrogel in the present invention was uniform and transparent regardless of the inorganic content, and no aggregation of clay minerals was observed.
  • the final clay mineral content is measured by thermogravimetric analysis (TGA), and the fine dispersibility is measured by transmission electron microscope (TEM) observation.
  • TGA thermogravimetric analysis
  • TEM transmission electron microscope
  • the organic / inorganic composite hydrogel obtained in the present invention is characterized by not only exhibiting excellent mechanical properties but also controlling the mechanical properties in a wide range.
  • FIG. 1 change in stress / strain curve in the tensile test of the organic / inorganic composite hydrogel when the clay concentration is changed
  • the organic / inorganic composite hydrogel almost always exhibits a breaking elongation of 500% or more.
  • polyethylene glycol hydrogel not complexed with clay minerals it showed a large extensibility.
  • tensile strength and elastic modulus physical properties were controlled in a wide range including those showing higher tensile strength and elastic modulus than polyethylene glycol hydrogel.
  • the organic-inorganic composite hydrogel of the present invention is obtained by using a compound having an ester group at least partially and a polyethylene glycol chain chemically crosslinked by an amide bond as the polymer compound (A).
  • This degradable gel has the property of degrading in water, in an aqueous inorganic salt solution such as physiological saline, in an aqueous solution containing an organic compound such as protein, and in an atmosphere such as high humidity or in vivo.
  • Decomposition rate varies depending on the atmospheric conditions (eg, atmospheric composition, pH, temperature, and retention time), and also depends on the gel composition (eg, polymer compound composition, clay mineral content, liquid content, medium composition). Change.
  • decomposability is often high.
  • Degradability is particularly high when the pH of the atmosphere is strongly acidic (for example, pH 3 or lower) and strong alkali (pH 10 or higher).
  • pH 3 or lower for example, pH 3 or lower
  • strong alkali pH 10 or higher
  • the degradation of the present invention also occurs under mild conditions centering on pH 7 during this period. This is a characteristic of degradable gels.
  • the composition of the degradable gel in the present invention as a general tendency, the larger the amount of clay mineral, the higher the degradability, and the higher the liquid content, the higher the degradability.
  • the gel containing no ester group has no or very low decomposability, and preferably the molar ratio of the ester group to the amide bond is 0.1 to 2.
  • the molar ratio of the ester group to the amide bond is 0.1 or less, the decomposability is often insufficient, and when the ratio is 2 or more, it is often difficult to form a uniform gel with high mechanical properties.
  • water or an aqueous solution is used as the medium composition of the gel.
  • a gel containing phosphoric acid or pyrophosphoric acid is preferably used in order to improve the decomposability.
  • pyrophosphoric acid may be unsuitable for applications that place importance on the biological safety (eg, cytotoxicity) of degradation products, and water or aqueous solutions containing phosphoric acid are preferred, with water being particularly preferred. .
  • the degradation mechanism of the degradable gel in the present invention is not necessarily limited, but it is presumed that the degradation is mainly at the ester group and / or amide group, particularly at the ester group. Further, when the clay mineral (B) is not included, it is presumed that the interaction between these functional groups and the clay mineral works effectively on the decomposability because it has no or very low degradability.
  • the organic / inorganic composite hydrogel in the present invention is preferably prepared by previously mixing an aqueous solution of a compound having a polyethylene glycol chain and a plurality of reactive functional groups in the same molecule with a layered exfoliated clay mineral aqueous dispersion, A compounding technique is used that promotes the crosslinking reaction of the compound. More preferably, in order to obtain an organic / inorganic composite hydrogel having excellent uniformity and mechanical properties, the following is performed which is different from the conventionally reported method for synthesizing polyethylene glycol hydrogel (see Non-Patent Document 2). (1) Use a solution prepared by adding hydrochloric acid to sodium pyrophosphate to adjust the pH.
  • a buffer using pyrophosphate is more effectively used than a commonly used phosphate buffer.
  • the pH adjusted by adding hydrochloric acid to sodium pyrophosphate also known as sodium diphosphate
  • Pyrophosphate is a dimer of phosphoric acid that is generally used.
  • phosphorus pyrophosphate is used to stably delaminate clay minerals and to improve the mechanical properties of the resulting polyethylene glycol hydrogel and organic / inorganic composite hydrogel. Works more effectively than acids.
  • pyrophosphoric acid is not always necessary. In particular, when the safety of decomposition products is important, it is preferably removed by washing with water.
  • a method of combining polyethylene glycol and layered exfoliated clay mineral it is preferable to use a mixture of polymer compound (A) and layered exfoliated clay mineral in advance, more preferably layered exfoliated clay mineral is more stable. It can be mixed with one reactive 4-chain polyethylene glycol (in the case of Non-Patent Document 2, amine-terminated polyethylene glycol) and then mixed with another reactive 4-chain polyethylene glycol and reacted. Used. In the above reaction, the use of pyrophosphoric acid as a buffer and the clay mineral / polyethylene glycol mass ratio of 0.03 to 3 are used in combination, and organic and inorganic having both excellent uniformity and mechanical properties. A composite hydrogel is obtained.
  • the degradable gels obtained in the present invention have in-vivo safety and biocompatibility, and can be used as degradable biological implant materials and drug delivery systems.
  • the degradable gel in the present invention can be used in various shapes depending on the purpose such as columnar shape, rod shape, film shape, and thread shape at the time of embedding. And the clay mineral concentration and liquid content).
  • the degradable gel obtained in the present invention can be used alone or in combination with a synthetic or natural biocompatible material or biodegradable material having other particle, fiber, film, mesh, or coating forms. When combined, it is effectively used as a bio-implanting material excellent in handleability, mechanical properties, biocompatibility, biodegradability, and safety after degradation.
  • the degradable gel in the present invention it is also possible to include compounds that are significant for the living body in the degradable gel and to release them gradually along with the degradation.
  • it is effectively used as a material taking advantage of degradability in the medical / pharmaceutical field other than the bio-implantation field, and in other industrial fields (for example, fields such as agriculture, industry, electronic materials, civil engineering construction, and packaging materials).
  • Example 1 The clay mineral is washed with a water-swellable synthetic hectorite (trademark Laponite XLG, manufactured by Rockwood) having a composition of [Mg 5.34 Li 0.66 Si 8 O 20 (OH) 4 ] Na + 0.66 Thereafter, it was lyophilized before use.
  • Reactive four-chain polyethylene glycol uses SUNBRIGHT PTE200GS (hereinafter abbreviated as PTE200GS) and SUNBRIGHT PTE200PA (weight average molecular weight 20000, hereinafter abbreviated as PTE200PA) (both manufactured by NOF Corporation) having a weight average molecular weight of 20000. It was.
  • Laponite XLG (0.064 g) was dispersed in 3 ml of an aqueous solution adjusted to pH 7.4 by adding hydrochloric acid to 100 mM sodium pyrophosphate (anhydrous) (also known as sodium diphosphate).
  • 240 mg of PTE200PA was added and mixed uniformly.
  • hydrochloric acid was added to 100 mM sodium pyrophosphate
  • 240 mg of PTE200GS was dissolved in 1 ml of an aqueous solution adjusted to pH 7.2.
  • the obtained PTE200PA / clay aqueous solution and PTE200GS aqueous solution were cooled in an ice bath, mixed, and stirred vigorously for 15 seconds.
  • the mixed solution was filled in an 80 ⁇ 50 ⁇ 1 mm glass container and reacted at 25 ° C. for 2 hours. As a result, a transparent and homogeneous hydrogel was obtained.
  • the obtained hydrogel was washed in 800 ml of water (20 ° C.) while changing water four times in the middle. As a result of fluorescent X-ray measurement, no outflow of polyethylene glycol and clay mineral was observed during the cleaning process. Further, after the hydrogel was dried, thermal mass analysis up to 800 ° C. (TG-DTA220 manufactured by Seiko Denshi Kogyo Co., Ltd .: air flow, temperature increase: 10 ° C./min) was performed to determine the clay content. The clay content (clay mineral / total solid content) was 12.8% by mass, which almost coincided with the calculated value (11.8% by mass) from the reaction solution composition.
  • Examples 2 to 4 The synthesis was performed in the same manner as in Example 1 except that 0.032 g (Example 2), 0.32 g (Example 3) and 0.64 g (Example 4) of clay mineral (Laponite XLG) were used. As a result, all have the same composition as calculated from the reaction solution composition, and the mass ratio of clay mineral / polyethylene glycol is 0.067 (Example 2), 0.67 (Example 3), 1.34 (Examples). 4) A uniform and transparent organic / inorganic composite hydrogel was obtained. However, Example 4 is translucent. The result of the tensile test measured in the same manner as in Example 1 is also shown in FIG. The elongation at break was controlled in a wide range from 700 to 1020%, the break strength from 50 to 170 kPa, and the elastic modulus from 28 to 12 kPa.
  • Example 9 The clay mineral is washed with a water-swellable synthetic hectorite (trademark Laponite XLG, manufactured by Rockwood) having a composition of [Mg 5.34 Li 0.66 Si 8 O 20 (OH) 4 ] Na + 0.66 Thereafter, it was lyophilized before use.
  • Reactive four-chain polyethylene glycol uses SUNBRIGHT PTE200GS (hereinafter abbreviated as PTE200GS) and SUNBRIGHT PTE200PA (weight average molecular weight 20000, hereinafter abbreviated as PTE200PA) (both manufactured by NOF Corporation) having a weight average molecular weight of 20000. It was. Note that PTE200GS contains one ester group in a single chain.
  • Laponite XLG 0.64 g of Laponite XLG was dispersed in 30 ml of an aqueous solution adjusted to pH 7.4 by adding hydrochloric acid to 100 mM sodium pyrophosphate (anhydrous) (also known as sodium diphosphate).
  • hydrochloric acid was added to 100 mM sodium pyrophosphate
  • 2.4 g of PTE200GS was dissolved in 10 ml of an aqueous solution adjusted to pH 7.2.
  • the obtained PTE200PA / clay aqueous solution and PTE200GS aqueous solution were cooled in an ice bath, mixed, and stirred vigorously for 15 seconds.
  • the mixed solution was filled in several 80 ⁇ 50 ⁇ 2 mm glass containers and reacted at 25 ° C. for 2 hours.
  • two reactive 4-chain polyethylene glycols were chemically cross-linked by amide bonds, and a transparent and homogeneous hydrogel was obtained.
  • the obtained hydrogel was washed in 800 ml of water (20 ° C.) for 20 hours while changing water four times in the middle. As a result of fluorescent X-ray measurement, no outflow of polyethylene glycol and clay mineral was observed during the cleaning process. Further, after the hydrogel was dried, thermal mass analysis up to 800 ° C. (TG-DTA220 manufactured by Seiko Denshi Kogyo Co., Ltd .: air flow, temperature increase: 10 ° C./min) was performed to determine the clay content. The clay content (clay mineral / polymer) was 13.0% by mass, which almost coincided with the calculated value (13.4% by mass) from the reaction solution composition.
  • FT-IR Fourier transform infrared absorption spectrum
  • the obtained washed organic / inorganic composite gel was cut into a size of 5 ⁇ 5 ⁇ 2 mm, and was washed in water at 20 ° C. (Example 9), 37 ° C. (Example 10), and 60 ° C. (Example 11). (100 g water / 1 g gel), and after a certain period of time, it is filtered with a SUS filter (mesh # 400), and the mass fraction (percentage) of the recovered gel solids to the solids of the first gel Degradability was evaluated. As a result, the mass fraction of the gel collected in 125 days in Example 9, 16 days in Example 10, and 4 days in Example 11 was almost 0%, and the organic / inorganic composite gel was decomposed under these conditions.
  • the liquid obtained by decomposing the gel is added to the medium (5% FBS, 1% sodium pyruvate, 1% P / S, MEM) so that the gel component contained in the medium is 0 to 1000 ppm.
  • the cytotoxicity test was conducted using V79 cells (125 cells / 5 ml / dish). None of Examples 9 to 11 showed cytotoxicity (the cytotoxicity evaluation results of Example 9 are shown in FIG. 2).
  • Example 12 The organic / inorganic composite gel obtained by synthesizing in the same manner as in Example 9 except that 0.48 g of Laponite XLG was used was cut into 80 ⁇ 10 ⁇ 2 mm, and then put into a glass hermetic container (50 ml), and 60 ° C. 5 hours (Example 12) and 24 hours (Example 13), after holding in a thermostatic chamber, the sample was taken out and subjected to a stretching test in the same manner as in Example 9. As a result, as shown in FIG. 3, compared to the gel that was not held in the 60 ° C. hermetically sealed container, the strength and elastic modulus decreased in Example 12, and in Example 13, the shape of the gel began to collapse, In addition, it was weak and could not be stretched.
  • Example 14 to 16 An organic-inorganic composite gel washed with water was obtained in the same manner as in Example 9 except that 1.6 g (Examples 14 and 15) or 3.2 g (Example 16) of Laponite XLG was used. Thereafter, in the same manner as in Example 9, the mass fraction of the recovered gel was measured by keeping it in water at 37 ° C. in Example 14 and Example 16, and in 60 ° C. in Example 15. As a result, the mass fraction of the recovered gel was 0% in 12.5 days in Example 14, 2.4 days in Example 15, and 9 days in Example 16, and the gel was almost decomposed. Was observed.
  • Example 17 The organic / inorganic composite gel synthesized in Example 9 and washed with water was kept in 37 ° C. physiological saline instead of in water, and the gel was decomposed in the same manner as in Example 9. evaluated. As a result, the mass fraction of the collected gel became 0% after 7.5 days.
  • Example 18 The organic-inorganic composite gel obtained by synthesis in Example 9 was washed in water for 20 hours, then in Example 18 in a 100 mM phosphate buffer aqueous solution (pH 7.4), and in Example 19 100 mM pyrophosphate. It was immersed in a buffer aqueous solution (pH 7.4) for 4 hours. The obtained gel was held in 37 ° C. water in the same manner as in Example 10 to evaluate the degradability of the gel. As a result, the mass fraction of the gel recovered after 14 days in Example 18 and 13 days in Example 19 was 0%, confirming faster degradability than in the case of only water washing (Example 10). .
  • Comparative Example 3 it was observed that the gel was degraded after 10 days. As a result of performing the cytotoxicity test of the obtained degradation solution in the same manner as in Example 11, cytotoxicity was observed. .
  • the results of the cytotoxicity test in Comparative Example 3 are shown in FIG.
  • Example 20 Instead of SUNBRIGHT PTE200GS containing one ester group in one ethylene glycol chain, the same procedure as in Example 9 was used except that SUNRIGHT PTE200HS (made by NOF Corporation) having the same molecular weight and no ester group was used.
  • An organic / inorganic composite gel was prepared to produce the organic / inorganic composite gel of Example 20.
  • Example 20 Thereafter, in the same manner as in Example 9, the decomposability in water of the washed organic-inorganic composite gel was evaluated. As a result, the gel of Example 20 could not be confirmed to be decomposed by evaluation for one month.
  • Example 21 Comparative Example 7
  • Example 21 the organic / inorganic composite gel synthesized in Example 9 and washed with water was used.
  • Comparative Example 7 the gel synthesized in Comparative Example 1 and washed with water was used.
  • Example 21 when the sample was taken under the skin of the goat and taken out one month later, it was decomposed to such a degree that a slight amount of gel residue was seen, whereas in Comparative Example 7, the same shape in the initial stage of implantation was firmly observed. A gel was obtained.
  • Example 22 The clay mineral is washed with a water-swellable synthetic hectorite (trademark Laponite XLG, manufactured by Rockwood) having a composition of [Mg 5.34 Li 0.66 Si 8 O 20 (OH) 4 ] Na + 0.66 Thereafter, it was lyophilized before use.
  • Reactive four-chain polyethylene glycol uses SUNBRIGHT PTE200GS (hereinafter abbreviated as PTE200GS) and SUNBRIGHT PTE200PA (weight average molecular weight 20000, hereinafter abbreviated as PTE200PA) (both manufactured by NOF Corporation) having a weight average molecular weight of 20000. It was.
  • Laponite XLG 0.16 g was dispersed in 3 ml of an aqueous solution adjusted to pH 7.4 by adding hydrochloric acid to 100 mM sodium pyrophosphate (anhydrous) (also known as sodium diphosphate).
  • hydrochloric acid 100 mM sodium pyrophosphate (anhydrous) (also known as sodium diphosphate).
  • HRP enzyme Horse radish peroxidase: hemeprotein: molecular weight 44000, EC No. 1.11.1.7
  • hydrochloric acid was added to 100 mM sodium pyrophosphate
  • 240 mg of PTE200GS was dissolved in 1 ml of an aqueous solution adjusted to pH 7.2.
  • 1 g of the obtained gel was cut out, put into 100 ml of water (37 ° C.) and held for 12 days. 200 ⁇ l of liquid was sampled at predetermined time intervals during 12 days, and enzyme activity was measured. In the measurement method, the sampled solution was kept at 37 ° C., and STE buffer (composition: 100 mM Tris-HCl, pH 8.0, 1 M NaCl, 10 mM EDTA) 5 ⁇ l, solution 45 ⁇ l, TMB (3, 3 ′, 5, 5 '-tetramethylbenzidine) substrate (50 ⁇ l) was added and reacted at 25 ° C. for 1 minute.
  • STE buffer composition: 100 mM Tris-HCl, pH 8.0, 1 M NaCl, 10 mM EDTA
  • Example 23, Comparative Example 10 The HRP enzyme-containing organic / inorganic composite gel obtained in Example 22 was transferred to a sealed container, and in Example 23, it was stored at 37 ° C. for 4 days. Thereafter, 1 g of the gel was immersed in 100 g of water (37 ° C.) and held for 1 day, and water was sampled to measure enzyme activity in the same manner as in Example 22. In Comparative Example 10, HRP was kept in 37 ° C. water for 4 days, and further kept in 37 ° C. water for 1 day, and then the water was sampled and the enzyme activity was similarly examined. In addition, the enzyme activity retention rate of Example 23 and Comparative Example 10 was displayed with the value measured before maintaining at 37 ° C. as 100%. Example 23 showed higher activity than Comparative Example 10.

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Abstract

La présente invention a pour objet un hydrogel macromoléculaire, contenant du polyéthylène glycol en tant que composant organique, et qui possède des propriétés mécaniques, une flexibilité, et une transparence améliorées et une résistance à la traction et un rapport d’étirement particulièrement améliorés. L’hydrogel macromoléculaire se décompose dans une atmosphère modérée mais les composants de post-décomposition sont non toxiques, l’hydrogel peut ainsi être utilisé en tant que matériau pour bio-implants. Aussi, des protéines peuvent être incorporées dans l’hydrogel macromoléculaire ; les protéines incorporées sont stabilisées thermiquement, et sont donc capables de fonctionner même à des températures supérieures. L’hydrogel composite organique/inorganique est un conjugué : d’un composé macromoléculaire (A), comprenant une structure ramifiée ou une structure en réseau, une pluralité de chaînes polyéthylène glycol étant chimiquement réticulées ; et d’un minéral argileux (B), qui a subi une séparation lamellaire. La présente invention concerne également un procédé de production pour l’hydrogel macromoléculaire.
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WO2019088289A1 (fr) * 2017-11-06 2019-05-09 国立大学法人群馬大学 Hydrogel autoportant et procédé de production associé

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WO2019088289A1 (fr) * 2017-11-06 2019-05-09 国立大学法人群馬大学 Hydrogel autoportant et procédé de production associé

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