JP2012024050A - Protein containing organic inorganic compound hydrogel, method of producing the same, and method of stabilizing protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein containing organic inorganic compound hydrogel in which thermal stability of a protein is raised, and which can demonstrate function also at a higher temperature, and to provide a method of producing the same, and a method of stabilizing a protein.SOLUTION: The stability of a protein is improved by using the protein containing organic inorganic compound hydrogel which contains a protein in a hydrogel or the decomposition product thereof in which a polymer which has a branching structure or a network structure in which two or more polyethylene glycol chains are chemically crosslinked and a clay mineral which carried out stratified peeling are compounded.

Description

  The present invention relates to a method for stably using a protein.

  A protein is a string-like polymer in which amino acids are linked one-dimensionally, and the string is folded to create a specific three-dimensional structure. As a result, 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. .

  In this way, 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.

  Heretofore, several methods have been studied to improve the heat resistance of proteins. For example, a method for obtaining an improved protein by substituting amino acid residues in a protein (Patent Document 1), a method for obtaining a thermostable protein by obtaining a mutant enzyme (Patent Document 2), a protein using a cationic surfactant And a method for thermally stabilizing (Patent Document 3) and the like. However, sufficient results are not always obtained for effective heat resistance improvement.

JP 2009-118749 A JP 2001-78786 A Special table 2004-500005

  The problem to be solved by the present invention is to provide a protein-containing organic-inorganic composite hydrogel that can increase the thermal stability of the protein and exhibit its function even at higher temperatures, a method for producing the same, and a method for stabilizing the protein. is there.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have uniformly dispersed finely dispersed clay minerals in a cross-linked polyethylene glycol so that they form a three-dimensional network. It has been found that the thermal stability of the protein is improved in the organic organic / inorganic composite gel containing the protein and the degradation product of the gel, and the present invention has been completed.

  That is, the present invention relates to a hydrogel in which a polymer compound (A) having a branched structure or network structure in which a plurality of polyethylene glycol chains are chemically cross-linked and a clay mineral (B) exfoliated in a layer form, or a decomposition product thereof. A protein-containing organic-inorganic composite hydrogel characterized by containing a protein (C).

  The present invention also provides a living body implant material using the protein-containing 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.
In the presence of the delaminated clay mineral and protein (C),
A compound (a1) having a polyethylene glycol chain and a plurality of reactive functional groups (Q1) in the same molecule;
Performing a step of reacting the compound (a2) having a plurality of reactive functional groups (Q2) capable of reacting with the reactive functional group (Q1),
A method for producing the protein-containing organic-inorganic composite hydrogel described above is provided.

  Furthermore, the present invention provides an organic-inorganic composite hydrogel in which a polymer compound (A) having a branched structure or network structure in which a plurality of polyethylene glycol chains are chemically cross-linked and a clay mineral (B) exfoliated in a layer form is combined. Provided is a protein stabilization method characterized by improving the stability of the protein (C) by containing the protein (C) in the decomposition product.

  According to the present invention, the thermal stability of the protein is improved, and for example, the effect that the enzyme activity of the enzyme is continued at a higher temperature for a long time can be obtained.

It is a figure which shows the time change of the activity of the enzyme (Horse radish peroxidase) in Example 1, Comparative Example 1, and Comparative Example 2. It is a figure which shows the change of the activity of the enzyme (Horse radish peroxidase) in Example 2 and Comparative Example 3.

  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. In particular, it is preferable that a part of the polymer chain has a functional group that causes 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.

  Among them, the chemical crosslinking of the polymer compound (A) is due to 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.

  Furthermore, 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.

  As the compound (a1) or compound (a2), for example, a compound represented by the following formula (4) or formula (5) can be used.

  In the above formula (4) and formula (5), R is a group represented by the following formula (3), formula (6) to formula (11), and n is an integer of 1 or more. Moreover, 50-1000 are preferable, as for the sum total of four n in 1 molecule, 100-800 are more preferable, and 150-500 are especially preferable.

  The weight average molecular weight of the compounds represented by the formulas (4) and (5) is preferably 1000 to 100,000, more preferably 5000 to 50000, and particularly preferably 5000 to 40000.

As a commercial product of such a compound,
(1) 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)
(2) R is the type PTE-100HS (weight average molecular weight 10,000) of the above formula (6), PTE-200HS (weight average molecular weight 20000), PTE-400HS (weight average molecular weight 40000)
(3) R is type PTE-100MA (weight average molecular weight 10,000) of the above formula (8), PTE-200MA (weight average molecular weight 20000), PTE-400MA (weight average molecular weight 40000)
(4) R is the type PTE-100PA (weight average molecular weight 10,000) of the above formula (9), PTE-150PA (weight average molecular weight 15000), PTE-200PA (weight average molecular weight 20000), PTE-400PA (weight average molecular weight 40000) )
(5) R is type PTE-050SH (weight average molecular weight 5000), PTE-100SH (weight average molecular weight 10000), PTE-200SH (weight average molecular weight 20000) of the above formula (11)
Etc.

  Any group selected from 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.

  Moreover, 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.

  As the polymer compound (A) used in the present invention, it is more preferable to use a compound that has been chemically cross-linked so that the molecular weight between cross-linking points is uniform. Examples of 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. Moreover, although the molecular weight between the crosslinking points of chemical crosslinking becomes 1/2 of the molecular weight of 4-chain polyethylene glycol, the value becomes like this. Preferably it is 1000-50000, More preferably, it is 2000-30000, Most preferably, it is 3000-20000. When the value of the molecular weight between cross-linking points is less than 1000, the flexibility is insufficient or the draw ratio is reduced. On the other hand, if it is larger than 50000, the elastic modulus is too low or the handleability is deteriorated.

  In the present invention, like the polyethylene glycol described in Non-Patent Documents 2 and 7, the compound (a1) is a compound represented by the following formula (1), and the compound (a2) is represented by the following formula (2). It is particularly preferable to use compounds that are both compounds and have a weight average molecular weight of 1,000 to 100,000.

（式中、Ｘは下記式（３）

(In the formula, X represents the following formula (3)

で表される基であり、ｎは整数である。）

And n is an integer. )

（式中、Ｙは-CH₂CH₂CH₂NH₂で表される基であり、ｎは整数である。）

(In the formula, Y is a group represented by —CH ₂ CH ₂ CH ₂ NH ₂ , and n is an integer.)

  The crosslinking reaction of the compound represented by the above formula (1) and the compound represented by the formula (2) is shown by the following reaction formula.

  As the clay mineral (B) used in the present invention, a swellable clay mineral that can be peeled in layers is used, and particularly preferably, it is molecularly dispersed (single layer) in water or layered within 1 to 10 layers for uniform dispersion. Possible clay minerals are used. For example, water-swellable smectite or water-swellable mica is used. Specifically, water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, water-swellable synthetic mica, etc. Can be mentioned.

  In the present invention, it is preferable that the layered exfoliated clay mineral (B) and the polymer compound (A) interact to form a three-dimensional network. As long as an effective three-dimensional network can be formed, 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. Good. Particularly preferably, 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.

  It should be noted that various organic or inorganic functional molecules or particles can be added for the purpose of improving or controlling physical properties, as long as such three-dimensional network formation is not hindered. For example, as inorganic particles, it is effective to use nanoparticles such as silica, titania, zirconia, palladium, silver, gold, and platinum together.

  In the organic / inorganic composite hydrogel in the present invention, 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. When 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.

  As the protein (C) used in the present invention, 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. .

  Specific examples of 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. In addition, an aqueous solution containing a salt or the like can be used as long as a uniform solution is formed.

  Examples of the organic solvent miscible with water 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.

  The protein-containing 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. In the present invention, it is confirmed by TGA that the total amount of clay used is contained in the composite, and TEM indicates that one or two to ten layers of exfoliated clay layers are uniformly dispersed. confirmed. Moreover, the protein-containing organic / inorganic composite hydrogel obtained in the present invention exhibited excellent mechanical properties, and in most cases, exhibited elongation at break of 500% or more. Further, the tensile strength and elastic modulus can be controlled in a wide range.

  In the present invention, the protein stability is improved by supporting the protein in the organic / inorganic composite gel. Specifically, for example, when 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 protein-containing 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, a layered exfoliated clay mineral aqueous dispersion, and a protein. Then, a composite method is used to advance the crosslinking reaction of the compound. More preferably, in order to obtain a protein-containing organic / inorganic composite hydrogel having excellent uniformity and mechanical properties, the following is performed.
(1) Use a solution prepared by adding hydrochloric acid to sodium pyrophosphate to adjust the pH.
(2) Either one of the above-mentioned compound (a1) and compound (a2) is added to the clay mineral aqueous dispersion which has been exfoliated in advance, and then the other compound is dissolved.
(3) The layered exfoliated clay mineral is adjusted so that the mass of the clay mineral / the total mass of the compound (a1) and the compound (a2) is 0.03 to 3.
More preferably,
(4) After synthesizing the protein-containing organic / inorganic composite hydrogel, pyrophosphate is removed by washing.

  As 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 is used to mix with one reactive 4-chain polyethylene glycol present and then mix with another reactive 4-chain polyethylene glycol to react. In the above reaction, pyrophosphoric acid is used as a buffer, protein is allowed to coexist, and a clay mineral / polyethylene glycol mass ratio of 0.03 to 3 is used in combination. A protein-containing organic / inorganic composite hydrogel having both mechanical properties can be obtained.

  Furthermore, many of the protein-containing organic / inorganic composite gels having degradability obtained in the present invention have in-vivo safety and biocompatibility, and can be used as degradable bio-implantable materials and drug delivery systems. is there. The protein-containing organic / inorganic composite gel having degradability according to 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. Physical properties can also be controlled over a wide range by gel composition (concentration of polymer compound or clay mineral, liquid content). Moreover, the protein-containing organic / inorganic composite gel having degradability obtained in the present invention can be used alone or can be decomposed and used as an aqueous dispersion.

  EXAMPLES Next, although an Example demonstrates this invention more concretely, this invention is not limited only to the Example shown below from the first.

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 0.16 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). Next, 240 mg of PTE200PA and 320 μg of HRP enzyme (Horse radish peroxidase: hemeprotein: molecular weight 44000, EC No. 1.11.1.7) were added and mixed uniformly. Separately, hydrochloric acid was added to 100 mM sodium pyrophosphate, and 240 mg of PTE200GS was dissolved in 1 ml of an aqueous solution adjusted to pH 7.2. Next, the obtained PTE200PA / clay / HRP 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 mm × 50 mm × 1 mm glass container and reacted at 25 ° C. for 2 hours. As a result, a transparent and homogeneous hydrogel was obtained.

  1 g of the obtained gel was cut out, put into 100 ml of water (37 ° C.), and held for 12 days. 200 μl 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 μl, solution 45 μl, TMB (3,3 ′, 5,5) were placed in a 96-well plate. '-tetramethylbenzidine) substrate (50 μl) was added and reacted at 25 ° C. for 1 minute. Then, 1 N hydrochloric acid (50 μl) was added to stop the enzyme reaction, and then the absorbance at 450 nm was measured with a plate reader. The enzyme activity was evaluated based on the absorbance (ABS / μL solution).

  The results are shown in FIG. Compared to Comparative Example 1 (HRP dispersed and held in 37 ° C. water) and Comparative Example 2 (HRP dispersed and held in 37 ° C. clay aqueous solution), it shows a high activity and is in a decomposition product of organic / inorganic composite gel. It was found that the enzyme contained in had improved thermal stability.

(Comparative Example 1)
HRP enzyme is not supported in the organic / inorganic composite gel, but 0.8 μg of HRP (along with the amount of HRP supported in Example 1) is placed in 100 ml of water (37 ° C.) and held for 12 days. 200 μl each was collected at the time of, and the enzyme activity was evaluated in the same manner as in Example 1. The result is shown in FIG. Showed low enzyme activity.

(Comparative Example 2)
Without supporting the HRP enzyme in the organic / inorganic composite gel, 0.8 μg of HRP (along with the amount of HRP supported in Example 1) was put in 100 ml of an aqueous clay solution (37 ° C.) containing 3.81 g of clay, The sample was held for 12 days, and 200 μl was collected at a predetermined time during that time, and the enzyme activity was evaluated in the same manner as in Example 1. The result is shown in FIG. Almost no enzyme activity was shown.

(Example 2, Comparative Example 3)
The HRP enzyme-containing organic / inorganic composite gel obtained in Example 1 was transferred to a sealed container, and in Example 2, 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 the enzyme activity in the same manner as in Example 1. In Comparative Example 3, HRP was kept in water at 37 ° C. for 4 days, and further kept in water at 37 ° C. 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 2 and Comparative Example 3 was displayed with the value measured before maintaining at 37 ° C. as 100%. Example 2 showed higher activity than Comparative Example 3.

Claims (12)

  Protein (C) in a hydrogel in which a polymer compound (A) having a branched structure or network structure in which a plurality of polyethylene glycol chains are chemically cross-linked and a clay mineral (B) exfoliated in layers is complexed or a decomposition product thereof A protein-containing organic-inorganic composite hydrogel comprising: The protein-containing organic-inorganic composite hydrogel according to claim 1, wherein a mass ratio ((B) / (A)) of the polymer compound (A) and the layered exfoliated clay mineral (B) is 0.03 to 3. The protein-containing organic-inorganic composite hydrogel according to claim 1 or 2, wherein the layered exfoliated clay mineral (B) is a water-swellable inorganic clay mineral. The protein-containing product according to any one of claims 1 to 3, wherein the chemical crosslinking of the polymer compound (A) is based on an amide bond, and (A) and (B) form a three-dimensional network. Organic inorganic composite hydrogel. The protein-containing organic-inorganic composite hydrogel according to claim 4, wherein the polymer compound (A) has an ester group, and the molar ratio of the ester group to the amide bond is 0.1-2. The polymer compound (A) has a compound (a1) having a polyethylene glycol chain and a plurality of reactive functional groups (Q1) in the same molecule, and a plurality of reactions capable of reacting with the reactive functional group (Q1). The protein-containing organic-inorganic composite hydrogel according to claim 1, 2 or 3, which is a compound obtained by reacting a compound (a2) having a functional functional group (Q2). The compound (a1) is a compound represented by the following formula (1), the compound (a2) is a compound represented by the following formula (2), and both have a weight average molecular weight of 1,000 to 100,000. The protein-containing organic-inorganic composite hydrogel described.

(In the formula, X represents the following formula (3)

And n is an integer. )

(In the formula, Y is a group represented by —CH ₂ CH ₂ CH ₂ NH ₂ , and n is an integer.) A biological implant material using the protein-containing organic-inorganic composite hydrogel according to claim 1. An aqueous dispersion of the clay mineral (B) is produced by layering the clay mineral (B) in an aqueous medium,
In the presence of the delaminated clay mineral and protein (C),
A compound (a1) having a polyethylene glycol chain and a plurality of reactive functional groups (Q1) in the same molecule;
Performing a step of reacting the compound (a2) having a plurality of reactive functional groups (Q2) capable of reacting with the reactive functional group (Q1),
The method for producing a protein-containing organic-inorganic composite hydrogel according to any one of claims 1 to 7. The method for producing a protein-containing organic-inorganic composite hydrogel according to claim 9, wherein the reaction between the compound (a1) and the compound (a2) is carried out in the presence of pyrophosphate. The compound (a1) or the compound (a2) is added to the aqueous dispersion of the clay mineral (B) and the protein (C), and then the other compound is added and reacted. The manufacturing method of protein containing organic-inorganic composite hydrogel of description.   An organic-inorganic composite hydrogel in which a polymer compound (A) having a branched structure or network structure in which a plurality of polyethylene glycol chains are chemically cross-linked and a clay mineral (B) exfoliated in a layer form a composite, or a protein in a decomposition product thereof A protein stabilization method comprising improving the stability of the protein (C) by containing (C).

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